Instituto Nacional de Ciência e tecnologia
de Biologia Estrutural e Bioimagem - INBEB
Sessões de pôsteres
Data das apresentações:
Trabalhos de número par: dia 28/11
Trabalhos de número ímpar: dia 29/11
Os resumos estão separados por titulação:
G – Graduandos
Ap – Aperfeiçoamento
Esp – Especialização
TG – Técnico Graduado
M – Mestrandos
D – Doutorandos
PD – Pós-doutorandos
P – Pesquisadores
E ordenados alfabeticamente pelo primeiro nome do apresentador.
Certificados:
Após a arguição dos avaliadores, serão entregues certificados de apresentação apenas
em nome dos apresentadores inscritos que forem avaliados. Os demais autores terão
um caderno de resumos como certificado de inscrição dos trabalhos no evento.
Graduandos:
G01 - Aline Miyoko Sakaguchi
Yamashita
G02 - Alvaro Carrier Ruiz
G03 - Ana Luiza Machado Torres
G04 - Ana Paula Miranda Mendonça
G05 - Andreza Fabiano de Almeida
G06 - Bárbara Rangel
G07 - Bernardo Jorge da Silva Mendes
G08 - Brunno Renato Farias Verçoza
G09 - Camila Hübner Costabile Wendt
G10 - Carolina Neves de Martins
G11 - Clarissa Werneck Ribeiro
G12 - Claudia Monteiro da Rocha
G13 - Dayana de Souza Freire
G14 - Eliã Barbosa Marins
G15 - Felipe Moraes dos Santos
G16 - Fernanda R. Figueiredo
G17 - Frederico Matheus de Angelis
Santanna Reis
G18 - Gabriela Veras de Moraes
G19 - Gabriele Machado Santos
G20 - Gabriellen Vitiello
G21 - Giulia Diniz da Silva Ferretti
G22 - Henrique Lisboa Mendes Borges
Soares
G23 - Ingrid Tavares Fragoso
G24 - Isabelle Ribeiro de Souza
Oliveira
G25 - Izabella Sodré Buty da Silva
G26 - Jefferson Cypriano
G27 - Juliana Vianna Lopes
G28 - Karol Torquato
G29 - Luiz Felipe Garcia e Souza
G30 - Luiza Helena Daltro Cardoso
G31 - Maiara de Oliveira Maia
G32 - Marcella Valentim Monteiro
Ferreira
G33 - Marcelo Pereira Rodrigues
G34 - Marcos Vinicius Fraga
G35 - Mariana Dias Carreiras
G36 - Marianne Melo Monnerat
G37 - Marina de Almeida Ferreira
G38 - Nathali Pereira da Costa
Campos
Aperfeiçoamento:
Ap01 - Isalira Peroba Rezende Ramos
Barbosa
G39 - Nathália Rocco Machado
G40 - Nicoli Cardoso Mortari
G41 - Paula L. de Souza Conceição
G42 - Pedro Ernesto Lopes Leão
G43 - Rodrigo Vieira Bernardo
G44 - Taiana da Silva Xavier
G45 - Thayana Araujo da Cruz
G46 - Tháyna Sisnande
G47 - Theo Ferreira Marins
G48 - Wesley Junio Alves da
Conceição
G49 - Wildon Novais de Mello
G50 - Yuri Komatsu Damas Abud
Especialização:
Esp01 - Cinthia Lima Rocha
Técnico com graduação:
TG01 - Marcelo Cardoso dos Reis Melo
Mestrandos:
M01 - Aline Yuri Murakami
M02 - Allan Bastos Lima
M03 - Amanda Anatácia Pinto Hage
M04 - Ana Clara Vicente dos Santos
M05 - Beatriz Ferreira de Carvalho
Patricio
M06 - Bruno Macedo da Silva
M07 - Camila Iansen Irion
M08 - Carlos Henrique Dumard
M09 - Carolina de Lima Alcantara
M10 - Danúbia Silva dos Santos
M11 - Felipe Lopes Brum da Silveira
M12 - Giselly da Silva Dias
M13 - Gloria Maria Castañeda
Valencia
M14 - Grazielle Suhett Dias
Doutorandos:
D01 - Adolfo Henrique de Moraes
Silva
D02 - Aline Araujo Zuma
D03 - Aline Lidiane Batista
D04 - Allan Cézar de Azevedo Martins
D05 - Amanda Karolina Soares e Silva
M15 - Jamila Monteiro dos Santos
M16 - Josineide Pantoja da Costa
M17 - Liliani Aparecida Sereno Fontes
M18 - Luciana Pereira
M19 - Luiz Carlos Saramago Gonçalves
M20 - Mônica Cruz
M21 - Raiana Andrade Quintanilha
Barbosa
M22 - Raquel Raick Pereira da Silva
M23 - Rayana Leal de Almeida Luna
M24 - Sara Teixeira de Macedo Silva
M25 - Thiago Britto Borges
M26 - Thiago Pereira de Abreu
M27 - Vanessa Lopes de Azevedo
Braga
D06 - Ana Paula Drummond Rodrigues
D07 - André Luiz Araujo dos Santos
D08 - Andréa Marins Damasceno
Bomfim
D09 - Angela Camila Orbem
Menegatti
D10 - Anne Cristine Silva Fernandes
D11 - Antonio Real Hohn Neto
D12 - Carlos Alberto Marques de
Carvalho
D13 - Caroline Madeira Moreira
D14 - Charles Vargas Lopes
D15 - Claudia Fernanda Dick
D16 - Claudia Maia Brigagão
D17 - Cristiane Latgé de Almeida e
Silva
D18 - Daniela Cosentino Gomes
D19 - Davi da Silva Barbirato
D20 - Elaine da Conceição Petronilho
D21 - Elaine Hilário de Souza
D22 - Elen Gomes Pereira
D23 - Erivan Schnaider Ramos-Junior
D24 - Estefania Pereira Cardoso
Azevedo
D25 - Eugenio Damaceno Hottz
D26 - Éverton Dias D'Andréa
D27 - Fabiana Oliveira dos Santos
Gomes
D28 - Fernanda Magalhães Ferrão
D29 - Fernando Antonio de Oliveira
Adnet
D30 - Fernando Pereira de Almeida
D31 - Gabrielle Moura do Valle
D32 - Glaucia Melina Squizato
Pinheiro
D33 - Guilherme Visconde Brasil
D34 - João Luiz da Silva Filho
D35 - Joseane Lima Prado Godinho
D36 - Juliana Amaral Passipieri
D37 - Karen Tavares Silva
D38 - Karla Patricia de Sousa Barbosa
D39 - Leandro Teixeira de Oliveira
D40 - Letícia Maria Zanphorlin
D41 - Louise Alessandra Mesentier
Louro
D42 - Luciana Elena de Souza Fraga
Machado
D43 - Luis Henrique Seabra de Farias
D44 - Luzia da Silva Sampaio
D45 - Marcelo Damião Ferreira de
Meneses
D46 - Mariana Acquarone
D47 - Mariana Aragão Matos Donato
D48 - Mariana Araya de Godoy
D49 - Michelle Bargas Rega
D50 - Milene Rangel da Costa
D51 - Moara Lemos
D52 - Moema Monteiro Batista
D53 - Myriam de Carvalho Monteiro
D54 - Naira Lígia Lima Giarola
D55 - Natália do Carmo Ferreira
D56 - Nathalia dos Santos Alves
D57 - Paulo André da Silva
D58 - Paulo Henrique Rosado de
Castro
D59 - Pedro Celso Braga Alexandre
D60 - Pedro Henrique Monteiro
Torres
D61 - Phercyles Veiga dos Santos
D62 - Priscila da Silva Figueiredo
Celestino
D63 - Rachel Santos de Menezes
D64 - Rafael Soares Lindoso
D65 - Raquel Rennó Braga
D66 - Reinaldo Souza de Oliveira
Júnior
D67 - Renata Travassos
D68 - Ricardo Luiz Luzardo Filho
D69 - Roberta Fernandes Pinto
D70 - Roberta Ferreira Cura das Neves
D71 - Rodrigo Amancio
D72 - Rosemberg de Oliveira Soares
D73 - Shana Priscila Coutinho Barroso
D74 - Sirlene Oliveira Francisco de
Azeredo
D75 - Sura Wanessa Santos Rocha
D76 - Susana Kelly de Abreu
D77 - Tácio Vinício Amorim Fernandes
D78 - Tais Hanae Kasai Brunswick
D79 - Tatiana Christina Paredes
Santos
D80 - Tatiana Kelly da Silva Fidalgo
D81 - Thais Russo Abrahão
D82 - Thaís Souza Silveira
D83 - Tiago Bortolotto
D84 - Vanessa Aparecida das Chagas
Moutinho
D85 - Victor do Valle Pereira Midlej
D86 - Viviane Cristina Heinzen da Silva
D87 - Wendell Girard Dias
Pós-doutorandos:
PD01 - Bruno Diaz Paredes
PD02 - Camila Zaverucha do Valle
PD03 - Carla Martins de Oliveira
PD04 - Carolina Galvão Sarzedas
PD05 - Catarina Rapôso Dias Carneiro
PD06 - Clarissa Rodrigues Nascimento
PD07 - Daniele dos Santos Andrade
PD08 - David Z. Mokry
PD09 - Diego Enry Barreto Gomes
PD10 - Eduardo José Lopes Torres
PD11 - Erica dos Santos Martins
Duarte
Pesquisadores:
P01 - Andréa Carla de Souza Góes
P02 - Claudia Vitoria de Moura Gallo
P03 - Erik Svensjö
P04 - Gustavo Miranda Rocha
PD12 - Felipe Dias
PD13 - Fernanda de Avila Abreu
PD14 - Joana da Costa Pinto d'Avila
PD15 - Lia Carolina Soares Medeiros
PD16 - Lisandra Marques Gava Borges
PD17 - Louise Moraes
PD18 - Marta Teixeira Gomes
PD19 - Nathalia Varejão
PD20 - Tuane Cristine Ramos
Gonçalves Vieira
PD21 - Viviane Silva de Paula
P05 - Karina Lidianne Alcântara
Saraiva
P06 - Liana Bastos Freitas-Fernandes
P07 - Theo Luiz Ferraz de Souza
Resumos
G01
CARACTERIZAÇÃO DA S-NITROSOGLUTATIONA REDUTASE EM HOMOGENATO DE MÚSCULO ESQUELÉTICO E CÉLULAS C2C12
1-YAMASHITA, A.M.S., 1-MOUSINHO, V.C., 1-FIGUEIREDO-FREITAS, C., 2-MERMELSTEIN, CS., 1-SORENSON, M.M.
1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2-Departamento de histologia e embriologia,
Universidade Federal do Rio de Janeiro.
Durante os últimos 10 anos, enzimas envolvidas no metabolismo do óxido nítrico (NO) têm sido estudadas intensamente. NO é
produzido fisiologicamente no músculo durante ciclos de contração. Entre as vias de transmissão de sinal encontra-se a oxidação
dos grupos tiol em proteínas, formando SNO-proteína. Para controlar o estado redox intracelular, todas as células sintetizam
antioxidantes como a glutationa (GSH). Excesso de NO pode levar a um aumento na produção de S-nitrosoglutationa (GSNO). Uma
enzima importante para controlar os níveis de GSNO é a S-nitrosoglutationa redutase (GSNO-R), que reduz GSNO para GSH. Tem
sido caracterizada a expressão e a atividade da GSNO-R em pulmão, fígado, rim, baço, timo e coração. No entanto, pouco se sabe
sobre essa enzima e seu papel no músculo esquelético. Objetivo: Caracterizar a expressão e a atividade da GSNO-R no músculo
esquelético. Métodos: Foi medida a atividade enzimática da GSNO-R em homogenatos (ratos Wistar) como descrito por Liu et al
(2001), com e sem o inibidor C3 (Sanghani et al, 2009). Western blots utilizaram anticorpos policlonais. Células C2C12 foram
cultivadas a 37°C com DMEM contendo 10% de soro fetal bovino para crescimento e 2% para diferenciação. Resultados: Foi
detectada a atividade da GSNO-R em homogenatos de todos os tecidos testados. A atividade no músculo esquelético foi
semelhante à de outros tecidos, com pequenas variações dependendo do tipo principal de fibras (rápido ou lento). O inibidor C3
(30 uM) inibiu a GSNO-R em ~85% em todos os tecidos testados. A atividade da GSNO-R em homogenatos das células C2C12
aumentou com o grau de diferenciação in vitro (2 a 8 dias). Conclusão:Foi encontrada atividade da GSNO-R em todos os tecidos
testados, inclusive os músculos esqueléticos. O inibidor apresentou ~85% de inibição da GSNO-R. A atividade da GSNO-R das
células C2C12 foi dependente do grau de diferenciação in vitro.
Apoio: INBEB, FAPERJ, CNPq
G02
BONE MARROW-CELLS DERIVED FIBROBLAST GROWTH FACTOR-2 INDUCES GLIAL CELL PROLIFERATION IN THE REGENERATING
PERIPHERAL NERVOUS SYSTEM.
1-CARRIER-RUIZ, A.; 2-LEMES, R.M.R.; 3-REIS, R.A.M.; 1-MENDEZ-OTERO, R.; 1-RIBEIRO-RESENDE, V.T.
1-Laboratório de Neurobiologia Celular e Molecular, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro; 2-Laboratório de Microbiologia Celular, Instituto Oswaldo Cruz, Rio de Janeiro; 3-Laboratório de Neuroquímica, Instituto
de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro.
Bone marrow derived cells can contribute to the regeneration of the PNS in many aspects. Collected data over five years have
shown that soluble factors released by BMDC can play important roles in fundamental steps for nerve regeneration. Our previous
work showed that bone marrow cells induce regeneration acting on both neuronal and glial cell population. FGF-2 is well
described as involved inducing glial cell proliferation during development and regeneration of PNS. We tested the possibility of
release of FGF-2 and his action on peripheral glial cells. Lister hooded rats had their right sciatic nerves transected and both
stumps were connected into a hollow silicon tube. A gap of 4mm was kept between the stumps. Control group received matrigel
diluted in PBS10mM, experimental group received 5,00x106 cells diluted in the same solution and neutralized group received the
same number of cells plus FGF-2 neutralizing antibody by an osmotic pump. Rats were perfused and their nerves, dorsal root
ganglion (DRG) and lumbar spinal cord were removed and frozen sections were obtained. Schwann cell line ST88-14, sciatic nerve
explants and embryonic dorsal root ganglia, were placed onto coverslips and were incubated with DMEM F-12+10% SFB, adding
FGF-2, conditioned medium of mesenchymal cells (CM) or CM plus FGF-2 neutralizing antibody. We observed an increased
expression of FGF-2 in Schwann cells, satellite cells and astrocytes, in rats that received BMMC. Neutralizing FGF-2 antibody was
able to reduce cell proliferation and survival, migration and neuritogenesis. Data were analyzed using one-way analysis of variance
(ANOVA) with Neuman-Keuls post-test for multiple comparisons. We provide evidence for FGF-2 released by BMMC acting on glial
cells of the PNS stimulating cell proliferation, survival and migration. Moreover, BMMC seem to increase FGF-2 expression by the
glia in the regenerating nerve and also act on neurons of the DRG stimulating neuritogenesis.
Support: Faperj, CNPq, CAPES.
G03
CARACTERIZAÇÃO BIOLÓGICA DE CÉLULAS MESENQUIMAIS DA MEDULA ÓSSEA MARCADAS COM NANOPARTÍCULAS DE ÓXIDO
DE FERRO E RASTREAMENTO POR IMAGENS DE RESSONÂNCIA MAGNÉTICA
1-TORRES, A.L.M.; 1-JASMIN; 1-ZAVERUCHA-DO-VALLE, C.; 2,3-TOVAR-MOLL, F.; 3-FERREIRA, F. G. M.; 1-PASSIPIERI, J. A.; 1CAMPOS DE CARVALHO, A. C.; 1-MENDEZ-OTERO, R.
1-Instituto de Biofísica Carlos Chagas Filho, UFRJ; 2-Instituto de Ciências Biomédicas, UFRJ; 3-Centro Nacional de Bioimagem
O uso de células-tronco tem sido descrito como terapia para o tratamento de diversas doenças. O rastreamento in vivo de células
transplantadas poderia responder questões relacionadas ao efeito destas células no tecido lesado. Técnicas como imagens de
ressonância magnética (RM) podem ser utilizadas com esta finalidade, sendo para isso necessária a marcação destas células com
agentes de contraste, como nanopartículas superparamagnéticas de óxido de ferro (SPIONs). Sendo assim, os objetivos deste
trabalho são analisar a eficácia da utilização de SPIONs na marcação de células mesenquimais (MSCs), a influência da utilização
desta técnica nas características biológicas destas células e o posterior rastreamento por RM. Metodologia: Obteve-se cultura de
MSCs de ratos e para marcação das células, estas foram incubadas por 4h ou 24h com Feridex - puro, associado à poli-L-lisina
(FePLL) ou a cloridrato de protamina (FeProt) -, Endorem associado a cloridrato de protamina ou FeraTrack. As taxas de
incorporação e proliferação foram avaliadas através de imunorreação com os anticorpos anti-dextran e Ki67, respectivamente, e a
viabilidade celular com o kit Live/Dead. A permanência de SPIONs nas células foi avaliada até 21 dias após a incubação. Além disso,
foi analisada a capacidade de diferenciação das MSCs. Células previamente marcadas com SPIONs foram injetadas em cérebros de
ratos e foram feitas imagens de RM dos animais. Resultados: Aproximadamente 95% das MSCs incorporaram Feridex nos grupos
contendo os facilitadores de incorporação. A proliferação foi reduzida após 24h de exposição a FePLL. Entretanto, MSCs incubadas
com FeProt mantiveram a capacidade proliferativa e viabilidade. Além disso, a capacidade de diferenciação das células não foi
alterada. Assim, o protocolo mostrou-se eficaz como ferramenta de marcação celular sem comprometer propriedades biológicas
das células. Além disso, foi possível a detecção das células marcadas com SPIONs nas imagens de RM até 7 dias após a injeção de
células.
Apoio: INBEB, FAPERJ, CNPq
G04
ALTERAÇÕES NO METABOLISMO ENERGÉTICO E REDOX EM GLIOBLASTOMAS INDUZIDAS POR FERRO
1- Mendonca,APM, 1-Oliveira MF.
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro.
O Acidente Vascular Cerebral hemorrágico (AVCh) representa um dos acometimentos no Sistema Nervoso Central (SNC) de maior
severidade e, é considerada a 3ª maior causa de morte no mundo, com múltiplas consequências tais como danos cognitivos e
motores permanentes aos pacientes. O dano tecidual causado pelo AVCh é iniciado quando ocorre o rompimento da barreira
hematoencefálica e o extravasamento do conteúdo sanguíneo, fazendo com que o parênquima cerebral seja exposto a uma
grande quantidade de células do sangue contendo hemoglobina, heme e ferro. Estas moléculas são potentes agentes próoxidantes e neurotóxicas, podendo acarretar em morte celular, caso estas não sejam devidamente metabolizadas. É nesse
contexto que este trabalho busca avaliar alterações no metabolismo energético e redox de células do SNC expostas ao ferro, ao
heme e a hemoglobina, já que, dados na literatura mostram que, em relação a neurônios, astrócitos possuem maior resistência à
toxicidade induzida pelo ferro e hemoglobina. Nosso modelo experimental consiste em uma linhagem tumoral (U-87) um
glioblastoma cultivado em meio DMEM - F12 (10% DE SFB). A abordagem experimental se baseia em incubações de 24 horas na
presença de ferro, hemoglobina e heme (1, 5, 10 e 50µM). Feito isso, verificamos a viabilidade celular por ensaio de MTT, ensaio
de extravasamento da enzima lactato desidrogenase (LDH) e exclusão do corante azul de tripan. Nossos dados mostram que essas
células possuem viabilidade de 100% até 50uM de concentração de ferro, heme e hemoglobina, mostrando que a U-87 é
resistente ao dano oxidativo sofrido. Além disso, verificamos o nível de peroxidação lipídica dessas células na presença de ferro
10µM e 50µM de ferro por 24h através do método de TBARS. Observamos que, a medida que a concentração de ferro presente no
meio aumenta, há maior peroxidação lipídica da U-87. Isso nos permite concluir que o dano oxidativo dessas células aumenta de
acordo com a concentração de ferro no meio. Por fim, essas mesmas células incubadas por 24h com 10 µM de ferro, foram
submetidas a respirometria. Esse resultado mostra que a U-87 possui uma redução global na taxa de consumo do oxigênio
(aproximadamente 37-47%) no consumo de oxigênio. Resultados similares são observados em células incubadas com 50µM de
ferro, apresentando uma inibição no consumo de oxigênio de 40-49%. Além disso, a exposição de ferro reduz a capacidade
respiratória das células (60-80%), influenciando na capacidade de síntese de ATP quando a célula precisa de uma demanda maior
de energia. Esses dados podem inferir que há uma disfunção mitocondrial nessas células e que as mesmas são afetadas no seu
metabolismo energético e redox.
Apoio: FAPERJ, CNPq.
G05
EFFECTS OF TOXOPLASMA GONDII ENCYSTATION IN THE HOST CELL ORGANIZATION
1,2- A. F. ALMEIDA ;1,2- T. C. PAREDES-SANTOS; 3- R.W.A. VITOR; 1,2,4- W. DE SOUZA; 1,2- R.C. VOMMARO
1- Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho; 2- Instituto Nacional de Ciência e Tecnologia
em Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro; 3- Universidade Federal de Minas Gerais,
Departamento de Parasitologia, Instituto de Ciências Biológicas; 4- Instituto Nacional de Metrologia, Normalização e Qualidade
Industrial.
Toxoplasma gondii is a protozoan parasite capable of infect all homoeothermic animals.The cyst form of Toxoplasma gondii has a
crucial role in the persistence of the infection as it is protected in the intracellular milieu by a cyst wall. The encystation process
and the following consequences for the host cells are poorly understood. Previous works have already shown that T. gondii can
recruit host organelles, as mitochondria, endoplasmic reticulum, Golgi complex and cytoskeleton filaments to the vicinity of the
parasitophorous vacuole, along the establishment of the infection. In this work we employed microscopy tools to evaluate the
organization of epithelial cell organelles, during the infection by the brazilian cystogenic strain of T. gondii, EGS. Epithelial cells of
the LLC-MK2 lineage were plated in coverslips in 24 well- plates and allowed to interact with tachyzoites, from the supernatant of
previously infected cell cultures, at ratios of 1:10 cell/parasite. To confirm the presence of mature cysts after 10days, infected
cells were stained with Dolichus Biflorus lectin (DBA-FITC), which recognizes the cyst wall N-Acetylgalactosamine residues. Host
cell structures were stained with anti α-tubulin (for microtubules), faloidin (for microfilaments) and anti pan-queratin (for
intermediate filaments). The coverslips were properly mounted with N-propylgalate and observed in a Axioplan Zeiss Microscope
or in a Leica Confocal Microscope SP5.Analysis of positive DBA infected cells showed no remarkable alteration on the
microfilaments organization of infected cells during the encystation process comparing to cells presenting parasitophorous
vacuoles or free of infection . Aggregates of the cyst wall elements can be seen in the periphery of immature cysts Staining with
anti pan-queratin showed that intermediate filaments are arranged surrounding the cysts, as it has been shown for T. gondii
parasitophorous vacuoles, as well as the microtubules, which appeared also arranged around the cyst structure. Experiments with
other fluorescent markers are being carried out to investigate the organization of other sub cellular structures as mitochondria
and the microtubule organization center of the host cell.
Support: CNPq, CAPES,FAPERJ
G06
NEURAL STEM CELLS LABELED WITH SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES: A TOOL TO TRACKING CELLS
TRANSPLANTED IN THE BRAIN BY MRI
1,2- AZEVEDO-PEREIRA, R.L.; 1,2- RANGEL, B.; 1,2- ZAVERUCHA-DO-VALE, C.; 2- MOLL, F.T.; 1,2- RACHID, R.; 1,2- CARVALHO, A.B.;
1,2- ATTIAS, M.; 1,2- MENDEZ-OTERO, R.
1- Instituto de Biofísica Carlos Chagas Filho; 2- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem –
INBEB; UFRJ.
Neural stem cells (NSCs) are a source of new neural cells with capacity to proliferate and differentiate into neurons and glia. This
capacity opens a possibility for replacement cell therapy in neurological disorders. Preclinical and clinical research will benefit from
reliable in vivo tracking of transplanted cells. Here, we investigated the potency of superparamagnetic iron oxide particles (SPION)
to label NSCs and how it affects: i) survival, ii) proliferation, iii) cell migration and iv) differentiation into neural cells. To label NSC,
dissociated neurospheres were incubated overnight with SPION (EndoreM®) and protamine sulfate. After 24 hours, SPION were
detected in neurospheres by immunochemistry and confirmed by transmission electron microscopy. The sizes of neurospheres
were analyzed at 24 and 48 hours after incubation with SPION and no differences were observed in the area of the neurospheres,
as compared to unlabeled neurospheres, indicating that SPION did not impair their growth. After 24 hours of labeling, the
neurospheres were dissociated and analyzed by FACS. We found about 40% of the cells labeled with SPION. Neurospheres labeled
with SPION were transferred onto coverslips previously coated with poly-L-lysine and laminin and cultured to induce
differentiation. After 3 days, SPION were detected in all three cells types and their quantification revealed that its incorporation
did not favor differentiation into neurons neither astrocytes. The percentage of cells that migrated out of the neurospheres after 3
days was similar in labeled and unlabeled neurospheres, showing that SPION incorporation did not affect migration. We were also
able to detect the presence of cells labeled with SPION after 3 weeks after transplantation in the mouse striatum by MRI. Taken
together, our data indicate that SPION can be incorporated by NSCs without affecting growth, differentiation and cellular
migration.
Support: Edital CT-Biotecnologia/MCT/CNPq/MS/SCTIE/DECIT, CAPES, FAPERJ and INCT.
G07
PERSPECTIVAS DE UM CORAÇÃO BIOARTIFICIAL ATRAVÉS DE UMA MATRIZ DESCELULARIZADA
1-PASSIPIERI,J.A.;2-MACIEL,L.;1-CARVALHO, A.B.;1-GOLDENBERG, R.C.S.;1,3-CARVALHO, A.C.C.
1-Laboratório de Cardiologia Celular e Molecular,Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro;
2-Laboratório de Eletrofisiologia Cardíaca Antonio Paes de Carvalho, Instituto de Biofísica Carlos Chagas Filho, Universidade
Federal do Rio de Janeiro; 3-Instituto Nacional de Cardiologia de Laranjeiras.
Somente nos Estados Unidos aproximadamente cinco milhões de indivíduos possuem insuficiência cardíaca e a cada ano
quatrocentos mil novos casos são diagnosticados. A insuficiência cardíaca é responsável por ocasionar morbidade aos pacientes,
ou seja, uma baixa qualidade de vida. Além de gerar altos custos governamentais. Os tratamentos tradicionais são baseados em
administração de fármacos como estatinas, beta-bloqueadores e inibidores da enzima conversora de angiotensina (ECA).
Entretanto a resolução desta enfermidade é o transplante cardíaco, o qual é limitado devido à ausência de doadores e as
complicações geradas pela terapia de imunossupressão. A criação de um coração bioartificial pode resolver esses problemas.Este
trabalho tem como objetivo produzir uma matriz cardíaca, na qual haja a preservação da estrutura geométrica tridimensional e da
vascularização deste órgão.Corações de ratos Wistar foram descelularizados em um sistema de Langendorff, através da perfusão
aórtica. Foi utilizado Dodecil Sulfato de Sódio (SDS) 1% por 12 horas e a matriz descelularizada foi fixada em paraformaldeído 4% e
emblocada em parafina ou em OCT. Em seguida as amostras foram seccionadas com 5 µm e submetidas ao tratamento com os
corantes hematoxilina-eosina e picrosirius. Em seguida, foi realizada a imunofluorescência, usando os anticorpos para os
componentes da matriz extracelular, colágeno I, colágeno III, fibronectina, e laminina. Corações não descelularizados foram
utilizados como controles positivos.A perfusão com SDS demonstrou ser um método eficiente para a remoção das células
cardíacas (n=5), ao preservar a matriz extracelular cardíaca de maneira intacta. A observação histológica foi capaz de demonstrar a
preservação dos vasos associado a este órgão. A imunofluorescência mostrou que as estruturas protéicas da matriz colágeno I,
colágeno III, fibronectina, e laminina se mantiverem inalteradas, concluíndo que a perfusão com SDS é um método eficiente de
produzir uma matriz extracelular cardíaca, e que possivelmente pode ser usada para a construção de um coração bioartificial.
Apoio: FAPERJ,CNPq,Capes, FINEP.
G08
EFFECTS OF A NOVEL HISTONE DEACETYLASES INHIBITOR ON LEISHMANIA AMAZONENSIS PROMASTIGOTES: AN
ULTRASTRUCTURAL STUDY
1,2,5- VERÇOZA, B. R. F.; 3-BRACHER, F.; 1,2,4- DE SOUZA, W.; 1,2,4,5- RODRIGUES, J. C. F.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro. 2- Instituto Nacional de Ciência e Tecnologia
em Biologia Estrutural e Bioimagem. 3- Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universitt
Mnchen, Munich, Germany. 4- Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Brasil. 5- Pólo Avançado de
Xerém, Universidade Federal do Rio de Janeiro.
Leishmaniasis is one of the most important tropical diseases caused by protozoan parasites of the Leishmania genus with high
prevalence around the world. This disease is divided in three main clinical manifestations: cutaneous, mucocutaneous and visceral
leishmaniasis. In Brazil, Leishmania amazonensis is one important species responsible for cutaneous and diffuse cutaneous
leishmaniasis, which is so difficulty to treat. The treatment is based on the use of pentavalent antimonials, amphotericin B or
pentadimidine. However they are extremely toxic for patients; thus, it is necessary studies in the field of chemotherapy trying to
develop new molecules or therapeutic regimen, which are more efficient and less toxic. Recently, histone deacetylases inhibitors
have been studied as potential target for the treatment of cancer. These enzymes act directly on the packaging of the DNA, thus
regulating the expression of various genes related to apoptotic cell death. Thus, the aim of our work is study the effects of novel
histone deacetylases inhibitors on Leishmania amazonensis. After treatment with TFMDI, the IC50 obtained was 2.0 μM. A potent
effect on the proliferation was observed in concentrations upper 3.0 μM. Scanning electron microscopy revealed important
alterations on the shape, where around 60% of promatigotes became more elongated and thinner comparing to the control
parasites. Alterations on the plasma membrane were also observed. In addition, transmission electron microscopy of treatedparasites showed a lipid accumulation, presence of protrusions on the plasma membrane similar to those observed in the
scanning electron microscopy, mitochondrial swelling with the appearance of vesicular cristae and alterations on the chromatin
condensation. These alterations are absent in control promastigotes. In conclusion, histone deacetilases inhibitors are effective
against Leishmania amazonensis promastigotes; however, other studies are necessary to describe the effects on intracellular
amastigotes and to define better the mechanisms of action.
Support: INBEB, FAPERJ, CAPES, CNPq.
G09
ALTERAÇÕES NO CITOPLASMA DO ERITRÓCITO INDUZIDAS PELO PLASMODIUM CHABAUDI
1- WENDT, C. 1-SOARES MEDEIROS, L. C. 1,2- De SOUZA, W. 1,2- MIRANDA, K.
1- Laboratório de Ultraestrutura Celular Hertha Mayer, Instituto de Biofísica Carlos Chagas Filho, UFRJ. 2-Diretoria de Programas,
Instituto Nacional de Metrologia, Normalização e Qualidade Industrial INMETRO.
A malária é uma doença parasitária causada pelos protozoários do gênero Plasmodium. A alta morbidade e mortalidade da
malária estão diretamente relacionadas ao ciclo de desenvolvimento intracelular do Plasmodium e à sua capacidade de alterar
radicalmente a membrana e o citoplasma da hemácea. O parasita invade e desenvolve seu ciclo infectivo nos eritrócitos, células
que são desprovidas de organelas e da maquinaria necessária para o tráfego de proteínas. Durante o seu desenvolvimento, o
Plasmodium falciparum estabelece estruturas membranosas no citoplasma do eritrócito, chamadas de rede tubulo vesicular, que
se estende desde a membrana do vacúolo parasitóforo; e de fendas de Maurer, que se encontram na periferia dos eritrócitos,
representando o principal mecanismo utilizado pelo para a exportação de proteínas para a superfície dos eritrócitos. Dentro das
espécies de Plasmodium que parasitam os eritrócitos de camundongos, o P. chabaudi é o que apresenta maior semelhança com o
P. falciparum, incluindo sua invasão preferencial de eritrócitos maduros e o seu desenvolvimento assexual sincronizado. Nosso
grupo recentemente encontrou evidencias da presença de estruturas similares a fendas de Maurer e a rede tubulo vesicular em P.
chabaudi. Os eritrócitos infectados com P. chabaudi foram observados por microscopia eletrônica de transmissão, onde notamos a
presença de dois principais tipos de perfis de membranas dispersos pelo citoplasma dos eritrócitos infectados. A presença de
grandes perfis de membranas conectados a membrana do vacúolo parasitóforo, e também de perfis menores, próximos a
membrana do eritrócito foi observada. Estas estruturas, quando comparadas a rede tubulo vesicular e as fendas de Maurer
caracterizadas em P. falciparum, parecem apresentar grandes semelhanças estruturais, sugerindo que provavelmente possam vir
a desempenhar funções análogas em P. chabaudi, participando de mecanismos de exportação de proteínas para a membrana do
eritrócito infectado.
Apoio: CNPq, FAPERJ.
G10
MORPHOLOGICAL ANALYSIS OF HABRONEMA SP. (NEMATODA:HABRONEMATIDAE) PARASITE OF HYDROCHOERUS
HYDROCHOERIS (RODENTIA: HIDROCHAERIDAE) BY SCANNING ELECTRON MICROSCOPY
1-MARTINS, C. N.; 1-ADNET, F.A.O.; 1-CHAGAS-MOUTINHO, V.A.; 1-GONÇALVES, J.P.; 1-ANJOS, D.H.S.; 2-OLIVEIRA-MENEZES, A.; 1DE SOUZA, W.
1-Instituto de Biofísica Carlos Chagas Filho, 2-Pólo Avançado Universidade Federal do Rio de Janeiro-Macaé.
The nematodes of the genus Habronema can be found parasite birds and horses but, several studies have reported this helminth
in Capybaras (Hydrochoerus hydrochaeri). In this work we realize the morphological analysis by scanning electron microscopy
(SEM) of nematodes from the stomach of this rodent. One specimen of H. hidrochoeris found dead was collected and donated
to Laboratório de Biologia de Helmintos Otto Wucherer for helminthes analysis. After necropsy, the nematodes found in the
stomach was washed in 0.9% NaCl and fixed in AFA. For identification the nematodes were observed on light microscopy Zeiss
Standard 20. For SEM, the nematodes were processed according Adnet et al., 2009 and observed by SEM Jeol JSM-5310. The
nematodes are elongated and cylindrical, with a single lateral ala and surface showing a transversal cuticular striation along of the
body. The morphology of anterior end is common to both sexes. In this region can be found a pair of derides in the lateral of the
body (one on each side). The cephalic end shows an oral aperture surrounded by two lateral lips, each with three digitiform
projections. Around the mouth, there are two lateral amphids, four papillae (two dorsolateral and two ventrolateral) and eight
pores (two ventrolateral, two dorsolateral and a pair in each lip). The posterior end of female is conical and has a pair of phasmids
at the tip.. The vulva is located at anterior third of body. The male posterior end is ventrally bent, with modifications on cuticular
striations, adopting a longitudinal standard. This region has a caudal ala well developed and several papillae, a cluster of small
papillae can be seen at the tip of the tail. A pair of unequal spicules is projected through the cloaca. The morphological analysis
allowed identifying the nematode parasite of Hydrochoerus hydrochoeri as Habronema sp.
Support: FAPERJ, CNPQ-PROTAX, CAPES.
G11
CULTIVO, CARACTERIZAÇÃO MORFOLÓGICA E FILOGENÉTICA DE BACTÉRIAS MICROAERÓFILAS PRESENTES EM AMOSTRAS DE
SEDIMENTOS DA BAIA DO ALMIRANTADO, ANTÁRTICA
1- Ribeiro, C.W.; 1- Abreu, F.; 1- Silva, K.T.; 2- Texeira, L.; 3- Kuhn, E.; 2- Peixoto, R.S.; 2- Rosado, A.S.; 3- Pellizari, V.H.; 1- Lins, U.
1 Laboratório de Biologia e Ultraestrutura de Procariotos, IMPPG, UFRJ. 2 Laboratório de Ecologia Molecular, IMPPG, UFRJ.3
Laboratório de Ecologia de Microorganismos Marinhos, IO, USP.
Estudos baseados em análises moleculares de amostras de sedimento marinho de regiões polares mostram a existência de uma
grande diversidade procariótica, sendo sua maioria desconhecida e pouco relacionada a organismos cultivados. Bactérias
microaerófilas estão amplamente distribuídas em ambientes aquáticos e sua densidade populacional é geralmente 1 ou 2 ordens
de grandeza menor que a população de aeróbios ou anaeróbios facultativos. Neste trabalho, pretendemos avaliar a diversidade
microbiana encontrada no sedimento costeiro da Antártica e obter uma descrição das bactérias microaerófilas presentes nas
amostras através do isolamento em cultura pura e caracterização morfológica e filogenética. O sedimento utilizado nesse projeto
foi coletado durante as XXVII e XXVIII Operações Antártica em fevereiro e novembro de 2009, respectivamente, como participação
no projeto “Microbial Diversity of Terrestrial and Maritime Ecosystems in Antartic Peninsula” (API-MIDIAPI). Amostras de
sedimento foram coletadas a 8 metros de profundidade na costa da Ilha de Rei George, em Punta Ullman (62°04’S e 58°21'W),
Baía do Almirantado, Antártica. O sedimento foi mantido em recipientes de polipropileno e armazenado a 4º C. O isolamento de
bactérias microaerófilas foi feito através de meio autotrófico. Sedimento e água da coleta foram agitados e após a deposição do
sedimento aproximadamente 200 microlitros foi inoculado no meio autotrófico e mantido a 4º C. O crescimento nos tubos foi
monitorado pela observação das bandas formadas no meio de cultura. As culturas estão sendo mantidas no laboratório. A
observação microscópica dos tubos iniciais mostrou vários morfotipos de bactérias tais como cocos, bastonetes e espirilos de
diferentes tamanhos e velocidades de nado. A técnica de shake tube foi então utilizada para obtenção de culturas puras através
do isolamento de colônias. As culturas que aparentam estar puras estão sendo caracterizadas morfológicamente por microscopia
eletrônica de transmissão e filogeneticamente pela amplificação e seqüenciamento do gene que codifica o rRNA 16S.
Apoio: INBEB, FAPERJ, PROANTAR, CNPq, MCT.
G12
INVESTIGATION OF YELLOW FEVER VIRUS-INDUCED ENDOPLASMIC RETICULUM STRESS
SANCHES, D.1; CAMPOS, S.P.C.1; ROCHA, C.M.1; GONÇALVES, B.S. 3; CHIARINI, L.B.3; GASPAR, L.P.2; FREIRE, M.S.2; SILVA, J.L.1;
GOMES, A.M.O.1 & OLIVEIRA, A.C.1
1-IBqM/UFRJ 2-ITI/FIOCRUZ/RJ 3-IBCCF/UFRJ
Flaviviruses cause diseases like Dengue and Yellow fever, and have a particular importance for public health mainly in South
America, Central America and Asiatic southeast. Virus-induced apoptosis is related to a cytopathological consequence of an
infection in vivo or in vitro. During apoptosis, some cellular mechanisms occur, such as DNA fragmentation and release of
messengers of the apoptotic pathways. The endoplasmic reticulum stress (ERS) can be triggered by an accumulation of unfolded
protein leading to stress response (UPR) with BIP-PERK complex dissociation. Once PERK is dissociated from Bip, it can lead to
eIF2α phosphorilation, inducing CHOP overexpression. CHOP is a nuclear factor that leads to expression and translocation of proapoptotic Bcl-2 proteins from the cytosol to the mitochondria, inducing apoptotic mitochondrial pathway activation. Yellow Fever
Virus (YFV) is endoplasmic reticulum (ER)-tropic viruses that are dependent on the host ER to translate, replicate and package their
genome. Here, we investigate the ERS induced by YFV. We infected Vero cells with YFV using a MOI=1 and analyzed the cell
viability using LIVE/DEAD kit assay and lactate dehydrogenase (LDH) activity assay. The expression of Bip, PhosphoeIF2α and CHOP
was followed by Western-blotting in a time-dependent manner. The YFV-induced ERS was confirmed by PhosphoeIF2α
overexpression 24 hours post infection. CHOP overexpression was observed after 48 hours post infection. We analyzed the role of
PERK pathway activation during YFV-induced apoptosis by using a PERK pathway inhibitor, 4 phenylbutyric acid (4-PBA). We
observed that even using 4-PBA inhibitor, YFV still induces apoptosis after 96 hours post infection. We also observed
mitochondrial pathway activation, after YFV-induced ERS, analyzing mitochondrial membrane potential transition. Our results
suggest that ERS could lead to apoptosis pathway activation through CHOP overexpression that leads to the intrinsic pathway
activation, but PERK activation does not seem to be necessary to YFV-induced apoptosis.
Support: CNPq, CAPES, FAPERJ, FINEP/CT-INFRA, INBEB, PRONEX.
G13
EFEITO DE DOSE SUB-LETAL DE MICROCISTINA-LR NA REABSORÇÃO DE SÓDIO E ALTERAÇÃO ESTRUTURAL DO TECIDO RENAL
DE RATOS WISTAR
1-FREIRE, D.S., CARDOSO, L.H.D., FERRÃO, F.M., da SILVA, R.T., VIEIRA-BEIRAL, H.J., VIEYRA, A., LOWE, J.
1-Laboratório Físico-Química Biológica Aída Hassón-Voloch; Instituto de Biofísica Carlos Chagas Filho (IBCCF), Universidade Federal
do Rio de Janeiro (UFRJ)
Introdução: A microcistina é uma toxina sintetizada por cianobactérias, onde a microcistina-LR (MCYST-LR) é a mais tóxica e talvez
por isso é a mais estudada. Estudos de exposição sub-letal indicam possíveis efeitos sobre o rim, podendo levar à falência renal em
pouco tempo. Objetivo: Investigar o efeito da dose sub-letal de MCYST-LR nos transportadores renais de Na+, estudando os
mecanismos cmoleculares envolvidos e analisar se uma única dose é suficiente para alterar o parênquima renal. Metodologia: Os
animais foram sacrificados após 24 horas da injeção i.p. de MCYST-LR 50ug/kg do peso corpóreo em ratos Wistar machos adultos.
O rim esquerdo dos grupos controle (CTRL) e toxina (MCYST) foram utilizados para análise histológica, onde foram processados e
os cortes corados com Picrosirius (PS), PAS(Ácido Periódico-Schiff) e Hematoxilina-Eosina (HE). Os rins direitos tiveram o córtex
dissecado, homogeneizado e centrifugado. A atividade da Na+-ATPase e Na+/K+-ATPase foi medida pelo método colorimétrico. A
atividade das proteínas cinases A e C foram medidas através da quantificação de 32P incorporado à histona. A análise da
expressão da Na+/K+-ATPase, PKA e PKC foram feitas através de SDS-PAGE, seguido de western blotting, utilizando anticorpos
específicos. O controle de carregamento foi realizado com anticorpo anti beta-actina. Resultados: Foi possível observar um
aumento do espaço intersticial renal (PAS) e do colágeno (PS) no grupo tratado. As atividades das bombas de Na+ foram inibidas
pela única dose de MCYST-LR, porém não foi observado aumento na expressão da Na+/K+-ATPase. Ainda, MCYST-LR não alterou a
expressão e atividade da PKC e PKA, logo a inibição de ambas ATPases não foi induzida por aumento da fosforilação destas
cinases. Conclusão: Apenas uma dose sub-letal de MCYST-LR é responsável por alterações morfológicas e bioquímicas no tecido
renal. Assim, propõe-se que o estado de fosforilação regulatória foi mantido nas bombas de sódio.
Apoio: INBEB, CNPq, FAPERJ.
G14
SELEÇÃO RÁPIDA DE FRAGMENTOS MOLECULARES NA BUSCA POR NOVOS FÁRMACOS
1 – MARINS, E. B.; 1- TINOCO, L.W. 2 -DA FROTA, L.C.R.M.; 2- GOMES, S; 2- DE SOUZA, C.C.; DE NOVAES, P.F.; 2-DE LIMA, E.C.; 2LEÃO, R.A.C.; 3- DA SILVA, A.J.M.; 4- DIAS, A.G.; 2-COSTA, P.R.R.
1-Laboratório de Análise e Desenvolvimento de Inibidores Enzimáticos e Laboratório Multiusuário de Análises por RMN; 2Laboratório de Química Bioinorgânica; 3- Núcleo de Pesquisas de Produtos Naturais, UFRJ; 4- Instituto de Química, Departamento
de Química Orgânica, UERJ
A estratégia de utilizar fragmentos moleculares vem mostrando sucesso em indústrias farmacêuticas por todo o mundo.1
Fragmentos são moléculas pequenas o suficiente para minimizar as chances de interações desfavoráveis, proporcionando uma
ligação eficiente. Foram testados 70 fragmentos moleculares, selecionados de acordo com a regra dos três2. Estes fragmentos,
foram analisados por RMN de 1H, a fim de gerar um banco de dados para testes de ligação com diferentes enzimas. As amostras
dos fragmentos foram preparadas em DMSO_d6 (50 mM), e analisadas a 25 °C em espectrômetro Agilent de 500 MHz. Após a
aquisição, os espectros foram processados usando o programa MestReNova e foi feita a atribuição de cada hidrogênio. Após a
análise, os compostos foram agrupados de acordo com seus deslocamentos químicos para evitar a sobreposição de sinais. As
misturas dos fragmentos foram preparadas em D2O e analisadas por RMN de 1H. Foram feitos testes de ligação com a proteína
nucleosídeo hidrolase de Leishmania donovani (expressa e purificada previamente)3 em uma relação de 1:1000. A supressão do
sinal da água em sua região característica foi feita usando a sequência WET. As análises de ligação fragmento-proteína foram
feitas por RMN usando a técnica de STD (Saturation Transfer Difference), na qual somente são observados no espectro sinais dos
hidrogênios da(s) molécula(s) que se liga(m) à proteína. De acordo com os testes de ligação, os ligantes identificados possuem em
média: peso molecular 136 Da, log P<0, área da superfície polar 62,5 e 1,3 ligações rotativas. Os fragmentos identificados que
apresentaram afinidade pela proteína serão testados em conjunto para identificarmos se a ligação ocorre no mesmo sítio ou em
sítios próximos. Paralelamente, são feitos estudos por modelagem molecular para o planejamento de uma molécula mais
complexa para que seja sintetizado um novo ligante. [1] Barelier, S; Pons,J. J. Med. Chem. 2010, 53, 5256–5266. [2] Congreve, M;
Carr, R; Murray, C. Drug Discovery Today 2003, 8, 876-877 [3] Tese de doutorado: Rennó, M. Avaliação da atividade inibitória de
protótipos sobre a nucleosídeo hidrolase de leishmania donovani. IME, 2009.
Apoio: INBEB, FAPERJ, CNPq E FINEP.
G15
SECRETORY TYPE GROUP V PLA2: POSSIBLE ROLE IN RENAL PHYSIOLOGY
1Moraes-Santos, F., 1Lemos, R. M. O., 1Landgraf, S. S., 2Samoto, V. Y., 1Zamith-Miranda, D., 1Diaz, B. L., 2Takiya, C. M., 1,3CarusoNeves, C.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Instituto de Ciências Biomédicas,
Universidade Federal do Rio de Janeiro; 3-INCT-INBEB/CNPq/MCT
Aim: The superfamily of phospholipase A2 (PLA2) includes four main types of proteins: secretory, cytosolic, calcium-independent
and platelet activating factor-acetyl hydrolases. It has been described that the group V PLA2 (GV sPLA2), a secretory member, has
a regulatory role in eicosanoid synthesis and its effects on renal physiology are unknown. Therefore, the aim of this work is to
assess the physiologic effect of GV sPLA2 on the renal functional parameters in GV sPLA2 knockout mice (GV sPLA2-/-). Methods
and Results: Urine and blood samples were collected from males C57BL/6 mice wild-type (WT) and GV sPLA2-/- and used to
determine renal function parameters. One kidney was used for histological studies and the other one was used to prepare the
cortical and medullar homogenate. (Na++K+)-ATPase activity was measured in the absence or in the presence of 1 mM ouabain
using a colorimetric method. Protein expression was evaluated by immunoblotting. In the hemodynamic study, urinary flow rate
of GV sPLA2-/- group was not significantly different from WT. The same profile was observed for UPCr. On the other hand, GGT
activity was increased in GV sPLA2-/- when compared with control. The GFR was decreased and FENa+ increased in GV sPLA2-/-.
Only the cortical (Na++K+)-ATPase activity and expression was decreased in GV sPLA2-/- while in the medulla remained
unchanged. The expression of (Na++K+) ATPase showed the same profile when compared to activity. In the histological analysis,
cellularity was not different in both superficial (subcapsular) and juxtamedullary glomeruli. Furthermore, interstitial space was not
changed in GV sPLA2-/-, however collagen deposition was 71% higher when compared with WT. Conclusion: The GV sPLA2 has a
stimulatory role on the expression and activity of cortical (Na++K+) ATPase and this regulation decreased the FENa+. In addition,
the GV sPLA2 absence induced a renal injury indicating a possible renoprotective role.
Support: FAPERJ, CAPES, INBEB, CNPq.
G16
Fernanda R. Figueiredo
BONE MARROW CELL THERAPY IN A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
1-FIGUEIREDO, F.R.; 1- GUBERT, F.G.; PEREIRA, I.B.; DECOTELLI-SILVA, A.L.B.; 1- SANTIAGO, M.F.; MENDEZ-OTERO, R.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease that affects selectively the motor neurons. The detail
mechanisms of selective motor neuron death remain to be unknown and no effective therapy has been developed. The aim of this
work is to study the therapy with bone marrow cells (BMC) in a mouse model of ALS (SOD1-G93A mice). We isolated 106 BMC
(mononuclear fraction) from wild type mice and inject them in the lumbar portion of the spinal cord of the SOD1-G93A mice. We
injected the cells at: 9 weeks (pre-symptomatic) and 14weeks (post-symptomatic). In each condition, we analyzed the progression
of disease and the lifespan of animals. We did not observed increase in the lifespan of the animals injected with BMC at 9weeks of
life, although there is slightly delay in the onset of the treated animals. When we injected the BMC at 14weeks, we observed only
a increase in the animals performance at the rotarod test when compared with the animals that receive saline injection one week
after the treatment. In this protocol, we also did not observe difference in the animal’s lifespan. We observed a significant
decrease in the number of motoneurons in the SOD1-G93A mice compared with the wild-type animals, but there was no
difference between the animals that were injected with BMC or saline. We observed an increase in the number of microglia in the
ALS mice, but there was no difference in this number between the animals that were injected with BMC or saline. The treatment
with BMC injected in the spinal cord of a mouse model of ALS, delayed the onset of the symptoms, but did not increase the
lifespan of the animals, indicating that is necessary more study to find an efficient treatment for this disease.
Support: INBEB, Protecel CNPq, CAPES, FAPERJ.
G17
ENVOLVIMENTO DE UM CIRCUITO AUDITÓRIO TEMPORO-FRONTAL NO MECANISMO DE PREDIÇÃO: IMPLICAÇÕES NA
ESQUIZOFRENIA
1- Angelis-Reis, F. M. S.; 1- Panizzutti, R.
1-Laboratório de Fronteiras em Neurociências, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro
Memória de trabalho se refere à habilidade do cérebro de manter informações durante pequenos períodos de tempo. Deficiência
na memória de trabalho é um sintoma central da esquizofrenia o qual pode levar pacientes a interpretar eventos diários
normalmente previsíveis como imprevisíveis, surpreendentes ou novos. Neste trabalho pretendemos estudar o circuito cerebral
envolvido na predição de um estímulo auditório esperado. Ratos foram treinados a reconhecer uma sequência alvo de dois tons
entre sequências não alvo de dois tons. Para conseguir reconhecer propriamente o alvo na tarefa, o rato deve preservar o
primeiro tom na memória durante o intervalo de silêncio entre os tons. Descobrimos que quando o segundo estímulo ocorre na
sequência alvo, os neurônios ativados sofrem uma maior excitação, mais cordenada e mais seletiva, sendo representados em uma
maior área cortical no córtex auditório primário. Em seguida, procuramos por estruturas no córtex frontal que pudessem estar
envolvidas neste aumento de resposta na predição ao segundo tom. Para identificar a região frontal que recebe projeções do
córtex auditório primário, nós injetamos o traçador anterógrado biotinylated dextran amine no córtex auditório primário.
Observamos em seguida abundantes fibras marcadas no córtex orbital ventral, indicando que o córtex auditório primário se
projeta diretamente para esta área. Finalmente, nós gravamos do córtex orbital ventral usando como estimulo as sequências alvo
e não alvo. Encontramos oscilações marcantes nas bandas teta e gama no córtex orbital ventral durante o intervalo de silêncio
entre tons quando o animal estava esperando o segundo tom na sequência alvo, que eram diferentes da presentes nas sequências
não alvo. Em sumário, nossos achados implicam o circuito temporo-frontal entre o córtex primário auditório e o córtex orbital
ventral no mecanismo de predição de estímulos auditórios esperados e nos mostram que áreas corticais não são estáticas, pelo
contrário, podem se alterar momento a momento no tempo como uma função de um estado de predição.
Apoio: CNPq, FAPERJ.
G18
OBSERVATION OF NEUTROPHIL EXTRACELLULAR TRAPS IN THE INTERACTION OF TOXOPLASMA GONDII AND HUMAN
NEUTROPHILS
1-VERAS, G M; 1-PAREDES-SANTOS,TC; 2-GUIMARÃES-COSTA, AB; 2-SARAIVA, EM; 1-ATTIAS M
1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil. 2-Instituto de Microbiologia Prof. Paulo
de Góes, Universidade Federal do Rio de Janeiro, Brasil.
Toxoplasma gondii is an obligate intracellular parasite that can actively invade any nucleated cell from warm blooded animals. It is
the causative agent of toxoplasmosis, a worldwide disease whose infection leads to severe consequences in pregnant women and
immunocompromised patients. Neutrophils participate in the innate immune response, acting in infections caused by
microorganisms. They are capable of destroying, through the release of their cytoplasmic granules, and phagocytose these
pathogens. Recently, a new mechanism of neutrophil death was described [1], in which occurs the release of neutrophil
extracellular traps (NET). Those consist of DNA associated with proteins such as histones and elastase, capable of imprisoning and
sometimes killing, fungi, bacteria and protozoa [2]. Thus, the aim of this work was to evaluate microscopy techniques to visualize
NETs induced by T. gondii. For this, human neutrophils were incubated with tachyzoites from the RH strain of T. gondii in different
proportions and the release of NETs tested by immunofluorescence microscopy (IFA), and scanning electron microscopy (SEM).
The samples were fixed with 4% formaldehyde in PBS for IFA and for scanning electron microscopy (SEM) with 2.5%
glutaraldehyde in sodium cacodylate buffer 0,1M. For visualization of NETs, the cells were stained with DAPI and anti-elastase,
anti-myeloperoxidase (MPO) and anti-histone. The samples for SEM were post-fixed with 1% of osmium tetroxide (OsO4) for 30
min, rinsed with the same buffer, dehydrated in crescent series of ethanol, critical point dried and covered with gold or chrome
and visualized in a JEOL JSM6340 Field-Emission Scanning Electron Microscope. In DIC light microscopy (Figure 1A), parasites were
seen associated to a filamentous material, that in Fluorescence microscopy was stained with DAPI, indicating the presence of DNA,
a NET component (Figure 1B). IFA assays also showed the presence of elastase, MPO and histone that have been described as
NET componente [3]. These observations were reinforced corroborated by FE-SEM where we visualized the ultrastructural
aspects of these extracellular traps in close contact with the parasite (Figure 1C). NETs are fragile and require careful preparation
of the samples. Those networks are not easily visible and can easily be confused with the actin filaments that compose the
cytoskeleton, but the combination of light and electron microscopy can show its true nature.
Support: INBEB, CNPq, FAPERJ.
G19
EXPRESSION OF GANGLIOSIDE 9-O-ACETYL GD3 ON EMBRYONIC STEM CELLS
1,2- AZEVEDO-PEREIRA, R.L.; 1,2- SANTOS, G.M.; 1-2, PAREDES, B.D.; 1- RODRIGUES, D.C.; 1,2- MENDEZ-OTERO, R.
1- Instituto de Biofísica Carlos Chagas Filho; 2-Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem –
INBEB; UFRJ
The ganglioside 9-O-Acetyl-GD3 (9AcGD3) is a natural variant of the disialoganglioside GD3 that is cell-type and developmentally
regulated and wildly expressed in the nervous system. During development, 9AcGD3 is expressed in migratory neurons and radial
glia. In the mammalian adult, the expression persists in a few areas of the brain, such as the subventricular zone around the
ventricular lateral wall. This region is known as a neurogenic area containing neural stem cells, making the ganglioside 9AcGD3 a
potential marker of neural stem cells. In this work, we investigated whether 9AcGD3 is expressed on undifferentiated mouse
embryonic stem cells (mESC), posing as a primordial marker of differentiation into neural stem/progenitor cells. For this purpose,
we co-cultured the 129S6/B6-F1/DsRed.T3 mESC line in DMEM high glucose supplemented with KnockOut Serum Replacement (KSR) and LIF, with irradiated mouse embryonic fibroblasts (iMEF). The expression of 9AcGD3 on undifferentiated mESC was
evaluated by immunocytochemistry and FACs, revealing that cells positive for Oct-3/4 also expressed 9AcGD3. Furthermore, we
found that 5-10% of 129S6/B6-F1/DsRed.T3 express 9AcGD3. The population expressing 9AcGD3 was purified by cell sorting and
we found that the levels of mRNA to nestin, a marker of neural stem cells, were upregulated in comparison to those in cells
negative for 9AcGD3 or in undifferentiated cells (mESC). We found that when, co-cultured with iMEF, the purified populations of
either 9AcGD3 positive or 9AcGD3 negative cells expressed Oct-3/4 and SSEA1. Intriguingly, the 9AcGD3 negative cells recovered
9AcGD3 expression within 48 hours. Our data suggest that 9AcGD3 is preferentially expressed in cells bearing high levels of early
neural markers.
Support: Edital CT-Biotecnologia/MCT/CNPq/MS/SCTIE/DECIT, CAPES, FAPERJ e INCT.
G20
DESENVOLVIMENTO DO MODELO DE CARDIOMIOPATIA CHAGÁSICA EM CAMUNDONGOS QUIMÉRICOS
1- VITIELLO, G.; 1- DURAN, D.B.; 1- CHRISTIE, B.B.; 1- IRION, C.I; 1- BRASIL, G.V.; 1- PAREDES, B. D.; 1- BRUNSWICK, T.H.K.; 2ROCHA, N.N.; 1- CUNHA, S.T.; 1-RAMOS, I.P.R.; 4- CANARY, P.C.V.; 1- CARVALHO, A.B.; 1,3- CARVALHO, A.C.C.; 1- GOLDENBERG,
R.C.S.
1- Laboratório de Cardiologia Celular e Molecular – Instituto de Biofísica Carlos Chagas Filho – Universidade Federal do Rio de
Janeiro; 2- Universidade Federal Fluminense; 3- Instituto Nacional de Cardiologia; 4- Serviço de Radioterapia do Hospital
Universitário Clementino Fraga Filho
A doença de Chagas é a principal causa de cardiomiopatia na América Latina. A doença é insidosa, manifestando os sintomas
característicos somente na fase crônica. O objetivo deste estudo é estabelecer o modelo experimental de doença de Chagas em
camundongos quiméricos para posterior realização da terapia celular. Foram utilizados 22 camundongos da linhagem C57BL/6
com dois meses de idade. Os animais foram submetidos à mieloablação e posterior transplante de 2x10E6 células da medulaóssea, oriundas de animais que possuem o gene da proteína verde fluorescente (GFP). Quinze animais foram infectados pela via
intraperitoneal com 3x10E4 formas tripomastigotas da cepa Brazil, a parasitemia foi monitorada por 30 dias e, o restante dos
animais, foi utilizado como grupo controle. A avaliação do desempenho cardíaco dos animais foi feita por eletrocardiograma e
ergometria mensalmente. Ao longo do estudo, os animais que morreram tiveram seus corações removidos, emblocados em
parafina e corados com o método hematoxilina-eosina para a observação ao microscópio óptico. A repopulação medular foi
analisada por citometria de fluxo, sendo incluídos no estudo aqueles animais que apresentaram grau de enxertia acima de 75%.
Dois meses após a infecção, 77% dos animais infectados e 40% dos animais não infectados morreram. Foram observados parasitas
no sangue periférico dos animais infectados a partir do quinto dia pós-infecção com pico de parasitemia por volta do 27º dia de
infecção. Na avaliação eletrocardiográfica, foi observado arritmia do tipo bloqueio atrioventricular de 1º grau após um mês de
infecção, assim como, foi possível observar diferença estatística significativa pela ergometria no 2º mês. Histologicamente foi
possível observar uma miocardite moderada com predomínio de células mononucleares e poucos polimorfonucleares com ninhos
de amastigotas nas células cardíacas. Logo, demonstra-se que camundongos quiméricos são susceptíveis à infecção. Entretanto,
esta quantidade de parasitas apresenta uma letalidade alta para esses animais quanto injetada via intraperitoneal.
Apoio: CNPq, FAPERJ, CAPES, Ministério da Saúde.
G21
O ENVOLVIMENTO DA MIRISTOILAÇÃO NA PROGRESSÃO DA LEUCEMIA MIELÓIDE CRÔNICA (LMC): REGULAÇÃO x
ANCORAMENTO
1 - FERRETTI, G.D.S, 1 - OLIVEIRA, G.A.P., 1 - FREITAS, A.P., 1 - CARVALHO, C.A.M., 1 - GOMES, A.M.O. E 1 - SILVA, J.L.
1 - Laboratório de Termodinâmica de Proteínas e Estruturas Virais Gregorio Weber, IBqM, UFRJ, Rio de Janeiro, RJ, Brasil.
Polipeptídios recém sintetizados sofrem mudanças que podem afetar sua estabilidade, localização e atividade biológica. No
câncer, diversas oncoproteínas sofrem modificações tornando-se ativas e funcionais. A proteína ABL, envolvida no
desenvolvimento da leucemia mielóide crônica (LMC) é expressa nas isoformas 1A (não miristoilada) e 1B que sofre incorporação
de um miristato no resíduo de glicina amino-terminal (G2). Em células leucêmicas, o gene bcr se fusiona ao gene abl, originando
um gene quimérico que codifica para uma proteína desregulada não miristoilada. Nossos objetivos visam investigar o impacto da
miristoilação na regulação e localização do ABL e sua importância na leucemia. Verificamos que o miristoil está envolvido na
localização do ABL na célula, porém, não é limitante para tal. Análises realizadas por microscopia confocal da super expressão, por
transfecção transiente, das isoformas ativas do ABL em células HEK293 evidenciaram marcação citosólica difusa e em regiões de
membrana plasmática para a isoforma miristoilada, e marcação pontual para a isoforma não-miristoilada. A inibição das
isoformas, via droga-dependente (10uM STI-571), revelam compartimentagem vesicular em regiões de retículo nos níveis
endógenos e após super expressão. Ensaios de mutagênese (G2A) da isoforma miristoilada, evidenciam papel da miristoilação no
ancoramento em membranas. Análises por western blotting indicam efeito pró-apoptótico da proteína Abl via BAX e p53
dependente. Diminuição nos níveis de RNAm de moléculas envolvidas em adesão (ADAMs) mediante inibição do ABL com STI-571
e alta expressão dessas moléculas em linhagem leucêmica K562 sugerem possível envolvimento do ABL em sua regulação e novo
mecanismo para perda de adesão ao estroma medular. Compreendendo melhor a função dessas modificações, teremos melhor
esclarecimento acerca dos mecanismos de ação durante o desenvolvimento de neoplasias e uma potente estratégia para futuras
intervenções terapêuticas.
Apoio: INBEB, FAPERJ, CNPq.
G22
MODELAGEM ESTRUTURAL DE PROTEÍNAS DE CANA-DE-AÇÚCAR DIFERENCIALMENTE EXPRESSAS DEVIDO A PRESENÇA DA
BACTÉRIA ENDOFÍTICA GLUCONACETOBACTER DIAZOTROPHICUS
1- SOARES, H. L.; 1- DA SILVA, M. L.; 1- BISCH, P. M.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de janeiro
Gluconacetobacter diazotrophicus é uma bacteria diazotrófica endofítica com papel importante no crescimento da cana-de-açúcar
(Saccharum officinarum). A bactéria é capaz de realizar o processo de fixação biológica de nitrogênio, cujo qual será aproveitado
pela planta, além de liberar fatores hormonais de crescimento para esta [Nogueira et al. Genetics and Molecular Biology, 24, 2001]
resultando em uma maior produtividade do cultivar. Estudos proteômicos listaram proteínas de cana-de-açúcar de duas
variedades, SP-70 e Chunee, tendo sua expressão aumentada durante a interação com G.Diazotrophicus [Lery et al. Mol Plant
microbe Interact, Dec 29, 2010]. Duas destas proteínas foram estudadas neste trabalho, devido à possibilidade de estarem
relacionadas com o processo de sinalização entre a bactéria e a planta: Uma tirosina cinase da variedade SP-70 de cana e uma
proteína CDC48-like da variedade Chunee. Nós trabalhamos para propor estruturas para estas proteínas, procurando em bancos
de dados sequencias similares às das duas proteínas e utilizando-as no método de modelagem comparativa. Com a utilização do
software MODELLER [Eswar N. et al. Current Protocols in Bioinformatics, Supplement 15, 2006.] e a proteína CDC48 (PDBid 1R7R)
de Mus muscullus (Camundongo) como molde, construímos então modelos estruturais para a proteína CDC48-like de cana-deaçúcar da variedade Chunee. Do mesmo modo, desta vez utilizando a tirosina cinase (PDBid 3HGK) de Solanum pimpinellifolium
(Tomate) como molde para construir a estrutura da tirosina cinase de cana de açúcar. Os modelos estruturais das duas proteínas
possuem bons resultados validativos e nos ajudarão a conhecer mais sobre a degradação de proteínas e fusão membranar
promovidas pela proteína CDC-like de Chunee e a resposta defensiva de aceitação da bactéria através do reconhecimento de
peptídeos liberados por esta, mediado pela tirosina cinase da cana-de-açúcar SP-70. Planejamos agora realizar estudos de docking,
dinâmica molecular e, em caso de bons resultados, experimentos in vitro para entender melhor o funcionamento destas proteínas
e encontrar papéis mais específicos destas na interação entre a cana e a bactéria.
Apoio: FAPERJ, CNPq, CAPES.
G23
THE EFFECTS OF DIETHYLCARBAMAZINE (DEC) IN A MOUSE MODEL OF CARRAGEENAN-INDUCED PLEURISY
1- RIBEIRO, E. L.; 1- FRAGOSO, I. T.; 1- BARBOSA, K.P.S.; 1- DONATO, M. A.,; 1- SILVA, B.S.; 1- OLIVEIRA, F.G.; 1- TORRES, D. O. C.;
1,2 PEIXOTO, C.A
1- centro de Pesquisas Aggeu Magalhães; 2- Centro de Tecnologias Estratégicas do Nordeste
Background: Carrageenan-induced local inflammation is commonly used to evaluation anti-inflammatory effects of non-steroidal
drugs and it is a useful model to assess the contribution of mediators involved in cellular alterations during the inflammatory
process. Clinical reports have described favorable results with the use of diethylcarbamazine(DEC) in bronchial asthma, reducing
the dosage of corticosteroid and bronchodilators. This drug has an important anti-inflammatory role since it interferes with
arachidonic acid metabolism. Objective: This study investigated anti-inflammatory effects of DEC in a mice model of acute
inflammation. Methodology: Male S. webster mice were separated in groups: control group(C), carrageenan group(CAR) and
carrageenan+DEC 50mg/kg group (CAR/DEC). DEC solutions were administered in dose 50mg/kg in water 3 days before
carrageenan administration. After Carrageenan(1% in saline) intrapeural injection(i.pl.) the animals were euthanized 4h later. Lung
fragments were processed for light microscopy and immunohistochemical assays. The exudates were collected to further
determine total numbers of leukocytes. Results: when compared with lung sections taken from control group, histological
examination showed that Carrageenan induced edema, tissue injury and presence of inflammatory cells. DEC significantly reduced
the degree of injury as well as the infiltration of inflammatory cells. Furthermore, injection of Carrageenan into the pleural cavity
of mice elicited an acute inflammatory response characterized by the accumulation of fluid that contained large amounts of
polymorphonuclear, which was significantly attenuated by DEC. Immunohistochemical analyses of tissue sections obtained from
animals at 4 h after Carrageenan injection demonstrated positive staining for TNF-a and IL1 β. In contrast, no staining for TNF-a
and IL1 β was found in the group treated with DEC, as well as observed in the control group. Conclusions: According to present
results DEC can be used as a potential anti-inflammatory drug for acute inflammation carrageenan-induced. Although this study
was undertaken in an experimental model, which may not be predictive of response to therapy in humans, the results suggest that
DEC may be useful for pulmonary disorders.
Support: INBEB, FACEPE, CNPQ, CPQAM.
G24
ATIVIDADE DO PEPTIDEO MELITINA SOBRE O TRYPANOSOMA CRUZI.
1,2- RIBEIRO, I.; 1,2- PAIS, J.; 1,2- ADADE, C.; 1,2- SOUTO-PADRÓN, T.
1- Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro; 2- Instituto Nacional de Ciência e Tecnologia
de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro.
A doença de Chagas, causada pelo protozoário Trypanosoma cruzi, afeta cerca de 16-18 milhões de pessoas nas Américas Central
e Sul, e o tratamento é baseado no uso do Benznidazol. Este, no entanto, possui eficácia variável, limitada à fase aguda da doença
e acarreta em diversos efeitos colaterais. Desta forma, novos agentes quimioterápicos obtidos de fontes naturais são uma fonte a
ser explorada. O veneno da abelha Apis mellifera é uma complexa mistura onde a sua fração majoritária é representada pela
melitina, objeto deste estudo. O presente trabalho avaliou a atividade da melitina sobre o T. cruzi e sua toxicidade sobre células
hospedeiras. Formas epimastigotas e tripomastigotas (clone CL-Brener) foram tratadas com melitina, e o seu efeito sobre o
crescimento dos epimastigotas e lise dos tripomastigotas foi avaliado por contagem em câmara de Neubauer. O IC50/ 24h de
inibição do crescimento dos epimastigotas foi 2,88 µg/ml e o DL50/ 24h para lise dos tripomastigotas foi 0,14 µg/ml. A viabilidade
dos parasitos foi avaliada através da incubação com iodeto de propídio e analisados por citometria de fluxo onde os epimastigotas
apresentaram marcação positiva de 70 a 99%, e os tripomastigotas exibiram de 62 a 69%. Os efeitos sobre a morfologia foram
avaliados por microscopia eletrônica, onde a ultraestrutura sugeriu fenótipos de morte distintos, onde epimastigotas estariam
morrendo por autofagia e tripomastigotas, por apoptose. A metodologia do MTS foi empregada para análise de citotoxicidade
sobre culturas de macrófagos peritoneais, tratados ou não com o peptideo por 48h. Somente o tratamento com 5 µg/ml gerou
citotoxicidade com relação às culturas controle. Estes dados demonstram que a melitina é eficaz sobre as formas epimastigotas e
tripomastigotas do T. cruzi em concentrações não tóxicas às células hospedeiras. Estudos estão em andamento para analisar os
possíveis alvos intracelulares do parasito e sobre o seu ciclo intracelular.
Apoio: INBEB, FAPERJ, CNPq, CAPES, Pronex.
G25
ISOLAMENTO E CARACTERIZAÇÃO DA PROTEÍNA RECOMBINANTE PSAA DE STREPTOCOCCUS PNEUMONIAE
1,3- ANO BOM, A.P.D. ; 1,3- SILVA, I.S.B. ; 1- SILVA, A. M. S. ; 2- ARGONDIZZO, A. P. C. ; 2- LARENTIS, A. L. ; 2- MEDEIROS, M. A. ; 3SILVA, J. L. ; 1- SILVA, J. G.
1- Laboratório de Macromoléculals LAMAM, Bio-Manguinhos, FIOCRUZ, Rio de Janeiro, Brasil; 2- Laboratório de Tecnologia
Recombinante, LATER, Bio-Manguinhos, FIOCRUZ, Rio de Janeiro, Brasil; 3- Laboratório de Termodinâmica de Proteínas e
Estruturas Virais Gragório Weber, LTPV, Instituto de Bioquímica Médica, CCS, UFRJ, Rio de Janeiro, Brasil
Streptococcus pneumoniae é uma bactéria Gram-positiva comensal encontrada no trato respiratório de uma variedade de
pessoas. É a maior causa de mortalidade e morbidade no mundo, por isso cada vez mais se procura o desenvolvimento de uma
vacina eficaz para erradicação desta bactéria. As principais doenças causadas por esse microrganismo é a pneumonia e a
meningite. O pneumococo possui vários fatores de virulência, dentre eles adesinas que permitem a adesão às células da faringe e
epitélio respiratório. A adesina Pneumococcal surface adhesin A ou proteína de superfície pneumocócica (PsaA), é uma
lipoproteína ligadora de metal, muito imunogênica e por isso se tornou foco nos estudos para desenvolvimento de vacinas.
Estudos demonstram que esta proteína é componente do sistema tranportador ligante de ATP (ABC) e tem especificidade por
manganes e zinco. Já foi verificado que a deficiencia em manganes em mutantes pode causar deficiencia na adesao e parece que a
ligação ao manganes é essencial para a Resistencia ao estresse oxidative. Neste projeto a PsaA foi clonada sem a porção lipídica,
permanecendo apenas com o domínio transmembrana protéico.
G26
INFLUÊNCIA DO FERRO NA BIOMINERALIZAÇÃO DE MAGNETOSSOMOS NO COCO MAGNETOTÁTICO CANDIDATUS
MAGNETOCOCCUS ITAPUENSIS, ISOLADO DA LAGOA ITAIPU.
Viviana Morillo1, Jefferson Cypriano1, Fernanda Abreu1, Ulysses Lins1*
1 Laboratório de Biologia e Ultraestrutura de Procariotos, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo
de Góes, UFRJ
As bactérias magnetotáticas são um grupo de bactérias definido pela presença de organelas denominadas magnetossomos, os
quais estão formados por cristais magnéticos envoltos por uma bicamada lipídica, que conferem às bactérias a capacidade de se
orientar e migrar ao longo de linhas de campos magnéticos. Neste trabalho se estudou a influência de três fontes de ferro em três
concentrações diferentes sobre a formação de magnetossomos e a magnetotaxia de um coco magnetotático isolado em cultura
pura da lagoa de Itaipu, RJ - Brasil. Os cocos magnetotáticos isolados por campos magnéticos e purificados pela técnica de dilução
em tubo foram crescidos em meio heterotrófico semisólido usando como fontes de ferro: citrato férrico, sulfato ferroso e quinato
férrico a concentrações de 20, 50 e 100 nM. As culturas foram incubadas 15 dias a 28o C. A magnetotaxia foi observada por
microscopia de luz, usando um campo magnético gerado por um magneto permanente. A presencia de magnetossomos nas
células foi determinada por microscopia eletrônica de transmissão. As medições de tamanho (largura e comprimento) e fator de
forma dos magnetossomos foram feitas usando o software AnaliSIS. Os resultados mostraram que independentemente das fontes
de ferro, as concentrações de este elemento influenciam na atividade magnetotática da bactéria, sendo que a concentrações de
50 e 100 nM se apresentam mais células procurando ativamente o sul de uma barra magnética enquanto que aquelas crescidas a
baixas concentrações, o nado parece ser menos ativo, com uma quantidade menor de células procurando o sul. As concentrações
e fontes de ferro usadas em este estudo não influenciam no fator de forma dos magnetossomos, porém se observam pequenos
aumentos no tamanho e número de magnetossomos quando a concentração é aumentada. Estes resultados permitiram ampliar
estudos envolvendo processos de biomineralização por bactérias magnéticas e melhoramento da produção de magnetossomos
para aplicações biotecnológicas futuras.
Apoio: CNPq, CAPES, FAPERJ.
G27
CROSSTALK BETWEEN ALBUMIN AND RENIN-ANGIOTENSIN SYSTEM IN RENAL SODIUM EXCRETION
1- LOPES, J.V.; 1- LANDGRAF S.S.; 1- PERUCHETTI D.B.; 2- TAKIYA C.M.; 1,3- CARUSO-NEVES C.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Instituto de Ciências Biomédicas,
Universidade Federal do Rio de Janeiro; 3- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem,
Universidade Federal do Rio de Janeiro
Aim: Recently, we observed that albumin modulates the proximal tubule (PT) (Na++K+)ATPase in low concentrations. Albumin also
increases angiotensina II, which is envolved in proinflammatory effects of overload of albumin in PT. Furthermore, Angiotensin II
can modulates the uptake of albumin, also the (Na++K+)ATPase activity in PT. Thus, the objective of this work was to verify the
existence of a crosstalk between albumin and Angiotensin II on the PT (Na++K+)ATPase modulation. Methods and Results: In vitro
model: it was used LLC-PK1 cells. We observed that 0.01 mg/mL albumin increased the (Na++K+)ATPase activity, while this effect
was lost with higher albumin concentration (20.0 mg/mL). 10-6M losartan, an AT1R antagonist, abolished the stimulatory effect of
0.01 mg/mL albumin while 10-8M PD123319, an AT2R antagonist, did not change it. Furthermore, 10-6M losartan abolished the
inhibitory effect of higher albumin concentration; In vivo model: it was used acute kidney injury (AKI) animal model, that is
characterized by PT albumin overload. AKI was induced in 14 weeks old male Wistar rats with intraperitoneal injection of
10g/kg/day bovine serum albumin during 7 days. The groups were treated or not with 30mg/kg/day losartan. Experiments
revealed that losartan treatment did not reverse the proteinuria, interstitial collagen deposition and glomerular lesion observed in
AKI group. On the other hand, the increase of renal sodium fraction excretion (FENa+) observed in AKI model was abolished with
losartan treatment. Was observed in AKI group: decrease of (Na++K+)ATPase activity and expression; decrease of AT1R expression
and increase of AT2R expression. However, the losartan treatment in the AKI group reverts: (Na++K+)ATPase activity and
expression and increases the AT2R expression. The ratio AT1R/AT2R was decreased in AKI model and treatment with losartan
partially restored this balance. Conclusion: Our data demonstrate that the effect of albumin on the PT (Na++K+)ATPase is
mediated by Angiotensin II/AT1R.
Support: FAPERJ, CAPES, CNPq.
G28
EXPRESSION AND PURIFICATION OF CHEMOKINE RECEPTOR CCR2 AND CCR6: MAPPING INTERACTION WITH HUMAN BETADEFENSINS BY NMR SPECTROSCOPY
KAROL TORQUATO*, MARIANA CARREIRAS*, FABIO C.L. ALMEIDA, ANA PAULA VALENTE, VIVIANE S. DE PAULA
Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Instituto de Bioquímica Médica, Universidade Federal do
Rio de Janeiro
Chemokine receptors (CCRs) belong to a class of integral membrane G-protein coupled receptors (GPCRs) that are expressed on
macrophages, immature dendritic cells and memory T cells. The activation of these receptors plays a pivotal role during the
immune reaction against bacterial or viral infections, attracting CCR-expressing inflammatory cells to the sites of infection.
Previous studies revealed the capacity of beta-defensin family members to chemoattract immature dendritic cells and memory T
cells through receptor CCR2 and CCR6 that are directly involved in HIV-1 infection, asthma and cancer metastasis. However, no
structural information about the beta-defensin-CRRs interaction is available, mainly because the difficulty of expression,
purification and solubilization of CCRs. In this study, we demonstrated the production and optimization in E. coli of CCR2 and CCR6
that will enable NMR analyses of the interaction between the full-length CCR2/CCR6 and human beta-defensins. The pET28a
plasmid was used as a vector for expression of fusion protein Mistic-GPCRs according to Kirpichnikov1 et al., 2010. The receptors
were solubilized in 1% lauroylsarcosine detergent and purified by affinity chromatography. In SDS-PAGE analysis, a major band in
~57 kDa (Mistic+CCR+HisTag) is observed. Purity of the CCR2 is >80% as judged from SDS-PAGE. Further experiments will be
performed to examine the conformational integrity of the CCRs and the interaction with beta-defensins. Petrovskaya et al., (2010)
Expression of G Protein Coupled Receptors in Escherichia coli for Structural Studies. Biochemistry (Moscow), 75, 881-891
Support: INBEB, FAPERJ, CNPq.
G29
THROMBIN TRIGGERS MITOCHONDRIAL FUNCTIONAL REMODELLING IN HUMAN PLATELETS BY TWO DISTINCT MECHANISMS
1-GARCIA-SOUZA, L.F.; 2-HOTTZ, E.D.; 3-MORTON, K.A.; 1-SANTIAGO, A.P.S.A.; 2-BOZZA, F.A.; 1-OLIVEIRA, M.F.
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil. 2- Fundação Oswaldo Cruz,
Rio de Janeiro, Brasil. 3 – Department of Radiology, University of Utah, Salt Lake City, EUA.
Platelet activation mediated by pro-coagulant factors trigger intracellular pathways and changes in the energy metabolism that
are not fully understood. Here, we investigated whether platelet activation would affect mitochondrial function. Human platelets
were collected from healthy volunteers, isolated in M199 medium, challenged by different thrombin concentrations and several
mitochondrial functional parameters were assessed by flow cytometry and high-resolution respirometry. We observed that
activation by low doses of thrombin (0-0.3 U/mL) increased glucose-induced oxygen consumption parallel to a reduction of
mitochondrial membrane potential (Δψm). This effect was strongly inhibited by cyclosporin A, implicating the opening of
mitochondrial permeability transition pore (MPTP) in this process. At higher thrombin doses (0.4-1.0 U/mL) changes in both
parameters were less pronounced, being undistinguishable from quiescent platelets at 1.0 U/mL thrombin. Curiously, in this
condition, Δψm maintenance involve the partial impairment of electron transport chain by nitric oxide and reversion of F1Fo ATP
synthase activity since the Δψm collapsed in the presence of oligomycin. Our data indicate that platelet activation promote
mitochondrial functional remodelling which, depending on the stimuli intensity, may involve MPTP opening or reversal of F1Fo
ATP synthase activity.
Support: CNPq, FAPERJ, ICGEB.
G30
PROTEÍNA CINASE C EPSILON MODULA ATIVIDADE DE ATP7B EM FÍGADO DE PORCO.
1,2- CARDOSO, L.H.D.; 1,2- HILÁRIO-SOUZA, E.; 1,2- VIEYRA, A.; 1,2- LOWE, J.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil; 2- Instituto Nacional de
Ciência e Tecnologia de Biologia Estrutural e Bioimagem (INBEB).
O cobre é um metal essencial para todos os organismos, embora em altas concentrações se torne tóxico. Sendo assim, o nível de
cobre nos organismos deve ser altamente regulado. ATP7B é uma ATPase transportadora de cobre expressa principalmente no
fígado e é de extrema importância para a homeostase do cobre. Estudos recentes de nosso laboratório já demonstraram que sua
atividade diminui quando fosforilada pela proteína cinase A (PKA). Neste trabalho investigamos se a via de sinalização da proteína
cinase C (PKC) também é capaz de modular a atividade desta ATPase. Em uma preparação de fígado de porco foram detectadas
diferentes isoformas de PKC (α, ε, ζ) por Western Blotting. Um ensaio colorimétrico foi utilizado para se determinar a atividade
Cu(I)-ATPásica na preparação. PMA (forbol, 12-miristato, 13-acetato) 10-8 M, um análogo de DAG e conhecido ativador de PKC,
aumentou a atividade de ATP7B em 55% em relação ao controle, enquanto calfostina C 10-8 M, inibidor de PKC, fez com que a
atividade diminuísse 40%. Adição de fosfatase λ após pré-incubação com PMA diminuiu a atividade Cu(I)-ATPásica ao mesmo nível
que quando adicionada apenas fosfatase λ. U73122 (10-7 M), inibidor de fosfolipase C também fez com que a atividade de ATP7B
diminuísse cerca de 65%. Quando Ca2+ 2 μM ou PMA/Ca2+ 2 μM foram adicionados não foi observada nenhuma alteração,
enquanto PMA/EGTA levou a um aumento da atividade Cu(I)-ATPásica, indicando o envolvimento de uma isoforma de PKC que
não necessita de Ca2+ para sua ativação. A atividade da Cu(I)-ATPase foi diminuída pelo inibidor específico de PKCε, mostrando
que esta isoforma de PKC é responsável pela estimulação da atividade da Cu(I)-ATPase de fígado de porco. Pode se concluir que
diferentes vias envolvendo fosfolipase C e ativação de PKCε em hepatócitos podem aumentar a atividade de ATP7B e assim alterar
o metabolismo do cobre.
Apoio: FAPERJ, CNPq, INBEB.
G31
EXPRESSÃO E PURIFICAÇÃO DA DIIDROFOLATO REDUTASE DE MYCOBACTERIUM LEPRAE
SARZEDAS, C. G.; TINOCO, L. W.
Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro
A Hanseníase é uma doença que acomete milhares de pessoas no mundo inteiro, conhecida por sua capacidade de gerar
deformidades permanentes; sendo assim considerada um problema de saúde mundial e inclusive nacional, já que o Brasil possui
alta prevalência da doença ocupando a segunda posição em números de casos no mundo (Boletim Epidemiologico 2010,
Ministério da Saúde). Esta doença é causada pelo bacilo Mycobacterium Leprae, contra a qual já existe tratamento. Apesar de
existir tratamento contra a hanseníase, já foram relatados casos de resistência aos antibiotícos usados (Diório, S. M. et al. Hansen
Int.2009, 34,1, 37), sendo necessária a busca por novos alvos terapêuticos, como por exemplo a enzima Diidrofolato Redutase
(DHFR). Frente à importância do processo patológico da hanseníase, este trabalho tem como objetivo expressar e purificar a DHFR
visando a busca de inibidores para esta enzima. A DHFR do M. leprae foi clonada pela primeira vez em nosso grupo de pesquisa.
Os testes de expressão desta enzima foram feitos em diferentes cepas de E. coli, diferentes temperaturas e concentrações de
agente indutor. Até o momento, a melhor condição de expressão encontrada para esta enzima é em E. coli BL21(DE3)-PlysE, com
temperatura de crescimento e expressão mantidas a 15 oC, de modo a evitar que a proteínas fosse para corpos de inclusão.
Contudo, como os níveis de expressão ainda são muito baixos estão sendo testadas novas condições de expressão e outras cepas
de E. coli.
Apoio: INBEB, CNPq, FAPERJ, FINEP.
G32
ANTIVIRAL ACTIVITY OF BOVINE LACTOFERRIN AGAINST YELLOW FEVER VIRUS INFECTION
1- Mendes, Y.S.; 1- Alves, N.S.; 1- Carvalho, C.A.M.; 2- Schwarcz, W.D.; 3- Silva, J.L.; 4- Gonçalves, R.B.; 1- Gomes, A.M.O.; 1Oliveira, A.C.1
1- LABEV - Instituto de Bioquímica Médica/UFRJ; 2- LATEV - Instituto de Tecnologia em Imunobiológicos/FIOCRUZ; 3- LTPV Instituto de Bioquímica Médica/UFRJ; 4- LAAB - Departamento de Bioquímica/UNIRIO.
Yellow Fever Virus (YFV) is an endemic arbovirus to tropical areas of Africa and South America and responsible for sporadic
outbreaks, having a global impact in public health. There are no effective antivirals currently available for the treatment of
flavivirus infection in humans. Lactoferrin (Lf) is a multifunctional iron-binding glycoprotein that is found in milk and other
secretions such as tears and saliva. Lf has numerous biological roles including iron metabolism, immune modulation and
antimicrobial activities, having a broad-spectrum primary defense activity against bacteria, fungi, protozoa and viruses. Here, we
aim to address the antiviral activity and the way by which bovine Lactoferrin (bLf) acts against YFV infection in Vero cells. MTT
assay indicated that bLf does not lead to cytotoxic effects in the cell line used over 7 days of treatment. Our data of plaque assays
show that the presence of bLf during the whole infection process was able to promote over 70% inhibition of YFV infection. In
order to investigate whether bLf interfere in the viral adsorption stage or on viral replication in the cell, bLf was added in different
steps of infection. Our results show that absence of bLf in adsorption step lead only to a slight inhibition (<15%) of viral infection.
Moreover, in order to investigate the decrease of viral load induced by treatment with bLf, we are now attempting to quantify the
viral RNA by RT-PCR, and evaluate whether bLf inhibits the amplification of genome in a dose-dependent manner. Our findings
suggest that bLf is capable of inhibiting YFV infection, mainly on its early steps. Thus, due to the absence of effective therapies
against this arbovirus, the finding of potent antiviral drugs is extremely important to minimize symptoms and mortality of this
disease.
Support: CNPq, CAPES, FAPERJ, PRONEX, INBEB.
G33
ESTUDOS DA AGREGAÇÃO DO MONÔMERO DA VARIANTE NÃO AMILOIDOGÊNICA DA TRANSTIRRETINA
1- RODRIGUES, M.P., 1- FONTES, L.A.S., 1- PALHANO, F.1- BRAGA, C. A., 1- FOGUEL, D.
1- Instituto de Bioquímica Médica
A Transtirretina (TTR) é uma proteína homotetramérica de 55 KDa, sintetizada principalmente pelo fígado, e em menor proporção
pelo plexo coróide. Presente no plasma e no fluido cérebro-espinal tem como função transportar a proteína ligadora de holo-
retinol, além de realizar o transporte secundário de tiroxina. Existem mais de cem mutações pontuais descritas na literatura
envolvidas com doenças amiloidóticas relacionados à transtirretina. A Polineuropatia Amiloidótica Familiar é uma doença
autossômica dominante caracterizada pelo depósito de agregado amilóide no sistema nervoso periférico. Esses agregados são
formados após a dissociação do tetrâmero e desenovelamento parcial do monômero. A mutação T119M foi identificada em uma
família com alta incidência da variante amiloidogênica V30M, suavizando os sintomas desta mutação agressiva. Assim, a
incorporação de uma subunidade monomérica de T119M em um tetrâmero com a mutação V30M (trans-supressão) pode ser uma
estratégia para estabilizar o heterotetrâmero e inibir a sua agregação. Nosso grupo vem desenvolvendo um modelo de transsupressão voltado para terapia dessas amiloidoses. Esse projeto conseguiu obter monômeros da variante TTR-T119M (M-T119M)
através da elaboração de um protocolo de dissociação com a combinação de baixo pH, baixa temperatura e alta pressão
hidrostática. No decorrer desta pesquisa, verificou-se que o M-T119M é suscetível a agregação Visto que estes monômeros
provêm de uma estrutura tetramérica estável, sua agregação mostrou-se como um importante objeto de estudo visando a
aplicação dos mesmos como terapia. Neste trabalho objetivamos estudar a via de agregação do M-T119M, assim como
caracterizar os agregados formados. Em condição de desnaturação parcial, pH 4.4, o M-T119M forma agregados, comportamento
observado por outras variantes monoméricas da transtirretina. De acordo com dados de cinética de agregação acompanhadas por
medidas de espalhamento de luz, após 16 horas, o M-T119M agrega 13 vezes mais quando submetido a pH 4.4, em relação ao
mesmo na sua forma solúvel (pH 3.0). Após vinte e dois dias, sem agitação a 37ºC, os agregados do M-T119M se mostram amorfos
iniciando a formação de protofibrilas. A fim de caracterizar sua estabilidade, estes agregados foram submetidos à alta pressão
hidrostática (42.000 psi) ao qual não foram resistentes. Os dados obtidos até aqui demonstram que a variante T119M tem sua
estabilidade estrutural afetada quando na forma monomérica, sendo importante a caracterização da agregação do M-T119M a fim
de aplicá-lo como uma terapia.
Apoio: CNPq, CAPES e FAPERJ.
G34
COMPARISON AMONG THREE EXPERIMENTAL MODELS OF HEPATIC DISEASES: BASIS TO DEVELOPMENT OF NEW THERAPIES.
1,2,3-QUINTANILHA,L.F; 1,2,4-CUNHA,S.T; 1,3- FRAGA,M.V; 1,2-SUHETT, G; 1-FACCIOLI,L; 1-PAREDES,B.D; 1-ORMONDE, J.V; 1BARGIONA,E; 1- HENRIQUES,A; 1-VILAS-BÔAS,T.S; 2-CANARY,P.C;2- RESENDE,C.M; 5-TAKIYA,C.M; 2- GUTFILEN,B; 1GOLDENBERG,R.C.
1- Instituto de Biofísica Carlos Chagas Filho – IBCCF/UFRJ, 2- Hospital Universitário Clementino Fraga Filho – HUCFF/UFRJ, 3Centro Universitário Geraldo di Biasi – UGB, 4- Faculdades Integradas Maria Thereza – FAMATH, 5-Departamento de Histologia e
Embriologia - ICB/UFRJ.
Introduction and Objectives: Several models of liver injury have been proposed in preclinical studies. However, the liver response
to injury differs according to its etiology. The aim of this study was compare three different models of experimental liver injury.
Methods and Results: Female transgenic mice (C57BL6–CAG-EGFP) were divided into four groups. Group 1 was irradiated directly
in the liver (dose: 20 Gy), group 2 was submitted to partial hepatectomy (1/3), Group 3 received injections of CCl4 (1.0 ml/kg i.p.),
3 times a week for 4 weeks and group 4 (control). Seven days after the end of each procedure, the animals were euthanized.
Histological staining by H&E and Sirius Red was performed to verify patterns of injury and fibrogenesis, respectively. Levels of
albumin, ALT, AST, alkaline phosphatase, glucose, cholesterol and total protein were measured to assess liver function and injury.
We also analyzed total bone marrow cells by flow cytometry and assessed TGF-β expression by Western Blot. H&E and Sirius Red
staining revealed higher tissue degradation and fibrogenesis in CCl4 group. The RAD group had marked decrease in serum albumin
(CTRL:2.35±0.126; RAD:1.4±0.22 g/dL) and alkaline phosphatase (CTRL:143±10.3; RAD:32.4±2.54 U/L). CCl4 and HPX groups had a
significant increase in serum alkaline phosphatase (HPX:189.0±2.2;
CCl4:192.0±15.6; CTRL:143±10.3 U/L) and AST
(CTRL:62.0±9.90; HPX:118.75±10.4; CCl4:96.0±9.60 U/L). The analysis revealed a decrease of circulating glucose in all groups
compared to control (CTRL:199.5±14.82; RAD:113.7±7.21; HPX:141.0±4.14 e CCl4:128.2±19,77 mg/dL) and the cholesterol level
increased in RAD group (CTRL:79.5±6,22; RAD:149.8±13,55 mg/dL). Flow cytometry analysis showed that CCl4 group presented a
reduction in the hematopoietic progenitors niche into bone marrow. TGF-β expression was increased in all groups. Conclusion:
These comparative results show similarities and differences among these liver injury models raising important aspects in selecting
relevant models for cellular therapy studies.
Support: CNPq, FAPERJ, CAPES, FINEP, Ministério da Saúde.
G35
EXPRESSÃO E PURIFICAÇÃO DA BETA DEFENSINA HUMANA HBD11
MARIANA DIAS CARREIRASa , VIVIANE SILVA DE PAULAa, FABIO C.L. ALMEIDAa, ANA PAULA VALENTEa
aCentro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Instituto de Bioquímica Médica, Universidade Federal do
Rio de Janeiro
Defensinas humanas fazem parte de um grupo de peptídeos antimicrobianos encontrados em mamíferos, apresentam de 30-40
aminoácidos e possuem uma importante função na imunidade inata. Em todas as defensinas seis resíduos de cisteínas
conservados formam pontes dissulfeto que estabilizam as três fitas betas. As defensinas humanas podem ser divididas em três
principais classes (α, β e θ- defensinas) de acordo com o espaço entre os resíduos de cisteína e as pontes dissulfeto. As αdefensinas são encontradas predominantemente nos neutrófilos e em células específicas no intestino delgado; as β-defensinas
são expressas em células epiteliais e da mucosa, sendo reguladas na resposta inata e adaptativa a estímulos inflamatórios e
infecciosos. Cada uma das β-defensinas caracterizadas tem a capacidade de matar ou inibir in vitro uma grande variedade de
bactérias e fungos, principalmente em baixas concentrações de sal e proteínas do plasma. Embora os genes para 28 beta
defensinas tenham sido identificados, a estrutura tridimensional de três (HbBD-1, HbD-2 e HbD-3) já foram resolvidas em solução
por Ressonância Magnética Nuclear (RMN). Neste trabalho, nós selecionamos a beta defensina humana 11 (HbD11) para sua
caracterização estrutural e funcional por RMN. A HbD11 foi subclonada em pET28a, obtida por expressão heteróloga em E. coli
Rosetta(DE3) e purificada por cromatografia líquida de alta performance. Posteriormente, iniciaremos as análises por RMN para
verificarmos se a proteína recombinante encontra-se enovelada corretamente.
Apoio: INBEB, FAPERJ, CNPq.
G36
MICROSCOPY AS A TOOL FOR EVALUATING THE OLEAGINOUS POTENTIAL OF YEASTS: THEIR USE AS FEEDSTOCK FOR BIODIESEL
PRODUCTION
1 -MONNERAT, M.M., 1-VIEIRA, V.S., 1-SANT’ ANNA, C., 3-FRASES, S., 1,2-GARCIA, E. S., 1,3-DE SOUZA, W., 1-MARTINS, J. L.
1-Laboratório de Biologia, Diretoria de Programas, Instituto Nacional de Metrologia, Qualidade e Tecnologia, Brazil
Biodiesel is one of the most prominent renewable energy resources. It is made mostly from vegetable oils but encounter
limitations regarding their availability and price. Recently, the exploration of microbial lipids as feedstock for biodiesel has
attracted considerable attention since the microbial oil productivity can greatly exceeds the vegetable productivity. Besides that,
oleaginous microorganisms, such as yeasts can accumulate high amount of triglycerides when grown in industrial and agricultural
residues, which reduce considerably the price of the biodiesel. The lipids accumulation is directly dependent on microorganism
cultivation conditions. Therefore, the investigation of lipids productivity in different growing conditions is important to evaluate
the viability of this alternative source of feedstock for biodiesel production. The aim of this work was to evaluate the oleaginous
potential of the yeasts using microscopy approaches. We used fluorimetric measurement of yeast stained with Nile Red (NR), a
lipid-specific fluorescent dye, as a fast method for evaluate lipid accumulation. The results were further confirmed by
morphometric quantification of lipid droplets (LD) volume in confocal microscopy. The LD morphology from best strains (those
who accumulated more lipids) was also analyzed by electron microscopy. By fluorimetric analysis, it was observed that lipids
accumulation increase with the time of growth in all selected strains. We observed by confocal microscopy large amounts of LD in
the cytoplasm of NR stained yeasts cells. In freeze-fracture images, LD was observed as concentric multilayered structures, as
previously reported in neutral LD from adipocytes. We found in freeze-substituted yeast cells several electron-dense citoplasmic
granules, probably the LD. The positive reaction in imidazole-buffered osmium tetroxide cytochemistry, confirmed the lipid nature
of these granules. We applied the combination of fluorimetry and morphometry in confocal images to evaluate lipid accumulation
in yeasts grown in different culture media. It was evidenced that some strains isolated from both sources accumulated 3-fold more
lipids in nitrogen limited medium. A high correlation between the NR fluorescence intensity and the morphometric quantification
was achieved.
Support: inmetro, INBEB, FAPERJ, CNPq.
G37
ANÁLISE DE ESFINGOLIPÍDIOS POR RESSONÂNCIA MAGNÉTICA NUCLEAR
SARZEDAS, C. G.; TINOCO, L. W.
Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro
A esfingosina e a esfingosina-1-fosfato são metabólitos bioativos que atuam como mensageiros para ativar ou inibir múltiplos
alvos para regular o crescimento celular, diferenciação e apoptose. A natureza anfifílica destes compostos leva a sua agregação e a
compreensão deste processo em diferentes condições pode contribuir para a compreensão dos processos de desordem no
armazenamento lisossomal dos glicoesfingolipídios [Sasaki, H. et al. Biophys. J. 2009, 96, 2727]. Este trabalho teve como objetivo
analisar o processo de agregação da esfingosina em solução aquosa por RMN. As amostras da D-eritro-esfingosina 8-20 mM
(Sigma Aldrich) foram preparadas em solução tampão fosfato salina 10 mM, pH 7,0 em D2O ou em D2O com NaCl 30 mM e 60
mM. Em todas as condições testadas foi observada a formação de pequenos agregados, insolúveis mesmo após intensa agitação.
Quando as amostras foram aquecidas houve a formação de uma suspensão e de agregados maiores. A parte solúvel destas
amostras foi analisada por RMN [VNMRS-500 (Agilent) 499,8 MHz para 1H] a 25 oC. Os sinais observados na região entre 0,7 a
2,15 ppm correspondem aos hidrogênios da metila (H18), da cadeia longa (H7-17) e ao hidrogênio vizinho à dupla ligação (H6). Os
picos entre 3,0 e 6,0 ppm, correspondem aos hidrogênios H1-H5. Com o aumento da concentração da esfingosina e de NaCl há
uma diminuição da intensidade desses sinais, indicando formação de novos agregados ou aumento do tamanho dos já existentes,
o que faz com que diminua o tempo de relaxação. Foram observados os sinais correspondentes a OH e NH2 em 7-8 ppm,
sugerindo formação de ligação de hidrogênio, já que estes hidrogênios sendo lábeis deveriam ser trocados pelo deutério. A
agregação da esfingosina varia conforme a sua concentração, a presença ou não de sal e tem sido descrita como dependente do
pH.
Apoio:INBEB, CNPq, FAPERJ, FINEP.
G38
ENVOLVIMENTO DA PROTEÍNA P53 NOS EFEITOS BIOLÓGICOS PROMOVIDOS POR RESVERATROL EM CÉLULAS TUMORAIS MCF-7
E H1299
1,2-CAMPOS, N.P.C, 1,2-COSTA, D.C.F, 1,3-CASANOVA, F.A., 1,2-SANCHES, D., 1,2-SANTOS, P. S., 3-FIALHO, E., 1,2-SILVA, J.L.
1- Instituto de Bioquímica Médica; 2- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem; 3- Instituto de
Nutrição Josué de Castro; Universidade Federal do Rio de Janeiro.
O resveratrol (RV), um polifenol encontrado principalmente nas cascas de uvas e no vinho tinto, possui inúmeras atividades
biológicas. A proteína p53 desempenha um papel essencial na prevenção do câncer, podendo induzir parada do ciclo celular ou
apoptose em resposta a diferentes estresses celulares. O objetivo deste trabalho foi estudar o efeito do RV em células tumorais de
mama (MCF-7), que expressam constitutivamente a p53, e de pulmão (H1299), que apresentam uma deleção no gene que codifica
esta proteína. O RV reduziu a viabilidade das células MCF-7 e H1299, de maneira tempo e dose dependente. Entretanto, as células
MFC-7 foram mais sensíveis que as células H1299 quando expostas a concentrações de resveratrol superiores a 100 µM por 24 h.
Adicionalmente, o RV aumentou os níveis de p53 em células MCF-7, sem alterar os níveis de RNAm desta proteína. Nas mesmas
condições, o RV estimulou a clivagem da proteína poli(ADP)ribose polimerase (PARP), utilizada como marcador de apoptose, o que
foi acompanhado pela ativação das caspases 7 e 9. Em células H1299, a citotoxicidade do RV foi menos pronunciada, de acordo
com os ensaios de MTT e LIVE/DEAD. Nestas células a morte celular não foi acompanhada pela ativação de caspases. Além disto,
as células MCF-7 foram positivamente marcadas para TUNEL após serem expostas a 100 µM de RV por 24 h, mas não as células
H1299. No entanto, a transfecção das células H1299 com um plasmídeo contendo a proteína p53 fusionada a GFP tornou as
mesmas mais sensíveis aos efeitos proapoptóticos do RV. Nossos resultados sugerem que a expressão de p53 parece ser
necessária para que ocorra apoptose induzida por RV na linhagem H1299. Dessa forma, a modulação da p53 por RV parece ser um
importante mecanismo pelo qual este composto bioativo exerce seus efeitos quimiopreventivos.
Apoio: INBEB, FAPERJ, CNPq.
G39
CARACTERIZAÇÃO DE UMA ATIVIDADE ECTO-FOSFATÁSICA NA SUPERFÍCIE DO FUNGO METARHIZIUM ANISOPLIAE
ROCCO-MACHADO, N., COSENTINO-GOMES, D., MEYER-FERNANDES, J.R.
Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, Brasil
Metarhizium anisopliae é um fungo entomopatogênico, com a capacidade de infectar uma variedade de artrópodes, desde
carrapatos e pragas agrícolas até vetores de doenças humanas. Os mecanismos que o fungo utiliza para reconhecer seu
hospedeiro ainda são desconhecidos, sabe-se que este processo envolve muitos fatores, como a secreção de proteases, quitinases
e lipases, que permitem sua penetração na cutícula do inseto. Em fungos que são patógenos humanos, como Candida parapsilosis
e Cryptococcus neoformans, as ecto-fosfatases são consideradas marcadores de patogenicidade. Neste contexto, este trabalho
tem como objetivo caracterizar uma atividade ecto-fosfatásica presente na superfície de conídios intactos do fungo M. anisopliae,
que pode estar relacionada ao processo de adesão do conídio na superfície do hospedeiro. A atividade desta enzima é linear de
acordo com o tempo e com o aumento do número de células. Apenas 20% desta atividade fosfatásica é secretada para o meio
extracelular. O pH ótimo da enzima encontra-se na faixa ácida. Metais divalentes como Cu2+, Cd2+ e Zn2+ são capazes de inibir a
atividade ecto-fosfatásica, enquanto que Co2+, Ca2+, Sr2+, Mg2+ e Fe2+ não apresentam qualquer efeito sobre a atividade.
Fluoreto de sódio, molibdato de amônio e ortovanadato de sódio, inibidores de fosfatases ácidas, são capazes de inibir a atividade
desta enzima, no entanto, a adição de levamizol, um conhecido inibidor de fosfatases alcalinas, não é capaz de modular a
atividade ecto-fosfatásica. O fosfato inorgânico, produto da reação catalisada por esta enzima, também é capaz de promover
inibição. Além disso, a inibição da atividade ecto-fosfatásica de M. anisopliae reduz a adesão dos conídios na asa do hemiptero
Dysdercus peruvianus, e consequentemente, a virulência de M. anisopliae. Os resultados aqui descritos mostram a importância da
atividade ecto-fosfatásica em conídios de M. anisopliae, e a primeira evidência de sua participação no processo de adesão e
infecção no inseto hospedeiro.
Apoio: INBEB, CNPq, CAPES e FAPERJ.
G40
AÇÕES DE CÉLULAS-TRONCO EM MITOCÔNDRIAS RENAIS NA LESÃO DE ISQUEMIA/REPERFUSÃO.
1,2- ADALBERTO VIEYRA;1,2- HELLEN VIEIRA-BEIRAL ; 3- ANTONIO GALINA; 3- CLARA RODRIGUES-FERREIRA;1,2- NICOLI
MORTARI;1,2- AMANDA FIGUEIREDO;2,3- MARTHA M. SORENSON; 2,3- CÍCERO FIGUEIREDO-FREITAS
1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Instituto Nacional de Ciência e Tecnologia
de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro; 3- Instituto de Bioquímica Médica, Universidade
Federal do Rio de Janeiro.
Introdução: A isquemia renal seguida de reperfusão (IR) é um processo sempre presente nos transplantes e na insuficiência renal
aguda (IRA) podendo resultar em insuficiência renal crônica irreversível onde o tratamento dialítico ou um novo transplante
constituem as únicas opções. Estas intervenções terapêuticas são custosas trazendo à tona discussões sobre terapias alternativas
incluindo a terapia celular. Objetivos: Investigar a influência de células mononucleares derivadas da medula óssea (CDMO) na
respiração, na geração do gradiente de potencial eletroquímico para H+ e de espécies reativas de oxigênio (ROS) em mitocôndrias
isoladas do córtex renal de rato. Metodologia: Foram utilizados ratos Wistar, machos adultos (180-200g). Sob anestesia geral, foi
realizado clampeamento bilateral das artérias renais por 30 min. Antes e após este procedimento foram injetadas
subcapsularmente as CDMO (107). A reperfusão foi de 24 h. As mitocôndrias foram isoladas e a respiração desta organela
investigada na presença de substratos e inibidores específicos para o complexo respiratório II. O gradiente de potencial para H+ foi
estimado usando o método de fluorescência da safranina O e a medida de ROS foi realizada pela oxidação da molécula de
resorufina do Amplex red ao composto fluorescente resorufina. Resultados: A IR inibiu a capacidade do complexo II de transferir
elétrons para o complexo III, diminui drasticamente a síntese de ATP, aumentou a produção de ROS e provocou colapso parcial do
gradiente de prótons. A administração de CDMO, 1 h antes da isquemia ou antes de iniciada a reperfusão, levou à prevenção da
ocorrência ou à recuperação completa de todos os parâmetros de funcionamento mitocondrial afetados pela IR. Conclusões: As
CDMO podem estar estimulando e mobilizando células progenitoras adultas a partir do seu nicho subcapsular e/ou agindo
diretamente na transferência de componentes mitocondriais para as células sobreviventes.
Apoio: INBEB, FAPERJ, CNPq, DECIT, CAPES.
G41
ANÁLISE POR RMN DA INTERAÇÃO DA PROTEÍNA MARCKS COM A ESFINGOSINA-1-FOSFATO
SARZEDAS, C. G.; TINOCO, L. W.
Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro
A proteína MARCKS - Myristoylated Alanine-Rich C Kinase Substrate, uma proteína de grande importância no desenvolvimento
cerebral. Recentemente, foi observado que a proteína MARCKS é conduzida à barreira endotelial pela esfingosina-1-fosfato (S1P),
e na ausência dessa proteína, a proteção concedida pela S1P à barreira endotelial é reduzida, desencadeando uma serie de
desordens provocadas pelo aumento da permeabilidade vascular que é vista em processos inflamatórios, metástases tumorais,
angiogêneses e arteriosclerose (Camerer et al. J. Clin. Invest. 2009, 119, 7, 1871-1879). A compreensão desse mecanismo de
interação poderá contribuir para o tratamento de diversas doenças. Sendo assim, o objetivo principal deste projeto é usar a RMN
como ferramenta para compreender como ocorre a interação entre a S1P e a proteína MARCKS identificando a região de ligação
da MARCKS responsável por esta interação. Começamos este estudo pela região da proteína MARCKS conhecida como Domínio
Efetor (ED). A região central da proteína, por ser essa a região responsável por todas as interações conhecidas até o momento da
proteína MARCKS com seus diversos alvos. Estão sendo feitas as análises dos espectros TOCSY e NOESY do domínio ED na ausência
e na presença da S1P, se esta interação ocorre e quais os aminoácidos envolvidos.
Apoio: INBEB, CNPq, FAPERJ, FINEP.
G42
PRODUÇÃO DE MAGNETOSSOMOS EM DIFERENTES CONDIÇÕES DE CULTIVO EM BIORREATOR DO TIPO TANQUE AGITADO
1-P.Leão, 1-K.T.Silva, 2-M.L.E. Gutarra, 1-U.Lins
1-Lab. de Biologia e Ultraestrutura de Procariotos, Instituto de microbiologia Paulo de Góes - UFRJ; 2- Escola de Quimica, UFRJ
Nanopartículas magnéticas são aplicadas em processos biotecnológicos, como no direcionamento magnético de drogas e na
separação magnética de células. Seu tamanho reduzido e as propriedades magnéticas são algumas das propriedades que tornam
possíveis tais aplicações. Porem, o processo químico de obtenção destes cristais é de baixa rentabilidade, gerando muitos cristais
defeituosos. Uma alternativa é o uso de partículas magnéticas bacterianas chamadas magnetossomos. Tais estruturas são
compostas por um cristal magnético de magnetita ou greigita, envolto por uma bicamada lipídica com tamanho entre 50-100nm.
Os magnetossomos são produzidos por bactérias magnetotáticas de forma biologicamente controlada, o que garante ótimas
propriedades magnéticas. O cultivo destas bactérias é um desafio, sendo necessários maiores estudos para a produção de
magnetossomos em larga escala. “Candidatus Magnetovibrio blakemorei” (MV-1), a bactéria alvo de nosso estudo, é uma
alfaproteobactérias e produz magnetossomos prismáticos de magnetita. O objetivo deste trabalho é observar o cultivo do MV-1
em biorreator e avaliar seus efeitos na fisiológia e na produção de magnetossomos. Acompanhamos o crescimento através de
espectrofotometria e analisamos: número de magnetossomos por célula e tamanho dos cristais utilizando eletromicrografias
obtidas em um microscópio eletrônico de transmissão (FEI Morgagni) operando a 80KV. Tais dados foram obtidos em dois
cultivos: 500 ml de cultura em frasco incubado a 28°C sem agitação e outro em biorreator de tanque agitado contendo 1,25L de
cultura incubado a 28°C, a 100 rpm. Verificamos uma diferença nas cinéticas do crescimento. Nos frascos após 48h as células
entram em fase estacionária, no biorreator após 48h a velocidade de crescimento é reduzida, mas ainda significativa. A produção
de magnetossomos, após 48h é de 2,29 x 1010 mag/mL no frasco e de 4,06 x 1010 mag/mL no biorreator. Assim, acreditamos ser
benéfica a passagem do cultivo para o biorreator. Os magnetossomos produzidos em biorreator têm bom tamanho e morfologia.
No futuro, o efeito da concentração de inoculo, oxigênio dissolvido e pH sobre o crescimento, serão avaliados.
Apoio: INBEB, FAPERJ, CNPq, CAPES.
G43
EFEITO DO HEME NA LIBERAÇÃO DE AMINOÁCIDOS INDUZIDO POR MEIO HIPOTÔNICO EM LEISHMANIA AMAZONENSIS
1,2-VIEIRA-BERNARDO, R; 1,2-PALETTA-SILVA, R; 1,2-MEYER-FERNANDES, J.R.
1-Instituto de Bioquímica Médica da UFRJ; 2-Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem,
Universidade Federal do Rio de Janeiro;
Introdução: Leishmania é um protozoário que durante seu ciclo de vida apresenta duas formas distintas: no hospedeiro
vertebrado, intracelular, denominada amastigota; nos hospedeiros invertebrados, denominada promastigota. Devido à exposição
a diferentes microambientes, nos quais apresentam variação nas concentrações de nutrientes, temperatura e pH, o parasito
necessita de mecanismos que permitam adaptação para desenvolvimento do seu ciclo. Um destes mecanismos é conhecido como
RVD, onde há uma liberação programada de osmólitos inorgânicos e/ou orgânicos como resposta a estresse hipotônico. Muitos
estudos demonstram que este mecanismo é regulado por proteína quinase C (PKC). Recentemente, foi demonstrado que a
molécula de Heme, é capaz de ativar a PKC de L. amazonensis e aumentar a atividade da bomba (Na+/K+)ATPase. Objetivo: Nossa
proposta foi de avaliar a participação da molécula de Heme como modulador de canais de aminoácidos ativados por estresse
hipotônico e a participação da bomba (Na+/K+)ATPase neste processo. Metodologia: Promastigotas de L. amazonensis foram préincubadas em meios hipotônicos por 10min, após exposição por 20min dos agentes efetores testados. Resultados: A molécula de
Heme e PMA foram capazes de aumentar em 50% a liberação de aminoácidos; ambas ativações foram revertidas na presença de
Calfostina C. A presença do inibidor de PLC, U73122, foi capaz de reverter a estimulação por Heme, mas não foi capaz de reverter
a estimulação por PMA. Ao realizar o ensaio com diferentes concentrações de Heme, observamos um pico de atividade em 50nM.
O próximo passo foi utilizar a Ouabaína, um inibidor da bomba (Na+/K+)ATPase. Foi observado, um efeito inibitório da estimulação
por Heme e PMA com o uso deste inibidor. Conclusão: Heme estimula a liberação de aminoácidos induzida por meio hipotônico
através de uma sinalização dependente de PLC levando a ativação de uma PKC. Esta estimulação ocorre com pico em 50nM e é
dependente da bomba (Na+/K+)ATPase.
Apoio: INBEB, FAPERJ, CNPq, CAPES.
G44
“INTERRUPTOR LIPÍDICO”: ESTUDO DAS MUDANÇAS CONFORMACIONAIS INDUZIDAS POR CONJUGAÇÃO LIPÍDICA DE UMA
PROTEÍNA REGULADORA DA AUTOFAGIA EM MAMÍFEROS
1- LAPIDO LOUREIRO, P.A.; 1- IGLESIAS JUNIOR, C.F.; 1- XAVIER, T.S.; 1- PASCUTTI, P.G.
1- Laboratório de Modelagem e Dinâmica Molecular, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro
Até recentemente, a autofagia era tida como um processo de autólise inespecífico, ocorrendo em células sob estresse metabólico.
Atualmente, começa-se a entender que a autofagia é, também, responsável pela degradação regulada de complexos protéicos
marcados com ubiquitina, organelas e microorganismos invasores. Este processo é compartimentalizado, ocorrendo em vesículas
de membrana lipídica dupla (autofagossomos). A proteína LC3 (de mamíferos, ortóloga à proteína ATG8 de fungos e,
estruturalmente, dividida num domínio C-terminal semelhante à ubiquitina e um domínio N-terminal) parece ser essencial à
formação das vesículas autofágicas através de fenômenos de fusão e tethering de membranas, além de servir de receptor para
proteínas marcadas para lise. Para ser ativada, LC3 é conjugada ao abundante fosfolipídio fosfatidiletanolamina (PE), em sua
glicina N-terminal [1]. Essa conjugação parece desencadear uma mudança conformacional em LC3, levando ao afastamento do
domínio N-terminal em relação ao C-terminal [2]. Por não se conhecer os eventos estruturais dessa ativação, realizamos
simulações por dinâmica molecular da proteína LC3 e LC3 conjugada a PE. Para isso, usou-se como ponto de partida a estrutura
derivada de cristalografia de RX de LC3 de camundongos [3], com modelagem por homologia [4] de 2 pequenos trechos nãoresolvidos (total de 7 aminoácidos). O sistema de LC3 complexada a PE foi construído in silico. No nosso conhecimento, este é o
primeiro estudo de simulação por dinâmica molecular que tenta fornecer subsídios de como LC3, uma proteína relativamente
pequena, participa em fenômenos aparentemente díspares como a hemifusão de membranas e ligação a proteínas da cascata da
autofagia. Serão analisadas em detalhe as mudanças conformacionais causadas pela complexação ao fosfolipídio. [1] Chem. Rev.
2009, 109, 1587–1598 [2] Cell 130, 165–178, July 13, 2007 [3] Genes to Cells (2008) 13, 1211–121 [4]
http://modbase.compbio.ucsf.edu/ModWeb20-html/modweb.html
Apoio: CNPq, INBEB.
G45
OBSERVATION OF THE CYTOSKELETON OF MOUSE MACROPHAGES AND LLC-MK2 CELLS BY SCANNING ELECTRON MICROSCOPY
1,2 - Cruz, T.A., 1,2 - Paredes-Santos, T.C., 1,2-Caldas, L.A., 1,2 - Attias, M.
1 Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, Brasil
Scanning Electron Microscopy (SEM) is the primary choice for observation of cell surfaces. With the improvement in resolution of
SEM, several methods have been developed for the stereological observation in nanometric scale, as is the case of microtubules,
microfilaments and intermediate filaments. However, the removal of the plasma membrane and simultaneous preservation of the
cytoskeleton is not very simple to achieve, varying with the cell type and the combination of detergents, buffers, time and
temperature used. For these reasons, we decided to establish protocols for the extraction of plasma membranes to expose and
preserve the cytoskeleton of two cell types widely employed, the LLC-MK2 lineage and mouse peritoneal macrophages (MPM).
LLC-MK2 cell and MPM were separately plated over round cover. Two extraction protocols were tested: 1- the culture was treated
with 1% Triton X-100 and 4% PEG for 5 minutes followed by fixation in 2% glutaraldehyde (Glut) in PHEM buffer, 0.1% tannic acid
solution and 0.1% uranyl acetate for 20 minutes each one. 2- The culture was treated with 1% TritonX-100 in PBS for 2 minutes.
Then, the sample was fixed in 2.5% Glut in 0.1 M sodium cacodylate buffer for 45 minutes and postfixed for 30 minutes. After that,
all the samples were dehydrated in ethanol series, dryed by critical point of CO2, coated with gold and observed in the SEM
microscope. MPM were better visualized with protocol 2, while LLC-MK2 cells with protocol 1. Through the micrographs, we
observe that the filaments of the cytoskeleton were exposed and preserved, showing that the methods were successful. We
observed different reactions to each type of treatment probably because the diferent composition of the plasma membranes of
each cell type. This strengthens that the establishment protocols for each cell type as it is essential for the application in several
studies of cell interaction with pathogens or drugs.
Support: CNPq, FAPERJ, Capes.
G46
AVALIAÇÃO ESTRUTURAL E FUNCIONAL DA INTERAÇÃO DA PROTEÍNA DO PRION COM ÍONS DIVALENTES E DNA
1- SISNANDE,T.; 1-CHAVES, J.A.P.; 1-MACEDO, B.; 2-BRAGA, C.A.; 1,3- CORDEIRO, Y.
1 - Faculdade de Farmácia, 2- Instutuo de Bioquímica Médica, Pólo Xerém; 3- Instituto Nacional de Ciência e Tecnologia de Biologia
Estrutural e Bioimagem
A proteína do prion (PrPC) é um constituinte normal da membrana de células de mamíferos, sendo expressa principalmente no
sistema nervoso central. A PrPC é sensível a proteases, possui um domínio N-terminal flexível e desestruturado e um domínio Cterminal globular bem enovelado [1]. Contido na região N-terminal está o domínio octarepeat que engloba de 4 a 5 repetições de
uma sequência de 8 resíduos de aminoácidos (PHGGGWGQ), compreendida entre os aminoácidos 51-90. Esta região está
envolvida na ligação a íons cobre (II) fisiologicamente; além disso, a PrP também pode interagir com cobre através das histidinas
96 e 111. As doenças de prion são desordens neurodegenerativas causadas por uma forma patogênica da PrP, denominada PrPSc.
A PrPSc apresenta um alto conteúdo de folhas-beta, é resistente à ação de proteases e forma agregados em fibras amilóides.
Sugere-se que a PrPC possa ser convertida espontaneamente em PrPSc; mas também é proposto que exista um catalisador que
diminua a energia de ativação da conversão. Nosso grupo acredita que moléculas de ácidos nucleicos atuem como catalisadores,
com base em diversos resultados experimentais [2]. Neste trabalho avaliamos a interação de íons divalentes e DNA para observar
a capacidade de ambos ligantes em modular a agregação de peptídeos hidrofóbicos da PrP (compreendendo a região 109-149) e
também da PrP recombinante de camundongo. Avaliamos estas interações através de medidas espectroscópicas de espalhamento
de luz (EL), fluorescência intrínseca do triptofano e extrínseca através da ligação à sonda tioflavina T. Verificamos, através do
acompanhamento do EL, que os domínios hidrofóbicos que contém a His-111 sofrem inibição da agregação na presença de íons
divalentes, com resultados mais pronunciados para o cobre (II). Além disso, as interações PrP:DNA:Cu2+ modificam a estrutura
secundária do peptídeo e da proteína, dados avaliados por dicroísmo circular. Avaliamos a morfologia dos agregados da PrP
induzidos pela interação com os íons divalentes e também na presença de DNA através de microscopia eletrônica de transmissão.
Verificamos que sequências de DNA alteram a estrutura da PrP e o perfil de agregação dos peptídeos na presença de Cu(II),
indicando que o DNA consegue exercer seus efeitos sobre a PrP mesmo quando esta encontra-se previamente carregada com íons
cobre. Estes estudos indicam que tanto Cu2+ quanto DNA, ligantes já caracterizados da PrP, não apresentam efeitos excludentes
na proteína e podem auxiliar na elucidação dos mecanismos envolvidos nas EETs.
Apoio: INBEB, FAPERJ, CNPq.
G47
EVIDENCE FOR COMPENSATORY PATHWAYS IN HUMAN CALLOSAL DYSGENESIS
1,2Marins, T., 1,2Queiroz, N., 1Bramati, I.E., 1,2Monteiro, M.C., 1,3Rodrigues, E., 2Lent, R., 1,2Tovar-Moll, F.
1D'Or Institute for Research and Education, Rio de Janeiro, Brazil, 2Institute of Biomedical Sciences, Federal University of Rio de
Janeiro, Rio de Janeiro, Brazil, 3 Augusto Motta University-UNISUAM, Rio de Janeiro, Brazil
Callosal dysgenesis (CD) is characterized by a developmental failure of formation of the major commissural fiber bundle in the
human brain, together with absence of a typical disconnection syndrome. So far, direct evidence for possible compensatory
interhemispheric connections through other commissures is lacking. Since diffusion tensor imaging (DTI) can quantify the integrity
and connectivity of major white matter bundles in vivo, we posed the objective of investigating, by DTI, the existence of possible
compensatory pathways through the other cerebral commissures in patients with CD. Volumetric anatomical and DTI images
were acquired (3T, Achieva Philips) in six patients (2 CD, 2 callosal hypoplasia and 2 partial CD) and seven healthy volunteers (HVs).
Anatomical analyses were carefully performed, with a special attention to the thalamic adherence (TA), the anterior commissure
(AC) and posterior commissure (PC). Regions-of-interest (ROIs) were placed in AC and PC on 3DT1 images in the sagittal plane. The
area, mean fractional anisotropy (FA), and mean diffusivity (MD) were quantified based on AC and PC ROIs. Comparisons between
groups were done using a non-parametric test (Mann-Whitney). Probabilistic tractography of AC and PC was also performed in
patients and HVs. Anatomical images showed a similar aspect of AC and PC in patients and controls, in size and general
topography. The TA was inconsistent between patients, making its measure impracticable. There was no difference in AC or PC
values of FA or MD between groups. However, probabilistic tractography of PC revealed a possible cortical connections in patients,
achieving parietal cortex bilaterally. No differences were found in AC or PC areas, values of FA or MD between CDs and HVs , based
on the ROIs analysis. However, color-coded-FA maps comparisons and tractography analysis showed an enlarged tract compatible
with PC in patients, as compared to HV, that seems to connect parietal lobes bilaterally.
Support: FAPERJ, CNPq and Capes.
G48
EFEITO ALOSTÉRICO DE LIGANTES NA PRPC POR MÉTODOS TEÓRICOS E EXPERIMENTAIS DE PREDIÇÃO ESTRUTURAL
MOLECULAR
1,2 - Wesley Alves; 1 - Rafael Linden; 1 - Pedro Pascutti; 2 - Yraima Cordeiro
1 - Instituto de Biofísica Carlos Chagas Filho; 2 - Faculdade de Farmácia
Embora a principal função da proteína prion (PrPC) permaneça controversa, uma série de ligantes desta proteína de superfície
celular já foram bem caracterizados. Estudos recentes sugerem que a PrPC sirva como uma pataforma de interação para outras
proteínas, modulando diversos processos de sinalização cujos efeitos podem ser traduzidos em diferentes consequências
funcionais. Proteínas que interagem com mais de um ligante podem sofrer alterações conformacionais mediante a ligação com um
primeiro ligante, aumentando ou diminuindo sua afinidade por um segundo ligante. Tais efeitos alostéricos podem ser
evidenciados pela ordem e/ou estequiometria de interação dos ligantes com a PrPC, podendo gerar respostas fisiológicas ou
patológicas. Dentre os ligantes de PrPC até então descritos podemos citar a co-chaperona hop/STI1, o precursor de receptor de
laminina-LRP, a molécula de adesão celular N-CAM, além de ligantes não proteicos como ácidos nucleicos e íons cobre. Nosso
estudo é dirigido aos ligantes proteicos, sendo que cada um desses possuem domínios mapeados de interação à PrPC, que por sua
vez também possui mapeados seus domínios de interação com os respectivos ligantes. Nesse contexto, nosso objetivo consiste
na determinação de mudanças conformacionais sofridas pela PrPC quando ligada a um ou mais de seus ligantes, por técnicas de
simulação computacional. Nessa etapa, usaremos o domínio C-terminal estruturado da PrPC humana e de camundongo, e faremos
um ancoramento molecular entre PrPC e cada domínio de ligação dos respectivos ligantes de forma sucessiva e em diferentes
ordens, acompanhando as mudanças conformacionais na PrPC por dinâmica molecular. Em uma segunda etapa, faremos série de
ensaios in vitro para validar a interação de PrPC com os mesmos domínios dos ligantes, foram obtidos por síntese em fase sólida.
Usaremos técnicas espectroscópicas para verificar o efeito alostérico dos peptídeos ligantes na PrPC, como dicroísmo circular,
anisotropia de fluorescência, e espalhamento de luz dinâmico.
Apoio: INBEB , CNPq , FAPERJ.
G49
CONTRIBUTION OF SCANNING ELECTRON MICROSCOPY TO THE STUDY OF THE COPULATORY POSITION IN Physaloptera
mirandai (NEMATODE: SPIRURID) AND THEIR INTERACTION WITH THE VERTEBRATE HOST
1,2 - MELLO, W.N.; 1,2 - LOPES TORRES, E.J.; 1 - ANJOS, D.H.S.; 1,2,3 - DE SOUZA, W.; 2,3 - MIRANDA, K.
1 - Laboratório de Biologia de Helmintos Otto Wucherer - IBCCF - UFRJ; 2 - Laboratório de Ultraestrutura Celular Herta Meyer IBCCF - UFRJ; 3 - Diretoria de Programas Instituto Nacional de Metrologia - INMETRO.
Species of the Physaloptera genus are nematodes that parasitize different carnivores and small mammals. Classical
helminthological taxonomy generally uses light microscopy (LM) for the characterization of morphological and morphometrical
parameters, although different groups have used scanning electron microscopy (SEM) to further characterize the detailed
structure of the surface nematodes. In this work, SEM was used to identify morphological characteristics of Physaloptera
nematodes with potential use for the redescription of the species. For that, Metachirus nudicaudatus, were captured in the
Biological Reserve Duas Bocas, Espírito Santo State, Brazil, and their stomachs collected. Nematodes found in the stomach were
washed in saline and fixed in 70% ethanol. For LM observation, samples were clarified in 50-90% phenol. For SEM, samples were
post-fixed in osmium tetroxide, dehydrated in ethanol series, critical point dried, mounted in a metal stub, sputtered with gold
and analyzed using a FEI-Quanta 250 or JEOL 5312. Physaloptera mirandai parasitizing the stomach of the M. nudicaudatus were
identified. Two semicircular pseudolips, each bearing a pair of papillae, one amphid, one tripartite structure and porous-like
regions were identified in the cephalic end. One pair of deirids was observed in the lateral line and excretory pore was observed in
ventral line. In addition, an imprint of these structures was observed in the fixation region of the nematode in the host stomach
wall. 21 papillae, three more than the original description, and two phasmids were observed in the posterior end of the male. In
the cuticular surface of the female vulva, one imprint of the posterior end of the male was also observed, where it was possible to
identify the copulation position. Taken together, the results reveal morphological characteristics that may contribute with novel
taxonomical information of the species P. mirandai, with further insights to the copulatory mechanisms of these parasites.
Support: CNPQ, CAPES-PROCAD, FAPERJ.
G50
LIGHT MICROSCOPY AS A TOOL TO INVESTIGATE PRETREATMENT EFFECT ON SUGARCANE
Yuri Abud1, 2, Ricardo Vilela1, Lilian T. Costa1, 2, Wanderley de Souza1, Celso Sant’Anna1
1 - Lab. de Biologia– Inmetro; 2- Pólo de Xérem/UFRJ
Sugarcane has received a prominent position since ethanol derived from it represents an important renewable source of biofuel.
Sugarcane vascular bundles have the highest degree of recalcitrance in plant tissue and a remarkable heterogeneity of cell wall
morphology. In this work, we quantified the distribution of vascular bundles in sugarcane internodal tissue by stereomicroscopy.
In addition, the effect of termochemical pretreatment and/ or cellulase hydrolysis on vascular bundles cell walls was evaluated by
confocal microscopy. Transverse section stained with toluidine blue was used to estimate the distribution and occupied area of
vascular bundles in sugarcane hind, intermediate region and pith (medulla). While 63% of vascular bundles were found in rind
region, the pith region concentrated 9%. Twenty eight percent was observed in the intermediate region. Estimative of occupied
area per region showed that vascular bundles accounted for 41%, 35%, and 24% of rind, intermediate region and pith,
respectively. These findings are indicative that rind is the highest recalcitrant region in sugarcane tissue and pith region is more
susceptible to cellulase action. Distinct vascular bundle morphologies were evidenced by confocal microscopy and lignin and
cellulose distribution was determined by incubation with Safranine O fluorescent staining. Red signal of lignin was more
concentrated in cell corner and compound middle lamella. The higher degree of recalcitrance of rind region when compared with
pith region was further confirmed by spectral analysis of sugarcane tissue stained with safranine. This way, we could show that
rind concentrates almost 3-fold more lignin (the key molecule responsible to recalcitrance of biomass) than pith. Quantitative
measurement of perimeter of vessels and fiber in the rind and pith lumen, as well as cell wall thickness of vessels, parenchyma and
fiber cells was performed in termochemical and/ or cellulase pretreated vascular bundles using confocal microscopy images. In
untreated samples we observed that average cell wall thickness of vessels and thin-walled parenchyma cells is similar, while the
thickness of thick-walled fibers is larger in rind. Termochemical treatment has no pronounced effect on cell wall in both regions.
Cell wall thickness in pith vessel, parenchyma and fibers was more susceptible to the termochemical treatment followed by
cellulase hydrolysis (data not shown). On the other hand, comparative measures of the perimeter in fibers and vessels either after
termochemical or termochemical treatment followed by cellulase hydrolysis is more evident in rind than pith region. Taken
together, our data point to the feasibility of relative quantification by light microscopy of plant tissue anatomical features and the
effectiveness of pretreatment on plant-based biomass.
Support: Inmetro, CNPq, FINEP.
Ap01
ADIPOSE TISSUE MESENCHYMAL CELLS PREVENT LATE RIGHT VENTRICULAR DILATATION IN AN EXPERIMENTAL MODEL OF
CHRONIC CHAGASIC CARDIOMYOPATHY
RAMOS, I.P.R. 1; MELLO, D.B.1; BRASIL, G.V.1; CARVALHO, A.B.1; ROCHA, N.N.2; GOLDENBERG, R.C.S.1; CAMPOS-DE-CARVALHO,
A.C.1,3.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro - UFRJ, 2- Universidade Federal Fluminense –
UFF, 3- Instituto Nacional de Cardiologia -INC.
Chagas’ disease is a leading cause of cardiomyopathy in Latin America. However, despite all the advances in the treatment of
cardiovascular diseases, there is no effective therapy for chagasic cardiomyopathy. Our objective was to evaluate the effects of
mouse adipose tissue-derived mesenchymal cell (mADSC) transplant in an experimental model of chagasic cardiomyopathy. Thirty
CD1 mice, 8-10 weeks of age, were used. Ten animals were used as controls (Cont) and 20 were infected with 3x104
trypomastigotes (Brazil strain, intraperitoneally – ip). The animals that survived the infection were divided into two groups:
infected-only (n=2) and infected+mADSC (n=3). Parasitemia was evaluated from 5th to 34th day post-infection (dpi) by parasite
count in the peripheral blood. Eight months post-infection (mpi) 1x106 cells were injected ip. Cells were isolated from
subcutaneous adipose tissue of transgenic mice expressing the GFP gene under the control of β-actin promoter through enzymatic
digestion and maintained until third passage. Electrocardiographic (ECG) recordings were made in non-anesthetized animals to
evaluate the presence of arrhythmias and alterations in duration and amplitude of segments and waves. Echocardiographic
(ECHO) studies measured left ventricle ejection fraction (EF) and right ventricular area (RVa) in anesthetized animals using 1.5%
isofluorane gas. Data were normalized by body weight and are shown as mean±sd. One-way ANOVA was used for statistical
analysis. Parasitemia was evaluated in all infected animals and reached peak in 27th dpi (61.00±58.08x105 trypomastigotes/ml of
blood). No changes were observed in ECG recordings in infected groups. ECHO analyses did not detect any difference in EF;
however, 3 months post-treatment (mpt), the injection of the cells prevented late RV dilatation in infected+mADSC compared to
Infected-only group (pre-Infection (all animals) 0.52±0.06; 3 mpt (11 mpi): Infected-only 0.81±0.03 vs infected+mADSC 0.56±0.19)
and this effect was sustained until the fourth month (4 mpt (12 mpi): Infected-only 0.76±0.40 vs infected+mADSC 0.58±0.18). Data
presented here shows that treatment with mADSC prevented late RV dilatation in an experimental model of chagasic
cardiomyopathy in mice showing that mADSC might be useful in Chagas’ disease treatment.
Capes, CNPq, FAPERJ, Miniterio da Saúde.
Esp01
SERUM TRANSTHYRETIN LEVELS IN BRAZILIAN PATIENTS WITH FAMILIAL AMYLOID POLYNEUROPATHY
2- FERREIRA, P. S.; 2- LIMA, C.; 1- CRUZ, MÁRCIA WADDINGTON ; 2- FOGUEL, D.
1- Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; 2- Instituto de Bioquímica Médica da Universidade Federal
do Rio de Janeiro, Brazil.
Familial amyloid polyneuropathy (FAP) is an autosomal dominant polyneuropathy of adult onset which used to lead to death
within 10 years on average after the first symptoms. Originally described by Andrade in Portugal, FAP was subsequently identified
throughout the world. Serum transthyretin (TTR) levels are reduced in familial amyloidotic polyneuropathy (FAP). A single study of
patients with senile systemic amyloidosis (SSA) in Sweden found that those individuals also had a significantly lower mean serum
TTR concentration than age- and gender-matched controls. It is noteworthy that there are no reports of levels of TTR in the serum
of patients in Brazil. The objective of this study is to assess the TTR serum levels of patients with FAP and compare these levels to
those of healthy patients. Another objective is to correlate these levels with the clinical development of the disease to assess the
correlation between the levels of TTR and disease progression. Initial patient data collection includes information on family and
medical history, physical and laboratory exam results, and patient reported outcomes. Results are shown for Brazilian patients
enrolled in THAOS. We compared the serum TTR levels, as determined by ELISA method. Some of the patients tested had low
levels of TTR in serum compared to controls. For this study the patients tested present a early and a more developed stage of
clinical disease to reach a more conclusive data.
Support: CNPq.
TG01
A NEW IMPLEMENTATION OF GENERALIZED SIMULATED ANNEALING FOR AB-INITIO PROTEIN STRUCTURE PREDICTION
1,2-Melo, M. C. R.;1-Fernandes, T. V. A.;1,2-Bernardi, R. C.;1-Pascutti, P. G.
1-Laboratório de Modelagem e Dinâmica Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil;2-Diretoria de
Programas, Instituto Nacional de Metrologia, Qualidade e Tecnologia , Rio de Janeiro, Brasil
Proteins are the building blocks of cells and the executioners of nearly all cellular functions. Their activity directly depends on their
specific three dimensional structure, determined by the folding of its amino acid chain. The folding process ultimately creates a
stable structure balancing internal contacts between amino acids and their occlusion to create the protein surface and the
hydrophobic core. In this work we explore a new design for applying Generalized Simulated Annealing (GSA) on protein structure
prediction, based on previous software developed by our group. The GSA is a stochastic search algorithm employed in energy
minimization and used in global optimization problems, such as gravity models, fitting of numerical data and conformation
optimization of small molecules. The software deploys a new way of updating the protein structure at each step of the simulation,
a different potential energy calculation function based on NAMD and parallel execution of simulations, granting a new take on abinitio protein structure prediction. The design of the software also allows for the inclusion of data derived from large scale analysis
of protein structures from the PDB, as the Solvation Free Energy, allowing us to use information already gathered by experimental
structure determinations. We present results on mastoparan-X, a 13 amino acid peptide. The chain folded with RMSD of 2,3 nm a
after 500.000 GSA steps. Structure prediction softwares allow us to study protein structures that cannot be experimentally
determined, by using data on chemical bonds, non-bonded interactions and protein solvation. Once approximately predicted, the
three dimensional structure can be refined by molecular dynamics simulations.
Support: INBEB, CNPq, FAPERJ.
M01
THE EFFECT OF HEPARIN LENGTH ON INTERACTION WITH THE PRION PROTEIN
1- Murakami, A.Y.; 1- Vieira, T.C.R.G; 1- Reynaldo, D.P.; 1- Gomes, M.P.B.; 1- Almeida, M.S.; 2- Cordeiro, Y; 1- Silva, J.L.
1- Instituto de Bioquímica Médica, IBqM-UFRJ, RJ; 2- Faculdade de Farmácia, FF, UFRJ, RJ, Brazil
Transmissible spongiform encephalopathies (TSEs) are a group of fatal diseases, which affect animals and humans, caused by an
abnormal isoform of the prion protein (PrP). Prions replicate in the host cell by the self-propagating refolding of the normal cell
surface protein, PrPC, into a b-sheet-rich conformer, the PrPSc. In addition, several lines of evidence suggest that
glycosaminoglycans (GAGs) and in particular heparan sulfate/heparin may play a role in the PrPC to PrPSc conversion process.
Recently we characterized the structural changes induced by low molecular weight heparin (LMWHep) on murine recombinant
prion protein (rPrP23-231) and showed that LMWHep interaction prevents protein aggregation induced by RNA (J Amer Chem Soc,
2011, 133: 334-344). However, it has been shown that GAGs can stimulate cell-free conversion. These paradoxical effects could be
related to a differential interaction dependent on heparin size. In the present work, we performed light-scattering, fluorescence
and nuclear magnetic resonance spectroscopy measurements in order to investigate this hypothesis. The interaction between
rPrP23-231 and full-length heparin showed a different stoichiometry from LMWHep. It induced PrP oligomerization/aggregation
with the same extent at pH 5.5 and pH 7.4. As we observed for LMWHep, full-length heparin also induces a transient aggregation.
NMR results showed marked differences in chemical shifts between free and Hep-bound samples corresponding to residues from
the N- and C-termini, revealing binding regions. On this basis it may be inferred that PrP interaction with full-length heparin exerts
different chemical and physical properties, and this difference may affect protein conversion.
Support: CNPq, INBEB, FAPERJ, CAPES.
M02
ESTUDOS POR ANCORAMENTO MOLECULAR DE POTENCIAIS QUIMIOTERÁPICOS PARA LEISHMANIOSE
1 - LIMA, A.B.; 1,2,3 - FIGUEROA-VILLAR, J.D.
1 - Seção de Engenharia Química - SE/5, Instituto Militar de Engenharia - IME; 2 - Associação de Usuários de Ressonância
Magnética Nuclear, AUREMN; 3 - Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem
Dentre as doenças tropicais negligenciadas – DTN apontadas pela Organização Mundial de Saúde – OMS [1], destaca-se nesse
estudo a leishmaniose, doença parasitária também conhecida como calazar. De acordo com dados da OMS, a leishmaniose
ameaça milhões de pessoas em vários países. Acredita-se que 12 milhões de pessoas estejam infectadas atualmente e estima-se a
ocorrência de 1 a 2 milhões de casos a cada ano [1]. Haja vista a complexidade e as dificuldades apresentadas no tratamento das
diferentes formas clínicas das leishmanioses, o desenvolvimento de novos fármacos mais seletivos e menos tóxicos é de extrema
importância. Nesse sentido, foram feitas simulações por ancoramento molecular de potenciais inibidores da Nucleosídeo
Hidrolase de Leishmania donovani - LdNH. Foram utilizados como parâmetros comparativos o substrato natural da enzima
(inosina) e dois inibidores teóricos propostos pelo grupo [2]. Os compostos comparados foram a riboflavina, que por irradiação
com luz ultravioleta apresentou resultados de inativação do patógeno de Leishmania donovani infantum [3], além de três novos
aciclonucleosídeos planejados pelo grupo que estão em etapa final de síntese. Resultados mostram que os aciclonucleosídeos
interagem com resíduos de aminoácido do sítio ativo da enzima e apresentam menor energia de ligação do que o substrato
natural da enzima. Com esse estudo, pode-se avaliar a interação de novos potenciais inibidores com a enzima LdNH. Aliados a
estudos por dinâmica molecular os resultados poderão indicar de maneira mais eficiente as potenciais interações do inibidor com
a enzima. Isso é, direcionarão os trabalhos de síntese orgânica e fornecerão uma possível prévia dos testes de atividade biológica.
Referências Bibliográficas [1] WORLD HEALTH ORGANIZATION – WHO. WHO | Infectious Diseases. 2010. [2] França, T. C. C.; et. al.
J. Braz. Chem. Soc., v. 19, p. 64-73, 2008. [3] Cardo, L.J. et al. Vox Sang. 90(2), 85-91, 2006.
Apoio: INBEB, CNPq, CAPES e FAPERJ.
M03
LIPIDS DROPLETS IDENTIFICATION IN Leishmania (Viannia) braziliensis PROMASTIGOTES CAUSED OF MUCOSAL LEISHMANIASIS
1,2-HAGE, A.A.P.; 1,2-RODRIGUES, A.P.D.; 1,2-FARIAS, L.H.S.; 1,2-SILVA, E.O.
1-Laboratório de Parasitologia e Laboratório de Biologia Estrutural, Instituto de Ciências Biológicas, Universidade Federal do Pará,
Belém, Pará, Brazil; 2-Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio
de Janeiro, Ilha do Fundão, Brazil
The American Tegumentary Leishmaniasis (ATL) is an infectious disease world wide spread, caused by a protozoan of genus
Leishmania. Leishmania (Viannia) braziliensis is the main etiologic agent of ATL. The strain and cellular immune response of the
infected host can develop the cutaneous or mucocutaneous forms of the diseases. The latter is the most severe type of clinical
manifestations. Lipid droplets are emerging as highly dynamic organelles that play crucial roles in mammalian cellular energy
homeostasis and lipid metabolism. Little is known about the relation between this specie of Leishmania and the lipid droplets and
it is important to a better understanding of a possible virulence factor that could be a therapeutic target. The present work
analyzed the presence of lipid droplets in L. (V.) braziliensis promastigotes in different days of the grown, early (7 days) and later
(10 days) stationary phase. In the present study we could observe using sudan black B dye and light microscopy that promastigote
forms cultivated until the later stationary phase showed more lipid droplets distributed in the cytoplasm than cells cultivated in
early stationary phase. The same result was observed by routine electron microscope and confirmed with osmium-imidazole
cytochemistry analysis. The largest distribution of lipid droplets in late stationary phase promastigotes was also observed using the
fluorescent BODIPY 493/503 labeling. The presence of lipids reserves can be related with parasite energetic necessities during the
stationary growth period when the parasite presents the higher capacity of infection. The identification of this structure is
important to understand the role of lipid reserves in Leishmania parasites. Moreover, those data are essential for future
identification of possible targets for action of therapeutic drugs. Keywords: L. (V.) braziliensis, mucosal leishmaniasis, lipids
droplets.
Support: CAPES, CNPq/UFPa, CNPq/MCT/CT-INFRA/CT-PETRO (Processo nº 620179/2008), MCT/CNPq/FNDCT/PROCAD-NF
CAPES/FAPERJ.
M04
ESTABILIDADE ESTRUTURAL DE UMA PLATAFORMA VACINAL PARA O VÍRUS DA IMUNODEFICIÊNCIA HUMANA (HIV-1)
1- VICENTE, A.C.S.; 1- BARROSO, S.P.C.; 2- GOMES, D.C.; 2- FERREIRA, D.F.; 3- PEABODY, D.S.; 1- OLIVEIRA, A.C.
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2- Instituto de Microbiologia e Imunologia,
Universidade Federal do Rio de Janeiro; 3- Department of Molecular Genetics and Microbiology, University of New México
Partículas semelhantes a vírus (VLPs) podem ser consideradas como arranjos densos repetitivos de uma ou mais subunidades de
uma proteína e esta característica confere propriedades que são altamente vantajosas para seu uso como plataformas de vacinas.
Neste projeto, utilizamos VLPs formadas pela proteína capsídica do bacteriófago MS2. Avaliamos a estabilidade estrutural desta
plataforma vacinal para peptídeos altamente imunogênicos relacionados ao ciclo infeccioso do HIV-1, submetendo tais partículas a
alta pressão hidrostática (APH) e a outros agentes desnaturantes químicos e físicos. Para essa avaliação, utilizamos medidas de
espalhamento de luz, fluorescência intrínseca e extrínseca e dicroísmo circular. Analisamos também a morfologia das VLPs por
microscopia eletrônica de transmissão. Os resultados foram obtidos com a VLP formada por um dímero de cadeia única da
proteína capsídica, duas construções do dímero de cadeia única com o epítopo Flag e com as VLPs que contêm peptídeos da alça
extracelular do co-receptor celular CCR5 e da alça V3 da proteína gp120 do HIV-1, peptídeos estes descritos por induzir a formação
de anticorpos com alto potencial antiviral. Os resultados de fluorescência e de espalhamento de luz indicam haver pequenas
mudanças na estrutura das VLPs com a inserção dos epítopos, com exceção dos resultados com APH, onde as construções com os
insertos obtiveram um maior desvio do centro de massa espectral. Medidas de dicroísmo circular indicam não ter havido mudança
na estrutura secundária entre o dímero de cadeia única e as construções com o epítopo Flag. Os estudos de estabilidade desta
nova forma de apresentação de peptídeos imunogênicos buscam contribuir com informações estruturais para o desenvolvimento
desta promissora plataforma vacinal.
Apoio: CAPES, FAPERJ, CNPq, PRONEX, INBEB.
M05
DEVELOPMENT OF 153SM-EDTMP NANORADIOPHARMACEUTICALS
1,2-PATRICIO,B.F.C.; 2- SANTOS-OLIVEIRA,R.; 1- WEISSMULLER, G.
1-Laboratório de Física Médica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Laboratório de
Nanorradiofármacos, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro.
Development of 153Sm-EDTMP Nanoradiopharmaceuticals Objectives: Nanoradiopharmaceuticals are the most promising
radiopharmaceuticals. In the case of bone metastases they represent an important advance. The Samarium-153 lexidronam
(153Sm-EDTMP) is an effective and well-tolerated treatment for this disease because its efficacy, low cost and low toxicity. In this
work we discuss the formation of such nanoradiopharmaceutical and its characterization using Atomic Force Microscopy (AFM).
Methods:Nanoparticles: Nanoparticles were produced according the double emulsion method. Two milliliters of a solution of 2,5%
of poly lactic acid (40-100kDa, Sigma-Aldrich) in dichloromethane were made. This was poured on 200µL solution of PVA 0,1% w/v
(85% hydrolyzed, Sigma-Aldrich) and EDTMP 4% w/v (IPEN) and then emulsified by sonication (ultrasonic processor, GEX600,
Sigma) for 1min (55W) producing a water/organic solvent (W/OS) solution. After that, this solution was emulsified with 4 mL of
PVA 0,7% w/v solution by sonication for 2min (55W) producing a W/OS/W emulsion. Then the organic solvent was eliminated by
evaporation under vacuum, 20min at 250C. The particles were recovered by centrifugation (Centrifugue 5424 Eppendorf) with
20,230xg for 20 min and washed twice with Milli-Q water. Nanoparticle surface morphology: Nanoparticle morphology was
characterized using MFP-3D-BIOTM (Asylum Research). Samples were prepared by deposition of particles suspension on freshly
cleaved mica. Topography was captured using TappingMode with AC240TS probe (spring constant of 2 N/m and cantilever length
240µm). Results: TappingMode images reveal that all nanoparticles have spherical shape and size dispersion ranging from 500200nm. The samples prepared for analyses on AFM shows nanoparticles aggregation with the large ones in the center and the
smaller ones on the edge. In the phase image it is possible to identify inhomogeneities corresponding to the different polymers
that compose the nanoparticle and that they are dispersed randomly. Conclusion: The nanoparticles were successfully formed by
this methodology and the characterization by AFM was established.
Support: INBEB, FAPERJ, CNPq.
M06
SELEÇÃO E AVALIAÇÃO DO EFEITO DE APTÂMEROS DE ÁCIDOS NUCLEICOS NA INTERAÇÃO E INIBIÇÃO DA CONVERSÃO
CONFORMACIONAL DA PROTEINA PRION
1- MACEDO, B.; 1,2- GOMES, M.P.B.; 2- BRAGA, C.A.C.A.; 2,3- SILVA, J.L.; 1,3- CORDEIRO, Y.
1- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro; 2- Instituto de Bioquímica Médica, Universidade Federal do Rio
de Janeiro; 3- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de
Janeiro.
O agente infeccioso das encefalopatias espongiformes transmissíveis (EETs) envolve uma proteína constitutiva do organismo,
chamada proteína prion (PrP). A proteína prion celular (PrPC) pode assumir uma conformação anormal e patológica, conhecida
como PrP scrapie, que pode sofrer agregação no sistema nervoso central e levar à neurodegeneração. Após um longo período de
incubação, surgem os primeiros sintomas clínicos de disfunção cognitiva e motora e os pacientes, invariavelmente, evoluem ao
óbito. Já foi visto que moléculas, como os ácidos nucleicos (NA), podem se ligar à PrP com alta afinidade e modular essa conversão
conformacional. O objetivo do projeto é selecionar aptâmeros de NA que se liguem a diferentes domínios da proteína prion e
avaliar os efeitos dessa interação. Os aptâmeros são oligonucleotídeos de fita simples (DNA ou RNA) que formam estruturas
tridimensionais definidas e se ligam aos seus alvos com alta afinidade e especificidade. Inicialmente, obtivemos aptâmeros
modificados (tioaptâmeros) baseados em seqüências de DNA que já foram identificadas por nosso grupo e por outros como
ligantes da rPrP. Tioaptâmeros são mais estáveis in vivo do que NA não modificados porque não são digeridos por nucleases.
Avaliamos a interação da PrP recombinante (rPrP23-231) com os aptâmeros, A22, A35 e SAF através das técnicas de
espalhamento de luz, fluorescência intrínseca e dicroísmo circular. Além disso, investigamos também o perfil de agregação da rPrP
na presença desses aptâmeros. A morfologia e citotoxicidade desses agregados foram avaliadas por MET e ensaios de disfunção
celular, respectivamente. Observamos que a interação da rPrP com esses tioaptâmeros promove alterações na estrutura
secundária e terciária da proteína e pode induzir sua agregação em diferentes níveis, dependendo da seqüência utilizada.
Interessantemente, os complexos PrP:Aptâmeros parecem não causar disfunção celular significativa em cultura de células de
neuroblastoma.
Apoio: CAPES, INBEB, FAPERJ, CNPq.
M07
THE DEVELOPMENT OF A CHAGASIC CARDIOMYOPATHY MODEL IN CHIMERIC MICE
CAMILA IANSEN IRION1; GUILHERME VISCONDE BRASIL1; BRUNO PAREDES1; DANIEL BARUSCO DURAN1, GABRIELLEN VITIELLO1;
LUIZA LAPOLLA PERRUSO1; TAIS HANAE BRUNSWICK1; SANDRO TORRENTES DA CUNHA1; NAZARETH ROCHA2; ISALIRA REZENDE1;
PAULO CÉSAR VENTURA CANARY4; ANTONIO CARLOS CAMPOS DE CARVALHO1,3; ADRIANA BASTOS CARVALHO1 ; REGINA COELI
DOS SANTOS GOLDENBERG1
1. Laboratório de Cardiologia Celular e Molecular – Instituto de Biofísica Carlos Chagas Filho – Universidade Federal do Rio de
Janeiro (UFRJ) 4-Serviço de Radioterapia do Hospital Universitário Clementino Fraga Filho (HUCFF),
Chagas’ disease is an important health concern in Latin America, as there is no effective treatment for the chronic infection.
Therapy with BMSCs indicated beneficial effects in a chagasic mouse model. Here we established a model of Chagas’ disease in
chimeric mice to study the role of bone marrow-derived cells in the heart after infection. Fifteen C57BL/6 mice were submitted to
total body irradiation, resulting in bone marrow ablation. Bone marrow mononuclear cells obtained from transgenic mice
expressing the green fluorescent protein gene were used for reconstitution. Before infection, 9 animals received 2x105 lysed
parasites of Brazil strain and after 22 days, they were infected with 3x104 trypomastigotes of Brazil strain by intraperitoneal route.
Parasitaemia was monitored during 60 days. Cardiac performance was evaluated by electrocardiogram and treadmild exercise.
Statistical analysis was performed by One-way ANOVA. Six animals were sacrificed and the hearts were studied using paraffinembedded tissue stained by hematoxylin-eosin and Sirius red. Heart cell populations were studied by flow cytometry in the other
remaining animals after 74 days of infection. Peripheral blood flow cytometry revealed that cell transplantation were effective in
generating chimeric mice with 88.5 ± 4.6% of GFP-positive cells. The parasitemia peak occurred between 33th and 43th day of
infection. Infected mice showed increased PR interval (0.042±0.012 vs 0.032±0.002) and PR segment (0.028±0.009 vs 0.019±0.002)
after 24 days of infection on ECG. After one month, infected mice had decreased treadmild exercise parameters. Distance (m)
(119.16±95.41 vs 448.10±216.9) and time (min) (12.57±7.15 vs. 33.83±11.90) were significantly lower in infected mice. Heart
tissue revealed myocarditis and presence of nests of amastigotes in infected mice. Heart cells from infected animals presented an
increase in citotoxic T lymphocytes (3422 vs 260 cells) and helper T lymphocytes (752 vs 130 cells) when compared to non-infected
mice. B lymphocytes were lower in infected mice (277 vs 1123 cells) and macrophages did not differ between groups. This data
suggest that C57BL/6 chimeric mice infected with Brazil strain of T. cruzi are susceptible to infection and might be a model to study
bone marrow contribution to heart repair.
Support: CNPq, CAPES, FAPERJ, Ministério da Saúde.
M08
USE OF HIGH HYDROSTATIC PRESSURE TO INACTIVATE AND STUDY THE STRUCTURAL STABILITY OF THE HUMAN INFLUENZA
VIRUS X-31
Dumard, C.H.1, Souza-Santos, P.1, Barroso, S.P.C.1, Couceiro, J.N.S.S.2, Ferreira, D.F.2, Oliveira, A.C.1, Silva, J.L.1
1- Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, Brazil. 2 - Instituto de Microbiologia Professor Paulo de Góes, UFRJ, Rio de
Janeiro, Brazil
Influenza is a respiratory disease caused by virus belonging to Orthomyxoviridae family. They are enveloped negative-stranded
RNA viruses that can be distinguished on the basis of antigenic differences in nucleocapsid (N) and matrix (M) proteins. The
haemagglutinin (HA), neuraminidase (NA) and M2 proteins are embedded in the envelope lipid and are critical for infectivity. To
inactivate and study the stability of virus we used high hydrostatic pressure (42 Kpsi) at different time points (3h, 6h and 12h).
Structural changes were accompanied by spectroscopy techniques, eletron microscopy and dynamic light scattering (DLS).
Inactivation were evaluated by hemagglutination title, neuraminidase activity, confocal microscopy and infectivity in cell culture.
Pressurized samples showed structural changes that were detected by a drop in fluorescense emission, and morphological
changes detected by eletron microscopy. A discret increase in average size of viral populations were observed by DLS.
Hemagglutination title at all time points showed a decrease, but no significant alteration of neuraminidase activity was found.
Infectivity in cells were abolished when analized by TCID50 and serial passage in cell culture. When analized by confocal
microscopy, virus showed a drop fusion activity in cells. High hydrostatic pressure showed to be an efficient method to study and
inactivate influenza virus. This method has an advantage to use whole virus particules that already was described to be more
immunogenic when compared with viral particles vaccines, that is currently used.
Support: CNPq, Capes, FAPERJ.
M09
MORPHOLOGICAL STUDIES OF THE TRYPANOSOMA CRUZI EPIMASTIGOTE EARLY ENDOSOMES NETWORK
1,2- ALCANTARA C. L., 1,2- VIDAL J. C., 1,2,3- DE SOUZA W. , 1,2- CUNHA-E-SILVA N. L.
1- Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro. 2- Instituto Nacional de Biologia Estrutural e Bioimagem (INBEB), 3- Diretoria de Programas, INMETRO
Epimastigotes of Trypanosoma cruzi are highly polarized cells that exhibit a corset of subpellicular microtubules that impairs
endocytosis and exocytosis except through the flagellar pocket and cytostome. After binding to one of these domains,
macromolecules are internalized and subsequently found inside a tubule-vesicular network, spread from the perinuclear region to
posterior tip of the protozoan. Budding from the network, vesicles fuse with reservosomes, where cargo is stored or degraded (De
Souza W et al, Prog. Histochem Cytochem, 2009, 44:67). The early endosomes of T. cruzi are poorly characterized, no molecular
markers were defined yet. In other cells, it is established that at 4°C the process of endocytosis doesn’t occur, while between 12°C
and 16°C endocytic tracers are retained in collecting tubules that correspond to early endosomes. Epimastigotes were submitted
to endocytosis using bovine transferrin (Tf) labeled with FITC or colloidal gold as tracers, at 4°C for 1 hour, when an aliquot was
collected. The remaining cells were transferred to 12°C and incubated for an additional hour, when an aliquot was collected.
Finally, the remaining cells were incubated at 28°C (physiological temperature of epimastigotes) for 1 hour. For each condition,
cells were fixed, the kinetic and morphological studies were analysed by fluorescence microscopy, electron microscopy and
tomography. We observed that after 1 hour at 4°C Tf was retained inside the bottom of the cytostome, which appeared enlarged.
At 12°C, the tracer were found distributed through a highly branched tubular-vesicular network but didn´t reach the
reservosomes. At 28°C it was possible to observe Tf inside some of the reservosomes, although at reduced their quantity, as
compared to control (endocytosis directly at 28°C for 30 minutes). Using this approach we conclude that the collecting tubules
could be characterizing kinetically as early endosomes. Using electron tomography, three-dimensional reconstructions revealed
the distribution of early endosomes.
Support: CAPES, CNPq, FAPERJ.
M10
EVALUATION OF GROWTH FACTORS PRODUCED BY HUMAN MENSTRUAL BLOOD-DERIVED MESENCHYMAL STEM CELLS FOR THE
MAINTENANCE OF HUMAN EMBRYONIC STEM CELLS
1-DANÚBIA SILVA DOS SANTOS, 1-VANESSA CARVALHO COELHO DE OLIVEIRA, 1-KARINA DUTRA ASENSI, 1-LEANDRO VAIRO, 1ADRIANA BASTOS CARVALHO,1,2-ANTONIO CARLOS CAMPOS DE CARVALHO, 1-REGINA COELI DOS SANTOS GOLDENBERG.
1-Laboratório de Cardiologia celular e Molecular, Instituo de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro;
2-Instituto Nacional de Cardiologia.
Growth factors produced by mouse embryonic fibroblasts (MEF) play an important role in maintaining the human embryonic stem
cells (hESC) in an undifferentiated state. Although this system of co-culture has been shown to be efficient, the use of animalderived feeder-layers is incompatible with the clinical setting. Hence, identifying the growth factors produced by human cells is
extremely important to use these cells as feeder-layers. The objective of this work was to investigate whether human menstrual
blood-derived mesenchymal stem cells (MBMC) produce factors comparable to MEF and can keep hESC in an undifferentiated
state. RNA samples from MBMC and MEF were obtained before and after inactivation. To test MBMC feeder-layer capacity, hESC
were grown over MBMC and MEF under standard conditions. hESC growth, proliferation, survival and maintenance of the
undifferentiated state were evaluated. Growth factors analysis revealed amplification of transcripts FGF-2, BMP4, TGF-β1, VEGF
and PEDF by RT-PCR in MBMC and MEF. IGF-1 was not amplified. hESC grown over MBMC had a similar morphology, expressed
Alkaline Phosphatase detected by commercial kit (Millipore), transcription factors Oct3/4, Sox2, Nanog and Klf4 by RT-PCR and
SSEA-4 and the transcription factor Oct3/4 in immunofluorescence assays. Furthermore, when cultured in suspension, we
observed similar embryoid body formation, size and morphology. However, the time course analysis of average colony size
showed that the colony was bigger when MBMC were used as feeder-layer compared to MEF (2 days: 632.09±79.06 vs
321.456±21.4; 3 days: 1439.110±28.72 vs 704.745±10.8; 4 days: 2021.058±21.93 vs 1224.67±222.8; 5 days: 2245.358±29.82 vs
1914.78±190.9 M±SD,n=3) in passage 4. In conclusion, growth factors found on MBMC are the same found on MEF and MBMC are
able to maintain hESC in an undifferentiated state with comparable efficiency to MEF. It should be noted that average colony size
was increased when hESC were grown over MBMC.
Support: CNPq, FAPERJ, CAPES, INCT e Ministério da Saúde.
M11
THE ENDOSYMBIONT OF Blastocrithidia culicis (Strigomonas culicis) DEPENDS ON THE HOST CELL TO DIVIDE
1- Brum-da-Silveira, F.L.; 2,3- Elias, M.C., 1,3,4- De Souza, 1,3- W. Motta, M.C.M.
1- Instituto de Biofísica Carlos Chagas Filho, Uiversidade Federal do Rio de Janeiro; 2- Laboratório de Parasitologia, Instituto
Butantan; 3- Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Universidade Federal do Rio de
Janeiro; 4- Instituto Nacional de Metrologia, Normalização e Qualidade Industrial
Blastocrithidia culicis is a monoxenic trypanosomatid that presents single-copy structures, as the nucleus, the kinetoplast and a
bacterium, which maintains an obligatory relationship with the host protozoan. Investigations of the cell cycle in symbiontharboring trypanosomatids suggest that the bacterium divides in coordination with other host cell structures. In this work we used
optical and electron microscopy approaches in order to study the symbiont division during the B. culicis cell cycle. During this
process, the symbiotic bacterium presents different formats and different positions in relation to other cell structures. Thus, at the
beginning of the protozoan cell cycle, the endosymbiont presents a constricted form that becomes more elongated until the
bacterium division that occurs before the kinetoplast and nucleus segregation. During cytokinesis, symbionts are positioned close
to nuclei to ensure that each daughter cell will inherit a single copy bacterium. After cytokinesis, the symbiont migrates to the
posterior end of the host and a new cell cycle begins. Protozoa treatment with aphidicolin, a DNA polymerase inhibitor that blocks
the nuclear DNA synthesis, promotes cell proliferation arrest in B. culicis. In aphidicolin treated cells the symbiont segregation is
inhibited, resulting in filamentous forms. These results indicate that somehow the host protozoan controls the number of bacteria
per cell in order to maintain the close symbiotic relationship.
Support: INBEB, CNPq, FAPERJ and FAPESP.
M12
UTILIZAÇÃO DE ANIMAÇÕES COMPUTACIONAIS A PARTIR DE DINÂMICA MOLECULAR NA DIVULGAÇÃO CIENTÍFICA E NO
ENSINO DE CIÊNCIA
Dias, Giselly.S.1, Pascutti, P.G..2 e Foguel, D.1
1-Instituto de Bioquímica Médica, 2-Instituto de Biofísica Carlos Chagas Filho
As tecnologias de informação e comunicação tem causado grandes e valiosas transformações em todas as áreas de conhecimento.
Diversas estratégias de aprendizagem de ensino, tais como animações em 3D, Web participativa, vídeo aulas e jogos interativos
tem se tornado cada vez mais necessário como auxilio no ensino de ciência e na divulgação científica. No entanto, a utilização
dessas estratégias ainda cresce de forma vagarosa, pois muitas instituições ainda utilizam métodos cartesianos
em sala
de
aula. A animação é uma ferramenta poderosa que possibilita a apresentação de ideias e conceitos complexos a uma audiência
diversificada, de diferentes áreas e especialidades. Estando o expectador imerso nas ideias e implicações do conteúdo da
animação, ele pode ter um maior nível de entendimento e retenção da informação. Diferente das ilustrações de livros, as
animações permitem um olhar intuitivo sobre os movimentos moleculares, permitindo ao expectador presenciar como ocorrem os
fenômenos biológicos. Nosso trabalho foi desenvolvido com o fim de ajudar na quebra deste paradigma, permitindo uma visão
holística do aluno e contribuir na popularização da ciência. Temas como a estrutura tridimensional de aminoácidos, e
biomembranas, estrutura e enovelamento de proteínas, são abordados. Desta forma a informação contida nos vídeos é
transmitida de maneira eficiente e cientificamente acurada ao mesmo tempo em que entretém o aluno. Com atenção especial
para a didática, a linguagem pretende ser acessível ao leigo, mas trazendo informações relevantes para alunos do ensino médio,
graduandos e especialistas. O conteúdo gerado estará disponível na página do LMDM para utilização em salas de aula Palavraschave: popularização da ciência, divulgação científica, animação molecular.
Apoio: INBEB, FAPERJ, CNPq.
M13
USO DE HPLC-RMN NA IDENTIFICAÇÃO DE CATEQUINAS E DERIVADOS EM EXTRATO BRUTO DE CHÁ VERDE DE CAMELLIA
SINENSIS
CASTAÑEDA-VALENCIA, G. M.; TINOCO, L. W
Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro
O chá é uma das bebidas mais antigas e populares consumidas no mundo (Seeram, N. et al. J Agric Food Chem. 2006, 1599). Os
polifenóis predominantes no chá verde Camellia sinensis (EGCG, EGC, ECG e EC) apresentam atividade antioxidante, estimulam a
termogênese e oxidação da gordura. Devido a falta de homogeneidade dos teores de catequinas na C. sinensis de diferentes
origens é necessário desenvolver uma metodologia rápida e eficiente para a sua caracterização. Foi preparada uma infusão
liofilizada de chá verde procedente do Brasil, Uruguai, Peru e Chile, com um rendimento de 16-18%. Estes chás apresentaram
cores e aromas diferentes nas infusões e depois de liofilizados. As análises foram feitas com 20 mg/mL extrato bruto liofilizado
para que fossem obtidos os espectros de RMN de 1H com melhor relação sinal/ruído. As foram feitas em coluna de fase reversa
C18 em metanol-água com 0,3% de ácido fórmico. Foram identificados espectros característicos das catequinas com banda I entre
226 a 236 nm e banda II entre 276 a 280 nm nos tempos de retenção de 12,3 min e 15,3 min. O acoplamento HPLC-RMN foi feito
com um espectrômetro VNMRS 500 (Agilent Thecnologies) a 499,78 MHz para 1H com uma sonda de fluxo de 60 uL. Os picos com
os tempos de retenção de 17,12 min. e 17,56 min. foram identificados de acordo com os dados de deslocamento químico como
sendo da (+)-catequina e (-)-epigalocatequina galato. Os chás do Brasil, Peru e Uruguai apresentaram perfis cromatográficos
semelhantes, somente variando nas intensidades dos picos. O chá do Chile, o único comprado a granel e sem Registro do
Ministério da Saúde, apresentou perfil cromatográfico muito diferente, o que evidencia como os produtos a granel são muitas
vezes adulterados ou falsificados, comprovando a necessidade em se desenvolver técnicas analíticas eficientes para atestar a
qualidade destes produtos.
Apoio: INBEB, CNPq, FAPERJ, FINEP.
M14
RADIATION-INDUCED LIVER DAMAGE IN MICE: AN EXPERIMENTAL MODEL TO STUDY TISSUE REGENERATION
SUHETT, GRAZIELLE DIAS1; HENRIQUES, ALINE1; VILAS-BÔAS, TIAGO SOUZA1; FACCIOLI, LANUZA ALABY PINHEIRO1; CUNHA,
SANDRO TORRENTES1; FRAGA, MARCOS VINICIUS1; QUINTANILHA, LUIZ FERNANDO1;PAREDES, BRUNO DIAS1; TAKIYA, CRISTINA
MAEDA2; CANARY, PAULO3; CARVALHO, ADRIANA BASTOS1; GOLDENBERG, REGINA COELI SANTOS1.
1 – Laboratory of Cellular and Molecular Cardiology - IBCCF, UFRJ, Rio de Janeiro, RJ. 2 Laboratory of Cellular Pathology - ICB, UFRJ,
Rio de Janeiro, RJ. 3 – Clementino Fraga Filho Hospital - UFRJ, Rio de Janeiro, RJ.
Introduction and Objectives: Despite major advances in cancer therapy by radiotherapy, damage to major organs and structures
adjacent to the tumor still shows high prevalence and morbidity. In this context, the liver injury arouses great interest because it is
a multifunctional and metabolic organ deeply affected by radiation. In this context, this study aims to establish the radiation ideal
dose to induce liver dysfunction in mice. Materials and Methods: Prior the irradiation three animals were used as controls to
identify liver abdominal position. These mice were anesthetized with ketamine (40mg/kg) and xylazine (8mg/kg) intraperitoneally
and submitted to abdominal computed tomography scans. Thirty C57BL/6 mice, males and females, weighting from 20 to 30g
were submitted to the radiation protocol. They were divided equally into the following groups: G0 - non-irradiated animals; G1 irradiated with dose of 10 Gy; G2 - irradiated with 15 Gy; G3 - irradiated with 24 Gy; and G4 - irradiated with 30 Gy. All mice
underwent biochemical evaluation of the peripheral blood prior to the procedure and 7 days after irradiation, and then sacrificed
for histology analysis (H&E and Sirius Red). Results: Abdominal tomography limited the liver area to perform directed radiation.
The animals of G4 did not survive to the irradiation protocol. The other groups tolerated the anesthesia and the procedure of
irradiation without complications or mortality. G1 and G2 did not present alterations in the biochemical and histological
parameters analyzed when compared to G0. However, G3 showed significant decrease in albumin levels (G0: 2.4 ± 0.25, G3: 1.4 ±
0.54 g / dL), total protein (G0: 3.8 ± 0.22; G3: 2.85 ± 0.47) and alkaline phosphatase (G0: 142.5 ± 20.62; G3: 32.4 ± 5.68) when
compared to the non-irradiated group. Moreover, G3 also presented degeneration, apoptotic cells and foci of necrosis in liver
tissue. Conclusion: Experimental model of hepatic injury in
Financial support: CNPq, CAPES, FAPERJ and Ministry of Health.
M15
HYPERTROPHIC CARDIOMYOPATHY MUTATIONS OF TROPONIN C ALTER ITS AFFINITY FOR THE THIN FILAMENT
1- MONTEIRO, J.; 1- VELTRI, T.; 1- REYNALDO, D. P.; 2- PINTO, J. R.; 1- SORENSON, M. M.
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2- 2Department of Molecular and Cellular
Pharmacology, Miller School of Medicine, University of Miami
INTRODUCTION: TnC is the Ca2+-binding subunit of the troponin complex that regulates striated muscle contraction and
mutations in this gene can lead to cardiomyopathy phenotypes. Recently, four new mutations that cause hypertrophic
cardiomyopathy were discovered in cTnC: A8V, in the N-terminus; C84Y, in the central helix; and E134D and D145E, in the Cterminus. Interestingly, three of these mutations increase the N-terminus Ca2+ affinity. Their effects on the affinity for the thin
filament itself, which depends on the C-terminus, are unknown. OBJECTIVE: Investigate the influence of these mutations on the
interaction between cTnC and the thin filament proteins. METHODS AND RESULTS: TnC dissociation and binding experiments were
performed in demembranated relaxed papillary fibers to evaluate TnC-thin filament affinity, as previously described. The results
show that the C84Y and D145E mutants dissociate more slowly than the control (t½ (min) 12.2 ± 2.0, control; 24.7 ± 1.3, C84Y*;
and 36.8 ± 0.6, D145E*; *p<0.05 vs. control). In binding experiments, where native TnC was extracted and the fibers were exposed
to increasing recombinant cTnC concentrations, 50% of the maximum reconstituted fiber tension in control was obtained with 0.3
µM cTnC, while this same concentration of D145E and C84Y resulted in 75% and 58% of the maximum, respectively, indicating that
both mutants have a higher TnC-thin filament affinity. In reconstituted thin filaments, Ca2+/ Mg2+ dissociation from cTnC was
measured using stopped-flow technology and terbium competition for the C-terminal sites. Mutant E134D had a higher affinity for
Mg2+ (~14 s-1 vs ~21 s-1 WT) and the other three mutants had higher Ca2+ affinity in sites III and IV (~23 s-1 vs ~27 s-1 WT).
CONCLUSION: C-domain mutations can alter TnC-thin filament affinity as well as ions binding to the C-terminus.
Support: INBEB, FAPERJ, CNPq.
M16
MORPHOLOGICAL ALTERATIONS ON HUMAN LEUCOCYTES PROMOTES BY 5-HYDROXY-2-HYDROXYMETHYL-GAMMA-PYRONE
(HMP), A BIOPRODUCT OBTAINED FROM ASPERGILLUS FUNGI
1,3-COSTA, J.P.; 1,3-FRADE, P.R; 1,3-RODRIGUES, A.P.D.; 1,3-FARIAS, L.H.S.; 1,3-SILVA, R.R.P.; 1,3-HAGE, A.A.P.; 1,3-SILVA, B.J.M. ;
2-SANTOS, A.S.; 1,3-SILVA, E.O.
1-Laboratório de Parasitologia e Laboratório de Biologia Estrutural, Instituto de Ciências Biológicas, Universidade Federal do Pará,
Belém, Pará, Brazil; 2-Laboratório de Desenvolvimento e Planejamento de Fármacos, Instituto de Ciências Exatas e Naturais,
Universidade Federal do Pará, Belém, Pará, Brazil; 3-Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e
Bioimagens, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Brazil
The 5-hydroxy-2-hydroxymethyl-g-pyrone (HMP) is a secondary metabolite synthesized by some species of fungi from Aspergillus,
Penicillium and Acetobacter genera. The HMP has several applications, being used as an antioxidant, tyrosinase inhibitor,
cosmetics, protective agent against radiation and antitumor. Recently, it was also shown that this metabolite acts as a
macrophage activator. However, the effect of HMP in human leucocytes is unknown. Thus, the aim of this study was to evaluate
the cell viability and morphological alterations in those cells. Human peripheral leucocytes isolation was performed using
HISTOPAQUE® 1077-density-gradient. Monocytes were treated for 24 and 48 hours and neutrophils were treated for 1h with 50
and 100 μg/mL of HMP. The ultrastructural analysis of treated monocytes showed spreading ability, high number of cytoplasmatic
projections and vacuoles, features that are often observed in activating cells. Treated neutrophils showed extensive lamellipodia
formation, pseudopodia extention and high spreading ability when treated with 100 μg/mL of HMP. In addition, the viability test
using JC-1, tetrazolium-dye (MTT) colorimetric method and neutral red showed that HMP has no citotoxicity effect on human
leucocytes when treated with 50 and 100 μg/mL of HMP. These results demonstrate that HMP promoted several morphological
changes that suggest cell activation without host cell injury. Keywords: Kojic acid; 5-hydroxy-2-hydroxymethyl-γ-pyrone; human
leucocytes
Support: CAPES, CNPq/UFPa, CNPq/MCT/CT-INFRA/CT-PETRO (Processo nº 620179/2008), MCT/CNPq/FNDCT/CAPES/FAPERJ.
M17
APRISIONANDO O MONÔMERO DA VARIANTE NÃO AMILOIDOGÊNICA DA TRANSTIRETINA: POSSÍVEL TERAPIA CONTRA A
POLINEUROPATIA AMILOIDOTICA FAMILIAR
FONTES, L.A.S., PALHANO, F., BRAGA, C.A. e FOGUEL, D.
Instituto de Bioquímica Medica Universidade Federal do Rio de Janeiro
A Transtirretina (TTR), um homotetrâmero de 55kDa rico em folha beta, é responsável pelo transporte de tiroxina e retinol no
plasma e fluido cérebro espinhal. Mais de 100 mutações pontuais na TTR foram associadas com a forma hereditária de amiloidose,
denominada Polineuropatia Amiloidótica Familiar (FAP), pelo qual é caracterizada por depósito de agregados no sistema nervoso
periférico. Esses agregados são formados após a dissociação do tetrâmero e posterior enovelamento parcial do monômero. A
mutação T119M foi identificada em uma família com alta incidência da variante amiloidogênica V30M, suavizando os sintomas
desta mutação agressiva. Desta forma, a incorporação de subunidades monoméricas de T119M em um tetrâmero com a mutação
V30M pode ser uma estratégia para estabilizar o heterotetrâmero e inibir a agregação. Neste presente estudo, alta pressão
hidrostática (HHP) foi utilizada como uma ferramenta para a produção de monômeros de T119M. Visto que a T119M é um
tetrâmero altamente estável, os experimentos foram feitos em pH 3.0, 3.000 atm e 1ºC. Objetivando o desenvolvimento de uma
nova forma de terapia contra FAP, é necessário manter este monômero solúvel em pH fisiológico (condição alcançada com pH
7.8). Em seguida, nós avaliamos o estado de enovelamento comparando o M-T119M com o monômero engenheirado enovelado
Wild-Type (M-TTR) utilizando alta pressão hidrostática como agente desnaturante. Após, investigamos a cinética de re-associação
desses monômeros em tetrâmeros a fim de identificar uma janela de tempo em que esses monômeros possam ser combinados
com os tetrâmeros altamente amiloidogênicos permitindo a troca de subunidades (trans-supressão in vitro). Nossos resultados
mostram que há retetramerização em aproximadamente 10 horas. Entretanto, ao ser colocado na presença de tetrâmeros da
variante amiloidogênica V30M, o M-T119M não foi capaz de diminuir a cinética de agregação desta variante, pois, ao ser
submetido a condição ideal para agregação (37°C, pH 4.4) formou agregados. Em seguida testamos a ação de possíveis inibidores
em bloquear a agregação deste monômero e investigamos se o protocolo de monomerização descrito neste trabalho poderia
estar formando espécies monoméricas estruturalmente alteradas. Os resultados mostram que os compostos testados não foram
capazes de inibir a agregação do M-T119M, assim como o protocolo de monomerização em pH 3.0 produz uma espécie
monomérica estruturalmente similar a espécie descrita pelo grupo. Como perspectivas, objetivamos alcançar uma condição em
que o M-T119M consiga retetramerizar sem formar agregados.
Apoio: FAPERJ, CNPq, CAPES
M18
METABÓLITOS SALIVARES DE PUÉRPERAS HIPERTENSAS COMO POTENCIAIS BIOMARCADORES
1-Pereira L*, 1-Fidalgo TKS, 1-Martins C, 1-Pomarico L, 2-Almeida F, 1-Freitas-Fernandes LB, 1-Souza IPR, 2-Valente AP
1-Faculdade de Odontologia, Departamento de Odontopediatria e Ortodontia, Universidade Federal do Rio de Janeiro; 2-Centro
Nacional de Ressonância Magnética Nuclear Jiri Jonas, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro
O objetivo do estudo foi avaliar o metaboloma salivar de puérperas saudáveis e hipertensas por Espectrometria de Ressonância
Magnética Nuclear (RMN). Coletou-se 5 ml de saliva total não estimulada de nulíparas (G1; idade média= 23,8 3,3; n=10),
puérperas hipertensas (G2; idade média= 24,7 7,6; n=08) e puérperas normotensas (G3; idade média= 22,1 4,3; n=12), logo
após centrifugadas por 60 minutos a 10.000g a 4°C foram armazenados a -80°C. Os espectros 1H de RMN foram obtidos por meio
de um espectrômetro Bruker 400 MHz. Para avaliar se houve distinção entre os grupos, utilizou-se a Análise dos Componentes
Principais (ACP), e ANOVA para as intensidades de cada metabólito salivar. Para cada hipótese nula rejeitada (p<0,05), foi utilizada
a comparação post hoc, aplicando-se o teste de Tukey com intervalo de confiança de 95%. A ACP foi capaz de separar o G1 do G2 e
G3. A maior intensidade de metabólitos no grupo de puérperas hipertensas: ácido graxo (G2>G1: p=0,018), fenilalanina (G2>G1:
p=0,019), (G2>G3: p=0,009), prolina (G2>G3: p=0,024), fosforiletanolamina (G2>G3: p=0,028), glicose (G2>G1: p=0,024), e no de
nulíparas: histidina (G1>G3: p=0,035), lisina (G1>G3: p=0,026), trimetilamina (G1>G2: p=0,005) e sarcosina (G1>G3: p=0,026;
G1>G2: p=0,016). Atualmente estamos validando os dados obtidos por análises complementares (ex. GS-MS) com o intuito de
avaliar a potencialidade destes metabólitos salivares como biomarcadores para a hipertensão.
Apoio: CEP-HFB 29/10,FAPS – FAPERJ N° 26/10001/2010.
M19
SENSIBILITY OF TRIOSEPHOSPHATE ISOMERASE FROM RHIPICEPHALUS MICROPLUS TO SULPHYDRIL REAGENTS AND
CONSTRUCTION OF A MUTANT IN RESIDUE 86
SARAMAGO, L.; FONSECA, R.; MASUDA, A.; VAZ JR., I.; LOGULLO, C.; MORAES, J.
1 Laboratório Integrado de Bioquímica Hatisaburo Masuda, UFRJ, IBqM and NUPEM, Macaé, RJ, Brazil; 2 Centro de Biotecnologia,
UFRGS, Porto Alegre, RS, Brazil; 3 Laboratório de Química e Função de Proteínas e Peptídeos, CBB, UENF, Campos dos
Goytacazes, RJ, Brazil
The hard tick Rhipicephalus microplus has become a major pest in tropical and sub-tropical agrosystems. Triosephosphate
isomerase (TIM) catalyzes the isomerization of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. TIM is a target for
drug design against parasitic diseases. The cysteine non-conserved aminoacids residues between the host and parasites were
tested to promote species-specific inhibition. The sequence comparison between RmTIM and bovine TIM (Bos taurus is R.
microplus preferential host) showed that the cysteine residues at positions 7, 25, 51, 86 and 143 are exclusively non-conserved.
Cysteine-reactive agents were able to disturb enzyme structure and activity. Methyl methanethiosulfonate (MMTS) induced 68%
of inactivation. However, 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) induced 82% of inactivation. By kinetic assays, it was verified
that RmTIM have 5 free cysteines per dimer. Structurally, most of the known TIMs are homodimers. Interestingly, TIM is active
only in its dimeric form. Therefore, residues of interface are usual candidates to drug design. In this context, RmTIM have a residue
present in the interface (Cys86). RmTIM cysteine 86 mutant (Alanine 86) was expressed in Escherichia coli (BL21DE3pLysS).
Purification was realized in niquel column and showed a single band of 27 kDa in SDS-PAGE electrophoresis. Kinetics parameters of
Cys 86 mutant like Km, Vmax and Kcat were also measured. Altogether, these exclusives cysteine residues of R. microplus could be
employed in the development of species-specific inhibitiors.
Supported by: FAPERJ, CNPq, CAPES.
FAPERJ CNPq, CAPES, FUNEMAC
M20
BREATHING PATTERN VARIABILITY ON PRESSURE SUPPORT VENTILATION
1 - CRUZ, M.; 2- CAMILO, L.; 2- CRUZ, L.; 1- JAPIASSÚ, A.; 2- RONCALLY, A.; 1- MEDEIROS, D.; 1- BOZZA, F.
1- Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz; 2 - Instituro de Biofísica Carlos Chagas Filho,
Universidade Federal do Rio de Janeiro
Breathing pattern variability (BPV) is inherent to physiology in healthy subjects and has been postulated as a weaning predictor
parameter. Besides this, artificially generation of BPV by the ventilator has been proposed as a method to improve gas exchange.
Pressure support (PS) ventilation is a spontaneous mode frequently used for weaning, but the impact of this ventilatory support
on spontaneous BPV is unknown. In this study we aim to investigate how the level of PS modifies BPV. Methods: Twelve patients
ventilated in PS mode were studied. All patients were initially ventilated with a PS level of 20 cmH2O form 10’. Thereafter, PS was
stepwise decreased to 5 cmH2O in steps of 5 cmH2O, 10 min per step. Esophageal and airway pressure, airflow, arterial blood
pressure and electrocardiogram were continuously acquired and arterial blood gases were collected at the end of the first 4 steps.
Transpulmonary pressure, inspiratory peak flow , tidal volume, respiratory rate, pressure time product (PTP) over the time and
their respective coefficients of variations (CV) were then calculated. Comparisons between PS levels were performed with the
Wilcoxon test (P<0.05). Results: Transpulmonary pressure, inspiratory peak flow and tidal volume, significantly decreased as PS
decreased from PS20 to PS15, PS15 to PS10 and PS10 to PS5, respectively. Respiratory rate and PTP significantly increased at lower
levels of PS. No significant changes on gas exchange and hemodynamic parameters were observed. No significant changes on CV in
all variables were observed. Conclusion: No evidences of increased levels of BPV were observed, even by reducing PS, in this small
cohort of patients. These data suggest that BPV is not a feasible index for weaning prediction when measured in PS ventilation and
support the use of an artificial BPV generated by the ventilator to improve lung function even during PS ventilation.
Support: FAPERJ, CNPq.
M21
ISOLATION AND CHARACTERIZATION OF CARDIOSPHERE DERIVED CELLS FROM HUMAN HEARTS
1- BARBOSA, R.A.Q.;1- KASAI-BRUNSWICK, T.H.; 1- ABREU, S.K.; 1- PASSIPIERI, J.A.; 1- ABREU, M.C.; 1- MESQUITA, F.C.P.; 1- DEL
CORSSO, C.; 1- CARVALHO, A.B.; 1- CAMPOS-DE-CARVALHO, A.C.
1- Universidade Federal do Rio de Janeiro
Introduction and Objectives: The regenerative capacity of the heart has been attributed to cardiac stem cells. The objective of this
study was to assess the in vitro properties of human cardiosphere-derived cells (CDCs) – one of the cardiac stem cell types
described so far – and their ability to differentiate into cardiomyocytes. Methods and Results: CDCs were obtained from human
atrium myocardial tissue discarded after surgery by enzymatic digestion with collagenase type II (0.4%). Ten to fifteen days after
isolation, small round phase-bright cells (PBC) appeared on top of the fibroblast-like cells. PBCs were collected and placed in a
non-adherent plate for 2 days where they formed cardiospheres, which were then transferred to adherent plates, giving rise to
CDCs. These cells are adherent to plastic and present a spindle-shaped morphology. Surface molecule expression was studied by
flow cytometry. CDCs were positive for the mesenchymal stem cell (MSCs) markers CD105, CD90, CD73 and negative for CD34,
CD45, CD31 and all hematopoietic lineage markers. Since CDCs expressed the core mesenchymal molecules found in bone marrow
(BM) MSCs, experiments were performed to test their ability to differentiate in osteogenic and adipogenic lineages, a well-known
property of BM MSCs. CDCs were unable to differentiate into these lineages. These cells had a population doubling time of
approximately 53.14 ± 5.63 hours. CDCs were then co-cultured for 72 hours with neonatal ventricular myocytes isolated from
-actin promoter. We detected the expression of
connexin 43, troponin T, myosin light chain 2a and myosin heavy chain by RT-PCR, suggesting that these cells are able to be
differentiated into cardiomyocytes. Immunofluorescence assays confirmed that these cells were able to express myosin and
connexin 43. Conclusion: CDCs are cells of mesenchymal origin, adherent to plastic and immunophenotipically similar to BM-MSCs.
However, their differentiation properties are quite different, as BM-MSCs differentiate in osteogenic, adipogenic and
chondrogenic lineages, while CDCs differentiate into the cardiac lineage. It still remains to be defined whether CDCs are able to
differentiate into the other cell types found in the heart, if the differentiated cells represent functional cells and if these cells have
the ability to generate cardiomyocytes in vivo.
Support: Capes, CNPq, Decit, Finep, Faperj.
M22
MORPHOLOGICAL ALTERATIONS IN MURINE MACROPHAGES CAUSED BY AQUEOUS EXTRACT OBTAINED FROM ROOTS OF
PHYSALIS ANGULATA
1,2-R.R.P. SILVA; 1,2-A.P.D. RODRIGUES; 1,2-FARIAS, L.H.S.; 3-G.N.T. BASTOS; 1,2-E.O. SILVA
1-Laboratório de Biologia Estrutural/Laboratório de Parasitologia, ICB, Universidade Federal do Pará. 2-Instituto Nacional de
Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro, Ilha do Fundão, RJ, Brazil 3Laboratório de Neuroquímica Molecular e celular, Universidade Federal do Pará, Brazil
Physalis angulata is extensively distributed in tropical and subtropical regions of the world and is used in traditional medicine as
analgesic, antirheumatic, anti-nociceptive, anti-diuretic and anti-inflammatory drug. In this work we examined the morphological
alterations of treated macrophages with aqueous extract of P.angulata. Cells were treated with 100 μg/ mL of the extract for 1
hour and cultivated for 24 hours. Then, untreated or treated cells were cultured on coated coverslips and fixed with
formaldehyde, incubated with Alexa Fluor® 594 Phaloidin for detection of actin filaments or incubated with polyclonal antibody
anti-tubulin for microtubules detection, incubated with Alexa Fluor®-labelled goat anti-rabbit IgG (except those labeled with
phalloidin) and DAPI for nuclei detection. Coverlips were examined under a Confocal Pascal LSM-510 microscope (Zeiss). The
results demonstrated that aqueous extract was able to activate and modify treated cells cytoskeleton. Control cells exhibited a
normal cell shape for actin filaments and microtubules, showing typical resident morphology. The cells treated with 100 µg/mL of
extract exhibited expressive alterations, labeled with phaloidin presenting increase of actin filaments polymerization and
cytoplasmatic projections; labeled microtubules exhibited enhanced polymerization, extending from the nucleus membrane to the
cell membrane. Those results were confirmed by transmission and scanning electron microscopy. Macrophages treated with 100
µg/mL of extract showed significant morphological changes. We observed an increase in membrane projections, cytoplasm
contained a high number of endoplasmatic reticulum, greater spreading and numerous cellular projections when compared with
control cells. Furthermore, the mitochondria exhibited normal morphology and no citotoxicity effects were observed on treated
cells. Thus, these preliminaries results demonstrate that macrophages treated with aqueous extract of P. angulata exhibit
expressive morphological alterations that suggest cell activation. Further studies are in progress to identify cytokines production
and phagocytic activity. Keywords: P. angulata, morphologycal alterations, macrophages
Support: CAPES, CNPq/UFPa, CNPq/MCT/CT-INFRA/CT-PETRO (Processo nº 620179/2008), MCT/CNPq/FNDCT/PROCAD-NF
CAPES/FAPERJ
M23
CYCLIC GMP PATHWAY REGULATES GLIAL CELL ACTIVITY IN THE CENTRAL NERVOUS SYSTEM
1- LUNA, R.L.A; 2- RAPÔSO, C.; 1,2- NUNES, A.K.S.; 2- SARAIVA, K.L.A.; 3- CRUZ-HÖFLING, M.A.; 1,2- PEIXOTO, C.A.
1- Laboratório de Ultraestrutura, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife-PE; 2- Laboratório de
Microscopia, Ministério de Ciência e Tecnologia, Centro de Tecnologias Estratégicas do Nordeste (MCT/CETENE), Recife-PE; 3Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas-SP
Glial cells play important roles in central nervous system physiology and in inflammatory/neurodegenerative diseases
development, but the control of these cells activation is poorly understood. It has been reported that the cyclic guanosine 3',5'monophosphate (cGMP)-dependent pathways protect myelin-forming oligodendrocytes against death. Moreover, mice lacking
iNOS exhibit more demyelination in demyelinating model. In cerebellum, the phosphodiesterases-5 and 9 (PDE5, PDE9) are the
cGMP-hydrolyzing enzymes, therefore, herein, we used Sildenafil (Viagra®), a PDE5 inhibitor, to induce cGMP accumulation in wild
type and iNOS-knockout mice, and investigate the NO-cGMP pathway role in myelination and glial cells activation. Five C57BL/6mice wild type (WT) and iNOS-/-, 21-day-old, were used/group. Treated groups received Sildenafil 25mg/kg/8 weeks, in the
drinking water, whereas the control groups received pure water. The cerebella were processed for Luxol Fast Blue-LFB dyeing,
western blotting-WB or frozen-immunofluorescence-IF. The GFAP, Iba1 and GST-pi expression (markers for astrogliosis,
microgliosis and mature oligodendrocytes, respectively) were detected by WB/IF, and the myelination was assessed by LFB.
Comparing to WT-control, WT-Sildenafil treated group showed a significant increase of GFAP, GSTpi and Iba1 expression. IF
showed thicker and more numerous astrocytic processes and much ramified microglia. LFB dyeing quantification showed
significantly more myelinated areas in WT-Sildenafil group, comparing with WT-control. The iNOS-/--control showed significantly
less labeling for GFAP, Iba1 and GST-pi, and weaker and vacuolated LFB-dyeing, comparing to WT-control. However, after
Sildenafil treatment, iNOS-/- animals showed GFAP, GSTpi and Iba1 increase, and LFB showed normal myelin, comparing with
iNOS-/--control. Although GFAP expression it remained lower than WT-control and WT-Sildenafil groups. We conclude that NO
produced by iNOS is important for glial cells activation and myelin integrity. But, cGMP accumulation induced glial activation and
myelin repair even in absence of NO produced by iNOS, suggesting that cGMP has a role independent of iNOS-NO in glia activity in
cerebellum.
Support: FACEPE, CNPq, MCT/CETENE, INBEB.
M24
STUDIES OF DIFFERENT ERGOSTEROL BIOSYNTHESIS INHIBITORS ON LEISHMANIA AMAZONENSIS
1,2- MACEDO-SILVA, S.T.; 3- URBINA, J.A.; 1,2,4- DE SOUZA, W.; 1,2,4,5- RODRIGUES, J.C.F.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro. 2- Instituto Nacional de Ciência e Tecnologia
em Biologia Estrutural e Bioimagem. 3- Instituto Venezolano de Investigaciones Científicas, Venezuela. 4- Instituto Nacional de
Metrologia, Qualidade e Tecnologia, Inmetro, Brasil. 5- Pólo Avançado de Xerém, Universidade Federal do Rio de Janeiro.
Leishmaniasis is a tropical disease caused by parasites of the Leishmania genus, which is associated with significant rates of
morbidity and mortality throughout the world. The mainstay of chemotherapy employed is based on pentavalent antimonials as
first line of compounds, and in special cases, on miltefosine, amphotericin B and pentamidine. However, they are very
unsatisfactory and there is an urgent need for safer and more efficacious anti-Leishmania agents. Trypanosomatids have an
essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells and the ergosterol
biosynthesis is an interesting pathway to search for new chemotherapeutic agents. Itraconazole and posaconazole are known
inhibitors of C14α-demethylase, an important enzyme of the sterol biosynthesis, with potent effects against fungus. Thus, we
decide to investigate the effects of posaconazole and itraconazole in the ultrastructure of L. amazonensis promastigotes and
intracellular amastigotes. Control and treated parasites were fixed and processed for fluorescence microscopy and scanning and
transmission electron microscopy. Treatment with posaconazole and itraconazole induced intense alterations on the morphology
of promatigotes that appeared rounded and swollen. Scanning electron miscrocopy also revealed the presence of cells presenting
more than one flagellum, which could be a result of cell cycle arrest. Fluorescence microscopy with Nile Red demonstrated a lipid
accumulation, which is dose-dependent. In addition, transmission electron microscopy confirmed the presence of lipid bodies. The
mitochondrion was also changed, presenting an intense swelling with loss of the matrix content. Presence of cells containing more
than one nucleus and autophagosome-like structures was also observed. These alterations were not observed on the control
promastigotes. Taken together, these results indicate that electron microscopy is an interesting tool to identify essential
organelles which are important targets during the treatment with new and promising compounds against Leishmania sp.
Support: INBEB, FAPERJ, CAPES, CNPq.
M25
CARACTERIZATION OF THE HEPATIC Cu(I)-ATPase FROM RATS.
1-BRITTO-BORGES, T.; 1-GOMES, F. ; 1-HILÁRIO-SOUZA, E.; 1-VALVERDE, R. R. F. H. ; 1-VIEYRA, A. ; 1-LOWE, J.
1- Instituto de Biofísica Carlos Chagas Filho/UFRJ, IBCCF/UFRJ
Objectives: Wilson disease is a monogenic inherited disorder characterized by liver failure and neurological symptoms. The
molecular basis of these events are related with mutations and consequent loss of function of ATP7B, one of the mammal the
hepatic Cu(I)-ATPases. Liver is responsible for copper plasma distribution and the excretion of its excess, being the most import
organ in mammals copper homeostasis. Likewise, this organ is central in energetic metabolism by controlling glucose levels when
signalized about of the nutrition state by insulin and glucagon, both hormones that trigger different kinase-mediated signaling
pathway including PKA, PKC and tyrosine kinases. Recent studies of our group demonstrated that PKA phosphorylates specific
residues that modifies catalytic behavior of Cu(I)-ATPases when heterologously expressed in a Sf9 cells (J. Biol. Chem. 286;6879,
2011) and in pig liver enriched Golgi fractions (Int. J. Biochem. Cell Biol. 43;358, 2011). This work concentrates not only in rat Cu(I)ATPase characterization, but an understanding in vivo animal model of interaction between energetic metabolism and copper
homeostasis. Methods and Results: Golgi-enriched membrane fractions were obtained from Wistar rat liver. ATP7B protein was
detected by Western Blotting. The Cu(I)-ATPase specific activity was evaluated by release of Pi from ATP. The results were given in
nmol × mg-1 × min-1 (means ± SE) and was measured by the difference in the absence and presence of 0.3 M BCS, a specific Cu(I)
chelator. Optimal temperature, time reaction and pH curve were determined, showing that the maximum Pi release was obtained
in acidic pH (5.0) at 37 °C. The Cu(I)-ATPase activity was 42.83 ± 6.39 nmol Pi × mg-1 × min-1 (n=5). Catalytic behavior from this
protein resembles Ccc2, the yeast Cu(I)-ATPase, with maximum catalytical phosphorylation in 45 s followed by dephosphorylation
after 3 min. Conclusions: Conclusion: This protein, a P-type ATPase, is responsible for active copper transport to the lumen of
trans-Golgi network. In vitro studies shows that insulin is responsible for stimulation of ATP7B activity and therefore the kinasesmediated regulatory phosphorylations are being studied in this in vivo model.
Support: FAPERJ and CNPq.
M26
AVALIAÇÃO MORFOFUNCINAL DO RIM EM CAMUNDONGOS INFECTADOS COM PLASMODIUM BERGHEI ANKA
1- Abreu T.P.,1,2- Ferreira-Da Silva, CT; 1,3-Souza-Silva, L, 2-Saraiva V.B., 3-Souza M.C., 3,1-Henriques M.G.,1,4-Landgraf, S.S., 1,5Caruso-Neves, C., 1,6-Pinheiro, A.A.S
1-Instituto de Biofísica Carlos Chagas Filho; 2- Instituto Federal de Educação, Ciência e Tecnologia Fluminense/IFFluminense; 3FARMANGUINHOS/FIOCRUZ; 4- Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem; 5- Instituto
Nacional de Ciência e Tecnologia para pesquisa translacional em Saúde e Ambiente na região Amazônica
Malária grave em humanos é caracterizada pelo desenvolvimento de patologias múltiplas que incluem a malária cerebral, anemia
aguda e insuficiência renal aguda. O modelo murino Plasmodium berghei ANKA permite contribuições valiosas para a
compreensão da patogênese da doença. Portanto o objetivo desse estudo é avaliar o dano renal causado pela infecção com
Plasmodium usando modelo murino. Camundongos C57BL / 6 machos foram submetidos à injeção intraperitoneal de solução
salina (grupo controle) ou 105 glóbulos vermelhos infectados com Plasmodium berghei ANKA (iRBC). Todos os grupos foram
alojados em gaiolas metabólicas para avaliar a função renal no dia 3 (grupo de baixa parasitemia) e dia 10 (grupo de alta
parasitemia) pós-infecção (pi). Neste ponto, os ratos foram sacrificados e os rins foram utilizados para preparação de homogenato
de córtex e medula, além de análises histológicas. A parasitemia alcançou 0,5% e 20% de eritrócitos infectados, respectivamente.
Somente grupo de alta infecção apresentou um aumento de 5,6 vezes na fração de excreção renal de sódio (FENa+) (0,5%
controle e baixa parasitemia contra 2,8% alta parasitemia). Assim, no mesmo grupo, a atividade (Na+ + K+) ATPase foi reduzida no
córtex e medula em 36% e 34% respectivamente, e não se alterou na baixa parasitemia em relação aos animais controle. O
número de células glomerulares aumentou progressivamente a partir de uma média de 36,3+- 0,39 células (controle) para 39,43 +1,12 (3d.p.i) e 45,8+- 0,5 (10d.p.i). Além disso, foi observada uma redução no tamanho do espaço de Bowman (7,6+- 0,54% de
controle; 5,56 +- 0,10% grupo de alta parasitemia). A densidade de superfície das fibras de colágeno aumentou apenas no dia
10p.i. (2,8+- controle 0,16% contra 6,3+- 0,91%). Os resultados apresentados neste trabalho sugerem que a infecção da malária
por P. berghei ANKA induz insuficiência renal revelada por alterações morfofuncionais.
Support: FAPERJ, CAPES, PRONEX e CNPq.
M27
INVESTIGATION OF THE MOLECULAR INTERACTIONS INVOLVED IN THE IN VITRO ASSEMBLY OF THE HEPATITIS C VIRUS
Braga,V.L.A.1, Mendes-Silva,A.1, Souza,T.L.F.2, Ferreira,D.F.3, Peabody,D.S.4, Silva,J.L.1, Gomes,A.M.O.1 & Oliveira,A.C.1
1PBE/IBqM/UFRJ/Brasil-2FF/UFRJ/Brasil-3IMPPG/UFRJ/Brasil-4UNM/USA.
Hepatitis C is a worldwide public health problem since around 3% of the population is infected by this virus and current therapies
are inefficient. Thus, there is a need to the development of more efficient drugs. The HCV core protein (HCVCP) is involved in
several processes. This work aims to understand the viral capsid assembly in vitro. We showed that the fusion of the HCVCP with
the Green Fluorescent Protein (GFP) (HCVCPGFP) does not inhibit the assembly process, since the addition of nonspecific nucleic
acids leads to formation of nucleocapsid-like particles (NLPs) as verified by electron microscopy. Spectrophotometric analysis
showed that NLPs formation depends on the protein and DNA concentrations. Gel shift assays showed a band of high molecular
weight suggesting capsid assembly in a cooperative process. Since the fusion with GFP does not inhibit the assembly process we
now are expressing the HCVCPGFP in HepG2 cells to obtain data about the cellular localization and the assembly process by
confocal microscopy and fluorescence correlation spectroscopy. We also have studied the physical chemical aspects of the
interaction with nonspecific nucleic acids and membrane models (micelles), such as sodium dodecyl sulfate (SDS) and n-octyl- -Dglucopyranoside (n-octyl), of three core protein regions important for the assembly (peptides 22-39, 50-67 and 85-102). In the
presence of SDS and n-octyl, only peptide 85-helix structure and the tryptophan residue showed to be in an
apolar environment, as verified by circular dichroism and fluorescence spectroscopy, respectively. We measured the calorimetric
parameters of peptide-micelles interactions and we found that the thermodynamics of the interaction of peptide 50-67 with
different DNAs was similar. Fluorescence polarization data showed that the addition of peptide 50-67 to nonspecific DNA does not
prevent NLPs formation promoted by core protein. Our data reveal a new approach to understand the HCV capsid assembly which
is a target for drugs against HCV.
Support: CNPq-CAPES-FAPERJ-INBEB.
D01
DETERMINAÇÃO DA ESTRUTURA EM SOLUÇÃO E DAS PROPRIEDADES DINÂMICAS DA PROTEINA ALERGÊNICA GAD M1 E DO
COMPLEXO GAD M1 – SCFV POR RESSONÂNCIA MAGNÉTICA NUCLEAR
1- MORAES, A. H.; 1- DE PAULA, V. S.; 2- BREITENEDER, H.; 3- FERREIRA,F.; 1- ALMEIDA, F.; 1- VALENTE, A. P.
1- Centro Nacional de Ressonância Magnética Nuclear , Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro,
Rio de Janeiro-RJ, Brasil; 2- Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology &
Immunology, Medical University of Vienna, Viena, Aústria; 3- Department of Molecular Biology, University of Salsburg, Salsburg,
Aústria
A construção de variantes hipoalergênicas, quimeras ou mutantes pontuais, são maneiras de chegar a tratamentos eficientes para
alergias. A caracterização estrutural dos alergenos e os estudos de interação com IgE são passos cruciais para viabilizar novas
estratégias para o desenvolvimento de vacinas alergeno-específicas. Neste estudo, pretendemos compreender os mecanismos de
interação proteínas-ligante. Especificamente, estudamos os detalhes da interação alergeno-IgE para a construção de moléculas
hipoalergênicas, ou seja, moléculas que mantém o reconhecimento pelas células T com um reduzido efeito da alergenicidade.
Neste estudo, utilizamos a proteína Gad m1, uma parvalbumina presente em bacalhau e principal responsável por reações
alérgicas ocasionadas pelo consumo desse espécie de peixe. Através da espectroscopia de RMN estamos resolvendo a estrutura
tridimensional da proteína e, concomitantemente, realizando ensaios de interação da proteína com os domínios variáveis dos
anticorpos IgE, conhecidos como scFv. Através de espectros de 1H-15N HSQC foi possível mapear 4 possíveis regiões de
reconhecimento da proteína ao scFv. Com excessão de resíduos de aminoácidos localizados no -C-terminal, os epitopos
observados concordam com os as regiões de reconhecimento visulaizadas, por outras técnicas, em parvalbuminas análogas. Para
analizar as propriedades dinâmicas da proteína e do complexo proteína-anticorpo, foram realizadados experimentos de relaxação
por RMN. Através dos parâmetros de relaxação R2 e R1, foi possível obervar que, apenas rezíduos localizados no C e N terminal da
proteína apresentam grau de mobilidade maior. O considerável decréscimo nos valores de R1 dos resíduos da proteína quando na
presença de scFv é um forte indicativo da formação do complexo Gad m1 e IgE. Com os dados obtidos será possível determinar a
estrutura em solução da proteína e mapear os epítopos de interação da Gad m1 com as moléculas de scFv.
INBEB, FAPERJ, CNPQ
D02
INSIGHTS INTO THE MECHANISM OF ACTION OF CAMPTOTHECIN, A TOPOISOMERASE I INHIBITOR, IN TRYPANOSOMA CRUZI
1,2- ZUMA, A.A.; 1,2- LACOMBE, O.K.; 1,2- SOUZA, W.; 1,2- MOTTA, M.C.M.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Instituto Nacional de Ciência e Tecnologia
de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro.
Trypanosoma cruzi, the aetiological agent of Chaga’s disease, presents a single nucleus and an unique mitochondrion with an
enlarged portion named kinetoplast, that contains the kDNA. The topological state of DNA is modulated by topoisomerases, that
revert supercoilings during replication, transcription, recombination and repair, thereby representing an interesting target in
chemotherapeutic studies. Topoisomerase I inhibitors have been widely tested in tumor cells, but few studies show its effects on
trypanosomatids, specially on Trypanosoma cruzi. Thus, in this work we evaluated the effects of different topo I inhibitors,
Camptothecin and its two derivatives, Topotecan and Irinotecan, on proliferation and ultrastructure of T. cruzi epimastigotes. For
this purpose, cells were cultivated in culture medium containing different drug concentrations. Samples were collected after each
24 hours (until 96 hours of cultivation) for counting on Neubauer’s chamber or for processing to transmission electron microscopy.
Cell proliferation was highly inhibited after treatment with Camptothecin, which presented a low IC50 value, whereas Topotecan
and Irinotecan did not cause significant growth impairment. Camptothecin led to cell cycle arrest in G2 phase, since there was a
significant increase in the number of protozoa at this cell cycle phase. Transmission electron microscopy analysis revealed that
Camptothecin promoted nuclear ultrastructural alterations, as a remarkable unpacking of the perinuclear chromatin. This
compound also induced higher levels of oxygen reactive species (ROS) and loss of mitochondrion membrane potential. Taken
together, our data emphasize the essential role of topoisomerase I on cell proliferation and nuclear ultrastructural organization
and reinforce the idea that topoisomerases constitute a promising target for antitrypanosomal chemotherapy.
CNPq, FAPERJ
D03
A SMART ALLERGEN: STUDIES OF BET V 1 DYNAMICS AND ITS INTERACTION WITH HUMAN IGE BY NMR
1-ALINE L. BATISTA, 1- ADOLFO MORAES1 , 2- MICHAEL WALLNER,1- VIVIANE DE-PAULA, 1-FÁBIO ALMEIDA, 2-FÁTIMA FERREIRA,
1- ANA PAULA VALENTE
1-Centro Nacional de Ressonância Magnética Nuclear de Macromloléculas,Instituto de Bioquímica Médica, Universidade Federal
do Rio de Janeiro, Rio de Janeiro-RJ 21941-902, Brasil 2-Christian Doppler Laboratory for Allergy Diagnosis and Therapy,University
of Salzburg, Salzburg, Austria
Allergens that lead to type I allergic reactions affect approximately 30% of adults and up to 40% of children in western societies.
Bet v 1 is among the major causes of pollinosis in the Northern Hemisphere. Bet v 1 is from Betula verrucosa (birch), with 159
residue and 17.5 kDa. The structure is organized in seven-stranded anti-parallel β-sheet that wraps around a long α-helix and by
two short ones. Its biological function is associated with the transport of hydrophobic molecules such as brassinosteroid and
cytokinin (plant hormones). In this study, we describe the interactions of IgE purified from patients sensitized and Bet v1, in order
to map possible epitopes by NMR. We performed experiments of 15N-HSQC of rBet v 1 free and with IgE. With these experiments
we were able to map a conformational epitope and at least 15 residues of the protein involved in this interaction. We also analyze
the interaction of the Bet v1 with deoxycholate (brassinosteroid like molecule). We observed that the protein binds 4
deoxycholate molecules, and the residues involved in this interaction are located mainly in the hydrophobic cavity. T1, T2 and
heteronuclear NOE were used to evaluate dynamic aspects of free Bet v1 and with ligands. The free protein underwent profound
conformational changes, which became more ordered in the presence of ligands. Since Bet v 1 showed complex dynamic
properties the identification of key residues for the IgE reactivity was much more complicate. Mapping the IgE epitope in the
presence of ligands might show the key residues involved in binding without the residues involved in the conformational exchange
process. This complex behavior had hampered the development of an efficient vaccine for this major allergen. Our strategy may
allow the development of hypo-allergenic variants for novel approaches to allergen-specific immunotherapy.
CNPq, ICGEB-Trieste, FAPERJ, CAPES, INBEB
D04
THE PRESENCE OF THE SYMBIOTIC BACTERIUM INFLUENCES THE O2 CONSUMPTION IN ITS HOST CELL, CRITHIDIA DEANEI
1- de Azevedo-Matins, A.C; 1- Machado Loyola, A.C; 2- Galina, A.; 3- Ciapina, L.; 3- Gonzaga, L.; 3- Vasconselos, A.T.; 1- de Souza,
W.; 4- Einiker-Lamas, M.; 1- Motta, M.C.M.
1 - Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofíosica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro; 2 - Insituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 3 - Labinfo, Laboratório Nacional de
Computação Científica; 4 - Laboratório de Físico-Química Biológica Aída Voloch, Instituto de Biofíosica Carlos Chagas Filho,
Universidade Federal do Rio de Janeiro.
Some trypanosomatids harbour a symbiotic bacterium which co-evolves with the host. This relationship constitutes an excellent
model in the study of the organelle origin and the cellular evolution. The presence of this symbiont promotes morphological
alterations in the host. Furthermore, the protozoan metabolism is altered and an intense metabolic exchange occurs between
both partners. The presence of the symbiont also inlfuences the host energetic metabolism, since the wild strain of Crithidia
deanei shows an increased O2 consumption when compared to the aposymbiotic strain. In order to characterize the mitochondrial
energetic metabolism in C. deanei, we tested the effect of different inhibitors and ionophore that specifically target the protein
complexes of the respiratory electron system. It was not observed a significant inhibition of oxygen consumption after addition of
oligomycin to the culture medium. This result suggests a minor contribution of complex V (FoF1 ATP synthase) to the ATP
synthesis in wild strain. Unexpectedly, oligomycin promoted a increase in O2 consumption in the aposymbiotic cells. The FCCP, a
proton ionophore, increased the O2 consumption in both strains to the same value, however the stimulation was more
pronounced in the aposymbiotic strain. The antimycin A and cyanide, potent inhibitors of complexes III and IV respectively,
completely abolish O2 consumption in both strains, but the aposymbiotic protozoa are more sensible to these compounds. The
TTFA, a potent inihbitor of complex II, partially blocks O2 consumption, whereas rotenone, a potent inhibitor of complex I showed
little effect in complex I. After searching sequences in C. deanei genome we found genes that encode subunits of the complexes I
and IV, but some important sequences are absent, as the subunit of complex I which is sensible to rotenone. Our next goal is to
explore the genome of C. deanei in order to construct the protozoan metabolic network.
Support: INBEB, FAPERJ, CAPES, CNPq, INCTEN.
D05
LPSF/GQ-02 REDUCES THE EXPRESSION OF MATRIX METALLOPROTEINASE-9 (MMP-9) AND EVOLUTION OF ATHEROSCLEROTIC
PLAQUE IN LDL RECEPTOR-DEFICIENT MICE
SILVA, A.K.S.1.; TORRES, D.O.C.1; ROCHA, S.W.S.1; GOMES, F.O.S.1; SILVA, B.S.1; NUNES, A.K.S.1; K.P.S., RAPOSO.2; LIMA, M. C.
A.3; GALDINO, S. L. 3.; PITTA, I. R.3.; PEIXOTO, C.A.;1,2
1. Centro de Pesquisas Aggeu Magalhães- CpqAM/FIOCRUZ, Brazil
Background: Experimental evidence suggests that matrix metalloproteinase-9 (MMP-9) may play a causal role in new-onset
cardiovascular disease. Changes in the expression of MMP-9 in vascular tissues have been implicated in the pathogenesis of
several cardiovascular conditions such as atherosclerosis, resulting in atherosclerotic plaque rupture. Objective: This study
investigated the effects of the thiazolidine derivative LPSF/GQ-02 and LPSF/GQ-16 on the expression of MMP-9 and evolution of
atherosclerotic plaque in RLDL-/-. Methodology: 32 male mice were divided into four groups: group 1-fed with high-fat diet (HFD)
(21% fat milk +1.25% cholesterol), 2–HFD+pioglitazone 20mg/kg/day, 3–HFD+LPSF/GQ-02 (30mg/kg/day) and 4–HFD+LPSF/GQ-16
(30mg/kg/day). The experiments were conducted for 10 weeks and in the last two weeks of HFD, the drugs were administered
daily by gavage. After experimental protocols, the animals were sacrificed and the aorta was quickly dissected and processed for
optical microscopy evaluation and stored at -80 °C for protein analysis by western blot. Results: The aortas from the HFD,
Pioglitazone and LPSF/GQ-16 group exhibited innumerous macrophages in the subendothelial space, containing lipid droplets in
their interior characterizing foam cells. LPSF/GQ-02, there were a smaller number of foam cells in the subendothelial space, with
the preservation of the tunic media. In the morphometric analysis the area of the atherosclerotic lesions, treatment with
pioglitazone did not reduce the area of injury compared with the HFD group. On the other hand, LPSF/GQ-02 reversed the
conditions caused by high-fat diet, reducing the atherosclerotic lesion. LPSF/GQ-16 injury increased considerably compared to all
other groups. Analysis of expression of MMP-9 showed that pioglitazone and LPSF/GQ-16 did not reduce the expression of MMP-9
compared with the HFD group. However, LPSF/GQ-02 was effective in reducing the expression of MMP-9 compared with the HFD
group. Conclusions: These results suggest that LPSF/GQ-02 is promising candidate for the treatment of atherosclerosis.
Support: INBEB,CNPq.
D06
MICROBICIDAL RESPONSE IN MACROPHAGES INFECTED WITH L. (L.) AMAZONENSIS AFTER TREATMENT WITH AQUEOUS
EXTRACT FROM ROOT OF PHYSALIS ANGULATA
1,3-SILVA, B.J.M; 1,3-SILVA, R.R.P.; 1,3-RODRIGUES, A.P.D.; 1,3- FARIAS, L.H.S.; 2-BASTOS, G.N.T.; 1,3-SILVA, E.O.
1-Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Pará,
Brasil; 2-Laboratório de Neuroquímica Molecular e Celular, Instituto de Ciências Biológicas, Universidade Federal do Pará, Brasil;.3Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro, Ilha do
Fundão, Brasil
Leishmaniasis an infectious diseases caused by protozoa of the genus Leishmania and transmitted by phlebotomine sandflies. This
protozoa multiply in phagocytic cells, mainly macrophages, which play an important role defending the organism from pathogens.
The production of superoxide radicals (ROS) and nitric oxide (NO) are defense mechanisms used by macrophages and are involved
in parasite destruction. The most effective treatment for leishmaniasis is the chemotherapy and besides the high cost, these drugs
are toxic and require a long period of treatment. Currently, some herbal products are considered an important alternative source
of a new leishmanicidal agent, which includes the plant Physalis angulata, widely used in popular medicine as analgesic,
antirheumatic and anti-inflammatory drug. Analysis by immunofluorescence microscopy detected the presence of inducible nitric
oxide synthase (iNOS) enzyme in infected and treated cells, with the concentrations of 50 and 100 µg/ml. However we did not
observed NO production, when nitrite levels in culture supernatants were measured by Griess reaction. The aqueous extract
promoted the activation of macrophages through the production of ROS in cells infected with L. amazonensis and treated with the
extract at a concentration of 100 µg/ml. No differences in phagocytic activity was observed between treated and untreated
macrophages. Analysis of cell viability by tetrazolium-dye (MTT) colorimetric method, Neutral Red and Mitochondrial Membrane
Potential Detection Kit (JC-1) showed that this compound presented no cytotoxic effects against host cells. Thus, this study
revealed that extract from Physalis angulata is able to activate macrophages through the production of ROS and has
antileishmanial properties. Keywords: microbicidal response; Physalis angulata; L. (L.) amazonensis.
Support: CAPES, CNPq/UFPa, CNPq/MCT/CT-INFRA/CT-PETRO (Processo nº 620179/2008), MCT/CNPq/FNDCT/PROCAD-NF
CAPES/FAPERJ.
D07
ECTO-PTPASE ACTIVITY FROM TRYPANOSOMA RANGELI IS RELATED TO RHODNIUS PROLIXUS SALIVARY GLANDS INTERACTION
1,4-DOS-SANTOS, A.L.A.; 1,2,4-DICK, C.F.; 1,2,4-SILVEIRA, T.S.; 1,4-FREITAS-MESQUITA, A.L.; 1,5-ALVES-BEZERRA, M., 3,4-SILVA,
P.A.; 3,4-VIEYRA, A; 1,5-GONDIM, K.C.; 1,4-MEYER-FERNANDES, J.R.
1 Instituto de Bioquímica Médica, UFRJ, Brazil 2 Instituto de Microbiologia Professor Paulo de Góes, UFRJ, Brazil 3 Instituto de
Biofísica Carlos Chagas Filho, UFRJ, Brazil 4 Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem (INBEB)
5 Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INEM)
PTPases have been reported as a virulence factor in different pathogens. The physiological role of these enzymes in
trypanosomatids had not been well established yet, although they are supposed to be involved in the virulence and cellular
differentiation. We report here the presence of an ecto-PTPase activity in T. rangeli epimastigotes and the role of this enzyme
during interaction between this parasite and R. prolixus salivary glands. In brief, salivary glands of R. prolixus were incubated in the
presence of short and long epimastigotes forms of T. rangeli (106 parasites per salivary gland). Three different strains of T. rangeli
were analyzed, namely strains H14, Choachi and Macias. The results demonstrated that long epimastigotes forms of H14 and
Choachi strains exhibit higher levels of ecto-PTPase activity and adhesion capacity to salivary glands. Furthermore, short
epimastigotes forms of all strains tested exhibit lower ecto-PTPase activity and adhesion to salivary glands compared to long
epimastigotes. We also verified that orthovanadate, a PTPase inhibitor, could significantly inhibit T. rangeli adhesion to the salivary
gland. The irreversible profile of PTPase inhibition produced by orthovanadate led us to study the effect of intracelomic infection
by T. rangeli in R. prolixus, when ecto-PTPase activity was both fully functional and inhibited by pretreatment with orthovanadate.
The inoculation of long epimastigotes pretreated for 1 h with different concentrations of orthovanadate impaired the T. rangeli
infection in triatomid bug. Taken together, the results suggest that the ecto-PTPase activity from T. rangeli may play a role in the
interaction with salivary glands of R. prolixus.
Support: CNPq, FAPERJ, CAPES, INBEB.
D08
CELLULAR ASSESSMENT AFTER UNILATERAL RAT SPINAL CORD INJURY: CORRELATION WITH FUNCTIONAL RECOVERY
1- BOMFIM, A.M.D.; 2-POTAS, J.R.; 3- CASTRO, N.G.; 1-MENDEZ-OTERO, R.
1- Laboratório de Neurobiologia Celular e Molecular, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro; 2- Medical School and Research School of Biology, Australia's national university; 3- Laboratório de Farmacologia
Molecular, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro
Objectives: To investigate a rat model of unilateral T10 spinal cord injury by comparing cellular and behavioral recovery ipsilateral
and contralateral to the injury. Methods and Results: Under local ethics guidelines, a laminectomy was performed on Wistar rats
followed by a hemitransection on the left side (n=6) or no lesion (sham control; n=3). Locomotor recovery was evaluated from 3
days after injury using the Basso Beattie Bresnahan Scale (BBB) for the duration of the recovery period (60 days). Immunostaining
was performed using the following markers: ED1 (CD68), glial fibrillary acidic protein (GFAP), neurofilament-200 and growth
associated protein 43 (GAP-43) marking macrophages, astrocytes, neuronal fibres and regenerating axons respectively. A
significant difference in the BBB locomotor score was found between the intact and injured sides within the first 15 days after
injury. A significant spontaneous post-injury period functional recovery was observed in the hemitransection group during the
course of recovery. Positive staining for macrophages were found at some considerable distance caudal to the level of
injury,restricted to the ipsilateral half of the spinal cord. There were morphological differences in reactive astroglyosis observed in
injured animals compared to the sham group above and below the injury. Regenerating axons were found bilaterally at and below
the level of injury. Conclusions: The findings that persistent changes occur well below the level of injury suggest that these distal
regions may influence to the functional recovery in the absence of treatment. This period may therefore provide a window of
opportunity for therapeutic intervention following spinal cord injury.
Support: CNPQ.
D09
CHALCONAS COMO INIBIDORES DAS PROTEÍNAS TIROSINA FOSFATASE A E B (PTPA/PTPB) DE MYCOBACTERIUM TUBERCULOSIS
1-MENEGATTI, A.C.O.; 1,2-CHIARADIA, L.D.; 1-ALVES MARTINS, P.G.; 2-CORDEIRO, M. N.S.; 1-ECCO, G.; 1- VERNAL, J.; 2-YUNES, R.
A.; 2-NUNES, R.J.; 1-TERENZI, H.
1-Centro de Biologia Molecular Estrutural-CEBIME, Universidade Federal de Santa Catarina; 2-Laboratório Estrutura e Atividade,
Departamento de Química, Universidade Federal de Santa Catarian
A bactéria Mycobacterium tuberculosis (Mtb) é o agente causador da tuberculose, uma doença grave, que tem se tornado um
grande desafio para a saúde pública, devido ao surgimento de cepas multirresistentes. Anualmente são diagnosticados 10 milhões
de novos casos, e cerca de 2 milhões de pessoas morrem em conseqüência da doença no mundo. A análise do genoma do Mtb
revelou a presença de duas proteínas fosfotirosinas fosfatases, PtpA e PtpB. Foi demostrado que ambas enzimas são essenciais à
virulência do Mtb, pois a deleção dos respectivos genes reduz o crescimento do patógeno nos macrófagos. Assim, a inibição da
PtpA e PtpB aparece como uma estratégia terapêutica promissora para a obtenção de fármacos anti-tuberculose. Para investigar
novos inibidores destas proteínas, 120 chalconas foram sintetizadas e testadas in vitro frente às enzimas recombinantes,
utilizando a concentração de 25µM de composto e pNPP (p-nitrofenil-fosfato) como substrato. Para os compostos que
apresentaram inibição ≥ a 30%, foram realizados ensaios para determinação da IC50. Por meio de estudos cinéticos,
determinamos o mecanismo de inibição das três chalconas mais potentes para PtpA e PtpB. Verificou-se que os inibidores atuam
por mecanismo do tipo competitivo, com valores de Ki entre 15 e 35 µM em PtpA e entre 8 e 13 µM em PtpB. Para detectar a
possível inibição cruzada com outras tirosina-fosfatases, os inibidores identificados para a PtpA e PtpB de Mtb foram ensaiados em
uma PTP humana, a PTP1B. A diferença de ação inibitória para as enzimas sugere que estas chalconas apresentam diferentes graus
de seletividade para a PtpA e PtpB. Dentre 120 compostos sintéticos, 3 chalconas foram identificadas como inibidores
competitivos da PtpA, sendo R6 seletiva para esta enzima e identificou-se, pela primeira vez, 3 chalconas como inibidores
competitivos da PtpB, sendo R32 seletiva para esta enzima.
Apoio: CAPES, CNPq, FAPSC, INCT, DQ-UFSC.
D10
IDENTIFICAÇÃO DE DIFERENTES FOSFATASES SECRETADAS EM Leishmania amazonensis
1-Fernandes ACS ,1- Soares DC , 1- Saraiva EM, 2-Meyer-Fernandes JR , 1-Souto-Padrón T 1
1Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil 2Instituto de Bioquímica
médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
Muitos patógenos possuem a capacidade de hidrolisar fosfatos orgânicos por fosfomonoesterases ácidas, as fosfatases ácidas,
presentes em organelas intracelulares, na superfície celular ou dispersa no meio extracelular. Leishmania sp apresenta intensa
atividade de fosfatase ácida localizada na membrana plasmática e secretada para o meio extracelular através da bolsa flagelar. As
fosfatases ácida secretadas, que são as proteínas de secreção mais abundantes de Leishmania, desempenham um importante
papel na infecção de vertebrados e na sobrevivência dentro do parasito no trato digestivo do inseto vetor. No presente estudo,
caracterizamos bioquimicamente e por citoquímica ultraestrutural a atividade fosfatásica secretada por L. amazonenses e sua
possível participação na interação e infectividade nestes parasitos. A atividade da fosfatase secretada mostrou-se afetada por
alteração no pH (faixa de 6,5-8,5). Duas diferentes atividades, uma ácida e outra alcalina, foram observadas. A atividade da
fosfatase secretada foi alterada, de acordo com o substrato, com uma maior atividade observada na presença de β-glicerofosfato
comparada com a obtida com p-nitrofenilfosfato nas mesmas condições de incubação. Ambas foram inibidas por tartarato de
sódio e vanadato. A análise citoquímica revelou a presença de atividade da fosfatase ácida na superfície do parasita (corpo celular
e no flagelo), em compartimentos intracelulares das vias exo/endocítica onde a intensidade variou de acordo com o substrato
utilizado, e uma marcação em organelas citoplasmáticas que tambem se mostrou alterada de acordo com o substrato hidrolizado.
Estudos de tomografia de cortes espessos estão sendo realizados para uma melhor observação da distribuição ultraestrutural
enzimática. A presença de diferentes fosfatase presente em promastigotas de Leishmania podem ser úteis para sua sobrevivência
nos ambientes hostis nos diferentes hospedeiros.
Apoio: CNPQ, FAPERJ, CAPES, INBEB.
D11
HUMAN RHINOVIRUS 14 RNA TRANSPORT DURING INFECTION OF HELA CELLS
2- Salerno, V. P.; 1- Gomes, A. M.
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2- Escola de Educação Física e Desportos, Universidade
Federal do Rio de Janeiro.
Rhinovirus is the causative agent of common cold and can exacerbate the symptoms of respiratory tract diseases. It belongs to the
Picornaviridae family comprising small viruses presenting icosahedral symmetry, a molecular weight of approximately 8.5 X 106
and a single strand positive sense RNA (about 7 kb). Human Rhinovirus (HRV) 14 belongs to the major group of Rhinoviruses and
use ICAM (intercellular adhesion molecule)-1 as its entry receptor. The mechanisms of entry used by rhinoviruses are relatively
well known for the minor group while many questions are still open for the major group. For example, the dynamics of delivery of
viral RNA to the replication site is still to be revealed. To investigate the RNA distribution in HRV14 infected cells (m.o.i.=10) we
used confocal microscopy imaging of total labeled RNA at different times of infection (30, 60, 90, 120, 150 minutes post infection).
We observed a redistribution of RNA inside the infected cells after 90 min of the infection process and this distribution seems to
go back to normal after 120 min. Immunolabeling of miosin Va allowed the observation of the distribution of this motor protein
during the infection process. Our results suggest that small clusters of RNA and myosin Va may appear during infection. Miosin Va
has been showed to be involved in RNA transport in different cell types. This suggests a role of myosin Va on the transport of viral
RNA in the infected cell and new experiments are being conducted to investigate this hypothesis. At the same time we are
investigating the role of myosin Va (known as a transporter of recently synthetized endogenous RNA) on the dynamics of viral RNA
entering the cell at the early stages of HRV14 infection cycle.
Support: CAPES, CNPq, FAPERJ, INCT-INBEB, PRONEX, FINEP.
D12.
AN EMERGING VIRUS “ON A HURRY”: HOW LONG DOES IT TAKE TO GET TO THE INNER OF THE CELL?
CARVALHO, C.A.M.; SILVA, J.L.; GOMES, A.M.O.
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro
Mayaro virus (MAYV) is an alphavirus widespread in South America in an endemic manner and represents an interesting case to
consider regarding the potential for urban emergence. Alphavirus entry into target cells is supposed to occur by receptormediated endocytosis followed by fusion between the viral envelope and the endosomal membrane, although non-endocytic
penetration of the viral genetic material into the cytoplasm without membrane fusion has also been suggested. The aim of this
work was to analyze the behavior of MAYV particles during their entry into host cells. MAYV was labeled with the lipophilic
fluorescent probe DiD without impairment to viral infectivity and the fluorescent signals were tracked in the host cells by laserscanning confocal fluorescence microscopy in real time. Our results show that MAYV entry into cells occurs by an endocytic
mechanism involving fast internalization of the endocyted cargo, since fluorescent signals from labeled virus particles could be
visualized inside the cell a few seconds after virus binding to receptors on the cell surface. Following DiD fluorescence
dequenching at the single particle level, we could capture the moment of the fusion between the viral envelope and the
endosomal membrane, that was shown to occur faster (around 3 min post-binding) than for other arboviruses. This work provides
unique kinetic insights into the entry of MAYV particles in living cells. Understanding the dynamics of virus infection may provide
important insights to the development of antiviral strategies.
Support: CAPES, CNPq, FAPERJ, FINEP, INBEB, PRONEX.
D13
INCREASED AΒ OLIGOMERS IN MILD ALZHEIMER'S DISEASE
1-MADEIRA C; 1-VARGAS-LOPES C; 1-VIEIRA MN; 3-SUEMOTO CK; 3-GRINBERG LT; 3-LEITE RE; 1,2-FERREIRA ST; 1-PANIZZUTTI R.
1-Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro; 2-Instituto de Bioquímica Médica, Universidade
Federal do Rio de Janeiro; 3-Departamento de Geriatria, Neurologia, Patologia, e Medicina Preventiva, Faculdade de Medicina da
Universidade de São Paulo
Objectives: Alzheimer's disease (AD) is characterized by progressive neuronal degeneration and accumulation of extracellular
deposits of β-amyloid peptide (Aβ). The soluble oligomers of Aβ are potent neurotoxins in the Central Nervous System that may
participate in the pathogenesis of AD. This study aims to study the levels of Aβ oligomers in postmortem brain tissue and CSF of
subjects with different stages of AD. Methods: Postmortem samples of CSF (N = 40), hippocampus (N = 35) and occipital cortex (N
= 27) are from the Brain Bank of the Brazilian Cerebral Aging of the USP. Postmortem cases were classified according to the Clinical
Dementia Rating (CDR). All cases received a histopathological diagnosis by an experienced neuropathologist. The levels of Aβ
oligomers were assessed by immunodetection of the binding of the Aβ oligomers antibody in samples pre-filtered in centricon of
100 KDa. Results: Levels of Aβ oligomers were significantly higher in subjects with mild AD than in controls in both CSF (F=3.33;
p<0.001) and hippocampus (F=1.94; p<0.01). In contrast, we found no difference in the levels of Aβ oligomers between subjects
with moderate/severe AD and controls. Levels of Aβ oligomers were not changed in occipital cortex, a region no affected in AD.
Conclusion: Levels of Aβ oligomers in the hippocampus and CSF are increased in mild AD, but not in moderate/ severe disease. The
detection of Aβ oligomers in CSF can be useful as a marker of AD progression.
Support: SUS/FAPERJ, FAPERJ, CNPq.
D14
CALCINEURINA REGULA A PRODUÇÃO DO NEUROMODULADOR D-SERINA
1- VARGAS-LOPES C.; 1- MADEIRA C.; 2- ALMEIDA S.; 2- MOULIN T.; 1-ROCHA-AMARANTE M.; 2- AMARAL O.; 1- PANIZZUTTI R.
1- Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro; 2- Instituto de Bioquímica Médica, Universidade
Federal do Rio de Janeiro.
Introdução: A D-serina é um co-agonista endógeno dos receptores ionotrópicos de glutamato do tipo NMDA, que participa de
vários processos fisiológicos e patológicos no sistema nervoso. Observamos que a proteína cinase C (PKC) fosforila a enzima serina
racemase (SR), que produz D-serina, e controla a disponibilidade de D-serina no cérebro. A calcineurina (CaN) é uma fosfatase que
defosforila proteínas fosforiladas pela PKC. Objetivo: Avaliar se a atividade da CaN regula os níveis de D-serina. Metodologia:
Inibimos a atividade da CaN em culturas de glioma e de neurônios, e no cérebro de camundongos, utilizando o inibidor
ciclosporina A e siRNA para CaN. Os níveis de D-serina foram monitorados por cromatografia líquida de alta performance (HPLC).
Resultados: Em células de glioma, observamos diminuição na formação de D-serina tanto com o tratamento com CsA (n=3; t=3,04;
P=0,005) como com o siRNA para CaN (n=3; t=4,88; P=0,0006). A inibição da calcineurina com CsA em neurônios reduziu
significativamente a concentração de D-serina (n=3; t=2,24; P=0,03) e aumentou em torno de 60% a fosforilação da SR em
resíduos de serina (n=3; t=4,07; P=0,006). Além disso, experimentos preliminares mostraram que camundongos tratados com CsA
têm a CaN inibida, o que pode regular os níveis de D-serina. Conclusão: Observamos que a atividade da calcineurina regula os
níveis de D-serina e a fosforilação da SR em cultura. Estamos investigando se isto ocorre também in vivo.
Apoio: FAPERJ, CNPQ.
D15
CAPTAÇÃO DE FOSFATO INORGÂNICO DEPENDENTE DE SÓDIO EM TRYPANOSOMA CRUZI.
1,2- DICK, C.F.; 2- DOS-SANTOS, A.L.A.; 3- MAJEROWICZ, D.; 4- KOELLER, C.M.; 4- HEISE, N.; 3- GONDIM, K.C.; 2- MEYERFERNANDES, J.R.
1- Instituto de Microbiologia Professor Paulo de Góes; 2- Laboratório de Bioquímica Celular, Instituto de Bioquímica Médica; 3Laboratório de Bioquímica e Fisiologia de Insetos, Instituto de Bioquímica Médica; 4- Instituto de Biofísica Carlos Chagas Filho,
UFRJ.
O Trypanosoma cruzi é o agente etiológico da doença de Chagas, uma doença debilitante crônica, com alta prevalência na América
Latina. Células vivas de T. cruzi, formas epimastigotas crescidas sob limitação de fosfato inorgânico (Pi) no meio de cultura são
capazes de transportar esse ânion com alta eficiência, através de um processo mediado por carreador. A dependência da
concentração de Na+ se mostrou com uma cinética de Michaelis-Menten para a captação de Pi, com valores de K0,5 aparente e
Vmáx de 2,7 ± 0,5 mM e 27,5 ± 0,9 pmol × min-1 × (107 células)-1, respectivamente. A adição do ionóforo de Na+, monensina,
reduz a acumulação de Pi, na presença de NaCl, a níveis comparáveis aqueles leveis observados na ausência de Na+. Tratamento
com valinomicina, um ionóforo de K+, e nigericina, um trocador K+-H+, inibem significativamente o transporte de Pi, resultado
consistente com o fato que os íons K+ e Li+ estimulam a captação de Pi. Dados de biologia molecular demonstraram que este
parasita expressa TcPHO89, um transportador simporter Na+:Pi de alta afinidade. Epimastigotas crescidos em meio LIT
suplementado com Pi, exibiram um influxo de Pi 46% menor quando comparado com aqueles crescidos em meio LIT com baixa
concentração de Pi, sem alterações na expressão de TcPHO89, mostrando que o “turnover” do simporter é estimulado pela baixa
concentração de Pi no meio de cultura. Além disso, durante seu ciclo de vida, T. cruzi apresenta quatro formas morfogenéticas.
Esse processo é altamente regulado e inclui mudanças na superfície desses parasitos. De fato, células crescidas em meio TAU,
meio de diferenciação de células epimastigotas para tripomastigotas metacíclicos, são ineficientes em captar Pi quando
comparados aqueles que cresceram em meio LIT. Nossos resultados sugerem a presença de um co-transportador Na+:Pi presente
em T. cruzi, contribuindo para a aquisição de Pi para o desenvolvimento das formas epimastigotas.
Apoio: MCT/CNPq, CAPES, FAPERJ (INCT/INBEB).
D16
CHARACTERIZATION OF PARAFLAGELLAR RODS IN GIARDIA DUODENALIS
Maia-Brigagão, C.1,2*, Gadelha, A.P.1,3, Rocha, G.M., de Souza, W.1,3
1 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. 2 Instituto de Bioquímica
Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. 3 Diretoria de Programas, INMETRO, Rio de Janeiro, Brazil.
Giardia duodenalis (syn. intestinalis, lamblia) is the etiological agent of giardiasis, an intestinal disease that has a widespread
distribution in the world, bringing to infected individuals symptoms like diarrhea, anemia, weight loss, among others. Giardia is a
flagellated protozoan that adheres to small intestine epithelium, and it has already been shown that flagellar movements are
important for parasite survival. Specifically, motility is required for the initiation and maintenance of giardial infection. In other
flagellates, like kinetoplastids, a structure called paraflagellar rod (PFR) is necessary for full flagellar motility and provides support
for metabolic regulators that may influence flagellar beating. G. duodenalis trophozoites present several flagella. In some of them
morphological studies have indicated the presence of not yet characterized structure associated to the flagellar axoneme,
especially in the portion of the flagella still in contact with the cell body. In the current work we used previously well characterized
antibodies associated with confocal laser scanning microscopy and transmission electron microscopy, high resolution scanning
electron microscopy and atomic force microscopy (AFM) to characterize the structure associated with the flagella of G.
duodenalis. Electron microscopy showed the presence of a continuous structure laterally located in relation to the anterior and
ventral pairs of flagella. It starts at the fagellar basis and runs up to the point where the flagella leaves the cell body and become
free. This structure was intensely labeled with antibodies recognizing PFR proteins 1 and 2 of trypanosomatids. Further
biochemical and ultrastructural studies are being performed to a better characterization of the PFR of G. duodenalis.
Support: CAPES, CNPq, FAPERJ.
D17
STRUCTURAL STUDIES WITH THE CEREBRAL DOPAMINE NEUROTROPHIC FACTOR (CDNF) AND ITS NEUROPROTECTIVE EFFECTS
AGAINST THE TOXICITY OF ALPHA-SYNUCLEIN OLIGOMERS
1Latgé, C., 1Rosa, A., 1Braga, C.A., 1Cabral, K., 2.Romão, L.,1Almeida, MS e 1Foguel, D.
1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2-Instituto de Ciências Biomédicas, Universidade
Federal do Rio de Janeiro
Parkinson's disease is characterized by loss of dopaminergic neurons in the substantia nigra of the brain. There is no available
therapy to treat this disease, which is the second most prevalent neurodegenerative disorder worldwide. Neurotrophic factors
promote survival, differentiation and maintenance of neurons in developing and adult vertebrate nervous system. A potent
neurotrophic factor for dopaminergic neurons recently described is the cerebral dopamine neurotrophic factor (CDNF). Little is
known about its structure and mechanism of action. The main goal of the present study is to solve the atomic structure and the
dynamics of CDNF in solution by NMR. We also aim to evaluate its neuroprotective effects against the cytotoxic synuclein
oligomers. Initially, CDNF (18kDa) was cloned, expressed and purified in a pure and soluble state as well as with 2H, 13C and 15N.
We observed that the purified protein undergoes spontaneous proteolysis releasing a fragment of 6kDa from its C-terminus; thus
we used a fragment of 12kDa for structural determination. We have already assigned all residues of CDNF by using triple
resonance NMR experiments and this structure will be presented. We are also investigating whether CDNF could alleviate the toxic
effects of alpha-synuclein oligomers added to SHSY-5Y cells in culture. Our data show an important protection of dopaminergic
neurons by CDNF pre-treatment. Cells previously treated with 10 µM of CDNF and then incubated with 10 µM of alpha-synuclein
oligomers (48h-old) showed a survival of approximately 80%. On the other hand, the cells treated with oligomers only presented
50% survival. Given these results, we intend to clarify the possible signaling pathways activated. Our data suggest that CDNF is a
well-folded protein with promising activity against alpha-synuclein oligomers. More studies are underway to unravel the exact
mechanism of action of CDNF, which would contribute as an alternative to treatment and elucidation of Parkinson's disease.
Support: INBEB, FAPERJ, CNPQ, CAPES.
D18
UNCOUPLING PROTEIN IN TRYPANOSOMA RANGELI AND ITS ROLE IN HYDROGEN PEROXIDE GENERATION
1,2- COSENTINO-GOMES, D., 3- FERNANDES, M.P, 1- KETZER, L.A., 1- GALINA, A., 3- VERCESI, A.E. AND 1,2- MEYER-FERNANDES, J.R
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; 2- Instituto Nacional de
Biologia Estrutural e Bioimagem; 3- Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de
Campinas, Campinas, SP, Brazil.
Uncoupling proteins (UCPs) form a subfamily within the mitochondrial carrier protein family, which catalyze a free fatty acidmediated proton recycling and can modulate the tightness of coupling between mitochondrial respiration and ATP synthesis. In
this work, respiration, oxidative phosphorylation and the mitochondrial membrane potential (Δψ) of short epimatigotes of the
protozoan parasites Trypanosoma rangeli were determined in situ in digitonin permeabilized cells. Mitochondria were able to
phosphorylate externally added ADP (100µM) in the presence of 0.05% BSA. T. rangeli mitochondria in situ generated and
sustained stable mitochondrial Δψ respiring on 5 mM succinate and presented a carboxyatractyloside-insensitive increase of Δψ
induced by the sequential additons of 500 µM GTP and 0.05% BSA. In contrast, the addition of 0.05 μM linoleic acid promoted a
significant Δψ decrease compatible with an UCP activity. This interpretation was further supported by respiration experiments and
by using antibodies raised against human UCP-1 homologue. In addition, inhibition of UCP activity by GTP significantly increased
H2O2 generation, while linoleic acid reduced this production. Together, these results suggest the existence of a mitochondrial
uncoupling protein in the Trypanosomatidae family, with a physiological role in protection against reactive oxygen species.
Support: INBEB, CAPES, CNPq, FAPESP e FAPERJ.
D19
O PAPEL DE HEME E BRADICININA NOS MECANISMOS DE INFLAMAÇÃO SUBGENGIVAL E REPARO TISSULAR EM
CAMUNDONGOS INFECTADOS POR PORPHYROMONAS GINGIVALIS
1-BARBIRATO, D.S.; 1-SCHARFSTEIN, J.; 2-BARJA-FIDALGO, T.C.
1 - Laboratório de Imunologia Molecular, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Departamento de Farmacologiae Psicobiologia, Instituto de Biologia Roberto Alcântara Gomes, Universidade Estadual do Rio de
Janeiro
Porphyromonas gingivalis, uma bactéria gram-negativa anaeróbia, é um dos principais agentes etiológicos da periodontite. Entre
outros fatores de virulência bem caracterizados, as cisteíno proteases denominadas gingipaínas R e K exercem potente atividade
pró-inflamatória ao processar cininogênios e hemoglobina, liberando respectivamente heme e bradicinina. Ao estudar o papel de
ligantes de TLR2 (LPS) e gingipaína R no mecanismo de inflamação subgengival induzido em camundongos infectados com
P.gingivalis, o nosso grupo demonstrou que (i) a liberação de cininas pela protease bacteriana é precedida pela difusão de
proteínas plasmáticas (inclusive de cininogênios) através de venulas pós-capilares (ii) o aporte de cininogênios para o espaço
extravascular por sua vez depende da ativação de neutrófilos/endotélio (pela via TLR2/CXCR2) (Monteiro et al., J.Immunol., 2009).
Além de atuar como uma enzima liberadora de cininas, a gingipaína é capaz de degradar hemoglobina, liberando heme, uma
substância extremamente tóxica e pró-inflamatória (Wagener et al., Blood, 2001; Lee et al., J. Periodontal Research, 2010). Neste
estudo, empregaremos mutantes de P.gingivalis deficientes de gingipaína R/K para determinar se heme e bradicinina podem agir
sinergicamente, intensificando o estresse oxidativo e subsequente injúria tecidual no tecido subgengival. Nossos resultados
preliminares indicam que existe produção transiente de heme com pico em 1,5h no tecido submandibular de camundongos
C57/B6 infectados localmente com Porphyromonas gingivalis. Experiências em andamento objetivam relacionar os efeitos da
liberação de heme/bradicinina pela gingipaína com variações no nível de expressão de iNOS e HO-1 nas adjacências dos sítios
microhemorrágicos provocados pela injeção de P.gingivalis no tecido submandibular. Wagener, F.A.D.T.G. et al. Blood, 2001, 98:
1802-1811; Mydel, P. et al. Plos Pathogens, 2006, 2(7): e76; Monteiro, A.C. et al. The Journal of Immunology, 2009, 183: 37003711; Lee, H.J. et al. Journal of Periodontal Research, 2010, 45: 367-374
Apoio: FAPERJ, CNPq, INBEB.
D20
HIDRAZONAS COMO INIBIDORAS DA ACETILCOLINESTERASE
1- PETRONILHO, E. C.; 2- CASTRO, N. G.; 3- PINTO, A. C.; 1- FIGUEROA-VILLAR, J. D.
1- Grupo de Química Medicinal, Departamento de Química, Instituto Militar de Engenharia; 2- Instituto de Ciências Biomédicas,
Universidade Federal do Rio de Janeiro; 3- Departamento de Química Orgânica, Centro de Tecnologia, Universidade Federal do Rio
de Janeiro.
A doença de Alzheimer (DA) é um dos principais problemas de demência e afeta cerca de 23 milhões de pessoas em todo o
mundo. Esta doença causa a redução da atividade dos neurônios colinérgicos, um problema que pode ser tratado aumentando a
quantidade do neurotransmissor acetilcolina (ACh). A melhor forma para aumentar a concentração de ACh é a inibição da enzima
acetilcolinesterase (AChE). Os inibidores da AChE utilizados para o tratamento da DA são donepezil, galantamina, tacrina e a
rivastigmina. A AChE é fortemente inibida por compostos organofosforados (OF), e pode ser reativada por oximas catiônicas como
a pralidoxima. Neste trabalho foram sintetizados novos análogos da pralidoxima (2-PAM) e testados como inibidores da AChE. Foi
dado ênfase a hidrazonas em função de resultados obtidos por modelagem molecular. A capacidade inibitória foi monitorada por
RMN e pelo método de Ellman. Os resultados obtidos pelos dois métodos confirmam que as hidrazonas catiônicas, que são uma
nova família de compostos, possuem um alto potencial como anticolinérgicos.
Apoio: INBEB, FAPERJ, CNPQ, CAPES-PRÓ DEFESA, MINISTÉRIO DA DEFESA.
D21
REGULATION OF WILSON DISEASE ATPASE (ATP7B) ACTIVITY BY CAMP-DEPENDENT PROTEIN KINASE
1,2- HILÁRIO-SOUZA, E.; 1,2- VALVERDE, R.H.F.; 1,2- BRITTO-BORGES, T.; 1,2- VIEYRA, A.; 1,2- LOWE, J.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2-Instituto Nacional de Ciência e Tecnologia
em Biologia Estrutural e Bioimagem (INBEB)
Copper homeostasis and molecular studies of the active transport of this metal are essential to understand the copper-related
diseases (Menkes and Wilson diseases). The main objective of this study was to analyze the modulation of the Cu(I)-ATPase
(Atp7b) activity in porcine liver by cyclic AMP-dependent protein kinase (PKA) and investigate the direct effects of this regulatory
phosphorylation on the catalytic cycle of Atp7b. The acyl-phosphate intermediate formation during the Atp7b catalytic cycle was
confirmed, indicating that it is a P-type ATPase and the catalytic phosphorylation was copper dependent. The Atp7b activity was
inhibited by 50% when PKA pathway was stimulated using forskolin (1 nM), cAMP (100 nM), cholera toxin (1 nM) or exogenous
PKA α -catalytic subunit (2000 U/mL). The Atp7b activity was increased by 50% when incubated with a specific PKA inhibitor (10
nM PKAi5-24 peptide). The addition of the exogenous PKA α -catalytic subunit increased the K0,5 for free copper (2.5 x 10-17 M
and 6.2 x 10-17 M in the absence and presence of PKA, respectively), although the Hilll coefficient for free copper was not changed
(2.7 ± 0.4 and 2.3 ± 0.7 in the absence and presence of PKA, respectively). The Vmax value found for the ATP curve with no
exogenous PKA was 69.1 ± 4.3 nmol Pi x mg-1 x min-1 and on the presence of PKA α -catalytic subunit, the Vmax decreased to 42.1
± 3.6 nmol Pi x mg-1 x min-1. The Km for ATP was the same when exogenous PKA was present (~ 1 mM). In conclusion, PKAmediated regulatory phosphorylation inhibited Cu(I)-ATPase activity by 50%. This phosphorylation did not change the affinity of
this enzyme for ATP and the Cu(I) ions cooperativity, but it affected the enzyme turnover because of the reduction of Cu(I)-ATPase
affinity for copper ions.
Support: FAPERJ, CNPq and CAPES.
D22
NUCLEAR MAGNETIC RESONANCE (NMR) STUDY OF A 22 kDa PROTEIN: NECROSIS AND ETHYLENE-INDUCING PROTEIN 2 (NEP2)
1- PEREIRA, E.G.; 2- DIAS, C.V.; 1- OLIVEIRA,G.A.P.; 1- DE PAULA, V.; 2- CASCARDO, J.C.M.; 1- DA SILVA, J.L.; 1-ALMEIDA, F.C.L. and
1- VALENTE, A.P.
1- Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Instituto de Bioquímica Médica, Universidade Federal do Rio de
Janeiro; 2- Departamento de Biologia, Universidade Federal de Santa Cruz
Witches' broom disease (WBD) of cacao (Theobroma cacao) is one of the most important phytopathological problems that afflict
the Southern Hemisphere in recent decades.1 In Brazil, the disease is endemic in the Amazon region, and in 1989 was introduced
into southern Bahia, the largest area of cacao production in the country.2 The gene enconding necrosis and ethylene-inducing
proteins (NEPs) are located at the same chromosome and the study of M. perniciosa genome led to identification of similar NEPs:
NEP1 and NEP2. Those proteins are involved in crucial steps in the plant disease and may be a target for drug design4. In this work,
we assigned the resonances of NEP2, a 22 kDa protein, using triple resonance strategy with a 2H, 13C and 15N sample and started
the structure calculation. We compared the NEP2 secondary structure evaluated by chemical shift index (CSI) with Nep1-like
protein (NLP) from the phytopathogenic oomycete Pythium aphanidermatum, obtained by X-ray with 1.35 Å resolution. The
analysis of CSI indicates that its secondary structure is majority in beta-sheet with two alpha-helix regions in the N- and Cterminals, in agreement with its homologous NLP deposited crystal structure. In addition, we observed that around 22 residues
that we did not found assignments are located in loop regions. We are now analysing the observed NOEs and we will pursue the
structure calculation. With the information about NEP2 structure and dynamics, we hope to understand its membrane interaction
process and therefore its pathogenesis. REFERENCES 1. Griffith G.W. and Nurnberger, T., Phytochemistry, 67, 1800 – 1807 (2003)
2. Pereira J.L., deAlmeida L.C.C., Santos S.M., Crop Protection, 15, 743 - 752 (1996) 3. Garcia O., Macedo J.A.N., Tibúrcio R.,
Zaparoli G., Rincones J., Bittencourt L.M.C., Ceita G.O., Micheli F., Gesteira A., Mariano A.C., Schiavinato M.A., Medrano F.J.,
Meinhardt L.W., Pereira G.A.G. and Cascardo J.C.M., Mycological Research III, 443 - 455 (2007) 4. Ottmann C., Luberacki B., Küfner
I., Koch W., Brunner F., Weyand M., Mattinen L., Pirhonen M., Anderluh G., Seitz H.U., Nürnberger T. and Oecking C., PNAS, 106,
10359 - 10364 (2009) Support: CAPES, CNPq.
D23
INTERAÇÃO ENTRE CÉLULAS SENTINELAS DO SISTEMA IMUNE INATO COM O PERIODONTOPATÓGENO PORPHYROMONAS
GINGIVALIS: MECANISMOS DE COOPERAÇÃO ENTRE TLR2, RECEPTORES DE ENDOTELINAS (ETRS) E RECEPTORES DE CININAS
(BKRS).
1 - RAMOS-JUNIOR E.S.; 1 - SCOVINO A.; 2 - MORANDINI A.C.F.; 1- SCHARFSTEIN, J.
1-Instituto de Biofísica Carlos Chagas Filho UFRJ ; 2- Faculdade de Odontologia de Bauru, Universidade de São Paulo.
A Periodontite, uma doença inflamatória crônica caracterizada pela destruição das estruturas de suporte dos dentes, resulta de
infecção persistente por bactérias gram-negativas anaeróbias do complexo vermelho, entre as quais a mais estudada é a
P.gingivalis. Empregando um modelo de infecção subgengival estabelecido em camundongos, o nosso grupo demonstrou que a
P.gingivalis induz (i) inflamação edematogênica e (ii) linfócitos T anti-Fimbriae produtores de IL-17 (Balb/c) e IFN-G (Balb/c e
C57BL/6) - através de mecanismos de cooperação entre dois bem caracterizados fatores de virulência bacteriana: LPS (agonista de
TLR2) e gingipaína (enzima liberadora de cininas, i.e., agonistas de BK2R). No presente estudo investigamos a relação funcional
entre TLR2 e GPCRs (BKRs e ETRs) no mecanismo de ativação de macrófagos (camundongos) e fibroblastos de gengiva (humana),
comparando o fenótipo de P.gingivalis selvagens com mutantes de gingipaína (R/K) versus mutantes de fimbria. Ensaios de
infecção realizados com a linhagem macrofágica (RAW) indicam que a infecção destes fagócitos com P.gingivalis mutantes duplos
negativos de PKR (i) resultaram em aumento de 6 vezes na infecção intracelular por P.gingivalis selvagens (ii) reduz
substancialmente os níveis de TNF-alfa secretado (14 vezes) por fagócitos PKR deficientes. Estudos adicionais realizados com
macrófagos J774 sugerem que o fenótipo pró-inflamatório de P.gingivalis (secreção de IL-1) é potencializado por ATP. Ampliando o
escopo destas investigações para o sistema humano, analisamos as respostas transcricionais de receptores de TLRs e GPCRs em
fibroblastos isolados de tecido gengival humano. Resultados preliminares (qPCR) sugerem que LPS de P.gingivalis estimula a
transcrição de ETRs nos fibroblastos humanos. Congruente com estes resultados, estudos farmacológicos em camundongos
infectados sugerem que a inflamação edematogênica induzida por P.gingivalis depende de cooperação funcional entre TLR2, BK2R
e ETRs.
Apoio: FAPERJ, CNPq e INCT-CNPq/INBEB.
D24
FIBRAS AMILÓIDES INDUZEM A LIBERAÇÃO DE REDES EXTRACELULARES DE NEUTRÓFILOS
AZEVEDO, E.P.C.1, GUIMARÃES-COSTA, A.B.2, TOREZANI, G.S.1, BRAGA, C.A.1,3, PALHANO, F.P.1, SARAIVA, E.M.2, FOGUEL, D.1
1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2-Instituto de Microbiologia Prof. Paulo de Góes,
Universidade Federal do Rio de Janeiro; 3-Pólo Xerém, Universidade Federal do Rio de Janeiro
Amiloidoses são doenças esporádicas ou transmissíveis caracterizadas pelo acúmulo de fibras amilóides em órgãos e tecidos. Estas
doenças afetam 50.000 pessoas mundialmente, todo ano. Uma potencial associação entre fibras amilóides e neutrófilos foi
sugerida quando proteases derivadas de neutrófilos, mas não células íntegras, foram encontradas associadas a depósitos
amilóides. No entanto, ainda não se sabe se o sistema imune possui um papel nas amiloidoses. Redes extracelulares de neutrófilos
(NETs) é uma consequência da ativação de um programa de morte celular no qual DNA decorado com proteínas, como a elastase e
histonas, é liberado. Utilizando uma abordagem in vitro, nós nos perguntamos se as NETs eram induzidas pelas fibras amilóides e
procuramos pelas NETs em tecidos de pacientes com amiloidose. Nós observamos que fibras amilóides eram capazes de induzir
NETs em um processo parcialmente dependente de espécies reativas de oxigênio (ROS), independentemente da proteína que
constituía as fibras. Adicionalmente, a elastase associada às NETs era capaz de digerir as fibras amilóides gerando espécies
citotóxicas. Finalmente, as NETs foram observadas em depósitos amilóides de pacientes com amiloidose sistêmica primária.
Nossos dados indicam fortemente que as NETs possuem uma relevância fisiológica nas amiloidoses e devem ser investigadas em
condições relacionadas com fibras amilóides.
Apoio: INBEB, FAPERJ, CAPES, CNPq.
D25
ACTIVATION, MITOCHONDRIAL DYSFUNCTION AND CELL DEATH IN DENGUE VIRUS INFECTED PLATELETS
1- HOTTZ, E.D.; 2- GARCIA-SOUZA, L.F.; 2- OLIVEIRA, M.F.; 3- BOZZA, F.A.; 1- BOZZA, P.T.
1- Laboratório de Imunofarmacologia, IOC, Fiocruz; 2- Instituto de Bioquímica Médica, UFRJ; 3- Instituto de Pesquisa Clínica
Evandro Chagas, Fiocruz
Dengue is the most important human arbovirus disease in the world. Infection can be asymptomatic or lead to sickness whose
intensity may vary since undifferentiated fever up to severe cases with bleeding and shock, in which thrombocytopenia is
frequently observed. Dengue virus (DENV) directly interacting with platelets may be an important mechanism of
thrombocytopenia, therefore, identification of mechanisms involved in this process plays a major role in determining markers of
severity or new therapeutic targets. This work aimed to characterize activation and mitochondrial response of platelets isolated
from patients with dengue or platelets exposed to dengue virus (DENV) in vitro. Our results showed a higher activation of platelets
isolated from dengue patients when compared to controls, principally in patients who were thrombocytopenic. Increased
zation
and phosphatidylserine exposition, both suggestive of cell death, were correlated in platelets isolated from dengue patients, and
higher levels of caspase-9 and caspas-3 were observed in platelets from patients than in platelets from health volunteers.
Furthermore, the phenotype observed in platelets isolated from patients with dengue, was reproduced by exposition of platelets
to DENV-2 in vitro, but not to heat inactivated DENV-2. Our results show that DENV interacting directly with human platelets
induces platelet activation, mitochondrial dysfunction and cell death, which may be involved with dengue-associated
thrombocytopenia.
Support: FAPERJ, CNPq, PRONEX Dengue.
D26
ESTUDOS DE INTERAÇÃO ENTRE A PROTEÍNA FKBP12 DE TRYPANOSOMA BRUCEI E COMPOSTOS ORGÂNICOS POR RMN
D’ANDRÉA, É.D.¹, AIDO-MACHADO, R.¹, PIOVESAN, L.A.², FLORES, A.F.C.², BARREIRO, E.J.³ E PIRES, J.R.M.¹
¹Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Instituto de Bioquímica Médica, Universidade Federal do Rio de
Janeiro, ²Núcleo de Química de Heterociclos, Universidade Federal de Santa Maria, ³Laboratório de Avaliação e Síntese de
Substâncias Bioativas, Universidade Federal do Rio de Janeiro
A proteína FKBP12 é uma peptidil-prolil-cistrans-isomerase, alvo clássico de fármacos imunossupressores como FK506 e
rapamicina em humanos. Em Trypanosoma brucei, estudos de interferência de RNA demonstraram que a existência de um gene
codificador de uma proteína homóloga à FKBP12 humana é essencial para o desenvolvimento deste parasita. Estes resultados
sugerem que fármacos análogos ao FK506 e à rapamicina poderiam ter um efeito anti-tripanossomatídeos. O entendimento da
função de macromoléculas requer o conhecimento de suas estruturas tridimensionais. Um dos métodos experimentais capazes de
fornecer uma descrição completa da estrutura de macromoléculas a nível atômico é a espectroscopia de ressonância magnética
nuclear (RMN). As análises por RMN são feitas em solução, possibilitando a investigação de interações entre macromoléculas e
pequenas moléculas orgânicas. Empregando-se a metodologia denominada relação estrutura atividade por ressonância magnética
nuclear (“SAR by NMR”), pequenas moléculas orgânicas que se ligam com baixa afinidade a sítios vizinhos de uma proteína são
identificados, otimizados e conectados para produzir ligantes de mais alta afinidade. Neste contexto, o objetivo do presente
trabalho é estudar interações entre a FKBP12 de Trypanosoma brucei e compostos orgânicos por RMN. Como resultados, foram
realizadas análises da FKBP12 com compostos provenientes de colaborações com os grupos NUQUIMHE-UFSM e LASSBio-UFRJ. A
análise dos resultados mostrou que os compostos interagiram de forma fraca com a FKBP12. Foi realizado o mapeamento destas
interações, mostrando que elas se realizam no sítio de ligação ao FK506. Foi também realizado o estudo da interação
FKBP12/FK506 e a dinâmica deste complexo, mostrando que este fármaco é um ligante forte. O mapeamento mostrou tanto
interações no sítio de ligação ao FK506, quanto em outras regiões da FKBP12.
Apoio: CNPq.
D27
THE EFFECTS OF CHRONIC TREATMENT WITH 5-PHOSPHODIESTERASE INHIBITORS ON THE PROSTATE OF MICE
Gomes, F.O.S.1; Carvalho, M.C.1; Silva, B.S.1; Rocha, S.W.S.1; Silva, A.K.S.-1; Ribeiro, E.L.-1; Barbosa, K.P.S.-1; Rodrigues, G.B-1;
Peixoto, C.A-1,2
1. Centro de Pesquisas Aggeu Magalhães- CpqAM/FIOCRUZ, Brazil
Background: Sildenafil is a selective inhibitor of phosphodiesterase-5 (PDE-5) used extensively for the treatment of erectile
dysfunction. Recent studies have shown that daily use with PDE-5 inhibitors improves symptoms of BPH and LUTS possibly as a
result of its relaxing action of NO via mechanisms inhibiting the proliferation of prostate stromal cells. Objective: Characterize the
action of sildenafil at a concentration of 25 mg / kg on the physiology of prostate of male C57BL/6 mice. Methodology: 20 male
C57BL/6 mice were separated in groups: control group (n=10) and treated group sildenafil 25mg/kg (n=10). Sildenafil solutions
were administered in dose 25mg/kg in drink bottle for two months. At the end of the treatment regimen, animals are euthanized
in CO2 chamber. Prostate fragments were processed for light microscopy, western blot and immunohistochemical assays. Results:
Prostate sections from control group presented normal prostate architecture, composed of cubic epithelial cells with basal nucleus
and dense stroma, with a predominance of fibromuscular tissue. Histological analyses of the Sildenafil group showed similar
regions showing small papillary invaginations, as well as changes in the epithelium ranging stratified and simple cubic, slightly
denser stroma and increase of secretion in lumen. Immunohistochemical analyses of the control group did not show substantial
PKA, PKG and sGC immunopositivity. This same result was evident in the group treated with Sildenafil in dose 25mg/kg. Western
blot analyses for guanylate cyclase (sGC) showed a no significant decrease expression in the treated group compared with the
control group. Quantification staining analyses were performed using the software Graph Pad Prism v.05. Conclusions: According
to our results Sildenafil caused no significant changes in concentration of 25mg/kg. Further assays are in development in our
laboratory in order to clarify the role of Sildenafil on the molecular mechanisms of the mice prostate. Keywords: Sildenafil,
phosphodiesterase-5, prostate.
Support: INBEB, FACEPE.
D28
ANGIOTENSINA II VIA MEMBRANA LUMINAL INDUZ A FORMAÇÃO DE HETERODÍMEROS AT1/AT2 PARA ESTIMULAR A
ATIVIDADE DA SERCA VIA PLC/PKC EM CÉLULAS LLC-PK1
1,3- FERRÃO, F. M.; 1,3- AXELBAND, F.; 1,3- DIAS, J.; 2,3- LARA, L.S.; 1,3- VIEYRA, A.; 1,3- LOWE, J.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brasil; 2- Instituto de
Ciências Biomédicas, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Jeneiro, Brasil; 3- Instituto Nacional de Ciência e
Tecnologia de Biologia Estrutural e Bioimagem (INBEB), Universidade Federal do Rio de Janeiro.
Angiotensina II (Ang II), que desencadeia vias de sinalização intracelulares dependentes de Ca2+, é encontrada em altas
concentrações no túbulo renal. Portanto, essa Ang II luminal pode ter efeitos na homeostasia do Ca2+ celular. O objetivo desse
trabalho é elucidar os mecanismos moleculares envolvidos nesse efeito. Células LLC-PK1 foram cultivadas em meio DMEM com
10% SFB a 37º C com 5% CO2. 5 x 105 células foram depositadas em garrafas de cultura de 25 cm2. Depois de atingir 90% de
confluência, as células foram tratadas com diferentes condições em meio DMEM sem soro, raspadas e lisadas em solução
contendo 1 mM EDTA, 20 mM Hepes-Tris pH 7,0, 250 mM Sacarose e 0,15 mg/mL de inibidor de tripisina. A concentração de
proteína foi medida como descrito em J. Biol. Chem. 193: 265, 1951. A atividade Ca2+-ATPásica foi medida com o método
colorimétrico e calculada pela diferença na ausência e presença de 2 mM EGTA (J. Biol. Chem. 202: 675, 1953). Com isso, foi
observado que a Ang II em baixas concentrações estimula a atividade Ca2+-ATPásica total em 65% (10-10 M) e não modula essa
atividade em altas concentrações (10-6 M). O efeito da Ang II na atividade da SERCA e PMCA demonstrou que a SERCA é a ATPase
alvo para a Ang II luminal regular a homeostase de Ca2+ em células renais, com um estímulo rápido (30 s) e persistente (30 min). A
ativação da SERCA promovida pela Ang II via membrana luminal foi bloqueada pelos antagonistas dos receptores de Ang II AT1 e
AT2, 10-10 M losartan e 10-7 M PD123319, e a imunoprecipitação seguida de Western blotting confirmou a formação de
heterodímeros AT1/AT2, induzida por Ang II. O inibidor da PLC (2 x 10-6 M U73122) e o inibidor da PKC (5 x 10-8 M calfostina C )
bloquearam a ativação da SERCA, e o tratamento com forbol éster [forbol 12-miristato 13-acetato (PMA)], que estimula a
atividade da PKC, mimetizou o efeito da Ang II via membrana luminal na atividade da SERCA. Esses resultados indicam que a Ang II
via membrana luminal induz a formação de heterodímeros AT1/AT2 para ativar a via PLC/PKC, aumentando a atividade da SERCA e
regulando assim a homeostasia de cálcio nas células renais.
Apoio: INBEB, CNPq, FAPERJ.
D29
STRUCTURAL ORGANIZATION OF THE CUTICLE OF THE NEMATODE Hassalstrongylus epsilon BY SEM OF FRACTURED SPECIMENS
ADNET, F.A.O.; ATTIAS, M.; DE SOUZA, W.
IBCCF
Hassalstrongylus epsilon é um nematóide parasito do intestino delgado do roedor Nectomys squamipes (dato d’água) que ocorre
na Mata Atlântica. H. épsilon pertence a superfamília Trichostrongyloidea, a qual incluiu parasitos gastrointestinais de rumintes de
interesse veterinário, tais como: Haemonchus e Trichostrongylus. Os membros desta superfamília têm como característica uma
bolsa copuladora desenvolvida e cristas cuticulares longitudinais ao longo do corpo que auxiliam na fixação ao trado digestivo
além de ser um importante caráter taxonômico. A proposta deste trabalho foi observar componentes ultra-estruturais da cutícula
de H. epsilon por microscopia eletrônica de varredura (MEV). Os parasitos foram coletados do intestino delgado de N. squamipes,
capturados no município de Sumidouro-RJ. Depois de recuperados do intestino os mesmos foram lavados em solução fisiológica
(NaCl à 0,9% em água destilada) e fixados em AFA (Etanol 70%, Formalina 37% e Ácido acético glacial) a 60ºC. A identificação
morfológica foi feita no microscópio de luz Zeiss Standard 20. Para MEV os nematóides foram processados segundo Adnet et al.,
2009. Contudo para realização da fratura dois suportes metálicos, já com as amostra montadas, forram levemente pressionados
face-a-face e depois puxados. Em seguida metalizados com ouro e observados ao microscópio eletrônico de varredura Quanta
250. A parede do corpo de H. episilon consiste de: cutícula, hipoderme e camada muscular. A cutícula apresenta um conjunto de
cristas cuticulares em sua superfície (Fig. 1). A fratura revelou a face interna da camada cuticular mostrando que as cristas
cuticulares são compostas internamente por estruturas de suporte, que não são contínuas, mas sim apresentam um padrão
repetitivo regular por sobre a crista e são interconectadas por estruturas fibrosas, que tanto as ligam uma nas outras como a
camada fibrosa. O uso de MEV associado a técnicas de fratura tem se mostrado uma valiosa ferramenta para elucidar a ultraestrutura desde grupo de helmintos.
Apoio: FAPERJ, CNPQ-PROTAX, CAPES.
D30
AVALIAÇÃO DA EFICÁCIA DO CONDICIONAMENTO DE SUPERFÍCIES COM NISINA E MAGNETOSSOMOS COMO ESTRATÉGIA PARA
A INIBIÇÃO DO DESENVOLVIMENTO DE BIOFILMES
1- ALMEIDA, F.P.; 2- GUTARRA, M.L.E.; 1- LINS, U.G.C.
1- Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro; 2- Departamento de Engenharia
Bioquímica, Escola de Química, Universidade Federal do Rio de Janeiro.
Biofilmes são associações de microrganismos aderidos a superfícies bióticas ou abióticas, envoltos em uma matriz de substâncias
poliméricas extracelulares. Os biofilmes são responsáveis por perdas econômicas em diferentes processos industriais e também
consistem em um problema de saúde pública devido a infecções causadas pelo crescimento de biofilmes em dispositivos médicos,
geralmente resistentes a antibióticos. A obtenção de materiais resistentes ao desenvolvimento de biofilmes é de grande
importância biotecnológica e duas estratégias adotadas nesse sentido consistem no condicionamento de superfícies com
compostos orgânicos biocidas ou com nanopartículas metálicas. A nisina, um peptídeo antimicrobiano produzido por Lactococcus
lactis, apresenta atividade biocida sobre bactérias Gram-positivas quando usado isoladamente e contra bactérias Gram-negativas
quando usado concomitantemente com substâncias capazes de desestabilizar a membrana externa bacteriana, como o ácido
etilenodiamino tetra-acético (EDTA). Magnetossomos são nanopartículas magnéticas de oxido e/ou sulfeto de ferro, envoltas em
membranas biológicas, produzidas por bactérias magnetotáticas. Magnetossomos purificados apresentam maior solubilidade
quando comparados a nanopartículas magnéticas sintéticas, devido à presença da membrana biológica, podendo ainda ser
manipulados com a aplicação de campos magnéticos. Já foi demonstrado que partículas de magnetita sintética envoltas em ácido
poli (γ-glutâmico) apresentam atividade biocida em solução. No presente trabalho, a eficácia do condicionamento de superfícies
de vidro com nisina/EDTA e magnetossomos foi avaliada através de diferentes técnicas de microscopia e da aplicação de softwares
para processamento e análise de imagens que possibilitaram o estudo qualitativo e quantitativo de biofilmes in situ. Os resultados
da quantificação de parâmetros como biovolume total, volume interno vazio e superfície externa vazia de biofilmes, obtida a partir
de séries de microscopia confocal de varredura a laser, demonstraram redução do crescimento de biofilmes tanto sobre as
superfícies condicionadas com nisina/EDTA, quanto sobre superfícies condicionadas com magnetossomos em comparação com o
controle (biofilmes crescidos sobre superfícies não condicionadas).
Apoio: INBEB, FAPERJ, CNPq, PETROBRAS.
D31
PURIFICATION AND PARTIAL CHARACTERIZATION OF A SULFATED POLYSACCHARIDE FROM SPERM OF SEA URCHIN LYTECHINUS
VARIEGATUS
1,2 - VALLE, G. M.; 1,2 - CINELLI, L. P.; 1,2 - MOURÃO, P. A. S.
1-Laboratório de Tecido Conjuntivo, Programa de Glicobiologia, HUCFF; 2-Instituto de Bioquímica Médica, UFRJ.
Fertilization is the result of a series of interactions between molecules located on egg´s and sperm´s surface. Each species of sea
urchin female synthesize a distinct type of sulfated fucan, located in egg jelly coat, which is responsible for species-specific
induction of the acrosome reaction in spermatozoa. The absence of concrete data about the sperm receptors for these ligands
motivated us to investigate if sulfated polysaccharide from male gametes could be involved in the acrosome reaction by
carbohydrate-carbohydrate interaction. In this study, we report for the first time the occurrence of sulfated polysialic acid in the
sperm of Lytechinus variegatus. The crude polysaccharides were applied to an ion exchange column and the fraction containing
sulfated polysaccharides eluted from the column with ~ 1.0 M NaCl. This fraction was resistant to enzymatic treatments and
revealed two sialic acid positive bands after hydrolysis followed by thin layer chromatography (TLC). Besides, the sulfated
polysaccharide interacted with a column containing EJC´s sulfated fucan extracted to the same species and eluted from the
column with ~ 0.5 M NaCl. Additional information regarding the characterization of these molecules and the existence and
involvement of carbohydrate-carbohydrate interactions in the sea urchin sperm´s acrosome reaction induced by EJC´s sulfated
polysaccharide, are being investigated.
Support: FAPERJ, CAPES, CNPQ.
D32
INSIGHTS ON THE INTERACTION OF SUGARCANE SMALL HEAT SHOCK PROTEINS WITH SUBSTRATES
1, PINHEIRO, G.M.S.; 1, TIROLI-CEPEDA, A.O.; 1, RAMOS, C.H.I
1- Instituto de Quimica - Unicamp
The small heat shock protein (sHsp) constitutes an important chaperone family which is linked to conformational diseases and
appears to have therapeutic properties. The diversity of small Hsps in plants is intriguing and characterization of their chaperone
activity is important to understand plant tolerance to heat stress. SHsps prevent protein aggregation by acting as thermosensors
and therefore enhancing cell stress tolerance. SsHsp17.2 and SsHsp17.9 are the most highly expressed class I sHsps in sugarcane.
Both proteins are dodecamers at room temperature and dissociated at high temperature, they are able to protect a wide range of
substrates from thermal aggregation, but have distinct substrate specificities. To gain further knowledge on the mechanism by
which class I sHsps protect client proteins from aggregation, we investigated the chaperone activity of these proteins in two ways:
isolation and identification of a stable complex formed between sHsp and substrate using size exclusion chromatography and cross
linking and partial recuperation of the activity of a model substrate in concert with Hsp70. Also, site-directed mutagenesis
experiments are underway in order to determine the region in sHsps involved with substrate interaction.
Support: INBEB.
D33
BONE MARROW TRANSPLANTATION: WHAT IS THE BEST DELIVERY SITE FOR ENGRAFTMENT IN MICE?
1- GUILHERME VISCONDE BRASIL; 1- CAMILA IANSEN IRION; 1- KARINA DUTRA ASENSI; 1- GABRIELLEN VITIELLO1; 1- DANIEL
BARUSCO DURAN;1- LUIZA LAPOLLA PERRUSO; 1-BRUNO DIAZ PAREDES; 1-TAÍS HANAE KASAI BRUNSWICK; 1- BRUNO LEONARDO
BARRANCO ESPORCATTE; 3- PAULO CÉSAR VENTURA CANARY; 1- ADRIANA BASTOS CARVALHO; 1- REGINA COELI DOS SANTOS
GOLDENBERG; 1,2- ANTONIO CARLOS CAMPOS DE CARVALHO
1- Laboratório de Cardiologia Celular e Molecular - Instituto de Biofísica Carlos Chagas Filho - Universidade Federal do Rio de
Janeiro (UFRJ); 2- Instituto Nacional de Cardiologia de Laranjeiras (INC); 3- Serviço de Radioterapia do Hospital Universitário
Clementino Fraga Filho (HUCFF) - Universidade Federal do Rio de Janeiro (UFRJ)
Different routes are used to deliver stem cells in pre-clinical studies. The spleen and the tail vein routes have been used for
delivering stem cells to repopulate the bone marrow; however the cells might be retained in the inoculum site. Therefore, other
delivery sites are being tested for bone marrow transplantation (BMT) in mice. In this study we compare four different routes: the
retro-orbital venous sinus, the left ventricle cavity, the jugular vein and the peritoneal cavity. Two months old C57BL/6 mice were
submitted to whole-body irradiation with 7Gy for bone marrow ablation. All mice received 1x106 bone marrow mononuclear cells
(BMMCs) harvested from transgenic mice expressing the green fluorescent protein gene (GFP). Twenty five animals received the
BMMCs through intraventricular injection guided by echocardiogram (group A), twenty five animals received the BMMCs by
injection into retro-orbital venous sinus (group B), eighteen mice received the BMMCs by injection into jugular vein (group C) and
four animals received the BMMCs by intraperitoneal injection (group D). Three weeks after the BMMCs delivery blood flow
cytometry was performed to evaluate the engraftment. Four animals from group A and three animals from group B died during
BMT and all animals from group D died 15 days after BMT. Peripheral blood flow cytometry revealed that cell transplantation was
effective in generating mice with 81.89 ± 1.97 % in group A, 78.51 ± 3.55 % in group B and 88.11 ± 3.91% in group C of GFP-positive
cells (MEAN ± SEM). No statistical difference was observed (p= 0.119). The retro-orbital venous sinus, the left ventricle cavity and
the jugular vein were equally effective to repopulate the bone marrow of myeloablated mice showing low mortality and
percentage of repopulation higher than 75%. In contrast, the peritoneal route was inefficient for bone marrow repopulation
causing 100% of mortality.
CNPq, CAPES, FAPERJ, Ministério da Saúde
D34
ANGIOTENSIN II: A NEW COMPONENT INVOLVED ON T LYMPHOCYTE –INDUCED MALARIA IMMUNOPATHOLOGY
1- SILVA-FILHO, J.L., 3- MORROT, A., 2- COSTA, M.F.S, 2- SOUZA, M.C., 2- HENRIQUES, M.G., 3- SAVINO, W., 1,5- CARUSO-NEVES,
C., 1,4- PINHEIRO, A.A.S.
1- Instituto de Biofísica Carlos Chagas Filho, IBCCF; 2- Instituto de Tecnologia em Fármacos, FIOCRUZ; 3- Departamento de
Imunologia, FIOCRUZ; 4- Institutos Nacionais de Ciência e Tecnologia, INCT/INPeTAm/MCT; 5- Institutos Nacionais de Ciência e
Tecnologia, INCT/INBEB/MCT
Malaria is one of the most serious infectious diseases in humans. The murine model Plasmodium berghei ANKA has provided
valuable contributions to the understanding of disease pathogenesis. Here, we verify the in vivo role of angiotensin II (Ang II) in
activated T lymphocytes as well as its contribution to malaria pathogenesis. C57BL/6 mice infected with P. berghei ANKA were
divided into groups treated with vehicle, losartan or captopril and euthanized at day 6 post infection for isolation of splenic T
lymphocytes. In vivo or ex vivo, losartan or captopril reduced the migration of T cells by 90% and 78%, respectively. Losartan or
captopril reduced the frequency of activated endothelial cells with adhered T cells by 43% and 51%, respectively. Both treatments
reduced rearrangement of actin cytoskeleton by contact with extracellular matrix proteins. FACS analysis of cells showed that Ang
II induced up-regulation of chemokine receptors such as CCR2 and CCR5. Treatment with these drugs reduced the percentage of
activated T cells by 50%. Effector memory T cells enhanced during infection but were reduced in treated mice and central memory
T cells showed the same profile. Foxp3+ T cells were reduced to control levels in treated mice. These data were supported by a
similar decrease in the frequency of IFN-γ-, IL-17- and IL-10-producing CD4+ T cells. Immunoblotting analysis revealed a 4.3- and
3.3-fold increase in the expression levels of AT1 and AT(1-7) receptors, respectively, in activated cells and in vivo losartan
treatment was able to suppress it. AT2 receptor levels were not modified. Moreover, serum TNF-α and IFN-γ was reduced by
losartan only. These results indicate, for the first time, a role of Ang II, through the AT1 receptor, in regulating T-cell functions. The
blockade of Ang II signaling can provides a strategy for adjunctive therapy to improve treatment outcomes of malaria disease.
Support: INBEB, INPeTAm, FAPERJ, CAPES, and CNPq.
D35
GLUCANTIME ON LEISHMANIA AMAZONENSIS: NEW CELLULAR APPROACHES FOR AN OLD DRUG-TREATMENT IN VITRO AND IN
VIVO
1,2- GODINHO, J.L.P.; 1,2- ATTIAS, M; 1,2,3- DE SOUZA, W., 1,2,3,4- RODRIGUES, J.C.F. 1,2,4
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro. 2- Instituto Nacional de Ciência e Tecnologia
em Biologia Estrutural e Bioimagem. 3- Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Brasil. 4- Pólo
Avançado de Xerém, Universidade Federal do Rio de Janeiro.
Leishmaniasis is one of the most important neglected tropical diseases caused by parasites of the Leishmania genus. In
Brazil, Leishmania amazonensis is responsible for cutaneous and diffuse cutaneous leishmaniasis. The chemotherapy currently
used in Brazil and other Latin American countries is based essentially on Pentavalent Antimonials such as Glucantime® (meglumine
antimoniate). Despite being used for a long time, the cellular targets of Glucantime are poorly explored and unknown. In this
work, we decide to investigate the ultrastructure of Leishmania amazonensis intracellular amastigotes after treatment in vitro and
in vivo with different doses of Glucantime®. Macrophage-infected cultures and infected-mice were treated for 72h and 21 days,
respectively, and them fixed and processed for transmission and scanning electron microscopy. The samples were observed under
a Zeiss 900 TEM and FEI-SEM Quanta 250. The ultrastructure of amastigotes after treatment with Glucantime in vitro at the
concentrations of 50, 100 and 150 mg/ml and in vivo at the doses of 50 mg/kg/day, was significantly altered. Treated-parasites
presented an intense cytoplasm vacuolization, increase in the number of lipid bodies, and alterations in the mitochondrion and
megasomes. An abundant number of glicosomes were also observed after treatment with Glucantime, which could be associated
with alterations on the glucose metabolism and fatty acid oxidation. Furthermore, presence of autophagosome-like structures,
concentric membranes in the cytoplasm, and phenotypes of apoptosis cell death were also found in treated-amastigotes. Taken
together, these results indicate that electron microscopy is an interesting tool to follow the cellular effects during treatment of
different models of Leishmania-infection with Glucantime and other molecules.
Support: INBEB, FAPERJ, CAPES, CNPq.
D36
TRANSPLANTATION OF HUMAN PLACENTA-DERIVED MESENCHYMAL STROMAL CELLS IMPROVES CARDIAC PERFORMANCE OF
IMMUNOCOMPETENT MICE SUBMITTED TO MYOCARDIAL INFARCTION.
1- PASSIPIERI, J.A.; 1- SUHETT, G.; 1- BRASIL, G.V.; 1- MELLO, D.B.; 1- KASAI-BRUNSWICK, T.H.; 1- MARTINS, A.B.; 1- RODRIGUES,
D.C.; 1- RAMOS, I.; 2- ROCHA, N.N.; 1- CARVALHO, A.B.; 1- GOLDENBERG, R.C.S; 1,3- CAMPOS DE CARVALHO, A.A.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Universidade Federal Fluminense; 3Instituto Nacional de Cardiologia
Introduction and Objectives: Death of cardiomyocytes due to myocardial infarction (MI) causes wall thinning and ventricular
dilatation, which diminishes the ability of the heart to pump blood, leading to heart failure. This study aims to evaluate the role of
placenta-derived mesenchymal stromal cells (pMSC) in the treatment of cardiac failure, as well as their survival after xenogenic
transplantation in mice. Methods and Results: pMSC were obtained from human placenta. They were characterized as plasticadherent, multipotent cells and they did not express hematopoietic or endothelial cells markers, but expressed bone marrow
derived MSC-associated molecules. pMSC also expressed pluripotency markers OCT-4, DMNT3b and KLF4, expressed in embryonic
stem cells, suggesting that their plasticity is superior to that of adult stem cells. Mice were submitted to MI. Two weeks after MI,
no statistical difference was observed in the ejection fraction (EF) of pMSC and placebo-treated groups. Mice were submitted to
weekly intramyocardial injections, guided by echocardiography, of 105 pMSC or placebo during 3 weeks. Functional parameters
were evaluated weekly by echocardiogram. Comparing the pMSC group (n=7) and placebo group (n=7), we observed significant
improvement of EF (37.8±9.8vs24.8±8.1, respectively; P<0.05) and end systolic volume (54.8±19.5vs77.2±15.9, respectively;
P<0.05) 21 days after the beginning of the treatment. No statistical difference was observed in the end diastolic volume of pMSC
and placebo-treated groups (70.2±17.1vs84.2±21.7, respectively). pMSC, stably transduced with a viral construct expressing
luciferase, were used in a bioluminescence assay. Cells were detected only at the injection site, suggesting that the injection
method is accurate. pMSC remained at injection site for 3 days after the first injection, but the survival period was reduced after
the 2nd and 3rd injection, suggesting immune rejection. Conclusions: pMSC transplantation might contribute to improvement in
cardiac performance after MI despite the reduced survival of the cells in the xenogenic setting. CNPq, FAPERJ, FINEP, Capes,
Ministério de Saúde
D37
PRODUÇÃO DE NANOPARTÍCULAS MAGNÉTICAS DE ORIGEM BACTERIANA PARA APLICAÇÕES EM BIOTECNOLOGIA
1- SILVA, K.T.; 2- GUTARRA, M.; 1- LEÃO, P.; 1- ABREU, F.; 3- BAZYLINSKI, D.A.; 2- FREIRE, D.M.G.; 1- LINS, U.
1- Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro 2- Instituto de Química, Universidade
Federal do Rio de Janeiro. 3- School of Life Sciences, University of Nevada at Las Vegas, USA.
Magnetossomos são nanocristais magnéticos produzidos por bactérias com potencial para aplicações biotecnológicas
apresentando diversas vantagens comparadas à nanopartículas artificiais, entre elas regularidade no tamanho, forma específica e
uma membrana biológica externa que pode ser funcionalizada. Nesse trabalho caracterizamos a produção de magnetossomos do
‘Candidatus Magnetovibrio blakemorei’ em meios de cultura com diferentes fontes de carbono e ferro através da microscopia
eletrônica de transmissão (MET). O MET é uma ferramenta interessante para a observação e análise das características dos
magnetossomos formados, pois pode avaliar diretamente as propriedades relacionadas à qualidade das nanopartículas como
forma, tamanho e volume. E ainda permite avaliar a quantidade de magnetosomos por célula, o que fornece uma informação
direta da produtividade da cultura. Os meios contendo succinato/acetato e succinato isoladamente foram os únicos que
promoveram o crescimento celular satisfatório. O aumento do ferro no meio aumenta o crescimento enquanto diferentes fontes
de ferro influenciam na produção de magnetossomos e no volume e forma. O quinato férrico levou a produção de
magnetossomos com um maior volume e menor variação no fator de forma. O meio SucQuin apresentou a melhor produtividade
e produção de magnetossomos, chegando a 3,94x1010 magnetossomos/ml em 73h, além de magnetossomos com volume maior e
formato mais uniforme. Esses magnetossomos têm qualidades que superam as nanopartículas artificiais e os tornam vantajosos
para aplicações biomédicas.
Apoio: INBEB, CNPq, CAPES, FAPERJ.
D38
ANTIINFLAMMATORY EFFECT OF THIAZOLIDINEDIONE DERIVATIVE RA-4 IN A MOUSE MODEL PLEURISY
1- BARBOSA,K.P.S.; 1- ROCHA, S.W.S.; 1- OLIVEIRA, F.G.; 1- RIBEIRO, E.L; 1- DONATO, M.; 3- SILVA, T.G; 3- LIMA, M.C.A.; 3- PITTA,
I.R.; GALDINO, S.L.; 2- PEIXOTO, C.A.
1- Centro de Pesquisas Aggeu Magalhães- CPqAM/FIOCRUZ; 2- Centro de Tecnologias Estratégicas do Nordeste- CETENE/MCT,
Universidade Federal de Pernambuco; 3- Departamento de Antibióticos - Universidade Federal de Pernambuco
Background: Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptors family. Recently,
there has been a great deal of interest in the involvement of PPARs in inflammatory processes. Objective: This study investigated
the anti-inflammatory effects of the new thiazolidinedione derivative in pleurisy model. Methodology: 30 male S. webster mice
were separated in groups: Saline group (n=10), carrageenan group (n=10) and CAR + RAanaesthetized and submitted to a injetion at the level of the left sixth intercostal space. Saline (0.1ml) or saline containing 1%
carrageenan (0.1ml) was injected into the pleural cavity. At 4 h after the injection of carrageenan, the animals were sacrificed
under CO2 vapors. Lung fragments were processed for light microscopy and immunohistochemical assays. Results: As expected,
no histological changes were observed in the lungs from the saline-treated animals. Immunohistochemical analyses did not show
substantial COX-2 immunopositivity. Histological analysis of the CAR group showed increased cellularity, emphysema. The CAR
group presented intense staining for cyclooxygenase (COX-2) compared with control mice. Increased COX-2 expression is
associated with increased levels of downstream enzymes required for prostanoid synthesis. The treatment with RA-4 before CAR
administration substantially reduced COX-2 expression, inflammatory cells infiltrates and pulmonary injury. The quantification
staining analyses were performed using the image program Gimp 2.6 software. Conclusions: According to present results RA-4 can
be considered as a promissory anti-inflammatory drug for lung acute inflammation. Therefore, assays are in development in our
laboratory in order to clarify the role of RA-4 on the molecular mechanisms involved in the inflammation.
Support: INBEB, FACEPE.
D39
SOLUBLE AMYLOID-Β PEPTIDE ACCUMULATION, INTRACELLULAR TRAFFICKING AND SIGNALING PATHWAY.
1,4- OLIVEIRA, L.T.; 1- MATOS, P.A.; 2- PROVANCE,D.W.; 3- MELLO, F.G.; 1- SORENSON, M.M.; 4- SALERNO,V.P.
1- Instituto de Bioquímica Médica - Universidade Federal do Rio de Janeiro, 2- Fundação Oswaldo Cruz, 3-Instituto de Biofísica
Carlos Chagas Filho - Universidade Federal do Rio de Janeiro, 4- Departamento de Biociências da Atividade Física – Escola da
Educação Física e Desportos - Universidade Federal do Rio de Janeiro.
Alzheimer’s disease (AD) is the most common neurodegenerative disorder afflicting the aging human population. It has been
recognized that β-amyloid, an important hallmark in AD, accumulates within neurons causing disruption of synapses and cognitive
function. Here we report the internalization of a fluorescently labeled β-amyloid peptide into cultured neurons, and we show that
the pattern of β-amyloid distribution during the time course of incubation is reminiscent of the endocytic pathway. The
internalization process is specific for the Aβ isoform that contains 42 amino acids, and exhibits characteristics of a cyclic process
without a specific point of saturation. Internalization of Aβ42 appears to be regulated by the Aβ40/Aβ42 balance: in the presence
of 1:1 Aβ40/Aβ42 concentration the Aβ42 internalization was not affected, but at an increased concentration of Aβ40,
internalization was reduced. Myosin Vb is an actin-based molecular motor that contributes to movement of various organelles by
its interactions with actin filaments and generation of force through ATP hydrolysis. The distribution of the internalized β-amyloid
peptide converges with that of myosin Vb and both relocalize from axons to cell bodies. Rab proteins are known as myosin-binding
partners, are essential for specific transport functions, and can be used in combination with myosin Vb as tools to assess
involvement of the endosomal pathway during intracellular trafficking of Aβ42 peptide. Rab 8 and Rab 10 also co-localized with
internalized β-amyloid after 30 min, suggesting that the internalized peptide was traveling along a characteristic recycling pathway
mediated by myosin Vb and those GTPases. These observations are consistent with the hypothesis that Alzheimer’s disease
proceeds as a result of an imbalance between β-amyloid production and β-amyloid clearance, suggesting a role for myosin Vb and
its binding partners in this process.
Support: CNPq, CAPES, FAPERJ, INCTBEB.
D40
SITE-DIRECTED MUTAGENESIS OF MYOGLOBIN HELPS TO UNDERSTAND THE EFFECT OF HYDROPHOBIC RESIDUES TO PROTEIN
STABILITY AND FOLDING
ZANPHORLIN, L.M; RAMOS, C.H.I.
1- Departamento de Química Orgânica, Instituto de Química, UNICAMP
Site-Directed Mutagenesis of Myoglobin Helps to Understand the Effect of Hydrophobic Residues to Protein Stability and Folding
Zanphorlin, L.M.; Ramos, C.H.I. Departamento de Química Orgânica, Instituto de Química, UNICAMP, SP, Brazil. Myoglobin has
had its function, structure and folding pathway extensively studied and is considered a model protein to study putative
intermediates formed in the protein folding. The existence of intermediates is considered an important step to reach its native
state and it has been the subject of intense investigation. In this work, we have analyzed the effect of three specific hydrophobic
mutations (A15G/A74G, A127F, V10F/M131F) in the stability of both native and intermediate states of myoglobin. The proposed
mutants were designed aiming to modify the geometry of the hydrophobic interactions within the myoglobin structure and it
intends to shed light in the role played by nonspecific hydrophobic collapse in the stability of apo and holo forms of the
myoglobin. The effect of these mutations was assessed by chemical unfolding probing secondary and tertiary structures by circular
dichroism and fluorescence spectroscopy, respectively. As expected, all mutants displayed CD spectra very similar to that of wildtype protein; however, they presented higher stability. Preliminary results showed the mutants in folded regions in the
intermediate appear also to affect the stability of the native state. These results support a model in which folded regions in the
intermediate have native-like folding. Keywords: apomyoglobin, folding, protein stability.
Supported by: FAPESP, CNPq and CAPES,
INBEB.
D41
MESENCHYMAL STEM CELL THERAPY IN A MODEL OF OPTIC NERVE CRUSH
1-MESENTIER-LOURO, LA; 1-ZAVERUCHA-DO-VALLE, C; 1-SILVA-JUNIOR, AJ; 1-TORRES, AL; 1-DIAZ-PAREDES, B; 1-MENDEZ-OTERO,
R; 1-SANTIAGO, MF
1- Instituto de Biofísica Carlos Chagas Filho
Previously, our group demonstrated that bone marrow mononuclear cells (BMMC) protect retinal ganglion cells and promote optic
nerve regeneration in a model of optic nerve crush (Cell Transplant. 20(3):391-406, 2010). Here we investigate the involvement of
mesenchymal stem cells (MSC) in the survival and regeneration of retinal ganglion cells. Adult rats had the optic nerve crushed and
received MSC (5x105 cells), or vehicle (saline) intravitreous. Retinal ganglion cell survival was analyzed by immunostaining to β-III
tubulin 16 days after optic nerve crush. In the group that received saline (n=6), cell count was equivalent to 21% of the mean of
cells in the control group, which is consistent to previous observations after optic nerve crush (Invest ophthalmol vis sci, 41(13),
4169-4174, 2000; Cell Transplant. 20(3):391-406, 2010). MSC therapy (n=4) significantly increased this percentage to 40% (P<0.05;
t-test). For regeneration assay, cholera toxin subunit B conjugated to Alexa 488 was injected 14 days after the injury. Labeled
axons at 0.75 mm beyond crush site were augmented ~3 fold in MSC treated group (n≥7, P<0.05; t-test). Considering our previous
observations (Cell Transplant. 20(3):391-406, 2010), we conclude that BMMC and MSC might have similar therapeutical potential,
both in neuronal survival and regeneration.
Support: INBEB, FAPERJ, CAPES.
D42
ROLE OF EXCITED STATES CHARACTERIZED BY NUCLEAR MAGNETIC RESONANCE IN THE CATALYTIC CYCLE OF YEAST
THIOREDOXIN 1 – THERMODYNAMIC CHARACTERIZATION OF WATER CHANNEL
1-CRUZEIRO-SILVA, C.; 1-RODRIGUES, N.L.; 2-NETTO, L.E.S.; 1-VALENTE, A.P.; 1-ALMEIDA, F.C.L.
1-Laboratório de RMN de Biomoléculas, Centro Nacional de Ressonância Magnética Nuclear, Instituto de Bioquímica Médica,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil; 2-Intituto de Biociências – Universidade de São Paulo, São Paulo,
Brazil
Thioredoxins (Trx) are small ubiquitous proteins present from archea to complex eukaryotes. Regions containing the active site
loop (29-33) and the loops 56-59, 70-73 and 90-93 are responsible for the molecular recognition. Trx are very efficient enzymes
with dithiol oxidoreductase and chaperone-like activity that enables the assessment of buried disulfide bonds. The later activity is
related to the molecular recognition but poorly understood. The aim of this work is to characterize the intermediate excited states
of thioredoxin 1. Trx1 displayed equilibrium between many conformational states. This breathing is essential for catalysis and
correlated well with the protonation of Asp24. The mutant D24N stabilizes an open conformational state, which enables the
entrance of water in the interior of the protein. We measured the dynamics of Trx1 at different temperatures by NMR and
obtained the conformational entropy at each temperature and an estimate the calorific capacity for each residue. The interacting
loops (especially Ala71) displayed higher calorific capacity while the secondary structure residues low calorific capacity. Rotational
correlation time decreased with temperature (7.65 ns/15oC and 3.86 ns/ 45oC). The active site residues are involved in
conformation exchange showing the typical energy absorptive behavior: decrease the exchange rate (Rex) with temperature. Most
residues showed linear variation of the N-H chemical shift with temperature. Remarkably, we also showed a significant decrease in
conformational exchange at 15oC and oxidized state, suggesting a stabilization of a conformational state. Keywords: Thioredoxin
1, dynamic, NMR.
Support: CAPES, CNPq, FAPERJ, INCT-INBEB.
D43
ANALYSIS OF THE ACTIVITY AND IMMUNOLOCALIZATION OF THE ENZYME CONSTITUTIVE NITRIC OXIDE SYNTHESIS (CNOS) IN
PROMASTIGOTES FORMS OF THE LEISHMANIA (VIANNIA) BRAZILIENSIS AT DIFFERENT GROWTH PHASES IN VITRO
1,2- FURTADO, R.R.; 1,2- RODRIGUES, A.P.D.; 1,2- FARIAS, L.H.S.; 1,2- SILVA, E.O.
1 - Laboratório de Parasitologia e Laboratório de Biologia Estrutural, Instituto de Ciências Biológicas, Universidade Federal do Pará;
2- Instituto de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro.
American Tegumentary Leishmaniasis (ATL) is a parasitic disease, widely spread in most countries of Latin America and caused by
different species of the genus Leishmania. This protozoan is an obligate intracellular parasite that developed mechanisms to
subvert the microbicidal activity of macrophages, such as regulation of superoxide and nitric oxide (NO) production. During
Leishmania infection, the nitric oxide plays a crucial role in the killing of parasites in vitro and in vivo. In this work, we analyzed the
constitutive Oxido Nitric Synthase (cNOS) expression and NO production by Leishmania (Viannia) braziliensis, during logarithmic
(LOG) and stationary phase (STAT). Leishmania cNOS was identified in promastigotes using indirect immunofluorescence assay by
confocal microscopy and immunolocalization by Transmission Electron Microscopy (TEM). These results showed that
promastigotes of L. braziliensis are able to express cNOS in both growth phases. Immunolocalization by TEM allowed observing
localization of cNOS in the cytoplasm. For detection of NADPH-diaphorasic activity, promastigotes were incubated with NADPH
and demonstrated that promastigotes in log phase showed a more intense reaction when compared to STAT growth phase. The
production of NO was measured in the supernatants of promastigotes cultures as nitrite form by adding Griess reagent. To
confirm the enzyme activity, nitrite measure showed that this parasite is able to produce NO and that Leishmania promastigotes in
LOG phase has a higher production of NO when compared to parasites in STAT phase. In conclusion, a correlation between the
expression of cNOS and NO production by L. braziliensis suggest a possible virulence factor, which is able to regulate the
mechanism of the NO production by the host cell, conferring parasites resistance to NO damages. Keywords: cNOS; Leishmania
(Viannia) braziliensis; nitric oxide Support: CAPES, CNPq/UFPa, CNPq/MCT/CT-INFRA/CT-PETRO (Processo nº 620179/2008),
MCT/CNPq/FNDCT/CAPES/FAPERJ.
D44
UNDERNUTRITION ALTERS THE BIOACTIVE LIPID PATTERN AND CHOLESTEROL CONTENT IN RENAL PROXIMAL TUBULES
1, 2- SAMPAIO, L. S., 1, 2- VIEYRA, A., 1, 2- EINICKER-LAMAS, M.
1Instituto de Biofísica Carlos Chagas Filho – UFRJ, Rio de Janeiro. 2Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural
e Bioimagem
In Brazil the highest rates of undernutrition are either in northeast of the country and in the periphery of the big cities. Several
studies using the Basic Regional Diet (BRD), an experimental diet of undernutrition, showed that it affects the renal tissue in
diverse levels, inducing changes in Na+ renal transporters’ activities that can be modulated by bioactive lipids. The aim of this work
was to study the formation of bioactive lipids in and the cholesterol content in the renal tissue from undernourished rats. Wistar
male rats were separated into control and undernourished group (BRD). Total membranes from kidney proximal tubules were
obtained by differential centrifugation. Total cholesterol was measured by colorimetric method, while the production of bioactive
lipids was acessed by thin layer chromatography. Our results showed fewer formation of PtdIns(4)P in BRD animals compared with
control even in the presence of sphingosine, a positive modulator of the phosphatidylinositol-4 kinase. Concerning diacilglicerol
kinase and ceramide kinase activity, our experiments showed an increase in DAG and ceramide availability, in the BRD group.
About LPA the results showed a decrease in the phospholipase A2 activity in the BRD model. It was also observed a decrease in the
membrane cholesterol content in the BRD group. The plasma membrane Ca2+ pump was assayed by western blotting and the
results showed a decrease on this protein in the BRD group. The reduction on the total cholesterol content probably induces a
disruption in the lipid rafts on BDR group. This hypothesis is reiforced by the observed reduction in the PMCA content, wich is
known to be localized and active in the rafts. Together these results showed for the first time that BDR alters the genertion of
important bioacive lipids which can explain the physiological alterations already reported for Na+ transporters.
Support: CNPq, FAPERJ, CAPES.
D45
EFFECT INDOMETACIN ON ARBOVIRUS REPLICATION
MARCELO, M. D. F,; REBELLO, M. A.; FERREIRA, D. F.
Instituto de Microbiologia Paulo de Góes
Indomethacin is an indol derivative classified as a non-steroidal anti-inflammatory drug. This compound has a potent activity on
cyclooxigenase, blocking prostaglandins biosynthesis. In addition it has effects in the uncoupling of oxidative phosphorylation and
in mucopolysaccharides metabolism. The aim of our study is to investigate the effect of indomethacin in Alphavirus (Mayaro) and
Flavivirus (Dengue) replication. The antiviral activity of indomethacin was investigated in Vero cells infected with Mayaro and
Dengue viruses (positive single-strand RNA). Our results shows that treatment of these cells with indomethacin, in non-toxic
concentrations (100 μM) inhibits the replication of both viruses in about 90%. In Vero cells the antiviral (Mayaro) effect does not
seem to result from modifications in virus proteins synthesis. Although the synthesis of constitutive cellular proteins is unchanged
after treatment with indomethacin, we observed an induction of the synthesis of stress proteins (HSP90 and HSP70) in these cells.
Our results suggest a possible participation of stress proteins on the antiviral activity of indomethacin. Other results from our lab
already demonstrated that Mayaro virus replication is sensitive to MAPk-p38 modulation. The treatment with an activator of the
phosphorilation patyway (anisomicin) decreased the viral titer by 99%. In contrast, the inhibition of this pathway enhances virus
titer by 200%. These results are very interesting and we are now beginning to investigate the role of Indomethacin in the MAPk38
metabolic pathway that regulates Mayaro virus replication.
Support: INBEB, FAPERJ, CNPq.
D46
MITOTIC INACTIVATION OF UNDIFFERENTIATED EMBRYONIC STEM CELLS INDUCE BEHAVIORAL IMPROVEMENT IN A MURINE
MODEL FOR PARKINSON DISEASE
1- ACQUARONE, M.; 2- FERREIRA, F.G.M.; 2- PAIVA, F.F.; 1,2- TOVAR-MOLL, F.; 1- HOUZEL, J.C.; 1- REHEN, S.K.
1- Instituto de Ciências Biomédicas da UFRJ; 2- Instituto D'Or de Pesquisa e Ensino
Parkinson’s disease (PD) is a progressive motor disorder resulting from the degeneration of the dopaminergic (DA) nigrostriatal
dopaminergic (DA) pathway. To date, cell replacement strategies in humans have focused mainly on transplantation of either DA
neurons derived from fetal ventral mesencephalon, which are difficult to obtained, or mesenchymal stem cells (MSCs). Fetal cells
are difficult to be obtained, while MSCs , which release neurotrophic factors but are unable without the ability to differentiate into
DA neurons. As an alternative, eEmbryonic stem cells (ESCs) may differentiate into DA neurons in vitro and in vivo and also
improve motor function. in PD animals, hHowever, the; risk of tumorigenesis precludes the clinical applicationtransfer of the
technology. Here, we propose to reduce the tumorigenic potential ofpreviously halt the mitotic ability of ESCs with an antitumoral
agent as an alternative for reducing the tumorigenic potential of pluripotent stem cells before transplanting themed into the
striatum of mice with unilateral 6-hydroxydopamine lesion PD mice. First, we confirmed that our murine ESCs (mES) are able to
differentiate in vitro into DA neurons in vitro, with or without a previous antitumoral incubation with the antitumoral, as well as .
These cells are also able to release dopamine in vitro, as assessed by HPLC. Second, we verified that mES treated with different
antitumoral concentrations were efficient in preventing tumorigenic activity of mES injected intramuscularly into immunodeficient
"nude" mice without forming tumors, as assessed by gadolinium-contrasted 7T magnetic resonance imaging (MRI). Third, Mitotic
inactivated undifferentiated antitumoral-treated mES were then transplanted into the striatum of PD mice, which, 2 months after
intrastriatal injection with 6-hydroxydopamine (6-OHDA). PD animals received either saline (n=6), 50,000 GFP-mES (n=6), or
50,000 antitumoral-treated GFP-mES previously treated with antitumoral (n=12). Our results indicate that naïve mES or mitotic
inactivated mES, but not saline, restore motor function, as assessed weekly by: apomorphine-induced rotational counts, open
field, gait analyses, Rotarod® and balance beam walking tests. The observation of Our results indicate that GFP-mES are able to
release dopamine when properly stimulated to become DA neurons in vitro and also in vivo as showed by ttyrosine hydroxylase-
(TH)positive immunohistochemistry. GFP-mES positive for TH into the transplanted striatum reinforces mice brain point out the
potential use of undifferentiated, mitotic inactivated mES to restore DA innervation in PD mice, with no risk of tumorigenesis.
Support: FAPERJ, CAPES, CNPq, INCTC.
D47
EFFECTS OF SILDENAFIL TREATMENT IN OVARIAN MORPHOLOGY
1, 2 - DONATO, M. A. M.; 1, 2 - RIBEIRO, E. L. ; 1, 2, 3 - PEIXOTO, C. A.
1Programa de Pós-Graduação do Centro de Ciências Biológicas, UFPE, Recife (PE), Brasil 2 Laboratório de Ultraestrutura, Centro de
Pesquisas Aggeu Magalhães (FIOCRUZ), Recife (PE), Brasil 3Laboratório de Microscopia e Microanálise,CETENE,Recife (PE), Brasil,
Sildenafil is a noted drug used in chronic treatment of pulmonary hypertension, which mechanism of action is based on the
inhibition of phosphodiesterase 5, resulting in elevated intracellular cGMP. For pulmonary hypertension patients, this relaxes
pulmonary artery and beneficiates clinical status. Recent studies have shown that Sildenafil can also interact with nitric oxide
synthases, augmenting NO production (KIM, et al, 2008). It is necessary to evaluate if chronic treatment with Sildenafil may affect
folliculogenesis. Adult female C57BL/6 mice and female C57BL/6 knockout for inducible nitric oxide synthase, 45-day-old, were
used in all experiments, divided in 4 experimental groups: G1: C57BL/6 control; G2: C57BL/6 treated; G3: knockout C57BL/6
control; G4: knockout C57BL/6 treated. Treated groups received 25mg/kg body weight of Sildenafil for 60. After treatment animals
were killed and ovaries were excised. Follicle stages were defined using the classification proposed by Pedersen and Peters (1968).
Data from follicle account was analyzed with ANOVA/Tukey's range test. Morphological analyses showed no difference between
control and treated groups and wildtype and knockout animals. However, in treated groups (G2 and G4), cells in corpora lutea
appeared to be diminished in size. This result agrees with previous studies that showed a diminished diameter and number of lipid
inclusions in luteal cells from animals treated with phosphodiesterase inhibitor (DONATO, et al, 2009). Data analyses showed no
statistical difference in follicle account between groups in primary, secondary and antral follicles, and corpora lutea numbers. For
primordial follicles and size of ovaries, there was no statistical difference between control and treated groups; however, between
types of animals (C57BL/6 mice and knockout C57BL/6 mice) statistical difference was observed. This suggests that knockout
animals, deficient in nitric oxide synthases, have smaller ovaries and that inducible nitric oxide synthase is necessary for activation
of primordial follicles.
Support: INBEB.
D48
TRATAMENTO COM CÉLULAS-TRONCO MESENQUIMAIS DA MEDULA ÓSSEA EM MODELOS IN VITRO DA DOENÇA DE ALZHEIMER
1-GODOY, M.A.; 2-SARAIVA, L.; 1-DE VASCONCELOS, A.; 2-GOTO, L.; 1-SINIS, L.; 1-ROCHA, L.; 1-MONTEIRO, V.; 2-DE FELICE, F.G.; 2FERREIRA, S.T., 1-MENDEZ-OTERO, R.
1-IBCCF/UFRJ; 2- Instituto de Bioquímica Médica/UFRJ
A doença de Alzheimer é uma doença neurodegenerativa cuja incidência deverá aumentar com o envelhecimento da população
mundial. Até o momento, não existem alternativas terapêuticas eficazes. Diante disso, a terapia celular surge como uma
perspectiva de tratamento para diversas neurodegenerativas. Na medula óssea podem ser encontradas células-tronco
hematopoiéticas (HSC), que dão origem a toda a linhagem hematopoiética, e as células-tronco mesenquimais (MSC) ou do
estroma, que formam as células do tecido conjuntivo da medula óssea. A principal hipótese sobre o mecanismo de ação das
células mesenquimais é sua ação parácrina, liberando fatores tróficos que poderiam atuar na estimulação da neurogênese
endógena, na sobrevivência neuronal e/ou na modulação de fenômenos inflamatórios. O objetivo deste trabalho é avaliar os
efeitos do tratamento com células mesenquimais da medula óssea em culturas hipocampais expostas aos oligômeros abeta. Em
experimentos piloto observamos que os oligômeros foram capazes de se ligar às MSCs após 24h de exposição, sem alterar sua
viabilidade durante todo período analisado (máximo de 72h de exposição). Além disso, os oligômeros não geraram estresse
oxidativo nas MSCs após 24h de exposição, ao contrário do observado nos neurônios. Em análises complementares, pretendemos
avaliar o efeito do tratamento com as células mesenquimais sobre o estresse oxidativo, ativação da microglia e integridade
sináptica nas culturas hipocampais.
Apoio: INBEB, FAPERJ, CNPq.
D49
TERAPIA CELULAR EM MODELO DE LESÃO CEREBELAR: MODULAÇÃO DA EXPRESSÃO E FUNÇÃO DO GANGLIOSÍDEO 9-O-ACETIL
GD3
1,2- BARGAS-REGA, M.; 1,2- ZAVERUCHA-DO-VALLE, C.; 1,2- GUBERT, F.; 1,2- JASMIN.; 1,2- VIANA, K.L.; 1,2- MENDEZ-OTERO, R.;
1,2 SANTIAGO, M.F.
1-Instituto de Biofísica Carlos Chagas Filho, UFRJ. 2-Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem
O gangliosídeo 9-O-acetil GD3 é altamente expresso durante o desenvolvimento do sistema nervoso, e sua expressão é
correlacionada à migração neuronal e extensão neurítica. Na animal adulto, a expressão deste gangliosídeo persiste somente em
regiões restritas como o cerebelo e a retina. Além disso, foi observado o aumento da expressão desse gangliosídeo em modelo de
lesão do nervo ciático. Neste trabalho pretendemos caracterizar a expressão do gangliosídeo 9-O-acetil GD3 em um modelo de
lesão cerebelar. Além disso, pretendemos avaliar se o transplante de células mononucleares de medula óssea altera o padrão de
expressão do 9-O-acetil GD3 e/ou promove melhora funcional nos animais lesados. Utilizamos ratos adultos, entre três e cinco
meses de idade, da variedade Lister-hooded que foram divididos nos seguintes grupos experimentais: animais normais que não
sofreram nenhum tipo de intervenção; animais normais que receberam injeções na jugular contendo somente veículo (Sham);
animais que foram submetidos a lesão cerebelar e receberam as injeções contendo apenas o veículo (Lesados); e animais que
sofreram a lesão e receberam células da fração mononuclear de medula óssea (tratados). A lesão cerebelar foi realizada através
de coordenadas estereotáxicas de forma a lesar o cerebelo entre o vermis e o hemisfério direito. Quatro horas após a lesão, os
animais tratados receberam edovenosamente 3x107 células mononucleares de medula óssea que foram obtidas previamente
através de um gradiente de ficoll. Já os animais do lesados receberam apenas 500µL de salina. Para análise imunohistoquímica os
animais foram perfundindos com paraformaldeído à 4% e seus cerebelos processados para a análise 3, 7 ou 21 dias após a lesão.
Antes de serem sacrificados, os animais de todos os grupos experimentais foram submetidos ao teste do cilindro para avaliar o
desempenho motor nos mesmos tempos de sobrevida. Utilizamos o mAb Jones que reconhece o gangliosídeo 9-O-acetil GD3,
permitindo assim, a caracterização da expressão desse gangliosídeo por técnicas imunohistoquímicas e bioquímicas. Nossos
resultados indicam que sete dias após a lesão existe uma supra-regulação de aproximadamente 40% da expressão do gangliosídeo
9-O-acetil GD3, principalmente na camada molecular do cerebelo. Após 21 dias essa expressão volta aos níveis normais. Através
de duplas marcações demonstramos que a expressão desta molécula está associada com as fibras de glia de Bergmann e com as
células de Purkinje. O transplante intravenoso de células da fração mononuclear da medula óssea causou uma melhora no
desempenho motor dos animais subemtidos ao teste do cilindro e uma diminuição na expressão do gangliosídeo 9-O-acetil GD3
após 7 dias de lesão. Através desses resultados, é possível sugerir um possível papel desta molécula na modulação de processos de
plasticidade/regeneração no sistema nervoso central.
Apoio: INBEB, CAPES, CNPq, DECIT, FAPERJ, INBEB.
D50
ANÁLISE PROTEÔMICA LABEL-FREE DE ALTA DEFINIÇÃO NA INVESTIGAÇÃO DOS EFEITOS MEDIADOS POR CÉLULAS-TRONCO
MESENQUIMAIS NO REPARO DE LESÕES RENAIS
1,2- COSTA, M.R.; 3,4- PIZZATTI, L.; 1,2- LINDOSO, R.S.; 3- ROCHER, B.; 3,4- ABDELHAY, E.; 1,2- VIEYRA, A.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, RJ, Brasil; 2-Instituto Nacional de Ciência e
Tecnologia de Biologia Estrutural e Bioimagem, RJ, Brasil.; 3- Laboratório de Célula Tronco, Instituto Nacional do Câncer, RJ,
Brasil.4- Rede Proteômica do Rio de Janeiro.
Introdução: Embora diversos estudos demonstrem o potencial terapêutico das células-tronco mesenquimais (MSC) no tratamento
das doenças renais, os mecanismos envolvidos ainda não são totalmente compreendidos. Neste trabalho, estratégias proteômicas
multidimensionais em larga escala, que são amplamente utilizadas para identificação e quantificação de marcadores e
mecanismos em diferentes doenças, serão empregadas para identificação de moléculas e potenciais vias de sinalização afetadas
pelo tratamento com MSC. Objetivo: Investigar os mecanismos celulares e moleculares envolvidos no reparo renal pela terapia
com MSC derivadas da medula óssea utilizando proteômica quantitativa de alta definição. Metodologia: Células tubulares renais
humanas (HK-2) confluentes foram submetidas à lesão de isquemia por 30 minutos pelo tratamento com meio contendo
antimicina A 50µM e livre de substratos e glicose. Após este período, o meio foi substituído por meio DMEM e a cada placa foi
adicionado um inserto de co-cultura com porosidade de 0,4µm, contendo 1 × 106 MSC humanas. Após 3 horas, as células renais
foram coletadas e as proteínas extraídas. As amostras foram concentradas 16x com colunas de 3k amicon (Millipore), digeridas em
solução e analisadas por cromatografia multidimensional (MudPIT) no sistema de espectrometria de massas 2D-NanoESI-MSE
(MudPIT) utilizando o espectrômetro de massas Synapt HDMS (Waters). As proteínas foram identificadas e quantificadas
utilizando o software Proteinlynks global Server (Waters) com a ferramenta Expression E. As interações protéicas foram preditas
com o software Ingenuity®. Resultados: Foram identificadas 365 proteínas, das quais 264 e 50 encontravam-se “superexpressas”
ou “downreguladas”, respectivamente, nas células renais tratadas com MSC. Este tratamento levou ao aumento da expressão de
proteínas envolvidas em vias de sinalização que regulam a síntese e modificação de proteínas, crescimento e apoptose celular,
resposta ao estresse oxidativo, metabolismo de carboidratos, organização do citoesqueleto e potencial de membrana
mitocondrial. Conclusão: O tratamento com MSC desencadeia respostas celulares associadas ao reparo tecidual.
Apoio: INBEB, FAPERJ, CAPES, CNPQ, Ministério da Saúde, FINEP.
D51
ISOLATION AND MAINTENANCE OF FISH TRYPANOSOMES FROM THE BRAZILIAN ARMORED CATFISH HYPOSTOMUS AFFINIS
(STEINDACHNER, 1977) AND HYPOSTOMUS LUETKENI (STEINDACHNER, 1977)
LEMOS, M.; SOUTO-PADRÓN, T.
Instituto de Microbiologia Prof. Paulo de Góes, UFRJ.
Fish trypanosomes have a worldwide distribution and are transmitted by hematophagous leeches in which the parasites multiply
and undergo morphological transformations. This study provides the first record of the isolation and in vitro maintenance of
trypanosomes found in naturally infected armored catfish from Brazil. Fish were collected on the Pomba River in Guarani City, MG,
Brazil. Blood samples were inoculated into 35 different combinations of hemocultures. Trypanosomes were characterized by
Giemsa stained smears, Nomarski Differential Interference Contrast (DIC), and fluorescence microscopy by incubation in Hoechst
fluorescent nucleic acid dye analyzed by epifluorescence microscopy using a Zeiss Axioplan II light microscope equipped with a
Color View XS digital video camera. Trypanosomes were also analyzed by scanning electron microscopy. Trypanosomes were
isolated from 9 individuals of Hypostomus affinis and 6 individuals of Hypostomus luetkeni, resulting in 51 isolates. Bloodstream
trypomastigotes transformed into pyriforms to elongated epimastigotes from 24 h post-inoculation for up to 10 days in different
biphasic media. Pleomorfic trypomastigotes and spheromastigotes were also observed in vitro. Trypanosomes usually clustered
into rosettes of varying sizes. Division of trypanosomes in culture media gave rise to two identical or two distinct developmental
forms. Trypanosomes were sequentially subcultured in vitro for 17 months, and cryopreserved parasites were successfully thawed
and subcultured for up to 8 months.
Support: CNPq, FAPERJ, CAPES, INBEB.
D52
SIMULAÇÕES DE DINÂMICA MOLECULAR COM O PEPTÍDEO DERMADISTINCTINA K: COMPORTAMENTO EM AMBIENTE AQUOSO
E TFE-ÁGUA
1-BATISTA, MOEMA M.; 2-FERANDES, TÁCIO VINÍCIO AMORIM; 1-SANTORO, MARCELO MATOS; 2-PASCUTTI, PEDRO GERALDO
1-Instituto de Ciências Biológicas - Universidade Federal de Minas Gerais 2- Instituto Carlos Chagas Filho - Universidade Federal
do Rio de Janeiro
A descoberta de novos princípios ativos é o grande objetivo de indústrias farmacêuticas. O Brasil é dotado de uma das maiores
faunas e flora do mundo sendo cobiçado pela disponibilidade de um banco de dados de novas substâncias. Esse trabalho tem por
objetivo investigar por simulação computacional a relação estrutura-dinâmica-função de peptídeos antimicrobianos de atividade
comprovada, sintetizados na Universidade Federal de Minas Gerais. Atualmente a amostra estudada é do peptídeo
Dermadistinctina K (DD K) produzido na pele do anfíbio Phyllomedusa distincta. Em que acredita-se ser o principal causador do
desequilíbrio da membrana bacteriana. Como padrão esse tipo de peptídeo não age em receptor específico e sim em membranas
de bactérias promovendo um desequilíbrio osmótico. Esse mecanismo de ação não está totalmente elucidado exigindo um estudo
estrutural e dinâmico mais aprofundado, objetivando otimizar o peptídeo para futuros biofármacos contra doenças infecciosas. As
simulações de Dinâmica Molecular (DM) são realizadas na UFRJ no Laboratório de Modelagem e Dinâmica Molecular (LMDM).
Submetemos o peptídeo a solvente aquoso (H2O) e a ambiente misto apolar/polar TFE/H2O(50:50, v/v) de forma a mimetizar a
conformação do mesmo quando se acopla a uma membrana, expondo possivelmente uma região hidrofóbica. A solução mista de
TFE-água (50:50, v/v) demostrou estabilização da estrutura helicoidal do peptídeo justamente devido ao equilíbrio eletrostático
entre os resíduos carregados e pela predominância dos grupos apolares das moléculas de TFE na região dos átomos
eletronegativos da cadeia principal. Além disso, os resultados observados na simulação aquosa corroboram com resultados
experimentais, no qual o peptídeo apresenta-se desestruturado em ambientes aquosos.
Apoio: CNPq, FAPEAM.
D53
FUNCTIONAL CORRELATES OF ABERRANT BUNDLES IN CALLOSAL DYSGENESIS
1LAZAREV, V.V., 1,2,3MONTEIRO, M.C., 2OLIVEIRA-SOUZA, R., 1DEAZEVEDO, L. C., 2, 4MOLL, J., 3, 4LENT, R., 2,3, 4TOVAR-MOLL, F.
1Fernandes Figueira Institute, FIOCRUZ, Rio de Janeiro, Brazil; 2D'Or Institute for Research and Education, Rio de Janeiro, Brazil;
3Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 4National Institute for Translational
Neuroscience, INNT.
Callosal dysgenesis (CD) is characterized by a developmental failure of formation of the major commissural bundle. Previous
studies demonstrated structural evidence of abnormal white-matter pathways (Probst and Sigmoid bundles) in CD. Here we
combined diffusion tensor imaging (DTI) with quantitative electroencephalography in order to address, in vivo, the functional
correlates of these pathways and the relationship between structural and functional neuroplasticity in human CD. Volumetric
anatomical (3DT1) and DTI (2,5mm isotropic voxel) images were acquired (3T, Achieva Philips) in 5 patients and 7 healthy
volunteers. Neurophysiological assessment was performed in all patients and 15 controls by 16-electrode eletroencephalogram
(EEG) recording (Bio-logic, USA) and analysis of the coefficients of coherence (CChr) (Brainsys-Neurometrics, Russia) in five EEG
frequency bands. Group comparison was performed using parametric (t-test) or non-parametric test (Mann-Whitney in
neurophysiological assessment). In patients, conventional images showed partial dysgenesis (n=2), agenesis (n=2) or hypoplasia
(n=1) of the corpus callosum (CC). In addition, DTI and tractography confirmed the presence of two abnormal white-matter tracts:
(i) the Probst bundles (PB) connecting intrahemispheric cortical regions in patients and, (ii) the Sigmoid bundles, connecting the
frontal lobe with the contralateral occipitoparietal cortex (n=3, except for agenesis). General analysis of CChr showed an increased
intrahemispheric and decreased interhemispheric coherence in patients. However, in patients with partial CD or CC hypoplasia, a
relatively higher interhemispheric coherence in the frontal areas was observed. Analysis of CChr showed an increased
intrahemispheric coherence in all patients, particularly in cases of total and partial CD. This finding may be related to the presence
of the Probst bundles. The anatomical connectivity of the Sigmoid bundle may underlie the higher contralateral coherence
between anterior (particularly right) and centroparietal (particularly left) in partial CD and hypoplasia. The changes in
electrophysiological connectivity observed in CD might be the functional counterpart of the abnormal white-matter connections.
Support: CAPES, CNPq, FAPERJ
D54
POSSIBLE RELATIONSHIP BETWEEN ECTO-ENZYMES AND DRUG TRANSPORT IN RESISTANT LEISHMANIA
1-Giarola, N. L. L. ;2- Costa, N. A.,1- Silveira, T.S.;2- Rossi-Bergmann, B.;3- Almeida-Amaral, E. E.;1-Meyer Fernandes, J. R.1
1- Universidade Federal do Rio de Janeiro (UFRJ) - Instituto de Bioquímica Médica 2- Universidade Federal do Rio de Janeiro (UFRJ)
- Instituto de Biofísica 3- Fundação Oswaldo Cruz – IOC/RJ
Ecto-nucleotidases are surface enzymes able to hydrolyze extracellular nucleotides. Some functions are suggested for these
proteins: cell adhesion, purine acquisition, protection against cytotoxic effects of extracellular ATP and, recently, MDR
phenomenon. P-glycoprotein (P-gp) is involved in the removal of drugs, most of them cations, from the plasma membrane and
cytoplasm. P-gp is also associated with movement of ATP, an anion, from the cytoplasm to the extracellular space. The central
question of this study is to establish the relationship between the ecto-ATPase activities and a possible mechanism of resistance to
vinblastine in Leishmania. Leishmania promastigotes were selected by gradual increasing concentrations of the vinblastine from
10µM to 100µM, and the cells were maintained continuously under drug pressure. The cells were used to determine Mg+2
dependent ectocto-phosphatase, ecto-5’
and ecto-3’-nucleotidases were assessed by colorimetric method by the complex formation of Pi released with ammonium
molybdate (Fiske and Subbarow, JBC, 1925). Our results show that ecto-ATPase activity from resistant Leishmania presented a
250% higher activity rate compared with wild-type Leishmania. This increase is progressive with the increasing of the drug
concentrations. In addition, other ecto-enzyme activities did not change significantly with drug pressure. We compared the ectoATPase activity in cells resistant to another drug and evaluated this activity in resistant L. amazonensis and L. donovani.
Modifications morphological were observed in resistant cells. Furthermore, rhodamine transport in resistant cells is higher than in
control cells. These results suggest a possible relationship between resistance and ecto-ATPase activity and this can contributes to
elucidate the mechanism of drug transport.
Financial Support: CNPq, FAPERJ
D55
AVALIAÇÃO DA ATIVIDADE DE MODULADORES DA AGREGAÇÃO DA PROTEÍNA PRION: ABORDAGENS TERAPÊUTICAS
1-FERREIRA, N.C.; 1-MARQUES, I.; 2-MASCARELLO, A.; 2-NUNES, R.; 1-CORDEIRO, Y. J.
1- Faculdade de Farmácia, Universiidade Federal do Rio de Janeiro; 2- Departamento de Química, Universidade Federal de Santa
Catarina
A proteína príon (PrP) é o agente etiológico das Encefalopatias Espongiformes Transmissíveis (EETs), que compreendem um grupo
de doenças neurodegenerativas fatais que podem ser transmitidas para humanos e animais. Os indivíduos infectados apresentam
sintomas clínicos de disfunção tanto cognitiva quanto motora e, em geral, os pacientes morrem cerca de doze meses depois do
surgimento dos primeiros sintomas. A PrP está majoritariamente presente na membrana de células nervosas na sua forma celular
(PrPC) e até o presente momento, sua função fisiológica não foi elucidada. Para o aparecimento das EETs, é necessário que
aconteça a conversão da forma celular (PrPC) para uma forma anormal, a proteína prion scrapie (PrPSc).A PrPC, que possui um
rico conteúdo em alfa-hélices sofre uma conversão estrutural que resulta na PrpSC, rica em folhas-beta em um processo póstradução. Apesar do grande número de estudos na área, não há até o momento terapia para estas doenças. Trabalhos no âmbito
terapêutico com o uso de derivados de quinolinas e acridinas se mostraram eficazes em sistemas in vitro, porém não
apresentaram ação anti-prion in vivo. Estes dados reforçam a necessidade de se buscar novos compostos que sejam capazes de
controlar a conversão PrPC-PrPSc; de inibir a agregação da PrPSc e, finalmente, de atravessar a barreira hematoencefálica. Neste
trabalho descrevemos os efeitos de diferentes compostos orgânicos na modulação e no controle da agregação do peptídeo da PrP
de hamster sírio (Sha 109-149), que compreende um domínio hidrofóbico da PrP e agrega prontamente em solução em pH 5.0.
Sua agregação foi avaliada através da monitoração dos valores de espalhamento de luz (EL), na presença e na ausência dos
compostos selecionados. Além disso avaliou-se a redução na formação de agregados amilóides através da ligação ao Vermelho do
Congo.
Apoio: INBEB, FAPERJ, CNPq, CAPES.
D56
UNRAVELING THE STRUCTURE AND FUNCTION OF A MEMBRANE-ACTIVE PEPTIDE OF THE HEPATITIS C VIRUS: IS IT A POSSIBLE
FUSION PEPTIDE?
1- ALVES, N.S.; 1-MENDES, Y.S.; 2-SOUZA, T.L.F.; 1-BIANCONI, M.L.; 3-ANOBOM, C.D.; 1-VALENTE, A.P.; 1-SILVA, J.L.; 1-GOMES,
A.M.O.; 1-OLIVEIRA, A.C.
1-Programa de Biologia Estrutural, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2-Faculdade de
Farmácia, Universidade Federal do Rio de Janeiro; 3-Instituto de Química, Universidade Federal do Rio de Janeiro
It has been estimated that 3% of the world population is infected with the Hepatitis C Virus (HCV). Hepatitis C is an important liver
illness and the most common indication for liver transplantation. Unfortunately it is still difficult to treat this disease due to
therapy adverse effects and its high cost. The entry of HCV is a slow and complex process, triggered by low pH and involves
multiple steps that still have to be elucidated. Recent evidences indicate that different membranotropic segments of the envelope
glycoproteins, E1 and E2, seem to be required for virus-cell fusion but the location of the fusion peptide is still unknown. In this
study we aim to characterize the structure, the possible interaction with model membranes and the membrane disrupting activity
of a membranotropic peptide (HCV421-445) present in HCV E2 glycoprotein. With this aim, we used biophysical methodologies
such as photometry, fluorescence spectroscopy, circular dichroism (CD), calorimetry and nuclear magnetic resonance (NMR).
Spectroscopic analysis, through the increase of Trp emission energy and acrylamide quenching; and calorimetric analysis suggest
that peptide-micelle interaction is enthalpically driven and involves Trp residues. Furthermore, this peptide is also able to induce
vesicle aggregation. The TOCSY and NOESY spectra of the peptide free in solution indicate the presence of few ordered structure
in this condition, but addition of TFE and micelles leads to conformational changes of the peptide, also confirmed by CD data.
Furthermore, this peptide is also able to induce vesicle aggregation only in acidic pH. Although HCV421-445 has very low hemolytic
activity, hemolysis only occurs in acidic pH, thus suggesting that HCV421-445 peptide participates in the entry process of HCV.
These structural studies are important to determine structural identities among the enveloped viruses, which should help in the
development of new antiviral therapies.
Support: Capes, CNPq, FAPERJ, PRONEX, INBEB.
D57
ANGIOTENSIN II ANTAGONIST PROMOTE A RENAL AND CARDIAC PROTECTOR EFFECT IN RATS SUBMITTED TO CHRONIC
UNDERNUTRITION
1,2- SILVA, P. A.; 1,2- CAHLI, G. M.; 1,2- LUZARDO R.L.; 3- VIEIRA-FILHO, L. D.; 1,2- EINICKER-LAMAS, M.; 1,2- MEDEI, E.; 1,2-VIEYRA,
A.
1- INBEB/Universidade Federal do Rio de Janeiro; 2- Instituto de Biofísica Carlos Chagas Filho; 3- Departamento de Fisiologia e
Farmacologia, Universidade Federal de Pernambuco
Epidemiological studies in the Northeastern region of Brazil and in the periphery of big cities show an association between
hypertension and undernutrition, especially in areas where protein-deficient diets are combined with high salt intake. Wistar rats
submitted to the Northeast basic regional diet (BRD) from weaning to the 13th week of life presented an increase in plasma
volume, and in the activity of Na+-ATPase from proximal tubule membranes. Na+-ATPase activity lost its physiological response to
angiotensin II (Ang II). These changes if present in cardiac tissue could lead to a cardiac electrical remodeling, facilitating the
appearance of arrhythmias and sudden death. The aim of this work was to evaluate whether the treatment of chronically
undernourished rats with losartan (an antagonist of angiotensin II type 1 receptor): (i) preserves the cardiac electrical activity, (ii)
recovers the normal Na+ reabsorption mediated by Na+-ATPase, and (iii) restores the normal plasma volume. Male Wistar rats
were divided in four groups: control (C) (fed with standard chow after weaning), D (undernourished fed with BRD), CL and DL
(control and undernourished treated with losartan (30 mg/kg/day from weaning to 13rd weeks of age). The plasma volume was
measured using the Evans Blue dye (100 μg/100 g body weight). The Na+-ATPase activity of proximal tubule membranes were
measured as the difference in Pi released from *γ-32P]ATP in absence and presence of furosemide (2 mM). The electrocardiogram
was assessed in anesthetized animals and the folowing parameters were measured: RR, QT and QTc interval, and QRS complex.
After 13 weeks the D group showed an increase in the heart weight/body weight ratio (3.56 ± 0.03) (in g/mg; means ± SEM) when
compared with the C group (3.45 ± 0.01; P<0.01); losartan had no influence on the relative decrease of cardiac mass (CL = 3.45 ±
0.01; DL = 3.45 ± 0.02). The increased plasma volume (ml/100 g body mass) in D group (7.85 ± 0.63) returned to normal values in
losartan-treated animals (DL = 5.60 ± 0.44 and C = 5.70 ± 0.35; P<0.05 with respect to D). The rats given BRD presented an
increased the Na+-ATPase activity (in nmol Pi × mg-1 × min-1) (D = 148.5 ± 12.7) that was prevented by losartan (DL = 62.2 ± 4.7; C
= 56.6 ± 4.8; P<0.005 vs D). In the electrocardiogram, both RR interval and QRS complex were similar among grou ps. However, the
ventricular repolarization parameters, QT (C: 47.4 ± 1.0 vs D: 57.0 ± 2.7 ms; P<0.05) and QTc (C: 100.7 ± 2.7 vs. D: 120.6 ± 6.2;
P<0.001) intervals, were longer in D when compared to C group. Losartan was able to prevent the cardiac electrical remodeling
maintaining both, QT (DL = 46.0 ± 1.8; P<0.01 vs. D) and QTc interval (DL = 93.8 ± 3.2; P<0.001 vs. D) values similar to CL and C
groups. The chronic undernutrition promotes increases in plasma volume, renal Na+-ATPase activity and induced a ventricular
cardiac repolarization disturbances reflected by a significant longer QT and QTc interval. The treatment with losartan was capable
of preserve the normal Na+-ATPase activity (and Na+ reabsorption), the plasma volemia and the cardiac electrical remodeling. The
results indicate that Ang II plays an important role during this alterations caused by chronic undernutrition via its AT1 receptors.
Support: INBEB, MCT-CNPq-MS- DECIT-FAPERJ , CAPES-PROCAD.
D58
BIODISTRIBUTION OF TRANSPLANTED BONE MARROW MONONUCLEAR CELLS IN SUBACUTE STROKE PATIENTS
1- Rosado de Castro, P.H.; 1- Schmidt, F.R.; 1- Gutfilen, B.; 1- Souza, S.A.L.; 1- de Freitas, G.R.; 1- Battistella, V.; 1- Silva, R.M.; 1Wajnberg, E.; 1- Maiolino, A.; 2- Kasai-Brunswick, T.H.; 2- Vairo, L.; 2- Goldenberg, R.C.S.; 1- Pereira, B.B.; 1- Nascimento, E.M.;
Alves-Leon, S.; 1- Gasparetto, E.L.; 1- Andre, C.; 2- Mendez-Otero, R.; 1- Barbosa da Fonseca, L.M.
1- Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro; 2- Instituto de Biofísica Carlos Chagas
Filho, Universidade Federal do Rio de Janeiro.
Background: The main goal of this study is to compare two routes of administration of bone marrow mononuclear cells (BMMC)
labeled with 99mTechnetium (99mTc) in patients with ischemic stroke in the territory of the middle cerebral artery (MCA) within
90 days after the beginning of symptoms. Methods: Twelve patients between 24 e 68 years have been included in the study, from
19 to 89 days after the stroke. Seven patients received cells by intra-arterial (IA) and 5 by intravenous (IV) route. Bone marrow
aspiration with BMMC separation was performed and approximately 10% of the cells were labeled and injected in the MCA or
intravenously. Whole body and planar scintigraphies and single photon emission computed tomography (SPECT) were acquired
after 2h and 24h. Results: Cells migrated to the brain in all patients and quantification of SPECT images at 2h showed that uptake
was higher in the lesioned hemisphere when compared with the contralateral side (68,1±14,6% of the total uptake in the brain in
the IA group and 53,4±3,8% in the IV group). In comparison to IV administration, IA route led to greater uptake in the liver, spleen
and kidneys and lower uptake in the lungs at 2h and 24h after cell therapy. The uptake in the brain was below 1,2% in 11 of 12
patients. Conclusion: Our results indicate that cell therapy with 99mTc labeled cells is safe and allows monitoring of cell
biodistribution and homing for up to 24h in patients with subacute ischemic stroke. There were significant differences in organ
distribution and in relative uptake between hemispheres when comparing IA and IV groups. However, total uptake in the brain
was low and without statistical difference between both groups, and these findings may have important implications for choosing
the route of administration in future larger clinical studies.
Support: CAPES, CNPq, FAPERJ, INBEB.
D59
SIMVASTATIN PROTECTS COGNITIVE IMPAIRMENT IN ANIMAL MODEL OF SEPSIS
1 - ALEXANDRE, P.C.B., 1 - REIS, P.A., 1 - PITOMBO, M.M., 1 - CASTRO-FARIA-NETO, H.C., 2 - BOZZA, F.A..
1 - Fundação Oswaldo Cruz; 2 - Instituto de Pesquisa Evandro Chagas
Sepsis is one of the most serious public health problems worldwide, with mortality rates are between 20% and 80% and
characterized by a systemic inflammatory response. In patients who has survived from infection it has been described a neuronal
dysfunction in the CNS, leading to a framework of cognitive impairment. Statins have the ability to block the cascade of cholesterol
formation by acting on the enzyme HMG-CoA reductase, reducing the synthesis of endogenous cholesterol. Recently it has been
observed that anti-inflammatory properties of statins, with decreased production of cytokines, endothelial adhesion molecules
and oxidative stress. The aim of this study is to evaluate the ability of statins to reduce the systemic inflammatory response and
thus the potential to protect against neurocognitive damage. Male Swiss Webster mice (25 g, n=10/group) were divided in four
groups (saline, saline + simvastatin, feces and feces + simvastatin). Feces were extracted from large intestine and diluted in saline,
centrifuged in 1100rpm (10 min). Supernatant was collected and 0.5 ml injected the animals feces groups. Control animals
received 0.5 ml of saline. The animals were treated at 6, 24 and 48 hours after that with antibiotic imipenem and of 1.0 ml saline.
The simvastatin groups were treated 1 h before to 48 hours after the infection (20 mg/kg b.w., p.o.). After 15 days we analyzed the
cognitive damage using the inhibitory avoidance task. Percentage of survival was higher in animals treated with simvastatin (80%)
compared to feces without adjuvant therapy (33.3%). The inhibitory avoidance shows that animals that received simvastatin were
able to keep the avoidance memory, that was missed in untreated infected mice. We concluded that simvastatin protected the
animals from septic cognitive damage at the level of aversive memory, and an essential tool for future strategies against
neurocognitive damage generated by the disease.
Support: INBEB, FAPERJ, CNPq
D60
STRUCTURAL ANALYSIS OF THE N-TERMINAL FRAGMENT OF THE ANTIANGIOGENIC PROTEIN ENDOSTATIN: A MOLECULAR
DYNAMICS STUDY
1 - Torres, P.H.M.; 2 - Limaverde, G.S.C.S.; 1 - Pascutti, P.G.
1 - Carlos Chagas Filho Biophysics Institute - IBCCF, Federal University of Rio de Janeiro - UFRJ; 2 - National Cancer Institute of
Brazil - INCA
Endostatin is a potent antiangiogenic protein derived from the non-collagenous domain 1 (NC1) of collagen XVIII. The mechanism
by which endostatin exerts its antiangiogenic effect is still incompletely understood. It has been shown that the 27 amino acid Nterminal fragment of murine endostatin has antitumor, antimigration and antipermeability activities comparable to the full soluble
protein. In order to understand how this peptide can exert such elaborate function, we performed structural analysis using
molecular dynamics to evaluate the behavior of this fragment in aqueous environment. Here we show that the N-terminal peptide
of murine endostatin is able to assume a well-defined structure, folding into a zinc-dependent beta-hairpin conformation.
Analyzing the folding mechanism, we were able to understand why the N-terminal peptide of human endostatin with the same
length failed to acquire a stable conformation. Conversely, we were able to predict the successful folding of the R4Q mutant and
of a shorter form of the human peptide with 25 residues. Finally, we show that the beta-hairpin conformation assumed by the
zinc-bound peptide of murine endostatin has a high structural similarity with fragments of another family of angiogenesis
inhibitors: the integrin-binding portion of the NC1 domain of collagen IV.
Support: INBEB, CNPq, FAPERJ.
D61
ACTIVITY OF 3-ARILIDENEINDOLIN-2-ONAS (TFMDI), A SIRTUINS INHIBITOR AGAINST TRYPANOSOMA CRUZI
1- VEIGA-SANTOS, P.; 2- BRACHER, F.; 1- DE SOUZA, W.; 1- CARVALHO, T.M.U.
1 - Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro, Brazil; 2 - Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität at München,
Butenandtstrasse, Munich, Germany.
Trypanosoma cruzi is the etiological agent of Chagas disease. At present, treatment of Chagas disease involves the use of
compounds such as nifurtimox and benznidazole which are poorly tolerated and unsatisfactory because of the frequent toxic side
effects. Therefore, it is important to find new active compounds as well potential parasite targets. The sirtuins are histone
deacetylase enzymes present in prokaryote and eukaryote cells, which are associated with stress resistance, longevity, genomic
stability and energy metabolism. In this study we evaluated the effect of 3-arilideneindolin-2-onas (TFMDI), a sirtuins inhibitor,
against T. cruzi. This compound inhibited proliferation of epimastigote and amastigote forms, showing IC50 of 7 µM and 1.1 µM,
respectively. Against trypomastigote form, the compound exhibited a LD50 of 1.1 µM. TFMDI demonstrated low potential toxicity
to peritoneal macrophages, with CC50 of 90 µM, being more selective for amastigote (around 81 times) than macrophages. This
compound induced alterations of kinetoplast size in treated epimastigote forms as observed by light microscopy. Moreover, it
inhibited parasite cell division, especially during cytokinesis. These alterations also were confirmed by scanning electron
microscopy. Thin sections of treated epimastigotes observed by transmission electron microscopy (TEM) revealed a loss of
chromatin condensation, presence of several electron-lucent and autophagic vacuoles, Golgi apparatus and kDNA disorganization.
The same kDNA alterations were observed in treated trypomastigote. Furthermore, loss of cytoplasm organelles, presence of
swelled mitochondrion and myelin-like figures also were visualized. Taken together, our observations show that TFMDI is a potent
inhibitor of T. cruzi proliferation (in epimastigote and amastigote forms) interfering mainly on cellular cycle, and display lytic
activity against trypomastigote forms.
Support: CNPq, CAPES, FINEP, FAPERJ.
D62
INTERACTION PROFILE OF SMALL INHIBITORS COMPLEXED WITH FALCIPAIN-2 AND FALCIPAIN-3 PLASMODIAL CYSTEINE
PROTEASES
1-CELESTINO, P. S. F.; 1- GOMES, D. E. B.; 1- PASCUTTI, P. G.
1- Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
About 500 million cases of malaria occur globally each year, leading to 1 million deaths, mainly caused by Plasmodium falciparum
specie. P. falciparum cysteine proteases Falcipain-2 (FP2) and Falcipain-3 (FP3) act in the hemoglobin degradation pathway,
parasite’s main source of aminoacids. The use of cysteine proteases inhibitors interrupts this pathway and some of them lead to
cure of the disease in infected mice. Many FP2 or FP3 inhibitors have been described, this work focused on the complexes with
some of the classes described as potent and specific inhibitors: a vinyl-sulfone peptide-based (v1b); three peptidomimetics, with a
pyridone ring scaffold (v5b) and benzodiazepine scaffold (et2b and et4c); and two non-peptide inhibitors (des4 and zhu2k). Our
goal is to characterize the enzimes-ligand interactions to support the rational design of new compounds. To accomplish that,
molecular docking and molecular dynamics simulations were performed for the complexes FP2/FP3-inhibitors.The analysis of the
intermolecular contact area and the hydrogen bond showed that the introduction of non-peptidic scaffold in the backbone of the
peptidomimetic inhibitors did not interfere with the stabilization of the complexes. Highly prevalent hydrogen bonds involving the
inhibitors and critical residues of the active site of the enzymes were found in higher number s for FP2 complexes, suggesting
more specificity to FP2. The non-peptidic zhu2k showed highly prevalent bonds with both enzymes, suggesting that it can be a
candidate for common inhibition. Analysis of binding energy confirmed the better interaction of v1b with FP3 followed by v5b,
des4 and zhu2k. For FP2, et2b and et4c together with zhu2k showed the smallest energy values. Based on intermolecular contact
area and hydrogen bond network, we also performed an analysis of the chemical groups derived from the inhibitors, highlighting
individual portions that would best fit the subsite cavities of FP2 and FP3 active sites.
Support: CAPES, CNPq, INBEB.
D63
CARACTERIZAÇÃO ESTRUTURAL DE PROTEÍNAS HIPOTÉTICAS CONSERVADAS EM CINETOPLASTÍDEOS
1- MENEZES, R.S.; RAMOS, T. ; CASTILLA, A. L. ; PIRES, J. R. M.
1- Centro Nacional de Ressonância Magnética Nuclear, Instituto de Bioquímica Médica, CCS, UFRJ
A doença de Chagas, doença do sono, e leishmanioses estão entre as doenças tropicais negligenciadas, que costumam afetar
populações mais pobres e têm pouca prioridade na saúde pública. Recentemente foram sequenciados os genomas dos agentes
etiológicos destas doenças, os cinetoplastídeos Trypanosoma cruzi, Trypanosoma brucei e Leishmania major, ampliando-se assim
as perspectivas de pesquisa por novos fármacos. Em cada um dos genomas cerca de 10.000 genes foram identificados e por volta
de 50 % destes codificam proteínas hipotéticas com função ainda desconhecida. O objetivo do nosso grupo é realizar a
determinação das estruturas tridimensionais destas proteínas, abrindo novos caminhos para a inferência de função (pela
homologia estrutural), ou de novos padrões de enovelamento (auxiliando na modelagem da estrutura de várias proteínas
homólogas). Para o desenvolvimento do projeto genoma estrutural será utilizada como principal ferramenta a espectroscopia de
Ressonância Magnética Nuclear (RMN), que além da determinação estrutural nos permite ainda avaliar a dinâmica da proteína em
solução. Inicialmente foi realizada uma análise bioinformática do genoma de Trypanosoma cruzia a fim de selecionar proteínas de
interesse e que sejam passíveis de serem estudadas por RMN: proteínas específicas de cinetoplastídeos (baixa homologia de
sequência primária com proteínas de mamíferos), conservadas nos genomas dos três tripanossomatídeos citados (alvo comum
contra os três parasitas), menores que 30 kDa, sem regiões transmembrana, e com evidência de expressão (proteína e/ou RNA
detectados). Cerca de 200 genes foram selecionados, e 17 destes foram adquiridos de forma sintética em plasmídeo comercial
pUC 57 através da GenScript. Os genes estão sendo subclonados em vetores de expressão, e as proteínas expressas e purificadas
para preparação de amostras necessárias na aquisição de espectros de RMN. Algumas destas proteínas já foram expressas e se
apresentaram enoveladas como mostrado pelos respectivos espectros de RMN de1H.
Apoio: CAPES, INBEB, CNPq.
D64
BONE MARROW-DERIVED STEM CELLS SUPPORT RENAL RECOVERY AFTER INJURY: A MUTUAL PARACRINE INTERACTION.
1,3- LINDOSO, R.S.; 1,3- ARAUJO, D.S.; 1,3- SANT´ANNA, J.F.; 1,3- ADÃO-NOVAES, J.; 2- FRAGEL-MADEIRA, L.; 1- MARIANTE, R.M.;
1,3- CARUSO-NEVES, C.;1- LINDEN, R.; 1,3- VIEYRA, A.; 1,3- EINICKER-LAMAS, M.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro; 2- Instituto de Biologia,
Universidade Federal Fluminense, Niterói; 3- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem.
During ischemic injury the proximal tubule cells are particularly affected, thus impairing kidney function. Bone marrow-derived
cells (BMDC) and, more specifically, mesenchymal stem cells (MSC) can be mobilized to sites of injury where they play an
important role in tissue recovery. In this study we aimed to investigate the interaction between BMDC/MSC and tubular cells, by
comparing their paracrine potential to protect and to stimulate renal cells proliferation. BMDC/MSC were obtained from murine
and were co-cultured with LLC-PK1 renal epithelial cells using a porous membrane insert (4 µm pore diameter) to separate the two
cell populations, thus allowing communication only by secreted factors. Renal cells proliferation was evaluated by counting viable
cells or by PCNA immunofluorescence analysis. Cell death was determined by picnotic nuclei analysis, activated caspase-3
immunofluorescence or propidium iodide staining. Conditioned media were obtained by culturing BMDC/MSC for 72 h in serum
free medium. The results showed that renal cells proliferated more rapidly when cultured with increasing concentrations of BMDC
to produce a more effective stimulus. MSC are more effective in stimulating renal cells proliferation. The renoprotective effect was
observed by the reduction of apoptosis in the presence of BMDSC/MSC. Again, increasing BMDC amounts decreases cell death,
and MSC leads to a more marked reduction in cell death, indicating their higher paracrine potential. Proliferative and protective
effects were not observed when renal cells were cultured with BMDC/MSC conditioned media. The results indicate that the
crosstalk between BMDSC/MSC and renal cells is mediated by a paracrine mechanism that leads to protection and proliferation of
renal cells.
Support: CNPq; DECIT-MS; CAPES; FAPERJ.
D65
LIPOSSOMAS PARA A LIBERAÇÃO CONTROLADA DE AMILINA HUMANA
1. BRAGA, R.R.; 2. SILVA, D.; 3. GADELHA, G.; 2- SOLA-PENNA, M.; 1- GARCIA, S.; 1- LIMA, L.MAURICIO T.R.
1-Laboratório Química Biológica Medicinal – pbiotech/BiotecFar, Faculdade de Farmácia, UFRJ 2-Laboratório de Enzimologia e
Controle do Metabolismo - LabECoM, Faculdade de Farmácia, UFRJ 3-Instituto Nacional de Metrologia – INMETRO
INTRODUÇÃO: Amilina, também conhecida como polipeptídeo pancreático, é uma proteína de trinta e sete resíduos de
aminoácidos cosecretada com insulina pelas cellulas beta. A variante humana é insolúvel em meio aquoso, podendo formar
depósitos amilóides in vivo e in vitro, e tem seus níveis plasmáticos bastante reduzidos em diabéticos. Visando obter uma
formulação para liberação controlada de amilina humana nativa (hAMYwt), testamos o potencial de uso de liposomos como
sistema de nanoconfinamento do hormônio de modo a promover sua estabilização, evitando a pronta agregação e, sua liberação
controlada in vivo de modo a evitar alta concentração localizada e gradual absorção e distribuição. MÉTODOS: Vesículas
multilamelares constituídas de fosfatidilcolina de soja foram preparadas pelo método de hidratação em filme lipídico. A
preparação foi normalizada em tamanho (400 nm) e o material não incluso foi removido por gel-filtração. A avaliação
farmacológica in vivo foi avaliada por monitoramento da glicemia em camundongos suíços em jejum conscientes e livres, após
administração subcutânea, através de sangue total caudal. A análise morfológica foi realizada por microscopia eletrônica de
varredura utilizando a técnica de criofratura. A Determinação do tamanho de partículas foi realizado por espalhamento de luz
dinâmico. RESULTADOS: Os lipossomas obtidos resultaram em 66 % de incorporação de hAMYwt. A análise microscópica revelou a
estrutura de vesículas multilamelares. A preparação apresentou tamanho médio (MD) de vesículas em torno de 255 nm, com
polidispersividade de 3,9. A avaliação farmacológica da preparação revelou sua atividade hipoglicemiante em comparação com
controle. CONCLUSAO: A preparação lipossomal de amilina apresentou atividade hipoglicemiante sugerindo o potencial uso
terapêutico da preparação desenvolvida.
Apoio: CAPES.
D66
STRUCTURAL STABILITY IN AMYLOIDOGENIC AND NON-AMYLOIDOGENIC VARIANTS OF THE TRANSTHYRETIN PROTEIN BY MD
SIMULATIONS UNDER HIGH PRESSURE.
1,2-OLIVEIRA JR., R.S.; 1,3-FONTES, L.; 1,3-PALHANO, F.L.; 1,3-FOGUEL, D.; 1,2-PASCUTTI, P.G.
1-LMDM, Instituto de Biofísica Carlos Chagas Filho, UFRJ; 2-INBEB/UFRJ; 3-LAPA, Instituto de Bioquímica Médica, UFRJ.
Structural Stability in Amyloidogenic and Non-amyloidogenic Variants of the Transthyretin Protein by MD Simulations under High
Pressure. Reinaldo S. de Oliveira Júnior (PG)†, Liliani Fontes (PG)*, Fernando L. Palhano(PQ)*, Debora Foguel(PQ)*, Pedro G.
Pascutti(PQ)† Biophysics Institute Carlos Chagas Filho – IBCCFº/UFRJ†, Medical Biochemistry Institute – IBqM/UFRJ* – Federal
University of Rio de Janeiro, Rio de Janeiro, Brazil. Key-words: Amyloid, High Pressure, Molecular Dynamics Simulations,
Transthyretin Conformations. Introduction: The formation of insoluble amyloid fibers is a characteristic of many diseases known
as amyloidosis. The transthyretin (TTR) is a homo-tetramer protein of 55 kDa and 127 residues per monomer. Over of one
hundred mutations have been described for the structure of transthyretin related to amyloid diseases such as Familial Amyloid
Polyneuropathy (FAP), characterized by the deposition of amyloid aggregates in the peripheral nervous system. Methodology: The
dissociation and denaturation of TTR variants have been studied in different conditions of temperature, pH and pressure. In the
previous studies our group has described that the non-amyloidogenic variant as T119M has great stability under 3.0 Kbar, pH 7.5,
and 1 °C (ref. 1) compared with amyloidogenic. We used Gromos96_53a6 Force Field, Reaction Field (radius 1.4 nm) in
electrostatic treatment; SPC/E water model, cubical box, 5 ns simulation for stabilization the hydrated layer of protein and ions.
Consecutive simulations totalize 40 ns, 5 ns for each pressure step of 0,5 kbar, temperature of 1 °C, pH 3,0, and pressure
enhancement from 1,0 bar ≅ 1,0 atm to 3,5 kbar. Results and Discussion: Multiple and sequential Molecular Dynamics (MD)
simulations of WT - and V30M-TTR dimmers were performed at high pressure (up to 3,5 kbar) and in explicit water to assess the
structural stability of transthyretin. It was explored the conformational space available to the polypeptide chain upon protein
unfolding, and identified potential structural changes leading to amyloid assembly. The analysis of molecular properties such as
secondary structure, hydrogen bonds, and solvent accessible surface area along the MD unfolding trajectories clearly demonstrate
that V30M-TTR has a much higher tendency to unfold than WT-TTR. These results are in agreement with previously published
experimental data on the conformational stability of dimmers of several TTR variants. References: 1. Foguel D. et al.; J Mol Biol.
(2003); 328(4):963-74. 2. Chiti F, Dobson, CM (2006). Annu Rev Biochem.; 75:333–366. 3. Feller SE, Zhang Y, Pastor RW, Brooks
BR (1995). J Chem Phys 103:4613–4621.
CNPq, INBEB, FAPERJ, CAPES
D67
CRIO-MICROSCOPIA ELETRÔNICA DE VARREDURA COMO FERRAMENTA DE ESTUDO E OBSERVAÇÃO DE AMOSTRAS
HIDRATADAS E CRIO-FIXADAS.
1,2- TRAVASSOS, R.;1-2 LOPES TORRES, E.J.; 1,2- ATTIAS, M.
1- Laboratório de Ultraestrutura Celular Herta Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro; 2- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro;
A preservação de amostras para observação em microscopia eletrônica busca mantê-las o mais próximo do estado in vivo. A
fixação química introduz intrinsecamente artefatos, especialmente decorrentes de variações osmóticas durante a fixação e
desidratação. Assim, é constante a busca pelo desenvolvimento de novas técnicas de preparo e observação de amostras. Neste
sentido, as técnicas que incluem a fixação física das amostras têm apresentado resultados superiores quando comparadas às
técnicas de fixação química: extração de lipídeos reduzida, menor deslocamento de conteúdo citoplasmático, ausência de choques
osmóticos, dentre outras. Além disso há a possibilidade da observação dessas amostras ainda congeladas, permitindo que a
mesma seja observada hidratada, num estado mais próximo do in vivo. Conjugando as técnicas de congelamento por alta pressão,
criofratura e observação em crio-microscopia, observamos a organização intracelular dos parasitos Trypanosoma cruzi e
Toxoplasma gondii e de ovos embrionados de Trichuris muris. A observação das amostras é feita em um microscópio eletrônico de
varredura de emissão de campo que possui um crio-estágio, responsável por manter a amostras sob baixas temperaturas (-120).
Observamos que as amostras apresentam membranas mais lisas, não são observadas células ou organelas com artefatos de
osmovariação, como células murchas ou inchadas e a criofratura das amostras expõe o citoplasma e suas organelas. Entretanto,
por se tratar de uma amostra hidratada, a técnica torna a amostra muito mais sujeita a danos irreversíveis decorrentes da
interação com o feixe de elétrons do microscópio. O problema pode ser minimizado pelo balanço entre o tempo de exposição da
amostra ao feixe e uma baixa aceleração da voltagem. A grande vantagem dessa técnica, se encontra na rapidez com que a
mesma pode ser executada, é possível fazer todo o processo desde o congelamento até a observação das amostras em um mesmo
dia, obtendo resultados de forma rápida e com excelente fidelidade de preservação da amostra.
Apoio: Capes, CNPq, INBEB.
D68
UNDERNUTRITION DURING LACTATION LEADS TO MORPHOFUNCTIONAL AND NA+ RENAL HANDLING ALTERATIONS WITH
ESTABLISHMENT OF CARDIOVASCULAR AND RENAL DISEASES IN ADULT LIFE
1,2- LUZARDO R.L.; 1,2- SILVA, P. A.; 4- VIEIRA-FILHO, L. D.; 1,2- EINICKER-LAMAS, M.; 3- Lara, L.S.; 1,2- VIEYRA, A.
1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Instituto Nacional de Ciência e Tecnologia
de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro; 3- Instituto de Ciências Biomédicas, Universidade
Federal do Rio de Janeiro; 4- Departamento de Fisiologia e Farmacologia, Universidade Federal de Pernambuco.
Several studies have correlated maternal undernutrition during lactation witch cardiovascular and renal diseases in adulthood,
giving support to the programming hypothesis. This thesis aimed to investigate programming-induced morphofunctional
alterations intrarenal tissue with the late onset of hypertension in adult life. Dams received a hypoproteic (8%) diet throughout
lactation. Control and programmed offspring consumed a diet containing 20% protein after weaning. Programmed rats aged 60
days present with reduced: (i) number of nephron (35%), (ii) area of bowman’s capsule (30%) and (iii) capillary tuft (30%). These
alterations were accompanied by increased RFG (70%) and proteinuria (400%), reveling a serious damage in the filtration barrier.
Increased collagen deposition (175% in cortex; 700% in medulla) and expanded cortical interstitium (130%) were the other
evidences of tissue damage. Functional alterations were increased urinary excretion of Na+ (80%), K+ (100), urea (200%) and
water (50%). At a molecular level, programmed rats presented with hypometilation of the promoter region of the Agt1a gene the
codifies for the AT1a type of Ang II receptors. Late onset of hypertension (35% increase in mean arterial pressure) followed a
progressive increased in plasma volume (16%). Early investigation (at weaning) of alterations in Na+-transporting ATPases revealed
a 50% and 75% increased in (Na++K+)ATPase and ouabain-insensitive Na+-ATPase, respectively. Expression of AT1 and AT2 type
Ang II receptors increased 40% and decreased 17%, respectively. PKC – that couples AT1 receptors to Na+-transporting ATPases
also increased 50% in programmed animal, together with O2 free radical species. Late hypertension in programmed rats gives
support to the idea that enhanced Na+ reabsorption – despite compensatory distal loss – and of its regulatory machinery, K+
depletion, plasma volume expansion and the serious morphometric alterations are critical events that culminate with
hypertension and possible renal illness.
Support: INBEB, MCT-CNPq-MS- DECIT-FAPERJ , CAPES-PROCAD.
D69
LIPID DETECTION IN MICROALGAE GROWN UNDER DIFFERENT LIGHT INTENSITIES
Pinto, R.F. 1, 2, Sant´Anna, C.1, Martins, J.L. 1, Azevedo, S.M.F.O. 2, Bessa, L. B. 2, Miranda, C.T. 2, Vinícius, D. 2, De Souza, W.1, 2
1 LABIO,DIPRO,INMETRO, Rio de Janeiro, Brazil
The environmental impact of fossil fuels and the increasing necessity for renewable energy have stimulated researches in biodiesel
production from microalgae. These microorganisms have a fast growing rate, high photosynthetic efficiency and they are able to
accumulate lipids. Recently, studies are focused on microalgae field analyzing the presence of triacylglicerol, a neutral lipid
important for biodiesel production. The aim of our work was to compare the lipid production in three microalgae genera
(Ankistrodesmus sp., Scenedesmus sp. and Chlorella sp.) under different light intensities. Microorganisms isolated from fresh
water were grown in ASM-1 medium, with an artificial illumination photoperiod of 12 h with intensities of 200, 500 and 1000 µmol
photons m2.s-1. Triacylglycerol detection was done with Nile Red (NR), a lipophilic fluorescent dye, used as a rapid screening
method for lipid production in oleaginous microorganisms. Quantitative measurement of lipid content was evaluated by
fluorimetric assays in conjugation with confocal microscopy. NR showed affinity for the lipid content in the microalgae tested,
indicating the presence of neutral lipids. Fluorimetric analysis showed that Ankistrodemus sp, exhibit the highest lipid content
when compared to Scenedesmus sp and Chlorella sp. Lipid production in Ankistrodesmus sp was confirmed by confocal
microscopy of the NR stained cells, where the highest amount of lipid droplets was observed disperse in the cytoplasm. When
algae were submitted to variable light intensities, results showed that 500 µmol photons m2.s-1 was the most effective intensity
to increase the lipid content in Ankistrodesmus sp. This analysis could be a standard method to evaluate microalgae lipid
production and it could be applied in studies focused on biodiesel production.
Support: CNPq, FAPERJ, INBEB, INMETRO.
D70
ULTRAESTRUTURA DAS VESÍCULAS DE “SHEDDING” LIBERADAS PELAS FORMAS AMASTIGOTAS INTRACELULARES DO
TRYPANOSOMA CRUZI
1-NEVES, R.F.C.; 1-SOUTO-PADRÓN, T.
1-Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro
Eventos de vesiculação ocorrem tanto na membrana plasmática (vesículas de shedding) ou no interior de estruturas endossomais
(exossomos) e a liberação por patógenos podem conter fatores de virulência podendo alterar a resposta imune ou ação de células
hospedeiras. Tripomastigotas do T. cruzi, liberam constitutivamente vesículas de 30-300nm dependendo de energia, temperatura,
tempo e cepa ou clone. Estas vesículas são denominadas vesículas de shedding e foram descritas inicialmente na forma
tripomastigota do parasito. Nesse trabalho, analisamos a liberação de vesículas de shedding por formas amastigotas intracelulares
do clone CL-Brener do T. cruzi. Células hospedeiras 4-5 dias pós-infecção foram fixadas e processadas para análise por microscopia
eletrônica de transmissão (MET) e varredura (MEV). Por MET observamos formas amastigotas intracelulares liberando vesículas de
40-170nm com uma camada de glicocálice espessa semelhante à superfície das formas amastigotas, bem como vesículas com um
coat menos denso e mais regular de aproximadamente 40nm no interior e proximidades da bolsa flagelar do parasito. Para
observamos o conteúdo citoplasmático da célula hospedeira por MEV, realizamos a técnica de clivagem a seco que consiste na
secagem de células aderidas pelo método do ponto crítico seguida da remoção da membrana com o auxílio de uma fita adesiva e
metalização com ouro (10 nm de espessura) e observação ao microscópio de varredura FEI Quanta. Observamos no citoplasma da
célula hospedeira, amastigotas apresentando vesículas aderidas no corpo celular e região de saída da bolsa flagelar. Vesículas,
possivelmente liberadas por esses parasitos, também foram observadas livres no citoplasma e aderidas ao citoesqueleto da célula
hospedeira. Assim como as formas tripomastigotas, as amastigotas também parecem liberar constitutivamente essas vesículas e
que poderiam de certa forma participar no processo de infecção.
Apoio: INBEB,CNPq, CAPES, FAPERJ, PRONEX.
D71
EVALUATION OF NEUROCOGNITIVE DISORDERS IN HIV ASYMPTOMATIC BRAZILIAN PATIENTS: NEUROLOGICAL EVALUATION,
CONVENTIONAL MAGNETIC RESONANCE IMAGING AND INFLAMMATORY MEDIATORS
AMANCIO RT, MOTTA E, LIMA D, SILVA MT, GRINSZTEJN, TOVAR-MOLL F, BOZZA FA
Instituto de Pesquisa Clínica Evandro Chagas; Instituto D'Or de Ensino e Pesquisa; INCT/INBEB - Laboratório de Inflamação e
Metabolismo
BACKGROUND With recent increase in survival of HIV patients clinicians face new challenges, including the control of chronic
diseases. Therefore cognitive dysfunction is now a significant cause disability in HIV patients. The prevalence of neuro-cognitive
disorders is not well-established in low and middle income countries as Brazil. The pathophysiological mechanisms involved in
neurological changes in HIV-infected patients are not well established. Current evidence suggests that cognitive changes in HIV
patients result from a neuronal dysfunction resulting from the combined action of viral proteins, activation of glia and mocroglia,
and inflammatory mediators. METHODS Sixty asymptomatic seropositive patients, not exposed to HAART, were included in a
prospective cohort-study of a Regional Reference Center for HIV treatment. Patients were submitted to clinical, laboratorial and
3.0 Tesla brain magnetic resonance imaging (MRI) evaluation, including single voxel proton spectroscopy in two different regions
(posterior cingulum and frontal lobe). Fourteen age-matched healthy volunteers (HV) were included as a control group.
Spectroscopy metabolites of interest included N-acetylaspartate (NAA), creatine (Cre), choline (Cho) and myo-Inositol (mI). Mann
Whitney test was used for group comparisons (p<0.05). The HIV-associated dementia diagnosis was based in criteria of American
Academy of Neurology AIDS Task Force. Blood samples were collected for measurement of inflammatory mediators in the
inclusion. RESULTS Patient`s mean CD4 count was 597 cells/mm3 (IQR: 434-806). 63% has no cognition alteration; 26% was
observed mild cognitive impairement and 11% was diagnosed HIV-associated dementia. Analysis of spectroscopy metabolites did
not show difference between groups (HIV vs non-HIV). CONCLUSION Our cohort has 11% prevalence of HIV-associated dementia.
Conventional MRI and spectroscopy did not shown signifcant brain changes in a HIV assintomatic middle-low income population,
without prior use ARV. Additional non-coventional MRI tecniques might be necessary to detect changes in the normal apparent
brain tissue in assintomatic HIV population. We are analysing the inflammatory markers and its use as possible biomarkers and its
role in neurocognitive disorders in HIV population.
Support: INBEB; CNPq, FAPERJ, FIOCRUZ.
D72
HOW CO-EVOLUTION OF NELFINAVIR-RESISTANT HIV-1 PROTEASE AND THE CLEAVAGE SITE CONTRIBUTE FOR BETTER
CATALYTIC EFFICIENCY
1- SOARES, R. O.; 2- BERNARDI, R. C.; 1- PASCUTTI, P. G.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Instituto Nacional de Metrologia.
Introduction: The HIV protease (HIV-PR) is an important target for development of therapies antiretroviral (ARV) in the treatment
against AIDS. However The emergence of resistance has been a major concern in the ARV treatment that use protease inhibitors
(PIs). The mutation in the Gag and Gag Pol cleavage sites may also to occur together to the resistance mutation in the protease, a
mechanism so called co-evolution. Further studies had shown that mutations in the p1-p6 substrate co-evolve with the
D30N/N88D drug-resistant mutation in the HIV-1 protease (HIV-PR). However a atomic level, it is not clear how theses mutations
affect the interaction between HIV-PR and p1-p6 substrate. To elucidate that, we carried out molecular dynamics (MD) studies for
both wild-type and D30N/N88D mutant of the HIV1-PR, investigating the binding of the p1-p6 substrate, as well as the binding of
the substrate p1-p6 with Lp1’F mutation to the HIV-PR mutant. Methods and Results:The three system suggested Prwt-SubWt,
PRD30N/N88D-SubWt and PRD30N/N88D-SubLp1'F were simulated in an aqueous environment for 100ns using the GROMACS
package. Analysis of the residue fluctuation suggests that the mutation in HIV-PR together with the mutation on substrate
decreased meaningful the structure flexibility of the HIV-PR. Furthermore, analysis of the prevalence of hydrogen bonds shows
that two of these bonds were less stable, about half of time, when only the HIV-PR was mutated. Analysis of the surface of
interaction between HIV-PR and p1-p6 shows a lower value in the region P1’ for the PRD30N/N88D-SubWT system. In addition,
analysis of catalytic residue/substrate distance indicates that the dyad catalytic in the co-mutant system is closer to the central
region of the substrate. Conclusion: These findings suggest that the mutation in the cleavage site p1_p6 might be a molecular
mechanisms to better the efficiency of the catalysis when the HIV-PR present the mutation D30N/N88D.
Support: CNPq, CAPES, FAPERJ, INBEB.
D73
STUDIES OF THE IMMUNOGENIC CAPACITY OF AVIAN INFLUENZA VIRUS (H3N8) INACTIVATED BY HIGH HYDROSTATIC PRESSURE
BARROSO, S.P.C.1, NICO, D.2, VICENTE, A.C.S.1,COUCEIRO, J.N.S.S.2, PALATNIK-DE-SOUSA, C.B.2, SILVA, J.L.1 & OLIVEIRA, A.C.1
1Instituto de Bioquímica Médica - Universidade Federal do Rio de Janeiro (UFRJ);2Instituto de Microbiologia Professor Paulo de
Góes - UFRJ, Rio de Janeiro, Brasil
H3N8 is an avian influenza virus that was originally isolated from birds, later found in horses and dogs. Here, we used 12 hours of
incubation under high hydrostatic pressure (HHP) for virus inactivation without damage to its hemagglutinin and neuraminidase
activities. Our goal is to assess the immunogenic and protective capacity of pressurized virus with and without adjuvant (saponin)
in mice. For such Balb / c mice were treated by the intranasal route, with 3 doses of 104.5 TCID50 of virus, or 104.5 TCID50 virus +
saponin, saponin or saline. After vaccination the mice were challenged and monitored for: virus-specific antibodies (ELISA), CD4+
and CD8+ virus-specific T cells producing cytokines (double staining) and clinical symptoms. After immunization there was an
increase of IgG1 and IgG2a in the serum of viruses and virus groups + saponin. In these same groups had increased production of
IgA in nasal lavage. The cell analysis shows increased production of IL-6 and IFNincrease in the production of antibodies and interleukins 2, 4, 6, and TNFmore clinical signs of disease (lethargy, weight loss and huddling) that vaccinated animals (viruses and virus + saponin). The results
indicate that the animals are having a satisfactory response after vaccination and be protected against challenge. Currently we are
checking whether antibodies produced are neutralizing. We will also evaluate viremia in mice and if the immune response is long
lasting. Our work reaffirms the use of HPP with a tool in the development of viral vaccines at low cost and good immune response.
Support: CAPES, PRONEX, INBEB, CNPQ, FAPERJ.
D74
SÍNTESE, CARACTERIZAÇÃO E AVALIAÇÃO ANTIBACTERIANA DE GUANIL HIDRAZONAS DERIVADAS DO FENANTRENO.
1-DE AZEREDO, S.O.F.; 1-HABU, S.;1- FIGUEROA-VILLAR, J.D.
1-INSTITUTO MILITAR DE ENGENHARIA
As guanil hidrazonas são formadas por um grupo de compostos com diversas estruturas, sendo caracterizadas por conter o grupo
amidina (guanil) ligado ao grupo hidrazona. As guanil hidrazonas são moléculas amplamente estudadas biológica e quimicamente.
Entre as diversas ações biológicas conhecidas temos depressores do sistema nervoso central, antihipertensivos, inibidores da
agregação plaquetária, antitumorais, antileucêmicos, antivirais, bactericidas, antimalariais. Neste trabalho foram sintetizadas,
caracterizadas e testadas duas guanil hidrazonas derivadas do fenantreno frente às bactérias E. coli IB – ATCC 35218 e Klebsiella
pneumonia. As estruturas dos compostos foram caracterizadas por Pf, IV, RMN de ¹H e ¹³C. Das duas guanil hidrazonas
sintetizadas e testadas, a que apresentou maior potencial antibacteriano foi a guanil hidrazona da 9,10-fenantrenoquinona. Sendo
assim, será feito um CMI para determinar a menor concentração capaz de inibir o crescimento bacteriano frente às cepas da E. coli
IB – ATCC 35218, a qual apresentou um maior halo de inibição.
Apoio: INBEB, MINISTÉRIO DA DEFESA, CAPES.
D75
DIETHYLCARBAMAZINE (DEC) REDUCES INFLAMMATORY MARKERS IN LIVER ALCOHOL-INDUCED IN C57BL/6 MICE
1-Rocha, S.W.S., 1-Silva,B.S, 1-Gomes, F.O.S., 1-Silva, A.K.S., 1-Barbosa, K.P.S., 3-Raposo, C., 2-Santos, A.C.O, 2-Torres, D.O.C., 1,3Nunes, A.K.S. 1,3-Peixoto, C.A.
1-Laboratório de Ultraestrutura, Centro de Pesquisas Aggeu Magalhães – FIOCRUZ-PE. 2- Laboratório de Bioquímica, Universidade
de Pernambuco. 3-Laboratório Microscopia e Microanálise, Centro de Tecnologia Estratégicas do Nordeste.
Alcoholic liver disease (ALD) is a collective term for the pathophysiological changes caused by chronic alcohol consumption, which
include oxidative stress generation, liver steatosis and inflammatory response fibrosis, and cirrhosis. Several pharmacotherapeutic studies have been undertaken to cure alcoholic hepatitis. However, these treatments are associated with an increase
of infections and death. Some pharmacological studies showed that diethylcarbamazine (DEC) interferes with the arachidonic acid
metabolism, acting as an anti-inflammatory drug, blocking in both cyclooxigenase (COX) and lipoxygenase pathways. The present
work analyzed the anti-inflammatory effect of DEC on hepatic cells of alcoholic mice. Thirty-two male C57BL/6 mice were equally
divided in the following groups: a) DEC- treated group, which received 50 mg/Kg for 12 days (DEC50); b) the control group (C),
which received only pure water, c) the alcoholic group (EtOH), submitted to alcohol for 5 weeks; and d) the alcohol-DEC treated
group (EtOH50), submitted to DEC treatment after the induction of chronic alcoholism. Biochemical analyses were performed and
liver fragments were processed for light microscopy and transmission electron microscopy, immunohistochemical and western
blot. The level of AST increased significantly in alcoholic group compared with the control group, conversely a significant reduction
of serum AST was detected in the EtOH50 group. Histological analysis of alcoholic group showed evident hepatocellular damage,
which was strikingly reduced in the alcoholic DEC-treated group. Ultrastructural analysis confirmed the histopathological data as
well-preserved organelles were observed in the EtOH50 group. Immunohistochemistry results revealed highly expression of
inflammatory markers as IL-6, VCAM and ICAM by the hepatic cells of the EtOH group; however no immunoreactivity for any of
these cytokines was detected after DEC treatment. Western blot analyses showed increased MCP-1 and iNOS expression in EtOH
group, which was significantly inhibited by DEC treatment. According to the present results, DEC is a potential drug for the
treatment of chronic inflammation induced by chronic alcoholism.
Support: FACEPE, INBEB, CNPq.
D76
NESTIN-GFP TRANSGENE REVEALS THE PRESENCE OF MULTIPOTENT STEM CELLS IN ADULT HEART
1- ABREU, S.K.; 1- CARVALHO, A.B.; 1- DEL CORSSO, C.; 3- FRENETTE, P.S.;1,2- CAMPOS DE CARVALHO, A.C.; 4- SPRAY, D.C.
1- Institute of Biophysics Carlos Chagas Filho, UFRJ; 2- National Institute of Cardiology, Rio de Janeiro; 3- Ruth L. and David S.
Gottesman Institute for Stem Cell and Regenerative Medicine Research, Bronx, New York; 4- Dominick P. Purpura Department of
Neuroscience, Bronx, New York
Nestin, a class VI intermediate filament protein is used to identify neuronal precursor cells and constitutes most of the
cytoskeleton. Many investigators consider detection of nestin expression as a proof of identification of stem/progenitor cells. The
presence of nestin cells has been reported in the bone marrow, dermis, hair follicles and other tissues. The goal of this work was
to verify if there are nestin+ cells in the heart of Nes-GFP transgenic adult mice (Nestin-GFP) and if these cells exhibit
characteristics of multipotent stem cell population. For that purpose, the primary cells were isolated of adult heart mice (NestinGFP) after digestion of small pieces of the heart in 420U/ml collagenase II for 20 minutes at 37ºC. Experimental procedures were
approved by the Animal Care and Use Committee of Mount Sinai School of Medicine. The cardiac cells expressing the green
fluorescent protein (GFP) under the control of the nestin promoter constitute a non-haematopoietic cell population (44,14 ±
3,72% of the CD45- population, n=5) by fluorescence-activated cell sorting (FACS). These cells were positive for Stem Cell Antigen1 (Sca-1) representing the majority of nucleated cells (97,94 ± 0,36%). A rare subset of Nes-GFP+ cells also expressed the receptor
for Stem Cell Factor, c-kit (0,008 ± 0,004%) in isolation and c-kit/Sca-1 simultaneously (0,48 ± 0,05%). To evaluate the clonogenicity
of Nestin-GFP cells, these cells were sorted and plated at low density in plates treated with poli- HEMA. After 7 days in culture,
both Nestin-GFP+/Sca-1+ cells (n= 18) and Nestin-GFP+/c-kit+/Sca-1+ formed clonal spheres and generated secondary spheres
after dissociation, which was not observed in Nestin-GFP+/Sca-1-/c-kit- cells. To study their differentiation potential, the NestinGFP+ cells sorted were subjected to conditions that induce the adipogenic, osteogenic, and chondrogenic differentiation.
Differentiated phenotypes of Nestin-GFP+/Sca-1+ and Nestin-GFP+/Sca-1+/c-kit+ cells were observed after staining with Oil red O
(adipocytes), Alizarin red (osteoblasts) and Alcian blue (chondrocytes), results not observed in Nestin+/Sca-1-/c-kit- cells. Our
studies suggest that nestin+cells are clonogenic with self-renewal capacity and multipotent in vitro.
Support: Ministério da Saúde, NIH, CNPq, CAPES, FAPERJ.
D77
PROTEIN PREDICTION COMBINING GENERALIZED SIMULATED ANNEALING AND MOLECULAR DYNAMICS
1- FERNANDES, T. V. A.; 1- PASCUTTI, P. G.
Instituto de Biofísica, Universidade Federal do Rio de Janeiro
The amino acids sequence defines not only its three-dimensional structure, but also its function. Thus understanding the
relationships between sequence and structure remains a primary focus in structural biology. Advances on genome mapping and
sequencing are producing an exponential number of amino acid sequences of new proteins, and the comprehension of these
protein structures becomes a crucial extension to these progresses. This makes protein structural prediction (PSP) a central
problem for the development of post genomic era. Currently, the determination of protein structures is achieved through two
main experimental techniques: X-ray Crystallography and Nuclear Magnetic Resonance (NMR). However, due to limitations in
these techniques have been developed several PSP methods in the past few years. A combined of Generalized Simulated
Annealing (GSA) and Molecular Dynamics (MD) was used in this work. Initially, we used the GSA to enhance conformational
sampling, seen that it is a powerful method for this. The simulations start at high temperature to overcome barriers followed by
gradual cooling to reach low energy structures. Once approximately predicted, the three dimensional structure can be refined by
MD simulations. This new approach was tested initially with two folds models, the mastoparan-X, a peptide of 14 residues that
folds in alpha-helix conformation in water-TFE environment, and the Trp-cage, a 20 residues mini protein used in several folding
studies. The results showed that starting from extended structures, the proteins folds to conformations with RMSD about 3Å,
using GSA. For the MD refinement we used the lowest energy structure found by GSA. After of 200ns MD optimization for
mastoparan-X and 1ms for Trp-cage the RMSD decreases to about 1 and 2Å respectively. Therefore the use of GSA followed by MD
methods proved to be a powerful strategy for the small proteins prediction making it attractive to use larger proteins.
Support: CNPq, FAPERJ and INBEB
D78
ISOLATION OF CLONOGENIC C-KIT POSITIVE CELLS FROM HUMAN HEARTS
TAIS H. KASAI-BRUNSWICK1; RAIANA A. Q. BARBOSA1; ANDREA R. DA COSTA1; JULIANA A. PASSIPIERI1; BRUNO D. PAREDES1;
CRISTIANE DEL CORSSO1; REGINA C. S. GOLDENBERG1; ANTONIO CARLOS CAMPOS DE CARVALHO1; ADRIANA BASTOS
CARVALHO1
1Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Introduction: Cardiac stem cells in the adult human heart have recently been described as c-kit positive cells and their isolation
and expansion constitute a great challenge for future clinical trials. Objective: It was to obtain human c-kit positive cells with high
purity using an efficient methodology. Methods and Results: Discarded human myocardial samples were submitted to enzymatic
digestion with collagenase type II (0.4%). Plating of the digestion product resulted in growth of adherent fibroblast-like cells in
monolayer. The percentage of c-kit+CD45- cells in these monolayers was 0.13±0.05%. CD45 expression was used to exclude the
presence of bone marrow-derived cells. Enrichment of c-kit positive cells was attempted by two methods: a) sorting using
magnetic beads conjugated to anti-c-kit antibodies and b) fluorescence activated cell sorting. Sample purity was checked by flow
cytometry immediately after sorting. We obtained a 4.1±0.09% enrichment of c-kit positive cells by magnetic sorting and
99.2±0.1% by FACS. Therefore, FACS was used to separate cells in all other experiments. Cells were plated after sorting and the
expression of c-kit was confirmed by confocal microscopy after 1 passage. All cells remained c-kit positive. Cells were then
submitted to single cell sorting into 96-well plates in order to evaluate their clonogenic potential. Surprisingly, we obtained 398
clones from 768 wells (51.83%). We are currently expanding these clones to perform IMF and check for the presence of the stem
cell factor receptor c-kit in the cloned cells. Conclusion: Although c-kit positive cells constitute a rare population in the adult
human heart, we were able to separate these cells successfully by FACS. Clonogenic potential of c-kit positive cells after single cell
sorting was extremely high, although this result is still preliminary and requires further investigation. It remains to be defined if
these cells can be stably expanded in vitro and differentiated into cardiomyocytes.
Support: Capes, CNPq, Finep, Faperj, Ministério da Saúde.
D79
BIOLOGY AND CYSTOGENESIS OF A BRAZILIAN TOXOPLASMA GONDII STRAIN.
T. C. PAREDES-SANTOS1,2*, E. S. MARTINS- DUARTE1,2, R.W.A. VITOR3, W. DE SOUZA1,2,4, M. ATTIAS1,2 AND R.C. VOMMARO1,2
1-Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho , 2- Instituto Nacional de Ciência e Tecnologia
em Biologia Estrutural e Bioimagem, 3-Universidade Federal de Minas Gerais, Departamento de Parasitologia, Instituto de Ciências
Biológicas, 4- Instituto Nacional de Metrologia, Normalização e Qualidade Industrial
Toxoplasma gondii is the protozoan agent of toxoplasmosis. The tachyzoites forms reside in a parasitophorous vacuole (PV) and
possess a fast cell division cycle, which is the cause of the tissue damage in acute stage. The bradyzoite is a latent state, replicates
slowly and resides in intracellular cyst, which maintains the parasites protected from the immune destruction by undetermined
time. Thus, the conversion between the two stages is a crucial step for the progression of the toxoplasmosis. In this work we
employed microscopy tools to evaluate the cystogenesis and the behavior of the Brazilian strain of T.gondii, EGS, which was
isolated from human amniotic fluid. Host cells of the lineage LLC-MK2 were infected with parasites from the supernatant of
infected cell cultures. To evaluate the percentage of cyst formation along 12 days of infection, cells were stained with DBA-FITC,
which recognizes the cyst wall and the anti-SAG1, which recognizes the tachyzoite surface. The samples were observed in an
Axioplan Zeiss Microscope or in a Leica Confocal Microscope SP5. Electron microscopy (TEM and SEM) was used to evaluate the
formation of cyst wall and matrix along the 12 days of infection. Tachyzoites of the EGS strain encysted spontaneously in the cell
cultures. The peak of cyst conversion appeared after 4 days of infection, when 30% of the infected cells observed contained cysts.
The majority of infected host cells contained both PVs and cysts. The anti-BAG-1 was used to specifically recognize the bradyzoite
form, permitting the discerning from immature to totally differentiated cysts. By TEM we observed that the thickness and electron
density of the cyst wall increased according to the maturation level of the cysts. A dense cyst matrix was also observed, confirming
that this strain forms cysts spontaneously in cell culture, as had been observed in mice. The EGS strain has shown to be an useful
in vitro tool to obtain and to study the behavior of T. gondii cysts INBEB, FAPERJ, CNPq, CAPES, PRONEX
D80
USO DE RMN PARA AVALIAÇÃO DE METABÓLITOS SALIVARES: COMPARAÇÃO ENTRE INDIVÍDUOS SAUDÁVEIS E COM CARIE
DENTAL
1-FIDALGO, T.K.S.; 1- FREITAS-FERNANDES, L.B; 1- PEREIRA, L.; 2- MUNIZ, A.M.; 3- ALMEIDA, F.; 3- VALENTE, A.P.; 1- SOUZA, I.P.R.
1- Departamento de Odontopediatria e Ortodontia, Faculdade de Odontologia, Universidade Federal do Rio de Janeiro; 2- Núcleo
de pós-graduação, Escola de Educação Física do Exército Brasileiro - EsEFEx; 3- Centro Nacional de Ressonância Magnética Nuclear
Jiri Jonas, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro.
O interesse pelo estudo dos metabólitos salivares é cada vez mais crescente e envolve ciência básica e clínica médicaodontológica. O objetivo do presente estudo foi avaliar o metaboloma salivar de indivíduos em diferentes faixas etárias. A amostra
foi constituída de 71 indivíduos saudáveis e 15 crianças com cárie dental. Coletou-se 0,5 ml de saliva total não estimulada de
bebês (meses médios= 9,5 ± 7,3; n=12) e 5ml de crianças em dentição decídua (idade média= 4,27 ± 1,27; n=16), crianças em
dentição mista (idade média= 7,94 ± 2,09; n=18), crianças em dentição mista com cárie dental (idade média= 7,20 ± 2,01; n=15),
adolescentes em dentição permanente (idade média= 10,88 ± 1,05; n=15) e adultos em dentição permanente (idade média= 26.0
± 2,2; n=10). As amostras foram centrifugadas por 1 hora a 10.000g a 4ºC. Os sobrenadantes foram submetidos à RMN e
obtivemos espectros de 1H (Bruker 400 MHz). Para avaliar se houve distinção entre os grupos, utilizou-se a Análise dos
Componentes Principais (ACP), e para avaliar as intensidades de cada metabólito foi utilizado o ANOVA e o teste de Tukey
(p<0,05). A ACP não demonstrou diferença entre os grupos etários saudáveis avaliados. O teste de Tukey demonstrou maior
intensidade de acetato (p=0,046), propionato (p=0,036) e ácido graxo (p=0,033) em adolescentes em comparação aos bebês. ACP
demonstrou tendência à separação do grupo não careado do careado em alguns componentes. Demonstrou-se maior intensidade
no grupo sem cárie de galactose (p<0,001), glicolato (p<0,001), glucose (p=0,012), fosfocolina (p=0,029), taurina (p=0,029),
succinato (p<0,001), glutamato (p=0,025) e hidroxibutirato (p=0,019), já o grupo com cárie apresentou maior intensidade dos
metabólitos 4-hidroxiprolina (p=0,025), glucose (p=0,025), sacarose (p=0,025), n-butirato (p<0,001) e lactato (p=0,025). Conclui-se
que a homogeneidade entre os grupos de pacientes saudáveis e mudança no perfil metabólico em pacientes com cárie dental.
Apoio: FAPERJ, CNPq.
D81
CAPTAÇÃO DE FOSFATO INORGÂNICO EM LEISHMANIA CHAGASI: CARACTERIZAÇÃO, EXPRESSÃO E REGULAÇÃO DO
TRANSPORTADOR DE FOSFATO INORGÂNICO
1,3,4-RUSSO-ABRAHÃO, T., 1,3,4- DICK, C.F., 2,5, ALVES-BEZERRA, M. 2,5- MAJEROWICZ, D. 2,5- GONDIM, K.C., 2,3,4-MEYERFERNANDES, J.R.
1-Instituto de Microbiologia Professor Paulo de Góes, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, RJ, Brasil 2-Instituto de Bioquímica Medica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, RJ, Brasil 3-Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Centro de Ciências da Saúde,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil 4-Laboratório de Bioquímica Celular, Instituto de Bioquímica
Médica 5-Laboratório de Bioquímica e Fisiologia de Insetos
Neste trabalho, mostramos, pela primeira vez, a presença de um transportador de fosfato inorgânico (Pi) em Leishmania chagasi,
protozoário causador de leishmaniose visceral. O transporte de Pi aumenta de acordo com o tempo e o número de células, no
entanto, não há modulação por variação do pH. O transporte de Pi mostra uma cinética de Michaelis-Menten com valores de K0,5
e Vmáx de 0,016 + 0,002 mM e 9,415 + 0,301 pmol x min-1 x 10-7 células, respectivamente. Esses valores incluem o transportador
de Pi de L. chagasi no grupo de transportadores de alta afinidade, como os conhecidos Pho89 e o Pho84 presentes em
Saccharomyces cereviseae. FCCP, um conhecido ionóforo de prótons, e valinomicina, um ionóforo K+, inibem o transporte de Pi.
Além disso, sabe-se que as mudanças ambientais são percebidas como sinais pelas células, o que gera respostas na expressão e
atividade de proteínas. Com base no genoma conhecido de Saccharomyces cerevisiae, encontramos uma seqüência homóloga a
PHO89 no genoma de L. major. A proteína codificada por essa seqüência, Pho89, tem grande afinidade para Pi e é modulada
(tanto a atividade da proteína, como a expressão gênica) por variações ambientais de Pi. Esses fatos levaram-nos a crer que o
mesmo ocorre para L. chagasi. Células cultivadas por 3 dias em baixa concentração de Pi, têm maior taxa de transporte de Pi e de
expressão do gene PHO89 do que as células cultivadas por 3 dias em alta concentração de Pi, enquanto as células cultivadas por 6
dias em condição de baixa concentração de Pi têm maior taxa de transporte de Pi e de expressão do gene Pho89 do que as células
cultivadas por 6 dias na presença de altas concentrações de Pi. Além disso, as células cultivadas por 3 dias têm maior expressão do
gene PHO89 do que células cultivadas por 6 dias, tanto na concentração baixa de Pi quanto em alta. Esses dados indicam a
importância do transportador de fosfato inorgânico para o crescimento de células em diferentes condições ambientais. Os
resultados aqui descritos confirmam a presença de um transportador Pi em L. chagasi, capaz de contribuir para a aquisição de
fosfato inorgânico, o crescimento e a sobrevivência de formas promastigotas de L. chagasi.
Apoio: CNPq, CAPES e FAPERJ.
D82
ATIVIDADE ECTO-FOSFATÁSICA DA MICROALGA EUGLENA GRACILIS
1,2,3- SILVEIRA, T.S.; 2,3- MEYER-FERNANDES, J.R.
1- Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro; 2- Instituto de Bioquímica Médica,
Universidade Federal do Rio de Janeiro; 3- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem,
Universidade Federal do Rio de Janeiro.
As ecto-enzimas são enzimas de membrana com seu sítio catalítico voltado para o meio extracelular, e não para o citoplasma. As
ecto-fosfatases são um grupo de ecto-enzimas capazes de gerar fosfato inorgânico a partir de substratos fosforilados. Atualmente,
diversos parâmetros da biologia de Euglena gracilis são analisados em bioensaios de toxicidade, entretanto, ainda não foi definido
um parâmetro ideal. Aqui, caracterizamos uma atividade ecto-fosfatásica de E. gracilis visando seu potencial uso como ferramenta
em ensaios toxicológicos. A atividade ecto-fosfatásica foi determinada em ensaios colorimétricos medindo-se a taxa de pnitrofenolato (p-NP) produzido pela hidrólise do substrato artificial p-nitrofenolfosfato (p-NPP). Células íntegras foram incubadas
por 1 h a 25°C em meio de reação contendo 15 mM de tampão MES e 5 mM de p-NPP. Diferentes pHs foram testados, e os
experimentos foram realizados no pH do início do cultivo (5,5), exceto quando citada outra condição. Outros compostos, como
inibidores de fosfatases e metais foram adicionados a esse meio para caracterizar essa atividade. E. gracilis possui uma atividade
de 2,32 ± 0,96 nmol p-NP/(10e6 células•h), que é estimulada em três vezes por cobre e inibida em 50 % por zinco,
aproximadamente. A hidrólise do substrato foi linear com o tempo por pelo menos 1 h e com o aumento da densidade celular até
pelo menos 5•106 células. A atividade enzimática é ácida, com a redução de cerca de 80 % da hidrólise em pH 7,0 comparado com
pH 4,0. Os inibidores de fosfatase NaF, vanadato e molibdato, assim como o fosfato inorgânico, produto da reação, foram capazes
de inibir essa atividade. Os fluoretos de alumínio e lítio foram capazes de inibir a atividade enzimática de forma dose-dependente.
Esses resultados sugerem que essa atividade pode ser inibida por outros metais pesados, um dos poluentes mais comumente
testados por parâmetros biológicos de E. gracilis.
Support: CNPq, CAPES, FAPERJ, INBEB.
D83
THE DNA CLEAVAGE PROMOTED BY A NEW EUROPIUM(III) COMPLEX IS STRICTLY DEPENDENT ON UV-A LIGHT IRRADIATION
1- SILVA, P.R.; 1- CAVALETT, A.; 1- BORTOLOTTO, T.; 2- CONTE, G.; 2- GALLARDO, H.; 1- TERENZI, H.
1-Centro de Biologia Molecular Estrutural, Departamento de Bioquímica; 2-Departamento de Química, Universidade Federal de
Santa Catarina, Brazil.
Rare earth ions and their complexes are able to hydrolyze nucleic acids and oligonucleotides with great efficiency. However,
recent studies have shown that lanthanide complexes can also promote the cleavage of DNA by production of reactive oxygen
species (ROS) under photo-irradiation, which is unexpected for this class of metal ions. Herein we report the ability of the
europium(III) complex, Eu(tdzp)(amfpp)3 (where tdzp is [1,2,5]thiadiazolo[3,4-f][1,10]phenanthroline and amfpp is 4-acyl-5hydroxyl-1-phenyl-3-trifluoromethylpirazolone), to efficiently cleave DNA under UV-A light irradiation, without any activity under
dark conditions. The DNA cleavage was examined following the conversion of the supercoiled form of plasmid DNA into its cleaved
forms using agarose gel electrophoresis. In neutral pH, 5 minutes of UV-A light (λ = 365 nm) exposure is sufficient to generate
more than 70% of cleaved plasmid DNA in the presence of complex at 100 µM. In addition, the complex activity seems to be
concentration-dependent. Without UV-A light irradiation, however, no cleavage is observed in 5 minutes or even in long
incubations times (up to 24 h). These results lead us to consider that the complex activity is strongly controlled by light irradiation.
Further assays suggested that the cleavage process under UV-A light is based on an oxidative mechanism, where ROS are involved,
such as hydroxyl and superoxide anion radicals, peroxo-species, singlet-oxygen and carbon-derived radicals. The addition of
sodium chloride did not alter the complex activity, which indicates that electrostatic interactions between the complex and DNA
are not necessary to achieve the nucleic acid cleavage. The great efficiency of DNA cleavage under strictly controlled light
condition indicates this complex as a potential prototype for an antitumor agent targeted to photodynamic therapy (PDT).
Support: CNPq, CAPES, MCTI, FINEP, FAPESC, INCT de Biologia Estrutural e Bioimagem – INBEB.
D84
ULTRASTRUCTURAL ANALYSIS OF THE WOLBACHIA ENDOSYMBIONT ON THE FEMALE REPRODUCTIVE SYSTEM OF
LITOMOSOIDES CHAGASFILHOI
1,2 - CHAGAS-MOUTINHO, V. A; 1,3 - OLIVEIRA-MENEZES, A.; 2 - DE SOUZA, W.; 2 - MOTTA, M. C. M
1 - LABORATÓRIO DE BIOLOGIA DE HELMINTOS OTTO WUCHERER - INSTITUTO DE BIOFÍSICA CARLOS CHAGAS FILHO,
UNIVERSIDADE FEDERAL DO RIO DE JANEIRO; 2 - LABORATÓRIO DE ULTRAESTRUTURA CELULAR HERTHA MEYER, INSTITUTO DE
BIOFÍSICA CARLOS CHAGAS FILHO, UNIVERSIDADE FEDERAL DO RIO DE JANEIRO; 3 - PÓLO AVANÇADO MACAÉ, UNIVERSIDADE
FEDERAL DO RIO DE JANEIRO
Genus Wolbachia comprises Gram-negative ɑ-proteobacteria that infects a wide range of arthropods and filarial nematodes. In
arthropods, Wolbachia bacteria are considered reproductive parasites, that display cytoplasmic incompatibility and promotes sexratio distortion on the host. In nematodes, such bacteria maintain obligatory symbiotic associations with the host, thus
characterizing mutualistic relationships, since endobacteria are essential for embryogenesis and worm survival. Most of the filarial
nematodes contain Wolbachia. Recent studies have shown the importance of the Wolbachia symbiotic relationship on
development and survival of nematode hosts, however extensive studies are still required to characterize the association between
worm and bacteria. The purpose of this work is to analyze ultrastructural aspects of the endosymbiotic bacterium present on
female reproductive system of the filarial nematode Litomosoides chagasfilhoi, to obtain better comprehension of symbiotic
relationship. The females of Litomosoides chagasfilhoi were collected from the abdominal cavity of experimentally infected
gerbils (Meriones unguiculatus, rinsed in 0.9% NaCl solution, and fixed in a solution of 2.5% glutaraldehyde, with 4% freshly
prepared paraformoldehyde in 0.1 M cacodylate buffer, pH 7.2. For TEM, fixed female were post-fixed in 1% OsO4 and 0.8%
K4[Fe(CN)6], dehydrated in series of acetone and embedded in Epoxy resin. Thin sections were collected on copper grids, stained
with uranyl acetate and lead citrate, and then observed in a Jeol 1200 TEM. Ultrastructural analysis showed that transversal
sections of the uterine wall consisted of an epithelium underlying the basal lamina and the cytoplasmic bridges. Early stage
embryos were observed on the anterior portion of the uterus, whereas completely developed microfilariae, surrounded by the
eggshell, were observed on the posterior portion. Embryos and microfilariae presented numerous bacteria that were observed in
cytoplasmic vacuoles. Such symbiotic bacteria are enclosed by two unit membranes and were usually observed close to the host
cell nucleus and to the endoplasmic reticulum.
Support: FAPERJ, CNPq, CAPES.
D85
IDENTIFICATION OF A NOVEL CARBOHYDRATE VESICLE DURING ENCYSTMENT OF GIARDIA LAMBLIA
1,2- MIDLEJ, V.; 1- MEINIG, I.; 1- BENCHIMOL, M.
1- Universidade Santa Úrsula, Rio de Janeiro, Brazil; 2- 2 Programa de pós-graduação em Ciências Morfológicas, Universidade
Federal do Rio de Janeiro, Brazil.
Differentiation from one cycle stage into another is an elegant adaption by which many parasites ensure their transmission and
survival. The protozoan Giardia lamblia is a major cause of waterborne diarrheal disease [1]. This parasite exhibits two forms in its
life cycle that includes trophozoite and cyst. Encystation is a crucial process for establishment of Giardia infection. The cyst wall
(CW) of the parasite is known to be composed by carbohydrates and proteins and it provides the resistance of the cyst. During the
process of encystation ESVs (encystation specific vesicles) are observed, these ESVs transport cyst wall proteins (CWPs) to the cell
surface for the formation of the cyst wall [2]. Furthermore, the CW is also composed by carbohydrates [3] and its association with
CWPs provide the resistance of the cyst. To address the question of from where the carboydrates of cyst wall originates we
performed fluorescent and cytochemistry assays using lectins to trace sugar molecules. Here we analyzed the origin of
carbohydrate components of the cyst surface by transmission electron microscopy, using cytochemistry for carbohydrate
detection, as well as by applying gold-labeled and fluorescent lectins. Immunocytochemistry and immunofluorescence with antiCWPs and carbohydrate detection using DBA and WGA lectins, which are known to interact with N-acetylgalactosamine and Nacetylglycosamine, respectively, were performed in a double labeling assay. Interestingly, a different cell compartment, positive
for carbohydrates was found in encysting cells. This different cell compartment is an electron-lucent compartment present only in
encysting cells, but not in mature cysts or trophozoites. It is distinct from the ESV (encystation specific vesicles) in four ways: (1) it
is electron lucent, with a milky appearance, whereas the ESV is electron dense; (2) it did not react with antibodies against cyst wall
proteins; (3) its contents were positive for carbohydrates, whereas the ESVs displayed a negative reaction; (4) these cell
compartments exhibited positive labeling for DBA and WGA lectins, indicating N-acetylgalactosamine and N-acetylglycosamine
presence, whereas the ESVs were negative. The vesicles positive only for carbohydrate, differently from the already well
previously described ESV, were observed in encysting cells and could represent structures involved in cyst wall formation.
Support: INBEB, AUSU, CNPq, Faperj, PRONEX.
D86
CLONING, PURIFICATION AND CHARACTERIZATION OF A 90 KDA HEAT SHOCK PROTEIN HSP90 FROM SUGARCANE
1,2-SILVA,V.C.H, 1,2-CAGLIARI, T.C., 2-RAMOS, CHI
1-Instituto de Biologia, Unicamp; 2- Instituto de Química, Unicamp
Hsp90 is an ubiquitous and highly abundant molecular chaperone (up to 2% of all cellular protein) that is crucial to maintain
cellular homeostasis. Hsp90 has a key role in regulation and activation of numerous client proteins involved in diverse functions
such DNA repair and synthesis, cell division, proliferation, differentiation and apoptosis. Besides its housekeeping function, Hsp90
also is an important stress protein, and can act to prevent protein aggregation. Although some Hsp90 plant homologues have been
studied, little information concerning structural and functional characteristics is available. The sugarcane Hsp90 homologue,
SHsp90.3, was annotated in the sugarcane EST genome project (SUCEST) and cloned. We present results on the expression,
purification and characterization of SHsp90.3 and give information on the conformation of the protein and on its role in protecting
against aggregation. SHsp90 was purified folded as investigated by circular dichroism and intrinsic fluorescence. SHsp90 was a
dimer in solution as measured by size exclusion chromatography multi-angle light scattering (SEC-MALS) experiment. Finally, our
results showed that SHsp90 was capable to prevent the aggregation of citrate synthase at 47°C. Altogether our results add to the
comprehension of the structure and function of sugarcane Hsp90.
Support: FAPESP, CNPq, CAPES, INBEB.
D87
ELECTRON TOMOGRAPHY ANALYSIS OF THE CONTRACTILE VACUOLE COMPLEX OF TRYPANOSOMA CRUZI SUBMITTED TO
HYPOSMOTIC TREATMENTS
1- GIRARD-DIAS, W.; 1,2- DE SOUZA, W.; 1,2- MIRANDA, K.
1- Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho and Instituto Nacional de Ciência
e Tecnologia em Biologia Estrutural e Bioimagens – Universidade Federal do Rio de Janeiro, Rio de Janeiro; 2- Diretoria de
Programas, Instituto Nacional de Metrologia, Normalização e Qualidade Industrial – Inmetro, Rio de Janeiro.
During the course of the infection, Trypanosoma cruzi faces environments where extreme variations in the concentration of ions
and osmolytes in the extracellular milieu are found. To cope with these fluctuations, the parasite has developed adaptation
mechanisms that involve signaling pathways and remodeling of parasite organelles, including the contractile vacuole complex
(CVC) and acidocalcisomes. The CVC of some trypanosomatids is formed by several tubules and vesicles forming a multi-tubular
structure named spongiome, connected to a central vacuole located near the flagellar pocket. This organelle has a mechanism for
fluid secretion that is achieved by cooperation with acidocalcisomes, which are electron-dense acidic organelles rich in calcium,
polyphosphate and other cations, and shown to be involved in several functions as calcium homeostasis, phosphate metabolism
and osmoregulation. The structural modifications that take place in the CVC during hyposmotic treatment have not been yet fully
characterized in T. cruzi. In this work we used cryotechniques and 3D electron tomography reconstruction to study the CVC of T.
cruzi submitted to hyposmotic treatment. Results revealed a CVC in T. cruzi with the central vacuole positioned docked to a
specific domain of the flagellar pocket, presenting an electron dense aspect, and associated with a spongiome. The tubules of the
spongiome were connected to the contractile vacuole preferentially in a region opposite to the kinetoplast and parallel to the
flagellum, suggesting that this structure has a polarized organization. Serial electron tomography revealed fusion of
acidocalcisome with the central vacuole. Local modulation of the acidocalcisome volume after hyposmotic stress could be
observed. Altogether, the results suggest that the spatial organization of CVC is defined by specific domains of the spongiome and
the contractile vacuole, which may be modulated during events of regulatory volume decrease in T. cruzi.
Support: CNPq, FAPERJ, FINEP, CAPES.
PD01
CHARACTERIZATION OF FRESHLY ISOLATED NON-MYOCYTE CELL POPULATIONS FROM MOUSE HEARTS
BRUNO DIAZ PAREDES1;LEONARDO MOREIRA ALVES1; CAMILA IANSEN IRION1; BRUNO BORGES DE SÁ1; GUILHERME SUAREZ
POMPEO1; TAÍS HANAE KASAI-BRUNSWICK1; CRISTIANE DEL CORSSO1; REGINA COELI DOS SANTOS GOLDENBERG1; ANTONIO
CARLOS CAMPOS DE CARVALHO1,2; ADRIANA BASTOS CARVALHO1.
1-Instituto de Biofísica Carlos Chagas Filho, UFRJ; 2-Instituto Nacional de Cardiologia
Introduction and objectives: Several types of cardiac stem cells have been described in the literature. Since it is unlikely that the
heart possesses multiple non-overlapping cardiac stem cell types, the objective of this work was to investigate the phenotype of
freshly isolated non-myocyte cells found in mouse hearts by flow cytometry. Methods and Results: Non-myocyte heart cells were
isolated from C57Bl/6 mice through digestion with 0.2% collagenase, a process known to eliminate cardiomyocytes. Before
digestion, the heart was thoroughly perfused with PBS to remove blood-derived cells. Cell suspensions were blocked with 0.5%
BSA and Fc receptor blocker (anti-CD16/CD32) for 5 minutes at 4°C and subsequently stained with the following antibodies: CD3,
CD4, CD14, CD19, CD31, CD45, CD133, Flk-1, Sca-1 and c-kit. Staining was performed at 4°C for 20 minutes and cells were washed
once with PBS. DAPI was used to exclude dead cells and isotypes were used to control for unspecific binding of primary antibodies.
Data collection and analysis were performed in BD FACSAria IIu and FlowJo software respectively. CD45 staining was used to
define cell origin: bone marrow cells were identified as CD45+ and heart-derived cells as CD45-. Bone marrow-derived cells
constituted 8.4% of the cells present in the heart. More specifically, these cells were identified as B lymphocytes (CD19+ 38.5%),
macrophages (CD14+ 18.1%), helper T lymphocytes (CD3+CD4+ 13.5%) and cytotoxic T lymphocytes (CD3+CD4- 12.4%). Regarding
the CD45- population, we found no expression of CD133, Flk-1 or c-kit. Sca-1 was widely expressed comprising 79.0 ± 4.2% of
heart-derived non-cardiomyocyte cells. A large portion of Sca-1 positive cells was also positive for CD31 (54.1 ± 8.7%), while 26.5 ±
5.4% were Sca-1+CD31-. We were able to enrich the Sca-1+CD31- population using Ficoll gradient. Comparing the mononuclear
layer with the pellet, we obtained 72% and 13% of Sca-1+CD31- cells respectively. Conclusion: We were unable to reproduce data
published in the literature showing the presence of CD45-ckit+ cells in the mouse heart. However, we cannot exclude that c-kit
expression was lost due to collagenase digestion, an effect that has been previously reported. The Sca-1+CD31- subset has been
shown to possess cells with dye efflux capability (side population) that present cardiomyogenic potential. The differentiation
potential of these cells needs to be further investigated in our samples.
Support: CNPq, CAPES, FAPERJ, Ministério da Saúde, INCT.
PD02
THERAPEUTIC EFFECT OF BONE-MARROW MONONUCLEAR CELLS IN THE OPTIC NERVE REGENERATION
1,2,3-ZAVERUCHA-DO-VALLE, C.; 1,2,3-GUBERT, F.; 1,2,3-MESENTIER-LOURO,L.; 1,2,3-BARGAS-REGA, M.; 1,2,3-Diaz-Paredes, B.;
4- MENCALHA, A.L.; 4-ABDELHAY, E.; 1,2,3-SANTIAGO, M.F.; 1,2,3-MENDEZ-OTERO, R.
1-Programa de Terapias Celulares. 2-Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagens, Universidade
Federal do Rio de Janeiro. 3-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro. 4-Laboratório de
Células Tronco, CEMO, INCa.
In adult mammals, the regeneration of the optic nerve is very limited and to date there are no efficient therapies to generate
neuroprotection and axon outgrowth after injuries. Using the optic nerve crush of rats as a model for CNS injury, we investigated
the effect of intravitreal transplantation of syngeneic bone-marrow mononuclear cells (BMMCs) on retinal ganglion cell (RGC)
survival and on the axonal regeneration. We demonstrated an increase of 1.6 fold in RGC survival and an increase of 3.7 fold in the
axon outgrowth 14 days after injury, besides a reduced Müller glia activation. We also showed an increase of 5.2 fold in the axon
outgrowth 28 days after lesion, but the effect on RGC survival was not sustained. A new protocol with two injections of BMMCs
didn’t prolong RGC survival. We also demonstrated an increase in the number of CD11b+ cells in the vitreous body one day after
BMMCs transplantation. Analysis by qRT-PCR revealed an increase in levels of fibroblast growth factor 2 (FGF-2) mRNA in treated
animals 1 and 14 days after injury. To investigate whether the regenerated axons could reach visual targets, we have retrograde
labeled the RGCs. We have also analyzed the expression of NGFI-A in the superficial layers of the superior colliculus to assess
glutamate release by the RGCs axons. We found evidences that a higher number of RGCs were able to reach the superior colliculus
after the treatment. We have also shown that NGFI-A expression was higher in the treated animals 60 days after injury. These
results demonstrate that BMMCs transplantation can promote neuroprotection and neuroregeneration 14 days after injury but
the effects on RGC survival were not sustained. Our results also suggest that the BMMC effects may be related to FGF-2 release or
macrophage/monocyte activation.
Support: CNPq, CAPES, FAPERJ, DECIT, INBEB.
PD03
CORRELAÇÃO ENTRE AÇÚCARES DO LEITE MATERNO E DA SALIVA DE LACTENTES POR RMN
1- MARTINS C.; 1- FIDALGO T.K.S.; 1- BASTOS V.A.S.; 1- FREITAS-FERNANDES L.B.; 2- ALMEIDA F.; 1- POMARICO I.; 2- VALENTE A.P.
1- Departamento de Odontopediatria e Ortodontia da Faculdade de Odontologia da Universidade Federal do Rio de Janeiro; 2Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Instituto de Bioquímica Médica, Universidade Federal do Rio de
Janeiro
O objetivo do presente estudo foi analisar a correlação entre os açúcares presente no leite materno, saliva materna, e na saliva de
lactentes através de Ressonância Magnética Nuclear (RMN). Foram coletados 5 ml de leite materno, 5 ml de saliva total não
estimulada de 13 lactantes (26,1 ± 7,0 anos) e 0,5 ml de saliva total não estimulada de seus respectivos lactentes (9,5 ± 7,3 meses).
As amostras de saliva foram centrifugadas por 1 hora a 10.000 g a 4 ºC e as de leite por 5 minutos a 1.000 g. Os espectros 1H de
RMN foram obtidos em um espectrômetro Bruker 400 MHz. Os espectros mostraram que, entre os metabólitos presentes no leite
materno, os açúcares são majoritários e que aparentemente isso se reflete na saliva do bebê em relação a saliva materna.
Atualmente, estamos assinalando o espectro para posterior avaliação da separação entre os grupos por análise dos Componentes
Principais entre outros testes estatísticos. CEP-IPPMG (UFRJ): 038/2010.
Apoio: CAPES e FAPERJ.
PD04
ESTUDO DA INTERAÇÃO ENTRE A PLASMINA DE YERSINIA PESTIS E O PLASMINOGÊNIO HUMANO POR DICROÍSMO CIRCULAR E
RESSONÂNCIA MAGNÉTICA NUCLEAR
1,2,3-SARZEDAS, C.G.; 1,2,3-VIDAL, T.J.; 1,2,3-TINOCO, L.W.
1-Laboratório de Análises e Desenvolvimento de Inibidores Enzimáticos, UFRJ , 2-Núcleo de Pesquisas de Produtos Naturais, UFRJ,
3- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, UFRJ
A “peste bubônica”, cujo agente etiológico é a bactéria Yersinia pestis, causou grandes surtos ao longo da história e dizimou
populações inteiras. A bactéria Y. pestis produz na sua membrana externa a proteína plasmina (plaYp) que após a infecção tem
como atividade a clivagem (ativação) do plasminogênio humano (Plg) para gerar plasmina (Pla), causando hemorragias em vários
órgãos. Estudos de atracamento e dinâmica molecular entre a plaYp e o Plg humano mostraram que o Plg se encaixa entre as
alças da plaYp. No Plg foi identificado o peptídeo PK2: PKKCPGRVVGGCV, que representa a região específica onde acontece a
interação com a plaYp. Com o objetivo de analisar estruturalmente a interação plaYp-Plg, este trabalho iniciou com a clonagem,
expressão e purificação da plaYp para que fossem feitas as análises de interação com o PK2 por CD e RMN. . Os espectros de CD
da plaYp mostraram características compatíveis com uma proteína em folha-β, como mostra a estrutura por cristalografia (Eren, et
al, 2010) Com a adição do peptídeo PK2, observou-se uma modificação estrutural da plaYp, mostrando que ocorre interação
plaYp-Plg. O estudo do PK2 livre em solução por RMN mostra o peptídeo com tendência estrutural em folha- ou estendida,
compatível com um peptídeo cíclico devido à ponte de dissulfeto entre as cisteínas 4 e 12. As análises estruturais do PK2 na
presença da plaYp fornecerão informações importantes para o planejamento de peptídeos miméticos com potencial para inibir a
interação plaYp-Plg, e assim anular o processo invasor da Y. pestis em células de mamíferos.
Apoio: INBEB, CNPq, FAPERJ.
PD05
SILDENAFIL (VIAGRA®) PREVENTS DEMYELINATION AND NEUROINFLAMMATION IN A MULTIPLE SCLEROSIS MODEL
1- RAPÔSO, C.; 1,2- NUNES, A.K.S.; 2- LUNA, R.L.A.; 3- CRUZ-HÖFLING, M.A.; 1,2- PEIXOTO, C.A.
1- Laboratório de Microscopia, Ministério de Ciência e Tecnologia, Centro de Tecnologias Estratégicas do Nordeste (MCT/CETENE);
2- Laboratório de Ultraestrutura, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife-PE; 3- Departamento de
Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas-SP.
Neuroinflammation plays a crucial role in the multiple sclerosis (MS) pathogenesis, an autoimmune disorder characterized by
demyelination and progressive psychomotor impairment. The current MS-treatment options are partially effective and need a
parenteral route of administration. Sildenafil (Viagra®) induces cyclic 3'5'guanosine monophosphate (cGMP) accumulation through
phosphodiesterase-5 (PDE5) inhibition. Cerebellum presents constitutively PDE5, and it has been shown that cGMP-pathways
modulates micro- and astroglial (neuroinflammatory resident cells) reaction. Sildenafil despite having an excellent tolerability
profile, it has used only for erectile dysfunction and currently for pulmonary hypertension treatment. Here, the effects of sildenafil
in the neuroinflammation were investigated in a MS animal model. Five C57BL/6 mice, 7-weeks-old, were used/group. The groups
received: 1) Cuprizone (0.2%) mixed into a chow/4 weeks, 2) Cuprizone into a chow while sildenafil (Viagra®) 3, 25 or 50 mg/kg in
the drinking water, or 3) Controls received pure chow/water. After perfusion, the cerebella were processed for western blotting,
immunohistochemistry/immunofluorescence and luxol fast blue staining. Cuprizone significantly increased the GFAP levels and
decreased myelination staining intensity. Sildenafil-25/50 mg/Kg groups showed GFAP-astrocytic expression close to baseline;
there was no demyelination. There was a significant COX-2, IL-2 and IL-1β increase after cuprizone treatment, compared to control
group. Cuprizone did not induce changes in IFN-γ and TNF-α expression. After sildenafil administration, all cytokines evaluated and
COX-2 expression significantly decreased, compared with control/cuprizone groups. Iba1 levels increased in the cuprizone-treated
animals compared with control, indicating microglial activation. Animals treated with sildenafil-25 mg/Kg showed decreased Iba1
expression, compared to cuprizone group. The increased levels of cGMP, by PDE5 inhibition, probably act as a neuroinflammation
modulator, regulating cytokine levels and protecting myelin, astrocytes and microglia. Therefore, after well-designed clinical trials,
Sildenafil may be a future drug compatible with daily oral administration for people with MS and other
neuroinflammatory/neurodegenerative diseases, providing additional benefits to current treatments.
Support: FACEPE, CNPq, INBEB, MCT-CETENE.
PD06
ATIVADORES NATURAIS DO SISTEMA CALICREÍNA-CININAS: UMA NOVA ROTA PROTEOLÍTICA INTERLIGANDO INFLAMAÇÃO AOS
MECANISMOS DO SISTEMA IMUNE
1*NASCIMENTO, C.R., 1WERGLES, J., 2JULIANO, L., 3MONTEIRO, R.Q., 1SVENSJO, E., 1SCHARFSTEIN, J.
1Laboratório de Imunologia Molecular, Instituto de Biofísica Carlos Chagas Filho, 3 Instituto de Bioquímica Médica, Universidade
Federal do Rio de Janeiro, Brasil; 2 Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo,
Brasil
A conversão do zimógeno FXII em FXIIa é a primeira etapa proteolítica do processo que culmina com a ativação da via intrínseca
de coagulação, frequentemente referida como Sistema Calicreína Cinina (SCC). Após ativação recíproca entre FXII e calicreína
plasmática (PKa), esta ultima (também serino protease) cliva 2 seqüências internas do Cininogênio de Alto Peso Molecular (HK),
liberando o nonapeptídeo bradicinina (BK). As cininas (BK e seus metabólitos) induzem vasodilatação arterial, aumento de
permeabilidade vascular, aumentam a nocicepção e modulam o sistema imune mediante ativação de receptores BK2R, expressos
por células dendríticas (DCs). Recentemente, polifosfatos (secretados por plaquetas ativadas) e heparina (secretada por
mastócitos ativados) foram descritos como substâncias de origem endógena capazes de ativar o SCC, liberando BK. No presente
trabalho, empregamos substratos de fluorescência apagada (Abz-XXX-EDDnp) contendo as sequências de HK flanqueadoras da BK
como ferramentas de detecção de proteases “liberadoras” de cininas. Ensaios enzimáticos mostram que a adição de (i) dextran
sulfato 500 kDa (DXS-500, mas não DXS-5) (ii) polifosfatos de alto peso molecular (polímeros com 45 e 65 resíduos), ao plasma
humano resulta na formação de “S-1125 hidrolases”. Em contraste, não houve hidrólise quando empregamos plasma deficiente de
FXII, nem tampouco quando adicionamos aprotinina ao plasma normal. Estes dados sugerem que PKa é a enzima responsável pela
referida clivagem. Consistente com Oschatz et al., (2011), verificamos que a adição de heparinas (PM>17 kDa) ao plasma gera S1125 hidrolases. Complementando os estudos bioquímicos, testamos o efeito da aplicação tópica de DXS-500 sobre o leito
vascular da bolsa da bochecha do hamster (Svensjo et al., ver resumo INBEB). Após 20 min sem índicos de alteração
microcirculatória, observamos uma intensa reação de extravasamento de plasma, mediada por cininas. Estudos em andamento
visam esclarecer se a via [mastócito/heparina/FXII-PK>>BK] tem impacto sobre a função de DCs, interligando a inflamação ao
sistema imunitário.
Apoio: FAPERJ, CNPq e INCT-CNPq/INBEB.
PD07
CHAGAS HEART DISEASE: BLUNTING INFECTION-ASSOCIATED MYOCARDITIS AND HEART FIBROSIS THROUGH THE BLOCKADE OF
ENDOTHELIN AND BRADYKININ RECEPTORS
1*ANDRADE, D., 1SERRA, R.R., 1MANEIRAS, L. M.V., 1SVENSJO, E., 2VAIRO, L., 2BRASIL, G.V., , 3FORTES F., 4CARVALHO-PINTO,
C.E., 2CARVALHO, A.C.C.,2COELI R.G., 1SCHARFSTEIN, J.
1Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Laboratório de Imunologia Molecular,UFRJ,
Brazil; 2Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Laboratório de Eletrofisiologia Cardíaca
“Antonio Paes de Carvalho”, Brazil. 3Centro Universitário Estadual da Zona Oeste, Rio de Janeiro, Brazil 4 Departamento de
Patologia, UFF, Rio de Janeiro, Brazil
Acting at the interface between microcirculation and immunity, the parasitic protozoan Trypanosoma cruzi induces inflammatory
edema via an activation pathway forged by sequential engagement of (at least) six cell surface receptors: Toll-like 2 receptors
(TLR2), the chemokine receptor CXCR2, bradykinin receptors (BK2R and BK1R) and endothelin receptors (ETaR and ETbR) (Andrade
and Scharfstein, 2011). In a recent paper (Andrade et al., 2011), we advanced the hypothesis that T. cruzi may take advantage of
the transient availability of plasma-borne proteins (eg. kininogens) in heart interstitial spaces to proteolytically generate infectionpromoting signals, such as bradykinin (BK). The concept that the interstitial edema may bring about reciprocal benefits to the hostparasite relationship is underscored by the findings that T. cruzi trypomastigotes invade cardiovascular cells through cooperative
signaling of ETRs and BKRs. In the present study, we investigated whether ETRs/BKRs may influence heart pathology (intracardiac
edema, myocarditis and heart fibrosis) in mice (BALB/c or B6) infected by T. cruzi trypomastigotes via the intramyocardial (echoguided) route. Control experiments showed robust leakage of TRITC-dextran in the pericardium/myocardium of C57BL/6 mice
injected 2 h earlier with trypomastigotes. In contrast, BK2R-deficient mice exhibited a blunted intracardiac edema response. Of
further interest, we found that the infection-associated leakage response in the heart was markedly reduced in C57BL/6 mice
pretreated either with (i) Bosentan (ETAR/ETBR antagonist) (ii) HOE-140 (BK2R antagonist) or (iii) B9858 (BK1R antagonist). As ET-1
has been established as a key mediator of myocardial fibrosis in heart diseases of autoimmune etiology, we then checked if ETR
and BKR antagonists could attenuate the severity of chagasic myocardiopathy, assessed 30 d.p.i.. Strikingly, we found a marked
reduction of intramyocardial inflammatory infiltrates and collagen deposition in mice pretreated either with Bosentan, HOE-140 or
B9858. Ongoing studies may elucidate the role of the ETRs/BKR axis in the immunopathogenesis of Chagas heart disease.
Supported by funds from CNPq, FAPERJ and INCT-CNPq/INBEB.
PD08
TROSY NMR REVEALS THE STRUCTURE OF SIS1 IS GOVERNED BY REGIONS OUTSIDE THE GLYCINE METHIONINE RICH DOMAIN
1- MOKRY, DAVID Z., 1- RAMOS, CARLOS H.I., 2- ALMEIDA, FABIO C.L.
1- Instituto de Química, Unicamp 2- Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Instituto de Bioquímica Médica,
Universidade Federal do Rio de Janeiro
In the cell, a sophisticated system of protein chaperones is responsible for maintaining the structural integrity of proteins. The
ubiquitous Hsp40 proteins are important components of this system, as they bind and present substrates for refolding to Hsp70
through a redundant J-domain, which aids in stimulating ATP hydrolysis and protein refolding. Despite this common feature,
Hsp40s represent the largest and most diverse sub-group of the HSP family, not only by the variety of substrates specific for each
member, but also by their implications involving other cellular processes. The most studied of the Hsp40s are the type I and type II
classes, which share several key features. However, both types are able to distinguish between different substrates, possess
unique cellular activities, and display dramatic differences in the orientation of their J-Domains. One hypothesis for these
variations is the substitution of the zinc finger in type I with a glycine/methionine (G/M) rich region in type II. Validation of this
hypothesis is complicated by the fact that a complete high resolution Hsp40 structure is not available in the Protein Data Bank. In
this work, we performed NMR TROSY on a type II Hsp40 from yeast named Sis1, as well as a functional deletion mutant with the
G/M rich region removed. Our results provide the first insight into the atomic-resolution of full length Sis1, and suggest a structure
with two highly ordered domains connected by a linker region possibly in conformational exchange. Interestingly, no major
differences in the spectra between the wild-type and mutant were observed, indicating that regions outside the G/M domain are
responsible for the structure and function of type II Hsp40s.
Support: FAPESP.
PD09
PROTEIN-PROTEIN DOCKING USING NORMAL MODES AND GENETIC ALGORITHM
1,2- GOMES, D.E.B; 3- SCOTT, L.P.; 1- PASCUTTI, P.G.; 1- BISCH P.M.; 2- PERAHIA, D
1- Universidade Federal do Rio de Janeiro; 2- École Normale Supérieure de Cachan; 3- Universidade Federal do ABC
Protein-protein docking (PPD) is a multidimensional problem unexplainable by the classical lock-and-key mechanism. In the
binding processes extensive conformational changes may occur that extend beyond local structure rearrangements. A realistic and
computationally efficient assessment of these changes remains a fundamental challenge. The linear combination (LC) of normal
modes (NM) emerges as an effective approach to reproduce the directions of protein’s conformational changes and capture their
structural transition from unbound to bond form. Still, the number of NM and their amplitudes necessary to reproduce the
transition remain essential parameters to be determined. Here we propose a new PPD method based on: i. An evaluation of the
NM space by a genetic algorithm (GA) to create an ensemble of structures for docking (both receptor and ligand). In every
generation, structures are produced by molecular dynamics simulations targeted to refine LCs of random NM amplitudes. ii. A GA
to orchestrate the docking of structures from both receptor and ligand populations. In every generation, the best docking results
are re-ranked by an improved scoring function (SF). The method was tested on a subset of the PPD benchmark 4, and was
implemented as a highly parallel program. The GA rapidly evolved the candidate structures towards low energy conformations,
although only some matched the structural changes upon binding. The docking algorithm should find the correct binding partners.
The GA-conducted docking also quickly converged. However, the near correct binding conformations were fairly sampled,
indicating that docking population must be enriched. But when selected those are poorly ranked, even with a sophisticated re-
scoring function, as a result, the incorrect ones prevail. That is indeed a limitation of current SFs, addressed by the CAPRI
competition. Combination of SFs was found to improve protein-ligand docking and could be considered as an alternative to
enhance PPD.
Support: INBEB, CAPES, FAPESP.
PD10
NEW APPROACHES OF BACILLARY GLANDS OF TRICHURIS MURIS USING ENVIRONMENTAL SCANNING ELECTRON MICROSCOPY
AND CRYOFIXATION
1,2 - LOPES TORRES E.J.; 2,3 - DE SOUZA, W.; 2,3 - MIRANDA, K.
1- Laboratório de Biologia de Helmintos Otto Wucherer IBCCF UFRJ; 2- Laboratório de Ultraestrutura Celular Hertha Meyer IBCCF
UFRJ; 3 - Instituto Nacional de Metrologia – INMETRO.
Trichuris genus comprises nematode species parasite of humans and other mammals. The structure of the bacillary band has not
been yet fully characterized and only a few reports that describe its basic characteristics have been published. In this regard, the
comprehension of the morphological and structural details of cuticle surface and bacillary band structures, therefore, may be
important for the understanding of parasite-host interaction. In this work, we report the use of an improved protocol for
Environmental, field emission scanning and transmission electron microscopy analyses of the surface properties of Trichuris muris.
The worms recovered of the mice large intestine, fixed with Karnovsky’s solution or freezing using HPM-010 and freeze
substituted. The microscopy analyses obtained using a FEI-Quanta 250 and Jeol JSM 6340F. High magnification images obtained by
FESEM analyses showed two patterns of the bacillary glands openings. The inside of glandular chambers presented clusters of the
spherical structures. Images obtained using environmental mode allowed the observation of unfixed and freshly obtained
nematodes, which presented the bacillary openings with a prominent pattern. TEM results showed that the bacillary glands
presented two parts: the cell proper and its secretory product. The lamellar system separates the cell proper from a secretory
product. This secretory product when they are spilling the line of the cuticle present the prominent pattern, but when leach out,
the pattern is as an empty pore. The material cryofixed and freeze substituted, present the better cuticular and secreted product
preservation. In this material are possible to observe the secretory products formed for vesicles in the glandular openings and on
the cuticle surface. Surface analysis using different techniques in unfixed samples has shown promising results that in association
with immunological and biochemical studies may provide new insights on the comprehension of the host-parasite interaction.
Support: CAPES, CNPq, UEZO, CEMIB-UNICAMP and FIOCRUZ.
PD11
EFFECT OF CIPROFLOXACIN DERIVATIVES AGAINST TOXOPLASMA GONDII
1,2-MARTINS-DUARTE, E.S.; 3,4- DUBAR, F.; 3-BIOT, C.; 1,2,5- DE SOUZA, W., 1,2-VOMMARO, R.C.
1-Instituto de Biofísica Carlos Chagas Filho-UFRJ; 2-Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem;
3-Université Lille Nord de France, Université de Lille1, Unité de Glycobiologie Structurale et Fonctionnelle; 4-Université Lille Nord
de France, Université de Lille1, Unité de Catalyse et Chimie du Solide; 5-Instituto Nacional de Metrologia e Qualidade Industrial Inmetro, Rio de Janeiro, Brazil
Toxoplasma gondii is one of the most important pathogen affecting immunocompromised patients. However, there are few
options of treatment against toxoplamosis which is often associated to side effects. Thus, the discovery of new compounds against
Toxoplasma gondii is extremely important. Fluoroquinolone is a known class of topoisomerases II and IV inhibitors in prokaryotes,
but has demonstrated a broad spectrum of activity against many other pathogens, including T. gondii. Herein we evaluated the
antiproliferative effect of four new ethyl-ester derivatives of ciprofloxacin against the tachyzoites of T. gondii in vitro. The
treatment of infected cultures with concentrations of ciprofloxacin up to 20µM for 24 and 48h reduced modestly the parasite
proliferation. On the other hand, the four novel derivatives were very active and inhibited parasite proliferation with IC50 ¬at
concentrations lower than 3µM after 24 and 48h. In addition, two derivatives were able to inhibit parasite proliferation with IC50
at nanomolar range. The study of the cytotoxic effect of the compounds against LLC-MK2 cell line showed a wide therapeutic
index. The cellular effects evaluated by transmission electron microscopy of parasites treated with the most two active
compounds showed that ciprofloxacin derivatives caused mainly cell division arrestment. Compound FD-6 also caused Golgi
complex cisternae disorganization and endoplasmic reticulum enlargement. Accordingly to the results obtained, chemical
modifications on ciprofloxacin structure were efficient to improve the anti-T. gondii activity of the new derivatives. Reference:
MedChem Comm 2011, 2, 430 *[email protected]
Support: INBEB, CNPq, FAPERJ.
PD12
IN VIVO MONITORING COLONIZATION OF RHODNIUS PROLIXUS BY T. CRUZI USING BIOLUMINESCENCE AND FLUORESCENCE
IMAGING
1-DIAS, F.; 1- VIEIRA, L. R.; 2-GUERRA, B.; 1-GANDARA, A. C.; 3-LARA, F. A.; 2-MEDEI, E.; 1-DE OLIVEIRA, P. L.; 1-SALMON, D.
1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, 2-Instituto de Biofísica Carlos Chagas Filho,
Universidade Federal do Rio de Janeiro, 3-Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro.
T. cruzi, ethiologic agent of the Chagas disease, represents a model of choice to study metacyclogenesis, a cellular differentiation
process which occurs in the hindgut of the reduviid insect vector (Triatomine). Although the key aspects of the T. cruzi life cycle
were described more than one century ago, the development and the interactions of T. cruzi with its vector are poorly
characterized. By dissection at regular time intervals (e.g. 7 days) of different compartments of the alimentary tract of the
Triatomine (i.e. anterior and posterior midgut and rectum), we evaluate the trypanosome development within the bug using a
transgenic parasite GFP-tagged for epifluorescence microscopy observation. Similarly, using luciferase-tagged trypanosomes, we
analyze trypanosomal colonization and clearance dynamics in real-time in vivo. In this study we show that both fluorescence
imaging and bioluminescence imaging (BLI) can be used for different purposes which can be complementary to each other. These
two methods combined would provide information on the timing of insect colonization by the parasite and determine accurately
the critical steps necessary to establish vector infection.
Support: CAPES, CNPq, DECIT, FAPERJ, INBEB, FINEP.
PD13
CRYO-ELECTRON TOMOGRAPHY OF THE MAGNETOTACTIC VIBRIO ‘CANDIDATUS MAGNETOVIBRIO BLAKEMOREI’
1- ABREU, F.; 2- SOUSA, A.A.; 2- ARONOVA, M.A.; 2- LEAPMAN, R.D. ; 3- ANDRADE, L.; 3- KACHAR, B.; 4- BAZYLINSKI, D.A.; 1- LINS,
U.
1-Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, Brasil. 2Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, 3Section on Structural Cell Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of
Health, Bethesda, MD 20892, USA. 4-School of Life Sciences, University of Nevada at Las Vegas, Las Vegas, NV 89154-4004, USA
Magnetotactic bacteria are a diverse group of microorganisms capable of alignment along magnetic field because of the presence
of intracellular nano-sized particle called magnetosomes. Each magnetosome consists of a magnetite (Fe3O4) or greigite (Fe3S4)
crystals enveloped by a lipidic membrane. In this membrane, proteins modulate the biomineralization of the magnetic crystals
inside vesicles and provide magnetosome chain organization. In Magnetospirillum species, the magnetosome organization is
provided by a filamentous network which runs parallel to the cell membrane and extends to the cell pole. This structure is
composed of MamK filaments, which is an actin-like protein. Here, we used cryotomography to determine the characteristics of
the magnetosome chain positioning and the molecular aspects of putative cytoskeleton elements and cryofracture to characterize
magnetosome membrane in ‘Candidatus Magnetovibrio blakemorei’, a marine magnetotactic vibrio which produces magnetite
magnetosomes. Cryotomography of ‘Ca. M. blakemorei’ showed filamentous structures consisting of a continuous structure with
multiple lengths near the magnetosome chain. To improve visualization of the filaments and its association with magnetosomes,
magnetic crystals were removed digitally in the tilt image series, and the tomogram was reconstructed from the processed tilt
series. Thus, the strong white artifacts around the crystals were avoided and the filaments could be observed in association with
the magnetosome chains. Sometimes tubular filamentous structures similar to “double membrane” profiles were observed
associated with the cell surface. Spherical and elongated empty magnetosome vesicles and immature magnetosome, which
consists of a small magnetic particle enclosed by a vesicle, were observed. Magnetosome membrane was not observed in mature
magnetosomes by cryotomography, but it was identified very closely associated with the crystal in a mature magnetosome by
freeze-fracture. The structural characterization of cytoskeleton elements and magnetosome membrane presented here is pivotal
for the understanding of the complex structural patterns of the cell biology of magnetic field orientation.
Support: INBEB, FAPERJ, CNPq.
PD14
BRAIN METABOLIC ADAPTATIONS IN RESPONSE TO SYSTEMIC INFLAMMATION
D'AVILA, J.C.; CARNEVALE R.; REIS, P.A.;GARCIA-SOUZA, L.F.; CASTRO-FARIA-NETO, H.C.; OLIVEIRA, M.F.; BOZZA, F.A.
1 Laboratório de Inflamação e Metabolismo - Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, UFRJ;
2Laboratório de Imunofarmacologia - Instituto Oswaldo Cruz, FIOCRUZ; 3Laboratório de Bioquímica Redox – Instituto de
Bioquímica Medica, UFRJ..
Aims: Complex metabolic changes are common in sepsis, including hypoglycemia, insulin resistance hypoxemia and
hyperlactatemia. During sepsis oxygen utilization and mitochondrial function are affected and associated with organ dysfunction
and mortality. Oxidative stress occurs in brain tissue and may be related to short and long term cognitive impairment. The goal of
this study was to characterize brain metabolic adaptations associated to sepsis and systemic inflammation. Methods: We used
murine models of endotoxemia and polymicrobial sepsis to access different aspects of brain energy metabolism and
neuroinflammation during experimental systemic inflammation. For glucose uptake study we used Positron Emission Tomography
(PET) with fluordeoxyglicose (FDG), glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG)
uptake by brain slices, and Hexokinase enzymatic activity in brain cytoplasmic and mitochondrial fractions. Mitochondrial function
was evaluated by oxygraphy; tissue oxidative stress was measured by protein immunoblot (dot-blot) for 4-hydroxynonenal.
Protein expression was measured by Western blotting. Inflammation was evaluated by ELISA and by immunohystochemistry; and
cognitive impairments were accessed by behavior tests. Results: Rodents with experimental sepsis induced by fecal peritonitis
present lower blood pressure, hyperlactatemia, renal and hepatic dysfunction, increased blood cytokines (IL-6, IL-1β and MIP),
brain oxidative stress and cognitive impairments. Brain slices from septic mice uptake more glucose and consume more oxygen
non-associated to ATP-synthesis 6h after fecal peritonitis induction, compared to slices from healthy animals. We detected a rapid
increase in brain FDG uptake in rats with endotoxemia in vivo by PET and increased expression of Hypoxia Inducible Factor-1 (HIF)
in the brain. Brain glucose uptake took place before peripheral organs, in 2h and peaked around 6h after LPS injection. Conclusion:
Our findings suggest that increased brain tissue oxidative stress has non-mitochondrial origin. We identified a new metabolic
phenotype that occurs in the brain in response to systemic inflammation, which is characterized by a rapid increase in glucose
uptake along with mitochondrial dysfunction that might be associated with tissue hypoxia.
Support: INBEB, FAPERJ, CNPq.
PD15
VISUALIZATION OF THE STRUCTURAL CHANGES ON THE SURFACE OF THE PLASMODIUM CHABAUDI INFECTED ERYTHROCYTE
USING DIFFERENT MICROSCOPY TECHNIQUES
1,2-SOARES MEDEIROS, L.C.; 3-ROCHA, G.M.; 1,2,3-DE SOUZA, W.; 1,2,3-MIRANDA, K.
1- Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro; 2 - Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro;
3- Laboratório de Biotecnologia, Diretoria de Programas, Instituto Nacional de Metrologia, Normalização e Qualidade Industrial
(INMETRO).
Plasmodium is an obligatory intracellular parasite that interacts with its host cell through several complex processes that include
insertion of membrane profiles in the cytoplasm of the erythrocyte, known as Maurer’s clefts and tubovesicular network (TVN),
responsible for protein transport. Several of the proteins transported through these membrane profiles have been localized on the
surface of the infected erythrocyte, suggesting an association of these membrane profiles and the alterations on the surface of the
red blood cells. The membrane of the infected erythrocyte often presents protrusions called knobs, which are structures
associated with cytoadherence and cerebral malaria. Here we characterized the surface and cytoplasm of erythrocytes infected
with P. chabaudi, a rodent malaria model for cytoadherence studies in the absence of knobs. We used different microscopy
techniques, including 3D reconstruction from electron tomography and serial images obtained with FIB-SEM, freeze-fracture TEM
and atomic force microscopy. Results showed that alterations in the intracellular organization of erythrocytes induced by infection
with P. chabaudi included insertion of membrane profiles of the parasites in the cytoplasm of the red blood cells that resemble the
TVN of P. falciparum infected erythrocytes. Three-dimensional (3D) visualization of these structures using images of FIB-SEM
microscopy showed that they represent 2% of the total volume of the infected red blood cells. Moreover, budding of micro and
small vesicles that are spread throughout the cytoplasm of the erythrocyte were also three-dimensionally observed. Atomic force
microscopy high resolution images showed the presence of knob like structures on the surface of the infected red blood cells.
These results provide insights of the structural modifications upon Plasmodium infection and exemplify how front-end microscopy
techniques can provide a better understanding of the mechanisms underlying the interaction of P. chabaudi with the host cell.
Support: CNPq, Capes e FAPERJ.
PD16
ON THE INTERACTION OF HSP90 WITH TOM70, A TPR CO-CHAPERONE
1,2- GAVA, L.M.; 1,2,3- GONÇALVES, D.C.; 4- BORGES, J.C.; 1,2- RAMOS, C.H.I.
1- Institute of Chemistry, University of Campinas (UNICAMP); 2- Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e
Bioimagem; 3- Institute of Biology, University of Campinas (UNICAMP); 4-Institute of Chemistry of São Carlos, University of São
Paulo.
Molecular chaperones play a key role acting as auxiliary protein on protein folding, refolding and dissociation of protein
aggregates. Hsp90 is one of the most important molecular chaperones, is essential for cell viability in eukaryotes and is usually
associated with proteins involved in cell cycling and cell signaling, which makes these chaperone a very interesting targeting for
therapeutic approaches for several diseases. Hsp90 acts in several biological processes in the cell, like protein translocation to
mitochondria, where Hsp90 and Hsp70 are involved with several co-chaperones, proteins that participate and also modulate the
Hsp90 activity. One of these co-chaperones is Tom70 - a TPR protein -that acts with Hsp90 to receive and translocate the cytosolic
preproteins into mitochondria. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90
chaperones. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we
used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif
that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer
of Tom70 per dimer of C-Hsp90 with a KD of 360 ± 30 nM, and the stoichiometry and thermodynamic parameters obtained
suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins
investigated.
Support: INBEB, FAPESP, CNPq.
PD17
POTENCIAL TERAPÊUTICO E RASTREAMENTO DE CÉLULAS-TRONCO MESENQUIMAIS DA MEDULA ÓSSEA MARCADAS COM
NANOPARTÍCULAS EM MODELO MURINO DA DOENÇA DE HUNTINGTON
1-MORAES, L.; 1-DE VASCONCELOS, A.;1-SANTANA, F.C.; 1-GODOY, M.A.; 1,2-ROSADO, P.R.; 1-AZEVEDO, R.; 1-JASMIN; 1,3-CINTRA
W.M.; 1,2-GASPARETTO,E.L.; 1-SANTIAGO, M.F., 1-MENDEZ-OTERO, R.
1-Instituto de Biofísica Carlos Chagas Filho da UFRJ; Departamento de Radiologia (HU, UFRJ); 3-Farmacologia UFRJ.
Neste trabalho, investigamos o potencial terapêutico de células mesenquimais de medula óssea (MSCs) em um modelo
experimental de Doença de Huntington (injeção intraestriatal de ácido quinolínico). As MSCs foram marcadas com nanopartículas
superparamagnéticas e implantadas no corpo estriado lesado. A detecção estas células foi realizada por ressonância magnética
(RM) e técnicas histológicas. Análises de neurodegeneração por Fluoro-Jade-C demonstraram que o tratamento com MSCs diminui
significativamente o número de neurônios mortos no estriado 7 dias após a lesão. Não houve variação significativa da
neurodegeneração comparando as diferentes doses utilizadas (1 x 106 e 2 x 106 MSCs). A terapia celular também reduziu a
ventriculomegalia e aumentou a expressão de FGF-2 no estriado lesado. As MSCs permaneceram viáveis após marcação com
nanopartículas e produziram sinais visíveis em imagens de RM por pelo menos 60 dias após o transplante. Nossas observações
indicaram que as células transplantadas no corpo estriado migram em direção ao corpo estriado lesado contralateral. Estes dados
apontam o potencial das MSCs em reduzir a degeneração estriatal e a possibilidade da utilização de nanopartículas no
rastreamento celular seguro e não invasivo.
Apoio: CNPq.
PD18
3-BROMOPIRUVATO: NOVA ESTRATÉGIA PARA INIBIÇÃO DE ENZIMAS GLICOLÍTICAS EM TRYPANOSOMA CRUZI E LEISHMANIA
AMAZONENSIS
1,2- GOMES, M.T.;1,2- COSENTINO-GOMES,D.; 1- GALINA, A.; 1,2- MEYER-FERNANDES, J.R.
1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2- Instituto Nacional de Ciências e Tecnologia de
Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro
A Doença de Chagas e a Leishmaniose são importantes doenças negligenciadas brasileiras. Embora estas doenças já tenham sido
descobertas a muitos anos atrás, não há vacinas disponíveis e a quimioterapia utilizada apresentam uma alta toxicidade e uma
eficácia variável, tornando-se urgente a procura por um tratamento mais eficaz e menos agressivo. Neste contexto, levando em
conta que tripanossomatideos tem diferenças importantes e fundamentais no processo de geração de energia quando
comparados aos eucariotos superiores, em nossos estudos pretendemos utilizar este mecanismo como estratégia antiparasitária.
Dentre os vários inibidores que afetam as enzimas chave da via glicolítica identificados, pesquisas recentes demonstraram que 3Bromopiruvato (3-BrPA) atua como um potente inibidor da enzima hexocinase, exercendo uma atividade anticancer promissora in
vitro e in vivo. Nossos resultados demonstram que 3-BrPA é capaz de inibir a proliferação de formas epimastigotas de
Trypanosoma cruzi (cepa Y e Dm28c) e formas promastigotas de Leishmania amazonensis. Observamos que a incubação de formas
amastigotas de T. cruzi (cepa Y) com 3-BrPA é capaz de reduzir em 50% a viabilidade celular. Em Dm28c observa-se uma inibição
da diferenciação de formas não infectivas em formas infectivas. Além disso, o tratamento prévio desses parasitos com essa droga
é capaz de diminuir a infecção dos parasitos a macrófagos peritoneais de camundongos. Quando observamos o efeito do 3-BrPA
sobre as enzimas glicolíticas desses organismos, verificamos uma redução de 53% na atividade da enzima hexocinase quando os
parasitos são cultivados por 4 dias na presença desta droga. A incubação dos parasitos por curtos intervalos de tempo (15, 30 e 60
minutos) não afetam as atividade desta enzima. Esses pequenos intervalos de tempo de tratamento com 3-BrPA também não são
capazes de induzir a morte celular por apoptose nesses parasitos. Mais estudos estão sendo realizados para melhor compreender
os aspectos bioquímicos e moleculares envolvidos no fenômeno.
Apoio: INBEB, CAPES, FAPERJ, CNPq.
PD19
HOW STABLE IS AN ENZYME FROM A THERMOPHILIC ORGANISM? DENATURATION STUDIES WITH THE ESTERASE FROM
PYROCOCCUS FURIOSUS – THE ROLE OF CHARGE-CHARGE INTERACTIONS
1-PAZ, N.V.N., 1-ALQUÉRES, S.M.C., 2-ALMEIDA, R.V., 1-ALMEIDA, M.S., 1-MARTINS, O.B., 1-FOGUEL, D.
1-IBqM, UFRJ, Brazil; 2-PEQ, COPPE, UFRJ, Brazil
Archaeal hyperthermophiles species grow optimally at temperatures between 80 and 110 oC. Enzymes from these organisms that
are more rigid than their mesophilic homologues developed unique structure-function properties of high thermostability and
Pyrococcus furiosus in Escherichia coli. This esterase showed to be active after boiling in the presence of SDS (under PAGE
conditions). Here, we accessed the unfolding of this heat-stable enzyme by fluorescence spectroscopy measurements using urea,
guanidinium hydrochloride (GndHCl) and high hydrostatic pressure (HHP) as denaturants. Ours results p
is strongly resistant to urea-induced denaturation ([Urea1/2]=7.1M) while GndHCl is three times more efficient
([GndHCl1/2]=2.3M) suggesting that ion pairs are important stabilizing factors on its structure. Similar results were found for two
HHP up to 3.1 kbar at 6 or 25oC, even by staying 120 min under very high pressure (2,500 bar), suggesting that protein is also
pressure-resistant. Interestingly, the combination of HHP with a subdenaturating concentration of urea (1M) displaced the center
of mass by ≈ 7nm after 40min at 2,500 bar, indicating a large exposure of tryptophan residues at this condition. Since HHP
enhances electrostriction, this result reinforces the crucial contribution of salt bridges in stabilize this enzyme. The binding of the
fluorescent probe bis(8-anilinonaphthalene-1- and 3-fold after treatment with 2M
GndHCl or under HHP combined with urea. These data suggest that these treatments convert the enzyme into a partially folded
intermediate with exposed hydrophobic regions. To our knowledge, this is the first time that HHP is used to access the ion pair
contributions to the stability of a hyperthermophilic esterase.
Support: FAPERJ.
PD20
HEPARIN BINDING INCREASES MURINE PRION PROTEIN STABILITY
1,2- VIEIRA, T.C. R. G.; 1,2- GOMES, M.P.B.; 3- CORDEIRO, Y.; 1,2- SILVA, J.L.
1- Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Instituto de Bioquímica Médica, Universidade Federal do Rio de
Janeiro; 2- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro;
3- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro.
The conversion of PrP into scrapie PrP is the central event of prion diseases. A series of molecules have been considered to work
as adjuvants in the conversion process, including glycosaminoglycans (GAGs). Our group recently reported that low molecular
weight heparin (LMWHep) does not induce recombinant murine prion protein (rPrP23-231) conversion, protecting rPrP23-231
from RNA-induced aggregation. We asked whether LMWHep increases murine prion protein stability, changing its ability to
aggregate. Here we show that formation of LMWHep-rPrP 23-231 complex increases rPrP 23-231 stability, decreasing
temperature-induced aggregation and amyloid fibril formation. Our findings may explain the protective effect of these molecules
in different models.
Support: INBEB, FAPERJ, CNPq.
PD21
-DEFENSIN 6 UPON BREAST CANCER CELL MEMBRANE INTERACTION
VIVIANE S. DE PAULA1, LUIZE G. LIMA2, ROBSON Q. MONTEIRO2, FABIO C.L. ALMEIDA1, ANA PAULA VALENTE1
1Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Instituto de Bioquímica Médica, Universidade Federal do
Rio de Janeiro. 2Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro
Defensins are small (30–45 amino acid residues) cationic proteins with broad antimicrobial activity against many bacteria and
fungi, some enveloped viruses, and other activities such as chemo attraction of a range of different cell types to the inflammation
sites. Human beta-defensins (hBD) selectively chemoattract human immature dendritic cells and memory T cells through specific
seven transmembrane G-protein-coupled receptors, the chemokine receptor CCR2 and CCR6. Recently, it has been described the
involvement of beta-defensin 1 (HbD1) in human cancer. This protein seems to be a potent tumor suppressor since it is able to
control transcription and induce apoptosis in renal cell carcinoma and its expression is decreased in some tumor types (Sun et al.,
2006). Our aim is to compare the binding of several types of tumorigenic breast cells and membrane models, searching for
correlations among immunological, structural, dynamics and binding properties of beta-human defensins. In this work, we present
the results of structural and dynamic studies by NMR of human beta-defensin 6 (HbD6) and its interaction with microvesicles
produced by breast cancer cells (cell lines: MCF-7 and MDA-MB-231) and membrane models. Because tumor microvesicles are
often rich in phosphatidylserine (PS) and thereby contribute to tumor escape mechanisms, we investigated HbD6 binding to PScontaining microvesicles. Our data showed a discrete interaction between HbD6 and MDA-MB-231 microvesicles, suggesting a
correlation with the high invasiveness of these cells. On the other hand, HbD6 exhibits enhanced binding to MCF-7, as compared
to MDA-MB-231. Furthermore, this interaction is partially reverted by Annexin V, indicating that the interaction could also involve
others receptors besides PS. Further experiments will be performed to examine the relationship between immunological and
binding properties with these microvesicles.
Support: INBEB, FAPERJ, CNPq.
P01
GENOMIC INSTABILITY AT 13Q31.1 LOCUS CORRELATES WITH TP53 MUTATION AND TUMOR AGGRESSIVENESS IN SPORADIC
BRAZILIAN BREAST CANCER CASES
1- GÓES, A.C.S.; 1- SANTOS JUNIOR, G.C.; 2- VITTO, H.; 1- MOREIRA, C.C.; 1- LEVY, C.; 3- AVVAD, E.; 2- RUMJANEK, F.D.; 1- MOURAGALLO, C.V.
1- Departamento de Genética, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro; 2Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 3- Departamento de Patologia, Instituto Fernandes
Figueira, FIOCRUZ.
Genomic instability is a hallmark of malignant tissues. Breast cancer, which is characteristically a heterogeneous disease, shows
diverse types and levels of genomic alterations. The present work aims to characterize nuclear allelic imbalance and mitochondrial
instabilities in a cohort of Brazilian breast cancer cases. Thus, we analyzed 64 matched pairs of breast cancer and adjacent noncancerous breast samples, by genotyping 13 STR loci, namely, D2S123, TPOX, D3S1358, D3S1611, FGA, D7S820, TH01, D13S317,
D13S790, D16S539, D17S796, BRCA1 (intron 12) and TP53 (intron 1) and by direct sequencing HVI and HVII mitochondrial regions.
About 60% of the cancer tissues presented, in some extent, nuclear allelic imbalance. The most frequently affected locus was the
D13S790 (29.85%) while the D2S123 locus didn`t presented altered cases. We observed that 49.2% of cases were mitochondrial
unstable, being 23 due to mitochondrial instability, 15 originated by somatic mutation and 5 with both genetic events. The advent
of nuclear allelic imbalance and Elston grade III (p=0.0001) were strongly correlated. Only the instabilities in 13q31 region were
associated with TP53 mutations (p=0.01), mostly in ductal infiltrating carcinomas (p=0.002). This data highlights the huge
importance of 13q31 region in development of more agressive tumors. Moreover, our results suggest that nuclear and
mitochondrial instabilities are independent events.
Support: FAPERJ, INBEB.
P02
PESQUISA DE MUTAÇÕES GERMINATIVAS NO TP53 EM MULHERES DE ALTO RISCO PARA O CÂNCER DE MAMA E INVESTIGAÇÃO
DO PAPEL DA P53 MUTANTE EM LINHAGENS LINFOBLASTÓIDES.
1,4-FERREIRA, E.S.; 2- CASTRO, V.R.M.; 2- PAIXÃO, J.C.; 3-PAGNONCELLI, D.; 1,4-DE MOURA GALLO, C.V.
1-Departamento de Genética, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro; 2Laboratório de Biologia Molecular, Instituto Fernandes Figueira – Fiocruz; 3-Departamento de Ginecologia, Instituto Fernandes
Figueira – Fiocruz; 4- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio
de Janeiro
A p53 é um fator de transcrição considerado como um regulador “master” envolvido no controle de inúmeras vias celulares
ativadas por estresse genotóxico. Mutações germinativas em seu gene (TP53) predispõe ao desenvolvimento precoce de vários
tumores, sendo o câncer de mama o mais frequente. O presente trabalho teve como objetivo a pesquisa de mutações no TP53 a
partir do DNA genômico de mulheres de alto risco para o câncer de mama e análise funcional da p53 mutante, através do
desenvolvimento de linhagens linfoblastóides (LFBs) originadas de indivíduos portadores de mutação. Também foi pesquisada a
presença de mutações em BRCA1 e BRCA2 em algumas destas famílias, selecionadas segundo o risco. Assim, foi realizada a análise
de 24 famílias do ambulatório de Alto Risco do Instituto Fernandes Figueira, Rio de Janeiro. Após a extração do DNA genômico de
sangue periférico, foi realizado o sequenciamento direto dos exons dos genes acima citados (Plataforma Genômica do PHL-UERJ).
Através dos resultados obtidos foi identificada uma mulher de alto risco, não afetada, portadora da mutação p.R337H e não
portadora de mutação em BRCA1 e 2. Esta mutação está localizada no domínio de oligomerização da p53 e é especialmente
presente na população brasileira. Resultados preliminares com o ensaio do MTT, para avaliação da viabilidade celualr, após
irradiação com raios gama de duas LFBs-selvagem e da LFB-mutante, mostraram que esta última apresenta comportamento
diferente dos controles. Outros parâmetros da fisiologia celular estão sendo avaliados nestas linhagens.
Apoio: INBEB; FAPERJ; CNPq; UERJ.
P03
HISTAMINE-PRIMING OF THE MICROVASCULATURE: A NEW PHYSIOLOGICAL PATHWAY LEADING TO THE EXTRAVASCULAR
ACTIVATION OF THE KALLIKREIN-KININ SYSTEM.
SVENSJÖ, E., NASCIMENTO, C.R., SERRA, R.,SCHARFSTEIN, J.
Instituto Biofísica Carlos Chagas Filho
It is well-established that intravenous injection of the contact system activator dextran sulfate (500 kDa) (DxSO4) induces
cardiovascular hypotension through the release of bradykinin (BK) (Siebeck et al. 1994). In the past few years, there has been a
resurgence of interest of the Kallikrein-Kinin System (KKS) owing to discoveries implicating FXII/PK-dependent pathways of fibrin
deposition on platelets to the pathogenesis of thrombosis. Here we tested whether the topical application of DxSO4 on the noninflamed microvasculature of the hamster cheek pouch (HCP) also could result in BK-induced plasma leakage. Anesthetized
hamsters prepared for intravital microscopy using FITC-dextran as a marker of plasma leakage were subjected to continuous
topical application of DxSO4 (0.2 mg/ml) on HCPs. Although no measurable changes in plasma leakage could be detected during
the first 30 min of DxSO4 superfusion, the microvasculature consistently responded at the end of the silent lag phase. Local
application of HOE-140 (0.5 μM) blocked the delayed plasma leakage elicited by DxSO4, implying that the reaction was elicited by
BK, acting via BK2R. We then addressed the possibility that presence of the contact system plasma components in the
extravascular tissues is a pre-requisite for the peripheral generation of kinins in the HCP exposed to DxSO4. This hypothesis was
tested by exposing the HCP to histamine 4 µM in the presence/absence of DxSO4 in the superfusate. Strikingly, we found that
histamine induced a very potent leakage reaction in HCP-DxSO4 (4 X higher than the reaction caused by histamine alone).
Although captopril (0.01 mg/ml) did not interfere with histamine-induced leakage in HCP controls, the application of the ACE
inhibitor in the presence of DxSO4 further enhanced (nine times) histamine-induced plasma leakage whereas HOE-140 (BK2
antagonist) abolished these effects. Ongoing studies should determine whether endothelium “priming” by histamine, a mast cell
secretion product, might render the HCP extravascular tissues increasingly sensitive to KKS activation by negatively charged
macromolecules of exogenous (pathogens) or endogenous (polyphosphate or heparin) origin.
Support: FAPERJ, CNPq/INBEB.
P04
REFERENCE MATERIAL OF GOLD NANOPARTICLES: ANALYTICAL MEASUREMENT
1,3ROCHA, G.M., 1,2,3FRASÉS, S., 1,3FONTES, G., 1,2CORDEIRO, L.S., 1,2,3DE SOUZA,W., 1,3SANT’ANNA, C.
1Lab. de Biologia (LABIO), Diretoria de Programas (DIPRO), INMETRO., 2Lab. de Ultra-Estrutura Celular Hertha Mayer, Instituto de
Biofísica Carlos chagas Filho, UFRJ, 3Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, UFRJ
Reference Materials and Certified Reference Materials are classified as homogeneous materials or substances with wellestablished functions, which are used by laboratories and industries to calibrate their instruments, to develop reliable testing
methods and to perform regular quality controls. The inappropriate standards for characterization of nanostructured products
generates bad quality measurements and therefore economical lost by rejection of products that cannot offer any reliability. The
use of gold nanoparticles to therapeutics and diagnostics analysis must be preceded by standardization methodologies. Our group
aims to apply image-based and nonimage-based standardization methodologies to produce a reference material of nominal 15 nm
citrate-stabilized colloidal gold nanoparticles (CGN) to be used according to internationally technical specification as laid down in
the ISO (International Organization for Standardization). The CGN were characterized by size, distribution and aggregation
potential in different pH (3.0, 5.0, 6.0, 7.0 and 9.0) and temperatures (4, 25 and 37oC) by using analytical measurement
techniques, such as Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM), Dynamic Light Scattering (DLS) and
Zeta Potential (ZP). CGN observed by TEM and AFM showed that they are approximately spherical in shape. TEM and AFM results
revealed that nanoparticle average size is 12 nm. CGN were well dispersed at pH 5 and 250C, which seems to be the optimal
condition to use. By TEM, AFM and DLS large aggregate of CGN was observed in low (3.0) and high (9.0) pH in all temperature
analyzed. In addition, ZP analyses demonstrated the ability of these nanoparticles to aggregate depend on the solvent
electronegativity. In all conditions tested CGN presented high potential to aggregate. The relevance of this study relies on the fact
that the production of a Brazilian reference material to be applied in the field of Nanotechnology is missing. Therefore, these CGN
would be the first national nanotechnological reference material.
Support: Inmetro, INBEB, CNPq.
P05
SILDENAFIL INHIBITS NITRIC OXIDE SIGNALING TO STIMULATE LEYDIG CELL STEROIDOGENESIS
1- SARAIVA, K.L.A.; 2- WANDERLEY, M.I.; 3- GOMES, F.O.S.; 3- RIBEIRO, E.L.; 1,3- PEIXOTO, C.A.
1-Laboratory of Microscopy and Microanalysis, Strategic Technologies Center of Northeast (CETENE); 2-Department of Physiology
and Pharmacology, Federal University of Pernambuco; 3-Laboratory of Ultrastructure, Aggeu Magalhães Institute (IAM/FIOCRUZ).
Background: Sildenafil citrate(Viagra®), a cGMP-selective phosphodiesterase (PDE) inhibitor, is widely used to treat erectile
dysfunction and pulmonary arterial hypertension. Little is known about the action of this drug on nitric oxide (NO) pathway and its
involvement on testicular steroidogenesis. This study was designed to assess the effects of prolonged Sildenafil treatment on NOdependent signaling and steroidogenic function of male mice Leydig cells. Methods: The treated group received Sildenafil (25
mg/kg body wt) for eight weeks administered in the drinking water. The control group received only pure water. After the
experimental design, the animals were anesthetized, before blood collection. Serum was separated and stored at –70 ºC for
radioimmunoassay of testosterone hormone. Testes were quickly dissected and fixed for transmission electron microscopy
evaluation. In addition, Leydig cells were isolated to detect NO synthases by immunocytochemical procedures, and to ex vivo
assay for determination of nitrite and testosterone concentration in cell culture supernatant. Results: After the prolonged
treatment, the ultrastructure of Leydig cells showed portions of vesicular smooth endoplasmic reticulum (VSER), enlarged
mitochondria with dilated cristaes and whorles membranes of SER. All of these alterations are typical of an activated cell. The
levels of testosterone were increased in serum, as well as in Leydig cells incubation medium. The immunocytochemical labeling for
NOS2 significantly decreased. Additionally, the expression of NOS3 remained unchanged. The nitrite concentration was reduced,
even in the presence of SNAP, a NO donor. Conclusion: It has been reported that chronic treatment with Sildenafil enhances
testosterone production via PDE inhibition and cAMP/cGMP signaling pathway stimulation. The present study demonstrated that
increased steroidogenic capacity is also related to the reduction of NO, since this messenger molecule acts as a modulator of
testosterone production. In summary, Sildenafil downregulates NO-dependent signaling in Leydig cells to stimulate
steroidogenesis.
Support: CETENE, CNPq, INBEB and FIOCRUZ.
P06
ANÁLISE DO EFEITO DE ANTI-HISTAMÍNICO NOS METABÓLITOS SALIVARES POR RMN.
FIDALGO TK 1,FREITAS-FERNANDES LB 1, ALMEIDA F 2, POMARICO I 1,VALENTE A-P 2.
1- Odontopediatria e Ortodontia Faculdade de Odontologia, Universidade de Federal do Rio de Janeiro, 2- Centro Nacional de
Ressonância Magnética Nuclear Jiri Jonas, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro
O efeito de medicamentos líquidos na superfície do esmalte dental é amplamente abordado na literatura. Entretanto, poucos
estudos avaliam sua influência nos metabólitos salivares. Objetivou-se avaliar in vitro e in vivo o efeito local de um xarope antihistamínico sobre metabólitos salivares por meio de espectroscopia de RMN. Foram recrutados 5 voluntários saudáveis com idade
variando de 24 a 30 anos (26,6 ± 2,2) para avaliação antes e após o uso do anti-histamínico (Claritin®). Para a interação in vitro, 5
ml de saliva total não estimulada foram coletados e misturados in vitro com o anti-histamínico. Para o estudo in vivo, antes da
coleta da amostra, os mesmos voluntários foram instruídos a bochechar com Claritin® por 20 segundos previamente a
expectoração da saliva. A mistura in vitro e in vivo foram centrifugadas a 40C, 3.000 g durante 3 min e os respectivos
sobrenadantes foram análisados por espectro 1H de RMN (Bruker 400 MHz). Os dados foram submetidos à Análise de
Componentes Principais (APC) e teste t pareado (p<0,05). O APC demonstrou diferença entre antes e após a interação. Os
metabolitos acetato, histidina, ɣ-aminobutirato, glutamato, isoleucina, fenilalanina, trimetilamina, propionato e valina
apresentaram menor concentração após a interação anti-histamínico (teste t; p<0.05). Existem diferenças entre os dados obtidos
in vitro quanto in vivo. Atualmente, estamos analisando os dados obtidos com o anti-histamínico na forma de comprimido para
analisarmos a importância da formulação nas alterações observadas.
Apoio: INBEB, FAPERJ, CNPq.
P07
THE HEPATITIS C VIRUS CORE PROTEIN: NEW INSIGHTS ON THE STRUCTURE, FUNCTION AND VIRUS CAPSID ASSEMBLY
1- SOUZA, T.L.F.; 2- BRAGA, V.L.A.; 3- LIMA, S.M.B.; 4,5- FERREIRA, D.F.; 6- PEABODY, D.S.; 2- BIANCONI, M.L.; 2,5- GOMES, A.M.O.;
2,5- SILVA, J.L.; 2,5- OLIVEIRA, A.C.
1- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Brasil; 2- Programa de Biologia Estrutural, Instituto de
Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brasil; 3- Instituto de Tecnologia em Imunobiológicos, Fundação
Oswaldo Cruz, Rio de Janeiro, Brasil; 4- Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Brasil; 5Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Brasil; 6University of New Mexico, USA.
Hepatitis C virus (HCV) is a worldwide public health problem because more than 3% of the population is infected by this virus and
therapies are inefficient so far. Hepatitis C Virus (HCV) core protein have been describe be involved in several cellular processes,
including lipid metabolism, microRNA and mRNA homeostasis, oxidative stress and apoptosis, playing a critical role in HCV
pathogenesis. The molecular basis of these mechanisms is not well understood. The C-terminal truncated HCV core protein
(HCVCP124) is intrinsically unstructured and has been found in patients with chronic HCV infection. In this work, we examine the
propensity of the HCVCP124 to folding in different conditions and investigate new physical chemical aspects of its interaction with
short unspecific nucleic acids (18 and 21 nucleotides that are equivalent size to cellular microRNA). For this purpose we used
spectrophotometry, circular dichroism, fluorescence spectroscopy, fluorescence correlation spectroscopy, transmission electron
microscopy and calorimetry. Only in the presence of trifluoret
-helix structure
stabilized. Interestingly, we found that the truncated HCV core protein multimerize itself into empty nucleocapsid-like particles
(NLPs) when is submitted at pH values close to the isoelectric point (pH ≈ 12). This result showed, for the first time, that the
electrostatic repulsion among the positive charges of the basic residues is the only an energy barrier that avoids protein
multimerization, what indicates that unspecific polyanions can initiate the process. In addition, data obtained from fluorescence
correlation spectroscopy indicate that HCVCP124, in nanomolar range, is able to interact and to sequester short unspecific acid
nucleic into NLPs in a process highly cooperative. We suggest that HCVCP124 can sequester cellular microRNA and mRNA into
NLPs. Our results can shed light on the molecular basis of secondary effects of the C-terminal truncated core protein on the HCV
pathogenesis.
Support: INBEB, FAPERJ, CNPq, CAPES, PRONEX.