Instituto Nacional de Ciência e tecnologia de Biologia Estrutural e

Transcrição

Instituto Nacional de Ciência e Tecnologia de
Biologia Estrutural e Bioimagem - INBEB
O Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem (INBEB) é uma iniciativa
pioneira com a missão de criar e consolidar uma infraestrutura técnico-científica que permita o estudo das
estruturas de sistemas biológicos, desde o nível macromolecular até o organismo inteiro, lançando mão das
técnicas mais avançadas e de mais alta resolução possível.
O INBEB promove a interdisciplinaridade, fazendo com que áreas convencionais como biofísica, parasitologia,
microbiologia, imunologia, bioquímica, farmacologia, química e computação tenham suas fronteiras
estendidas. Isto permite a maior interação entre grupos para solucionar diversos problemas biológicos. Outro
objetivo fundamental do Instituto é contribuir para o treinamento de pesquisadores em Biologia Estrutural e
Bioimagem em vários níveis.
É neste âmbito que apresentamos no Programa do IV Encontro Anual do INBEB a programação do evento,
assim como os resumos dos 163 pôsteres inscritos no encontro. Esperamos que todos possam aproveitar a
oportunidade para interagir, aperfeiçoar seus trabalhos e estabelecer novas parcerias.
Prof. Jerson Lima da Silva
Prof. Wanderley de Souza
Coordenador do INBEB
Vice-coordenador do INBEB
Apoio:
IV Encontro Annual do INBEB – www.inbeb.org.br
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Índice
Programação .......................................................................................................................................................................... 3
Quarta-feira - 08/05/2013 .................................................................................................................................................. 3
Quinta-feira - 09/05/2013 .................................................................................................................................................. 5
Sexta-feira - 10/05/2013..................................................................................................................................................... 7
Sessões de pôsteres ............................................................................................................................................................... 9
Data das apresentações ...................................................................................................................................................... 9
Certificados ......................................................................................................................................................................... 9
Listas de apresentadores .................................................................................................................................................. 10
Graduandos................................................................................................................................................................... 10
Mestrandos ................................................................................................................................................................... 12
Doutorandos ................................................................................................................................................................. 13
Pós-doutorandos .......................................................................................................................................................... 14
Pesquisadores ............................................................................................................................................................... 15
Resumos ............................................................................................................................................................................... 16
Graduandos....................................................................................................................................................................... 16
Mestrandos ....................................................................................................................................................................... 30
Doutorandos ..................................................................................................................................................................... 39
Pós-doutorandos .............................................................................................................................................................. 58
Pesquisadores ................................................................................................................................................................... 66
IV Encontro Anual do INBEB – www.inbeb.org.br
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Programação
Quarta-feira - 08/05/2013
Auditório Hélio Fraga, CCS/UFRJ
9:00 - 9:30h
Cadastramento e entrega dos materiais.
9:30 - 10:00h
Apresentação - Prof. Jerson Lima da Silva, IBqM / UFRJ / Coordenador do INBEB.
10:00 – 10:30h
LA 14: “Potencial imunomodulador de compostos naturais com propriedades leishmanicida”. - Profa.
Edilene Oliveira da Silva, UFPA.
10:30 – 11:00h: Coffee-Break.
11:00 – 11: 30h
LA 2: “O papel das microglias nas amiloidoses”. - Profa. Débora Foguel, IBqM/UFRJ.
11:00 – 11:30h
LA 3: “BEX3 (Brain expressed x-linked), an apoptotic protein with intriguing structural behaviour”. - Prof.
Fabio Almeida, IBqM/UFRJ.
11:30 – 12: 00h
LA18: “Trypanosoma cruzi, a flagellated protozoan surfing on a proteolytic tzunami: an adaptive strategy
leading to persistent infection in the edematous heart?”. - Prof. Julio Scharfstein, IBCCF/UFRJ
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12:00 – 13:30h: Almoço.
13:30 – 15:00h
Mesa redonda:
“PESQUISA TRANSLACIONAL: COLABORAÇÃO IDOR – INBEB”
Chair: DRA. LÉA MIRIAN/FACULDADE DE MEDICINA/UFRJ
1.Dra. Fernanda Tovar Moll, Profa. Adjunta/ICB/UFRJ, Responsável pela RMN do CENABIO/UFRJ e Diretora
Científica do Instituto D'Or de Pesquisa e Ensino: “POSSIBILIDADES DE APLICAÇÃO DA RESSONÂNCIA MAGNÉTICA DE 7T E
3T”
2.Dr. Jorge Moll, Diretor de Pesquisa do Instituto D'Or de Pesquisa e Ensino: “RESSONÂNCIA MAGNÉTICA
ESTRUTURAL E FUNCIONAL NA INVESTIGAÇÃO DE DOENÇAS PSIQUIÁTRICAS”
3.Dr. Fernando Bozza, Pesquisador da Fundação Oswaldo Cruz e Chefe do Centro de Terapia Intensiva do
Instituto de Pesquisa Clínica Evandro Chagas/FIOCRUZ: “ESTUDO DA INFLAMAÇÃO PULMONAR, AVALIADA ATRAVÉS DE
MODELOS CLÍNICOS E EXPERIMENTAIS”
15:00 – 15:30h
LA 6: “Protein phosphatases: model systems for biomimetics”. - Prof. Hernán Terenzi, UFSC.
15:30 - 16:00h
LA 15: “Caracterização de novos fármacos anti-inflamatórios”. - Profa. Christina Peixoto, Fiocruz/INTCETENE.
16:00 – 16:30h
LA 8: “Planejamento in silico de candidatos à fármacos contra doença tropicais negligenciadas”. - Prof.
Marcelo Castilho, FF/UFBA.
16:30 – 17:0h
LA 10: “Novos microscópios de força atômica do inbeb” - Prof. Gilberto Weissmuller, IBCCF/UFRJ.
17:00 – 19:00h: Sessão de Pôsteres / Coquetel.
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Quinta-feira - 09/05/2013
9:30 – 10:00h
LA 9: “Em busca de compostos mais eficientes e menos tóxicos para quimioterapia das leishmanioses". Profa. Juliany Cola Fernandes Rodrigues, IBCCF/UFRJ.
10:00 – 10:30h
LA 1: “Structural biology and bioimaging studies on cancer, neurodegenerative and viral diseases”. – Prof.
Jerson Lima da Silva, IBqM/UFRJ.
10:30 – 11:00h: Coffee-Break
11:00 – 11:30h
LA 4: “A venômica e antivenômica revelando novas moléculas com potencial biotecnológico para o desenho
de novos fármacos e controle de qualidade de soros antiofídicos”. - Profa. Lina Zingali, IBqM/UFRJ e Dr.
Carlos Correa Netto, Pós-doc IBqM/UFRJ.
11:30 – 12:00h
LA 16: “Abordagens experimentais no estudo das células-tronco”. - Profa. Regina Goldenberg, IBCCF/UFRJ.
12:00 – 13:00h
Conferência Magna:
- Dr. Kurt Wuthrich (Prêmio Nobel de Química em 2002), Prof. Visitante do IBqM/UFRJ/INBEB: “Historical
Development and Current Trends of NMR in Structural Biology and Biotechnology“.
13:00 – 14:30h: Almoço
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14:30 – 16:00h
Mesa Redonda
“DIVULGAÇÃO CIENTÍFICA NO INBEB: INTERAÇÃO UNIVERSIDADE E ESCOLA”
Chair: PROFA. MARTHA SORENSON/IBQM/UFRJ
1.Dr. Emiliano Medei/Prof. Adjunto/IBCCF/UFRJ: “CONTEXTUALIZAÇÃO CIENTÍFICA PARA ALUNOS DA EDUCAÇÃO BÁSICA DE
UMA REGIÃO RURAL DO ESTADO DO RJ”
2.MsC. Josineide Pantoja/Profa. da Rede Municipal/Igarapé Miri/PA: “ATIVIDADES CIENTÍFICAS NA CIDADE DE
IGARAPÉ-MIRI/PA”
3.Dra. Patricia S. dos Santos, Coordenadora do Núcleo de Educação e Divulgação Científica do INBEB:
“ATIVIDADES DO NEDICI/INBEB”
16:00 – 16:30h
LA13: “Biogênese do flagelo de leishmania amazonensis durante a diferenciação celular”. – Dra. Ana Paula,
INMETRO.
16:30 – 17:30h: Reunião do Comitê Gestor do INBEB
17:00 – 19:00h: Sessão de pôsteres/Coffee-break.
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Sexta-feira - 10/05/2013
10:00h: Coffee-break
10:00 – 10:30h
LA 12: “Identificação de uma nova estrutura que participa na formação da parede cística em Giardia
intestinalis”. - Profa. Marlene Benchimol, USU.
10:30 – 11:00h
LA 19: “Terapia celular em modelos animais de doenças neurológicas”. - Prof. Marcelo Santiago, IBCCF/UFRJ.
11:00 – 11:30h
LA 5: “Novos métodos em cálculos estruturais e dinâmicos de biomoléculas: aplicações em biomembranas
e enzimas”. - Prof. Pedro Pascutti, IBCCF/UFRJ.
11:30 – 12:00h
“Caracterização do padrão de captação de 18-FDG na lesão pulmonar aguda”. - Prof. Alysson Roncally, IBCCF
COPPE/UFRJ.
12:00 – 13:30h: Almoço
13:30 – 14:00h
LA 7: “Eukariotic molecular chaperones: sturctural and cell biology”. - Prof. Carlos Ramos, IQ/Unicamp.
14:00 – 14:30h
LA 11: "Trypanosoma abeli: um novo Trypanosoma de peixes". – Dra. Moara Lemos, Pós-doc/IMPPG/UFRJ.
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14:30 – 15:00h
LA 17: “Uma visão do realizado no biênio 2011-2012 no laboratório 17 do INBEB”. - Prof. Adalberto Vieyra,
IBCCF/UFRJ.
15:00 – 15:30h
Conferência de encerramento:
-Premiação dos melhores pôsteres
-Resultado do concurso do logo do INBEB
-CENABIO: O novo Órgão Suplementar da UFRJ – Apresentação do Diretor.
15:30 – 17:00h: Confraternização.
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Sessões de pôsteres
Data das apresentações
Trabalhos de número par: dia 08/05
Trabalhos de número ímpar: dia 09/05
Certificados
Após a arguição dos avaliadores, serão entregues certificados de apresentação apenas em nome dos
apresentadores inscritos que forem avaliados. Os demais autores terão este caderno de resumos como
certificado de inscrição dos trabalhos no evento.
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Listas de apresentadores
Os resumos estão separados por titulação e ordenados alfabeticamente pelo primeiro nome do apresentador.
Graduandos
G 1
- Ana Beatriz Padilha de Figueiredo
G 17 - Geovana Vargas Silva
G 2
- Arthur Daniel Rocha Alves
G 18 - Gerson Duarte Guercio
G 3
- Brunno Renato Farias Verçoza
G 19 - Giselle Souza Moreira
G 4
- Camila Cristina da Silva
G 20 - Gusthavo figueira Barbosa
G 5
- Carolina Andrade
G 21 - Hortencia Almeida da Silva Monteiro
G 6
- Caroline Lauritzen da Costa
G 22 - Jéssica Monteiro de Oliveira
G 7
- Caroline Mendes Ferreira
G 23 - João Paulo Bortot Soares
G 8
- Cássia Netto de Araújo
G 24 - José Eduardo Teixeira Pinto Junior
G 9
- Clarice de Souza Cerqueira Machado
G 25 - Juliana Santos Santana
G 10 - Claudia Monteiro da Rocha
G 26 - Leandro Braga Rodrigues
G 11 - Daniel Antônio Shimizu Kitagawa
G 27 - Lilian Goulart Schultz
G 12 - Daniel Meira dos Anjos
G 28 - Lilianne Gomes de Magalhães LAmeira
G 13 - Eliã Barbosa Marins
G 29 - Marcella Valentim Monteiro Ferreira
G 14 - Elias Ataide Mendonça
G 30 - Marina Chao Campello
G 15 - Felipe Henrique Souza daSilva
G 31 - Matheus Esteves Ferreira
G 16 - Gabriel Nascimento dos Santos
G 32 - Mayara Gil de Castro Santos
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G 33 - Mayra de Amorim Marques
G 40 - Thais Cordovil
G 34 - Michelle Guimarães Furtado
G 41 - Thaís de Oliveira Silva
G 35 - Nathali Pereira da Costa Campos
G 42 - Veronica da Silva Ferreira
G 36 - Neilton Cesar Araujo da Cruz
G 43 - Wesley Júnio Alves da Conceição
G 37 - Nicoli Cardoso Mortari
G 44 - William Yutaka Ohashi
G 38 - Roger Borges dos Santos
G 45 - Yasmin Lemos Rollemberg Cruz Machado
G 39 - Tarcísio Nascimento Corrêa
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Mestrandos
M 1
- Aline Miyoko Sakaguchi Yamashita
M 17 - Gabriela Veras de Moraes
M 2
- Almir Jordão da Silva Junior
M 18 - Jorge Augusto Leão Pereira
M 3
- Alvaro Carrier Ruiz
M 19 - Júlia Araújo de Freitas
M 4
- Ana Clara Vicente dos Santos
M 20 - Laise Aline Martins dos Santos
M 5
- Ana Paula Miranda Mendonça
M 21 - Luiz Carlos dos Santos Ribeiro
M 6
- André Roberto de Oliveira Fredi
M 22 - Luiz Felipe Garcia e Souza
M 7
- Beatriz Bastos Fonseca
M 23 - Luiza Fernandes
M 8
- Bruno José Martins da Silva
M 24 - Maria Eduarda Rocha de França
M 9
- Camila Carane Bitencourt Brito
M 25 - Paula Cristina Rodrigues Frade
M 10 - Camila Silva Gonçalves
M 26 - Pedro Ernesto Lopes Leão
M 11 - Carolina De Lima Alcantara
M 27 - Rachel de Pinho Rachid
M 12 - Caroline Martins Almeida
M 28 - Tatiana Kelly da Silva Fidalgo
M 13 - Cinthia Lima
M 29 - Wilma Helena de Oliveira
M 14 - Clara Simões
M 15 - Diego de Souza Gonçalves
M 16 - Gabriel Barros Rodrigues
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Doutorandos
D 1 - Adolfo Henrique de Moraes Silva
D 20 - Edlene Lima Ribeiro
D 2 - Alane Bernardo Ramos
D 21 - Erivan Schnaider Ramos Junior
D 3 - Aline Lidiane Batista de Amorim
D 22 - Estefania Pereira Cardoso Azevedo
D 4 - Amanda Karolina Soares e Silva
D 23 - Éverton Dias D'Andréa
D 5 - Ana Karolina de Santanna Nunes
D 24 - Fabian Gilberto Villalta Romero
D 6 - Andréa Marins Damasceno Bomfim
D 25 - Fernando Antonio de Oliveira Adnet
D 7 - Anne Cristine Silva Fernandes
D 26 - Franco Henrique Andrade Leite
D 8 - Aracelys López Castilla
D 27 - Gilson Costa dos Santos Junior
D 9 - Bruna Santos da Silva
D 28 - Glaucia Melina Squizato Pinheiro
D 10 - Bruno Macedo
D 29 - Gloria Maria Castañeda Valencia
D 11 - Camila Salata
D 30 - Gustavo Monnerat Cahli
D 12 - Carlos Alberto Marques de Carvalho
D 31 - Ivone Rosa de Andrade
D 13 - Carlos Henrique Dumard
D 32 - Joseane Lima Prado Godinho
D 14 - Carolina Catta Preta
D 33 - Josineide Pantoja da Costa
D 15 - Caroline Rezende Guerra
D 16 - Cherley Borba Vieira de Andrade
D 17 - Cícero Figueiredo Freitas
D 36 - Juliana Cunha Vidal
D 18 - Daniel Sanches
D 37 - Juliana Pandini Castelpoggi
D 19 - Denise Cristian Ferreira Neto
D 38 - Leandro Teixeira de Oliveira
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D 39 - Letícia Maria Zanphorlin Murakami
D 51 - Samir pereira da costa campos
D 40 - Luana Borba
D 52 - Sara Teixeira de Macedo Silva
D 41 - Marcelo de Lima Sant Anna
D 53 - Sirlene Oliveira Francisco de Azeredo
D 42 - Marco Antonio Vidal Jimenez
D 54 - Sura Wanessa Santos Rocha
D 43 - Mariana Araya de Godoy
D 55 - Tatiana Christina Paredes Santos
D 44 - Matheus Pinto de Oliveira
D 56 - Tiago Bortolotto
D 45 - Natália do Carmo Ferreira de Araújo
D 57 - Tiago Veltri Ormastroni da Trindade
D 46 - Nathalia dos Santos Alves
D 58 - Vanessa Lopes de Azevedo Braga
D 47 - Rayana Leal de Almeida Luna
D 59 - Victor do Valle Pereira Midlej
D 48 - Renata Travassos de Lima
D 60 - Viviane Cristina Heinzen da Silva
D 49 - Ricardo Santanna Oliveira
D 50 - Roberta Ferreira Cura das Neves
Pós-doutorandos
PD 1 - Bruno Kaufmann Robbs
PD 7 - Catarina Raposo
PD 2 - Camila Marques Adade
PD 8 - Clarissa Rodrigues Nascimento
PD 3 - Camila Zaverucha do Valle
PD 9 - Danielly Cristiny Ferraz da Costa
PD 4 - Carlos Correa Netto
PD 10 - Fabiana Pestana Albernaz
PD 5 - Carolina Galvão Sarzedas
PD 11 - Fabio Mendonça Gomes
PD 6 - Caroline Madeira Moreira
PD 12 - Fernanda de Avila Abreu
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PD 13 - Fernanda Gubert
PD 19 - Nathalia Varejão
PD 14 - Joana da Costa Pinto d'Avila
PD 20 - Patricia Alves Reis
PD 15 - Juliana M. F Dutra Santiago
PD 21 - Priscila Ferreira
PD 16 - Leandro Lara de Carvalho
PD 22 - Shana Priscila Coutinho Barroso
PD 17 - Mariana Pierre de Barros Gomes
PD 23 - Tuane Cristine Ramos Goncalves Vieira
PD 18 - Moara Lemos
PD 24 - Viviane Silva de Paula
Pesquisadores
P 1
- Ana Carolina Oliveira
P 4
- Marcela Cristina de Moraes
P 2
- Gisele Cardoso de Amorim
P 5
- Mariana Aragão Matos Donato
P 3
- Karla Patricia de Sousa Barbosa
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Resumos
Graduandos
G1.
ANÁLISE POR RESSONÂNCIA MAGNÉTICA DA PERMANÊNCIA DE CÉLULAS
DE MEDULA ÓSSEA NO VÍTREO APÓS LESÃO DO NERVO ÓPTICO
1,2- FIGUEIRÊDO, A.B.P.; 1,2- MESENTIER-LOURO, L.; 1,2- ZAVERUCHA-DO-VALLE, C.;
1,2- NASCIMENTO-DOS-SANTOS, G.; 2- TEIXEIRA, C.; 2- TOVAR-MOLL, F.; 1,2- MENDEZOTERO, R.; 1,2- SANTIAGO, M.F.
1- Instituto de Biofísica Carlos Chagas Filho; 2- Instituto Nacional de Ciência e Tecnologia
de Biologia Estrutural e Bioimagem – INBEB
"Em mamíferos adultos, lesões no nervo óptico provocam degeneração retrógrada das
células ganglionares da retina e morte progressiva, especialmente por apoptose. Várias
abordagens terapêuticas vem sendo estudadas para aumentar a sobrevida neuronal e
regeneração axonal daqueles neurônios em diferentes modelos de lesão, incluindo a
terapia celular. Nesse sentido, em trabalhos anteriores demonstramos que tanto as
células mononucleares quanto as células mesenquimais derivadas da medula óssea
geram neuroproteção e aumentam a extensão axonal após lesão no nervo óptico. No
presente trabalho, procuramos, então, analisar o destino das células transplantadas in
vivo por imagem através de ressonância magnética e ex vivo através de reações
histoquímicas. Para isso, utilizamos ratos adultos da variedade Lister-hooded e as
células derivadas da medula óssea foram marcadas com nanopartículas
superparamagnéticas (SPIO - Feratrack) e injetadas no vítreo dos animais
imediatamente após lesão no nervo óptico. Os animais foram então analisados por
ressonância magnética 1 dia e 2, 14 e 18 semanas após o transplante. Demonstramos,
então, que por ressonância magnética foi possível observar um sinal hipointenso
derivado das células marcadas em todos os períodos analisados e não houve redução
evidente de sinal ao longo do experimento. Além disso, foi possível identificar células
marcadas com SPIO ex vivo 2 e 18 semanas após a injeção através da reação de azul da
Prússia. Dezoito semanas após a injeção, observamos uma concentração de células
marcadas no vítreo próximas à região do disco óptico. Isso sugere a permanência a
longo prazo das células injetadas nos olhos dos animais hospedeiros, demonstrando a
eficiência do transplante e levantando novas questões sobre o possível efeito
terapêutico dessas células."
CNPq, FAPERJ, CAPES, INBEB
G2.
PREHISTORICAL
PEDICULUS
HUMANUS
CAPITIS
INFESTATION:
QUANTITATIVE DATA AND LOW VACUUM SCANNING MICROSCOPY
1 - ALVES, A. D.; 1 - DUTRA, J. M. F.; 1 - PESSANHA, T.; 2 - RACHID, R.; 2 - SOUZA, W.; 3 LINARDI, P. M.; 1 - FERREIRA, L. F.; 1 - SOUZA, S. M.; 1 - ARAUJO, A.
1 - Laboratório de Paleoparasitologia - Escola Nacional de Saúde Pública Sérgio Arouca,
Fundação Oswaldo Cruz, Rio de Janeiro; 2 - Laboratório de Ultraestrutura Celular
Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro; 3 - Departamento de Parasitologia, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais.
Há décadas um escalpo peruano, datado do período pré)colombiano, foi examinado por
um pesquisador da Fundação Oswaldo Cruz. O Professor Olympio da Fonseca Filho
descreveu lêndeas e adultos fixos a fios de cabelos e fez comentários sobre a origem da
infecção por piolhos na espécie humana. Este mesmo escalpo foi enviado ao nosso
laboratório e é objeto deste artigo. Sua análise mostrou maciça infestação, com 9
lêndeas/ cm2 em impressionante número de adultos muito bem preservados. O tempo
de infestação foi estimado em cerca de 9 meses antes da morte, baseado na maior
distância entre lêndeas e o couro cabeludo, levando em consideração uma taxa média
de crescimento capilar de 1cm por mês. Um pequeno pedaço de tecido tradicional
peruano foi encontrado associado ao escalpo, provavelmente pertencente ao conjunto
de peças usado no ritual funerário. Aqui, apresentamos alguns aspectos morfológicos
de adultos e lêndeas visualizados por microscopia eletrônica de varredura de baixo
vácuo.
FAPERJ
G3.
EVALUATION OF A NOVEL HISTONE DEACETYLASES INHIBITOR AGAINST
LEISHMANIA AMAZONENSIS
1,2,3 – VERÇOZA, B. R. F.; 4 - HUBER, K.; 4 - BRACHER, F., 1,2,3,5 - DE SOUZA, W.;
1,2,3,5 - RODRIGUES, J. C. F.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Polo Avançado de Xerém, Universidade Federal do Rio de Janeiro; 3- Instituto Nacional
de Ciência e Tecnologia de Biologia Estrutural e Bioimagem; 4- Department of
Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universitt Mnchen, Germany;
5- Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro.
Leishmaniasis is one of the most important neglected tropical diseases caused by
protozoan parasites of the Leishmania genus. The treatment is based on the use of
antimonials, miltefosine, amphotericin B and pentamidine. However, all these drugs
cause several side effects for the patients. In this context, there is an urgent necessity to
develop new compounds safer, more efficacious and accessible. Histone desacetilases
inhibitors are new compounds that inducing alterations on the gene expression that
promoting apoptosis in the treated-cells. Thus, the aim of this work is study the effects
of a novel histone desacetilases inhibitors, against Leishmania amazonensis
promastigotes and intracellular amastigotes. The effects induced by TFMDI were
evaluated using different techniques such as: growth curve, immunofluorescence
microscopy, scanning and transmission electron microscopy, western blotting, and
fluorimetry. For promastigotes, the IC50 value was 2μM. Against intracellular
amastigotes, the IC50 value was around 3μM. Immunofluorescence, DIC and scanning
electron microscopy revealed an alteration on the promastigote' shape, that presented
elongated and thinner, and increased expression of acetylated tubulin. Furthermore,
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transmission electron microscopy revealed several ultrastuctural alterations. These
results together indicated that this compound is a promising molecule against
leishmaniasis, however new studies are necessary to understand better this mechanism
of action.
CNPq, CAPES, FAPERJ
G4.
CAMPTOTECINA, UM INIBIDOR DA TOPOISOMERASE I, QUE PODE SER
USADO COMO FERRAMENTA PARA O ESTUDO DA BIOLOGIA CELULAR DE
TRIPANOSOMATÍDEOS
1,2 - ZUMA, A.A.; 1,2- DA SILVA, C.C.; 3- ELIAS, M.C.; 1,2,4- DE 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 Imagem; 3 - Instituto
Butantan; 4 - Instituto Nacional de Metrologia, Qualidade e Tecnologia - Inmetro
Os protozoários Trypanosoma cruzi e Strigomonas culicis pertencem à família
Trypanosomatidae, ordem Kinetoplastida. O primeiro é um parasita heteroxênico e o
agente etiológico da Doença de Chagas, enquanto que o segundo é um monoxênico
não-patogênico que abriga uma bactéria simbiótica em seu citoplasma. A organização
do DNA do cinetoplasto, assim como a do DNA nuclear, conta com a participação das
topoisomerases, enzimas que atuam nos processos de replicação, transcrição e reparo.
Inibidores de topoisomerases vêm sendo amplamente usados em estudos
quimioterápicos, porém também podem ser empregados para estudar a biologia celular
destes protozoários. Neste trabalho nós avaliamos os efeitos da camptotecina, um
inibidor da topoisomerase do tipo I, na proliferação, no ciclo celular e na ultraestrutura
de T. cruzi e S. culicis. Para isto, as células foram cultivadas na presença de diferentes
concentrações do inibidor e a cada 24h, uma alíquota foi retirada para contagem em
câmara de Neubauer e para análises por microscopia óptica de fluorescência e por
microscopia eletrônica de transmissão. A proliferação de ambas as espécies diminuiu na
presença da camptotecina, sendo S. culicis mais sensível ao inibidor. A camptotecina
também afetou o ciclo celular de ambos tripanosomatídeos, promovendo em T. cruzi a
parada na fase S/G2 e induzindo em S. culicis a formação de simbiontes filamentosos.
Do ponto de vista ultraestrutural, observamos que células tratadas apresentam a
heterocromatina nuclear fortemente descompactada e inchaço mitocondrial. Além
disso, houve um aumento do número de corpos lipídicos, especialmente no
tripanosomatídeo com simbionte, como verificado em análises por microscopia de
fluorescência e fluorimetria. Juntos, nossos resultados mostram que estas duas espécies
podem ser usadas como modelos comparativos para a melhor compreensão da biologia
celular da família Trypanosomatidae.
CNPq e FAPERJ
G5.
SERUM TRANSTHYRETIN LEVELS IN BRAZILIAN PATIENTS WITH FAMILIAL
AMYLOID POLYNEUROPATHY.
1-LIMA, C.; 1-FERREIRA, P. S.;1- ANDRADE, C.; 2-CRUZ, MÁRCIA WADDINGTON;1FOGUEL, D.
1- Instituto de Bioquímica Médica-UFRJ Brazil. 2- Hospital Universitário Clementino
Fraga Filho, 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. Serum transthyretin (TTR) levels are reduced in familial amyloidotic
polyneuropathy (FAP) in others populations. 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. It is noteworthy that there are no
reports of levels of TTR in the serum of patients with PAF in Brazil. The objective of this
work is to assess the TTR serum levels of patients with FAP and compare these levels to
those of healthy patients and to correlate these levels with the clinical development of
the disease. 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. Our data shows that patients that presents a late onset of
disease has lower levels of serum TTR in comparison with the patients with early onset
of symptoms. In addition we also verified that the levels of serum TTR decreases with
clinical disease progression. These data lead us to believe that serum levels of TTR can
be used as markers for the clinical development of the disease.
INBEB, FAPERJ, CNPq, CAPES
G6.
A NEW MECHANISM OF ACTION FOR PRIMA-1 ON P53 REACTIVATION
1,2- LAURITZEN, C.; 1,2- RANGEL, L.P.; 1,2- SILVA, J.L.
1- 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.
Introduction: p53, among other functions, acts as a transcription factor and is involved
in cell cycle control, leading to apoptosis or to the arrest of the cell cycle for DNA
damage repair. It is mutated in around 50% of all tumors. In general, mutations occur on
the DNA-binding domain, leading to its loss of function as a transcription factor. The
mutation in a single allele leads to the complete loss of function due to a phenomenon
called negative dominance. Prima-1 is a classical drug described to reestablish the
structure and function of p53, leading to apoptosis. Our group has previously described
that p53 is able to form amyloid aggregates, in which the mutant form of the protein is
responsible for the conversion of the wild type form into oligomers and amorphous
aggregates [1, 2]. Material & Methods: Recombinant p53 central core domain (p53C)
was submitted to aggregation at 37oC for 1 h in the presence of increasing
concentrations of Prima-1 and 2-methylene-3-quinuclidinone hydrate (MQ) and the
fluorescence of ThT was measured at 440 nm (excitation) and 480 nm (emission). For
immunostaining, cells were incubated with anti-p53 antibody DO-1 and anti-oligomer
antibody A11 for posterior confocal microscopy visualization. Results and discussion: In
the present work, we used Prima-1 and its active metabolite, MQ, and both of them
demonstrated the ability to inhibit mutant p53C aggregation. The WT form has been
protected in a lower degree. Also, we have observed the ability of Prima-1 to revert p53
aggregation in MDA-MB-231 cells, which express mutant p53. Conclusion: These results
might lead to a different understanding on the controversial mechanism of action of
Prima-1. We suggest that this compound is able to recover p53 from aggregates
accumulated into cancer cells.
CNPq, FAPERJ , MS-DECIT, INBEB.
G7.
THE ENEMY WITHIN: IMPAIRMENT OF HEME CRYSTALLIZATION BY A
QUINOLINE DRUG DRIVES BIOCHEMICAL, CELLULAR AND PHYSIOLOGICAL SHIFTS ON AN
INSECT VECTOR
Página 17
Ferreira, C.M.¹, Stiebler, R¹, Gandara, A.C.P.¹, Nuno,A.B.W.¹, Paiva-Silva, G.O.¹, MenaBarreto, R.B.², Oliveira, M.F.1
1 - Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro 2 - Fundação
Oswaldo Cruz, FIOCRUZ
Introduction: Hematophagous organisms digest hemoglobin and release large amounts
of heme in their digestive tracts. When free, heme is toxic and promotes redox
imbalance and membrane destabilization. Thus, blood-feeding organisms require
protective mechanisms to deal with excessive heme. Hemozoin (Hz) is a heme crystal
produced by Plasmodium sp, Schistosoma sp and triatomine insects as the main heme
detoxification mechanism. Previous evidence demonstrated that quinoline drugs are
antimalarial and antischistosomal agents by inhibiting Hz formation. Here, we
investigated the effect of quinidine, an antimalarial quinoline, in R. prolixus. Material
and methods: Hz was extracted from posterior midgut of R. prolixus by differential
solubilization. Urate and hemoglobin levels were determined by colorimetric assays.
Lipid peroxidation was determined by the TBARS assay in hemolymph. Total heme levels
were measured by the alkaline-pyridine method. Reactive species in posterior midgut
were analyzed by fluorescence microscopy using the dihydroethidium (DHE) probe. The
hemolymph levels of Rhodnius heme binding protein (RHBP) were determined
spectrophotometricaly and transcript levels assessed by real time PCR in fat bodies.
Posterior midguts were fixed with glutaraldeyde and submitted to transmission electron
microscopy routine. Results and discussion: Quinidine did not affect blood ingestion or
digestion, but severely affected Hz formation. Heme levels in the hemolymph were
increased by quinidine, which promoted lipid peroxidation, reactive species generation
and reductions in urate levels. As a compensatory mechanism, protein and transcript
levels of RHBP increased by quinidine. Ultrastructural analyses of posterior midgut
indicate general loss of cellular organization with reduced densities of mitochondria,
together with the presence of myelin figures as well as numerous electron dense
structures (haemoxysomes), indicating autophagic pathways. Although there were no
changes on insect viability after quidinine treatment, the most striking physiological
observation was a decrease in eggs laying. Conclusion: Impairment of heme
crystallization within the Rhodnius midgut results in severe physiological changes
involving redox imbalance and autophagy.
G8.
ANTIPROLIFERATIVE AND ULTRASTRUCTURAL EFFECTS OF BFDI, A HISTONE
DESACETILASES INHIBITOR, IN LEISHMANIA AMAZONENSIS
ARAÚJO, C. N.1,2,3, VERÇOSA, B. R. F1,2,3., BRACHER, F4.,DE SOUZA, W1,3,5.,
RODRIGUES, J. C. F. R.1,2,3,5
1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,
Brasil. 2- Polo Avançado de Xerém, Universidade Federal do Rio de Janeiro 3- Instituto
Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Brasil 4Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universitt
Mnchen, Germany 5- Instituto Nacional de Metrologia, Qualidade e Tecnologia,
Inmetro, Brasil.
Leishmaniasis is one of the most important neglected tropical disease distributed
around the world, and is caused by protozoan parasite of the Leishmania genus. In
Brazil, endemic areas are mainly concentrated in the North and Northeast regions,
however there is a significant increase in other regions more developed regions. 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, miltefosine, amphotericin B and pentamidine.
Therefore, there is a urgent necessity to develop new drugs 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 a novel histone deacetylases inhibitor on Leishmania
amazonensis. The present study focuses on the effect of BTFDI in promastigote forms.
The IC50 value found was 2 μM, with total inhibition of the growth after treatment with
4μM. DIC and Immunofluorescence microscopy with anti-α-tubulin antibody showed
that the promastigotes became swollen mainly in the nuclear region of the cell body
after treatment with concentrations up 1 μM. Scanning electron microscopy confirmed
this alteration on the cell body. Furthermore, transmission electron microscopy was
carried out to identify the organelles and structures affected by the treatment. Several
alterations were observed, such as: mitochondrial swelling, presence of many lipid
bodies, and changes in the chromatin condensation. These initial results obtained
suggest that BTFDI is a potential new compound against Leishmania, which needs to be
tested in intracellular amastigotes and in murine model of leishmaniasis, thus
demonstrating its real efficacy. New experiments also have to be carry out to
understand better this mechanism of action.
CNPq, CAPES and FAPERJ.
G9.
ANÁLISE DAS INTERAÇÕES ENTRE A PROTEÍNA PRION E DIFERENTES
LIGANTES ATRAVÉS DE DOCKING MOLECULAR
1-MACHADO, C. S. C.;1- ARAUJO, N. C. F.;1- ALVES, W.;2- MASCARELLO, A. 2CHIARADIA, L. D.;2- NUNES; 2- R. J. YUNES, R.3- CAUGHEY, B.; 1-CORDEIRO, Y.
1- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro; 2- Departamento de
Química, Universidade Federal de Santa Catarina; 3- National Institutes of Health,
Hamilton, Montana
As Encefalopatias Espongiformes Transmissíveis (EETs), compreendem um grupo de
doenças neurodegenerativas que acometem seres humanos e animais. A proteína príon,
agente causador das EETs, na sua forma celular (PrPC) está majoritariamente presente
na membrana de células nervosas, organizada em três alfa-hélices, 1 fita beta
antiparalela e uma região desestruturada e, até o presente momento, sua função
biológica não é elucidada. As EETs desenvolvem-se após a conversão da PrPC em uma
isoforma patológica, scrapie (PrPSc) que pode formar agregados que se depositam no
sistema nervoso central. Quando comparadas por espectroscopia óptica as estruturas
secundárias da PrPC e da PrPSc se mostram bastante distintas, a primeira é rica em
alfas-hélices, enquanto a segunda apresenta um maior conteúdo de folhas-beta. Em
nosso laboratório foram identificados 46 compostos eficazes em diminuir os níveis de
PrPSc em células de neuroblatoma ScN2a. Neste trabalho nos propomos a analisar os
tipos de interações entre a PrP e esses compostos. Utilizamos a técnica de “docking”
molecular, que permite pesquisar potenciais de interação entre proteína e ligante,
buscando os complexos moleculares menos energéticos, levando em consideração os
aspectos geométricos e eletrostáticos das moléculas envolvidas. Através do SwissDock,
um servidor online, realizamos o “docking” da PrPC com os compostos, o qual
forneceu informações sobre as forças, sítios e estequeometria dessas interações.
Observamos diferentes padrões de afinidade para os complexos, mas sempre com
energias favoráveis. Para todos os “dockings”, verificou-se que uma cavidade presente
entre a hélice 2 e as fitas betas da PrPC, sítio preferencial da interação, com maior
número de clusters conformacionais para todos os ligantes. Uma vez que esses
compostos são possíveis candidatos a fármacos, objetivamos também realizar testes in
silico com o intuito de predizer diferentes propriedades desses compostos, como sua
capacidade de atravessar a barreira hematoencefálica e seus possíveis efeitos tóxicos.
CAPES, FAPERJ, INBEB, CNPQ
Página 18
G10.
INVESTIGATION OF YELLOW FEVER VIRUS-INDUCED ENDOPLASMIC
RETICULUM STRESS
SANCHES, D.1; ROCHA, C.M. 1; CAMPOS, S.P.C.1 ; GASPAR, L.P.2; FREIRE, M.S.2;
GONÇALVES, B.S. 3; CHIARINI, L.B.3; SILVA, J.L.1; GOMES, A.M.O.1 & OLIVEIRA, A.C.1
1Instituto de Bioquímica Médica/UFRJ 2ITI/FIOCRUZ/RJ 3Instituto de Biofísica Carlos
Chagas Filho/UFRJ
INTRODUCTION:The yellow fever is a hemorrhagic disease of great relevance in Africa
and South America, where it occurs as small outbreaks. It is caused by the yellow fever
virus (YFV), a flavivirus such as the Dengue Virus, both being transmitted by mosquitos.
During its life cycle, the YFV uses the endoplasmic reticulum for translation of viral
proteins and assembly of new viral particles. The accumulation of misfolded proteins in
this organelle triggers the endoplasmic reticulum stress (ERS), which leads to the
dissociation of the binding immunoglobulin protein (BiP) from ATF6, PERK e IRE1. Once
released, these factors become active and start to mediate the ERS. ATF6 is transported
to Golgi, where is cleaved. PERK and IRE1 homodimerize, are phosphorylated and
become active. PERK phosphorylates and inactivates eIF2a. IRE1 is a RNAse that
alternatively splices XBP1 mRNA, leading to the production of a response for ERS. One
of these responses is the overexpression of the nuclear transcription factor CHOP, which
regulates the expression of pro and anti-apoptotic genes. MATERIAL AND METHODS: In
this study, we investigate the ERS induced by the infection of VERO cells by YFV through
western-blotting and fluorescence microscopy. We infected VERO cells with YFV, using a
multiplicity of infection of 1. RESULTS AND DISCUSSION: We analyzed cell viability by the
LIVE/DEAD assay and we observed that by 72 hours post infection, cells undergo cell
death. The ERS induction was noticed by CHOP overexpression. Moreover, we observed
phosphorylation of eIF2a, ATF6 cleavage and spliced XBP1 18 hours post infection. BiP
expression levels remained unaltered. Apoptosis was analyzed by the TUNEL assay and
it was observed 72 hours post infection. CONCLUSION: Our results suggest that the YFV
induces ERS in VERO cells through PERK, ATF6 cleavage, XBP1 splicing and CHOP
overexpression.
Capes, CNPq, FAPERJ, Pronex, INBEB
G11.
SYNTHESIS AND EVALUATION OF AROMATIC HYDRAZONES, GUANYL
HYDRAZONE AND OXIMES WITH ELECTRON-WITHDRAWING SUBSTITUENTS AS
POTENTIAL AGENTS AGAINST CHEMICAL WARFARE
1- PETRONILHO, E. C.; 1- KITAGAWA, D. A. S.; 1- FIGUEROA-VILLAR, J. D.
1- Group of Medicinal Chemistry, Department of Chemistry, Military Institute of
Engineering, Square Gen. Tiburcio, 80, Red Beach, 22290-070, Rio de Janeiro, RJ, Brazil.
The neurotoxic chemical warfare agents are a major threat to humanity, those deserve
special attention by the high lethality and danger. Most neurotoxic agents are
organophosphorus compounds (OP) that act primarily by inhibiting acetylcholinesterase
enzyme which is essential to control the transmission of nervous impulses. When
inhibited by OPs, AChE may be reactivated by cationic oximes such as pralidoxime (2PAM) and other analogs, however, none are effective against all known neurotoxic
agents. To deal with OPS intoxication efficiently it is necessary to find out the OP used, a
long process that generally leads to death of the victim. This highlights the need for
development of potential new drugs that are potent and effective against all OPs and
low toxicity. In previous research it was found that the more acidic oximes have greater
efficiency in the reactivation of AChE. With this objective in this study were synthesized
and characterized new aromatic hydrazones, guanyl hydrazones and oximes that have
electron-withdrawing groups to assist the reactivation process. These compounds were
tested as reactivators of AChE Electrophorus electricus inhibited by paraoxon-ethyl,
using as positive reference pralidoxime (2-PAM), through the Ellman method.
INBEB, CAPES, FAPERJ, CNPQ, MINISTÉRIO DA DEFESA
G12.
PRION
PROTEIN
AGGREGATION
TRIGGERED
BY
NEUTROPHIL
EXTRACELLULAR TRAPS (NETS)
1- DOS ANJOS, D.M.; 1- AZEVEDO, E.P.; 1- VIEIRA T.C.R.G.; 1- FOGUEL, D.; 2- SARAIVA,
E.M.; 1- SILVA, J.L.
1- Inst. de Bioquímica Médica, IBqM-UFRJ, RJ, Brazil; 2- Inst. de Microbiologia, IMPPGUFRJ, RJ, Brazil
"INTRODUCTION: Prion diseases are fatal neurodegenerative protein-misfolding
diseases related to a protein known as prion protein (PrP). The constitutive cellular
isoform of PrP (PrPC), present in cell surface, can be converted into its abnormal, βsheet-rich isoform (PrPSc) that undergoes aggregation. PrPSc can be transmissible and
infectious. The evidences so far suggest that adjuvant factors, such as
glycosaminoglycans and nucleic acids, may play a role in the conversion process. Our
group previously reported that DNA can convert PrPC into the β-sheet conformation
leading to aggregation. Neutrophil extracellular traps (NETs) are large DNA webs
decorated with histones and granule proteins released by neutrophils that trap and kill
pathogens. Recently, NETs were found in association with amyloid fibrils in tissues of
patients with other protein-misfolding diseases. Furthermore, amyloid fibrils triggered
the release of NETs [1]. Following peripheral exposure, the replication of prions within
lymphoid tissue has been shown to be important for the efficient spread of the disease
to the brain. Moreover, NETs were reported of being released in lymphoid tissues.
Regarding these observations and considering the large quantity of DNA provided by
NETs we asked if NETs could induce PrPC aggregation in vitro. MATERIALS AND
METHODS: Murine recombinant PrP (23-231) was added into the supernatant from
activated human neutrophils containing NETs. Aggregation was measured by
turbidimetry (330 nm) and electron microscopy. RESULTS AND DISCUSSION:
Aggregation was detected and reached a maximum after the first 10 minutes of
incubation and then gradually declined remaining detectable after 1 hour. The
aggregates showed amorphous morphology. This results report that NETs can trigger a
stable aggregation of PrPC. However, fibrillar structures were not detected.
CONCLUSION: Our data suggest that NETs are an intriguing factor that must be
appraised in the studies concerning prion propagation mechanisms.
[1] Azevedo EP, Guimarães-Costa AB, Torezani GS, Braga CA, Palhano FL, Kelly JW,
Saraiva EM, Foguel D. (2012) J. Biol. Chem. 287, 37206-37218."
FAPERJ,
CNPq, PRONEX, CAPES, IMBEBB
G13.
SELEÇÃO DE INIBIDORES DA ACETILCOLINESTERASE EM EXTRATOS BRUTOS
DA FLORA FLUMINENSE
1- MARINS, E. B. ; 1- TINOCO, L. W. ; 2- HAMERSKI, L.; 2- PINTO, A.C.
1- Laboratório de Análise e Desenvolvimento de Inibidores Enzimáticos, NPPN, CCS,
UFRJ; 2 - NPPN, UFRJ
"O sistema mais comprometido no portador do Mal de Alzheimer é o colinérgico1. Uma
das soluções é prolongar o tempo de permanência do neurotransmissor acetilcolina na
fenda sináptica, através da inibição da enzima acetilcolinesterase(AChE). Este projeto
tem como objetivo identificar novas moléculas que inibam a acetilcolinesterase a partir
da prospecção direta de extratos de plantas da Mata Atlântica do Rio de Janeiro. A
atividade da acetilcolinesterase (Eletric eel -TipoVI-S-SIGMA) foi medida pelo método de
Ellman.2 A enzima hidrolisa o substrato iodeto de acetiltiocolina(ATCI), produzindo
tiocolina e acetato. A tiocolina e o reagente de Ellman(DTNB) reagem, produzindo dois
Página 19
ácidos detectados a 405 nm. Os extratos etanólicos(folhas, galhos, frutos e/ou flores)
das espécies de plantas coletadas foram submetidos ao teste de inibição enzimática. O
ensaio foi realizado com 100 µL do substrato ATCI 15mM, 500 µL de DTNB 3mM, 200 µL
de tampão Tris HCl 50mM pH8 com BSA 0,1%, 100 µL de amostra em metanol 1% na
concentração final de 0,1mg/uL. Antes da adição da enzima, mediu-se absorbância 5
vezes a cada 13 segundos. Em seguida adicionou-se 100 µL da enzima AChE 0,22U/mL, e
a absorbância foi medida 8 vezes a cada 13 segundos. Os inibidores Eserina e Carbacol
foram os controles positivos da inibição. Dos 20 extratos testados até o momento, 3
mostraram atividade inibitória. Após os testes de inibição, foram feitos ensaios de
interação ligante-proteína com estes 3 extratos através da diferença da transferência de
saturação
(STD-SaturationTransferDifference)
em
um
espectrômetro
AgilentVNMRS500MHz a 25°C. As soluções estoque dos extratos de foram preparadas
em DMSO_d6 e diluídas em D2O para uma concentração final de 0,02mg/uL da amostra
e 10% DMSO_d6. Após a análise dos extratos, adicionou-se AChE para uma
concentração final de 1:100(proteína:ligante). Através dos espectros STD foi possível
observar os picos dos componentes do extrato que interagem com a acetilcolinesterase.
1 - MINETT, T.S.C. & BERTOLUCCI, P.H.F. Neurociências 8(1): 11-14, 2000.
2 - RHEE, MEENT, INGKANINAN, VERPOORTE, Journal of Chromatography A, 915 (2001)
217–223"
INCT-INBEB, FAPERJ, CNPq
G14.
EMBRYONIC STEM CELLS LABELED WITH SUPERPARAMAGNETIC IRON
OXIDE NANOPARTICLES ARE DETECTED BY MAGNETIC RESONANCE IMAGING IN THE
MURINE HEART
1- ATAIDE-MENDONCA, E.; 1- BRASIL, G. V.; 1- SILVA DOS SANTOS, D.; 2- VASCONCELOSDOS-SANTOS, A; 3- AZEVEDO, C. F.; 3,4- TOVAR-MOLL, F.; 2- MENDEZ-OTERO, R.; 1GOLDENBERG, RC.; 1,5- DE CARVALHO, ANTONIO C.
1- Laboratory of Cellular and Molecular Cardiology - Carlos Chagas Filho Institute of
Biophysics (IBCCF) - Federal University of Rio de Janeiro (UFRJ); 2- Laboratory of Cellular
and Molecular Neurobiology - Carlos Chagas Filho Institute of Biophysics (IBCCF) Federal University of Rio de Janeiro (UFRJ); 3- D’Or Institute for Research and Education
(IDOR); 4- National Institute of Science and Technology for Structural Biology and
Bioimaging (INBEB - CENABIO); 5- National Institute of Cardiology (INC)
"Background: Embryonic stem cells (ESC) differentiate into several cell types and have
been considered a promising source for cellular therapy. However, the lack of noninvasive tracking of cells has represented a serious obstacle to understand the
mechanisms responsible for functional improvement of host tissue. Cellular magnetic
resonance imaging (MRI) is a rapidly growing field that aims to visualize and track cells
in living organisms. Objectives: To label beating cells derived from an ESC with
superparamagnetic iron oxide nanoparticles (SPIONs) and track these cells in the murine
heart by MRI after intramyocardial injection. Methods: Mouse ESC line E14TG2A was
cultured and differentiated in embryoid bodies (EB) by the hanging drop method. On
the fifth day, the EB were plated on adherent dishes and incubated with SPIONs
(FeraTrack™). After 2-days, labeled beating cells were trypsinized and 3x105cells were
injected into mice hearts using echocardiography to guide the closed chest
intramyocardial injection. Then, heart images were obtained by MRI. Results: ESC
expressed undifferentiated markers (Oct-3/4 and SSEA-1) in immunofluorescence
assays. Beating cells were observed after 7-days of EB cultivation. Labeling efficiency
was evaluated by dextran immunofluorescence and prussian blue reaction in vitro. The
labeled cells were detected by MRI. Conclusion: The ESC derived beating cells were
efficiently labeled with FeraTrack™ and detected in the murine heart by MRI showing
that this approach is suitable for in vivo cell tracking of transplanted cells in the host
organism. Approved by the Committee for the Use of Experimental Animals (UFRJ)"
CNPq, Faperj, CAPES and Ministério da Saúde
G15.
ESTUDOS ESTRUTURAIS DO INIBIDOR DA COAGULAÇÃO SANGUÍNEA
IXOLARIS
1-SILVA, F. H. S.; 2-FRANCISCHETTI, I. M. B.; 1- MONTEIRO, R. Q.; 1- VALENTE, A. P.; 1DE PAULA, V.S.
1- Intituto de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do
Rio de Janeiro, Rio de Janeiro, Brasil; 2- Section of Vector Biology, Laboratory of Malaria
and Vector Research, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Rockville, Maryland, USA
"A cascata de coagulação sanguínea é um mecanismo fundamental para a manutenção
da homeostasia do corpo humano. Diversos estudos demonstram que pacientes com
câncer apresentam alterações no sistema de coagulação sanguínea que levam a um
quadro de hipercoagulabilidade, conduzindo ao acontecimento de eventos trombóticos
que são os principais responsáveis pelo óbito de pacientes com câncer. Em virtude
disso, tem-se feito o uso de anti-coagulantes a fim de diminuir o avanço do tumor e
melhorar o prognóstico do paciente. O Ixolaris é uma proteína de 140 aminoácidos
presente na saliva do carrapato Ixodes scapularis. O Ixolaris se liga ao fator Xa e
posteriormente forma um complexo quaternário com o fator tecidual e fator VIIa,
resultando na inativação do complexo tenase extrínseco. Alguns estudos demonstram
que o Ixolaris possui propriedades anti-coagulantes e anti-tumorais, caracterizando-o
como um possível alvo terapêutico no tratamento do câncer. O modelo proposto para o
Ixolaris mostra uma proteína com dois domínios bastante homólogos chamados
domínio kunitz 1 e kunitz 2 (K1 e K2). O objetivo deste trabalho é mapear os sítios de
interação no Ixolaris com o complexo binário FVIIa-Fator Tecidual através de estudos
estruturais por RMN e calcular sua estrutura tridimensional. Até o momento, foi
possível definir um protocolo eficiente para expressão e purificação das proteínas
recombinantes na forma solúvel. Estes dados foram confirmados através de análise por
espectrometria de massas e western blotting. Os espectros de 1H de RMN mostram
linhas finas e boa dispersão de deslocamento químico, indicando que as proteínas estão
enoveladas. Ensaios de coagulação sanguínea estão sendo realizados para verificar a
atividade das proteínas purificadas, como também a obtenção das proteínas
isotopicamente marcadas com para iniciarmos o assinalamento de 1H, 13C e 15N,
utilizando experimentos de tripla ressonância para o cálculo da estrutura."
CNPq
G16.
MESENCHYMAL STEM CELLS PROMOTE NEUROPROTECTION AND AXONAL
REGENERATION IN A MODEL OF OPTIC NERVE CRUSH
1,2- MESENTIER-LOURO, L.; 1,2- ZAVERUCHA-DO-VALLE, C.; 1,2- FIGUEIRÊDO, A.B.P.;
1,2- NASCIMENTO-DOS-SANTOS, G.; 1,2- SILVA JÚNIOR, A.J.; 1,2- TORRES, A.L.; 1,2PAREDES, B.D.; 1,2- MENDEZ-OTERO, R.; 1,2- SANTIAGO, M.F.
Bone marrow derived cells have been used in different animal models of neurologic
diseases. In this study we have investigated the therapeutic potential of mesenchymal
stem cells (MSC) injected into the vitreous body in a model of optic nerve injury. Adult
(3-5 months old) Lister rats underwent unilateral optic nerve crush followed by MSC or
vehicle injection into the vitreous body. Sixteen and 28 days after injury, RGC survival
was evaluated assessing the number of Tuj1 or Brn3a-positive cells in flat-mounted
retinas, and optic nerve regeneration was investigated after anterograde labeling of the
Página 20
optic axons with cholera toxin B conjugated to Alexa 488. Cell therapy with MSC
increase the number of Tuj1 and Brn3a positive cells in the retina and the number of
axons distal to the crush site both at 16 and 28 days after optic nerve crush. In
summary, MSC protects RGC and stimulates axon regeneration after optic nerve crush.
The long permanence of the transplanted cells in the eye may account for the sustained
effect observed. Further studies are necessary to elucidate mechanisms of action of
MSC in the visual system.
INBEB, FAPERJ, CNPq, CAPES.
G17.
STRUCTURAL AND DYNAMIC PROPERTIES OF THE GLYCOPROTEIN E –
DOMAIN III OF DENGUE VIRUS FREE AND IN COMPLEX WITH SPECIFIC SINGLE CHAIN
FRAGMENTS (SCFV) BY NMR SPECTROSCOPY
1- SILVA, G. V.; 1- MORAES, A.H.; 1- ALMEIDA, F.C.L.; 2- VARANI, L. ; 1- VALENTE, A.P.
1- Centro Nacional de Ressonância Magnética, IBQM-UFRJ,RJ, Brazil; 2- Medical Institute
for Research in Biomedicine, Bellinzona, Switzerland
The dengue virus belongs to the family of flavivirus and is responsible for more than 100
million cases annually. The viral envelope glycoprotein E is the major constituent of
dengue virus surface and is the main target for antibody response against DENV. The
crystal structures of the glycoprotein E demonstrate this protein as dimer, where each
monomer is composed of three domains: DI, DII and DIII. The development of a dengue
vaccine has been hampered by the fact that there are four distinct serotypes of dengue
(DENV 1 - 4), and due to a poorly understood process: antibody depended enhancement
(ADE). In an ADE, antibodies generated against a dengue serotype, facilitate the
infection by a different one later, even leading to dengue hemorrhagic fever, a lethal
form of the disease. In this work, we characterized the dynamics and the structural
properties of the domain III of serotypes 1 and 4 of protein E using NMR spectroscopy.
We are mapping the residues that participate of the interaction with scFv using
chemical shift perturbation. The molecular dynamics on ps-ns time scale, which is
related to thermal flexibility, is being accessed using 15N relaxation experiments. Also,
movements on us-ms time scale, which are associated to conformation exchange
events, are being studied using relaxation dispersion experiments based on CPMG pulse
sequences. This work will present the characterization of glycoprotein E-domain III
dynamics free and with scFv, along with a structural description of DIII-scFv interaction.
This information set will be crucial for a better understanding of domain III-antibody
interaction and might help the development of a dengue virus vaccine.
FAPERJ, CNPq, CAPES and INBEB
G18.
D-SERINA REVERTE DÉFICITS COGNITIVOS E NA INIBIÇÃO POR PRÉ-PULSO
CAUSADOS PELO ESTRESSE AGUDO: RELEVÂNCIA PARA A ESQUIZOFRENIA
1- GUERCIO, G.D.; 1- BEVICTORI, L.; 1- MADEIRA, C.;1- VARGAS-LOPES, C.;1- MELLO, I.;1PANIZZUTTI, R.
1- Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro
"A esquizofrenia é um grave transtorno que acomete cerca de 0,7 % da população.
Apesar de vários genes terem sidos relacionados com o transtorno, estudos com
gêmeos homozigotos mostram que fatores ambientais podem estar envolvidos com sua
etiologia. Dentre eles, destaca-se o estresse. Nosso objetivo é investigar se o estresse
em camundongos provoca alterações bioquímicas e comportamentais observadas na
esquizofrenia e desenvolver estratégias para revertê-las. Após 90 minutos de estresse,
sacrificamos camundongos adultos e dissecamos o cérebro. Animais controles foram
sacrificados após serem retirados de suas caixas. Os animais estressados possuíam
menos D-serina no córtex pré-frontal (média ± erro padrão, 5,78 µM ± 0,18 controle,
5,04 µM ± 0,13 estresse, t test p < 0,005). Testamos os animais na tarefa de
reconhecimento de objetos, na qual eles são expostos a dois objetos iguais (treino) e
após 24h são reexpostos a um objeto familiar e outro novo (teste). Animais controle
exploram mais o objeto novo independentemente se recebem D-serina (1g/kg, I.P.) ou
salina (razão de exploração do objeto novo/ exploração total, 0,64 ± 0,03 e 0,66 ± 0,03,
respectivamente), mas animais estressados que recebem D-serina após o treino
exploram mais o objeto novo quando comparados a animais que receberam salina (0,65
± 0,3 e 0,54 ± 0,3, p < 0,05 ANOVA de duas vias seguida de Bonferroni post test). No
teste de inibição por pré-pulso, animais estressados possuem inibição menor do que
animais controle (-0,68% ± 8,5 e 31,77% ± 5,54, p < 0,005), o que é prevenido pela Dserina (-0,68% ± 8,5 e 34,36% ± 4,19, p < 0,005). Em suma, o estresse é capaz de
diminuir o nível de D-serina no córtex pré-frontal, e a administração de D-serina
recupera déficits comportamentais causados pelo estresse. Este trabalho pode ajudar a
entender como o estresse pode ser relevante para a esquizofrenia."
FAPERJ, CNPq
G19.
ESTUDOS DE INTERAÇÃO ENTRE OS RECEPTORES DE QUIMIOCINA CXCR4 e
CCR6 E β-DEFENSINA 6
1 - MOREIRA, G.S., 2 - ALMEIDA, F.L.C., 3 - VALENTE, A.P., 4- DE PAULA, V.S.1
Centro Nacional De Ressonância Magnética Nuclear, Instituto de BioquímicaMédica,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
Os receptores de quimiocina pertencem a família de receptores acoplados a proteína G
e possuem importante papel na resposta inflamatória e estão associados em câncer
metastático e infecção por HIV-1.O domínioN-terminal destesreceptores, localizados na
região extracelular, tem sido empregado em estudos de interação quimiocina-receptor,
entretanto atualmente nenhum dado de interação foi mostrado com -defensinas.Estes
receptores normalmente apresentam uma sulfatação nos resíduos de tirosina no Nterminal, aumentando ainteração com quimiocinas.As -defensinas humanas são
proteínas antimicrobianas importantes no sistema imunológico, produzidas
principalmente por leucócitos e células epiteliais e apresentam atividade quimiotática
para diferentes tipos de células nos sítios de inflamação, através dos receptores CCR2 e
CCR6.Aβ-defensina 6 (hBD6) possui uma estrutura tridimensional constituída de uma
alfa-hélice e três fitas beta, estabilizadas por pontes dissulfeto entre as cisteínas
conservadas, e uma região C-terminal flexível. O objetivo deste trabalho é mapear os
sítios de interação entre os peptídeos N-terminal do CCR6 e CXCR4 e a hBD6 através da
Ressonância Magnética Nuclear (RMN). Nós estamos produzindo peptídeos
correspondentes ao N-terminal do CXCR4 (resíduos 1-30) e CCR6 (resíduos 1-28) através
de síntese em fase-sólida. O resíduo C28 do CXCR4 foi substituído por uma alanina para
prevenir a formação de dímero e o resíduo tirosina sulfatadafoi introduzido na posição
21.A defensina hBD6foi expressa marcada isotopicamente com 15N e purificada por
cromatografia de afinidade a níquel e fase reversa.Atualmente, estamos otimizando o
protocolo de purificação dos peptídeos para posteriormente mapearmos através de
RMN as regiões de interação na 15N-hBD6como também medirmos a dinâmica do
complexo.
G20.
INJEÇÃO INTRAESTRIATAL DE ALFA-SINUCLEÍNA CAUSA DÉFICITS MOTORES
EM CAMUNDONGOS
1- Barbosa, G.F.; 2 - Melo, I.; 2 - Foguel, D.; 2 - Braga, C.A.; 3- Romão, L.
1 - Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro; 2- Instituto
de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 3 - Universidade Federal
do Rio de Janeiro - Campus Macaé
Página 21
"A doença de Parkinson é a segunda desordem neurodegenerativa mais comum no
mundo, sendo superada apenas pelo Mal de Alzheimer e acometendo de 1 a 2% da
população mundial com mais de 65 anos. No alicerce da patologia está a proteína alfasinucleína, que pode sofrer mutações pontuais do tipo missense, gerando a forma
mutante A30P que está propensa a transitar de alfa-hélice para folhas-beta, agregar e
formar fibras amiloides. Eventualmente, na cinética de agregação, diferentes espécies
oligoméricas são extremamente deletérias acarretando em morte de neurônios
dopaminérgicos. O objeto deste estudo foi determinar se a injeção intraestriatal de
oligômeros de alfa-sinucleína selvagem e a forma mutante A30P em camundongos é
capaz de desenvolver a morfofisiologia da doença de Parkinson em humanos - tendo
como controles animais injetados com veículo e 6-OHDA. Utilizamos o teste de campo
aberto para analisar a atividade locomotora e o pole teste para mensurar bradicinesia.
Para estudar assimetria lateral foi feito o teste do cilindro, assim como a discriminação
olfatória para análise de perda de olfato. A morte de neurônios dopaminérgicos,
rearranjo sináptico e migração astrocitária serão visualizados por imunohistoquímica
após diferentes intervalos de tempo da cirurgia. Nossos resultados sugerem que na
primeira semana pós-cirugia, tanto os animais injetados com a alfa-sinucleína selvagem,
quanto os injetados com a forma mutante A30P apresentam redução de 40% da
atividade locomotora. Quanto a redução do controle motor fino, embora ela aconteça
em ambas as condições experimentais, ela é mais acentuada nos animais injetados com
o mutante A30P. Além disso, ao longo das 8 semanas de experimento, nenhum dos
grupos apresentou assimetria lateral, porém existe uma substancial perda de olfato,
levando crer que este possa ser um bom modelo para pesquisas e testes de fármacos
em estágios precoces da patologia."
FAPERJ, CNPq, PIBIC-UFRJ
G21.
IDENTIFICAÇÃO E QUANTIFICAÇÃO DE CAPSAICINA POR RMN EM EXTRATOS
DE HÍBRIDOS DE Capsicum chinense E C. annum
1-MONTEIRO H. S. M.; 2-ANDRADE V. C. J.; 1-TINOCO L. W.
1-Núcleo de Pesquisa de Produtos Naturais da UFRJ; 2-Departamento de Agronomia –
UFVJM-Diamantina-MG
"A capsaicina (8-metil-N-vanilil-trans-6-nonenamida) é um metabólito secundário
encontrado nas pimentas, sendo facilmente reconhecida pela sua pungência e de
grande importância econômica e medicinal. Sendo uma substância de alto valor
econômico, há um grande interesse pelo aumento da sua produção por hectare. Para
isto, vem sendo desenvolvido o cultivo de plantas híbridas que produzam capsaicina em
maiores quantidades. Este trabalho teve como objetivo quantificar o teor de
capsaicina produzida pelos diferentes híbridos de Capsicum chinense e C. annum
fornecidos pelo grupo do Prof. Valter da UFVJM. A partir das análises dos espectros de
RMN 1D e 2D da amostra padrão (15 mg de Nonivamide - SIGMA em 600uL de MeOD)
foi feita a atribuição completa dos deslocamentos químicos dos hidrogênios da
capsaicina, para que fosse possível identificar os sinais correspondentes à capsaicina
diretamente nos extratos. Foram usados extratos dos frutos com e sem semente e das
sementes dos 32 híbridos previamente secos e pulverizados. Os extratos foram
preparados com 0,2 g de cada amostra em 1mL de MeOD, sonicados e centrifugados. O
sobrenadante (600uL) foi usado para as análises de quantificação feita através da
integração do sinal em 2,20 ppm em relação ao do padrão interno (3 L de dioxano sinal em 3,67 ppm). Para a quantificação, os espectros de RMN de 1H (499,79 MHz –
VNMRS-500 Agilent) foram adquiridos com 48 scans, d1 de 20 s e com pulsos de 90o. O
processamento dos espectros foi feito com o programa MestRenova. Foi possível
observar que o teor de capsaicina nos frutos inteiros, nos frutos sem semente e na
semente varia de acordo com o tipo de híbrido. Em 16 híbridos o teor de capsaicina foi
maior no fruto sem semente, em 7 no fruto inteiro e em apenas dois híbridos o maior
teor foi na semente."
FAPERJ, CNPq, FINEP
G22.
ESTRUTURA E FUNÇÃO DE β-DEFENSINAS HUMANAS E INTERAÇÃO COM
GLICOSAMINOGLICANO
de Oliveira, J.M., Almeida, F.L.C., Pomin, V., Valente, A.P., De Paula, V.S.
¹ Centro Nacional De Ressonância Magnética Nuclear, Instituto de Bioquímica Médica
UFRJ
β-defensinas humanas são proteínas catiônicas, com estruturas em folha β estabilizada
por três pontes dissulfeto intramolecular. Embora 28 genes de β-defensinas humanas
tenham sido identificados, apenas seis foram caracterizadas até o momento. Estas
proteínas apresentam atividade antimicrobiana frente a bactérias Gram positiva e
negativa, e também apresentam efeito anti-HIV. Além disso, apresentam atividade
quimiotática para diferentes tipos de células nos sítios de inflamação, através dos
receptores de quimiocina CCR2 e CCR6. A estrutura tridimensional de três β-defensinas
(hBD1, hBD2 e hBD3) já foram resolvidas em solução por RMN. O nosso objetivo inicial é
expressar e purificar β-defensinas, e posteriormente determinar a estrutura
tridimensional de uma delas por meio da técnica de Ressonância Magnética Nuclear
(RMN). Neste trabalho, nós realizamos um screening de seis β-defensinas testando
solubilidade e enovelamento, afim de selecionarmos as melhores candidatas para o
cálculo de sua estrutura tridimensional. A partir do testes realizados, três defensinas
foram selecionadas (hBD4, hBD5 e hBD18) e foram expressas marcadas isotopicamente
com 15N. O espectro de 1H,15N HSQC para hBD4 mostra linhas finas e boa dispersão de
deslocamento químico, indicando que a proteína está enovelada. Nós também
utilizamos RMN para mapear os sítios de ligação na 15N hBD6 na presença de
pentassacarídeo sintético fondaparinux (Arixtra®), um mimético da heparina altamente
sulfatado. Neste estudo nós identificamos um sítio de ligação para o fondaparinux e
analisamos o complexo utilizando experimentos de relaxação. Iniciaremos o
assinalamento de 1H, 13C e 15N da hBD4, utilizando experimentos de tripla ressonância
para o cálculo da estrutura tridimensional. A partir do cálculo da estrutura, poderemos
realizar experimentos de interação com seus alvos biológicos e assim poderemos
mapear na sua estrutura os sítios de interação e comparar com os dados da hBD6 a fim
de compreender melhor o seu papel fisiológico e a sua relação com certos tipos de
patologias.
PIBIC-UFRJ
G23.
THE “SWEET” SIDE OF VIRUSES: HOW GLYCOSYLATION AFFECTS THE
BIOLOGICAL ACTIVITY OF AN EMERGING VIRUS?
Carvalho, C.A.M.; Bortot, J.P.S.; Piazza, T.; 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 with regards to potential for
urban emergence. The viral envelope proteins mediate cell recognition and entry and
present both an N-glycosylation motif along their primary structures. These motifs are
conserved amongst other alphaviruses, what suggests that they play an important role
for the virus particle. The aim of this work was to address the role of glycans N-linked to
MAYV envelope proteins on its infectivity, via specific cleavage by N-glycosidase F. Our
results showed that digestion with N-glycosidase F promotes a shift in the
electrophoretic mobility of MAYV envelope proteins E1 and E2, but not in the capsid
protein C. Cleavage of N-linked oligosaccharides also interfered with MAYV infectivity.
Analysis of MAYV morphology by negative-staining electron microscopy revealed that
Página 22
removal of N-linked glycans from MAYV envelope proteins led to an unusual viral
structure. Further experiments are under course in order to evaluate possible effects of
N-deglycosylation on MAYV entry into host cells. Our preliminary results points to virus
glycosylation as an important issue in virus biology.
CAPES, CNPq, FAPERJ, FINEP, INBEB, PRONEX
G24.
RABBITS AND THEIR RESISTANCE TO PRION INFECTION: A MATTER OF
ALTERED PRP- NUCLEIC ACID INTERACTIONS?
Casali, S.1 , Teixeira, J.E1. , Macedo, B.1 , Silva, J.L. , Gomes2, M. P. B.1 , Cordeiro, Y.1
1Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
2Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro,
Brazil
"Prion protein (PrP) is known for its vulnerability to suffer structural changes. The
conversion of its native conformation (PrPC) into a misfolded form (prion scrapie, PrPSc)
is associated to neurodegenerative diseases, such as bovine spongiform encephalopathy
(BSE), Creutzfeldt-Jakob disease and Gerstmann-Sträussler- Scheinker Syndrome (GSS).
PrP is highly conserved among mammals, but some aminoacid variations can lead to
differences in protein structure and stability. Previous studies have indicated that rabbit
prion protein (raPrP) is more stable and resistant to structural conversion than other
mammalian PrPs, due to differences in its primary sequence. It has been shown that
murine PrP binds to RNA and DNA molecules and that this interaction may trigger the
misfolding process. However, nucleic acid (NA) interaction with rabbit PrP has not been
investigated yet. In this work, we evaluated the effects of small nucleic acid molecules
(DNA and RNA) in recombinant raPrP tertiary and secondary structures. These NA
sequences were previously shown to induce murine recombinant PrP (mPrP)
conformational changes leading to aggregation and cytotoxicity. Static light scattering
(LS) data indicate that both selected DNA and RNA sequences (21bp DNA sequence and
a 21mer RNA sequence) change the tertiary structure of raPrP similarly to mPrP.
Moreover, intrinsic fluorescence emission of recombinant PrPs was equally suppressed
by NA interaction. In general, our preliminary results indicate that, although raPrP
seems to be resistant to conversion into PrPSc, its interaction with nucleic acids parallels
previously reported mPrP:NA binding. It remains to be established if raPrP interaction
with nucleic acids results in aggregated species also toxic to cells in culture and/or with
distinct biophysical properties in comparison with mPrP:NA complexes. Keywords:
prion, protein, nucleic acids, spectroscopy, cytotoxicity"
CAPES, CNPq and FAPERJ
G25.
STRUCTURAL CHARACTERIZATION OF M-TTR AMYLOID FIBRILS BY SOLIDSTATE NMR APPROACHES
ANDRADE, F. G.1, SANT’ANNA, R.O.1, SANTANA, J.S. 1, CORRÊA, D.H A., GAVA, L.M.,
DIEHL, A.2, BRAGA, C.A.1, RAMOS, C.H., OSCHKINAT, H.2, FOGUEL, D.1 AND FREITAS
M.S.1
1.University Federal of Rio de Janeiro, Medical Biochemistry Institute, The National
Institute of Science and Technology in Structure and Bio-image. Rio de Janeiro, Brazil. 2.
Leibniz-Institute für Molekulare Pharmakologie, Structural Biology, Berlin, Germany.
Amyloidosis is a clinical disorder caused by extracellular deposition of proteins that are
normally soluble in their native conformation, but suffer conformational modifications
resulting in insoluble, abnormal fibrils that impair organ function. Parkinson’s disease
(PD) and Alzheimer’s disease (AD) are the two major common neurodegenerative
diseases. In this work we are using transthyretin (TTR) fibrils formed from the
monomeric construction of TTR protein (MTTR). The solid state NMR data has shown
narrow peaks as an indication of well ordered fibrils. However, only few peaks were
observed in comparison to the length of the protein that suggest us different degrees of
mobility on fibril structure. In this way, a combination of solution and solid-state NMR
has been applied in this work. Amyloid fibrils are interesting polymers whose the
structure use to adopt a cross-b-sheet organization, indicating a common core structure
shared by them. On the other hand, the protein-protein association seems not to be
working in a promiscuous fashion, but it requires amino acids specificity. This behavior
can be illustrated in a cross-seeding approach, in which an inter-species barrier can be
observed, as in the case of prion protein. Applying atomic resolution techniques is
possible to observe differences in fibrils structure at the quaternary level. For instance,
X-ray diffraction map can provide slights differences in the diffraction pattern among
different b-sheet structure conformations. In general, there exist a signal rising up at
4.7A and another at 10A that are regarding to the distances inter-strands and intersheets, respectively. The X-ray information taken together Solid-state NMR results had
innovated the field of protein structure. The dipolar-dipolar interaction has added
precise distance measurements between two spin-systems. This parameter has been
reintroduced by application of some pulse sequences such as Transferred Echo Double
Resonance (TEDOR). Indeed, 1H back exchanged samples with different levels of
deuterium in combination to Radiofrequency Driven dipolar Recoupling (RFDR) has
showed up as a nice tool to observe mobile parts of fibrils structure. Our work has
collecting several structural information concerning fibrils formation and organization,
as well, structural mobility that could help a future drug design to stop or force non
toxic species of oligomers.
Alexander von Humboldt, FAPERJ, CNPq, UFRJ, DAAD
G26.
TEMPO DE FORMAÇÃO, ESTABILIDADE ESTRUTURAL E INCORPORAÇÃO DE
PROTEÍNA SOLÚVEL EM AGREGADOS FORMADOS A PARTIR DOS PRODUTOS DE
DISSOCIAÇÃO DAS FIBRILAS DE TRANSTIRRETINA.
RODRIGUES, L.B.; SUAREZ, M. C. AND FOGUEL, D.
Instituto de Bioquímica Médica, UFRJ
"Muitas doenças amilodoigênicas são causadas por agregação de proteínas celulares
solúveis. Entretanto, as amiloidoses não são doenças de perda de função. Em nenhuma
das doenças amiloidogênicas descritas, há evidências que uma quantidade suficiente de
proteína é removida de fluidos fisiológicos a ponto de comprometer sua função. A
hipótese amilóide considera que o processo de formação de fibrilas é a principal causa
das doenças amiloidogênicas. Apesar das inúmeras evidências a favor desta hipótese,
falta um entendimento detalhado de como a auto-associação de uma proteína
malformada leva a disfunções de órgãos e às características neurodegenerativas.
Diversos estudos apontam que intermediários precocemente associados são muito mais
tóxicos que intermediários de alto peso molecular ou fibrilas. A proteína tetramérica
transtirretina (TTR), encontrada no plasma humano e fluido cérebro-espinhal é
responsável pelo transporte de retinol e tiroxina. Esta proteína está envolvida em duas
doenças amiloidogênicas: a polineuropatia amilóide familiar, que afeta
aproximadamente 1 em 100.000 pessoas e a amiloidose senil sistêmica, que afeta 25%
das pessoas com mais de 80 anos. A primeira delas é causada por variantes da TTR ao
passo que a proteína tipo selvagem tem sido encontrada nas fibrilas de pessoas
acometidas pela forma senil da doença. Em trabalhos anteriores verificamos que fibrilas
da TTR selvagem e do variante L55P podem ser dissociadas pelo emprego de alta
pressão hidrostática. Neste trabalho, utilizando estas mesmas proteínas constatamos: 1)
que a estabilidade estrutural das fibrilas diminui à medida em que elas se tornam mais
“velhas”; 2) as espécies geradas a partir da dissociação das fibrilas podem sofrer
reassociação após descompressão e “sequestrar” proteína solúvel marcada com
Página 23
acrilodan adicionada ao meio antes da pressurização. Tais resultados parecem
redimensionar o papel das fibrilas amilóides no desenvolvimento das patologias."
FAPERJ, CNPq
G27.
STRUCTURAL INSIGHTS ON ONE FLAGELLAR CHAPERONE FROM
XANTHOMONAS AXONOPODIS PV. CITRI
1-SCHULTZ,L.G.; 2-FATTORI,J.; 3-PRANDO, A.; 2-ASSIS, L.H.P.; 4-APARICIO, R.; 1-TASIC, L.
1- Laboratório de Química Biológica, Universidade Estadual de Campinas; 2LNBio,LNLS,CNPEM,Campinas; 3- Syngenta Braul, São Paulo; 4- Laboratório de Biologia
Estrutural e Cristalografia, Universidade Estadual de Campinas.
"The Gram-negative bacterial pathogens use several secretion systems to deliver
effectors to eukaryotic cells. The type III secretion system (T3SSs) is the most complex of
all, due to a kind of a needle that is formed in on the pathogenic cell capable to deliver
virulence proteins directly into eukaryotic cells. These processes require customized
chaperones whit high specificity for binding partners, thus providing the secretion to
occur. The flagellar system, where our protein of interest acts, is considered a precursor
of the type III secretion system (T3SSs). Due to the low similarity of sequence and
annotation of secretion chaperones their identification is a very difficult task. However
they present some common characteristics as being small (<20 kDa), have low pI,
amphipathic helix and have a tendency to dimerize, which make the work less arduous.
Concerning about this, herein, we present structural features on one flagellar secretion
chaperone belonging to plant pathogen Xanthomonas axonopodis pv. citri and suggest
how low resolution models based on Small Angle X-ray Scattering (SAXS) patterns can
provide new structural insights that could be very helpful in their analysis and posterior
classification."
INBEB
G28.
ACTIVITY OF SQ-109, A SQUALENE SYNTHASE INHIBITOR, AND ANALOGUES
AGAINST TRYPANOSOMA CRUZI
1 - LAMEIRA, L.; 1 - VEIGA-SANTOS P; 2 - LI, K.; 2 - OLDFIELD, E.; 3 - URBINA, J.A.; 1 - DE
SOUZA, W.; 1 - CARVALHO, T.M.U.
1Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos
Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil, 2Departments of
Chemistry and Biophysics, University of Illinois at Urbana-Champaign, USA, 3Emeritus
Investigator, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones
Cientiﬁcas, Caracas, Venezuela.
Chagas’ disease is caused by the protozoan Trypanosoma cruzi. Currently available
treatments use compounds such as nifurtimox and benznidazole, which have a wide
range of side effects and an unsatisfactory response during the chronic phase. Thereby,
it is important to find new safer and more active drugs. Previous studies have validated
the ergosterol biosynthesis pathway as a chemotherapeutic target in T. cruzi and
Leishmania spp. In the present work we investigated for the first time the anti-T. cruzi
activity of SQ-109 (N-[(2E)-3,7-dimethyl-2,6-octadienyl]-N’-tricyclo[3.3.1.13,7]dec-2-yl1,2-ethanediamine), a compound in clinical development for the treatment of drugresistant tuberculosis, which has also been shown to inhibit T. cruzi ’s squalene
synthase. We also evaluated the effects of SQ-109 and three analogs (1281, 1283 and
1304) against both proliferative stages of T. cruzi, cultured in vitro. All compounds
inhibited the proliferation of epimastigote forms with IC50 values (144 h of exposure),
of 4.2 µM for SQ-109 and 6.9 µM, 9.3 µM, and 6.1µM, respectively, for the analogs. The
compounds also inhibited the proliferation of intracellular amastigote in cultured
murine peritoneal macrophages, with IC50 values (96 h of exposure) of 1.4 µM for SQ109, and 1.6 µM, 1.9 µM and 1.7 µM, respectively, for the analogs. The selectivity index
of SQ-109 activity was 12. Analyses by scanning electron microscopy revealed that SQ109 caused depressions of the plasma membrane and rounding of the cell body. Thin
sections of treated epimastigotes observed by transmission electron microscopy
showed drastic changes in the Golgi complex, mitochondrial swelling, formation of
myelin-like figures and cytoplasmic vesiculation. Taken together, our observations show
that SQ-109 and analogs are promising selective inhibitors of T. cruzi proliferation.
Further studies will evaluate their potential antiproliferative synergism with
posaconazole.
CNPq, CAPES, FINEP, FAPERJ, USPHS
G29.
A LACTOFERRINA INIBE A ENTRADA DO VÍRUS DA FEBRE AMARELA VIA
LIGAÇÃO À SUPERFÍCIE CELULAR
1- Ferreira, M.V.M.; 1- Mendes, Y.S.; 1- Alves, N.S.; 1- Carvalho, C.A.M.; 1- Campos,
S.P.C.; 2- Schwarcz, W.D.; 3- Silva, J.L.; 1,4- Gonçalves, R.B.; 1- Gomes, A.M.O.; 1Oliveira, A.C.
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.
O vírus da Febre Amarela (YFV) é um Flavivírus endêmico em regiões tropicais,
principalmente África e América do Sul, provocando uma doença febril aguda de grande
impacto na saúde pública. Ainda não existem tratamentos específicos para esta
infecção, tornando a busca por antivirais um alvo de grande importância médica. A
lactoferrina bovina (bLf), uma glicoproteína presente em diversas secreções, como leite,
lágrima e saliva, apresenta diversas funções biológicas, incluindo modulação da resposta
imune e defesa contra diversos patógenos, como diferentes vírus de importância
médica e socioeconômica. O objetivo deste estudo é avaliar a atividade antiviral da bLf
contra a infecção pelo YFV. Nossos resultados mostram que a bLf apresenta uma
atividade de inibição viral de aproximadamente 70%, sem provocar efeitos citotóxicos
em nosso modelo celular. Buscando investigar quais etapas e que mecanismos estão
envolvidos nesta inibição, nossos dados indicam que, ao pré-tratarmos a célula com bLf
ou adicionarmos somente na etapa de ligação ao receptor celular (adsorção viral), a
infecção é inibida em torno de 60%. Em contrapartida, a presença da bLf apenas após os
processos iniciais de infecção (pós adsorção e internalização viral) leva a uma inibição
inferior a 10%. Além disso, ao avaliarmos a capacidade da bLf em se ligar às partículas
virais, notamos que não houve alteração significativa no título viral. Juntos, nossos
resultados fortemente sugerem que a bLf apresenta atividade antiviral, atuando
majoritariamente sobre os eventos iniciais do ciclo de infecção do YFV por se ligar à
superfície celular e possivelmente dificultar a interação vírus-célula. O presente estudo
pode ajudar na melhor compreensão do ciclo, além de auxiliar importantes estratégias
para o desenvolvimento de antivirais eficazes contra a infecção por diferentes flavivírus.
CNPq, CAPES, FAPERJ, PRONEX, INBEB
G30.
POLARIDADE MAGNÉTICA EM COCOS MAGNETOTÁTICOS CULTIVADOS DA
LAGOA DE ITAIPU, RJ
1 - CAMPELLO, M.; 1- KARINA, V.; 1- LINS, U.
1 - Instituto de Microbiologia Paulo de Goes, Universidade Federal do Rio de Janeiro.
Bactérias magnetotáticas formam um grupo diverso de procariontes aquáticos que
produzem organelas compostas de cristais magnéticos de magnetita (Fe3O4) ou greigita
(Fe3S4), envoltos por uma bicamada lipídica, conhecidos como magnetossomos. Os
magnetossomos conferem às bactérias a capacidade de orientação ao longo de linhas
de campos magnéticos através da movimentação por flagelos. As bactérias no
Página 24
hemisfério Norte nadam preferencialmente para o norte geográfico (tipo N), enquanto
as do hemisfério Sul nadam para o Sul (tipo S). Como a componente vertical do campo
geomagnético aponta para baixo no Hemisfério Norte e para cima no Hemisfério Sul, as
bactérias magnetotáticas nadam para baixo nos dois hemisférios devido á polaridade
invertida de seus flagelos. Contudo, há relatos de bactérias magnetotáticas com
comportamento semelhante ao tipo S no hemisfério Norte. Neste trabalho, será testada
a influência da inversão do campo magnético sobre a polaridade de um grupo de cocos
magnetotáticos isolado em cultura axênica da lagoa de Itaipu, RJ. As bactérias foram
crescidas em tubos de ensaio com meio de cultura semi-sólido heterotrófico com
gradiente de oxigênio, usando quinato férrico como fonte de ferro e succinato de sódio,
acetato de sódio e bicarbonato de sódio como fontes de carbono. As culturas serão
crescidas em duas condições: a) em um campo magnético Norte, gerado por uma
bobina magnética e (b) sob a influência do campo geomagnético Sul. A cada 24 horas, o
padrão de formação das bandas no meio de cultura será observado. Por microscopia
óptica, conferiremos a polaridade das bactérias magnetotáticas crescidas sob o campo
magnético Norte e Sul e, por Microscopia Eletrônica de Transmissão, a produção de
magnetossomos e outras características morfológicas na célula. Os resultados obtidos
permitirão uma melhor compreensão do papel dos magnetossomos e da magnetotaxia
para a sobrevivência dos micro-organismos magnetotáticos no ambiente.
Financiamento: CNPq, CAPES, FAPERJ
G31.
ESTUDOS DE INTERMEDIÁRIOS DE AGREGAÇÃO DA PROTEÍNA SUPRESSORA
DE TUMOR P53
1,2- FERREIRA, M. E.; 1,2- Rangel, L.P.; 1,2- Silva, J.L.
1-Instituto de Bioquímica Médica UFRJ 2-Instituto Nacional de Ciência e Tecnologia de
Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro,
RJ, Brasil.
"Introdução A proteína supressora de tumor p53 é uma das principais proteínas
regulatórias do ciclo celular, responsável por levar a célula à apoptose caso ocorra um
dano irrevesível no material genético. Aproximadamente 50% dos tumores humanos
estão relacionados a mutações dessa proteína. Estudos com a p53 selvagem e sua
forma mutante R248Q levam a acreditar que a p53 quando sofre mutação pode formar
agregados e flibrilas oligoméricas, de forma parecida com o que ocorre no prion, o que
levariam a uma mudança conformacional e a perda de função. O objetivo desse
trabalho é estudar a agregação da p53 selvagem e tentar caracterizar possíveis
intermediários relacionados com esse processo. Métodos Como metodologia de estudo,
foram utilizadas técnicas de espectroscopia para analisar a fluorescêcia e o
espalhamento de luz da p53 quando submetida a desnaturação por alta pressão. Foi
analisado também o efeito de diferentes concentrações de guanidina (0 a 1M) na
reversibilidade da desnaturação da p53. A desnaturação por pressão é resultado da
força hidrostática que leva a entrada de moléculas de água nas cavidades hidrofóbicas
de uma proteína quando essa é submetida a pressões na ordem de Kpsi. O espectro de
fluorescência de resíduos de triptofano, normalmente não expostos, foi usado como
forma de quantificar o grau de desnaturação da p53. Estudos de espalhamento de luz
foram usados para analisar a agregação proteica. Resultados e discussão Estudos iniciais
mostram que há uma reversibilidade da desnaturação da p53 por pressão quando
exposta a diferentes concentrações de guanidina. Apesar de reversível, é possível
perceber uma diferença na cinética de desnaturação quando a p53 volta para seu
estado nativo, podendo então mostrar a presença da formação de intermediários após
o desenovelamento."
INBEB
G32.
PRODUÇÃO DE PHA E MAGNETOSSOMOS EMPREGANDO BACTÉRIAS
MAGNETOTÁTICAS
1-SANTOS, M.G.C.; 1- LEÃO, P..E.L;1- CORREA,T.N.;2-GUTARRA, M.L.E; 3- LINS, U.G.C.
1- Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro; 2Escola de Química, Polo Avançado de Xerém, Universidade Federal do Rio de Janeiro; 3Instituto de Química, Universidade Federal do Rio de Janeiro
"As bactérias magnetotáticas são microrganismos encontrados em ambientes de água
doce e salgada, são caracterizadas por produzirem nanocristais magnéticos compostos
de magnetita ou greigita envoltos por uma membrana, denominados magnetossomos.
Além dos magnetossomos, algumas bactérias magnetotáticas têm a capacidade de
produzir grânulos lipídicos intracelulares geralmente em condições de desequilíbrio da
razão carbono/nitrogênio no meio. Esses grânulos são utilizados como reserva
energética por esses microrganismos, nesse trabalho analisamos a produção de
grânulos lipídicos e magnetossomos pela bactéria Magnetovibrio blakemorei (cepa MV1). A bactéria foi cultivada em meio específico contendo: solução de minerais, succinato
de sódio, cloreto de amônio, hidrolisado de caseína, tampão fosfato, cisteína, solução
de vitamina e sulfato ferroso, à 24°C, 100 rpm e em pH 7,5 por 168h. As amostras foram
retiradas em intervalos de 24h para análise de crescimento celular em
espectrofotometria à 600nm. A analise e quantificação da produção de grânulos
lipídicos e magnetossomos foi realizada por microscopia eletrônica de transmissão. Com
o sobrenadante foi analisado o consumo da fonte de nitrogênio pela determinação de
FAN (Free Amino Nitrogen) e o consumo de succinato de sódio por HPLC. Como
resultado observamos que mesmo em condições de desequilibrio das concentrações de
carbono e nitrogênio o MV-1 apresentou cinética de crescimento semelhante à
observada nas condições do meio controle (1,69x109 células/ml em 96h). Em 96h
observamos 4,23x109 magnetossomos/ml. Após 168h de cultivo ocorreu o consumo de
61% das fontes de nitrogênio. Estes resultados demonstram que esta condição
empregada foi eficiente para induzir o acumulo de grânulos lipídicos no interior das
células. Novas análises de consumo de carbono estão sendo realizadas para caracterizar
a produção e a natureza do lipídeo produzido."
CNPq, CAPES, FAPERJ
G33.
ESTUDOS DE ENOVELAMENTO PROTEICO NOS DOMÍNIOS –N E –C DA
TROPONINA C MEDIANTE LIGAÇÃO A IONS DE CÁLCIO
1-MAYRA A. MARQUES, 1-GUILHERME A. P. DE OLIVEIRA, 2-CRISTIANE B. ROCHA, 3YRAIMA CORDEIRO, 1-MARTHA M. SORENSON, 1-DÉBORA FOGUEL, 1-JERSON L. SILVA E
1-MARISA C. SUAREZ
1-Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, RJ, Brasil 2-Centro de Ciências
Biológicas e da Saúde, Instituto Biomédico – IB, Departamento de Bioquímica, Rio de
Janeiro, Brasil. 3-Faculdade de Farmácia, UFRJ, Rio de Janeiro, RJ, Brasil.
A Troponina C (TnC), é uma subunidade do complexo Troponina encontrado no músculo
vertebrado esquelético e cardíaco; possui dois domínios estruturalmente homólogos: o
domínio N e C terminais conectados entre si por uma alfa hélice central e é capaz de se
ligar a íons cálcio (Ca+2) através de uma estrutura denominada “EF-hand”. Ensaios de
mutagênese sítio dirigida tem possibilitado a produção de mutantes, da construção
inteira da TnC e dos seus domínios isolados, com resíduos de fenilalanina (Phe)
substituídos por Triptofano (Trp). Estudos anteriores, utilizando esses mutantes
submetidos à alta pressão hidrostática, propõem que o domínio C-terminal, na ausência
de cálcio é menos estável que o domínio N-terminal. Este não parece exercer efeito na
estabilidade do C-terminal. Analisamos a estabilidade da construção completa da F29W
TnC, utilizando abordagens estruturais, sob condições desnaturantes de ureia e pressão;
F29W TnC é um mutante fluorescente, na qual a Phe 29, localizada no domínio N foi
Página 25
substituída por Trp. Foram calculados parâmetros termodinâmicos ( V e G atm) que
regem o enovelamento da TnC F29W intacta, na presença ou ausência de Ca+2. Os
dados sugerem que o domínio C tem um pequeno efeito sobre a estrutura do domínio
N, na ausência de Ca+2. No entanto, usando espectroscopia de fluorescência, foi
demonstrada uma queda significativa na estabilidade do domínio N ligado a Ca+2
quando, os sítios III e IV do domínio C estavam ligados ao Ca+2. Observou-se um
decréscimo na estabilidade termodinâmica do domínio N promovendo uma redução de
G atm, em termos absolutos, além da afinidade do domínio N pelo Ca+2 mostrarse alterada. Dados de espalhamento de raios-X a baixos ângulos revelam que a
interação entre os domínios C e N pode ser mediada pela hélice central, que possui
menor volume e aparenta maior rigidez e estabilidade, após a ligação do Ca+2 aos sítios
EF-hands.
INBEB, FAPERJ, CNPq
G34.
ANÁLISE DA EXPRESSÃO DO GANGLIOSÍDEO 9-O-ACETIL GD3 NA ZONA
SUBVENTRICULAR
1-FURTADO, M; 1-MORTARI, N; 1-GUBERT, F;1- ZAVERUCHA-DO-VALLE, C;1- SANTIAGO,
MF;1- MENDEZ-OTERO, R
1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
"As células-tronco neurais são uma esperança para o tratamento de doenças
neurodegenerativas. Desta forma, a descoberta dos mecanismos envolvidos com a
proliferação e o recrutamento dessas células para áreas de lesão é essencial para que se
possam desenvolver terapias celulares baseadas no estímulo as células-tronco
endógenas. Em cultura, as células-tronco neurais são caracterizadas pela capacidade de
formar neuroesferas em placas não-aderentes na presença dos fatores EGF e FGF-2,
mas in vivo ainda não existem marcadores eficazes para que se possa distinguir
claramente uma célula-tronco neural de um progenitor já comprometido. Nesse
trabalho estamos propondo a análise do gangliosídeo 9-O-acetil GD3 como um possível
marcador de células-tronco neurais. O gangliosídeo 9-O-acetil GD3 está presente no
sistema nervoso central e periférico de roedores durante o desenvolvimento e foi
descrito como uma molécula associada a eventos de migração celular e extensão axonal
nesse período. Em ratos adultos, o gangliosídeo 9-O-acetil GD3 deixa de ser expresso na
maior parte do sistema nervoso central, sendo observado apenas na zona
subventricular, na via migratória rostral, na retina e no cerebelo. Como a zona
subventricular é a região onde encontram-se as células-tronco neurais em mamíferos
adultos, o objetivo desse trabalho é analisar uma possível associação do gangliosídeo 9O-acetil GD3 com as células-tronco neurais. Para isso, analisamos a expressão do
gangliosídeo 9-O-acetil GD3 na zona subventricular de ratos adultos e de ratos em idade
pós-natal 21 (P21) e embrionários (E16), por imunohistoquímicas combinado o
anticorpo contra o gangliosídeo 9-O-acetil GD3 com anticorpos específicos para os
diferentes tipos celulares da região. Comparamos também a população de células da
zona subventricular positiva para o gangliosídeo 9-O-acetil GD3 com a população
negativa em ensaio de formação de neuroesferas utilizando as mesmas idades."
G35.
CYTOTOXIC AND PROAPOPTOTIC EFFECTS OF PTEROSTILBENE ON BREAST
CANCER CELLS
Campos, N.P.C.1; Costa, D.C.F.1; Fialho, E.2, Silva, J.L1
1Instituto de Bioquímica Médica, IBqM-UFRJ, 2Instituto de Nutrição Josué de Castro,
INJC-UFRJ.
"Pterostilbene, a dimethyl ester derivative of resveratrol, is a bioactive food compound
that mediates many cellular targets involved in cancer signaling pathways. The p53
tumor suppressor protein plays an essential role in preventing cancer development by
inducing cell cycle arrest or apoptosis in response to cellular stress. This protein has
been suggested to have a role in the anticancer properties of resveratrol and its
structural analogues. Thus, the present study was aimed to check the cytotoxic and proapoptotic effects of pterostilbene on MCF-7 cells, a p53-positive human breast cancer
cell line. MTT reduction cell viability assay showed that pterostilbene (10–200 µM)
promoted a cytotoxic effect on MCF-7 cells in a dose- and time-dependent manner. In a
concentration of 50 µM, this compound was able to impair approximately 30% and 75%
of cell viability after 24 and 48h of treatment, respectively. These effects were more
pronounced than those induced by resveratrol in MCF-7, at the same experimental
conditions. Furthermore, cell treatment with 100 µM of pterostilbene for 24h increased
the exposition of phosphatidylserine on cell surface, which is suggestive of apoptosis.
This effect was partially prevented when cells were pretreated with pifithrin-α, a
specific p53 inhibitor. Taken together, our results indicate that pterostilbene can be
suggested as a promising chemopreventive agent and the cytotoxicity promoted by this
compound in tumor cells possibly requires p53 function. Word Keys: resveratrol
derivatives, pterostilbene, MCF-7, p53, apoptosis."
Supported by: INBEB, FAPERJ and CNPq.
G36.
EFFECTS
OF
THE
COMBINATION
THERAPY
BETWEEN
ALQUILPHOSPHOLIPIDS AND DINITROANILINES AGAINST LEISHMANIA AMAZONENSIS
NEILTON CESAR ARAUJO DA CRUZ- 1; JOSEANE LIMA PRADO GODINHO - 2; CHISTINA
MAEDA TAKIYA - 3; WANDERLEY DE SOUZA - 4; JULIANY COLA FERNANDES RODRIGUES 5.
1.POLO AVANÇADO DE XERÉM, UNIVERSIDADE FEDERAL DO RIO DE JANEIRO;2,3.INSTITUTO DE BIOFÍSICA CARLOS CHAGAS FILHO, UNIVERSIDADE FEDERAL DO RIO
DE JANEIRO; -4.UFRJ,IBCCF,IMETRO,INBEB; -5.POLO AVANÇADO DE
XERÉM,UFRJ,IBCCF,IMETRO,INBEB.
Protozoan parasites of the Leishmania genus are able to cause leishmaniasis around the
world. In Brazil, Leishmania amazonensis is responsible for cutaneous and diffuse
cutaneous leishmaniasis. The current chemotherapy is based on pentavalent
antimonials, amphotericin B, miltefosine and pentamidine. Nowadays, miltefosine has
been implemented as oral treatment for visceral and cutaneous leishmaniasis in several
countries. Trifluralin, is a dinitroaniline that displays important effects in some
protozoan parasites, and its main action is specific against parasite tubulin. In this work,
we evaluated the effect of miltefosine and trifluralin alone or in combination against L.
amazonensis in vitro and in vivo. For promastigotes in vitro, the IC50 values found were
around 25 µM and higher than 20 µM for miltefosine and trifluralin, respectively. Both
compounds, when tested alone against intracellular amastigote, showed IC50 values
higher than 50 µM. Microscopy techniques showed alterations on the shape of
promastigotes and the presence of lipid bodies. The first combination assay was done in
Balb/C-infected mice with L. amazonensis. After the development of the lesions at the
basis of the tail, the mice were divided in different groups: Glucantime group (50
mg/kg/ day), Trifluralin groups (30, 50 mg/kg/day), Miltefosine-treated groups (20; 30;
40; 50 mg/kg/day), and combination of Miltefosine/Trifluralin [(20 x 30 mg/kg/day;(20 x
50 mg/kg/day). Miltefosine and trifluralin were administrated by oral gavage, while
Glucantime by intraperitoneal route, during 21 days of treatment. The efficacy was
evaluated by measuring the size of the lesions, presence of parasite in lesions stained
with Giemsa and histopathological analysis. The results obtained with miltefosine
showed a significant dose-dependent decrease in the lesion size, which was not
observed after treatment with trifluraline alone. Combination of trifluralin with
miltefosine resulted in an effect similar to those obtained for miltefosine alone.
Página 26
Histophatological analysis showed an important immune response in the groups treated
with miltefosine alone or in combination. Reduction of the number of foam
macrophages and resident inflammatory cells was observed. Thus, this study suggests
that miltefosine alone or in combination with trifluralin could be an alternative
treatment for cutaneous leishmaniasis caused by L. amazonensis.
CNPq, CAPES, and FAPERJ.
G37.
ANÁLISE DA NEUROGÊNESE NA ZONA SUBVENTRICULAR DE
CAMUNDONGOS NOCAUTE PARA A ENZIMA GD3 SINTASE
2- NICOLI MORTARI ; 2- MICHELLE GUIMARÃES 2; 1,2- CAMILA ZAVERUCHA-DO-VALLE;
1-2- FERNANDA GUBERT; 1,2- MICHELLE BARGAS REGA, 1,2- MARCELO F. SANTIAGO;
1,2- ROSALIA MENDEZ-OTERO.
1- Programa de Terapias Celulares; 2- Instituto de Biofisica Carlos Chagas Filho,
Universidade Federal do Rio de Janeiro.
"Gangliosídeos são glicoesfingolipídeos que contêm ácidos siálicos e estão presentes na
membrana plasmática celular, com altas concentrações no sistema nervoso central. Eles
desempenham importantes papéis no desenvolvimento e diferenciação do sistema
nervoso de vertebrados. O gangliosídeo 9-O-acetil GD3 é formado a partir da acetilação
do gangliosídeo GD3 e está presente em eventos de migração celular e extensão axonal
durante o desenvolvimento. Em roedores adultos, esse gangliosídeo deixa de ser
expresso na maior parte do sistema nervoso central, mantendo-se expresso, entretanto,
na zona subventricular, principalmente. Como a zona subventricular é o principal nicho
neurogênico em mamíferos adultos, procuramos avaliar uma possível relação entre o
gangliosídeo 9-O-acetil GD3 e a neurogênese em animais adultos. Para isso, utilizamos
camundongos nocaute para a enzima GD3 sintase, responsável pela síntese do
gangliosídeo GD3 – precursor do gangliosídeo 9-O-acetil GD3 – e crítica para a formação
de todos os gangliosídeos da série b. Desta forma, ao analisar a neurogênese nos
animais nocaute para a enzima, estamos analisando não só a perda do gangliosídeo 9-Oacetil GD3, mas de todos os gangliosídeos da série b. Dissecamos a zona subventricular
de camundongos adultos nocaute para a enzima GD3 sintase (GD3KO) e de
camundongos selvagens e comparamos as duas populações em ensaios de formação de
neuroesferas. Para isso, as células foram mantidas em condições não-aderentes por 7
dias. Conseguimos estabelecer um protocolo para o ensaio de formação de
neuroesferas e observamos que as células dos dois animais são capazes de formar
neuroesferas primárias e secundárias. Observamos nos dois casos a diferenciação das
esferas em astrócitos e neurônios. Desta forma, demonstramos que as células da zona
subventricular dos camundongos GD3KO são capazes de formar neuroesferas, mas
temos evidências de que a neurogênese nesse animais possa estar de alguma forma
alterada."
INBEB, FAPERJ, CAPES, CNPq, DECIT, PROTECEL
G38.
INHIBITOR OF APOPTOSIS PROTEIN XIAP: A STRUCTURAL AND
THERMODYNAMIC ANALYSES OF ITS INHIBITION FOR COMPOUNDS SMAC MIMETICS.
1-SANTOS, R.B. , 2- OLIVEIRA, A.C. & 1- SOUZA, T.L.F.
1-Faculdade de Farmácia, UFRJ, Rio de Janeiro,Brazil. 2-Programa de Biologia Estrutural,
IBqM, UFRJ, Rio de Janeiro,Brazil.
Apoptosis (programmed cell death) resistance can be generated by high expression of
inhibitor of apoptosis proteins (IAPs) family, which are responsible by the inhibition of
effector proteases. Smac/DIABLO, is an endogenous inhibitor of XIAP due to its
interaction on the XIAP-BIR3 domain. In cancer, the high expression of XIAP leads to
apoptosis resistance and Smac/DIABLO becomes inefficient. Smac mimetics have been
considered potential drug candidates, owing to its capacity to bind on XIAP-BIR3 and to
sensitize apoptosis in cancer cell. The goal of this work is better understand of the
mechanisms of the XIAP-BIR3 inhibition. We used circular dichroism (CD), fluorescence
spectroscopy and isothermal titration calorimetric (ITC) to perform structural and
thermodynamic analyses of the effects of the binding of Smac mimetics on XIAP-BIR3.
Compounds with similarities in structure, but with different activities, were selected.
Our data showed that this domain has a high stability and that the chemical
denaturation occurs with two transitions. In the presence of the different compounds, a
differential stabilization of the domain was observed and the chemical denaturation,
differently, occurred with a single transition. Additionally, the Smac mimetics promote
to a significant change in the CD spectra of free XIAP-BIR3 domain, indicating changes in
the secondary structure content. Results obtained by ITC revealed that, although
present different affinities, the interaction is entropically and enthalpically favored to all
compounds. In conclusion, we found that the XIAP-BIR3 domain undergoes significant
changes in the structure, stability and thermodynamic parameters of binding when
associated to different ligands, what may be a relevant factor to be considered in
affinity optimization of anti-IAP drug candidates.
CNPq, CAPES, FAPERJ, INBEB
G39.
CONSUMO DE ÓXIDO NITROSO RELACIONADO AO CRESCIMENTO DE
MAGNETOVIBRIO BLAKEMOREI EM BIORREATOR DE BANCADA
1- CORREA, T.N.; 1- LEÃO, P.E.L.; 1- SANTOS, M.G.C.; 2- GUTARRA, M.L.E.; 3- LÓPEZ, J.A.
1- LINS, U.G.C.
1- Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro; 2Escola de Química, Polo Avançado de Xerém, Universidade Federal do Rio de Janeiro; 3Instituto de Química, Universidade Federal do Rio de Janeiro
Bactérias magnetotáticas apresentam a capacidade de produzir nanopartículas
magnéticas denominadas magnetossomos. Formados por cristal de magnetita ou
greigita envolto por uma membrana biológica, os magnetossomos possuem inúmeras
aplicações no campo biomédico, tais como no carreamento de drogas. A bactéria alvo
de nossos estudos é o vibrião marinho Magnetovibrio blakemorei, que produzem
cristais prismáticos de magnetita, exibindo uma grande versatilidade metabólica,
podendo utilizar diferentes compostos como aceptores finais de elétrons. A maior
produção de magnetossomos por célula, porém, é atingida no crescimento com óxido
nitroso (N2O) como aceptor final de elétrons. Pouco se conhece, entretanto, sobre a
fisiologia desta bactéria e a relação entre o óxido nitroso e a produção de
magnetossomos. Neste trabalho, o consumo de óxido nitroso pelo Magnetovibrio
blakemorei foi medido durante o cultivo em biorreator de bancada e correlacionado
com o crescimento celular e o consumo dos demais nutrientes presentes no meio. O
meio de cultivo empregado continha succinato de sódio (11,3 g/L) e hidrolisado de
caseína (3,3 g/L) como fonte de carbono e nitrogênio respectivamente, sulfato ferroso
(130µmol/L) como fonte de ferro para o crescimento e formação dos magnetossomos,
além de encontrar-se saturado com N2O (10mmol/L). Em 48h de crescimento,
aproximadamente 50% do óxido nitroso presente no meio havia sido consumido, sendo
que ao fim de 120h sua concentração havia se esgotado, assim como o ferro (II). A
exaustão destes dois componentes no meio causou uma redução na produção de
cristais por célula, observada através da contagem por microscopia eletrônica de
transmissão.
UFRJ
G40.
INTRACARDIAC INJECTION OF DM28C TRYPANOSOMA CRUZI PROVIDES A
MODEL OF INFECTION-ASSOCIATED MYOCARDITIS AND HEART FIBROSIS THAT DEPENDS
Página 27
CRITICALLY ON COOPERATIVE ACTIVATION OF THE KALLIKREIN/KININ SYSTEM AND THE
ENDOTHELIN PATHWAY
ANDRADE D1., SERRA RR1., CORDOVIL, T1., BRASIL, G.V1., RAMOS-JUNIOR E.S1.,
CARVALHO-PINTO, C.E2., VAIRO L1., FORTES F3., GOLDENBERG, R1., CARVALHO A.C.C1.,
SVENSJÖ, E1, SCHARFSTEIN J1.
1Instituto de Biofísica Carlos Chagas Filho, UFRJ, CCS, Bl. D sala 7, Cidade Universitária,
CEP 21944-900, Rio de Janeiro; 2 Instituto de Biologia - Departamento de Imunologia –
UFF, Niterói, RJ; 3CentroUniversitário Estadual da Zona Oeste -UEZO
In this study, we tested the hypothesis (Andrade et al., 2012; Scharfstein and Andrade,
2011) that kinins released intramyocardiacally by Dm28c trypomastigotes, acting
cooperatively with cardiac endothelins, infect heart tissues through the activation of
bradykinin (BKRs) and endothelin receptors (ETRs). Guided by high-resolution
echocardiography, we injected tissue-culture derived trypomastigotes (TCTs) in the left
ventricle of C57BL/6 BK2R+/+(wt), BK2R-/- or Balb/c (naïve). One hour before parasite
inoculation, wt mice were treated with a single dose (systemic) of (i) HOE-140 (BK2R
antagonist) (ii) BK1R antagonist (iii) Bosentan (ETAR/ETBR antagonist). The functional
parameters analyzed p.i. were intracardiac oedema (2 h p.i.); parasite load and mRNA
levels of chemokines/cytokine in heart tissues (qPCR); myocarditis and fibrosis at 30 d
p.i. As predicted, Dm28c TCTs evoked an early-phase intracardiac oedema in wt mice. In
contrast, plasma extravasation was virtually abolished in the heart of BK2R-/- mice, or in
infected wt mice that were pretreated with a single dose of BKR antagonists or with
bosentan. Parasite load in the heart tissues (3 d p.i.) was markedly reduced by these
GPCR antagonists. Combined, these results suggest that Dm28c TCTs might exploit the
transient availability of pro-edematogenic peptides in the interstitial spaces to
efficiently invade cardiovascular cells through the signaling of
GPCRs. We then
checked whether the benefits brought about by the pharmacological interventions, all
of which made at the onset of infection, mitigated subsequent development of heart
pathology. Our results (30 d p.i.) showed that myocarditis and fibrosis were virtually
absent in infected mice that were pretreated with BKR or ETR antagonists whereas
control mice exhibited conspicuous pathology. Additional studies are required to
evaluate whether these anti-inflammatory drugs might ameliorate heart pathology
during the chronic stage of Dm28c infection.
CNPq, FAPERJ, INBEB
G41.
INIBIÇÃO DO SISTEMA TIOREDOXINA POR POLIFENÓIS NO TRATAMENTO
DO CÂNCER
1- SILVA, T.; 2- ALMEIDA, F.; 3- AMORIM, G.;
1- Universidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica, Centro
Nacional de Ressonância Magnética Nuclear 2- Universidade Federal do Rio de Janeiro,
Instituto de Bioquímica Médica, Centro Nacional de Ressonância Magnética Nuclear 3Universidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica, Centro
Nacional de Ressonância Magnética Nuclear
"As tioredoxinas (Trxs) são enzimas antioxidantes ubíquas de baixo peso molecular
(12kDa) que estão ligadas ao controle da homeostasia oxido-redutora das células e
desempenham um importante papel em diversos processos celulares relacionados ao
equilíbrio saúde-doença. Em diversas patologias, incluindo o câncer, o balanço oxidoredutor das células é afetado. Células tumorais possuem alta taxa metabólica e,
consequentemente, nível elevado de espécies reativas de oxigênio (ROS). Dessa forma,
interferir na homeostasia redox destas células representa uma abordagem promissora
na terapia do câncer. Devido a seu papel essencial na regulação redox, as tioredoxinas
representam um alvo importante na busca por novos tratamentos quimioterápicos.
Muito já foi feito no sentido de mostrar a eficácia da inibição do sistema tioredoxina no
tratamento do câncer. Na busca por novos inibidores, os flavonóides foram
identificados como potenciais agentes quimioterápicos, e seu mecanismo de ação é
provavelmente mediado pelo sistema tioredoxina. O principal objetivo deste trabalho
de pesquisa é selecionar compostos naturais, especialmente flavonóides, com potencial
ação inibitória do sistema tioredoxina, e estudar as interações do complexo enzimainibidor com enfoque bioquímico e estrutural. Alguns dados já foram obtidos, dentre os
quais foi possível identificar uma amostra de flavonóide que mostrou-se capaz de
interagir com a tioredoxina. Os possíveis resíduos envolvidos na interação já foram
identificados. Estes resultados serão utilizados no desenho racional de drogas, visando a
obtenção de moléculas mais eficazes e menos tóxicos na terapia contra o câncer, que
estarão na base do desenvolvimento de futuras abordagens terapêuticas."
FAPERJ, CNPq
G42.
SELEÇÃO DE MICROALGAS OLEAGINOSAS PARA A PRODUÇÃO DE BIODIESEL
1 - FERREIRA, V.S.; 1,2 -PINTO, R.F; 1- SANT'ANNA, C.B
1 -Inmetro (Instituto Nacional de Metrologia Normalização e Qualidade Industrial) - 2Instituto de biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
O uso de combustíveis fósseis será futuramente insustentável devido à depleção de
suas fontes e ao acúmulo de gases do efeito estufa gerado por seu consumo. O
biodiesel é um combustível derivado de biomassa renovável e seu uso traz substancial
redução na emissão de gases estufa. Atualmente, este é produzido a partir de óleos
vegetais ou gorduras animais, entretanto o uso de micro-organismos oleaginosos têm se
mostrado interessante para produção de biodiesel devido ao rápido crescimento e
menor gasto de energia e espaço para produção. As microalgas, organismos
fotossintéticos, são promissoras fontes de lipídeos para a produção de biodiesel devido
às altas taxas de produção de biomassa e à síntese de óleos em altas quantidades,
podendo alcançar 60-70% de lipídeos em relação ao seu peso seco total. O objetivo
deste trabalho é fazer a bioprospecção de microalgas capazes de produzir e armazenar
grande quantidade de lipídeos. As microalgas foram coletadas de ambientes aquáticos
diversos, e isoladas por estriamento em placa de ágar. A morfologia das espécies
isoladas foi observada por microscopia óptica convencional, microscopia eletrônica de
transmissão e varredura. Para a detecção de lipídeos neutros nestes microorganismos
foi utilizado um corante lipofílico fluorescente, o Nile Red, e a observação foi realizada
em um microscópio confocal de varredura a laser. Foram feitas as curvas de
crescimento das células na presença e ausência de nitrogênio, e em seguida a presença
e quantia de lipídeos foi comparada por citometria de fluxo. Pudemos observar que
todas as cepas isoladas são capazes de armazenar lipídeos, e que há redução no
crescimento de células que foram cultivadas na ausência de nitrogênio, no entanto,
estas cepas tiveram uma maior produção de lipídeos.
CNPq, Inmetro
G43.
CONFORMATIONAL ENSEMBLES OF GLYCOSYLATED PRION PROTEIN: A
STRATEGY FOR MAPPING INTERACTIONS WITH PHYSIOLOGICAL AND THERAPEUTIC
LIGANDS
1,2- ALVES, W. J. C.; 2- MACEDO, B.; 2- GONÇALVES, K. M.; 1- OLIVEIRA JUNIOR, R. S.; 1TORRES, P. M.; 3- POL-FACHIN, L.; 3- VERLI, H.; 1- LINDEN, R.; 2- CORDEIRO, Y.; 1PASCUTTI, P.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Faculdade de Farmácia, Universidade Federal do Rio de Janeiro; 3- Centro de
Biotecnologia, Universidade Federal do Rio Grande do Sul
Introduction: The prion protein (PrPC) is ubiquitously expressed in mammals, and
anchored by GPI on the outer leaflet of the plasma membrane. Three alpha-helices, one
Página 28
short anti-parallel beta-sheet, a large highly flexible domain, and two glycans compose
the structure of PrPC. The functional properties of PrPC are controversial, although a
number of its natural ligands have been described. Laminin receptor precursor(LRP),
stress inducible protein-1(STI1) and neural cell adhesion molecule(NCAM), are three
such proteins ligands for which cognate interaction domains have been mapped onto
each pair of ligands. Our goal is to test whether the main function of PrPC may be to
scaffold signaling modules at the cell surface, through allosteric interactions with its
protein ligands, thus leading to effects on cell proliferation, differentiation and death.
Materials/Methods: We use techniques of molecular modelling and dynamics,
generalized simulated annealing and docking, to generate conformational ensembles
including PrPC with its flexible N-terminus, and to stabilize structural models of
interaction of PrPC together with its ligands. In addition, we use circular dichroism(CD),
X-ray small angle scattering, and calorimetry for in vitro assays of binding and
conformational effects among recombinant mouse PrPC and synthetic cognate peptides
from each ligands. Results/Discussion: An initial CD experiment showed apparent
conformational changes undergone by PrPC in the presence of cognate peptides added
in a particular order, consistent with results from docking, and suggesting that the
affinity of the globular domain of PrPC for a given peptide is distinct, depending on the
previous binding of another ligand peptide. A novel protocol of molecular dynamics was
produced, specific for the generation of conformational ensembles based on PrPC,
including parameters for glycans. Conclusions: Promissory data are consistent with the
hypothesis that PrPC scaffolds cell surface protein ensembles, and additional
spectroscopy and simulation will provide further testing of allosteric regulation of PrPC
structure.
FAPERJ, CNPq, INBEB
G44.
1H NMR METABOLIC INTERACTION EVALUATION BETWEEN Citrus cinensis
AND Cadidatus Liberibacter asiaticus
1- OHASHI, W.; 1- ESPINDOLA, A.; 2- FERREIRA, M.; 3- COLETTA, H.; 1- TASIC, L.
Universidade Estadual de Campinas - Instituto de Química – 1- Laboratório de Química
Biológica, 2- Laboratório de Quimiometria Teória e Aplicada. 3- Centro APTA Citros
Sylvio Moreira IAC – Cordeirópolis.
"Candidatus liberibacter spp. is the pathogen associated with the Huanglongbing (HLB),
a high economic impact disease present all over the world’s mayor citrus producing
areas. The infection diagnosis can be confirmed by PCR after visual inspection. Our
objective was to evaluate pathogen induced changes in the metabolic profile shown in
1H NMR spectra obtained from different polarity orange leaf extracts. By comparing
these spectra of heathy, symptomatic and asymptomatic Citrus cinensis leafs using
chemometric tools, such as, Principal Component Analysis (PCA) and Hierarchical
Cluster Analysis (HCA), we attempted to visualize clustering on the chemometric models
indicating the possibility of creating a HLB diagnostic method. The leaf samples were
processed by liquid nitrogen grinding, separated in four fractions for extraction with
phosphate buffer (A), methanol (B), chloroform/methanol (1:1, v v-1, C) and chloroform
(D), evaporated and diluted in DMSO d-6 for 1H NMR analysis on a Bruker Avance III 600
MHz spectrometer. Resultant spectra were standardized, normalized and processed
with Infometrix Pirouette® software. Initial results show that we were able to classify
healthy and sympthomatic samples with a variance of PC1: 60.85%; PC2: 28.75% and
PC3: 3.70%. Further analysis are currently being made for a wider statistical significance.
Keywords: Citrus sinensis, Liberibacter asiaticus, Huanglongbing, plant-pathogen
interaction, metabolomics, 1H NMR."
INBEB
G45.
ANÁLISE E DETECÇÃO DE MUTAÇÕES NO GENE TP53 EM CASOS DE CÂNCER
DE MAMA EM MULHERES DO ESTADO DO RIO DE JANEIRO E ESTUDO DE SUA PROTEÍNA
MUTANTE P53 EM LINHAGENS CELULARES DE CARCINOMAS MAMÁRIOS
1- MACHADO, Y.L.R.C.; 1- ALMEIDA DA SILVA, A. P.; 2- PORTARI, E.A.; 3- SILVA JL; 1- DE
MOURA GALLO, C.V.
1- Departamento de Genética, IBRAG, Universidade do Estado do Rio de Janeiro; 2Instituto Fernandes Figueira, Fundação Oswaldo Cruz; 3-Instituto de Bioquimica Medica,
Universidade Federal do Rio de Janeiro
O câncer de mama é o tipo de neoplasia maligna mais freqüente entre mulheres, com
incidência de mais de um milhão de novos casos/ano no mundo. O gene TP53
apresenta-se mutado em 50% dos carcinomas humanos e em torno de 25% no câncer
de mama, cujas alterações localizam-se majoritariamente nos éxons 5-8, domínio de
ligação da proteína com o DNA. Além disso, verificou-se que a proteína p53 quando
mutada apresenta uma tendência à agregação, exibindo um comportamento dominante
negativo. O objetivo deste trabalho foi detectar mutações neste gene, associando-as
com a agressividade do tumor, obtido dos prontuários médicos, e observar o
comportamento da proteína p53 em linhagens celulares de carcinomas mamários. Foi
realizada a análise da presença de mutações nos éxons 4-10 do TP53, em amostras de
câncer de mama de 98 pacientes do IFF-FIOCRUZ e de 68 pacientes do INCA. Para
avaliar a existência de co-localização da proteína p53 com agregados protéicos, utilizouse linhagens celulares de carcinomas mamários e anticorpos A11 e DO-1 para
reconhecimento de agregados protéicos e da p53, respectivamente. Após extração de
DNA genômico, realizaram-se as técnicas de PCR e seqüenciamento automático para
verificação da presença de mutações. As linhagens MDA-MB231 (p53 mutante
p.R280K), T47-D (p53 mutante p.L194F), MCF-7 (p53 selvagem) e 21-NT (p53 nula)
foram cultivadas em meio de cultura contendo 10% FBS, a 37oC e atmosfera de 5% de
CO2, tratadas com os anticorpos conjugados a fluoróforos. Dos casos analisados,
mutações no gene TP53 foram detectadas em 18 (20,45 %) dos 98 casos do IFF, sendo
12 (66,66 %) do tipo missense; já para os casos do INCA, foram detectadas apenas 3
mutações (4,41%), sendo 2 do tipo missense. Nas linhagens celulares, foi observada por
análise em microscopia confocal co-localização entre a p53 mutante e os agregados
protéicos, onde observaram-se diferentes perfis de agregação.
FAPERJ
Página 29
Mestrandos
M1.
O AUMENTO DA FUSÃO E PROLIFERAÇÃO DE MIÓCITOS MODULADO PELA
GSNO-R
1- Yamashita, A.M.S., 1- Figueiredo-freitas, C., 2- Possidonio, A.C., 2- Soares, C.P., 2Mermelstein, C.S., 1- Sorenson, M.M.
1Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio
de Janeiro, Brasil, 2- Departamento de histologia e embriologia, universidade Federal do
rio de Janeiro, rio de janeiro, Brasil.
"O óxido nítrico (NO) é produzido fisiologicamente no músculo durante a contração.
Uma das vias de sinalização por NO 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 promovendo a denitrosilação de proteínas. Diversos tecidos expressam GSNOR, porém muito pouco se sabe sobre essa enzima no músculo esquelético. O objetivo é
caracterizar a expressão e a atividade da GSNO-R no músculo esquelético (peitoral) de
embrião de galinha (11 dias) e analisar o papel durante a diferenciação celular, usando
um inibidor específico para GSNO-R (Sanghani, 2009). A cultura primária de galinha foi
crescida por 24h, seguido por 48h de tratamento com DMSO (veículo) ou inibidor (10 ou
30µM) e o controle foi crescido por 72h. Foi medida a atividade enzimática da GSNO-R
no homogenato de miócitos (Liu, 2001). Foi feito Western blot para GSNO-R das
amostras de fibroblasto e miócito (72h e 96h). Imunofluorescência para núcleos DAPI,
anti-GSNO-R e anti-desmina, permitiram quantificar o número e a espessura dos
miotubos, número de células mononucleadas e multinucleadas (Image J Fiji)
acompanhando a diferenciação. A atividade, expressão e imuno-marcação da GSNO-R
na cultura primária de miócito foi detectada. Não foi encontrada diferença no número
de miotubos entre os grupos. Um aumento da espessura dos miotubos tratados com
inibidor sugerindo um aumento na fusão. O número de núcleos associado aos miotubos
não aumentou, porém o número de células mononucleadas do grupo com inibidor
aumentou 40%, sugerindo um aumento da proliferação. Os dados sugerem que a
atividade da GSNO-R pode modular a proliferação e fusão celular."
M2.
EFEITOS DA ADMINISTRAÇÃO SISTÊMICA DE GUANOSINA EM MODELO DE
LESÃO NO NERVO ÓPTICO
1- SILVA-JUNIOR, AJ; 1- MESENTIER-LOURO, LA; 1- ZAVERUCHA-DO-VALLE, C; 1MENDEZ-OTERO, R e 1- SANTIAGO, MF.
1- Institudo de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
As lesões no nervo óptico podem levar desde a perda parcial da visão à cegueira total.
Estas lesões afetam especialmente as células ganglionares da retina (CGRs). Nosso
grupo obteve resultados positivos com a terapia celular utilizando tanto a população de
células mononucleares da medula óssea, quanto as células-tronco mesenquimais da
medula óssea., após lesão no nervo óptico. Outra terapia potencial é a administração
sistêmica de guanosina. O tratamento com guanosina tem demonstrado efeito
neuroprotetor em diferentes modelos. Neste trabalho nós investigamos se a
administração sistêmica de guanosina tem um efeito neuroprotetor e regenerativo
sobre as CGRs após lesão no nervo óptico. Ratos da variedade Lister-Hooded passaram
por um modelo de lesão no nervo óptico seguida de administração intraperitonial de
guanosina (7,5 mg/kg) ou veículo, separados em grupos que receberam uma única
injeção, ou uma injeção diária por 7 dias. Quatorze dias após este procedimento foram
analisadas a sobrevivência das CGRs por imunomarcação para Tuj-1 (β-III-tubulina) ou
Brn3a em montagens planas de retinas, e sua extensão axonal por imunomarcação para
GAP-43 no nervo óptico. Nossas análises não demonstram diferenças significativas na
sobrevivência das CGRs nos grupos de animais tratados em comparação aos grupos nãotratados, em todos os protocolos utilizados. Foi observada diferença significativa no
número de axônios se estendendo além de 1,5mm do sítio de lesão no grupo que
recebeu uma única injeção i.p. de guanosina em comparação ao grupo não-tratado. A
avaliação da extensão axonal nos grupos que receberam 7 injeções ainda está em curso.
Até o presente momento, pode-se concluir que o tratamento utilizando estas doses de
guanosina não foi eficaz para aumentar o número de CGRs sobreviventes após a lesão
no nervo óptico.Entretanto, as células sobreviventes são capazes de emitir um maior
número de axônios através do nervo óptico em regeneração.
M3.
BIOLOGICAL BEHAVIOR OF MESENCHYMAL STEM CELLS ON POLI-ΕCAPROLACTONE FILAMENTS AND A POTENTIAL STRATEGY FOR TISSUE ENGINEERING OF
PERIPHERAL NERVES
1- CARRIER-RUIZ, A.; 1- VASCONCELOS-DOS-SANTOS, A.; 1- CARVALHO, L.R.P.; 1MENDEZ-OTERO, R.; 1- RIBEIRO-RESENDE, V.T.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
"Peripheral human nerves fail to regenerate across long tube implants, probably
because implants lack the microarchitecture of native nerves. Bone marrow
mesenchymal stem cells (MSC) contribute to the regeneration of the peripheral nervous
system (PNS) in many aspects under cell therapies approaches. To optimize tubular
implants by integrating artificial bands of Bungner and a biological component capable
of enhancing that regenerative capacity, we study the interaction between polycaprolactone (PCL) filaments and MSC. Rat’s MSC were placed on PCL filaments treated
with plasma O2, poli-D-lysine and laminin. Cells were plated in three different
concentrations, 2x10^5, 2x10^6 and 8x10^6 cells/ml, and, after 48h of incubation, the
adhesion profile, viability and proliferation capacity were analyzed. We also plated rat’s
Schwann cells at the concentration of 5x10^5 cells/mL on PCL filaments covered with
MSC, 24h after the MSC’s plating, and let the co-culture for another 24h to analyze the
feasibility of the system. Embryonic rat’s dorsal root ganglia (DRG) were plated in
contact with PCL filaments, with or without MSC. The samples were incubated for 4
days then we analyzed the neurite extension of DRG’s neurons. MSC showed an
alignment along the longitudinal microstructure, however, as we increased the
concentration of cells, the rate of alignment gradually decreased. MSC showed high
viability and their proliferation capability was not completely inhibited by the filaments.
Schwann cells were able to adhere to the filaments plated with MSC maintaining also
high viability. Neurites were able to grow and extend over the surface of the PCL
filaments when they were previously covered with MSC. Data were analyzed using
ANOVA with Neuman-Keuls post-test for multiple comparisons. We provide evidence
for the interaction between MSC, Schwann cells and PCL filaments, as they can
constitute a stable system permissive for neurite extension and possibly events for the
regeneration of the PNS."
CNPq, CAPES, FAPERJ, INBEB
M4.
STRUCTURAL ANALYSIS OF A VACCINE PLATFORM BASED ON MS2 VIRUSLIKE PARTICLES
Página 30
1- VICENTE, A.C.; 1- BARROSO, S.P.C.; 2- PEABODY, D.S.; 3- FERREIRA, D.F.; 4- DE
MESQUITA, J.F.; 1- SILVA, J.L.; 1- 0LIVEIRA, A.C.
1- Inst. de Bioquímica Médica, IBqM-UFRJ, RJ, Brazil; 2- Department of Molecular
Genetics and Microbiology, University of New Mexico; 3- Inst. de Microbiologia, IMPPGUFRJ, RJ, Brazil; 4- Universidade Federal do Estado do Rio de Janeiro.
Virus-like particles (VLPs) can be considered as dense arrays of one or more repetitive
subunits of a protein and this characteristic confers highly advantageous properties for
their use as vaccines platforms. The platforms used here are VLPs of the bacteriophage
MS2, an E. coli phage. We evaluated the structural stability of VLPs containing highly
immunogenic peptides related to the infectious cycle of HIV-1 by submitting them to
high hydrostatic pressure (HHP) and other chemical and physical denaturant agents and
evaluating the changes by means of light scattering, intrinsic and extrinsic fluorescence
and circular dichroism (CD). We analyzed the morphology of VLPs by transmission
electron microscopy. In addition, the structure prediction of the coat protein with
peptide insertions was done by homology modeling approach. The results obtained so
far were performed with VLPs formed by a single chain dimer of the coat protein, the
native coat protein, two constructions with the Flag epitope and with VLPs containing
peptides of the extracellular loop of CCR5 cell co-receptor and the V3 loop of gp120 of
HIV-1. The spectral center of mass and light scattering data indicate that there are
differences in the structure and stability of VLPs with insertion of the epitopes, except
for results using HHP, in which the construction with inserts showed the largest shift of
the center of mass. CD measurements indicate no changes in secondary structure
between dimer and native protein, but single chain constructs with Flag epitope had a
different behavior. Coat proteins with peptide insertions show small RMSD when
aligned with the native protein. Our results demonstrate that the VLPs assembled from
coat protein containing peptides insertions behave differently from the ones assembled
from native coat protein, however they showed structural stability under most of the
conditions used, suggesting that these particles are very promising for application as a
vaccine platform.
CAPES-FAPERJ-CNPq-PRONEX-INBEB
M5.
METABOLIC CHANGES IN A GLIOBLASTOMA CELL LINE INDUCED BY IRON
Mendonça,A.P.M; Amoedo, N.D.;Oliveira, M.F.
Instituto de Bioquímica Médica, IBqM-UFRJ, RJ; Brasil
"Introduction: The intracerebral hemorrhage (ICH) represents one of most severe
events in the CNS with poor prognosis and outcome. ICH is caused by the extravasation
of blood into the brain parenchyma as a result of blood brain barrier disruption. Blood
derived products (BDPs) such as hemoglobin, heme and iron, are powerful toxic
components which are released and metabolized in cerebral parenchyma after the ICH
onset. Here, we investigated the effects of iron on energy and redox metabolism in U-87
glioblastoma cells. Material and methods: U-87 cells were cultured for 24 hours in the
presence of iron (up to 50 mM) and several cellular and biochemical parameters were
evaluated. Cell viability was assessed by the MTT reduction assay. Redox imbalance was
determined by measuring the TBARS levels, whereas mitochondrial physiology
assessment was carried out by measuring the oxygen fluxes on intact cells in glucose
containing medium by high-resolution respirometry (O2k). Citrate synthase activity was
determined spectrophotometrically. Results and discussion: Iron promoted lipid
peroxidation without affecting cell viability. Mitochondrial content, determined by the
activity of citrate synthase, was also not affected by iron up to 50 μM. High-resolution
respirometry analyses showed that iron caused an overall reduction of the oxygen
consumption rates, regardless the mitochondrial metabolic states. Conclusion: Iron
triggered a signaling cascade that promote functional mitochondrial remodeling in a
glioblastoma cell line, which may represent a cellular survival mechanism in response to
the BDPs during the ICH events.
Keywords: mitochondria, oxidative stress, stroke"
Faperj, CNPq
M6.
ANÁLISE DO PROCESSO DE AGREGAÇÃO DA ESFINGOSINA POR MEDIDAS DE
RELAXAÇÃO E EFEITO DE TROCA H/D.
1- ANDRÉ ROBERTO DE OLIVEIRA FREDI; 1- LUZINEIDE W. TINOCO
1- Núcleo de Pesquisa de Produtos Naturais, Universidade Federal do Rio de Janeiro
A esfingosina (2S,3R,4E)-2amino-4-octadeceno-1,3-diol) é um derivado da cadeia de
formação de esfingolipídios e atua como um mensageiro estando envolvida nos
processos de inibição do crescimento celular e na estimulação da apoptose. A natureza
anfifílica destes compostos leva a sua agregação, com a formação de micelas. A
compreensão deste processo, em diferentes condições, pode contribuir para o
entendimento da desordem no armazenamento lisossomal dos glicoesfingolipídios. Este
trabalho teve como objetivo analisar o processo de agregação da esfingosina em função
do pH através da análise dos espectros de RMN de 1H e de medidas do tempo de
relaxação longitudinal. As amostras da D-eritro-esfingosina 1 mM (Sigma Aldrich), foram
preparadas em solução tampão fosfato pH 5,0 e 7,2, em 10% de D2O e 100% D2O e
analisadas por RMN [VNMRS-500 (Agilent) 499,8 MHz para 1H] a 25°C. A intensidade
dos sinais e os valores de T1 sofrem uma significativa variação em função do pH e do
percentual de D2O usado. Interessantemente, os sinais que, provavelmente,
correspondem aos hidrogênios da amina (~8 ppm) somente são observados nos
espectros adquiridos em 100% de D2O. Além disso, os hidrogênios da cabeça polar da
esfingosina apresentam valores baixos de T1 que os da cadeia carbônica,
principalmente na região da dupla ligação. Assim como o efeito de troca H-D, as
medidas de T1 fornecem resultados opostos ao esperado. Uma vez que, os hidrogênios
da cabeça polar estando de em uma região maior mobilidade deveriam ter valores de
T1 mais altos. Isto sugere que o processo de troca H-D e os tempos de relaxação dos
hidrogênios da cabeça polar devam ser mais bem investigados. Estes dados poderão
fornecer informações importantes acerca do processo de agregação através da
formação de ligações de hidrogênio intra e intermoleculares.
CNPq, INBEB, FAPERJ, FINEP, CAPES
M7.
COMPARATIVE ANALYSIS OF DIFFERENT CHEMICAL FIXATIVES FOR
SCANNING ELECTRON MICROSCOPY TO STUDY FUNGAL BIOFILM STRUCTURE FORMED
BY CANDIDA ALBICANS.
1- FONSECA, B. B., 1- VILA, T.V.M., 2- ISHIDA, K., 1- ROZENTAL, S.
1- Laboratório de Biologia Celular de Fungos, Instituto de Biofísica Carlos Chagas Filho,
Universidade Federal do Rio de Janeiro. 2- Departamento de Microbiologia, Instituto de
Ciências Biomédicas, Universidade de São Paulo.
"Biofilms are frequently related to invasive fungal infections and are reported to be
more resistant to antifungal drugs than planktonic cells. Biofilm cells are known to
synthesize a polymeric extracellular matrix (ECM). In this work, we make a comparative
analysis of different chemical fixatives for scanning electron microscopy (SEM) to study
fungal biofilm. Candida albicans biofilms were formed on 5 mm sections of central
venous catheters (CVC) and fixed by 3 different chemical fixatives: (a) Routine fixative
method: glutaraldehyde (2.5%) formaldehyde (4%) in cacodylate buffer; (b) Sucrose
fixative method: glutaraldehyde (2.5%) in cacodylate buffer, then a post-fixative with
0.2 M sucrose and 2 mM MgCl2; (c) Ruthenium red fixative method: glutaraldehyde
(2.5%) and ruthenium red (0.5 mg/mL) in cacodylate buffer; all the samples are
processed as routine and observed under a scanning electron microscope (FEI Quanta
Página 31
250, Japan). Utilization of both routine and sucrose fixative methods allowed a good
preservation of cell distribution inside the biofilm. However, the ECM was extracted
from the biofilm. Although the sucrose fixative method seemed to preserve some ECM
filaments interconnecting cells, the absence of a post-fixation step with OsO4 may be
responsible for the reduced quality of images obtained from these samples. Ruthenium
red fixative method leaded to the formation of a large amount of precipitate upon the
biofilm structure. This result was confirmed by incubating uncultivated CVC and isolated
C. albicans yeasts with ruthenium red as controls. Ruthenium red protocol was also
adapted for lower concentrations (0,1mg/mL) but even so precipitates was formated.
Routine and sucrose fixative methods were helpful to analyze cell distribution inside the
biofilm but did not preserve the ECM. Unhappily, addition of ruthenium red to chemical
fixative solutions did not improve biofilm ECM preservation. Other methods as
cryothecniques should to be used to evaluate these structures."
FAPERJ, CNPq, CAPES
M8.
IMMUNOMODULATORY EFFECTS OF AQUEOUS EXTRACT OF ROOT PLANT
Physalis angulata ON CELL OBTAINED OF BONE MARROW
SILVA, B. J. M 1,2; RODRIGUES, A. P. D. 1,2,3; FARIAS, L.H.S. 1,2; HAGE, A, A, P1,2.; SILVA,
E. O. 1,2
¹ Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências
Biológicas, Universidade Federal do Pará, Brazil. 2Instituto Nacional de Ciência e
Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro,
Ilha do Fundão, Brazil 3Laboratório de Microscopia, Instituto Evandro Chagas, secretaria
de vigilância em saúde, ministério da saúde, Belém, Pará, Brazil.
"The bone marrow is a hematopoietic tissue that in the presence of cytokines and
growth factors generates all circulating blood cells. These cells are important to protect
the organism against pathogens and for establishing an effective immune response.
Some studies in literature have been showing action of immunomodulatory effects of
different products obtained of plant extracts. Thus, this study aim to evaluate the
immunomodulatory properties of the root extract from Physalis angulata (REPa), a plant
widely used in popular medicine. Bone marrow cells were obtained by flushing femurs,
and maintained in cultures treated with REPa at a concentration of 100 µg/mL. It was
observed by optical microscopy that REPa stimulates the process of cell adhesion,
increase of cytoplasm, spreading ability, cytoskeleton alterations and high number of
cytoplasmatic projections. Furthermore, REPa did not promote the proliferation of
lymphocytes and polymorphonuclear leukocytes, however promotes increased the
number of macrophages in the culture. Immunophenotyping was performed by
immunofluorescence and flow cytometry. Labeling CD11b and F4/80, a marker specific
for mononuclear phagocytes revealed that REPa seems to stimulate differentiation of
bone marrow cells in macrophages. Labeling using a specific dendritic cells CD11c
marker, showed that REPa did not stimulate differentiation of the treated cells into
dendritic cells. No cytotoxic effect was observed in cells treated with REPa when
compared to the untreated control. Thereby, these results demonstrate that REPa can
promote the differentiation of bone marrow cells into macrophages in just 96 hours
culture. These results demonstrate that REPa can be used as an immunomodulator
agent.
Keywords: Cell proliferation and differentiation, Bone marrow, Physalis angulata."
Supported by CAPES, CNPq/UFPa, CNPq/MCT/CT-INFRA/CT-PETRO (Processo nº
620179/2008), MCT/CNPq/FNDCT/PROCAD-NF CAPES/FAPERJ.
M9.
TRIAGEM IN VITRO E PLANEJAMENTO IN SILICO DE INIBIDORES DE
SUPERÓXIDO DISMUTASE DE TRYPANOSOMA BRUCEI (TbSOD)
1- BRITO,C.C.B.; 1- CASTILHO, M.S.
1- Faculdade de Fármacia, Universidade Federal da Bahia
A Doença de chagas, causada pelo Trypanosoma cruzi, ainda é um problema de saúde
publica e uma das maiores causas de morbidade e mortalidade da América Latina. O
sistema de defesa antioxidante é uma importante rota na sobrevivência deste parasita
no hospedeiro e as enzimas chaves envolvidas nesse processo são vitais para o
crescimento e desenvolvimento do parasita. Por essa razão, a enzima superoxido
dismutase (SOD), que dismuta radicais superóxido em oxigênio (O2) e peróxido de
hidrogênio (H2O2), é um alvo interessante para o desenvolvimento de fármacos
antichagásicos. Devido a identidade sequencial elevada, a enzima do parasita T. brucei
pode ser utilizada como modelo para identificação de moléculas que inibam a enzima
de T. cruzi. A fim de alcançar esse objetivo, realizou-se a avaliação biológica de 33
derivados de tiossemicarbazonas, sendo a fenantrenoquinona -4 fenil
tiossemicarbazona identificada como um inibidor de Tbsod em ensaio de dose única. O
IC50 calculado para essa molécula foi em torno de 200µM. Para verificar o modo de
interação proteina-ligante utilizou-se a técnica de acoplamento molecular, através dos
programas SurflexDock e o AutoDock Vina. Os resultados do acoplamento sugerem que
a Fenantrenoquinona 4-fenil tiossemicarbazona interage num bolsão que não faz parte
do sítio ativo de Tbsod, sugerindo um modo de inibição não competitiva. As
informações provenientes dos experimentos in silico guiarão o planejamento de
inibidores mais potentes que possam ser considerados como compostos protótipos para
o desenvolvimento de fármacos contra a doença de Chagas.
INBEB
M10.
TÉCNICAS DE MICROSCOPIA COMO FERRAMENTA PARA ESTUDO DO DNA
MITOCONDRIAL DE TRIPANOSOMATÍDEOS
1-CAMILA S. GONÇALVES, 1-MARCELO ZOGOVICH, 1-GABRIEL FELIPE B. D. da SILVA, 1LUANA PORTELLA, 1-DANIELA L. GONÇALVES, 1,2-WANDERLEY de SOUZA e 1DANIELLE P. CAVACALTI
1-Instituto Nacional de Metrologia, Qualidade e Tecnologia, INMETRO; 2-Instituto de
Biofísica Carlos Chagas Filho, UFRJ
Os tripanosomatídeos possuem estruturas peculiares, como o cinetoplasto, que está
localizado dentro da matriz mitocondrial e contêm parte do genoma do protozoário. O
DNA mitocondrial ou kDNA é a mais complexa e incomum forma de DNA extranuclear
encontrada na natureza, sendo composto de milhares de moléculas circulares, os
minicírculos e os maxicírculos, que se encontram topologicamente relaxadas e
interligadas formando uma única rede. O objetivo do nosso trabalho é caracterizar
ultraestruturalmente o cinetoplasto e a rede de kDNA de diferentes membros da
família. Análises por Microscopia Eletrônica de Transmissão (MET) demonstram que, na
maioria dos tripanosomatídeos, as fibrilas de kDNA apresentam-se bastante
compactadas e contidas em um cinetoplasto em forma de disco. Já na forma
tripomastigota de T. cruzi e nos tripanosomatídeos que contêm endossimbionte, as
fibrilas de DNA estão organizadas em um arranjo mais largo e frouxo, que preenche
toda a matriz do cinetoplasto. Além disso, nosso grupo desenvolveu uma metodologia
para analisar a rede de kDNA isolada utilizando a Microscopia de Força Atômica (AFM).
Os resultados obtidos mostram que as redes de kDNA de Crithidia fasciculata,
Angomonas deanei, Strigomonas culicis, Leishmania amazonensis e Trypanosoma cruzi
apresentam um padrão de organização muito similar, sendo possível identificar
minicírculos interligados e um agrupamento de moléculas de DNA na periferia da rede,
formando rosetas. Recentemente, utilizamos o AFM para analisar a ação de drogas
intercalantes de DNA sobre a rede e entender melhor o mecanismo de ação destes
Página 32
compostos. O próximo passo do trabalho é obter modelos tridimensionais do
cinetoplasto dos diferentes tripanosomatídeos utilizando a tomografia de elétrons.
INBEB, FAPERJ, CNPq.
M11.
EXPLORING THE ULTRASTRUCTURE AND FUNCTION OF THE CYTOSTOMECYTOPHARINX COMPLEX OF TRYPANOSOMA CRUZI EPIMASTIGOTES
1,2- ALCANTARA, C.L.; 1,2- VIDAL.J.C; 1,2,3- DE SOUZA, W.; 1,2- CUNHA-E-SILVA, N.
1- Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos
Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373,
bloco G subsolo, Cidade Universitária, Ilha do Fundão, Rio de Janeiro 21941-902, Brazil.
2- Instituto Nacional de Biologia Estrutural e Bioimagem (INBEB), 3- Diretoria de
Programas, INMETRO"
The cytostome is an aperture at the plasma membrane close to the flagellar pocket (FP)
followed by a profound invagination called cytopharinx. This complex is the major portal
for endocytosis in T. cruzi epimastigotes. Data from literature has shown that this
region presents a prominent glycocalix continuous to the preoral ridge (POR), the
membrane domain between the cytostome and the FP, and an uncharacterized set of
microtubules (MTs) and vesicles along the cytopharinx. Little is known about its detailed
structure and the correlation with the endocytic function. In this work we focused on
the 3D structure of the cytostome-cytopharinx complex to describe how endocytic
cargo passes through and leaves it. Epimastigotes were submitted to endocytosis of
transferrin-gold particles with short incubations times. The material was processed to
electron microscopy and serial semi-thin sections were observed by electron
tomography. The structures of interest were segmented using appropriated software.
We observed seven MTs supporting the opening of cytostome and the cytopharinx.
These MTs could be separated in two sets: a quartet coming from the FP that also
sustains the POR and a triplet coming from the subpellicular microtubules. The MTs
arrangement around the cytopharinx leaves a free side, where aligned vesicles were
seen. As the invagination becomes deeper and thinner, two MTs finish, one from the
quartet and one from the triplet. Moreover, the MTs continue even beyond the end of
the cytopharinx and accompany vesicles and tubules that seem to be originated from
the cytopharinx. We also used horseradish peroxidase as a tracer to follow the
cytopharinx in FIB-SEM preparations. We measured the cytopharinx length in 25 whole
cells and found out that the length is independent of cell cycle or differentiation stage.
Together these data provides a new vision about the structure and dynamics of
endocytic pathway of epimastigotes.
INBEB, CNPQ, CAPES
M12.
KOJIC ACID , A SECONDARY METABOLITE OBTAINED FROM ASPERGILLUS
FUNGI, INDUCES THE DIFFERENTIATION OF MONONUCLEAR CELLS OBTAINED FROM
MOUSE BONE MARROW IN VITRO
ALMEIDA, C. M. 1,4; SILVA, B. J. M 1,4; RODRIGUES, A. P. D. 1,3,4; FARIAS, L.. H. S. 1,3;
HAJE, A. A. P. 1,4; SILVA, E. O. 1,4
Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências
Biológicas, Universidade Federal do Pará, Brasil. ² Laboratório de Neuroinflamação,
Instituto de Ciências Biológicas, Universidade Federal do Pará, Brasil. 3 Laboratório de
Microscopia, Instituto Evandro chagas, Pará, Brasil. 4 Instituto Nacional de Ciência e
Ilha do Fundão, Brasil
"Bone marrow is a tissue responsible for hematopoiesis. Cell proliferation and
differentiation depend of a complex interaction between the stromal cells, cytokines,
growth factors and extracellular matrix that compose the bone marrow
microenvironment. The most of the cells generated in the bone marrow give rise to
circulating blood cells that play a important role in the immune response against
pathogens. In this context, the research for drugs that enhance the innate immune
response is needed to restore the homeostasis and the immune response in
immunocompromised patients. Thus, in this study we evaluated the
immunomodulatory effects of Kojic Acid (KA), a secondary metabolite obtained from
Aspergillus fungi, on the bone marrow cells of mice. These cells were obtained by
flushing femurs, and maintained in cultures treated with KA at the concentration of 50
and 100 µg/mL at four different times of culture. Analysis by optical microscopy
revealed that KA promoted increased cell adhesion, spreading ability and high number
of cytoplasmatic projections and vacuoles in cytoplasm of bone marrow mononuclear
cells. These cells seem represent mature macrophages as indicated by the presence of
mature-cell markers like CD11b (specific marker of mononuclear phagocytes). In
addition, the absence of CD11c (a dendritic cell marker) showed that KA did not
stimulate the differentiation of the treated bone marrow cells into dendritic cells. Also,
no cytotoxic effects were observed in cells treated with KA when compared to the
untreated bone marrow cells. Thus, these results suggested that KA can promote the
differentiation of mononuclear cells obtained from mouse bone marrow in vitro.
Keywords: Cell proliferation and differentiation, Kojic Acid, Bone marrow."
Supported by CAPES, PRONEX/FAPESPA/CNPq, CNPq/MCT/CT-INFRA/CT-PETRO
(Processo nº 620179/2008), MCT/CNPq/FNDCT/PROCAD-NF CAPES/FAPERJ.
M13.
DESCRIPTION OF A NOVEL TRANSTHYRETIN VARIANT WITH CARDIAC
INVOLVEMENT: A STUDY OF STRUCTURAL PREDICTION
1-FERREIRA, P. S.; 1-VAREJÃO, N.; 1-LIMA, C;1-SANT'ANNA, R. O.;2-CALDEIRA, C. M.;1FRANKLIN, D. W.;3-CRUZ, MARCIA WADDINGTON; 1-FOGUEL, D.
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro.2LABORATÓRIO SONDA-UFRJ; 3- HOSPITAL UNIVERSITÁRIO CLEMENTINO FRAGA FILHOUFRJ
Mutations in the TTR gene are known to destabilize the structure of protein and
facilitate the aggregation process causing an amyloidosis which can be characterized by
the involvement of peripheral nerves, cardiac function and other disorders. Here we
report a patient from Santa Catarina, state in the south region of Brazil, and his family is
from Swedish/German origin, with a rare mutation in exon 2 of TTR gene where we
have a substitution of a Ala for a Asp at the codon 19, causing a severe compromise of
cardiac function characterizing the Familial Amyloidotic Cardiomyopathy (FAC). To
predict the stability of this mutant we use bioinformatics tools how FoldX and PDBSum
to analyze the thermodynamic stability of this mutant. We predicted that the tetramers
of A19D presented a decreased thermodynamic stability, when compared to the WTTTR, and is more amyloidogenic than V30M tetramers. The PDBSum analyses
demonstrated that the strongest interface of TTR (AB) loses two hydrogen bonds and
drastic changes in the orientation of some amino acids in C2 axis can facilitate the
dissociation process in this interface. Taken together, all the structural changes imposed
by the mutation A19D to the tetramers resulted in a destabilized protein that presents
alterations in the monomer-monomer and dimer-dimer interface. Indeed the patient
that presents this mutation has a severe cardiomyopathy which developed very fast in a
short period of time. We describe for the first time in Brazil this mutation.
INBEB, FAPERJ, CAPES, CNPq
M14.
CONFORMATIONAL
ANALYSIS
OF
9,10-PHENANTHRENEQUINONE
GUANYLHYDRAZONE BY NUCLEAR MAGNETIC RESSONANCE AND MOLECULAR
MODELING.
Página 33
1 -SIMOES, C.; 2 -AZEREDO, S.O.F.,3- VILLAR, J.D.F
1,2,3 - Instituto Militar de Engenharia, Seção de Engenharia Química – Praça General
Tibúrcio, 80, 22290-270, Praia Vermelha, Rio de Janeiro, RJ, Brasil.
The novel compound 9,10-phenantrenequinone guanylhydrazone is a promising
molecule with potential biological activity such as antibiotic and anticancer (through
interactions with DNA). This is possible because yours structural features (fused
aromatic rings, Intracellular hydrogen bond with an oxygen atom and amines terminals).
The structure has a rotation angle that allows changes in amine terminal nevertheless
was observed there is a minimum energy to obtain this rotation angle and this occurs
when the compound is submitted to a specific temperature. The objective of this work
is study the 9,10-phenantrenequinone guanylhydrazone conformational changes versus
temperature via NMR and molecular modeling to calculate the minimum energy value
to achieve the rotation angle. The experimental conformational changes were studied
via Nuclear Magnetic Resonance (Spectrometer Varian 600 MHz) through 1H
experiments to elucidate the structure after submitted it to a different temperatures.
The theoretical energy values were obtained through molecular modeling studies
(Spartan 06). The NMR study showed that the rotation angle is achieved when the
compound is submitted to a temperature above 34 °C, and this data was confirmed by
quantum mechanics. This information is important to understand the form of
interaction of this compound with DNA and with enzymes of microorganisms."
INBEB
M15.
SERINA PROTEASES EM CANDIDA ALBICANS: IDENTIFICAÇÃO E PROCURA
POR FUNÇÃO BIOLÓGICA
Gonçalves D.S1, Braga-Silva L.A1, Gandra R.M1, Seabra S.H2, Sodré C.L1, Santos A.L.S1
1 Laboratório de Estudos Integrados em Bioquímica Microbiana (LEIBM), UFRJ Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo de Góes,
Centro de Ciências da Saúde - Bloco E-subsolo 2 Universidade Estadual do Estado do Rio
de Janeiro- UEZO
"Introdução: O aumento expressivo das infecções fúngicas nas últimas décadas pode ser
atribuído ao grande número de casos de pacientes imunocomprometidos /
imunossuprimidos. Candida albicans é responsável por 43% das infecções causadas por
leveduras, fato atribuído a seus fatores de virulência e sua capacidade de se adaptar a
diversos nichos ecológicos. Nosso grupo descreveu a secreção de serina peptidase em C.
albicans, capaz de degradar componentes proteicos essenciais do hospedeiro.
Objetivos: Avaliar a presença de serina protease secretada em diferentes isolados
clínicos e estudar os efeitos de diferentes inibidores de serina protease sobre múltiplos
aspectos da biologia celular e virulência de C. albicans. Resultados: Sobrenadantes de
cultivo de seis diferentes cepas de C. albicans, cultivadas em meio BHI por 48h a 37°C,
foram concentradas 40 em sistema AMICON e analisados em gelatina-SDS-PAGE. Dois
perfis de protease foram detectados: (1) 90kDa e 50kDa (cepas ATCC10231, ATCC38688,
2A e 72) e (2) 50kDa (ATCC24433 e 11). A protease de 50kDa foi inibida por PMSF, um
inibidor de serina proteases. A cepa ATCC10231 foi selecionada para os testes com
inibidores de serina protease. Dos inibidores de serina proteases (n=7) testados a
100 M, somente TPCK e TLCK foram capazes de inibir significativamente a proliferação
celular em 100% e 62% respectivamente. O tratamento de C. albicans (103 leveduras)
com TPCK gerou um IC50 de 11,5µM. A inibição da proliferação também foi dependente
da densidade celular. A análise ultraestrutural evidenciou alterações na superfície das
leveduras bem como células rompidas. TPCK apresentou uma redução significativa na
diferenciação (transformação de levedura a tubo germinativo) e na formação do
biofilme. Conclusão: Os resultados sugerem a importância da serina protease na
biologia celular de C. albicans, uma vez que funções como crescimento e diferenciação
foram sensivelmente alterados quando expostas a inibidores de serina proteases.
Suporte Financeiro: ." CNPq, CAPES & FAPERJ
M16.
EVALUATION
OF
ANTI-INFLAMMATORY
POTENTIAL
OF
DIETHYLCARBAMAZINE COMPARED TO CELECOXIB IN MODEL OF LIVER INFLAMMATION
INDUCED BY ALCOHOL
1-RODRIGUES, G.B., 1-ROCHA, S.W., 1-SILVA, B.S., 1-GOMES, F.O.S., 1-BARBOSA,K.P.S.,
1-RIBEIRO, E.L., 1-SANTOS, L.A.L.M., 1-FRANCA,M.E.R., 1,2-PEIXOTO,C.A.
1-Centro de Pesquisas Aggeu Magalhães – FIOCRUZ, Recife; 2-Centro de Tecnologias
Estratégicas do Nordeste - MCT, Recife.
"Iintroduction: The Diethylcarbamazine (DEC) presents several different biochemical
effects on enzyme systems and potential anti-inflammatory properties. The Celecoxib is
part of the class of nonsteroidal anti-inflammatory drugs (NSAIDs). This study aims to
evaluate the potential anti-inflammatory of DEC compared with celecoxib in liver
inflammation induced by alcoholism in C57BL/6J mice wild. Materials and Methods:
Solutions of DEC and celecoxib (concentration 50 mg / kg both) were administered by
gavage for 12 days. Forty strain C57BL/6J male mice were divided into four groups: (I)
control group that received only water, (II) alcoholic group, (III) alcohol + DEC (50 mg /
kg) and (IV) alcohol + celecoxib group (50 mg / kg). Ethanol was supplied in the drinking
water at crescent concentrations of 10%, 15% and 20%. The liver fragments were
processed according to routine protocol and stained with hematoxylin-eosin (HE) and
picro-sirius red for histopathological analysis and evaluation of collagen fibers.
Immunohistochemistry was performed for markers: IL-1β (1:100, Genway), IL-17 (1:
100, Abcam) and malondialdehyde (1:50, Abcam). Results/Discussion: In alcoholic group
several histological changes were observed, which were reduced in the groups III and IV.
By picro-sirius staining was noted increased production of collagen in alcoholic group (II)
compared to control (I), and this production was decreased only in group + DEC alcohol
(III). By immunohistochemistry analysis intense staining of all inflammatory markers in
group II was observed. There was a significant decrease in labeling of these markers in
groups treated with DEC (III) and celecoxib (IV). Conclusion: Due to lack of efficacies
therapies for the treatment of advanced stages of alcoholic liver disease, DEC shows up
as a possible therapeutic alternative for the treatment of the condition compared to
other drugs used already.
Key-words: Diethylcarbamazine, inflammation, celecoxib."
INBEB, CAPPES
M17.
ASPECTOS ULTRAESTRUTURAIS DA INFECÇÃO POR TOXOPLASMA GONDII
EM INTESTINO DE FELINOS
VERAS, G. M¹. DUBEY, JP, DE SOUZA, W¹, ATTIAS, M.¹,
¹ Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,
Brasil.
A toxoplasmose é uma doença de distribuição mundial causada pelo protozoário
Toxoplasma gondii, um parasito intracelular obrigatório capaz de invadir e infectar
qualquer célula nucleada de todos os animais homeotermos. A transmissão da doença
pode ocorrer por via placentária, pela ingestão de carne crua ou mal cozida contendo
cistos teciduais e ainda por alimentos e água contaminada com oocistos esporulados. O
ciclo sexuado deste parasito acontece apenas nos felinos com a formação de oocistos,
que são eliminados junto com as fezes e liberados no ambiente, onde se tornam
infecciosos. Neste trabalho objetivamos visualizar por microscopia eletrônica de
varredura as formas enteroepiteliais no intestino dos felinos, recorrendo à criofratura
destes tecidos. Para isto, fixamos nossas amostras com glutaraldeído, pós-fixamos com
Página 34
tetróxido de ósmio e ferrocianeto de potássio e desidratamos em uma bateria crescente
de etanol. A seguir, congelamos em nitrogênio líquido e criofraturamos
longitudinalmente o epitélio intestinal e finalmente, secamos no ponto crítico. As
primeiras observações mostraram a presença do parasito em diversos estágios
exclusivos da porção mais superficial das vilosidades intestinais, local em que ocorre a
descamação do epitélio. Merozoítas, esquizontes, gamontes e oocistos puderam ser
observados.
INBEB, FAPERJ, CNPq
M18.
EFFECTS OF 5-HYDROXY-2-HYDROXYMETHYL-GAMA-PYRONE (HMP) IN
FILAMENTOUS FUNGAL Curvularia pallescens
1- PEREIRA, J.A.L.; 1,2,4 - RODRIGUES, A.P.D.; 1,4 - FARIAS, L.H.S.; 1- OLIVEIRA, D.M.S.; 3SANTOS, A.S.; 1,4- SILVA, E.O.
1 Laboratório de Parasitologia e Biologia Estrutural, Instituto de Ciências Biológicas,
Universidade Federal do Pará, Brasil; 2 Instituto Evandro Chagas, Laboratório de
Microscopia Eletrônica; 3- Universidade Federal do Pará, Laboratório de
Desenvolvimento e Planejamento de Fármacos; 4- Instituto Nacional de Ciência e
Ilha do Fundão, Brasil.
"The species Curvularia pallescens is a entophytic fungi and producer of melanin that
occasionally causes a variety of human infections. Nowadays, there are some studies
about antifungi effects of bioproducts which have low toxicity. The hydroxy-2hydroxymethyl-γ-pyrone (HMP) is a secondary metabolite synthesized by some species
of fungi from Aspergillus genera. The HMP has several applications mainly as tyrosinase
inhibitor, enzyme that works in biosynthetic pathway for melanin formation in various
mammalian and fungi cells. This study evaluated the HMP effect in C. pallescens
morphology cultivated and treated with 25, 50, 100 e 200 µg/ml of HMP. The
morphological analysis by optical microscopy, scanning electron microscopy and
transmission electron microscopy of treated cells showed accumulation of many
cytoplasmic vesicles like lipid droplets as well external vesicles. In addition, it was
showed conidia became wilted after HMP treatment. These results suggested HMP
caused many morphological alterations in C. pallescens that could be involved in fungi
death. Further studies are needed to identify the mechanisms that induce these
alterations.
Keywords: HMP; Fungal; Morphological alterations; Curvularia pallescens."
CAPES, CNPq/UFPA, CNPq/MCT/CT-INFRA/CT-PETRO (Processo nº 620179/2008),
MCT/CNPq/FNDCT/PROCAD-NF CAPES/FAPERJ.
M19.
ENVOLVIMENTO DO FATOR DE TRANSCRIÇÃO NFAT NA DOENÇA DE
PARKINSON: CONTRIBUIÇÃO PARA A INFLAMAÇÃO E APOPTOSE.
1- DE FREITAS, J.A; 1- ROBSS, B.K.; 1- FOGUEL, D.
1- Instituto de Biquímica Médica
"A Doença de Parkinson (DP) é uma doença neurodegenerativa, progressiva e
irreversível associada a um déficit da função motora. Em termos patológicos, a DP é
descrita através da perda de neurônios dopaminérgicos. O mecanismo que leva a perda
de função dos neurônios na DP consiste de um acúmulo anormal de uma proteína
denominada α-sinucleína e posterior formação de agregados proteicos intracelulares
chamados de corpúsculos de Lewy. Estudos demonstraram que agregados da αsinucleína são capazes de induzir influxo de Ca+2 e levar a ativação da fosfatase
calcineurina A (CnA). Aparentemente, tanto o influxo de Ca+2 quanto a ativação da CnA
estão fortemente ligados com os processos de neurodegeneração. Apesar do fator de
transcrição nuclear de célula T ativada (NFAT) ser um dos alvos da ação da via de
sinalização de Ca2+/CnA, pouco sabe-se sobre sua contribuição para a DP. As proteínas
NFAT regulam diretamente a expressão de genes envolvidos no controle da morte
celular por apoptose como Fas-L, TNF-α, c- Nur-77, FLIP, TRAIL, assim como genes
envolvidos no processo inflamatório como IL1β, Cox-2, TNF-α, MCP1, iNOS. Tendo em
vista que a desregulação dos processos de inflamação e de morte celular programada
das células do sistema nervoso possui um papel central na neurodegeneração e que um
dos eventos importantes para o processo é o aumento do Ca2, o objetivo principal do
projeto é avaliar o envolvimento do NFAT no processo de neurodegereração induzido
pelos agregados de α-sinucleina. Para isso serão avaliados a capacidade do agregado da
α-sinucleína de ativar o fator de transcrição NFAT, através de ensaios que avaliam os
processos de desfosforilação, transativação e translocação do NFAT, além de
determinar a relevância da ativação da proteína NFAT para os processos de morte
celular e desintegração de neuritos e por fim caracterizar quais genes são ativados pelo
NFAT em modelo de DP."
INBEB, CNPq, FAPERJ
M20.
DIETILCARBAMAZINA (DEC) CONFERE PROTEÇÃO ANTI-INFLAMATÓRIA EM
MODELO MURINO DE PLEURISIA INDUZIDA POR CARRAGENINA
SANTOS, L.A.M.; 1- RIBEIRO, E. L.; 1- FRAGOSO, I. T.; 1- BARBOSA, K.P.S.; 1- DONATO, M.
A.; 1- ROCHA, S.W.S.; 1- OLIVEIRA, W.B.; 1- FRANÇA, M.E.R.; 1- SILVA, A.K.S.; 1RODRIGUES, G.B.; OLIVEIRA, F.G.; SILVA, B. S.; 1,2 - PEIXOTO, C.A;
1- Centro de Pesquisas Aggeu Magalhães 2- Centro de Tecnologias Estratégicas do
Nordeste
A Dietilcarbamazina (DEC) é um fármaco utilizado no tratamento da filariose linfática.
Apesar de seu longo período de utilização, desde 1947, seu mecanismo de ação
permanece obscuro. Alguns estudos mostram que a DEC detém provável ação antiinflamatória por interferir sobre o metabolismo do ácido araquidônico e atuar no
sistema imune do hospedeiro. Nesta perspectiva, a DEC pode ser útil no tratamento da
inflamação aguda pulmonar, grave problema de saúde mundial, segundo a OMS. O
objetivo deste trabalho foi avaliar o efeito protetor da DEC sobre o dano tecidual, a
produção de mediadores pro-inflamatórios e infiltração leucocitária em modelo murino
de pleurisia induzida por carragenina. Metodologia: Os camundongos foram distribuídos
em três grupos experimentais; Grupo 1- Controle SHAM (n=10); Grupo 2- Carragenina
CAR (n=10); Grupo 3- Dietilcarbamazina, DEC (50 mg/kg dissolvida em água, n=10). Os
animais do grupo 3 receberam o tratamento acima por três dias consecutivos antes da
administração de carragenina intrapleural (CAR 1%, i.pl.); O grupo SHAM, recebeu uma
injeção de salina 0,9% i.pl.; 4hs após a indução, os animais foram eutanaziados em
câmara de CO2. Em seguida, recolheu-se o exsudato pleural para quantificação dos
níveis de óxido nítrico (NO) e contagem celular total. Os fragmentos do pulmão foram
processados para análise histológica, imunohistoquímica (iNOS, IL-1β). O resultado
sobre a análise histológica demonstra o efeito protetor da DEC sobre as alterações
teciduais, com redução de áreas de dano alveolar difuso, acompanhada de significativa
redução da expressão de iNOS e IL-1β (p < 0,05) quando comparados ao grupo CAR.
Paralelamente, houve significativa redução dos níveis de NO (p <0,05) e recrutamento
de leucócitos no grupo DEC (p < 0,001) confirmando o efeito anti-inflamatório da DEC
neste modelo experimental.
FACEPE, INBEB, CNPQ
M21.
INVESTIGATION ON THE BEHAVIOR OF TRICHOMONAS TENAX
1,2- Ribeiro, L. C. S. and 1- Benchimol, M.
1- Laboratório de Ultraestrutura Celular da Universidade Santa Úrsula; 2- Instituto de
Biofísica Carlos Chagas Filho, UFRJ
Página 35
T. tenax is a commensal of the human mouth found under conditions of poor oral
hygiene or associated with periodontal diseases. There are some controversies
concerning the pathogenicity of this protozoan. T. tenax is very similar to Trichomonas
vaginalis, a parasite that inhabits the human genitourinary tract. This parasite is known
to induce the disruption of various mammalian cells. Currently, there is a discussion
whether T. tenax would be a genetic variant of T. vaginalis. The aim of this study was to
investigate the capable of T. tenax to cause damage to mammalian cells and to compare
their cytotoxicity with T.vaginalis. For this, the detection and distribution of the proteins
membrane of T. vaginalis were verified in T. tenax by immunofluorescence using the
antibodies anti-P270 and anti-AP65. In addition, interaction assays of this protozoan
with MDCK and HeLa cell lines were performed with the ratio of 5:1. The samples were
examined by scanning electron microscope (SEM) and the cytotoxic effects of the
protozoa were analyzed by MTT assay. Our results show several similarities between the
species. T. tenax and T. vaginalis displays four anterior flagella and one recurrent
flagellum and the same proteins membrane distribution. Moreover, the interaction
assay showed that both trichomonad species were able to adhere on mammalian cells.
Interestingly, T. tenax provoked injury to mammalian cells in the same way of T.
vaginalis. Take together, these results indicated that T. Tenax could be a parasite, not a
commensal, but further experiments are needed to confirm its pathogenicity.
AUSU, FAPERJ, CNPq, PRONEX
M22.
THROMBIN TRIGGER MITOCHONDRIAL FUNCTIONAL REMODELING IN
HUMAN PLATELETS INVOLVING 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., 1Oliveira M.F.
1-Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, Brasil. 2-Fiocruz, Rio de Janeiro,
Brasil. 3-University of Utah, Salt Lake City, USA.
Introduction Evidence has indicated that pro-coagulant factors modulate platelet energy
and redox metabolism pathways. However, the involvement of mitochondria during
platelet activation remain poorly understood and was investigated in the present work.
Material and Methods Human platelets were collected from healthy volunteers, isolated
in M199 medium, and subsequently challenged with different thrombin concentrations.
Several parameters were analyzed in activated platelets such as P-selectin
externalization, respiration, reactive species generation, mitochondrial membrane
potential, lactate and NO production. These were assessed by specific probes
fluorescence detection by flow cytometry or by high-resolution respirometry. Results
and Discussion When platelets were exposed to low doses of thrombin (0-0.3 IU/mL)
increased oxygen consumption parallel to a reduction of mitochondrial membrane
potential (Δψm) was observed. 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 IU/mL) changes in both parameters were less
pronounced, being undistinguishable from quiescent platelets at 1.0 IU/mL thrombin. In
this condition, nitric oxide (NO) is implicated in the partial impairment of electron
transport system (ETS) resulting in a decrease in maximum oxygen consumption.
Maintenance of Δψm seems to involve the reversion of F1Fo ATP synthase activity,
since the presence its inhibitor oligomycin collapsed the Δψm when the electron
transport system is impaired by antimycin a. Conclusions Our data indicate that platelet
mitochondrial function is affected by thrombin activation and seems to involve two
distinct mechanisms, depending on the stimuli intensity. The dominant mechanism
during low activation seems to be the mitochondrial permeability transition, whereas at
high activation, reversibility of ATP synthase prevails.
FAPERJ, CNPq and CAPES
M23.
ENTENDENDO A TOXICIDADE DA PROTEÍNA Α-SINUCLEÍNA E O EFEITO DE
POSSÍVEIS COMPOSTOS ANTI-PARKINSONIANOS
1- FERNANDES, L ; 1- MIRANDA, M.C.; 1 - FOGUEL, D.; 1- BRAGA, C.
1 - Instituto de Bioquimica Médica, Universidade Federal do Rio de Janeiro
Doença de Parkinson (DP) é a segunda desordem neurodegenerativa mais comum em
humanos, caracterizando-se, principalmente, por perda de neurônios dopaminérgicos
na substância nigra (SN) e presença de inclusões proteicas intracelulares, denominadas
Corpos de Lewy (CL). Os CL são majoritariamente compostos por agregados amilóides
de alfa-sinucleína (α-sin), uma proteína abundantemente expressa no cérebro e
localizada nos terminais pré-sinápticos. A função fisiológica da α-sin ainda é
desconhecida e seu papel na degeneração, observada na DP, foi ressaltado após serem
identificados mutantes dessa proteína (A53T, A30P e E46K) envolvidos em uma forma
precoce e hereditária da doença. Acredita-se que a agregação de α-sin desencadeie
estresse oxidativo e processos inflamatórios, causando dano aos neurônios
dopaminérgicos. No entanto, os mecanismos moleculares pelo qual a agregação
contribui para a neurodegeneração e as vias de sinalização afetadas não foram
elucidados. A selegilina (Sel) é um composto inibidor de Monoamina Oxidase B (MAO-B)
utilizado na terapia da DP e a Edaravona (Ed) um composto antioxidante utilizado em
isquemias cerebrais, ambos tem demonstrado efeito neuroprotetor em modelos da DP
induzida por drogas como a 6-hidroxidapamina (6-OHDA) ou 1-metil-4-fenil-1,2,3,6tetraidropiridina (MPTP). Além disso, estudos do nosso grupo demonstram que tanto
Sel quanto ED modulam a agregação da α-sin in vitro e levam à formação agregados não
tóxicos aos neurônios em cultura. No presente estudo, pretendemos elucidar o
mecanismo de neuroproteção destes compostos contra a toxicidade de agregados de αsin. Utilizaremos culturas celulares de neuroblastoma submetidas à pré-tratamento com
esses compostos e, posteriormente, à presença de diferentes espécies oligoméricas da
α-sin. Será, então, avaliado o papel do pre-tratamento com os compostos frente à
toxicidade dessas da proteína através de testes de viabilidade celular como MTT, Tunel
e Live/Dead.
INBEB, FAPERJ, CNPq
M24.
EVALUATION
OF
ANTI-INFLAMMATORY
ACTIVITIES
OF
DIETHYLCARBAMAZINE ON ACUTE HEPATIC INFLAMMATION IN MICE
1- FRANÇA, M.E.R. ; 1- ROCHA, S.W.S. ; 2- ARAUJO, S. M. R. ; 2- SILVA, J.C. ;1- BARBOSA,
K.P.S. ; 1- RODRIGUES, G.B. ;1- RIBEIRO, E.L. ; 1- SPINOLA, M.G.D. ; 1- OLIVEIRA A.C. ;
1,2- PEIXOTO, C.A.
1. Centro de Pesquisas Aggeu Magalhães- CpqAM/FIOCRUZ. 2. Centro de Tecnologias
Estratégicas do Nordeste- CETENE/MCT.
Pharmacological studies showed that Diethylcarbamazine (DEC) interferes with the
arachidonic acid metabolism, acting as an anti-inflammatory drug. This study
investigated the anti-inflammatory effects of DEC on the CCl4-induced hepatotoxicity in
Swiss mice. 20 male S. Webster mice were separated in groups: control group (C) (n=6),
CCl4 group (CCl4) (n=6) and CCl4 + DEC 50mg/kg group (CCl4DEC) (n=8). DEC solutions
were administered in dose 50mg/kg in drink bottle for 3 days before CCl4
administration. After 24h of intraperitoneal injection (i.p.) of CCl4 (0,1µl/kg, in olive oil),
the animals were euthanized. Liver fragments were processed for light microscopy and
immunohistochemical assays. Liver sections from control group presented normal liver
architecture, showed well preserved tissue, composed of radially arranged cords of
hepatocytes distributed in hepatic lobules. Histological analyses of the CCl4 group
showed large areas of extensive necrose, mainly pericentral, loss of hepatic
architecture, lipid accumulation, perivenular hemorrhage and inflammatory cell
Página 36
infiltration. The treatment with DEC before CCL4 administration completely prevented
liver necrosis reduced the inflammatory infiltrates and hemorrhagic foci. In fact, the
DEC+CCL4 group showed minimal hepatic damage. Immunohistochemical analyses of
the control group did not show substantial TNF- immunopositivity. On the other hand,
strong TNF-expression was found in the CCl4 group. TNF- immunoreactivity was
observed mainly in mononuclear infiltrates and necrotic areas, predominantly in
pericentral areas. No apparent TNF-expression was detected in nonparenchymal cells.
DEC+CCl4 group substantially reduced TNF-expression. Quantification staining analyses
were performed using the image program Gimp 2.6 software. According to present
results DEC can be used as a potential anti-inflammatory drug for acute hepatic
inflammation. Further assays are in development in our laboratory in order to clarify the
role of DEC on the molecular mechanisms involved in the inflammation signaling
network.
INBEB, FACEPE, CNPq.
M25.
EFFECTS OF 5-HYDROXY-2-HYDROXYMETHYL-GAMMA-PYRONE (HMP), A
BIOPRODUCT OBTAINED FROM ASPERGILLUS FUNGI, ON HUMAN NEUTROPHILS IN
VITRO
1,3 - FRADE, P. C. R.; 1,3 - COSTA, J. P.; 1,3,4 - RODRIGUES, A. P. D.; 1,3 - FARIAS, L.H.S.; 2
- SANTOS, A.S. ; 1,3 - SILVA, E.O.
1 - Laboratório de Parasitologia e Laboratório de Biologia Estrutural, Instituto de
Ciências Biológicas da Universidade Federal do Pará; 2 - Laboratório de
Desenvolvimento e Planejamento de Fármacos, Instituto de Ciências Exatas e Naturais
da Universidade Federal do Pará; 3 - Instituto Nacional de Ciência e Tecnologia em
Biologia Estrutural e Bioimagem da Universidade Federal do Rio de Janeiro; 4 Laboratório de Microscopia Eletrônica, Instituto Evandro Chagas, Secretaria de
Vigilância em Saúde do Ministério da Saúde.
Neutrophils are cells that participate in innate immune system as professional
phagocytes to eliminate pathogens. The mainly mechanisms that cells use are mediated
by reactive oxygen species and proteolytic enzymes. The 5-hydroxy-2-hydroxymethylgamma-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 a food additive, tyrosinase inhibitor, antitumor agent and macrophage
activator. Thus, this study evaluated the in vitro effect of HMP in functional properties
related to human neutrophils activation, such as morphological changes, production of
ROS (reactive oxygen species) and phagocytosis. Human peripheral leucocytes were
obtained from blood bag donated from Fundation Hemocenter of Para State. Cells
isolation was performed using HISTOPAQUE® 1077-density-gradient. Neutrophils were
treated for 1 hour with 50 and 100 μg/mL of HMP. Cytometric analysis was performed
for measurement of neutrophils viability by Mitochondrial Membrane Potential
Detection Kit (JC-1) and Propidium Iodide (PI) exclusion test. These results showed that
HMP has no citotoxicity effect on neutrophils. The morphological analysis by optical
microscopy and electron microscopy of treated neutrophils showed extensive
lamellipodia formation, pseudopodia extention, high spreading ability and increase of
cell volume, features that are often observed in activating cells. The increased
cytoplasmic area was confirmed by morphometric analysis. Regarding the microbicidal
activity, HMP increased the production of ROS and phagocytosis activity, but not NO
(nitric oxide) production. In conclusion, this study demonstrates a role for HMP as a
human neutrophil activator.
INBEB, CAPES, CNPq/UFPa.
M26.
PERFIL DE PRODUÇÃO DE MAGNETOSSOMOS PELA BACTÉRIA
MAGNETOVIBRIO BLAKEMOREI AO LONGO DE UM CULTIVO EM BATELADA SIMPLES.
1-PEDRO E. LEÃO, 1-TARCÍSIO CORREA, 2-MAYARA G.C. SANTOS, 2-MELISSA GUTARRA E
1-ULYSSES LINS
1-Instituto de Microbiologia Paulo de Goes, Universidade Federal do Rio de Janeiro; 2Escola de Química, Universidade Federal do Rio de Janeiro, Xerém
Nanopartículas magnéticas são estruturas de grande interesse biotecnológico. O
principal desafio para uma maior aplicação dessas partículas é a sua obtenção em larga
escala. Os processos físico-químicos de síntese destas estruturas apresentam um baixo
rendimento e um elevado custo. Uma alternativa é o uso de nanopartículas magnéticas
de origem biológica. A bactéria Magnetovibrio blakemorei (cepa MV-1) é capaz de
sintetizar em seu interior nanocristais magnéticos pseudo-hexagonais prismáticos de
magnetita, com 50-70nm de diâmetro, envoltos por uma bicamada lipídica. Tais
estruturas são chamadas magnetossomos. Neste trabalho foi analisado o perfil da
produção de magnetossomos pela cepa MV-1, quando cultivado em um biorreator de
tanque agitado com uma estratégia de batelada simples. O meio de cultura utilizado
disponibiliza acetato de sódio e succinato de sódio como fontes de carbono, hidrolisado
de caseína com fonte de nitrogênio e óxido nitroso como aceptor final de elétrons da
cadeia respiratória. Amostras foram retiradas a cada 24h de cultivo para
acompanharmos a cinética de consumo dos nutrientes e para a observação do tamanho
e da forma dos magnetossomos produzidos. Os resultados mostram que após 3 dias de
cultivo o meio de cultura está esgotado, em relação a disponibilidade de ferro. Nas 24
horas seguintes o número de magnetossomos por célula se mantêm inalterado (14
magnetossomos/célula). Após mais 24 horas, no quinto dia de cultivo, ocorre uma
queda (8 magnetossomos/célula). Eletromicrografias dos magnetossomos mostram
que alem de estarem em menor número nas células, os magnetossomos produzidos
após 96h de cultivo são menores do que aqueles observados no inicio da cinética. Estes
cristais menores e em menor quantidade podem comprometer tanto a produtividade
quanto a qualidade destas nanopartículas. Uma nova cinética, envolvendo uma
estratégia de suplementação de ferro esta em andamento. Com esta estratégia de
batelada alimentada, pretendemos obter cristais maduros durante toda a cinética de
crescimento celular.
M27.
ESTABELECIMENTO DE METODOLOGIAS DE CRIOFIXAÇÃO E SUBSTITUIÇÃO
A FRIO PARA ANÁLISE POR MICROSCOPIA ELETRÔNICA DE PROTOZOÁRIOS PARASITAS
RACHID,R.P.; MIRANDA, K. R
Instituto de Biofísica Carlos Chagas Fillho, Universidade Federal do Rio de Janeiro
A malária persiste ainda como uma doença responsável por altos níveis de mortalidade
no mundo. Esta é causada por protozoários parasitos do gênero Plasmodium, entre os
quais o P. falciparum e o P. vivax são os de maior importância médica. Parasitos deste
gênero apresentam estruturas peculiares, não encontradas em células de mamíferos e
constituindo, portanto, interessantes modelos de estudo tanto do ponto de vista da
biologia celular quanto para o desenvolvimento de fármacos. A maior parte do
conhecimento sobre a estrutura e organização morfoestrutural de protozoários se deu
com a utilização de técnicas de microscopia eletrônica. Estas requerem uma serie de
etapas no preparo de amostras, que notadamente provocam significativas alterações na
sua estrutura. O desenvolvimento de métodos menos evasivos, como técnicas de
fixação física e metodologias complementares, surgiu então como alternativa de
preparo para manter a integridade estrutural o mais próximo do nativo. Neste trabalho,
utilizamos técnicas de criofixação de protozoários parasitas como método de preparo
para microscopia eletrônica. Células de Plasmodium chabaudi foram criofixadas por
Página 37
congelamento ultrarrápido por alta pressão e em seguida submetidas à substituição a
frio. Os resultados mostraram maior preservação estrutural em células submetidas às
criotécnicas, quando comparadas às submetidas a técnicas de fixação química. Em geral,
estruturas com organização menos rugosa foram observadas em diferentes domínios da
célula hospedeira e dos parasitas, como a membrana do vacúolo parasitóforo e o
conjunto de membranas do parasito, indicando sua acentuada preservação estrutural, e
estrutura íntegra dos cristais de hemozoína. Em conjunto, os resultados mostram um
papel potencial de métodos de preparo menos invasivos para a compreensão detalhada
sobre a organização estrutural destes parasitas, contribuindo assim para diversos
estudos que envolvem a biologia da malária.
CNPq, FAPERJ, FINEP, CAPES, INBEB.
M28.
SALIVARY METABOLITE SIGNATURES OF CHILDREN WITH AND WITHOUT
DENTAL CARIES LESIONS
1-FIDALGO, T. K. S. ; 1-FREITAS-FERNANDES, L. B. ; 2-MUNIZ, A. M. S. ; 3-ANGELI, R. ; 4GONÇALVES, E. ; 1-PINHEIRO, R. ; 5-NADAL, J. ; 3-ALMEIDA, F. ; 3-VALENTE, A. P. ; 1SOUZA, I. P. R.
1-Faculdade de Odontologia, Universidade Federal do Rio de Janeiro; 2-Escola de
Educação Física do Exército; 3- Centro Nacional de Ressonância Magnética Nuclear,
Universidade Federal do Rio de Janeiro; 4-Escola de Física, Universidade Federal do Rio
de Janeiro; 5-COPPE, Universidade Federal do Rio de Janeiro
A metabolomic approach was used to analyze endogenous metabolites and to correlate
with a specific biological state. The analysis of salivary metabolites is a growing area of
investigation with potential for basic and clinical applications. Analyses of children’s
saliva in different dentitions and with or without caries could potentially reveal a
specific profile related to oral disease risk. Nuclear Magnetic Resonance (NMR) is well
suited for mixture analysis followed by Principal Component Analysis combined with
Linear Regression (PCA-LR) statistics and was used to identify differences in the salivary
metabolites. The classificatory analysis was performed using PCA-LR based on 1,000
cross-validation bootstrap runs from both classifiers in order to increase the data
information from a small sample size. The PCA-LR presented a statistically good
classificatory performance for children with and without caries with an accuracy of
90.11 % (P < 0.001), 89.61 % sensitivity (P < 0.001), and 90.82 % specificity (P < 0.001).
Children with caries lesions presented higher levels of several metabolites, including
lactate, fatty acid, acetate and n-butyrate. Saliva from subjects with different dentition
stages was also analyzed. Although the salivary samples were poorly classified,
permanent dentition presented increased levels of acetate, saccharides and propionate.
The NMR data and PCA-LR were able to classify saliva from children with or without
caries, with performance indexes comparable to the partial least-squares regression
discriminant analysis (PLS-DA) results also performed. Our data also showed similar
salivary metabolite profiles for healthy subjects despite the differences in their oral
hygiene habits, socioeconomic status and food intake.
FAPERJ, CNPq.
M29.
THE AMPK-eNOS PATHWAY IS INVOLVED IN THE NEUROPROTECTIVE ROLE
OF SILDENAFIL (VIAGRA®) IN CUPRIZONE-DEMYELINATION MODEL
4- OLIVEIRA, W.H., 4- NUNES, A.K.S.; 3- RAPÔSO, C.; 1- ARAÚJO, S.M.R.; 2- OLIVEIRA,
A.G.V.; 4- ROCHA, S.W.; 1- BARBOSA, K.P.S.; 4- LUNA, R.L.A.; 3- HÖFLING, M.A.; 1, 2PEIXOTO, C.
1Laboratório de Ultraestrutura - Centro de Pesquisas Aggeu Magalhães - FIOCRUZ;
2Departamento de Microscopia - Centro de Tecnologias Estratégicas do Nordeste
(CETENE ) – MCT; 3Departamento de Histologia e Embriologia, Instituto de Biologia,
Universidade Estadual de Campinas (UNICAMP); 4 Centro de Ciências BiológicasUniversidade Federal de Pernambuco
It was recently demonstrated that sildenafil (Viagra®) has neuroprotective role,
inhibiting inflammation and demyelination in the cerebellum, in cuprizonedemyelination model. However, the mechanisms of sildenafil neuroprotection are
unclear. AMPK is a regulatory protein of lipid and glucose metabolism and it activation
modulates inflammatory processes, possibly through eNOS activation. The present work
investigated if sildenafil acts through AMPK-eNOS pathway, modulating
neuroinflammation. Five male mice (C57BL/6), 6 weeks-old, were used per group. The
groups received for four weeks: 0.2% Cuprizone (CPZ) mixed into the chow; CPZ into the
chow plus sildenafil (Viagra®, Pfizer, 25 mg/kg) in the drinking water, starting
concomitantly (sild-T0) or fifteen days (sild-T15) after initiation of CPZ; Controls received
pure chow/water. Cerebella were processed for western blotting, immunofluorescence,
transmission electron microscopy and Luxol Fast Blue staining (LFB); serum was used for
nitrite dosage by ELISA. Results showed demyelination after CPZ treatment, with
decreased MBP (myelin basic protein) expression, light labeling for LFB and damaged
myelin sheath ultrastructure, comparing to control group. The inactive-AMPK
(unphosphorylated) expression increased and eNOS levels decreased after CPZ
administration. Sildenafil treatment (T0 and T15) showed significant increase of MBP
expression and more intense LFB staining, comparing to CPZ group. Sildenafil-T0, but
not -T15, improved ultrastructural aspect of myelin sheath, significantly increased the
eNOS expression and decreased inactive-AMPK levels. These data suggest that sildenafil
activates the AMPK-eNOS signaling pathway. The sildenafil treatment beginning
concomitantly to CPZ was more effective then beginning 15 days after CPZ. This work
clarifies the mechanism of sildenafil neuroprotection in a demyelinating condition.
INBEB, FACEPE, CNPq
Página 38
Doutorandos
D1.
STRUCTURE AND DYNAMICS OF ALLERGEN GAD M 1 FREE AND IN COMPLEX
WITH SINGLE CHAIN VARIABLE FRAGMENT BY NMR SPECTROSCOPY
1- MORAES, A.H.; 2- ACKERBAUER, D.; 2- KOSTADINOVA, M.; 2- BUBLIN, M.; 3FERREIRA, F.; 1- ALMEIDA, F.C.L.; 2- BREITENEDER, H.; 1- VALENTE, A.P.
1- Centro Nacional de Ressonância Magnética, IBQM-UFRJ,RJ, Brazil; 2- Medical
University of Vienna, Vienna, Austria; 3- University of Salzburg, Salzburg, Austria.
Allergenic proteins are able to stimulate an inappropriate IgE production in atopic
individuals, which results in manifestations of clinical symptoms such as asthma, rhinitis
and atopic dermatitis. Gad m 1 is the major allergen from Atlantic cod and belongs to bparvalbumin protein family, which are the most important fish allergens and their high
cross-reactivity is the cause of the observed polysensitization in allergic patients.
Despite extensive efforts, the complete elucidation of b-parvalbumin-IgE complexes has
not been achieved yet. In this work, we solved the solution structure of Gad m 1 using
NMR spectroscopy. Concomitantly, scFv interaction studies were performed, using
chemical shift perturbation assays. We compared our results with other sites mapped in
homologous parvalbumins: Cyp c 1, Sco j 1 and Gad c 1. Residues around positions 30
to 40 matched epitopes mapped for the three allergens. Residues around position 50 to
60 were only observed for Gad m 1 and Gad c 1, while residue around 80 was mapped
on Cyp c 1. On the other hand, the N-terminal was only mapped in Gad m 1 and our
results did not showed the participation of residues around position 90. The dynamic
properties of Gad m 1 in the free state and in the complex with scFv were obtained from
relaxation experiments. Residues 31-33 and 75-78 exhibit increased R2/R1 ratios. These
residues are located in two regions mapped by CSP. The big ratios observed may be due
to chemical exchange between bound and free forms. This work will present for the first
time the solution structure of a b-parvalbumin as well as the characterization of Gad m
1-scFv interaction as a first and crucial step in the development of hypoallergenic
vaccine and to an understanding of the molecular interactions between allergens and
IgE’s.
FAPERJ, CNPq, CAPES and INBEB
D2.
EFFECT
OF
BONE
MARROW
MONONUCLEAR
CELLS
IN
NEURODEGENERATION, NEURONAL SURVIVAL AND REACTIVE MICROGLIOSIS IN
HIPPOCAMPAL CA1 LAYER AFTER GLOBAL CEREBRAL ISCHEMIA
1- RAMOS, A.B.; 1- CHAN, J.M.; 2- CINTRA, W.M.; 1- MENDEZ-OTERO, R.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Instituto de Ciências Biomédicas , Universidade Federal do Rio de Janeiro
"Introduction: The pyramidal neurons of the hippocampal CA1 layer are essential for
cognitive functions and selectively destroyed after global cerebral ischemia. Bone
marrow mononuclear cell (BMMC) therapy has shown positive results in preclinical
models of focal cerebral ischemia, but has not been studied in transient global ischemia.
Aim: The aim of our study was to investigate whether BMMC treatment could reduce
the neurodegeneration and the inflammation observed in CA1 layer of ischemic
animals. Methods: Transient forebrain ischemia was performed by four vessels
occlusion method. To establish the time course of CA1 cell death, the rats were
transcardially perfused 3, 7, 14, 21 and 28 days after ischemia (DAI). To analyze the
effect of BMMC therapy in neurodegeneration, neuronal survival and reactive
microgliosis in CA1 layer, ischemic animals received 3x107 BMMC 3 DAI, in the left
carotid, and were sacrificed different days after ischemia. For quantification of neuronal
degeneration, survival and reactive microgliosis, we counted Fluoro-Jade C, NeuN and
ED-1 positive cells in CA1 layer, respectively. Results: We observed a greater number of
FJC positive cells in CA1 layer of ischemic animals 7 DAI when compared with the others
days. The time course of neuronal survival shows a reduction of pyramidal neurons 7
DAI and in the days after. In the analysis of the effect of BMMC in neurodegeneration
and neuronal survival, we observed a significant reduction of FJC cells and increase of
NeuN cells in CA1 of animals injected with BMMC when compared to the saline group.
Furthermore, we observed a lower number of ED-1 positive cells in ischemic animals
treated with BMMC compared with ischemic animals that received only saline.
Conclusion: We suggest that the BMMC therapy in transient global ischemia has a
neuroprotective effect in CA1 layer that was followed by reduction of microgliosis in this
region."
CNPq; FAPERJ; CAPES;
D3.
STRUCTURAL DYNAMICS AFFECT THE ALLERGENIC POTENTIAL OF BET V1
1- A.L. BATISTA, 1- C. ASAM, 1- A. MORAES, 1- F.C.L. ALMEIDA, 2- F. FERREIRA, 2- M.
WALLNER AND 1- A.P. VALENTE
1- CNRMN, Dep de Bioquímica Médica, IBqM-UFRJ, Brasil; 2- Christian Doppler
Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, Salzburg, Austria
Bet v 1 is the major allergen of birch pollen with molecular mass of 17.5 kDa. It is
estimated that 100 million people are sensitized to Bet v 1, in Northern American and
Western Europe. Bet v 1 secondary structure arrangement is β-α2-β6-α where seven
anti-parallel β-sheet surround a long C-terminal α-helix. Also, a Y-shaped hydrophobic
pocket is present inside the protein. Its biological function is associated with the
transport of hydrophobic molecules such as brassinosteroid (plant hormone). In this
work we used Na-deoxycholate (Doc) that is structurally similar to plant steroids, in
order to analyze the influence of ligand binding on dynamics, conformation and IgEbinding. Recombinant 15N isotope labeled Bet v 1 was produced in E. coli and purified.
We used NMR spectroscopy to probe dynamics aspects using R1, R2 and 1H - 15N-NOE
for free and bound state. To further understand the free state dynamics we performed
15N CPMG relaxation dispersion experiments at 800 and 600 MHz. Moreover, we
performed experiments to map IgE epitopes in the free and Doc-bound state of Bet v 1
using purified human IgE antibodies from birch pollen allergic patients. Bet v 1 is a
dynamic protein with several residues in conformational exchange. The epitopes for
three patients were slightly different but several residues are in common. Doc
interaction changed slightly the protein conformation and profoundly the dynamics.
These changes did not affected the IgE epitope but there was an increase in antibody
afinity. Understanding the structural dynamics of Bet v 1 and its interaction with IgE
with or without ligands may help to develop hypoallergic derivatives for
immunotherapy and as well as understand the correlation between dynamics of
allergens and the forces that drive the allergenicity.
INBEB, FAPERJ, CNPq, CAPES.
D4.
EFFECTS OF LPSF/GQ-02 ON NONALCOHOLIC FATTY LIVER DISEASE
1– SILVA, A.K.S., 1– SPINOLA, M.G.D., 1- GOMES, F.O.S., 1- OLIVEIRA, A.C., 1OLIVEIRA,W. B., 1– SANTOS, L.A.M., 1- DONATO, M.A.M., 1,2- PEIXOTO, C.A.
Página 39
1 - Centro de Pesquisas Aggeu Magalhães- CPqAM/FIOCRUZ, Brasil. 2 - Centro de
Tecnologias Estratégicas do Nordeste- CETENE/MCT, Brasil.
Nonalcoholic fatty liver disease (NAFLD) defines a wide spectrum of liver diseases that
extend from simple steatosis to nonalcoholic steatohepatitis. Although the
pathogenesis of NAFLD remains undefined, it is recognized that insulin resistance is
present in almost all patients who develop this disease. The Thiazolidinediones act as an
insulin sensitizer and have been used in the treatment of patients with type 2 diabetes
and other insulin-resistant conditions, including NAFLD. Hence, therapy of NAFLD with
insulin-sensitizing drugs should ideally improve the key hepatic histological changes, but
should also reduce cardiometabolic and cancer risk. The aim of our study was to
evaluate the therapeutic effects of LPSF/GQ-02 on the liver the LDLR-/- mice after a diet
rich in fat. 40 male mice were divided into four groups: 1 - received a standard diet of
laboratory, 2- fed with high-fat diet (HFD), 3–HFD+pioglitazone, 4–HFD+LPSF/GQ-02.
The experiments were conducted for 10 weeks and in the last two weeks the drugs
were administered daily by gavage. After experimental protocols, the liver was
processed for optical microscopy, Oil Red O and immunohistochemistry for interleukin-6
(IL-6). The control group showed well-preserved tissue with low lipid content and a low
reactivity for IL-6. The HFD group presented steatosis, inflammatory infiltrates and a
high reactivity for IL-6. Treatment with pioglitazone was not effective in reversing the
changes caused by high-fat diet, showing disorganization in liver tissue, with
accumulation of lipid and a strong reactivity for IL-6. However, treatment with LPSF/GQ02 improved organization of liver tissue with a significant decrease of lipid inclusions
and cytokine IL-6 levels. This study showed that pioglitazone, a drug used to treat type 2
diabetes mellitus was unable to reverse the condition caused by the diet rich in fat.
However LPSF/GQ-02, showed to be effective in reducing the accumulation of lipid and
also reduced significantly IL-6 expression in liver of LDLR-/- mice.
INBEB, CNPq, FACEPE
D5.
SILDENAFIL PREVENT MICRO AND ASTROGLIOSIS POSSIBLY VIA NFKB IN
MODEL OF DEMYELINATION
1,2- NUNES,AKS.;1,3- RAPÔSO C.;1,2- LUNA RLA.; 2- ARAÚJO SMR.;1- OLIVEIRA WH.; 2OLIVEIRA AGV.;1- BARBOSA KP.; 1- ROCHA SWS.;3- Cruz-Hofling MA.;1,2- PEIXOTO C.
1- Laboratório de Ultraestrutura - Instituto de Pesquisas Aggeu Magalhães - FIOCRUZ; 2Departamento de Microscopia - Centro de Tecnologias do Nordeste (CETENE ) – MCT; 3Departamento de Histologia e Embriologia,Universidade Estadual de Campinas UNICAMP
Sildenafil (Viagra ®) induces cGMP accumulation by PDE5 inhibition. Recent studies have
demonstrated that sildenafil inhibits demyelination process, decreases micro- and
astrogliosis and proinflammatory cytokines expression in cuprizone EM-model. The NFКβ plays an important role in the regulation of expression of inflammatory mediators.
This study investigated whether treatment of chronic and late Sildenafil inhibit the
activation of microglia and astrocytes via NFkB pathway in a model of demyelination.
Five C57BL/6 mice, 6-weeks-old, were used/group. The groups received: 1) Cuprizone
(CPZ) 0,2% mixed into a chow/4 weeks, 2) CPZ in chow while Sildenafil (Viagra®)
25mg/kg in the drinking water (T0) , or 3) CPZ in chow while Sildenafil (Viagra®)
25mg/kg in the drinking water after 15 days administration the CPZ (T15), 4) Controls
received pure chow and water. The cerrebellums were processed for western blotting
(WB), immunohistochemistry (IH) and immunofluorescence (IF) in frozen sections.
Results showed treatment with CPZ increased GFAP levels and induced morphological
changes indicating activation of astrocytes compared to the control group. Sildenafil
(T0) decreased levels of expression of GFAP compared with the group CPZ. However
treatment with Sildenafil (T15) was not able to significantly reduce levels of GFAP.
Double staining with Iba-1 and NFkB in the control group showed cytoplasmic labeling in
the resting state indicating microglia cells and inactivation of NFkB. CPZ treatment
showed labeling of NFkB nearest the core of microglial cells indicating activation.
Corroborant these results, WB data showed that treatment CPZ inhibit NFkB inhibitory
protein (IKβα) and increased expression indicating NFkB translocation from the
cytoplasm to the nucleus and activate transcription of genes for pro-inflammatory.
Sildenafil (T0) was effective in decreasing expression and increased NFkB levels IKβα.
Consequently the marking to Iba-1 was decreased, and prolongations with thinner and
branched. However late treatment with Sildenafil was not effective in reducing the
marking and Iba-1 expression levels of NFkB. The increase in cGMP levels by inhibiting
PDE5, probably acts by inhibiting NFkB via gene transcription and pro-inflammatory
activation of microglial cells and prevent astrocytes in a model of demyelination.
Keywords: Microglia, inflammation,sildenafill
CNPq, CAPES, FAPESP, FACEPE, INBEB
D6.
EFFECT OF BONE MARROW MONONUCLEAR CELLS THERAPY IN A MODEL
OF SPINAL CORD IN HEMITRANSECTION: CORRELATION MORPHOLOGICAL AND
FUNCTIONAL
1- BOMFIM, A. M. D.; 1- RAMOS, A. B.; 2- CASTRO, N. G. D.; 1- MENDEZ-OTERO, R.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro
"Aim: The aim of our study was to evaluate the potential of bone marrow mononuclear
cells (BMMC) therapy in a rat model of unilateral T10 spinal cord injury (SCI). Methods:
Wistar rats were divided into 3 groups: false-operated group, underwent laminectomy,
SCI + saline group and SCI+ BMMC group. The locomotor recovery was assessed by
Basso Beattie Bresnahan scale (BBB) from 3 days after surgery. In histochemical analysis,
immunostaining was performed using markers as ED-1 (CD68), GFAP and NF-200
marking macrophages and microglia, astrocytes and axons respectively. Results:
Functional analysis showed that the treated group (SCI + BMMC) showed better
locomotor recovery through the BBB scale compared with SCI + saline group. In the
histochemical analysis of bone marrow mononuclear cells were found 3, 7 and 14 days
after injury (DAI). The SCI + BMMC group showed more axons in all time windows
observed and less activated microglia and macrophages at 7 and 14 DAI, compared with
SCI + saline group. Morphological differences associated with reactive astrogliosis was
also observed in the treated group. Conclusion: Our data suggest that injection of bone
marrow mononuclear cells after spinal cord injury provided better locomotor recovery
associated with a higher presence of axons and reduction of reactive microglia in
different time windows."
CNPq
D7.
ENVOLVIMENTO DA IPLA2 NA ADERÊNCIA, INTERNALIZAÇÃO E
INFECTIVIDADE DE LEISHMANIA AMAZONENSIS EM MACRÓFAGOS PERITONEAIS
Fernandes ACS, Soares DC, Saraiva EM, Souto-Padrón T*
1Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, Brasil *[email protected]
"O processo de interação parasito hospedeiro depende do reconhecimento mútuo de
moléculas e receptores. A exposição de moléculas na superfície de ambas as células ou
sua secreção para o meio extracelular dependem das vias endo / exocíticas, onde a
fusão entre seus diferentes compartimentos é mediada por múltiplos fatores, entre eles
a ação das fosfolipases. Nosso grupo vem investigando a participação da fosfolipase A2
Página 40
independente de cálcio (iPLA2) na fusão de compartimrntos das vias endo / exociticas
de Leishmania amazonensis e no processo de interação com macrófagos peritoneais.
Promastigotas de L. amazonensis foram mantidos em meio de Schneider durante 144h
e tratados durante 1 h com 2,5 M de bromenolactone (BEL), um inibidor irreversível
da iPLA2. Após o tratamento, parasitas controle e tratados foram incubados com
macrófagos. Observamos os processos de adesão, internalização e sobrevivência do
parasito por até 48 horas no interior da célula hospedeira. BEL reduziu
aproximadamente em 80% a adesão de promastigotas à superfície de macrófagos. Ao
contrário do controle, parasitos tratados com BEL aderiram-se aos macrófagos
exclusivamente pela região do flagelo. BEL também reduziu a percentagem de
macrófagos infectados e o número de parasitas no interior do vacúolo parasitóforo (PV)
em 80 e 90%, respectivamente, em um período de até 48 h após a infecção. O PV em
macrófagos infectados com parasitos previamente tratados com BEL apresentaram
diferenças significativas em relação a parasitos controle. Observamos um atraso na
diferenciação de promastigota em formas amastigotas, redução na acidez do PV e
alteração no processo de fusão com de lisossomos ao PV. Nossos resultados indicam
que a inibição da iPLA2 em promastigotas de L. amazonensis reduz a adesão e a
infecção na célula hospedeira com inibição da diferenciação do parasito, inibição na
multiplicação de formas amastigota e alteração na formação e morfologia do PV."
Apoiado pelo CNPq, CAPES, FAPERJ, Pronex, CONCEA (11.794/08).
D8.
NMR STRUCTURAL CHARACTERIZATION OF Q4D059 AND Q4DRX8,
CONSERVED HYPOTHETICAL PROTEINS FROM TRYPANOSSOMA CRUZI
1-LOPEZ-CASTILLA A.; 1-DE MENEZES R.; 1-DOS SANTOS T.; 1-PIRES J.R
1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro
Q4D059 and Q4DRX8 (Uniprot ID) are hypothetical proteins of 84 and 104 amino acids
respectively from T.cruzi. A PSI-BLAST search using Q4D059 and Q4DRX8 sequences as
query only identified hypothetical proteins from trypanosomatids (E-value above the
threshold), so failed to provide insight into the protein function. Both proteins are
conserved in the kinetoplastid genomes and show low-sequence homology with
mammal proteins; therefore are considered potential targets for drug development
against trypanosomatids parasites. Structural studies of these proteins could give
information about their function and facilitate drug screening. In this work, the genes
codifying for Q4D059 and Q4DRX8 were cloned into a pGEX expression vector and 13Cand 15N- NMR protein samples were overexpressed in Escherichia coli as a GST-fusion
protein. Protein purification was performed by GST-affinity chromatography. GST was
removed by thrombine protease digestion and as last step a size-exclusion
chromatography was carried out. 1D 1H and 15N-1H HSQC spectra of Q4D059 showed a
well-behaved and structured protein in solution. The NMR spectra required for protein
structure determination were recorded and the assignment process of all atoms in
Q4D059 is in progress.
CNPq
D9.
DIETHYLCARBAMAZINE REDUCED THE NF- B ACTIVATION IN MODEL OF
CHRONIC ETHANOL CONSUMPTION
1- SILVA, B.S.; 1- RODRIGUES, G.B.; 1- ROCHA, S.W.S.; 1- GOMES, F.O.S.; 1- RIBEIRO,
E.L.;1- SILVA, A.K.S.; 1- SPINOLA, M.G.D.; 1- OLIVEIRA, A.C.;1- OLIVEIRA, W.B.; 1,2PEIXOTO, C.A.
1- Centro de Ciências Biológicas, Universidade Federal de Pernambuco; 2- Laboratório
de Microscopia do Centro de Tecnologias Estratégicas do Nordeste (INT-CETENE Pernambuco)
Alcoholic liver disease (ALD) is considered as a complex and multifaceted pathological
process, involving oxidative stress, inflammation and excessive fatty acid synthesis.
Progression of disease involves various proinflammatory molecules such as interleukins,
cytokines, adhesion molecules and nuclear factor-kB (NF-kB). NF-kB is a transcription
factor that is implicated in inflammation and immune response and is activated by
oxidants and cytokines that play important roles in the inflammation and development
of ALD. Therapy for the treatment of ALD is limited and ineffective. Diethylcarbamazine
(DEC) has anti-inflammatory properties as a result of its interference with the
arachidonic acid metabolism and few studies focus their role in the pathophysiology of
inflammation. The objective of this work was to investigate the role of DEC on NF-kB
pathways in hepatic inflammation induced by alcoholism. Forty male C57BL were
separated in four groups (n=10): control group (C) that received just distilled water,
DEC-treated group (D50) that received 50 mg/kg DEC for twelve days by gavage,
alcoholic group (EtOH) that received ethanol and alcoholic plus 50 mg/kg DEC group
(EtOH50). After 5 weeks the alcoholism induction fragments of liver were processed for
immunohistochemistry and western blot for an antibody against the activated p65
subunit of NF-κB and their regulatory enzyme, IkBa. High level of NF-κB expression was
seen in hepatocytes exposed to alcohol, which was significantly decreased in the
hepatocytes of the EtOH50 group. Activation of NF-kB occurs secondarily to the
degradation of IkBa. Ours findings support a role for IkBa in alcoholic liver injury
because the activation of NF-kB was accompanied by a loss of IkBa in alcoholic group.
The inhibition of NF-kB activation in EtOH50 was accompanied by preservation of IkBa
expression. These results suggest that agents that prevent the activation of the
transcription factor NF-kB, will thereby prevent ALD.
INBEB, FACEPE, CNPq, UFPE
D10.
CROSS-TALK BETWEEN PRION PROTEIN AND NUCLEIC ACIDS: PROTEIN
MISFOLDING LEADING TO CYTOTOXICITY
1- MACEDO, B.; 2- MILLEN, T. A.; 1- FERREIRA, N. C.; 1- GOMES, M. P. B.; 2- BRAGA, C. A.
C. A.; 2- FERREIRA, P. S.; 2- SILVA, J. L.; 1- CORDEIRO, Y.
1- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, ²Instituto de
Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro; 2- Instituto
de Bioquímica Médica, Universidade Federal do Rio de Janeiro
"Prion proteins (PrPs) are involved in lethal neurodegenerative diseases, that affect
humans and other animals. So far, many issues remain unclear about PrP
physiopathological role. The misfolding of its normal cellular form (PrPC) into an
abnormal form (PrPSc) is thought to be the central event of these pathogenesis. Some
studies have suggested nuclear localization for PrPC and functions derived from its
interaction with nuclear components. Having a common local to cross-talk, nucleic acids
(NAs) are really intriguing PrP molecular partners, and have been proposed to work as
catalysts in the protein misfolding process. To clarify the biological relevance of PrP-NA
interactions, we worked with several oligonucleotides (RNA and DNA) and different cell
lineages (neuroblastoma - N2a - and kidney cells – HK2), using diverse cellular assays to
evaluate cytotoxicity and cell viability. We have found that interactions of PrP with NAs
can lead to different aggregates and neurotoxic species, depending on the nucleic acid
molecule (1). Interestingly, the number of N2a cells was reduced and their morphology
also changed in the presence of the cytotoxic PrP-NA complexes, which indicate that
they are indeed affecting cell signaling. We are currently investigating the mechanism
through which such complexes mediate cell death. Understanding the cellular and
structural effects observed for PrP and nucleic acid cross-talks may enlighthen the still
obscure prion physiopathology.
Página 41
Reference: (1) Macedo, B., Millen, T. A., Braga, C. A. C. A., Gomes, M. P. B., Ferreira, P.
S., Kraineva, J., Winter, R., Silva, J. L. and Cordeiro, Y. (2012) Biochemistry, 51:
5402−5413"
FAPERJ, CNPq, CAPES, INBEB
D11.
MOLECULAR EVALUATION OF THE RENIN ANGIOTENSIN SYSTEM
COMPONENTS BY RT-QPCR AFTER BREAST CANCER TREATMENT
1,2SALATA C; 1,3FERREIRA-MACHADO SC; 4MENCALHA AL; 1CAMPOS VMA;
1,5ANDRADE CBV; 2MANDARIM-DE-LACERDA CA; 1DEALMEIDA CE.
1. Laboratório de Ciências Radiológicas/UERJ/RJ, Brasil; 2. Laboratório de Morfometria e
Morfologia Cardiovascular/UERJ/RJ, Brasil; 3. Departamento de Biologia Geral/UFF/RJ,
Brasil; 4. Departamento de Biologia e Biometria/UERJ/RJ, Brasil; 5. Laboratório de
Ultraestrutura e Biologia Tecidual/UERJ/RJ, Brasil
"Introduction: New treatment protocols and technologies for Breast Cancer (BC) have
allowed that more women with early stage BC survive their disease. This increases their
chances of developing late cardiovascular effects, as a result of BC treatments,
chemotherapy and radiotherapy. It is known the relevance of the local cardiac Renin
Angiotensin System (RAS) in the pathofisiology of cardiac damage. The angiotensin II
(Ang II) in the heart can induce hypertrophy, inflammation and fibrosis, by activation of
AT1 receptors. The present study aims to evaluate the mRNA expression of different
components of RAS associate it with cardiac damage induced by breast cancer
treatment. Methods: Female Wistar rats, were divided into groups (5 animals per
group): control, chemotherapy (cyclophosphamide + docetaxel) + radiotherapy (TC+IR)
and radiotherapy only (IR). The animals were euthanized 5 months after the treatment.
RT-qPCR of angiotensinogen, renin, ECA, AT1R, TGF-ß1 and procollagen type I of left
ventricle tissue was performed. Values are expressed as (mean±SEM). This study was
submitted and approved by the UERJ ethical committee (n° CEUA/010/2012).
Results/Discussion: Results showed no mRNA expression of angiotensinogen in heart
tissue, as expected, because the angiotensinogen used in the local heart RAS is
produced in the liver. Renin was only expressed in treated groups, and not in control.
AT1R expression was different (p<0.001) between IR+TC and control, and between IR
and control, with p<0.05. Procollagen type I expression was different (p<0.001) between
TC + IR group and control. Results of TGF-ß1 showed significant difference (p<0.05)
between TC + IR group and control group. Parameters as TGF-ß1 and procollagen type I
were up-regulated, probably because of AT1R activation. These preliminary results
indicate that the association of chemotherapy and radiotherapy cause more injuries in
the heart tissue than radiotherapy alone. We will confirm these results using another
techniques such stereology."
CAPES, FAPERJ
D12.
THE “INS” AND “OUTS” OF VIRAL INFECTION: DISSECTING THE DYNAMICS
OF ENTRY AND EXIT OF AN EMERGING VIRUS
CARVALHO, C.A.M.; VIGNOLI, K.; SILVA, J.L.; GOMES, A.M.O.
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 receptor-mediated
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. On the opposite way,
alphavirus exit from host cells is supposed to occur by budding from the plasma
membrane, although egress by exocytosis may also be observed depending on cell
lineage. The aim of this work is to analyze the behavior of MAYV particles and their
structural components during the entry into and exit from host cells. To study viral
entry, MAYV was labeled with DiD and the fluorescent signals were tracked in Vero cells
by laser-scanning confocal fluorescence microscopy (LSCFM) in real time. Our results
show that MAYV entry into cells occurs by a fast endocytic mechanism: following DiD
fluorescence dequenching at the single particle level, fusion between the viral envelope
and the endosomal membrane was shown to occur at around 3 min post-binding. To
study viral exit, Vero cells previously transfected with expression vectors for MAYV
structural proteins fused to fluorescent proteins were infected and the interaction
between these viral components was evaluated by LSCFM over viral infection. Our
results suggest that plasma membrane is the viral budding site in these cells and that it
seems to occur a rearrangement of the intracellular distribution of the fluorescent viral
structural proteins during infection. This work provides unique dynamic insights into the
entry and exit of MAYV particles in living cells. Understanding the dynamics of virus
infection may provide important insights to the development of antiviral strategies.
CAPES, CNPq, FAPERJ, FINEP, INBEB, PRONEX
D13.
HYDROSTATIC PRESSURE FULL INACTIVATED INFLUENZA VIRUS PRESERVES
BINDING AND MEMBRANE FUSION WITH GENERATION OF IMMUNE RESPONSE AND
PROTECTION OF MICES AGAINST INFECTION
1-DUMARD, C.H.; 1-SOUZA-SANTOS, P.; 1-BARROSO, S.P.C.; 1- OLIVEIRA, G.A.P.; 1CARVALHO, C.A.M.; 2- COUCEIRO, J.N.S.S.; 2-FERREIRA, D.F.; 1- SILVA, J.L.
1-Laboratório de Termodinâmica de Proteínas e Estruturas Virais Gregorio Weber,
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2- Instituto de
Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro
Whole inactivated vaccines (WIV) present higher immunogenicity then split and subunit
vaccines. Recent studies have demonstrated that WIV with preserved fusogenic activity
are more protective then non-fusogenic WIV. In this work, we show the inactivation of
human influenza virus X-31 by hydrostatic pressure (HP) and analyze the effects on the
structure by spectroscopic measurements, light scattering, and electron microscopy. We
also investigated the HP effects in the glycoproteins activity. Fusogenic activity was
evaluated by confocal microscopy. Immune response and disease were tested by Elisa
and weight loss respectively. Electron microscopy data show pore formation on viral
envelope but general morphology was preserved, and small variations were seen in the
particle structure. The activity of hemagglutinin in the process of binding and fusion was
affected in time dependent-manner but neuraminidase activity was not affected.
Infectious activities were abolished after three hours of pressurization as observed by
RT-PCR. Vaccinated mices presented high levels of IgG2a, IgG1 and IgA after
immunization and were protected from weight loss after infection. Our results show
fully viral inactivation with preservation of viral structure and maintenance of fusogenic
activity, with strong immune response and protection of mices against infection. Our
data strongly support the idea of applying the hydrostatic pressure to develop new
vaccines for influenza A and other viruses as well.
D14.
ENDOSSIMBIOSE EM TRIPANOSSOMATÍDEOS, A ASSOCIAÇÃO DA BACTÉRIA
COM OUTRAS ESTRUTURAS DO PROTOZOÁRIO HOSPEDEIRO
CATTA-PRETA, C.M.C. 1,2; MACHADO, A.C.L. 1,2; DE SOUZA, W. 1,2,3; MOTTA, M.C.M.
1,2
1 Laboratório de Ultraestrutura Celular Hertha Meyer, IBCCF, UFRJ 2 Instituto Nacional
de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, UFRJ 3 Instituto Nacional de
Metrologia, Qualidade e Tecnologia (INMETRO)
Página 42
O estudo da co-evolução entre dois seres primitivos fornece informações relevantes
sobre a origem de organelas na célula eucariota. Alguns protozoários monoxênicos da
família Trypanosomatidae mantém uma relação mutualística, e portanto obrigatória,
com uma bactéria simbiótica. A divisão do simbionte é coordenada com outras
estruturas do hospedeiro, de modo que cada célula filha herda uma única bactéria
simbiótica. Outro aspecto interessante desta relação são as intensas trocas metabólicas
que ocorrem entre os dois seres associados, como a cooperação na produção de
aminoácidos, vitaminas e grupamentos heme. O objetivo deste trabalho é verificar do
ponto de vista ultraestrutural, usando a tomografia eletrônica, a associação do
simbionte com organelas do hospedeiro, como o núcleo, o retículo endoplasmático e
organelas energéticas. Utilizando técnicas de preservação como congelamento em alta
pressão e crio-substituição é possível observar com precisão se as estruturas que
interagem somente se tocam ou se fundem. Nossos resultados mostram que o
envoltório do simbionte encontra-se justaposto à membrana nuclear ao longo do ciclo
celular do hospedeiro, o que pode estar relacionado com a simetria do eixo de divisão
do protozoário e com o controle do número de bactérias por célula. Também foram
flagrados toques entre as membranas do simbionte e dos glicossomos, organela
envolvida na produção de ATP, reforçando a ideia que a bactéria simbiótica se
beneficia da produção de energia do hospedeiro. Observamos também que o retículo
endoplasmático apresenta íntima associação com o simbionte, e que em alguns
momentos, estas estruturas parecem estar fundidas. Esta associação pode estar
relacionada com a importação pelo simbionte de lipídeos e glicoproteinas produzidos
pelo hospedeiro, como sugerem os nossos dados genômicos e bioquímicos.
INBEB, CNPq e FAPERJ
D15.
IMAGING CRYPTOCOCCUS NEOFORMANS-MACROPHAGE INTERACTION
1-GUERRA, C.R.; 2-SEABRA, S.H.; 1-ROZENTAL, S.
1-Laboratório de Biologia Celular de Fungos, Instituto de Biofísica Carlos Chagas Filho,
Universidade Federal do Rio de Janeiro; 2-Laboratório de Tecnologia em Bioquímica e
Microscopia, Centro Universitário Estadual da Zona Oeste.
"Cryptococcus neoformans is an encapsulated yeast that causes disease in
immunocompromised patients. Cryptococcosis is considered an AIDS-defining condition
and it is the third most frequent neurological complication in AIDS patients.
Macrophages represent first line of defense of cryptococcosis. During the last decade,
studies have focused on how this yeast can survive and proliferate within macrophages.
However, studies dealing with exact mechanisms used by the yeast to enter host cells
are still incipient. Therefore, we focus this study using cytoskeleton inhibitors to observe
C. neoformans interaction with peritoneal macrophages by confocal microscopy and
scanning electron microscopy. Four different C. neoformans strains differing in
serotypes and capsule size (ATCC28957, H99 and acapsular mutants CAP59 and CAP67)
were allowed to interact with peritoneal macrophages, previously adhered on glass
cover slips in a ratio of 50 yeast per macrophage, for 4 hours. Interactions occurred in
absence or presence of cytoskeleton inhibitors: Cytochalasin D or Nocodazole and then
cells were processed for flow cytometry (FACS), scanning electron microscopy (SEM) or
confocal microscopy (actin filaments were labeled with AlexaFluor 488 phalloidin and αtubulin was labeled with anti-α-tubulin AlexaFluor 546 conjugate). FACS experiments
revealed that actin and tubulin inhibitors diminished yeast internalization process by
macrophages. Confocal microscopy revealed the presence of actin surrounding
endosomes vacuoles containing yeasts, whereas α-tubulin apparently was less recruited
after yeast internalization. Macrophage membranes were extracted after interaction
and visualized by SEM demonstrating cytoskeleton attachment to yeast cells and its
important role upon host cell entry. Taken together, these results indicate a more
prominent role of actin recruitment than α-tubulin and helps us to better understand
how the initial process of C. neoformans-macrophage interaction may occur. Further
studies are ongoing."
CNPq, FAPERJ, Capes
D16.
ASSOCIAÇÃO ENTRE OSTEOPOROSE E QUIMIOTERAPIA PARA O
TRATAMENTO DO CÂNCER DE MAMA
ANDRADE, CBV1,2,3*; SALATA, C1,4; SILVA, CM1; FERREIRA-MACHADO, SC1,5; C. L.
MOTA7; A. PICKLER7; A. MANTUANO7; ALMEIDA, AP6; BRAZ D6; NOGUEIRA, LP7;
BARROSO, RCR7; DEALMEIDA, CE1
1. Laboratório de Ciências Radiológicas/UERJ/RJ 2. Universidade Severino
Sombra/USS/RJ 3. Ultraestrutura e Biologia Tecidual/UERJ/RJ 4. Laboratório de
Morfometria e Morfologia Cardiovascular/UERJ/RJ 5. Departamento de Biologia
Geral/UFF/ 6. Laboratório de Instrumentação Nuclear/COPPE/UFRJ 7. Instituto de
Fisica/UERJ
"Introdução: Uma das mais utilizadas estratégias de tratamento para o câncer de mama
(CM) é a quimioterapia. O regime TC (docetaxel e ciclofosfamida). TC é mais recente,
portanto existem poucos estudos relacionados a ele em relação a efeitos tardios, como
a osteoporose precoce. Mulheres na pré-menopausa submetidas à quimioterapia para o
tratamento do CM apresentam significante perda óssea a partir do primeiro ano após o
início do tratamento. A mais provável causa desta perda deve-se ao fato destes
quimioterápicos levarem a menopausa precoce. A diminuição da densidade mineral
óssea pode levar ao aumento do risco de fraturas na pós-menopausa. Sabendo-se que
alguns quimioterápicos para o tratamento do CM, aumentam o risco de menopausa
precoce, e subsequente osteoporose, espera-se neste estudo, determinar se o recente
regime quimioterápico TC pode induzir aos mesmos danos. Metodologia: Ratas Wistar,
com 3 meses, foram divididas em 2 grupos, quimioterapia (TC) e controle. Os animais
foram eutanaziados 5 meses após o término do tratamento. Foi realizada a coleta de
sangue, dos fêmures e dos úteros para posterior análise. As técnicas utilizadas foram:
aferição da massa uterina; concentração de estradiol sorológico por radioimunoensaio;
distribuição espacial dos elementos cálcio e zinco na matriz óssea através da técnica de
Microfluorescência de raios X (µXRF) Resultados/Discussão: Os animais do grupo TC
demonstraram redução significativa (p<0.01) da massa do útero em relação ao controle,
sugerindo atrofia uterina gerada pela baixa de estrogênio (p<0.05), devido à ação dos
quimioterápicos. Foram obtidas as distribuições bidimensionais de Ca e Zn dos grupos
controle e TC, pois ambos são elementos importantes na composição da matriz óssea.
Embora a concentração de Ca não tenha variado entre os grupos, a concentração de Zn
no grupo TC é significativamente menor comparado ao controle (p<0.001). Futuramente
serão realizadas análises por imunohistoquímica do número de osteoclastos e
osteoblastos."
CAPES, FAPERJ
D17.
S-NITROSYLATION DECREASES CA2+ SENSITIVITY AND ACTOMYOSIN ATPASE
ACTIVITY OF CONTRACTILE PROTEINS IN CARDIAC MYOFIBRILS
1,2,3Figueiredo-Freitas C.,4,5Foster M.W., 6Nogueira L., 3Liang J.S., 2Yamashita
A.M.S.,7Dulce R., 4,5Thompson J.W., 7Hare J.M., 4,5Moseley A., 2Sorenson M.M.,
1,3Pinto J.R.
1- Department of Biomedical Sciences, Florida State University, College of Medicine,
Tallahassee, FL, USA; 2- Instituto de BioquímicaMédica, UFRJ, Brazil; 3- Department of
Molecular and Cellular Pharmacology, University of Miami, Miller School of Medicine,
Miami, FL; 4- Pulmonary, Allergy and Critical Care Medicine, Duke University Medical
Center, Durham, NC; 5- Institute for Genome Sciences and Policy, Duke University
Página 43
Medical Center, Durham, NC;6-Division of Physiology, Department of Medicine,
University of California, San Diego, CA, USA, 7- Interdisciplinary Stem Cell Institute,
University of Miami, Miami, FL, USA.
Increases in muscle nitric-oxide (NO) production can be buffered by reaction with
intracellular glutathione, forming S-nitrosoglutathione (GSNO). GSNO has been shown
to S-nitrosylate Cys thiols of cardiac contractile proteins in vivo and in vitro, but effects
on maximal force, thin-filament Ca2+ sensitivity and actomyosin ATPase activity are
unknown. Here, we analyzed the targets of S-nitrosylation in mouse cardiac contractile
proteins, and examined the effects of these modifications on function in myocytes and
skinned cardiac myofibrils. S-Nitrosylation and denitrosylation were detected using
resin-assisted capture (SNO-RAC) and targets for S-nitrosylation were identified by
quantitative LC-MS/MS. Isolated cardiomyocytes treated with S-nitrosocysteine (CysNO,
500µM, 10min) showed an increase in total protein-SNO, followed by progressive
denitrosylation (30-60min with CysNO). At 10min, CysNO dose-dependently increased Snitrosylation of specific Cys thiols in myosin heavy chain, actin, TnC, TnI, myosin-binding
protein C and other muscle proteins. Myofibril thin-filament Ca2+ sensitivity decreased
(P<0.05) after in-vitro treatment with pharmacological GSNO concentrations (1, 10, 100
µM), but maximum force did not change (P>0.05). Loss of Ca2+ sensitivity was partially
reversed by the denitrosation agent, ascorbate. Relaxation kinetics of skinned fibers, as
measured by flash photolysis, were also significantly reduced by100µM GSNO (k1,15.33
to 11.68/s; k2 2.33 to 0.87/s; fit to double exponential).. Maximal myofibrillar ATPase
activity (pCa 5.0) was also dose-dependently inhibited (8, 15, 30%) by 50, 100 and
500µM GSNO, an effect that was reversed by ascorbate. The findings suggest that Snitrosylation of regulatory Cys thiol(s) can reversibly modulate cardiac muscle
contraction and may be able to protect the heart by reducing myosin ATPase demand
without affecting the maximal force of contraction.
CAPES, CNPq, FAPERJ
D18.
YELLOW FEVER VIRUS-INDUCED MITOCHONDRIAL DYSFUNCTION: CHANGES
IN MITOCHONDRIAL ENERGETIC METABOLISM AND APOPTOSIS INDUCTION
Sanches, D.1; Campos, S.P.C.1; Rocha, C.M. 1; Gaspar, L.P.2; Freire, M.S.2; Silva, J.L.1;
Gomes, A.M.O.1 & Oliveira, A.C.1
1Instituto de Bioquímica Médica – CCS – Universidade Federal do Rio de Janeiro, RJ,
Brasil.
Flaviviruses cause diseases like Dengue and Yellow fever. These viruses are transmitted
by mosquitoes mainly in South America, Central America and Asiatic southeast, where
they have a particular importance for public health. Virus-induced apoptosis is related
to a cytopathological consequence of an infection in vivo or in vitro. During apoptosis,
mitochondrial pathway has been described as a crucial step during viruses-induced
apoptosis. Once the mitochondrial pathway is activated, loss of mitochondrial
membrane potential (Δ m) occurs and caspases can be activated, culminating in
apoptotic process. Here, we investigate the role of mitochondrial cell death pathway
during Yellow Fever Virus (YFV) infection and its consequence to mitochondrial
energetic metabolism. We infected Vero cells with YFV using a MOI=1. We analyzed the
cell viability using Live/Dead and LDH assay. Apoptosis was analyzed by
PhosphatidylSerine (PS) exposure and TUNEL, while the role of mitochondrial pathway
was followed by Δ m through fluorescence microscopy. The importance of
mitochondrial pathway was investigated by Bongkrekic acid, an adenine nucleotide
translocator (ANT) inhibitor. The mitochondrial energetic metabolism was studied by
oxygraphy. Apoptosis was observed after 72 hours post infection (h.p.i.) through TUNEL
and PS exposure. The dependence of caspases activation during the apoptosis process
was also observed, using z-Vad-fmk, a pancaspase inhibitor. We also observed loss of
Δ m 72 h.p.i. demonstrating that the apoptotic mitochondrial pathway is being
activated and apoptosis is dependent of ANT activity. Oxygraphy results show a slighter
increase of routine respiration, but a significant increase of oligomycin-sensitive oxygen
consumption at 48 h.p.i., that indicate an increase in oxygen consumption rate coupled
to ATP synthesis. Our results suggest that the mitochondrial pathway is activated,
contributing partially for the caspase-dependent cell death process induced by YFV. Our
data also suggest changes on mitochondrial energetic metabolism associated to virus
infection.
CNPq, CAPES, FAPERJ, INBEB, PRONEX
D19.
INTERACTION STUDY OF BOVINE SERUM ALBUMIN (BSA) WITH
GUANYLHYDRAZONES BY NUCLEAR MAGNETIC RESONANCE
FERREIRA NETO, D.C.; AZEREDO, S.O.F.; PETRONILHO, E. C. AND FIGUEROA VILLAR, J. D.
Grupo de Química Medicinal, Instituto Militar de Engenharia, Seção de Engenharia
Química – Praça General Tibúrcio, 80, 22290-270, Praia Vermelha, Rio de Janeiro, RJ.
The interaction between biomolecules and ligands has aroused interest in recent years
and new researches have been developed aiming to study how these interactions
occurs. Albumin is a important protein, and its main function is to transport and bind to
substances in the blood. Substances that interact strongly with albumin present
difficulty in their pharmacological function. However, molecules that do not interact
with the albumin are likely to be destroyed by other proteins present in blood. Thus, to
take appropriate action, the drug must provide a medium interaction with albumin. The
objective of this study was to examine the possibility of interaction of bovine serum
albumin (BSA) with guanylhydrazones synthesized by our research group, via Nuclear
Magnetic Resonance (NMR). We used the experimental measurements of T1 and T2
(relaxation time), differences transferring saturation (Saturation Transfer Difference,
STD), Nuclear Overhauser Effect (NOE), and Diffusion Ordered Spectroscopy (DOSY).
Preliminary results by NMR using the techniques described, showed average changes in
the values of T1 and T2 of some guanylhydrazones, which interact with albumin. These
changes in relaxation times, which are confirmed by diffusion coefficient variations
indicate the interaction ligand-protein. These preliminary results indicate that the
tested guanylhydrazones are potentially stable drugs for cancer and bacterial infections.
CAPES/Pró-defesa, INBEB e ao Exército Brasileiro.
D20.
DIETHYLCARBAMAZINE
ATTENUATES
THE
DEVELOPMENT
OF
MONOCROTALINE-INDUCED LUNG INJURY IN MICE
1-Ribeiro E. L.; 1-Fragoso I. T.; 1- Silva A. K. S.; 1- Donato M.A.M.; 1-Olibeira A. C.; 1Oliveira W. B. 1, 2- Peixoto C. A.
1- Universidade Federal de Pernambuco; 2- Centro de Tecnologia Estrategicas do
Nordeste
The alkaloid monocrotaline (MCT) is a toxin from plants, mainly employed to establish a
model of pulmonary dysfunction. Clinical reports have described favorable results with
the use of diethylcarbamazine (DEC) in bronchial asthma. This drug has an important
anti-inflammatory role since it interferes with arachidonic acid metabolism. In the
present study, we investigated the efficacy of oral DEC treatment in mice model of
injury pulmonary. Forty C57/BL6 male mice, weighting 20-25g, were used in all
experiments. Four groups were studied: control; MCT7; MCT14; MCT14/DEC14
(50mg/Kg per day of DEC from 1 to day 14). MCT solution intraperitoneal injection
(600mg/kg once per week). The animals were anesthetized and collection of
bronchoalveolar lavage fluid. Lung tissues were collected and processed for light
microscopy and immunohistochemistry (COX-2 and F4/80). The histological analysis
revealed that the animals of group MCT7 and MCT14 exhibited discrete alveolar
Página 44
thickening due to increased cellularity, mild hemorrhage and congestion, inflammatory
cells, pulmonary edema and emphysema. No group MCT14/DEC14 there was a decrease
of injury and the infiltration of PMNs. The lungs in the group control exhibited
preserved morphological characteristics. The lung tissue sections obtained from mice in
the group MCT7 and MCT14 revealed considerable COX-2 expression, whereas COX-2
expression was significantly reduced in lung sections obtained from mice treated with
DEC. Lung sections from the mice in the group control expressed baseline levels of COX2. Lung sections obtained from mice in the group MCT7 and MCT14 revealed positive
staining for F4/80 in alveolar macrophages, whereas DEC treatment significantly
attenuated F4/80 expression. Little staining for F4/80 was observed in the lung tissue
obtained from the group control. In conclusion, the MCT induced pulmonary
dysfunction was attenuated by DEC treatment, probably via inhibition of a number of
steps in arachidonic acid pathway, thereby preventing the production of cyclooxygenase
or lipoxygenase metabolites, important inflammatory intermediates.
INBEB, FACEPE, CNPq, CPqAM
D21.
CROSS-TALK BETWEEN FIBROBLASTS AND DENDRITIC CELLS UPREGULATES
IL-23 PRODUCTION DURING PORPHYROMONAS GINGIVALIS INFECTION IN GINGIPAINDEPENDENT MANNER
1-RAMOS-JUNIOR, E.S., 1-NASCIMENTO, C.R.,1-MORANDINI, A.C., 1-SCHARFSTEIN, J.
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
Using a murine model of mucosal P. gingivalis (P.g) infection, a bacterium that causes
periodontal disease, we have demonstrated that bradykinin, a proinflammatory peptide
released by gingipain, upregulates generation of INF-γ and IL-17-producing T cells in
submandibular LN. IL-23 produced by dendritic cells (DCs) is essential for Th17
expansion. Fibroblasts contribute to the gingival microenvironment, and it is known that
DC/fibroblast interaction is important to cytokine modulation. Here we investigated the
interplay between DCs/gingival fibroblasts (g-FB) in innate responses elicited by P.g.
Mouse BMDCs were co-cultured with g-FBs and stimulated either with P.g (strain W83)
or a triple gingipain knockout P.g (KRAB), in the presence of IFN-γ. We found that gFBs/DCs exposed to W83 upregulated IL-23 in the supernatant as compared to DC alone
or g- FBs/DCs exposed to KRAB. IL-10 was exclusively produced by W83-stimulated DCs
(alone), and the levels were decreased upon g-FB co-culturing. IL-23 production is
dependent on IL-6, TNF-α and IL-1β. IL-6 was produced by g-FB exposed to W83, but not
by KRAB. TNF-α and IL-1β, were not observed in g-FB. DCs exposed to W83 or KRAB
upregulated same levels of TNF-α and IL-1β, and the presence of g-FB decreased the
levels of both cytokines. Furthermore, these differences were not related to DC
activation, since CD40 and CD86 surface expression were upregulated independently of
co-culturing with g-FB or bacteria strains. g- FBs and DCs are involved in a cellular
“cross-talk” leading to the upregulation of IL-23. Ongoing studies should clarify whether
bradykinin receptors and/or alternative molecular targets may account for the
gingipain-dependent induction of the IL-23/Th17 pathway.
FAPERJ, CNPq and INCT-CNPq/INBEB
D22.
THE ROLE OF MICROGLIA ACTIVATION IN SYNAPSE LOSS AND SHORT-TERM
MEMORY DEFICITS IN OCULOLEPTOMENINGEAL AMYLOIDOSIS RELATED TO
TRANSTHYRETIN
1-ESTEFANIA P.C.AZEVEDO, 1-JOSÉ HENRIQUE LEDO, 2-GUSTHAVO BARBOSA, 2MORGANA SOBRINHO, 2-LUAN DINIZ, 2-ANNA C. C. FONSECA, 2-FLÁVIA GOMES, 2,3LUCIANA ROMÃO, 1-FERNANDO L. PALHANO, 2-FLÁVIA R. S. LIMA, 1-SÉRGIO T.
FERREIRA AND 1-DEBORA FOGUEL
1-Instituto de Bioquímica Médica, 2-Instituto de Ciências Biomédicas, Universidade
Federal do Rio de Janeiro , 3-Pólo de Macaé, Universidade Federal do Rio de Janeiro,
Macaé
Oculoleptomeningeal amyloidosis (OA) is a fatal and untreatable hereditary disease
characterized by the accumulation of transthyretin (TTR) amyloid within the central
nervous system. The mechanistic basis of OA pathogenesis is still unknown, thus, we
aimed to understand how amyloid triggers neuronal damage in this context. Herein we
showed that amyloid formed by a TTR mutant, A25T, activates microglia leading to the
secretion of TNF-α, IL-6 and nitric oxide. Also, we observed that A25T amyloid induces
Akt activation culminating in NFκB translocation to the nucleus of microglia cells. A25T
fibrils are not directly toxic to neurons, however when exposed to the conditioned
media of fibrils-activated microglia, neuronal death via apoptosis was extensive. Prior,
the conditioned media of fibrils-activated microglia led to synapse loss in neuron in
culture. We tested A25T fibrils toxicity in vivo, using an i.c.v. procedure, and observed
microgliosis and cognitive dysfunction in mice that received fibrils as treatment. These
data indicates that A25T amyloid fibrils act mostly as inflammatory agents, activating
microglia and leading to bystander neuronal damage.
INBEB, FAPERJ, CNPq
D23.
DETERMINAÇÃO ESTRUTURAL DA PROTEÍNA HIPOTÉTICA Q4DY78
CONSERVADA EM CINETOPLASTÍDEOS POR RMN
1 - D'ANDRÉA, E. D.; 1 - HEREDIA, G.P.; 1 - PIRES, J.R.
1 - Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro
Os tripanossomatídeos Trypanosoma cruzi, Trypanosoma brucei e Leishmania major são
causadores das doenças negligenciadas como a doença de Chagas, a doença do sono e
leishmaniose, respectivamente. A partir do sequenciamento genômico destes
cinetoplastídeos foi possível identificar genes que codificam proteínas hipotéticas
específicas destes parasitas. A determinação estrutural de proteínas auxilia o processo
de caracterização funcional e validação dessas macromoléculas como alvo terapêutico.
O presente trabalho tem como objetivo primeiramente a seleção de genes codificadores
de proteínas hipotéticas específicas de tripanossomatídeos e posteriormente a sua
determinação estrutural através de ressonância magnética nuclear (RMN). A partir da
análise das estruturas espera-se obter informações sobre suas funções. Para a seleção
foi utilizado o banco de dados TritrypDB disponível na rede. O gene Q4DY78, que é
predito codificar uma proteína alfa/beta e apresenta homologia com outras proteínas
sem estrutura conhecida, foi obtido comercialmente clonado no plasmídeo pUC57. Ele
foi sub-clonado no plasmídeo de expressão pGEX-4T2, seguido da expressão e
purificação da proteína. A proteína se expressou solúvel e o espectro de RMN de
hidrogênio mostrou que ela é enovelada. A partir daí, os seguintes experimentos de
RMN foram obtidos: com uma amostra não marcada - 2D TOCSY, 2D NOESY (em H2O e
em D2O); com uma amostra uniformemente marcada com 15N - 15N HSQC, 15N HSQC
TOCSY, 15N HSQC NOESY, série de 15N HSQC com diversos tempos de relaxação para
medida de T1 e T2 de 15N e heteronuclear NOE; com uma amostra duplamente
marcada com 15N e 13C – 13C HMQC, 13C HSQC, CBCANH, CBCACONH, HCCH-COSY,
HNCO, HBHACONH, 13C HSQC NOESY, além de espectros utilizando metodologia de Fast
NMR, bCBCANH, bCBCACONH, bHNCO e bHNCOi. 15N HSQCs em diferentes
temperaturas foram feitos para verificar ligações de hidrogênio e se o aminoácido está
em estrutura secundária. O assinalamento do backbone da proteína está em
andamento.
CNPq
Página 45
D24.
IDENTIFICATION OF ZINC-BINDING GROUPS AS SPECIFIC SNAKE VENOM
METALLOPROTEINASE INHIBITORS
1- Villalta-Romero, F.; 1- Espíndola A. P.; Tasic. L.
Instituto de Química, UNICAMP
"Snakebite envenomation represents a highly relevant public health problem, mostly
affecting poor people living in rural settings of Asia, Africa and Latin America. The
majority of snakebite envenomations in Latin America are caused by the viperid species,
whose venom contains a high proportion of zinc-dependent metalloproteinases that
play a relevant role in the pathogenesis of hemorrhage characteristic of these
envenomations. Antivenoms have proved to be highly effective in the neutralization of
systemic effects induced by snake venoms; however, they are only partially effective in
abrogating the local pathological alterations induced by viperid snake venoms. The use
of broad MP inhibitor, such as Batimastat®, has shown to be effective in decreasing
hemorrhagic, dermonecrotic and edema-forming effects of various snake-venom
metalloproteinases. However, the difficulty of open public access to Batimastat®
legitimates the design of new inhibitors for SVMP. More recently, reports of matrix
metalloprotease inhibitors (MMPi) with novel zinc-binding groups for these inhibitors
(ZBGs) have been found to influence inhibitor potency and selectivity. These ZBG effects
have been attributed to hydrogen-bonding interactions with the protein and van der
Waals contacts. ZBGs figure as potent leads, which can be further optimized to improve
solubility and pharmacokinetics via traditional medicinal chemistry. Protein (MP) and
ligand interactions were monitored using Saturation Transfer Difference (STD) NMR
experiments. Screening different zing binding molecules in terms of their affinity to
target protein (BaP1) enabled us to discriminate investigated compounds and to
propose new and potent drugs. The desing of small molecules with affinity for the
metalloproteinase active site, coupled to zinc-binding groups offers promising
possibilities."
CAPES, INBEB, FAPESP
D25.
SEMI-CORRELATIVE MICROSCOPY OF THE CUTICLE OF THE PARASITIC
NEMATODE HASSALSTRONGYLUS EPISLON
Adnet, F. A. O.; de Souza, W.; Attias, M.
Instituto de Biofísica Carlos Chagas Filho-UFRJ
Hassalstrongylus epsilon (Travassos 1937), is a nematode parasite of the duodenum of
the water rat Nectomys squamipes, Brants, 1827 (Rodentia: Muridae). Hassalstrongylus
epsilon is a member of Trichostrongyloidea superfamily that includes gastrointestinal
parasites of ruminants such as Haemonchus and Trichostrongylus, which cause
important diseases and economic loss in ruminants. The members this superfamily have
longitudinal cuticular ridges that fix the parasite to the intestine or stomach of the host
and that are sustained by the so called struts. These cuticular structures have been used
as basis for taxonomic classification of trichostongylides. By observing in light
microscopy semithin transversal sections of H. epsilon fixed and embedded in epoxy
resin and comparing it with ultrathin sections subsequently taken at the same region
and observed by Transmission electron microscopy. These observations revealed that
each cuticular ridge can have a different appearance in cross section, indicating that this
is not a good parameter for taxonomical classification. Electron tomography of the
cuticle revealed a complex structure of the cuticle, with striated fibers associated with
the struts and several layers of varied morphology. This complex structure needs to be
further explored in order to establish its association with the muscular layer and its role
in the parasite motility.
INBEB, FAPERJ, CNPQ e CNPQ-PROTRAX
D26.
EFEITO DO ESTADO DE PROTONAÇÃO DO ÁCIDO ASPÁRTICO NA
ESTABILIDADE DA PTERIDINA REDUTASE DE LEISHMANIA MAJOR
1,2- LEITE, F. H. A.; 2- LACERDA, P. S.; 2- PITA, S. S. R.; 2- CASTILHO, M. S.
1- Programa de Pós-Graduação em Biotecnologia, Universidade Estadual de Feira de
Santana; 2- Faculdade de Farmácia, Universidade Federal da Bahia.
Segundo Organização Mundial de Saúde (OMS) a Leishmaniose é a segunda protozoose
mais importante em termos de prevalência e mortalidade. Entretanto, o repertório de
fármacos é limitado e apresenta, na maioria dos casos, baixos índices de eficácia e
segurança. Embora os protozoários do gênero Leishmania sejam auxotróficos para
folatos, inibidores da enzima Diidrofolato redutase (DHFR) são pouco eficazes, pois a
Pteridina redutase (PTR1) atua como via bioquímica alternativa. Diante desse cenário,
inibidores de PTR1 e DHFR poderão ser compostos promissores para o desenvolvimento
de novos fármacos. O conhecimento do mecanismo catalítico em nível molecular da
PTR1 pode auxiliar no planejamento de fármacos leishmanicidas. Portanto, simulações
de Dinâmica Molecular (DM) foram empregadas para investigar a influência do estado
de protonação do resíduo catalítico ASP181 sobre a estabilidade da PTR1 (APO), bem
como sobre a afinidade da enzima pelo cofator (NADPH). Os resultados mostram que
após 50 ns de simulação (programa GROMACS 4.5.5, campo de força GROMOS 53a6, T=
303 K, pH= 4,7 e p=1atm), o valor de desvio em relação a estrutura inicial da DM para o
sistema APO protonado foi 0,59±0,12 nm, enquanto que o sistema APO não protonado,
0,41±0,08 nm. Por outro lado, o valor de desvio para o sistema PTR1-NADPH protonado
foi 0,41±0,06 nm e o sistema PTR1-NADPH não protonado, 0,46±0,08 nm. Deve-se
ressaltar também que o sistema PTR1-NADPH protonado apresentou 2,8±0,8 ligações
de hidrogênio com uma vida média de 705ps, enquanto que o sistema PTR1-NADPH não
protonado apresentou 3,0±1,1 ligações de hidrogênio com uma vida média de 1305 ps.
As informações estruturais obtidas através da DM revelam que o estado de protonação
do resíduo ASP181 é essencial para estabilidade de PTR1 e modulação de sua afinidade
pelo cofator. Esses dados serão cruciais para guiar estudos de triagem virtual baseados
na estrutura tridimensional de PTR1 de L. major.
FAPESB
D27.
ESTUDO EPIGENÉTICO NA PROGRESSÃO TUMORAL MAMÁRIA
1- GILSON C. SANTOS JR; 1- ANA P. SILVA; 1- LUCAS FELDMAN; 1- JULIANA CONSTANTE;
1- CLAUDIA V. DE MOURA-GALLO
1- Departamento de Genética, Universidade do Estado do Rio de Janeiro (UERJ),
Instituto de Biologia Roberto Alcântara Gomes, Rio de Janeiro, 20550-013, Brasil.
A progressão tumoral mamária depende de uma série de alterações epigenéticas,
dentre as quais se destacam a metilação e a acetilação. A metilação pode ocorrer tanto
no genoma quanto em determinados tipos de histonas, a acetilação, por sua vez, ocorre
praticamente apenas nas histonas. No intuito de analisar as principais alterações
epigenéticas na progressão do carcinoma mamário, utilizamos as linhagens celulares da
série de progressão tumoral mamária 21T(H16N2, 21PT, 21NT, 21MT1 e 21MT2),
provenientes da mesma paciente, apresentando portanto o mesmo “background”
genético. Com os resultados obtidos, através da digestão pelas enzimas de restrição
MspI e HpaII, foi possível verificar um aumento significativo na metilação global
genômica nas linhagens metastáticas, assim como um aumento da trimetilação na lisina
9 da histona H3 (H3K9me3), através de microscopia confocal e western blot, marcador
de heterocromatina constitutiva. Também foi detectada uma diminuição da acetilação
na histona H4, marcadora de eucromatina, através dos mesmos ensaios descritos
anteriormente. Uma diferença significativa na distribuição da eucromatina e da
heterocromatina ao longo da progressão do carcinoma mamário foi observada. Estes
resultados ressaltam a importância das alterações epigenéticas na progressão da câncer
Página 46
e apontam para a extensa mudança espacial que ocorre na cromatina no núcleo das
células ao longo da progressão para malignidade.
FAPERJ, CAPES, CNPq
ser levado em consideração na avaliação dos ensaios de atividade biológica feitos com o
chá verde.
INBEB, FAPERJ, CNPq
D28.
D30.
BONE MARROW MESENCHYMAL STROMAL CELLS RESCUE CARDIAC
FUNCTION IN STREPTOZOTOCIN-INDUCED DIABETIC RATS
1-MONNERAT-CAHLI, G; 1-GUERRA, B; 1-MANSO, G; 1-FERREIRA, A; 1-GRAN DA SILVA,
D; 2-COUTINHO, D.C; 3-CARNEIRO-RAMOS, M.S.; 3-TRENTIN-SONODA, M; 1RODRIGUES, D; 1-CABRAL-DA-SILVA, M.C; 1-GOLDENBERG, R; 1-NASCIMENTO, J.H.M;
1,4-CAMPOS-DE-CARVALHO, A.C; 1-MEDEI, E
1 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio
de Janeiro, Brasil. 2 Department of Morphology, Institute of Biological Sciences, Federal
University of Minas Gerais, Belo Horizonte, Brazil; 3 Centro de Ciências Naturais e
Humanas, Universidade Federal do ABC, Santo Andre, Brazil; 4 Instituto Nacional de
Cardiologia. Rio de Janeiro. Brasil.
Diabetes Mellitus can lead to cardiac dysfunction. Several studies have shown the antidiabetic effects of bone marrow mesenchymal stromal cells (MSC). In the present study,
we investigate whether MSC transplantation can revert cardiac dysfunction in
streptozotocin-induced diabetic rats. The rats were divided in three groups: Nondiabetic, Diabetic (DM) and Diabetic-Treated (DM+MSC) that received 5x106 MSC 4
weeks after establishment of diabetes. Four weeks after MSC-therapy, systemic
metabolic parameters and cardiac function were assessed. MSC transplantation was
able to revert the hyperglycemia and increase the body weight of the animals, while
decreasing sera corticosterone levels without restoring insulin secretion and oral
glucose tolerance. Also, MSC improved electrical remodeling, shortening the QT and
QTc on the ECG and the action potential duration of left ventricle myocytes. MSC
rescued the cardiac beta-adrenergic sensitivity by increasing beta-1 adrenergic receptor
expression. Both alpha and beta cardiac AMPK and P-AMPK returned to baseline values
after MSC-therapy. Toll-like receptor signaling pathways are not involved in the
mechanism of MSC-improved cardiac function. The results indicate MSC-therapy was
able to rescue cardiac impairment induced by diabetes, normalize cardiac AMPK subunit
expression and activity and decrease corticosterone and glycemia.
CNPq, FAPERJ, INBEB
BIOPHYSICAL STUDIES OF SUGARCANE MITOCHONDRIAL SHSP22
PINHEIRO,G.M.S; TIROLI-CEPEDA,A.O.; RAMOS, C.H.I.
Instituto de Química, UNICAMP, Campinas, SP-Brasil.
"Heat shock proteins (HSPs) can be classified into six structurally conserved classes
according to their molecular weight, namely, HSP100, HSP90, HSP70, HSP60, small heat
shock proteins (sHSPs) and ubiquitin (8.5 kDa). SHSPs form large polydisperse oligomers
that are exceptionally dynamic and important for the functional ability of protecting
substrate proteins from aggregation. The diversity of sHSP in plants is intriguing and
characterization of their chaperone activity is important to understand plant tolerance
to heat stress. In the present work we describe the structural characterization of a
recombinant SsHSP22 using circular dichroism, intrinsic/extrinsic fluorescence and size
exclusion chromatography. The SsHSP22 was obtained 95% pure, and was folded as
deemed by CD and fluorescence emission spectra. The chaperone was purified as a
single species constituted of an oligomer with approximately 26 monomers. CD analyses
showed that SsHSP22 had a secondary structure consisting primarily of β-sheets (~45%)
due to the presence of a minimum at approximately 217 nm. Biophysical and
biochemical characterization of a recombinant mitochondrial SsHSP22 from sugarcane
are in progress and the results may be significant to the study of the ethanol production
as a renewable energy source. Keywords: small heat-shock proteins, protein folding,
chaperone activity, sugarcane, SsHSP22."
FAPESP and CNPq.
D29.
ANÁLISE POR RMN DAS INTERAÇÕES INTERMOLECULARES DAS
CATEQUINAS NO CHÁ VERDE (CAMELLIA SINENSIS)
1- CASTAÑEDA-VALENCIA, G; 2- TINOCO, L
1,2-Nucleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro
As catequinas são componentes fenólicos extraídos de plantas, possuem atividades
anticancerígenas, antioxidantes, etc. e são encontradas em alimentos e bebidas, como
no chá verde. Embora a agregação de compostos fenólicos já tenha sido descrita na
literatura, ainda há pouca informação sobre os processos de agregação das catequinas
diretamente no chá verde. As interações intermoleculares podem ser identificadas
como variações nos deslocamentos químicos, nos tempos de relaxação e nos
coeficientes de difusão translacional. A infusão do chá verde foi liofilizada e preparada
nas concentrações de 0,5; 1; 2 e 5 mg/mL em D2O. Foram feitos espectros de RMN de
1H a 499,79 MHz ( VNMRS-500 –Agilent) e medidos os tempos de relaxação T1 e T2 a 25
o C. Nos espectros de RMN de 1H foram identificados os sinais representativos dos
componentes catequinicos: Epigalocatequina galato (EGCG), Epicatequina (EC),
Epigalocatequina (EGC) e Catequina (CATE). Foi observado que há uma diminuição dos
deslocamentos químicos com o aumento da concentração, sendo esta variação mais
evidente para os sinais de H-3, H-2 e H-2´´ da EGCG, para H-2, H-6 e H-2´ da EGC, para H2´ da EC e para H-8, H-4α e H-2´ da CATE. Na amostra mais concentrada (5 mg/mL)
houve uma maior sobreposição e alargamento dos sinais, não sendo possível identificar
alguns dos hidrogênios, claramente identificados nas amostras de 0,1 e 1 mg/mL. Os
tempos de relaxação T1 e T2 para a maioria dos hidrogênios representativos da EC, EGC
e EGCG diminuem com o aumento da concentração, mas os hidrogênios da CATE
apresentaram uma variação irregular dependo do hidrogênio analisado. Estes
resultados demonstram que as catequinas, mesmo na presença dos demais
componentes presentes no chá verde, sofrem um processo de agregação, o que deve
D31.
VISUALIZATION OF CYTOSKELETON OF TRITRICHOMONAS FOETUS BY XHRSEM (EXTREME HIGH RESOLUTION SCANNING ELECTRON MICROSCOPY)
1,2- De Andrade Rosa, I; 3,4-De Souza, W.; 2,5 Benchimol, M.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2Universidade Santa Úrsula; 3- Laboratório de Ultraestrutura Celular Hertha Meyer –
Universidade Federal do Rio de Janeiro; 4- Instituto Nacional de Metrologia, Qualidade e
Tecnologia-Inmetro, Duque de Caxias, Rio de Janeiro; 5- Instituto Nacional de Ciência e
Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro
Tritrichomonas foetus is the causative agent of bovine trichomoniasis, one of the most
widespread sexually transmitted diseases in cattle. T. foetus is also studied because of
its particular cell structure such as the mastigont system. This system is formed mainly
for microtubular structures, the pelta and axostyle; the costa, a periodic rootlet; the
parabasal fibers and several other filaments. The goal of this study was obtain a better
understanding of T. foetus cytoskeleton using the extreme high resolution scanning
electron microscopy (HXR-SEM). For this, the plasma membrane of T. foetus was
extracted with detergent. Afterwards, the cytoskeleton was fixed with 2.5%
glutaraldehyde, post-fixed with 1% osmium tetroxide, dehydrated in graded ethanol,
critical point-dried in CO2, coated with carbon layer and observed on Magellan (FEI
Página 47
Company). The Magellan microscopy offer subnanometer resolution over the full 1 kV
to 30 kV electron energy range, effectively establishing a new performance category
known as Extreme High Resolution Scanning Electron Microscopy (XHR-SEM). The
results showed details of structures such as the costa and associated filaments,
microtubules of axostyle and their connections, the comb and infrakinetosomal body.
The costa was seen as a large striated root fibril that emerges from the anterior region
and its bands could be observed. In addition, a new filament associated to the costa and
connections between this structure and recurrent flagellum were also observed. A
higher magnification revealed that these connections are formed to filaments and
globular structures. The microtubule ribbons that form the pelta and axostyle were seen
as two distinct orientations of microtubules. The posterior part of the axostyle turns
upon itself forming a tube. Higher magnification of the axostyle allowed the observation
of the oriented microtubules that form this structure. Thus, the results obtained show
that XHR-SEM contributes to adding new data on the cytoskeleton of this protist.
AUSU, INMETRO, INBEB, CNPq, FAPERJ and PRONEX
D32.
A NOVEL ALKYL PHOSPHOCHOLINE-DINITROANILINE HYBRID MOLECULE
EXHIBITS BIOLOGICAL ACTIVITY IN VITRO AGAINST LEISHMANIA AMAZONENSIS
1 - GODINHO, J.L.P; 5 - GEORGIKOPOULOU, K.; 5 - CALOGEROPOULOU, T. ;1, 2, 3 - DE
SOUZA, W. ; 1 ,2, 3, 4 - RODRIGUES, J.C.F.
1Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,
Brasil. 2 Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Brasil. 3
Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Brasil 4
Polo Avançado de Xerém, Universidade Federal do Rio de Janeiro. 5National Hellenic
Research Foundation, Institute of Organic and Pharmaceutical Chemistry, Athens,
Greece.
Leishmaniasis is one of the most important neglected tropical diseases caused by
parasites of the Leishmania genus. The current chemotherapy is based on pentavalent
antimonials, miltefosine, amphotericin B and pentamidine. However, there is an urgent
need for safer and more efficacious drugs. An interesting approach in drug development
is the combination therapy using drugs with known activity against the parasites. Both,
trifluralin, a dinitroaniline herbicide, and miltefosine have activity against protozoan
parasites, but with high IC50 and cytotoxicity for host cells. In this work, we investigated
the effects of TC95 on Leishmania amazonensis. TC95 is a hybrid compound that
combines trifluralin and miltefosine in only one molecular scaffold. The antiproliferative
effects of TC95 against promastigotes were dose-dependent, with a total inhibition at
concentrations of 4 and 5 μM. Against intracellular amastigotes, the effect was also
dose-dependent after 24 h of treatment, however the effect was more pronounced
after 48 h of treatment for all concentrations tested [1, 5 and 10 μM]. The IC50 values
obtained after 48 h of treatment was 2.6 μM and 1.2 μM for promastigotes and
intracellular amastigotes, respectively. Cytotoxicity assays against murine macrophages
revealed that the CC50 was 60 μM, thus presenting a selective index of 24x. From 12 h
until 72 h of treatment, promastigotes displayed profound alteration in the shape
appearing rounded and swollen, with alterations in the cell cycle and in the cytoskeleton
constituted mainly by microtubules. Transmission electron microscopy revealed that the
mitochondrion is the main organelle affected, presenting an intense swelling with loss
of the matrix content and disorganization of the mitochondrial membranes. Sometimes,
lysis of the mitochondrion was also observed. Furthermore, alterations in the flagellar
membrane, Golgi complex, increase in the number of lipid bodies and appearance of
structures typically found in autophagic processes were also observed. Against
intracellular amastigotes, TC95 also induced significant alterations such as:
mitochondrial swelling with vesiculation of the mitochondrial membranes and
alterations in the kinetoplast; significant dilation of the trans-Golgi network; presence of
lipid bodies in the amastigotes and inside the macrophages; and changes in the flagellar
structure. Taken together, these results indicate that TC95 is a promising compound
against Leishmania sp.
CNPq, CAPES, and FAPERJ
D33.
IMMUNOMODULATORY EFFECT ON HUMAN MONOCYTES PROMOTES BY 5HYDROXY-2-HYDROXYMETHYL-GAMMA-PYRONE (HMP), A SECONDARY METABOLITE
OBTAINED FROM ASPERGILLUS FUNGI
Costa, J.P. 1,2*; Frade, P.C. R 1,2; Rodrigues, A. P. D.1,2,4; Farias, L.H.S. 1,2; Hage,
A.A.P1,2; Silva, B. J. M 1,2; Santos, A.S. 3; Silva, E.O. 1,2
1Laboratório de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do
Pará, Belém, Pará, Brazil 2 Laboratório de Biologia Estrutural, Instituto de Ciências
Biológicas, Universidade Federal do Pará, Belém, Pará, Brazil 3Laboratório de
Planejamento e desenvolvimento de Fármacos, Instituto de Ciências Exatas e Naturais,
Universidade Federal do Pará, Belém, Pará, Brazil 4Laboratório de Microscopia
Eletrônica, Instituto Evandro Chagas, secretaria de vigilância em saúde, ministério da
saúde, Belém, Pará, Brazil;
"The 5-hydroxy-2-hydroxymethyl-gamma-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 antioxidant, tyrosinase
inhibitor, 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 monocytes is unknown. Thus, the aim of this study was to evaluate the effects of
HMP on the cell viability and differentiation of human blood monocytes in vitro. Human
peripheral leucocytes were obtained from blood bag donated from Foundation
Hemocenter of Para State. Cell isolation was performed using HISTOPAQUE® 1077density-gradient. Monocytes were treated for 24, 48 and 72 hours 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. Immunofluorescence analysis of the expression of surface
protein specific for the macrophage (F4/80), demonstrated that human monocytes
treated with 50 and 100μg/mL for 48 and 72h showed the similar pattern of expression
of proteins to that of human monocytes differentiated by macrophage colonystimulating factor (M-CFS). The viability test used showed that HMP has no citotoxicity
effect on human monocytes when treated with 50 and 100 μg/mL of HMP. These results
demonstrated a new role for HMP as an immunomodulator agent, in inducing the
differentiation of monocytes into macrophages. Keywords: HMP; Macrophages
differentiation; Human monocytes."
Supported by CAPES, CNPq/UFPa, CNPq/MCT/CT-INFRA/CT-PETRO (Processo nº
620179/2008), MCT/CNPq/FNDCT/CAPES/FAPERJ.
D34.
PRODUÇÃO DE CREME NATURAL A PARTIR DE PLANTAS DA AMAZÔNIA
COM ATIVIDADE MICROBICIDA
*SANTOS, J.P2., COSTA, M.C2., PIMENTEL, B.W.M.2, COSTA1,2, J.P., JUNIOR, H.N.P2.,
SILVA, E.O2.
1Laboratório de Parasitologia e Laboratório de Biologia Estrutural, Instituto de Ciências
Biológicas, Universidade Federal do Pará, Belém, Pará, Brasil 2Colégio Estadual de
Ensino Médio Manoel Antônio de Castro, Secretaria Executiva Estadual de Educação
(SEDUC) Belém, Pará, Brasil.
As mãos constituem a principal via de transmissão de microrganismos, pois a pele é um
grande reservatório de diversos microrganismos que são transferidos de uma superfície
Página 48
para outra, por meio de contato direto ou indireto com a pele. Na camada mais
superficial da pele pode-se encontrar bactérias Gram-negativas, como a enterobactéria
Escherichia coli e bactérias não fermentadoras como a Pseudomonas aeruginosa, além
de fungos e vírus diversos. Assim, as mãos constituem uma importante maneira de
veicular doenças que variam desde uma simples gripe até uma infecção mais severa,
podendo levar a morte. Logo, o simples fato de higienizar as mãos, é um procedimento
de grande importância e muito eficaz para evitar a propagação de muitas doenças
contagiosas, sobretudo em ambientes frequentados por um grande numero de pessoas
como nas Escolas Públicas. De acordo com o Centro de Controle e Prevenção de
Doenças, dos EUA, a correta higiene das mãos é um dos mais importantes passos na
prevenção de doenças e no controle da proliferação de germes. Para a ANVISA, o álcool
em gel é o produto mais eficaz para higienização das mãos, pois é capaz de reduzir de
forma significativa o número de bactérias e fungos que parasitam a pele. Entretanto,
estudos mencionam que muitos microoganismos estão adquirindo resistência a vários
produtos sintéticos, havendo portanto uma grande tendência a utilização de produtos
de origem natural. Assim, este projeto tem como objetivo utilizar a rica biodiversidade
da Amazônia para produção de um cosmético que tenha ação microbicida similar a
verificada no álcool em gel. Para tanto, bactérias Staphylococcus aureus foram tratadas
com extratos aquosos, infusões e extratos alcoólicos de 13 diferentes espécies de
plantas na concentração de 0,1g/mL. Os ensaios preliminares mostraram que a o
extrato aquoso da folha do jambo apresenta considerável ação microbicida.
INBEB
D35.
DOENÇAS HELMÍNTICAS NO MUNICÍPIO DE IGARAPÉ-MIRI
2GOMES, D.A.O., 1,2 COSTA, J.P., 2JUNIOR, H.N.P.,1SILVA, E.O.
1Laboratório de Parasitologia e Laboratório de Biologia Estrutural, Instituto de Ciências
Biológicas, Universidade Federal do Pará, Belém, Pará, Brasil 2Colégio Estadual de
Ensino Médio Manoel Antônio de Castro, Secretaria Executiva Estadual de Educação
(SEDUC) Igarapé-Miri, Pará, Brasil.
"As enteroparasitoses, em especial as causadas por helmintos, constituem um grave
problema de saúde pública no Brasil, estando diretamente relacionadas com as
condições de saneamento básico, nível socioeconômico, grau de escolaridade, hábitos
de higiene, entre outras variáveis. O município de Igarapé-Miri apresenta características
favoráveis à ocorrência dessas doenças, pois apresenta saneamento básico precário,
além de outros fatores que também favorecem a ocorrência dessas parasitoses. Assim,
este estudo investiga quais helmintos encontram-se com maior frequência em
amostras fecais de moradores do município de Igarapé-Miri. O estudo foi dividido em
duas etapas: pesquisa de campo e pesquisa laboratorial, tendo como público alvo
moradores da comunidade escolar do Colégio Manoel Antônio de Castro. Para a
pesquisa de campo, foi realizada aplicação de questionários na comunidade escolar, a
fim de fazer o levantamento do grau de conhecimento que esses indivíduos detêm em
relação às parasitoses intestinais causadas por helmintos. Os resultados da pesquisa de
campo mostraram que 86% dos entrevistados não sabiam informar as formas de
transmissão de ao menos uma helmintíase 54% utilizam água sem nenhuma forma de
tratamento. Para pesquisas laboratoriais, utilizou-se 50 amostras fecais que foram
avaliadas através dos métodos coproscópicos Hoffman e Direto. Os testes laboratoriais
mostraram que 42% das amostras fecais apresentavam ovos de T.trichiuris, 18% ovos de
A. lumbricoides e 2% ovos de ancilostomídeo. Somente 38% das amostras, não
apresentaram nenhuma forma de ovos helmínticos. Assim, este projeto é de grande
relevância para a realização de um trabalho de educação preventiva no municpio de
Igarapé-Miri.
Palavras-chave: helmintíases, Educação preventiva, Municipio de Igarapé-Miri, Métodos
de Hoffman e Direto."
INBEB, UFPA
D36.
LOSS OF ENDOCYTIC ACTIVITY DURING TRYPANOSOMA CRUZI
METACYCLOGENESIS
1-NARCISA LEAL DA CUNHA-E-SILVA;1,2-WANDERLEY DE SOUZA,1-CAROLINA DE LIMA
ALCANTARA; 1- JULIANA CUNHA VIDAL
1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
Trypanosomatids require essential exogenous nutrients and growth factors in order to
survive and divide. Trypanosoma cruzi epimastigotes acquire them by endocytosis via
the cytostome and the flagellar pocket, both localized at the parasite’s anterior region.
The cytostome is an opening at the plasma membrane surface that deepens in a
membrane invagination called cytopharinx. This structure is responsible for 85% of
endocytic activity in epimastigotes, delivering cargo to reservosomes. T. cruzi loses its
endocytic ability somewhere during the transformation into trypomastigotes
(metacyclogenesis), as these infective forms are not able to uptake exogenous cargo
and do not present the cytostome-cytopharinx complex. Between epimastigotes and
trypomastigotes, three subsequent types of T.cruzi intermediate forms were described:
Ia, where the kinetoplast is close to the elongated nucleus, Ib where the kinetoplast is
located side by side with nucleus, and Ic that presents the kinetoplast posterior to the
nucleus. The latter is the form that just precedes the complete morphological
transformation into trypomastigotes (Ferreira et al., 2008). We have analyzed changes
in the ultrastructure of endocytic apparatus and the endocytic activity along in vitro
metacyclogenesis. After four hours in TAU3AAG, we have obtained 30% of intermediate
forms; using transmission electron microscopy we observed that Ic intermediate forms
still present a cytostome. Interestingly, this invagination maintained its typical
morphology, even after the migration backwards, accompanying the kinetoplast.
Moreover, the endocytic apparatus proved to be functional in Ic intermediate forms, as
they were able to uptake transferrin-gold offered after metacyclogenesis and store in
typical reservosomes. Our data indicate that the disassembly and loss of function of the
endocytic machinery is a very late event in metacyclogenesis.
INBEB, CAPES, FAPERJ, CNPq
D37.
INTRA-ABDOMINAL ABSCESSES IN MICE CHALLENGED BY THE COMMENSAL
BACTERIA
BACTEROIDES
FRAGILIS:
EVIDENCE
FOR
INVOLVEMENT
OF
INFLAMMASOMES/IL-1BETA PATHWAY
CASTELPOGGI, J.1, TORRES, M.E.W.1, PEREIRA, J.S.1, LOBO, L.2, FERREIRA, E.O.2,
DOMINGUES, R.M.C.P.2, ZAMBONI, D.3, BELLIO, M.2, SCHARFSTEIN, J.1 AND OLIVEIRA,
A.C.1
1- Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro; 2- Instituto de
Microbiologia Paulo de Góes, UFRJ, Rio de Janeiro; 3 - Faculdade de Medicina de
Ribeirão Preto, USP, São Paulo.
Bacteroides fragilis is a strictly anaerobic Gram-negative bacterium frequently isolated
from patients with clinical infections, such as intra-abdominal abscesses and
bacteremia. Its major source is the normal colonic microbiota where Bacteroides spp.
outnumbers aerobic and facultative anaerobic bacteria. When intestinal integrity is
disrupted, B. fragilis and colonic contents escape into the peritoneum. Although
proinflammatory cytokines (e.g. IL-1
TNF ) may enhance host inflammatory
response during sepsis and intra-abdominal abscess formation, these innate responses
are also require to ensure the containment and elimination of intestinal
microorganisms. Here we have evaluated the participation of IL-1 and inflammasome
Página 49
pathway in the formation of intra-abdominal abscesses in mice injected
intraperitoneally with B. fragilis plus sterile cecal contents (SCC). Although infected
C57BL/6 (WT) mice present an intense cellular infiltration into the peritoneal cavity, the
pathology was dramatically reduced in caspase-1-, ASC- and IL-1R-deficient mice.
Consistently, caspase-1, ASC and IL-1R knockout mice were protected from early
decrease on body weight and exhibit reduced inflammatory abscesses as compared to
WT and IL-18R-/- mice subjected to the same challenge. Of further interest, we found
that SCC alone induces in vitro IL-1 production by dendritic cells and macrophages in a
MyD88-dependent manner, whereas no effect was observed with B. fragilis alone. The
same results were obtained in vivo when we injected bacteria or SCC alone into
peritoneal cavity and the IL-1 production was analyzed. Taken together, our results
strongly suggest that the development of intra-abdominal abscesses containing this
commensal microbe is critically dependent on adverse activation of the
inflammasome/IL- pathway by colonic contents. Based on our mice studies, we
suggest that the development of such abscesses in susceptible patients might be
controlled by procedures that restore the epithelium barrier, preventing the
transposition of colonic contents to the peritoneal cavity, and/or by inhibitors of the
inflammasome/IL-1 pathway.
INBEB, CNPq
D38.
IMPACT OF SOLUBLE Β-AMYLOID PEPTIDE ACCUMULATION ON
INTRACELLULAR TRAFFICKING IN CULTURED NEURONS.
OLIVEIRA, L.T.1,4; LEON, G.V.O.4; PROVANCE, D.W.2; MELLO, F.G.3; SORENSON, M.M.1;
SALERNO V.P.1,4
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2- Centro
Desenvolvimento Tecnológico de Saúde, Fiocruz; 3- Instituto de Biofísica Carlos Chagas
Filho, Universidade Federal do Rio de Janeiro; 4- Departamento de Biociências da
Atividade Física - EEFD, Universidade Federal do Rio de Janeiro.
It is widely recognized that β-amyloid (Aβ) also accumulates within neurons causing
disruption of synapses and cognitive function. Here, we report the internalization and
transport of a fluorescently labeled Aβ peptide and suggest its impact on intracellular
trafficking. Using cultured chick neurons, we have shown previously that exogenous
Aβ42 is internalized and leads to the redistribution of Myosin Vb (MVb), a molecular
motor protein involved in intracellular traffic. The internalization process is specific for
the 1-42 isoform, exhibits characteristics of a constitutive process without a specific
point of saturation and depends on Aβ40/Aβ42 (Cytoskeleton. 69(3):166, 2012). Colabeling for caveolin, EEA1 and Lamp1 after 10 min with Aβ42 suggested that a portion
of the Aβ42 oligomers was internalized by caveolin-coated vesicles and localized with
primary endosomes, but not lysosomes. Internalized Aβ42 evolved from a diffuse to a
punctate distribution that shifted from the processes to the perinuclear region. These
changes in pattern of Aβ coincided with an increased coincidence with MVb. A similar
profile was observed for Rabs 8 and 10, which co-localized with internalized Aβ42 and
suffered a shift in distribution. The localization of MVb with Rabs 8 and 10 is consistent
with the early endosomes distribution suggested by labeling by with EEA1. It also
suggested that some of the Aβ42 may be transported to the Golgi network. These
observations are consistent with the hypothesis that AD proceeds as a result of an
imbalance between Aβ production and Aβ clearance. Furthermore, it suggests a role for
MVb and its binding partners in this process with the changes in MVb and Rab proteins
distribution as possible consequences of Aβ42 internalization that could impact
intracellular transport pathway(s) involving myosin Vb, thereby sequestering myosin Vb
away from its normal sites of action and compromising overall neuronal function.
INBEB, FAPERJ, CNPq, CNPq/INCT
D39.
INSIGHTS INTO INTERACTION BETWEEN THE C-TERMINUS OF HUMAN 90
KDA HEAT SHOCK PROTEIN HSP90 AND THE MITOCHONDRIAL TRANSLOCASE OF OUTER
MEMBRANE TOM70
ZANPHORLIN LM1, SQUILACE ALA1, FIGUEIREDO A1, GOZZO FC1, BARBOSA L2, RAMOS
CHI1.
1Instituto de Química, Unicamp, Campinas, SP, Brasil; 2Instituto de Física, USP, SP,
Brasil.
“The mitochondrial import receptor Tom70 (translocase of the mitochondrial outer
membrane 70) is a TPR-like protein, which interacts with chaperone–preprotein
complexes and Hsp90 is a crucial molecular chaperone required for folding. Although it
is known that the pentapeptide MEEVD at the C-terminus of Hsp90 is the motif involved
in the recognition of TPR proteins, the molecular mechanism by which Tom70 coordinates interactions with preproteins and chaperones is still elusive. The interaction
was investigated by cross-linking coupled with LC-MS/MS analysis, small-angle X-ray
scattering (SAXS), isothermal tritration calorimetry (ITC) and in silico methods. Mass
spectrometry (MS) analysis of the tryptic products of the complex revealed four
interpeptide cross-link sites in the complex, from which two were sites between Tom70
and C-Hsp90. The interpeptide cross-link sites were confirmed by ITC experiments in
which a model peptide of the region acted as a competitor for the complex interaction.
Structural modeling based on cross-linking MS analysis was employed to unveil the
molecular interfaces involved in the interaction and the complex model, which was
supported by SAXS analysis. This study constitutes the first direct mapping of interaction
sites in the C-Hsp90/Tom70 complex and is a significant contribution to the mechanism
of interaction between Hsp90 and co-chaperones.
Key Words: Hsp90, Tom70, interface mapping, crosslinking, ITC, SAXS.”
Financial Support from Fapesp, Cnpq and INBEB
D40.
DIFERENÇAS ULTRAESTRUTURAIS ENTRE LEVEDURAS DE SPOROTHRIX
SCHENCKII E SPOROTHRIX BRASILIENSIS
1-BORBA-SANTOS, L.P.;1- FONSECA, B., B.;2- ISHIDA, K.2; 3-LOPES-BEZERRA, L.M.;1ROZENTAL, S.
1-Laboratório de Biologia Celular de Fungos, IBCCF, UFRJ. 2-Departamento de
Microbiologia, ICB, USP. 3-Laboratório de Micologia Celular e Proteômica, UERJ.
"Espécies dimórficas do complexo Sporothrix schenckii são causadoras da esporotricose,
micose subcutânea mais frequente no Brasil e de grande incidência no Estado do Rio de
Janeiro. O objetivo deste estudo foi caracterizar aspectos ultraestruturais das leveduras
de Sporothrix schenkii e Sporothrix brasiliensis, que são as espécies mais virulentas do
complexo. Leveduras de dois isolados (ATCC MYA-4821 de S. schenckii e ATCC MYA4823 de S. brasiliensis) crescidas em meio RPMI 1640 por 24 horas à 35°C, foram
coletadas por centrifugação, lavadas em PBS e fixadas em solução de 2,5%
glutaraldeído, 4% paraformaldeído em 0,1M tampão cacodilato de sódio por 24 horas à
4°C. As células foram pós fixadas em 1% OsO4 e 1,25 M K4[Fe(CN)6].3H2O por 2 horas à
4°C, lavadas em tampão cacodilato e desidratadas em etanol. Amostras para
microscopia eletrônica de transmissão foram incluídas em resina Spurr e os cortes
ultrafinos foram contrastados em acetato de uranila e citrato de chumbo. As imagens
foram obtidas em um microscópio eletrônico de transmissão JEOL 1200 EX. Amostras
para microscopia eletrônica de varredura foram aderidas em lamínulas recobertas com
poli-L-lisina antes da pós fixação, desidratadas, secas em ponto crítico de CO2 e
metalizadas com ouro. As imagens foram obtidas em um microscópio eletrônico de
varredura FEI Quanta 250. Leveduras de S. schenckii exibiram morfologia alongada
enquanto leveduras de S. brasiliensis exibiram morfologia ovóide. Leveduras de S.
Página 50
schenckii apresentaram uma segunda camada da parede celular que não foi observada
em leveduras de S. brasiliensis. Por outro lado, leveduras de S. brasiliensis apresentaram
a parede celular e a camada microfibrilar mais espessa. Além disso, a parede celular de
leveduras de S. brasiliensis apresentou acúmulo de material elétron denso. Estes
estudos ainda são preliminares, entretanto, alguns aspectos ultraestruturais podem ser
considerados na distinção entre S. schenckii e S. brasiliensis."
Capes, FAPERJ e CNPq
D41.
CYSTATIN S/SA/SN IS SECRETED IN WORKLOAD-DEPENDENT MANNER
1,3-Sant’Anna, M.L.; 1,2-Almeida, A.C.P.A.; 1-Sorenson, M.M.; 2-Salerno, V.P
1-Instituto de Bioquímica Médica/UFRJ; 2-Escola de Educação Física e Desportos/UFRJ;
3-Centro de Instrução Almirante Sylvio de Camargo/RJ.
"Ain: Physical exercise generates multiple physical and biochemical changes in the
human body that can be monitored in various manners. The measurement of these
changes can be good indicators of workload, redox state and the state of the immune
system. To evaluate a group of cysteine protease inhibitors involved in several diseases,
we following the salivary cystatin secretion at rest and after four incremental bouts on a
cycle ergometer. Methods and results: The procedures in this study were approved by
the Ethics Committee of the Hospital Universitario Clementino Fraga Filho. The subjects
were 6 career enlisted men from the Brazilian Navy (age 25.8 ± 9.4 years). Exercise on
the cycle ergometer consisted of four bouts at different intensities (65%, 75%, 85% and
95% of heart rate reserve). Saliva was collected before and after each bout (5, 10 and 15
min), centrifuged, and the supernatant stored at – 20° C. Accutrend indicator strips
(Roche Diagnostic) were used to determine blood lactate concentration at rest and
immediately after each test, with significant increase from 3 to 13 mM. Cystatin
S/SA/SN was identified by Western blot and quantified by densitometry using a twochannel infrared scanner (Odyssey®). Cystatin increased significantly, 5 min after the
highest exercise efforts 54% and 81% compared to resting values and returned toward
normal values after 10 and 15 min post-exercise. Conclusion: Salivary cystatin secretion
is correlated with exercise workload but recovers within 15 min."
CAPES, CNPq, FAPERJ, INBEB/INCT, Centro de Instrução Almirante Sylvio de
Camargo/MB
D42.
CONTRIBUIÇÃO DOS SOROS DE PACIENTES CHAGÁSICOS CRÔNICOS NA
GÊNESE DE ARRITMIAS QUANDO PRESENTES EM UM AMBIENTE COM AUMENTADA
DISPERSÃO TRANSMURAL DA REPOLARIZAÇÃO
1- VIDAL, M.A.; 1- MACIEL, L.; 2- PEDROSA R.C.; 1- NASCIMENTO, J. H. M.; 1- CAMPOS
DE CARVALHO A. C; 1- CABRAL DA SILVA M. C.; 1- MEDEI, E.
1- Laboratório de Eletrofisiologia cardíaca Antonio Paes de Carvalho / Instituto de
Biofísica Carlos Chagas Filho / UFRJ; 2- Hospital Universitário Clementino Fraga Filho /
UFRJ.
"Introdução: A presença de anticorpos funcionais capazes de ativar receptores β1adrenergicos e muscarínicos acoplados a proteínas G em pacientes chagásicos crônicos
(PChC) foi descrita. Objetivo: Observar o efeito dos soros dos PChC na eletrogênese
cardíaca, quando colocados no modelo farmacológico (E-4031) da Síndrome do QT
longo do tipo 2 (SQTL2). Comparar a incidência de arritmias destes soros tanto em
corações sadios como no modelo da SQTL2. Métodos: Foram utilizados corações de
coelhos Nova Zelândia, machos. A SQTL2 foi induzida mediante perfusão do bloqueador
da corrente IKr, E-4031 (5 µM) ao longo do experimento. Foram utilizados os soros de
52 PChC, previamente, funcionalmente caracterizados como tendo efeito: muscarínico
(Soros-M; n=10), adrenérgico (Soros-β1; n=15) e ambos, muscarínico e adrenérgico
(Soros-Mβ; n=17). Os soros sem atividade na bioeletrogenese foram denominados de
soros não-reativos (Soros-NR; n=10). Foi analisado o efeito de cada um dos soros no
modelo farmacológico da SQTL2 visando estudar a incidências de arritmias. Resultados:
A incidência de arritmias pelos soros dos PChC no coração sadio e na SQTL2 foi
respectivamente: soros-β1: 0% vs. 75%, soros-M: 50% vs. 78% e os soros-Mβ: 67 vs.
64%. Já os soros-NR não induziram arritmias. Conclusão: Os soros-β1 em um coração
sadio não apresentam eventos arrítmicos, entretanto, quando presentes em um modelo
da SQTL-2 induziram arritmias em 75% dos casos. Um ambiente de maior dispersão
transmural da repolarização, também, favoreceu uma maior incidência de arritmias na
presencia dos soros-M. Todavia, os soros-Mβ apresentaram incidência semelhante de
arritmias em ambos os casos. Estes achados preliminares sugerem que, para os soros
dos PChC induzir arritmias ou aumentar a incidência destas, é necessário da presença de
um microambiente conspícuo."
FAPERJ, CNPq e CAPES
D43.
NEUROPROTECTIVE POTENTIAL OF BONE MARROW MESENCHYMAL STEM
CELLS IN A MODEL OF ALZHEIMER´S DISEASE
1-GODOY, M.A.; 2-SARAIVA, L.; 1-VASCONCELOS-DOS-SANTOS, A.; 1-BEIRAL, H.J.V.; 1SILVA, L.R.P.; 1-BRAGA, C.V.; 1-LEAL, R.B.; 1-SILVA, C.A.A.; 1-LIMA, A.P.C.A.; 1-VIEYRA,
A.; 2-DE FELICE, F.G.; 2-FERREIRA, S.T.; 1-MENDEZ-OTERO, R.
1-Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de
Janeiro, Brazil.2-Institute of Medical Biochemistry, Federal University of Rio de Janeiro,
Rio de Janeiro, Brazil.
"Alzheimer's disease is a neurodegenerative disease whose incidence is expected to
increase with the ageing of the world population. So far, there are no effective
therapeutic alternatives. Thus, cellular therapy is a perspective for treatment of this
neurodegenerative disorder. In bone marrow there are hematopoietic stem cells (HSC),
which give rise to hematopoietic lineage and mesenchymal stem cells (MSCs), which
form the connective tissue cells of bone marrow. The main hypothesis on the
mechanism of action of MSCs is their paracrine action, releasing trophic factors that
could act in the stimulation of neuronal survival, neurogenesis and modulation of
inflammation. In this work, we aim to evaluate the effects of treatment with bone
marrow mesenchymal cells in hippocampal cultures exposed to Aβ-derived diffusible
ligands (ADDLs), the main neurotoxins responsible for the adverse effects observed in
the beginning of disease. We found that ADDLs were able to bind to MSCs, without
change the proliferation after 24h, cell respiration until 48h and viability during the
whole period analyzed (maximum of 72h of exposure). In addition, MSCs were resistant
to the oxidative stress generated by ADDLs after 24h of exposure, unlike observed in
neurons. Furthermore, initial analysis suggests that the co-culture with mesenchymal
stem cells prevents oxidative stress in hippocampal cultures exposed to Aβ oligomers
(ADDLs). Thus, our data suggest that MSCs can represent a good therapeutic approach
for the treatment of initial signs of Alzheimer’s disease.
Key words: Alzheimer’s disease, mesenchymal stem cells, in vitro model"
INBEB, FAPERJ, CNPq, PROTECEL, CAPES
D44.
STUDIES ON THE ENERGY AND REDOX METABOLISM OF THE BLOOD FLUKE
SCHISTOSOMA MANSONI
1 - Oliveira, MP.; 1- -Oliveira, M.F
1 - Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro
The blood fluke Schistosoma mansoni is an intravascular parasite which causes human
schistosomiasis. Within the human host, the adult forms of S. mansoni depend on the
ingestion of large amounts of blood to supply their energy demands. Furthermore,
evidence indicates that important metabolic changes occur in worms in several stages
Página 51
of their life cycle, so that the cercariae present essentially an oxidative metabolism,
while the adult forms living within the vertebrate blood vessels have a more
fermentative metabolism. Furthermore, the activity and expression of antioxidant
enzymes increases during development of the worm in the vertebrate host which is
parallel with the ingestion of blood. Thus, we aim to investigate the redox and energy
metabolism in adult forms of S. mansoni. We observed that the oxygen consumption
associated with the Electron Transport System (ETS) is significantly lower in females
than in males. This phenomenon occurs independently of the type of substrate used by
worms. Glucose uptake was higher in females than in males suggesting increased
dependence on glucose metabolism in females. Evaluation of ETS complexes activities
showed that females have higher complex I-III and lower complex IV compared to
males. Hydrogen peroxide production was significantly higher in whole females than in
males. Pro-oxidant challenge with menadione showed that females are significant more
resistant than males. We therefore conclude that there is a difference in energy
metabolism and redox between adult female and adult male worms, which may be
relevant to allow these organisms to the blood feeding habit.
INBEB, FAPERJ, CNPq
D45.
AVALIAÇÃO DA ATIVIDADE DE MODULADORES DA AGREGAÇÃO DA
PROTEÍNA PRION: ABORDAGENS TERAPÊUTICAS
1- ARAUJO, N. C. F.;2- MASCARELLO, A.;2- CHIARADIA, L. D.;2- NUNES, R. J.;2- YUNES,
R.;1- MACEDO, B.;1- ALVES, W. J.;1- MACHADO, C. S. C.; 3- CAUGHEY, B.; 1-CORDEIRO, Y.
1- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro; 2- Departamento de
Química, Universidade Federal de Santa Catarina; 3- National Institutes of Health,
Hamilton, Montana
"As
Encefalopatias
Espongiformes
Transmissíveis
(EETs)
são
doenças
neurodegenerativas fatais que afetam humanos e animais. Normalmente 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. Essas doenças ocorrem quando a proteína príon celular (PrPC) é
convertida na sua isoforma infecciosa, a PrP scrapie (PrPSc). Essa conversão é
caracterizada por uma alteração na estrutura secundária dessa proteína, que passa de
uma proteína rica em alfa-hélices (PrPC) para uma proteína rica em folhas-beta (PrPSc),
que forma agregados amiloides que se depositam no sistema nervoso central. Apesar
do grande número de estudos na área, não há até o momento terapia para estas
doenças, reforçando a necessidade de se buscar compostos eficazes em inibir essa
conversão. Neste trabalho utilizamos como modelo de estudo o peptídeo da PrP de
hamster sírio (Sha 109-149), que compreende um domínio hidrofóbico envolvido nessa
conversão e que agrega prontamente em solução aquosa. Através da técnica de
espalhamento de luz estático avaliamos a capacidade de uma biblioteca de compostos
em inibir a agregação deste peptídeo. A redução da formação de agregados
estruturalmente organizados foi acompanhada através da técnica de fluorescência da
tioflavina T. A toxicidade desses compostos foi analisada através do ensaio de disfunção
celular por redução de MTT e, através da técnica de dot blot verificamos a capacidade
dos compostos em inibir o acúmulo da PrPSc em células de neuroblastoma infectadas
(ScN2a). Através do “docking” molecular foram gerados modelos de interação entre a
PrP e os compostos. Nossos resultados preliminares mostraram que alguns dos
compostos avaliados apresentaram atividade moduladora da agregação do peptídeo da
PrP e não mostraram toxicidade frente ao ensaio de viabilidade celular. Além disso, 47
compostos foram eficazes em reduzir o acúmulo da PrPSc em células ScN2a."
CAPES, FAPERJ, INBEB, CNPQ
D46.
THE PUTATIVE HEPATITIS C VIRUS FUSION PEPTIDE INTERACTS WITH
MODEL MEMBRANES AND GAINS HELIX CONTENT
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- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2- Faculdade
de Farmácia, Universidade Federal do Rio de Janeiro; 3- Instituto de Química,
Introduction: The Hepatitis C virus (HCV) is an enveloped RNA virus, and the most
common cause of chronic liver disease. Although much information has been gathered
in recent years, the exact mechanisms that lead to membrane fusion are poorly
understood. The envelope glycoprotein, E1 or E2, directly interacts with biological
membranes, throughout the fusion peptide (FP). As the localization of FP remains
unknown, we aim to characterize the interaction of a putative HCV FP, corresponding to
residues 421-445 (HCV421-445), present in E2, with different membrane models.
Methods: We used spectrophotometry, fluorescence spectroscopy, circular dichroism
(CD), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC),
dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) to evaluate
peptide-membrane interaction and conformational changes due to this interaction.
Results: ITC and fluorescence polarization data showed that HCV421-445 can interact
with large unillamelar vesicles (LUV). However, DSC revealed that the peptide cannot
change the phase transition temperature of multilamellar vesicles. Photometry analyses
revealed that the peptide can induce LUV aggregation, but DLS data revealed that the
peptide can either disrupt or aggregate the same vesicles. Since the interaction of fusion
peptides with membranes can lead to an important structural gain, we also aim to solve
HCV421-445 structure. NMR data of the peptide free in solution indicated low structure
content but addition of SDS micelles leaded to a structural gain, confirmed by CD data.
The preliminary structure calculation showed that the C-terminal of the peptide in the
presence of micelles presents a tendency to form an alpha-helix, which could be
stabilized by a helix stabilization motif, GXXXG. Conclusions: Altogether our results
indicate that HCV421-445 peptide is able to perturb lipid membranes, and becomes
structured. These studies are important to better understand the fusion mechanisms of
HCV, which could help in the development of new antiviral therapies.
Capes, CNPq, FAPERJ, PRONEX, INBEB
D47.
ANTI-INFLAMMATORY ACTION OF SILDENAFIL (VIAGRA®) TO PROTECT THE
PLACENTAL INTEGRITY IN ABORTION MODEL INDUCED BY LIPOPOLYSACCHARIDES
RAYANA LEAL DE ALMEIDA LUNA1, ANA KAROLINA SANTANA NUNES1, ANNE GABRIELLE
VASCONCELOS DE OLIVEIRA2, SURA WANESSA SANTOS ROCHA1, KARLA PATRÍCIA DE
SOUSA BARBOSA1, WILMA HELENA DE OLIVEIRA2, SHYRLENE MERY DA ROCHA, MARIA
DA CONCEIÇÃO CARVALHO2, CHRISTINA ALVES PEIXOTO1,2*
1Laboratório de Ultraestrutura, Centro de Pesquisas Aggeu Magalhães, Fundação
Oswaldo Cruz, Recife-PE, Brasil; 2Laboratório de Microscopia, Centro de Tecnologias
Estratégicas do Nordeste (MCT/CETENE
Infertility is an important problem of worldwide public health, but the mechanisms
related to gestational complications are not completely understood yet. Abortion is an
inflammatory and thrombotic process. Sildenafil plays an important role as vasodilator
and anti-inflammatory properties. This study investigated the action of Sildenafil on
placental integrity in a model of loss pregnancy induced by LPS, on treatment alone or in
coadjutant treatment with Heparin. Eight females Albino Swiss aged 60, and weighing
30g were used per group. Control-group: females did not receive any treatment neither
LPS administration; LPS-group: females received only the administration of LPS;
Sildenafil-group: treated with 50mg/Kg of Sildenafil (Viagra®) administrated through the
Página 52
drinking and received LPS administration; Heparin-group: treated with low weight
molecular Heparin 500UI/Kg (Fragmin®) per day and received LPS administration; the
Sild+Hep-group received both treatments and the LPS administration. Treatments were
realized since first day of pregnancy. The model was induced by LPS injection on the
15th day and after 2h the females were euthanized. Placentas were processed for
Histopathology, Electron Transmission Microscopy, immunohistochemistry and western
blotting to TNF-α. In placental labyrinth region, the LPS caused edema, congestion and
degeneration; all treatments resulted in the improvement of such conditions.
Ultrastructural analysis of the labyrinth showed that exposure to LPS induced drastic
alterations such as intense degenerations in the labyrinth cells. The treatments
protected in different levels the tissue. The LPS group increased immunostaining for
TNF-α, mainly in the labyrinth area. In contrast, the Sildenafil group reduced TNF-α
labeling, the treatment was effective in reducing inflammation. The Sil + Hep group had
the best results, showing significant immunostainnig reduction. These results were
confirmed by western blotting analysis. Further studies are necessary to establish if
Sildenafil can become a future treatment to female infertility especially those with
inflammatory component.
INBEB, FACEPE, Capes, CNPq
D48.
MODELAGEM TRIDIMENSIONAL DO COMPLEXO APICAL DE T. GONDII
1,2- TRAVASSOS, R.; 1,2- PAREDES-SANTOS, T.C.; 1,2- DE SOUZA, W.; 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;
Situado na região apical do Toxoplasma gondii, o complexo apical é constituído pelo
conóide e estruturas do citoesqueleto a ele associadas e pelas organelas secretórias,
róptrias e micronemas. Essas organelas liberam proteínas diretamente envolvidas com
os processos de reconhecimento, adesão e formação do vacúolo parasitóforo. As
róptrias e os micronemas possuem forma e distribuição peculiar dentro da célula. As
róptrias são alongadas e costumam estender-se desde a região próxima ao núcleo até o
interior do conóide. Os micronemas são menores em tamanho, mas estão em maior
quantidade e em geral se localizam na região abaixo do conóide. Pouco se sabe sobre a
forma e o local de secreção dessas organelas. Utilizando cortes ultrafinos e espessos de
amostras submetidas a congelamento por alta-pressão e crio-substituição e a elétrontomografia, buscamos demonstrar com maior precisão a localização dos micronemas
dentro do corpo do parasita e com isso demonstrar seu possível local de secreção.
Através das imagens por microscopia eletrônica de transmissão conseguimos observar a
distribuição dos micronemas, em forma radial, logo abaixo do anel polar apical. Essa
distribuição ocorre de forma constante em vários materiais observados. Nos relatos da
literatura, assume-se que a secreção dos micronemas e das róptrias se dá pelo interior
do conóide, entretanto com o uso da elétron-tomografia reconstruímos essa região do
parasita e não observamos a presença de micronemas dentro do conóide, todos sempre
se localizavam abaixo e em torno do mesmo. A tomografia eletrônica de taquizoítas
extraídos e contrastados negativamente, também acrescentou maior detalhamento da
estrutura do citoesqueleto apical.
CAPES, FAPERJ, CNPq
D49.
THE ROLE OF LYSINE 35 AS AN IMPORTANT TRANSTHYRETIN ANTIAMYLOIDOGENIC GATEKEEPER RESIDUE
1-SANT'ANNA,R.O.; 2-VENTURA, S.; 1-BRAGA, C.A.; 1-FOGUEL D.
1-INSTITUTO DE BIOQUÍMICA MÉDICA, UFRJ; 2-UNIVERSIDADE AUTONOMA DE
BARCELONA
The aggregation of proteins into amyloid fibrils maybe is a generic feature of most
polypeptides sequences, if not all, under appropriated conditions. Besides the ones
those find their biological function on the amyloid state, the great majority have to
remain on their soluble state. It seems that nature has raised mechanisms to counteract
the tendency for aggregation. An example of such strategies is the placement of
charged residues, known as gatekeepers, in key positions of the protein primary
sequence. This idea has been supported by numerous recent in silico studies using
whole proteomes as object of research. Here, we aim to explore if this evolutionary
constrain is acting on the amyloidogenic protein transthyretin (TTR).We choose as
model a stretch of TTR sequence that has been pointed out as decisive for the formation
of the TTR amyloidogenic intermediate as well as concentrates many known natural
amyloidogenic mutants. This segment comprises the residues 26 to 57. In fact, using a
variety of bioinformatic tolls we found that this stretch has a very high tendency for
aggregation in comparison to the entire protein sequence. Moreover, the presence of a
lys (gatekeeper) at position 35, flanking this prone to aggregation segment, encouraged
us to evaluate the importance of this residue in playing with the aggregation propensity
of the peptide and confirm its rule as a TTR gatekeeper. As this peptide has other lys
(position 48) we performed two rational changes by a highly aggregation favorable
residue leu (K35L and K48L). Our experimental data support this hypothesis and the
predictions of the algorithms. We found that secondary structure propensity vary
dramatically between the three sequences, being the K35L by far the most prone for
beta sheet formation. Analyzing amyloid formation by a lot of techniques we found that
the substitution at position 35 converts the WT peptide into a sequence that display all
classic characteristics of amyloid proteins, like seeding dependent aggregation,
presence of spherical nucleus on the pathway for fibril formation and highly ordered
fibrils that bind thioflavin-T. The K48L substitution did not trigger such behavior,
confirming the importance of the right position for a gatekeeper. The WT sequence is by
far the less prone to aggregation and only forms fibrils when induced by drastic
conditions like incubation with SDS and HCl for long periods. Its aggregation pathway
goes through a different intermediate, that displays alpha helical secondary content.
More interestingly, is the recovery of the amyloidogenic potency of the WT peptide
when it is in the presence of the physiologically relevant co-factor heparin, like has been
demonstrated for the entire protein. To further confirm the rule of residue 35 on
converting the sequence into aggregation prone, we performed limited proteolysis with
proteinase K to map the fibrils architecture and confirmed the presence of this residue
on fibrils core by mass spectrometry sequencing. These data together help to
understand how proteins have escaped from aggregation and shed light on the field of
rational design of proteins and formation of fibrils for nanotechnology.
INBEB, FAPER e CNPQ
D50.
INCORPORAÇÃO
DAS
VESÍCULAS
LIBERADAS
PELAS
FORMAS
TRIPOMASTIGOTAS PELA CÉLULA LLC-MK2 E ULTRAESTRUTURA DAS VESÍCULAS DE
AMASTIGOTAS DO CLONE CL-BRENER DO Trypanosoma cruzi 1NEVES, R.F.C., 1- SOUTO-PADRÓN, T.
1-Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio
de Janeiro, Brasil
Patógenos liberam para o meio extracelular fatores de virulência associados a vesículas,
oriundas de brotamentos (microvesículas) ou fusão de corpos multivesiculares com
pequenas vesículas (exossomos) com a membrana plasmática. Vesículas podem ser
incorporadas pelas células através de eventos de fusão entre as membranas vesículacélula-alvo ou endocitose pela célula recipiente, interferindo ou modulando a resposta
imune ou ação de células hospedeiras. Formas tripomastigotas do Trypanosoma cruzi,
Página 53
liberam constitutivamente vesículas de “shedding”. Sabendo da importância das
vesículas na comunicação celular, analisamos a interação das vesículas liberadas pelos
tripomastigotas de cultura do clone CL Brener do T. cruzi com a célula LLC-MK2 e a
ultraestrutura das vesículas de formas amastigotas desse parasito, que são importantes
para o estabelecimento da infecção no vertebrado. Formas tripomastigotas foram
incubadas em Hank’s sem soro por 3h-37°C e as vesículas, obtidas da ultracentrifugação
do sobrenadante, foram marcadas com análogo lipídico fluorescente (DiI). Células LLCMK2 internalizaram as vesículas, sendo a entrada não dependente de “lipid rafts”. Por
imunofluorescência, após 2h-37°C de interação, observamos vesículas direcionadas para
compartimentos endocíticos acídicos, lisossomos e endossomos tardios da LLC-MK2.
Assim como tripomastigotas, observamos por microscopia de transmissão, que
amastigotas liberam vesículas de 40-170nm para espaço extracelular apresentando
glicocálice semelhante ao observado na sua superfície. Nas amastigotas, observamos
estruturas semelhantes a corpos multivesiculares contendo múltiplas vesículas com
aproximadamente 100nm de diâmetro que também puderam ser encontradas no
interior da bolsa flagelar do parasito, sugerindo uma possível liberação das vesículas
oriundas de compartimentos intracelulares do parasito para o meio extracelular.
CNPq, CAPES, FAPERJ, Pronex
D51.
INTERACTION OF DENGUE AND YELLOW FEVER VIRUS WITH
MEGAKARYOBLASTS: ROLE IN HEMOSTATIC ALTERATIONS
1- CAMPOS, S.P.C.;1- CASTRO, M. G.;1- SANCHES; D.;1- ROCHA, C.M.;2- RODRIGUES, M.
F.;3- PAREDES, B. D.; 1- SILVA, J.L.; 1- GOMES, A.M.O. & 1- OLIVEIRA, A.C.
1- Laboratório de Termodinâmica de Proteínas e Estruturas Virais Gregorio Weber IBqM/UFRJ, 2- Laboratório de Biologia Molecular do Câncer - IBqM/UFRJ; 3- Laboratório
de Cardiologia Celular e Molecular – IBCCF/UFRJ
"Introduction: Dengue Virus (DENV) and Yellow Fever Virus (YFV) have great importance
in economy and public health in Africa, South America and Asia. They are the etiologic
agents of acute hemorrhagic fevers that are related to hemostasis dysfunction, with
coagulation factors consumption and thrombocytopenia. The low platelet count is
related to the evolution of the disease severity. Platelets play a crucial role in
hemostasis and are cytoplasmic fragments of megakaryocytes. Each megakaryocyte
produces from 5.000 to 10.000 platelets. The processes that lead to disease severity
evolution have not yet been elucidated. Aim: To better clarify the processes in which
viral infection leads to thrombocytopenia, we aim to study the interaction between
DENV and YFV with megakaryocyte precursors. Material and Methods: We infected
MEG-01 cells (Human Megakaryoblastic cell line) with DENV-2 and YFV 17 DD in a
multiplicity of infection of 1. Results and Discussion: We confirmed YFV infection by
detecting intracellular YFV proteins since 24h post infection (p.i.) by confocal
microscopy. We analyzed the production of infectious particles by plaque assay and
observed increasing production until 96h p.i. and followed by decrease. We analyzed
cell viability by extracellular activity of LDH and trypan blue exclusion. We observed
higher LDH activity from 96h p.i. with YFV but not with DENV-2. A decrease of cell
number was evident after 72h p.i. but we only observed an increase of cell mortality
from 120h p.i. for both viruses. We observed mitochondrial physiology changes during
DENV and YFV infection by measuring oxygen consumption. We also did not observe cell
differentiation profile changes from the control. Conclusion: Our data suggest that YFV
can infect and replicate in MEG-01 cells. Our data also suggest that DENV-2 and YFV
infections inhibit cell growth until 72h and induce cell death from 120h p.i., with
mitochondrial alterations without changing cell differentiation kinetics."
CAPES, CNPQ, FAPERJ, FINEP, INBEB, PRONEX
D52.
EVALUATION OF THE SYNERGIC EFFECTS OF ERGOSTEROL BIOSYNTHESIS
INHIBITORS IN LEISHMANIA AMAZONENSIS
1- MACEDO-SILVA, S. T; 2- URBINA, J. A.; 1- DE SOUZA, W.; 1- RODRIGUES, J. C. F.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,
Brazil; 2- Instituto Venezolano de Investigaciones Científicas, Venezuela.
Leishmaniases are among the most prevalent neglected tropical diseases and are
endemic in 98 countries. The treatments include the pentavalent antimonials,
miltefosine, amphotericin B, and pentamidine. However, such drugs are unsatisfactory
due to toxicity, limited efficacy, cost and administration. Therefore, there is an urgent
need to develop new drugs that are efficacious, safe, and more accessible.
Trypanosomatids have an essential requirement for ergosterol and other 24-alkyl
sterols, which are absent in mammalian cells. The inhibition of ergosterol biosynthesis
(EB) has been recognized as a promising target for the development of new
chemotherapeutic agents. Thus, the aim of this work was to investigate the effects of
E5700, a squalene synthase inhibitor, in combination with itraconazole (ITZ) or
posaconazole (POSA), two known inhibitors of the sterol C14α-demethylase (CYP51),
against Leishmania amazonensis in vitro. E5700, ITZ and POSA alone produced a marked
reduction in the viability of L. amazonensis promastigotes, with MIC (minimum
inhibitory concentration) values of 30 nM, 1 µM, and 1 µM, respectively. Several
combinations were tested and the most efficient was the combination of 1.25 nM
E5700 with 40 nM ITZ or 0.625 nM E5700 with 5 nM POSA, which resulted in FIC
(fractional inhibitory concentration) values of 0.082 for the combination of E5700 with
ITZ and 0.026 for E5700 with POSA, indicating a potent synergistic effects. Against
intracellular amastigotes, the MICs for E5700, ITZ and POSA alone were 30 nM, 1 µM,
and 1 µM, respectively. The results indicated again strong synergism, with MICs of 2.5
nM E5700 plus 20 nM ITZ (FIC=0.103) and 2.5 nM E5700 plus 2.5 nM POSA (FIC=0.085).
Differential interference contrast microcopy (DIC) revealed a significant alteration on
the shape of promastigotes after treatment with E5700 in combination with POSA, more
than that observed with ITZ. Transmission electron microscopy of treated-parasites
showed several alterations such as: 1) Presence of lipid bodies; 2) Intense mitochondrial
swelling followed by the loss of matrix content; and 3) Presence of autophagosome-like
structures. In summary, our results indicate that combinations of EB inhibitors acting at
different steps of the pathway have synergistic activity against L. amazonensis and open
up the possibility of a novel combination therapies for the treatment of leishmaniasis.
CNPq, CAPES, FAPERJ.
D53.
SYNTHESIS AND CHARACTERIZATION OF DERIVATIVES OF CHLORINATED
COMPOUNDS PHENANTHRENEQUINONE USING TRICHLOROISOCYANURIC ACID
1-AZEREDO, S,O,F; 1-BARRETO, R,B,L; 1-FIGUEROA-VILLAR, J.D.
1-Military Institute of Engineering
Halogenated aromatic compounds are used in the synthesis of natural products and
with important biological activity. Many of the methods of direct halogenation of
aromatic systems involve the use of elemental halogen or expensive transition metal
based catalysts. A reagent that has been used for chlorination of aromatic compounds
enabled or disabled is trichloroisocyanuric acid (TCCA), which is a reagent that provides
"Cl +". TCCA is easy to access and manipulate, being also stable and inexpensive.
Reactions of aromatic substitution electrophilic using trichloroisocyanuric acid can be
performed on solutions polar aqueous solution of sulfuric acid 50% (v / v) or using a
Lewis acid as catalyst . The objective of this work is to use acid trichloroisocyanuric for
chlorination phenantrenequinone and use chlorinated compounds obtained as
intermediates for the synthesis of compounds with potential biological activity.
CAPES, CNPq, FAPERJ, INBEB, MINISTÉRIO DA DEFESA
Página 54
D54.
DIETHYLCARBAMAZINE REDUCES THE INFLAMMATORY PROCESS IN
CHRONIC HEPATIC C57BL/6J MICE
1-ROCHA, S.W.S.; 1-FRANÇA, M.E.R; 1-BARBOSA, K.P.S.; 1-NUNES, A.K.S; 2-OLIVEIRA,
A.G.V. ; 2-OLIVEIRA, W.H.; 1-RODRIGUES, G.B.; 1-ARAÚJO, S.N.R.; 1,2-PEIXOTO, C.A
1- Centro de Pesquisas Aggeu Magalhães- CpqAM/FIOCRUZ, Brazil 2- Centro de
Tecnologias Estratégicas do Nordeste- CETENE/MCT, Brazil
Pharmacological studies show that DEC interferes in the arachidonic acid metabolism,
acting as an anti-inflammatory drug [1]. Rocha et al (2012) demonstrated that DEC can
be a potential drug for the treatment of chronic inflammation induced by chronic
alcoholism and as a hepatoprotective drug in reducing lesions in mice malnourished
[2,3]. In the present study we investigated the effect of DEC on chronic liver
inflammation induced by carbon tetrachloride (CCl4). Forty C57BL/6J male mice were
separated in groups (n=10): control group, DEC 50mg kg group, CCl4 group and CCl4 +
DEC group. DEC (50mg/kg) was administered in bottle for 12 days. CCl4 (0.5 ml/g) was
administered for 6 weeks (2 injections per week). Liver fragments were processed for
histological (HE and Sirius red), immunofluorescence and molecular analysis.
Histopathological findings in the control group and DEC 50mg/kg group showed no
change in their morphology pattern. In the group of animals exposed to CCl4 was
observed striking cytoplasmic and nuclear degeneration, with the presence of fibrosis,
inflammatory infiltrates and hemorrhagic foci. The animals treated with CCl4 + DEC
showed a decrease of all lesions observed in CCl4 group. In staining specific for collagen
CCl4 group intense staining was observed in fibrotic areas. However, CCl4 + DEC group
showed reduced collagen labeling. Results of immunofluorescence revealed strong
increase of inflammatory markers such as IL-1β and α-SMA in fibrotic areas and
mononuclear infiltrates, especially in perivenulares areas in CCl4 group. However, a
reduction of immunoreactivity of these markers was observed after treatment with DEC
50 mg/kg. Western blot analyzes showed increased expression of COX-2, IL-1β and NFkB in the group subjected to chronic liver injury and there was a significant decrease in
expression of these proteins after treatment with DEC 50mg/kg. According to the
present results, DEC is a possible alternative treatment for chronic liver inflammation
induced by CCl4.
INBEB, FACEPE, CNPq.
D55.
A TOXOPLASMA GONDII STRAIN THAT SPONTANEOUSLY FORM CYSTS IN
DIFFERENT CELL TYPES
PAREDES-SANTOTS T.C.1, TIBAU R. 1, DE SOUZA W. 1, ATTIAS M. 1, VOMMARO R.C.
1
Instituto de Biofísica Carlos Chagas Filho
Conversion of Toxoplasma gondii tachyzoites to the bradyzoite stage and tissue cyst
formation have crucial roles in the establishment of chronic toxoplasmosis. In this work
we investigated the in vitro cystogenesis and behavior of the EGS strain, isolated from
human amniotic fluid. We observed that tachyzoites of the EGS strain converted to
intracellular cysts spontaneously in LLC-MK2 epithelial cells, HSFS fibroblasts and C6 glial
cell lineage. The peak of conversion occurred in the LLC-MK2 cells after 4 days of
infection, when 72.3±15.9 of the infected cells contained DBA positive cysts. Using
specific markers against bradyzoite, tachyzoite and cyst wall components, we confirmed
stage conversion and distinguished immature from mature cysts. It was also observed
that the deposition of cyst wall components occurred before the total conversion of
parasites. Transmission electron microscopy confirmed the full conversion of parasites
presenting the typical characteristics of bradyzoites such as the posterior position of the
nucleus and the presence of amylopectin granules. A thick cyst wall was also present.
Furthermore, scanning electron microscopy revealed that the intracystic matrix had
tubules that were shorter than those from the intravacuolar network of the
parasitophorous vacuole and were immersed in a granular electron dense material. The
EGS strain spontaneously forms high burden of cysts in cell culture without artificial
stress conditions, and constitutes a useful tool to study this stage of the T. gondii life
cycle.
D56.
DNA BINDING AND CLEAVAGE ANALYSIS OF TWO NEW FEIIIZNII
COMPLEXES WITH PYRENE MOTIFS
1-BORTOLOTTO, T.; 2-CAMARGO, T.P.; 2-NEVES, A.; 1-TERENZI, H.
1- Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade
Federal de Santa Catarina, Santa Catarina, Brazil; 2- Departmento de Química,
Universidade Federal de Santa Catarina, Santa Catarina, Brazil.
Purple acid phosphatases (PAPs) are a group of metallohydrolases that contain a
heterovalentdinuclear FeIIIMII center (MII = Fe, Mn, Zn) in the active site, and which are
able to catalyze the hydrolysis of a variety of phosphoric acid esters and anhydrides.
Recently, we have demonstrated the DNA cleavage ability of FeIIIZnII complexes using
the unsymmetrical donor ligand 2-[N-bis-(2 pyridylmethyl)-aminomethyl]-4-methyl-6*N’-(2-pyridylmethyl)(2-hydroxybenzyl) aminomethyl]phenol (H2L-H) that mimics the
active site of red kidney bean PAP. To improve this ability, the complex ligand was
specifically altered by the addition ofone or twopyrene motifs that tightly binds to the
DNA structure, facilitating the nucleophilic attack of phosphodiester bonds. Herein, we
report the initial DNA binding and cleavage studies of the complex [FeIII-(μ-OH)ZnIILPyrene] (1) compared to its parent complex with two pyrene motifs [FeIII-(μ-OH)ZnIILPyrene2] (2).Thermal denaturation of DNA was used to evaluate the binding of the
complexes to the DNA structure and their ability to cleave DNA was examined following
the conversion of supercoiled form of pBSK-II plasmid into its cleaved forms, using
agarose gel electrophoresis. Both complexes showed to interact to dsDNA with similar
patterns on short DNA fragments (10-bp, 60% GC) or long fragments (CT-DNA, ~1-10kb,
42% GC). In terms of DNA cleavage, both complexes can cleave the DNA molecule
introducing single- and double-strand breaks in a time and concentration-dependent
manner. The pH-activity profile showed a peak of cleavage around 7.0 due to the
formation of nucleophilic hydroxyl group at the metal center, in agreement with the
hydrolytic pathway of strand scission. In conclusion, new pyrene derived metal
complexes seem to be interesting models to develop improved synthetic nucleases.
CNPq, CAPES, MCT, FINEP, FAPESC, INCT - Biologia Estrutural e Bioimagem.
D57.
INSTABILITY OF A CARDIOMYOPATHY MUTANT OF CARDIAC TROPONIN C
AMPLIFIES ITS CA2+-SENSITIZING EFFECT AT PHYSIOLOGICAL TEMPERATURES
1,2- VELTRI, T.; 1- Bienkiewicz, E.A.; 2- PALHANO F.L.; 2- SORENON, M.M.; 1- PINTO, J.R.
1- Department of Biomedical Sciences, Florida State University, College of Medicine; 2Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro
Although it has been linked to hypertrophic cardiomyopathy in humans, the cardiac
troponin C mutant D145E has never been investigated for its impact on the thin
filament at physiological temperatures. Here, we demonstrate that this C-domain
mutation causes generalized instability of the isolated protein and that this leads to
defective Ca2+ regulation in the N-domain. Circular dichroism of the isolated HcTnC
isoforms revealed that D145E was more sensitive to thermal unfolding, with Tmelt at
52-56oC for apo, Mg2+ and Ca2+ states compared to 67-78oC for the WT protein. Even
at 10-20oC in the presence of Ca2+ or Mg2+, D145E was almost completely unfolded by
3 M urea, while the WT protein remained intact up to 50o/60oC. Remarkably,
physiologically relevant temperatures were associated with progressive increases in
Página 55
Ca2+ affinity for D145E but not for the WT protein, as measured using a fluorescent
probe attached to Cys residues: pCa50 increased by 0.98 log units from 21o to 45oC,
compared to an increase of 0.26 units for WT . As expected, both proteins became more
sensitive to Ca2+ when incorporated into cardiac skinned fibers, but this increase was
greater near physiological temperatures. Maximal isometric force in HcTnCreconstituted fibers was essentially identical with both proteins at 21oC, but at 30oC the
force was 40% lower with the mutant, reflecting its instability. Thus, D145E destabilizes
regions of HcTnC that lie closer to the N terminus, especially near physiological
temperatures, where a higher Ca2+ affinity is correlated with loss of secondary
structure and becomes an important hallmark of its impaired regulatory function.
Future studies of cardiomyopathy mutants can benefit from including experiments at or
near physiological temperatures, where the full range of their functional defects is
exposed.
INCT-INBEB, CNPq, FAPERJ, CAPES, NIH
D58.
STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE HEPATITIS C VIRUS CORE
PROTEIN
BRAGA, V. L. A.1; MENDES-SILVA, A.1 ; CARVALHO, C. A. M.1; BIANCONI, M. L.1;
PEABODY, D.2; SILVA, J. L.1; GOMES, A. M. O.1; SOUZA, T. L. F.2& OLIVEIRA, A. C.1
1Programa de Biologia Estrutural/IBqM/UFRJ, Brasil. 2Department of Molecular
Genetics and Microbiology and Cancer Research and Treatment Center/UNMSM/USA.
3Faculdade de Farmácia/UFRJ/Brazil.
Approximately 170 million people are infected worldwide with the Hepatitis C Virus
(HCV), which clearly represents a public health problem. The HCV core protein (HCVCP)
is responsible for the interaction with the viral RNA and capsid assembly. Three regions
of the HCVCP are specifically important during nucleocapsid assembly: the sequences
containing
the
amino
acids
22-39
(VKFPGGGQIVGGVYLLPR),
50-67
(RKTSERSQPRGRRQPIPK) and 85-102 (PWPLYGNEGMGWAGWLLSPRG). To better
understand the structural and physicochemical aspects of their interactions with the
RNA and the viral envelope during HCV assembly we used membrane models (micelles),
such
as
sodium-dodecyl-sulfate,
n-octyl-b-D-glucopyranoside,
Hexadecyltrimethylammonium-bromide and n-dodecylphosphocholine, and non specific
nucleic acids. In the presence of different micelles, only the peptide 85-102 adopted an
alpha-helix structure as verified by circular dichroism. Analyses of tryptophan intrinsic
fluorescence and acrylamide quenching reveal that the interaction between peptide 85102 and micelles probably involves a partial internalization of the tryptophan residue.
Calorimetric measurements reveal different heat profiles for the interactions of micelles
and the different peptides and showed similar thermodynamics of the interaction of
peptide 50-67 with different DNAs. Fluorescence polarization data showed that, in the
range of concentrations used, the peptides do not prevent the formation of NLPs by the
interaction between core protein and nucleic acids. We also investigate the cellular
localization and the assembly process in HepG2 and Huh7. Confocal microscopy data
showed that 24 hours post transfection the HCVCP fused with the Green Fluorescent
Protein (GFP) (HCVCPGFP) is located in the nucleus. In Huh7 cells, HCVCPGFP seems to
be located on lipid droplets surface. Our data reveal a new approach to understand the
HCV assembly, which is a great target for the development of drugs anti-HCV.
CNPQ, CAPES, FAPERJ, FUJB, INBEB, PRONEX, FINEP
D59.
DIFFERENTIATION OF GIARDIA LAMBLIA: THE CYST WALL UNDER
CONSTRUCTION
1,2- Midlej, V.; 3, 4, 5- de Souza, W.; 1, 4, 5- Benchimol, M.
1- Universidade Santa Úrsula. Rio de Janeiro, Brazil; 2- Programa de Pós-graduação em
Ciências Morfológicas, Instituto de Ciências Biológicas, Universidade Federal do Rio de
Janeiro – Rio de Janeiro, Brazil; 3- Instituto de Biofísica Carlos Chagas FilhoUniversidade Federal do Rio de janeiro, Brazil;4- Instituto Nacional de Metrologia e
Qualidade Industrial – Inmetro, Rio de Janeiro, Brazil; 5- Instituto Nacional de Ciência e
Tecnologia de Biologia Estrutural e Bioimagem.
Differentiation from one life cycle stage into another is an elegant adaptation by which
many parasites ensure their transmission and survival. The protozoan Giardia lamblia is
a major cause of water-borne diarrheal disease. Nonetheless, the basic biology of this
protist is incompletely understood. This parasite exhibits two forms in its life cycle that
includes trophozoite and cyst. The formation of the resistant extracellular cyst wall
(CW), which is composed by proteins and carbohydrates, begins with intestinal signals,
where the cyst wall proteins (CWPs) are synthesized. All of the CWPs are transported via
encystation-specific secretory vesicles (ESVs) and are released at the site of CW
assembly. Although the presence of carbohydrates in cyst wall has been reported, the
detection and origin of the cyst wall carbohydrate-rich moieties is completely unknown.
Moreover, the changes that happen in the cyst wall (CW) during the excystation process
are poorly studied. Encystation and excystation are crucial processes for establishment
and maintenance of Giardia infection. Thus, the aim of this work is to better understand
the CW: verifying the origin of the carbohydrate components and observe the
ultrastructural modification during the differentiation process. Here we tested the
differentiation process in vitro and analyzed the formation and changes in the CW of the
parasite by complementary techniques, such as scanning and transmission electron
microscopy, cytochemistry for carbohydrates and immunocytochemistry.
Immunofluorescence microscopy using anti-cyst wall protein 1 (CWP1) and gold- and
fluorescent-conjugated DBA lectin were also used. A field emission scanning electron
microscopy (FESEM) was also used to compare the mature cysts and the beginning of
excystation process. Interestingly, encystation carbohydrate-containing vesicles (ECVs),
positive for N-acetyl-galactosamine, were found in the encysting cells. The ECVs are
distinct from the encystation specific vesicles (ESVs) because: (1) they are electron
lucent whereas ESVs are electron dense; (2) they do not react with antibodies against
cyst wall proteins; (3) their contents are positive for carbohydrates, whereas ESVs
display a negative reaction; and (4) this cell compartment exhibits a positive labeling for
DBA lectin indicating the presence of N-acetyl-galactosamine, whereas ESVs are
negative. During the excystation changes in the cyst form and the presence of electrondense vesicles near the CW were observed. The CW was seen in detailed using FESEM.
In mature cyst, the cyst wall microfibrils were seen in a tighter arrangement. However,
when excystation process begins this tight arrangement of the wall microfibrils is lost. In
conclusion, our results suggest that the ECVs may represent a new structure involved in
the cyst wall formation and alteration of CW fibrils is needed for the development of
excystation.
INBEB, AUSU, CNPq, FAPERJ, PRONEX
D60.
BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF A CYTOSOLIC
HSP90 FROM SUGARCANE
1,2- SILVA,V.C.H.;1,2- CAGLIARI, T.C.; 1- LIMA, T.B.;1- GOZZO, F.C.; 1,2,3- RAMOS, C.H.I.
1-Institute of Chemistry;2-Institute of Biology;3-Instituto Nacional de Ciência e
Tecnologia em Biologia Estrutural e Bioimagem
Hsp90s are involved in several cellular processes, such as signaling, proteostasis,
epigenetics, differentiation and stress defense. Although Hsp90s from different
organisms are highly similar, they usually have small variations in conformation and
function. Thus, the characterization of different Hsp90s is important to gain insight into
Página 56
the structure-function relationship that makes these chaperones key regulators in
protein homeostasis. This work describes the characterization of a cytosolic Hsp90 from
sugarcane and its comparison with Hsp90s from other plants. Previous expressed
sequence tag (EST) studies in Saccharum spp. (sugarcane) predicted the presence of an
mRNA coding for a cytosolic Hsp90. The corresponding cDNA was cloned, and the
recombinant protein was purified and its conformation and function characterized. The
structural conformation of Hsp90 was assessed by chemical cross-linking and
hydrogen/deuterium exchange using mass spectrometry and hydrodynamic assays,
which revealed regions accessible to solvent and that Hsp90 is an elongated dimer in
solution. The in vivo expression of Hsp90 in sugarcane leaves was confirmed by western
blot, and in vitro functional characterization indicated that sugarcane Hsp90 has strong
chaperone activity.
INBEB, CAPES, CNPq, FAPESP
Página 57
Pós-doutorandos
PD1.
ENVOLVIMENTO DO FATOR DE TRANSCRIÇÃO NFAT NAS DOENÇAS DE
ALZHEIMER E PARKINSON: CONTRIBUIÇÕES PARA A INFLAMAÇÃO E APOPTOSE
1- ROBBS, B.K.; 1- SPÍNDOLA, T; 1- DE FREITAS, J.A.; 1- LEDO, J.H.; 1- FERREIRA, S.T.; 1FOGUEL, D.
1- Instituto de Bioquímica Médica
Juntas, a Doença de Alzheimer (DA) e de Parkinson (DP) afetam mais de 7 milhões de
pessoas nos Estados Unidos e, são a primeira e segunda causas de demência na
população acima dos 65 anos de idade. Apesar do grande investimento para o estudo
dessas patologias, pouco sabe-se sobre os mecanismos de controle da expressão gênica
que levam à neurodegeneração. Aparentemente o processo de influxo de Ca2+ e
ativação da fosfatase calcineurina A (CnA) podem estar envolvidos no controle da morte
celular programada e do processo de inflamação crônica, que são mecanismos centrais
para a progressiva perda cognitiva e motora em pacientes com essas patologias. Um dos
principais fatores de transcrição ativado pela via de Ca2+/CnA é o fator nuclear de célula
T ativada (NFAT). As funções das proteínas da família NFAT foram inicialmente descritas
e caracterizadas no sistema imunológico. Contudo, o NFAT desempenha um importante
papel na regulação de genes envolvidos no controle da morte celular como TNF-α; cFlip; Fas-L; de mediadores inflamatórios como IL1β; Cox-2; TNF-α, além de estar
envolvido no desenvolvimento e manutenção do sistema nervoso central. Portanto,
postulamos que a regulação gênica mediada pelo NFAT poderia desempenhar um papel
fundamental na DA e DP. Nesta proposta, temos interesse em investigar os mecanismos
moleculares através dos quais as proteínas NFAT regulam a apoptose bem como a
inflamação, e a sua potencial relação com a patogênese dessas doenças
neurodegenerativas. Para tanto, avaliaremos a função das proteínas NFAT na indução
do processo de morte celular programada e inflamação; na regulação de genes
envolvidos nesses processos, e a relevância do NFAT na perda cognitiva e motora de
modelos animais. Os estudos propostos auxiliarão na caracterização dos mecanismos
moleculares que regulam a DA e DP e sua relação com a neurodegeneração, podendo
trazer inovações para definir novos biomarcadores e/ou podendo contribuir para o
desenvolvimento de novas terapias.
INBEB, FAPERJ, CNPq
PD2.
DIFERENTES VIAS DE MORTE CELULAR SÃO INDUZIDAS NO TRYPANOSOMA
CRUZI PELO PEPTIDEO MELITINA
1- ADADE, C.M.; 1- OLIVEIRA, I.R.; 1- PAIS, J.; 1- SOUTO-PADRÓN, T.
1- Instituo de Microbiologia Paulo de Góes
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.
FAPERJ, CNPq, INBEB, CAPES, Pronex
PD3.
SUSTAINED EFFECT OF BONE-MARROW MONONUCLEAR CELL THERAPY IN
AXONAL REGENERATION AFTER OPTIC NERVE CRUSH
1,2- ZAVERUCHA-DO-VALLE,C.; 1,2- MESENTIER-LOURO, L.; 1,2- SANTIAGO, M.F.; 1,2MENDEZ-OTERO, R.
"PURPOSE: In adult mammals, the regeneration of the optic nerve is very limited and at
the moment there are several groups trying different approaches to increase retinal
ganglion cell (RGC) survival and axonal outgrowth. One promising approach is cell
therapy using bone marrow cells. In previous work, we performed intravitreal
transplantation of syngeneic bone-marrow mononuclear cells (BMMCs) after optic
nerve crush in adult rats and we demonstrated an increase in RGC survival and axon
outgrowth 14 days after injury. In the present work, we investigated if these results
could be sustained for a longer period of time. METHODS: Optic nerve crush was
performed in Lister-hooded adult rats and BMMC or saline injections were performed
intravitreally shortly after injury. Neuronal survival and regeneration were evaluated in
rats' retina and optic nerve after 28 days. RESULTS: We demonstrated that there is an
increase of 5.2 fold in the axon outgrowth 28 days after lesion, but the BMMCs effect on
RGC survival was not sustained. In an attempt to prolong RGC survival, we established a
new protocol with two BMMC injections, the first one soon after the lesion and the
second one 7 days after the injury. Untreated animals received two injections of saline.
We observed that although the axonal outgrowth was still increased after the second
BMMC injection, the RGC survival was not prolonged. CONCLUSIONS: These results
demonstrate that BMMCs transplantation promotes neuroregeneration 28 days after
injury. However, the effects on RGC survival previously observed by us at 14 days were
not sustained at 28 days and could not be prolonged with a second dose of BMMC."
PD4.
THE ANTIVENOMIC CHARACTERIZATION OF ANTIBODIES AGAINST ELAPID
THREE FINGER TOXIN AND PLA2 MOLECULES FROM MICRURUS VENOMS.
Correa-Netto, C1,2., Araújo, R. T1,2., Lima L.M.T.R3., Perez-Payá. E4,5., Calvete, J. J4.,
Foguel, D & Zingali, R. B2.
1Instituto Vital Brazil; 2IBQM-UFRJ; 3Faculdade de Farmácia-UFRJ; 4Instituto de
Biomedicina de Valencia, CSIC, Espanha; 5CIPF, Valencia, Espanha.
Polyclonal antibodies have been used for over a century for the treatment of snake bite
envenoming. New strategies and approaches to understand how antibodies recognize
and neutralize snake toxins represent a challenge to improve the antivenoms.
Página 58
Previously we characterized the venom proteome of M. altirostris and M. corallinus
venom and Elapid 3FTx and PLA2 molecules are the major toxic compounds. These
molecules were purified from venoms and used as antigen to produce polyclonal and
monoclonal antibodies against them. Moreover some regions of 3FTx were used as
model to produce synthetic peptides. Three antisera against synthetic peptides were
generated following different procedures. The serum produced by peptides
encapsulated in liposome vesicles were the most reactive to 3FTxs molecules. However,
the antisera were unable to neutralize the neurotoxicity of these proteins in vivo.
Antivenomic analyses showed a poor recognition for many toxic compounds. We also
tested the generation of monoclonal antibodies against the most toxic components. M.
altirostris venom was fractionated, its major toxic proteins identified, and a fraction
containing 3FTx and PLA2 were used to generate a panel of nine monoclonal antibodies.
Six mAbs showed high affinity towards 3FTx and two against PLA2. We characterized the
specificity of all mAb by antivenomic approach and identified a pair of antibodies able to
recognize all PLA2 molecules of these venoms. These results showed that PLA2 of M.
altirostris venom share a pair of conserved antigenic regions and draw attention to use
these epitopes as antigen to generate antibodies against this class of protein.
Support: CNPq, CAPES, FAPERJ, INBEB, Ministerio de Ciencia e Innovación, Madrid,
Spain.
PD5.
INTERACTION STUDIES OF YERSINIA PESTIS PLASMIN (BUBONIC PLAGUE
AGENT) AND HUMAN PLASMINOGEN BY CIRCULAR DICHROISM AND NMR
1-Sarzedas, C.G.; 1-Vidal, T.J.; 1-Barreto, J.R., 1-Tinoco, L.W.
1-Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro
Yersinia pestis is the agent of bubonic plague classified in category A of bioterrorism
agents according with Center for Disease Control and Prevention, USA. Plasmin (Pla) of
Y. pestis is a membrane protein that cleaves/activates the human plasminogen in the
cell invasion process, destabilizing the fibrinolitic cascade causing bleeding. Our goal
was to characterize the pH effect in the Pla folding and their interaction with human
plasminogen peptide (PK2). The structural characterization of PK2-Pla interaction could
be useful to plan mimetic peptides to inhibit such interaction. Gel filtration
chromatography showed that Pla is dimeric at pH 6.5 and monomeric at pH 8.0. CD and
NMR analyses were performed to evaluate the pH effect in the Pla folding and in the
PK2-Pla interaction. CD studies showed structural changes of PK2 in the presence of Pla
at pH 8.0, indicating Pla-PK2 interaction in this condition. NMR spectra of PK2 in the
presence of Pla were acquired for the sample at pH 5.0. It was observed an increase in
the peaks intensities compared with the PK2 free spectra, suggesting less
conformational variability of PK2, probably due PK2-Pla interaction. However, a slight
chemical shift change was observed only for H of VV residues of PK2 in the presence
of Pla, suggesting that Pla-PK2 interaction is affecting specifically the RVV region. In the
plasminogen activation process, Pla cleaves the R561-V562 bond in the sequence
PGRVVGG. The absence of significant alteration on the NMR spectra suggests that PK2Pla interaction at pH 5.0 could be weak, probably because Pla in this condition is in the
dimeric form. These data show that Pla folding and aggregation state is sensitive to pH
changes and interfere in the PK2-Pla interaction.
INBEB, CNPq
PD6.
ALTERAÇÃO DOS NÍVEIS CEREBRAIS DE GLUTAMATO E GLUTAMINA NA
SEPSE EXPERIMENTAL
1- MADEIRA, C.; 1- VARGAS-LOPES, C.; 2- D'ÁVILLA, J.; 3- KURTZ, P.; 3- AZEVEDO, L.; 2BOZZA, F. e 1- PANIZZUTTI, R.
1- Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro; 2- Fundação
Oswaldo Cruz, FIOCRUZ, Rio de Janeiro; 3- Instituto Sírio-Libanês de Ensino e Pesquisa,
São Paulo.
A encefalopatia associada à sepse (EAS) é uma manifestação comum de quadros
infecciosos graves, acometendo até 70% dos pacientes. Alguns pacientes apresentam
disfunção cognitiva meses após o episódio de EAS. Apesar de sua enorme importância
clínica, a fisiopatologia da EAS ainda não é completamente esclarecida. Glutamato é o
principal neurotransmissor do cérebro e participa de diversos processos cognitivos. Nós
investigamos os níveis de glutamato e seu precursor glutamina durante a evolução da
sepse. Induzimos sepse em porcos através da injeção de fezes na cavidade abdominal e
monitoramos os níveis extracelulares dos aminoácidos utilizando um cateter de
microdiálise no hemisfério cerebral esquerdo. Nós observamos maiores níveis de
glutamina no fluido extracelular dos porcos sépticos nos tempos de 12 horas (p< 0,05) e
18 horas (p< 0,01) quando comparados aos porcos controles. Entretanto, não
encontramos diferença nos níveis de glutamato no fluido extracelular entre os porcos
sépticos e os controles. Após o experimento sacrificamos os animais e observamos que
os níveis de glutamato e glutamina totais no tecido estavam significativamente maiores
no córtex frontal dos porcos sépticos do que nos porcos controles (p= 0,017, t= 2,87,
para glutamato; p= 0,013, t= 3,01, para glutamina). Nossos resultados sugerem que
durante a evolução da sepse experimental pode ocorrer uma lesão neuronal ativa,
promovendo alteração nos níveis de glutamato e glutamina.
INBEB, FAPERJ, CNPq
PD7.
THE ROLE OF INOS ON THE GLIAL CELLS ACTIVATION AND
NEUROINFLAMMATION IN DEMYELINATING MODEL
1,2- RAPÔSO, C.; 2,3- NUNES, A.K.S.; 2,3- LUNA, R.L.A.; 3- ARAÚJO, S.M.R.; 1- CRUZHÖFLING, M.A.; 2,3- PEIXOTO, C.A.
1Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual
de Campinas/UNICAMP, Campinas-SP; 2Laboratório de Ultraestrutura CPqAM/FIOCRUZ, 3Centro de Tecnologias Estratégicas do Nordeste /CETENE, Recife-PE,
Brazil
The protective role of iNOS/NO system in neurodegenerative diseases has been matter
of debate. Recently, we have demonstrated that sildenafil protects the neural tissue in a
cuprizone(CPZ)-induced demyelination model in C57BL/6 wild-type (WT) mice. Sildenafil
accumulates cGMP by inhibiting PDE5, but the mechanism of neuroprotection is
unknown. Here, we used male iNOS-/- mice (6 week-old) treated with sildenafil to
investigate
the
nitric
oxide/cyclic
guanosine
3',5'-monophosphate/PDE5
(NO/cGMP/PDE5) signaling pathway to examine sildenafil protection. iNOS-/- mice
received for four weeks (n = 5/group): Pure chow and water (control group); 0.2% CPZ
into the chow; CPZ into the chow plus sildenafil (Viagra®, Pfizer, 25 mg/kg) in the
drinking water. Cerebella were processed for immunofluorescence or western blotting.
CPZ significantly increased GFAP, Iba-1, TNF-α, COX-2, IL-1β and IFN-γ expressions in
iNOS-/- mice. Comparison of eNOS level in WT and iNOS-/- animals showed that the
enzyme expression was higher in iNOS-/- than in WT mice in all treatments. In mice
lacking iNOS, sildenafil did not decrease GFAP, Iba-1, TNF-α and COX-2; however, IFN-γ
and IL-1β were diminished by sildenafil. All proteins were decreased by sildenafil in
previous study using WT mice. The present findings reveal that the absence of iNOS
limited the protective sildenafil effects. iNOS have an important feedback mechanism in
inflammatory conditions, when chronic increases of this enzyme is self-regulated and
induces decrease of some pro-inflammatory proteins. This feedback mechanism is coregulated by high concentration of cGMP. The results suggest that sildenafil may exert
its anti-inflammatory effect through iNOS inhibition by cGMP-iNOS feedback; in iNOS-/-
Página 59
mice the inflammatory regulation through iNOS-feedback would be absent. On the
other hand, the constant eNOS overexpression can generate high NO concentrations,
damaging the tissue. Further studies are required to explain the the NO/cGMP/PDE5
mechanism underlying sildenafil effect.
CNPq,CAPES,FACEPE,FAPESP, INBEB.
PD8.
A BI-PHASIC MODEL OF MICROVASCULAR LEAKAGE EXPLAINS HOW
ACTIVATION OF THE MAST CELL/CONTACT SYSTEM PROPAGATES INFLAMMATION
NASCIMENTO CR1, SERRA R1, JULIANO MA2, JULIANO L2, SVENSJÖ E1, SCHARFSTEIN J1.
1 Laboratório de Imunologia Molecular, Instituto de Biofísica Carlos Chagas Filho, UFRJ,
Rio de Janeiro; 2 Departamento de Biofísica, Escola Paulista de Medicina, UNIFESP, São
Paulo, Brasil
The contact system/Kallikrein-Kinin System (KKS) is activated by negatively charged
molecules released from mast cell (MC) granules. We investigated the functional
interplay between MCs and the KKS in a hamster model. Male hamsters (5 groups of n =
4 - 8) were anesthetized and their cheek pouches (HCPs) were prepared for intravital
microscopy, (Svensjö et al. 2012). HCPs were subjected to topical applications of the
contact system activator dextran sulfate 500 kDa (DXS) alone or in the presence of the
ACE-inhibitor captopril, BK2R-antagonist HOE-140, cromoglycate (a stabilizer of MC
granules) or PKSI-527 (a selective inhibitor of plasma kallikrein). Application of DXS
alone during 30 min caused no plasma leakage (indicated with FITC-dextran) but it
started to appear at 30 min. The dramatic increase in plasma leakage was blunted by
HOE-140 and cromoglycate evidencing bradykinin (BK) and MC involvement. The
leakage started in a few postcapillary venules suggesting that a discrete increase in
plasma leakage was required for contact system (DXS)-dependent intensification of
plasma leakage. We then found that leakage induced by histamine (4 μM) was not
inhibited by HOE-140, cromoglycate or PKSI-527. However, subtle increases in plasma
leakage (950 ± 490 RFU) provoked by histamine were greatly enhanced in the presence
of DXS, (3907 ± 533 RFU) and were further enhanced by DXS+captopril (11340 ± 733
RFU). Conversely, the plasma leakage induced by histamine combined with
DXS+captopril was almost completely inhibited by HOE-140, cromoglycate and PKSI-527
(924 ± 552, 1235 ± 497 and 2042± 879 RFU, respectively). Conclusions: plasma leakage
induced by histamine may intensify inflammatory edema through contact-system
dependent activation of the KKS and release of BK. Studies are underway to investigate
the interplay between MS/KKS and BKRs in infection by T. cruzi, Andrade et al. 2012, L.
chagasi, Svensjö, unpublished and bacteria (Porphyromonas gingivalis, Monteiro et al.
2009).
FAPERJ, CNPq e INCT-CNPq/INBEB
PD9.
RESVERATROL PREVENTS P53 CORE DOMAIN AGGREGATION
1,2- COSTA, D.C.F.; 1,2- CAMPOS, N.P.C.; 1,2- PEDROTE, M. M.; 2,3- ZANPHORLIN, L.;
2,3- RAMOS, C.H.I.; 1,2- SILVA, J.L.
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, Brazil; 2- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e
Bioimagem, Rio de Janeiro, Brazil; 3- Instituto de Química, Universidade Estadual de
Campinas, São Paulo, Brazil.
INTRODUCTION: p53 has an essential role in preventing cancer development by
inducing cell cycle arrest and/or apoptosis in response to cellular stress. Mutations in
the p53 gene are described in over 50% of all human cancers. Besides the mutations,
cellular aggregation of p53 can also inactivate the protein, leading to malignancy.
Resveratrol, a natural polyphenol found in grapes and red wine, is able to induce p53dependent cell death in a variety of cell lines. Although several indirect mechanisms of
p53 activation by resveratrol have been proposed, there is no evidence that this
bioactive compound can interact with p53. Thus, we investigated a possible interaction
between resveratrol and the p53 core domain (p53C). In addition, we tested the
potential of resveratrol in preventing wild-type and mutated p53 aggregation in vitro.
MATERIAL AND METHODS: Experiments were performed by using fluorescence
spectroscopy techniques. RESULTS AND DISCUSSION: Our data suggest that an
interaction between resveratrol and the wild-type p53C does occur. Additionally, we
found that resveratrol has the ability to inhibit the wild-type p53 core domain as well as
the R248Q p53 mutant to undergo aggregation. CONCLUSIONS: Our study provides
evidence that resveratrol can directly modulate p53 and may pave the way for a better
understanding of the mechanisms involved in p53 protein aggregation as a therapeutic
strategy for cancer treatment. Key words: resveratrol, cancer, p53, aggregation.
INBEB, FAPERJ, CNPq and Fundação do Câncer
PD10.
STRUCTURAL AND THERMODYNAMIC CHARACTERIZATION OF THE CAPSID
PROTEIN OF HEPATITIS C VIRUS AND ITS INTERACTION WITH THE TUMOR SUPPRESSOR
PROTEIN P53
1- Albernaz, F. P.;1- Braga, V. L. A.; 2- Souza, T. L. F.; 1- Rodrigues-Pinto, T.; 1- Rangel, L.
P.; 3- Peabody, D.;1- Silva, J. L. ; 1- Gomes, A. M. O.;1- Almeida, F. C. L.; 1- Oliveira, A. C.
1- Programa de Biologia Estrutural and Centro Nacional de Ressonância Magnética
Nuclear de Macromoléculas -Instituto de Bioquímica Médica - UFRJ, RJ, Brazil; 2Faculdade de Farmácia - UFRJ, RJ, Brazil; 3-Department of Molecular Genetics and
Microbiology and Cancer Research and Treatment Center, University of New Mexico
School of Medicine, USA
Hepatitis C virus (HCV) acts as a major agent of chronic hepatitis, cirrhosis, and
hepatocellular carcinoma. The HCV core protein (HCVcp) is involved with the
nucleocapsid assembly and with different cellular processes. The interaction of HCVcp
with the tumor suppressor protein p53 has been described in hepatocellular carcinoma
but the mechanism of this interaction remains unknown. In this work we use
thermodynamic and spectroscopic techniques to achieve the structural characterization
of HCVcp and its interaction with p53. Although HCVcp is an intrinsically unstructured
protein, our fluorescence spectroscopy results demonstrated a significant decrease in
the spectral center of mass of the protein in function of increasing concentrations of
chemical denaturants, indicating the presence of structural elements. These data are
corroborated by Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR).
HCVcp backbone assignments strategy based on projection spectroscopy, a way of
reducing the experiment time when recording high-dimensional NMR experiments and
Triple resonance experiments are in course. To evaluate the interaction between HCVcp
and p53, 1H/15N HSQC NMR spectra of labeled HCVcp were acquired in the absence
and in the presence of unlabeled p53. Changes in the chemical shift of some
resonances of the HCVcp were observed, showing changes in the chemical environment
of these amino acid residues and suggesting interaction with p53. Fluorescence
polarization assays of FITC-labeled p53 in the presence of HCVcp corroborate the
interaction in vitro between both proteins. Additionally to these studies, we are also
investigating the interaction of the HCVcp with the p53 in HepG2 and H1299 cells. We
constructed vectors to express both HCVcp and p53 fused to the proteins GFP and
DsRed-Monomer, respectively, in order to perform Fluorescence Resonance Energy
Transfer analyses and these studies are in progress. Our data reveal a new approach to
understand the HCVcp-p53 interaction.
FAPERJ, CAPES, CNPq and INBEB
Página 60
PD11.
IMAGEAMENTO POR ESPECTROMETRIA DE MASSA COMO FERRAMENTO DE
ESTUDO DA FISIOLOGIA DE INVERTEBRADOS
1,2 GOMES, F.M., 3 ZECCHI, R., 4 BARACCHI, D., 3 PIERACCINI, G., 2, 5 MIRANDA, K., 1,5
MACHADO, E.A., 3 MONETI, G., 4 TURILLAZZI, S.
1. Laboratório de Entomologia Médica, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, Brazil 2. Laboratório de Ultraestrutura Celular Hertha Meyer, Universidade
Federal do Rio de Janeiro, Rio de Janeiro, Brazil 3. Centro di Servizi di Spettrometria di
Massa dell’Università degli Studi di Firenze, viale G.Piaraccini, 50139, Firenze, Italy 4.
Università degli Studi di Firenze, Dipartimento di Biologia Evoluzionistica ‘Leo Pardi’, Via
Romana 17, 50125, Firenze, Italy 5. Divisão de Biologia Estrutural, Diretoria de
Metrologia Aplicada a Ciências da Vida, Instituto Nacional de Metrologia, Qualidade e
Tecnologia (INMETRO), Xerém, RJ, Brazil
O imageamento por espectrometria de massa acoplado a ionização e dessorção a laser
assistida por matriz (MALDI-IMS) é uma metodologia que vem se popularizado como
ferramenta de estudo da fisiologia de vertebrados. Sua utilização permite que, por meio
da análise por espectrometria de massa em modo de varredura de cortes de material
biológico, sejam geradas imagens da distribuição de moléculas. Assim, presente
trabalho pretendeu avaliar o potencial do MALDI-IMS como ferramenta de estudo em
invertebrados, utilizando cortes de larvas não-fixada da abelha europeia Apis mellifera.
Inicialmente, visou-se a otimização da composição de matriz a ser utilizado a fim de
maximizar a variedade de analitos detectados e a sua relação sinal-ruído.
Posteriormente, os padrões de segmentação de peptídeos no intestino e na hemolinfa –
o líquido circulante dos insetos - foram analisados. Nossos resultados sugerem um
protocolo inicial baseado na utilização de uma mistura contendo ácido alfa-ciano-4hidroxicinamínico (CHCA) e anilina, na presença de ácido trifluoroacético e acetonitrila.
Posteriormente, identificamos padrões que evidenciaram a segmentação do intestino
médio da abelha em quatro regiões não-sobrepostas. A análise da distribuição de
peptídeos na hemolinfa revelou a presença de gradientes previamente desconhecidos,
cuja categorização era mais facilmente realizada quanto a proximidade destes com o
intestino - sugerindo que este órgão atue na organização e, possivelmente na secreção,
desses componentes para a hemolinfa. Finalmente, pelo acompanhamento da variação
dos níveis de complexidade dos espectros ao longo do lúmen do intestino,
corroboramos a hipótese de que a rota digestiva de proteínas ocorra em um modelo de
contra-corrente organizado pela segmentação do lúmen em espaço ecto- e
endoperitrófico. Em conjunto, nossos resultados corroboram que a análise por MALDIIMS fornece uma valiosa ferramenta para o estudo da fisiologia de insetos e apontam
para a presença de níveis de complexidade previamente desconhecidos no intestino e
hemolinfa desses animais.
CAPES, CNPq, FAPERJ
PD12.
DESVENDANDO
A
FISIOLOGIA
DA
BACTÉRIA
MULTICELULAR
MAGNETOTÁTICA CANDIDATUS MAGNETOGLOBUS MULTICELLULARIS ATRAVÉS DA
ASSOCIAÇÃO DA ANÁLISE POR MICROSCOPIA E METAGENÔMICA
1- ABREU, F.; 1- MORILLO, V.; 1- NASCIMENTO, F.F.; 1- WERNECK, C.; 2- CANTÃO, M.E.;
2- CIAPINA, L.P.; 2- ALMEIDA, L.G.P.; 3- LEFÈVRE, C.T.; 4- BAZYLINSKI, D.A.; 2VASCONCELOS, A.T.R.; 1- LINS, U.
1- Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro; 2Departamento de Matemática Aplicada e Computacional, Laboratório Nacional de
Computação Científica; 3- UMR7265 Service de Biologie Végétale et de Microbiologie
Environnementale, CEA Cadarache/CNRS/Aix-Marseille Université; 4- School of Life
Sciences, University of Nevada at Las Vegas
Candidatus
Magnetoglobus multicellularis é um procarioto multicelular magnetotático não
cultivável formado por células Gram-negativas dispostas lado a lado, formando uma
esfera. Cada célula possui inúmeras inclusões ricas em ferro chamadas magnetossomos,
organelas responsáveis pela orientação ao longo do campo magnético. Devido à sua
capacidade de responder a campos magnéticos, é possível seu enriquecimento a partir
de amostra ambiental. A caracterização morfológica por microscopia eletrônica de
transmissão e varredura e a análise do comportamento do micro-organismo por
microscopia de luz é feita desde sua descoberta em 1983. Nesse trabalho, a análise das
sequencias geradas pelo pirosequenciamento de amostra enriquecida em Ca. M.
multicellularis foi feita tendo como base os dados observados por microscopia, de
forma que as características do micro-organismo descritas por microscopia garantissem
que genes detectados realmente pertencessem ao micro-organismo. Dessa forma,
características definidas por proteínas envolvidas na adesão e morfogênese
multicelular, divisão celular, produção de grânulos e cápsula, quimio-, foto- e
magnetotaxia, entre outras, foram identificadas, permitindo o entendimento da
fisiologia do micro-organismo. As conclusões obtidas a partir da integração dos dados
obtidos pelo emprego das técnicas de microscopia e dos dados genômicos levaram ao
desenvolvimento de um meio de enriquecimento para o micro-organismo, condição em
que alguns dados ultraestruturais foram confirmados, e ao entendimento das taxias
observadas, indicando que o deslocamento do micro-organismo no ambiente é
dependente não só da magnetotaxia como também da fototaxia e quimiotaxia, como
observado por experimentos de quantificação dos micro-organismos em diferentes
camadas de sedimento e da análise por vídeo microscopia do comportamento do microorganismo quando expostos a diferentes substratos em ambiente anaeróbico. Assim,
esse trabalho exemplifica como dados morfológicos, ultraestruturais e de análise de
comportamento obtidos por técnicas de microscopia podem ser utilizados para refinar,
ou mesmo, transformar dados brutos provenientes de sequenciamento em estudos de
caracterização detalhada de micro-organismos específicos.
INBEB, CNPq, CAPES e FAPERJ
PD13.
BONE MARROW CELL THERAPY DURING PRE AND POST SYMPTOMATIC
PHASE IN A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
1,2- GUBERT, F.; 1,2-PEREIRA, I.B.; 1,2-DECOTELLI-SILVA, A.; 1,2-ZAVERUCHA-DO-VALLE,
C.; 1,2-FIGUEIREDO, F.R; 2-TOVAR-MOLL, F.; 1,2-SANTIAGO, MF; 1,2-MENDEZ-OTERO, R.
1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 2Insituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem,
Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease that affects
selectively the motor neurons. The detail mechanisms of selective motor neuron death
remain unknown. and no effective therapy has been developed. The aim of this work is
to investigate the therapy with bone marrow cells (BMC) in a mouse model of ALS
(SOD1-G93A mice). We injected 106 BMC (mononuclear fraction) in the lumbar portion
of the spinal cord of the SOD1-G93A mice 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 disease onset in the
treated animals. When we injected the BMC at 14weeks, we observed only an increase
in the animal’s 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. Using different strategies to track the BMC,
we notice that these cells do not remain in the spinal cord after injected. One week
after the transplant, approximately 3% of BMC still could be observed, however we did
Página 61
not observe significantly BMC after a longer period. These results indicate that,
although the treatment with BMC in the spinal cord of a mouse model of ALS, delayed
the progression of the symptoms, it did not increase the lifespan of the animals,
probably because the BMC did not remain in the injected site.
INBEB, Protecel, FAPERJ, CNPq, CAPES
PD14.
BIOENERGETIC IMBALANCE AND OXIDATIVE STRESS IN THE
PATHOPHYSIOLOGY OF SEPTIC ENCEPHALOPATHY
1- D'AVILA, JC; 1- REIS, PA; 2- RODRIGUES, RS; 1- CASTRO-FARIA-NETO, H; 3- OLIVEIRA,
MF; 4- BOZZA, F.
1- Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz - FIOCRUZ; 2- Radiologia
do Hospital Universitário Clementino Fraga Filho, UFRJ; 3- Instituto de Bioquímica
Médica - UFRJ; 4- Instituto de Pesquisa Clínica Evandro Chagas - FIOCRUZ e Instituto
D'Or de Pesquisa e Ensino.
"Septic encephalopathy is a frequent complication in severe sepsis but its pathogenesis
and mechanisms are not fully understood. Oxygen supply and utilization are critical for
organ function, especially for the brain, a tissue extremely dependent on oxygen and
glucose. Disturbances in oxygen utilization are common in sepsis and a number of
mitochondrial dysfunctions have been described in different tissues in septic animals as
well as in septic patients. Our group described brain mitochondrial dysfunctions in
experimental sepsis. But mitochondria isolated from septic brains generated less ROS in
vitro. This led us to investigate the role of NADPH oxidase as a source of reactive oxygen
species in the brain during sepsis. Experimental sepsis was induced by endotoxemia (LPS
10mg/kg i.p.) in Sprague Dawley rats and by polymicrobial fecal peritonits in Swiss mice.
Brain glucose uptake was observed in vivo in endotoxemic rats using Positron Emission
Tomography (PET) with [18F]Fluorodeoxyglucose (FDG) and autoradiography with 2deoxy-14C-glucose. Mice with polymicrobial sepsis present hypoglycemia,
hyperlactatemia and long-term cognitive impairment. We observed a rapid increase in
the uptake of fluorescent glucose analog 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4- yl)
amino]-D-glucose (NBDG) in brain slices from septic mice in vitro. A similar increase in
brain glucose uptake was observed in vivo in endotoxemic rats. The brains of mice with
experimental sepsis presented neuroinflammation, mitochondrial dysfunctions and
oxidative stress, NADPH oxidase inhibitor apocynin prevented brain oxidative stress and
long term cognitive impairment in survivors from experimental sepsis. Our data indicate
that a bioenergetic imbalance and oxidative stress are
associated to the
pathophysiology of septic encephalopathy. We are observing a new metabolic
phenotype in the brain during sepsis, characterized by a rapid increase in glucose
uptake and mitochondrial dysfunctions that may be secondary to inflammation and
hypoxia."
FAPERJ, CNPq
PD15.
PALEOPARASITOLOGY REVISITED BY SCANNING ELECTRON MICROSCOPY
1-DUTRA JMF; 1-CAMACHO M;2- RACHID R; 2- DE SOUZA W; 1- ARAÚJO A.
1-Laboratório de Paleoparasitologia - Escola Nacional de Saúde Pública Sérgio Arouca,
Fundação Oswaldo Cruz; 2-Laboratório de Ultraestrutura Celular Hertha Meyer,
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro.
Microscopy techniques are used since 16th century to make visible what our eyes are
not able to distinguish. For paleoparasitology studies, coprolites – desiccated or
mineralized feces - are rehydrated to recover consistence of fresh feces before
observation. After applying recommended techniques to concentrate cysts and eggs of
parasites, samples are prepared for light microscopy analysis. Only in 1960 the first
study using electron microscopy described a method to identify intestinal parasites,
bones or injuries in mummified soft tissues. Until today, scanning electron microscopy
(SEM) in ancient samples is made following a routine protocol that passes through
rehydration, fixation, dehydration and critical point drying steps. We describe here
some alternative methodologies to prepare archeological samples for scanning electron
microscopy to improve paleoparasitological findings. Natural desiccated, rehydrated
samples and even parasites eggs trapped between glass slides and coverslip are
submitted to different techniques for SEM analysis.
CAPES, FAPERJ, CNPq.
PD16.
NEW
2-AMINO-5-BENZYL-4,6-DIHYDROXYPYRIMIDINES:
SYNTHESIS,
STRUCTURAL ELUCIDATION AND BIOLOGICAL ACTIVITY EVALUATION
1- CARVALHO, L.L.; 1- FIGUEROA-VILLAR, J.D.
1- Grupo de Química Medicinal, Departamento de Química, Instituto Militar de
Engenharia
In Brazil, the diseases such as dengue, malaria, leishmaniasis, among others, are
considered a serious public health problem. According to the National Institute of
Science and Technology for Innovation in Neglected Diseases (INCT-IDN), these diseases
prevail in poverty conditions and contribute to the maintenance of social inequality in
the country. Following this judging, today there is a priority to research new prototype
of drugs capable for more effective treatment of these diseases. Our group has
developed research on novel substances able to act as antifolates inhibiting the enzyme
dihydrofolate reductase (DHFR) present in malaria protozoa. Pyrimidine frameworks
such as pyrimethamine (PYR) and trimethoprim (TMP) are a good example of potent
and selective drugs capable to inhibit the dihydrofolate reductase present in the
Plasmodium falsiparum (pfDHFR) responsible to the most dangerous type of malaria.
Herein we wish to report our initial investigations on the synthesis and structural
elucidation of new 5-benzylpyrimidines derivatives. The synthetic route was initiated by
Knoevenagel condensation reaction between substituted aromatic aldehydes and a
malonic ester in 40-74% yield. The benzylidene malonates generated in the first step
were reduced easily from NaBH4 in 70-80% yield and, sequentially, the respective
benzyl malonates can react with guanidine to furnish the desired 2-amino-5-benzyl-4,6dihydroxypyrimidines, which are in process of the respective evaluation of their
biological activity as bactericidal, fungicide and antimalarial.
INBEB
PD17.
ISOLATION AND CHARACTERIZATION OF RNA MOLECULES BOUND TO
RECOMBINANT MOUSE PRION PROTEIN
Mariana P. B. Gomes1, Luciana P. Rangel2, Murilo S. Amaral3, Sergio VerjovskiAlmeida3, Yraima Cordeiro1 and Jerson L. Silva2
1Departamento de Fármacos, Faculdade de Farmácia, UFRJ; 2Instituto de Bioquímica
Médica, UFRJ; 3Departamento de Bioquímica, Instituto de Química, USP
It is accepted that the prion protein (PrP) interacts with nucleic acids (NA) and that
these interactions might be related to the early events in prion conformational changes
and subsequent appearance of prion diseases. Our group has been studying PrP:NA
interactions and our findings show that mouse recombinant PrP (rPrP) binds with high
affinity to DNA in vitro. Recently, we demonstrated that rPrP:DNA interactions lead to
different aggregated species and induce cell dysfunction depending on the DNA
sequence tested, which appears to be correlated with the biophysical properties of the
complex. Our studies with RNA showed that these molecules can also modulate rPrP
aggregation and form toxic species, depending on RNA source. In the present study we
use total RNA extracted from neuroblastoma cells (N2aRNA) to search for sequences
that would bind rPrP with high affinity, being responsible for the effects observed
Página 62
previously. N2aRNA was incubated with rPrP and after that, RNA was recovered using
different isolation protocols. The samples were analyzed to determine quality, integrity
and composition. In all conditions tested, large amounts of rRNA were recovered. When
rPrP:N2aRNA complex was treated with RNase, the obtained RNA was partially
degraded, confirming that rPrP-bound RNA is protected from digestion. The set of
molecules recovered is still heterogeneous, suggesting that more than one RNA
sequence contained in this extract can be responsible for inducing aggregation and
toxicity. This finding is in agreement with recent studies that show that PrP is part of a
ribonucleoprotein complex, as well as studies that identify large amounts of rRNA in
tissues infected with PrPSc. Despite these findings, we cannot rule out the possibility of
other RNA types being involved in this interaction. We believe that RNA encoded by the
host itself or derived of foreign sources, may be directly involved in the PrPC/PrPSc
conversion.
CNPq, CAPES, INCT-INBEB, FAPERJ
PD18.
CARACTERIZAÇÃO ULTRAESTRUTURAL DE TRIPANOSSOMAS ENCONTRADOS
EM PEIXES CASCUDOS, HYPOSTOMUS AFFINIS E HYPOSOTMUS LUETKENI
1-LEMOS, M. & 1-SOUTO-PADRÓN, T.
1-Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio
de Janeiro, Brasil.
Os tripanossomas que parasitam peixes são transmitidos por sanguessugas durante a
hematofagia. Formas metacíclicas presentes na probóscide de sanguessugas entram em
contato com a lesão provocada no peixe e alcançam a circulação sanguínea. Neste
estudo procedeu-se a caracterização ultraestrutural dos tripanossomas que infectam
peixes cascudos identificados como Hypostomus affinis e Hypostomus luetkeni,
procedentes do Rio Pomba (21º21'07“S, 43º02'49” O) Guarani, MG. Foi verificada a
presença de sanguessugas do gênero Haementeria, aderidas à superfície corporal dos
peixes, infectadas por tripanossomas. Os tripomastigotas foram isolados em 9 tipos
distintos de meios de cultura a partir do sangue de 9 peixes da espécie H. affinis e de 6
da espécie H. luetkeni. Os tripomastigotas sanguíneos e os epimastigotas,
esferomastigotas e tripomastigotas observados no estômago das sanguessugas e in
vitro foram analisados por microscopia eletrônica de varredura (MEV) e de transmissão
(MET). A caracterização ultraestrutural das formas sanguíneas por MEV revelou variação
morfológica sendo encontrados tripomastigotas de corpo alongado e largo e de corpo
curto e delgado. Nos cecos estomacais das sanguessugas foram observados
tripomastigotas de corpo alongado e delgado e epimastigotas piriformes. Por MET os
tripomastigotas sanguíneos apresentaram estruturas esféricas semelhantes a
acidocalcisomas e o cinetoplasto alongado em forma de barra com arranjo compacto
das fibrilas de kDNA. A ultraestrutura das formas de cultura revelou cinetoplasto
semelhante ao observado para os tripomastigotas sanguíneos e a presença de complexo
citóstoma-citofaringe em epimastigotas e tripomastigotas além de inclusões lipídicas,
estruturas semelhantes à acidocalcisomas, organelas relacionadas a lisossomas, corpos
multivesiculares.
CNPq, INBEB
PD19.
CRATYLIA MOLLIS SEED LECTIN (CRAMOLL 1) CAN CAMOUFLAGE
LEISHMANIA PROMASTIGOTES IMPAIRING THE RELEASE OF NEUTROPHIL
EXTRACELLULAR TRAPS (NETS): IMPACT IN CUTANEOUS LEISHMANIASIS
NATHALIA VAREJÃO1, PEDRO TOJAL1, THIAGO VIEIRA2, TEREZA CORREIA3, ELVIRA
SARAIVA2, HERBERT GUEDES4, DEBORA FOGUEL1
1Instituto de Bioquímica Médica, Federal University of Rio de Janeiro, Rio de Janeiro,
BR, 2Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, BR, 3Departamento de Bioquímica, Federal University of Pernambuco,
Recife, BR, 4Institutode Biofísica Carlos Chagas Filho, Federal University of Rio de
Janeiro, Rio de Janeiro, BR.
rCRAMOLL 1 is the bacterial recombinant form of lectin found in seeds of Cratylia mollis
(Leguminosae family, Diocleinae subtribe). We have demonstrated that this tetrameric
lectin (236 amino acids per monomer) retained sugar-binding activities of the plant
lectin counterpart that could result in agglutination of Trypanosome epimastigotes and
Leishmania promastigotes, and, possibly induction of Th1 cell response. So, we
hypothesized if rCRAMOLL 1 could promote a protective effect in a murine model of
American cutaneous leishmaniasis acting as a healing chemical in initial stages of
footpad lesions by intradermal or ointment applications or as an adjuvant molecule in
vaccination designs (oral, nasal). Surprisingly, our results pointed to an aggravation of
the lesions in the animals treated with rCRAMOLL 1 associated with enhanced parasite
burden. In 2009, Dr. Saraiva and cols. showed that neutrophils play an important role in
control of leishmanial infection by a peculiar cellular death named NETosis which is
trigger by interaction of neutrophils with lipophosphoglycan (LPG) present in
Leishmania surface that results in fibrilar DNA release that traps and kills the parasites.
Now, we are investigating how the interaction of rCRAMOLL 1 with L. amazonensis
promastigotes could affect NETs release and parasite survival. Interestingly, our data
show that neutrophils lost the ability to recognize and release the extracellular traps in
the presence of promastigotes that were previously incubated with subagglutinating
concentration of rCRAMOLL 1. On another hand, when the neutrophils were preincubated with our lectin they engulfed it and even so they can perfectly respond to the
presence of Leishmania, which indicates that rCRAMOLL 1 does not affect any
mechanism of NETs formation and release, instead it acts as a parasite camouflage.
Taken together our findings could help to explain the negative effects observed in the
model disease treatments and point an alert in the use of plant lectins in this field.
CAPES, FAPERJ, INBEB
PD20.
STATINS PREVENT BRAIN MICROVASCULAR ALTERATIONS AND COGNITIVE
IMPAIRMENT DUE TO SEPSIS
1-REIS, P.A.; 1-ALEXANDRE, P.C.B.; 2-ESTATO, V.; 2-TIBIRIÇÁ, E.; 1-BOZZA, F.A.; 1CASTRO-FARIA-NETO, H.C..
Lab. de Imunofarmacologia, Lab Invest. Cardiovascular -IOC-FIOCRUZ-Rio de Janeiro,
Brazil
Sepsis is a major cause of mortality in intensive care units, and brain dysfunction is
frequently observed as a consequence of changes in cerebral structure and metabolism.
Statins have been prescribed extensively for their cholesterol-lowering properties and
efficacy in cardiovascular disease and also present antiinflammatory and antioxidant
effects due to pleiotropic mechanisms. The objective of the present study was
investigate the effect of statins on the brain microcirculatory changes and on the
cognitive impairment due to sepsis. Feces were extracted (5 mg/g b.w.) from large
intestine of SW mice and diluted in saline, centrifuged and the supernatant was
collected and injected in the animals (n=5-8/group). Control animals received 0.5 ml of
saline. Animals were treated at 6, 24 and 48 hours after that with imipenem (10 mg/kg
b.w., 0.2 ml s.c.) and 1.0 ml of saline (s.c.). The statins healthy or septic groups were
treated 1 h before to 48 hours after the infection (20 mg/kg b.w., p.o.). Mortality was
observed for 96 h and the severity score was evaluated. After 24 hours, groups of
animals were anesthetized and intravital fluorescence videomicroscopy was performed.
Cognitive damage was evaluated by inhibitory avoidance task 15 days post-sepsis. Mice
developed a moderate sepsis 6 and 24 h post feces injection which is reduced in 48 h.
Treatment with atorvastatin or simvastatin did not reduced the clinical score during
Página 63
sepsis development or the mortality. Statins therapy reduced the leukocyte rolling and
adhesion induced by sepsis, and reversed brain functional capillary rarefaction. Animals
that received statins were able to keep the avoidance memory missed in sepsisuntreated mice. The present findings indicate that the treatment with statins was able
to protect the brain microvascular alterations and reverse the cognitive impairment
during sepsis.
FIOCRUZ, FAPERJ, CNPq, CAPES
blue leakage than the vaccinated group. In the same way differential cell counts in
bronchoalveolar lavage showed more cells in control group. HP-inactivated H3N8 virus
vaccine induced significant protection against the infection by H3N8 influenza virus. Our
work reaffirms the use of HP as an interesting tool in the development of viral vaccines
at low cost and good immune response.
Support:CAPES,PRONEX,INBEB,CNPq,FAPERJ.
PD23.
PD21.
DISSECTING THE MECHANISM OF CITOTOXICITY TRIGGERED BY
AGGREGATES OF TRANSTHYRETIN
1-FERREIRA, P. S.;1- OLIVEIRA, L. T.;1- FOGUEL, D..
1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro.
Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative autosomal dominant
disorder characterized by the extracellular deposition of transthyretin (TTR) fibrils in
several tissues. There is a debate in the literature concerning the identification of the
toxic species present in the pathway to fibril formation (native àprotofibrilà fibril) trying
to elucidate which species would be responsible for cellular damage caused in PAF. In
the present study we evaluated which are the most toxic species in the aggregation
pathway of TTR, and the mechanism by which they promote their citotoxic effects in
different cell lineages and in primary culture of retinal neurons. The viability assays
show that the oligomers formed during the aggregation process are the most toxic
species and that have a tissue specific toxicity. Our data also shows that these oligomers
trigger cell death by activating the caspase-dependent apoptotic pathway. Furthermore,
we verify the internalization of these oligomers only in cells which were toxic,
suggesting that perhaps this internalization has some correlation with toxicity. All
together these data show that oligomers of TTR were the most toxic species formed
during the aggregation process of TTR and account for much of the cell damage caused
by the TTR amyloidosis.
PD22.
VACCINATION WITH A PRESSURE INACTIVATED AVIAN INFLUENZA VIRUS
PROTECTS MICE AGAINST INFECTION WITH INDUCTION OF GOOD IMMUNE RESPONSE
AND PRESERVATION OF HA AND NA ACTIVITY
BARROSO, S.P.C.1, NICO, D.2, VICENTE, A.C.S.1, COUCEIRO, J.N.S.S.2, NASCIMENTO, D.3,
BOZZA, F.3, FERREIRA, DF2, PALATNIK-DE-SOUSA, C.B.2, SILVA, J.L.1 & OLIVEIRA, A.C.1
1Institute of Medical Biochemistry - Federal University of Rio de Janeiro (UFRJ);
2Institute of Microbiology Paulo de Goes - UFRJ, Rio de Janeiro; 3Oswaldo Cruz Institute
- FIOCRUZ
H3N8 is an avian influenza virus that was originally isolated from birds, and later found
in horses and dogs. Here, we used 12 h of incubation under hydrostatic pressure (HP) to
achieve this virus inactivation without damage to its hemagglutinin and neuraminidase
activities and study its protective capability in vaccination against H3N8 infection. Balb/c
mice were treated by the intranasal route, with 3 doses of the pressure-inactivated
virus. Mice were challenged with native H3N8 on fourth week, and monitored for:
virus-specific antibodies in serum, nasal lavage and faeces, CD4+ and CD8+ virus-specific
T cells, cytokine ELISA, clinical symptoms and inflammatory parameters. After
immunization and challenge we found an increase of IgG1, IgG2a, and IgA antibodies.
These antibodies found in serum are neutralizing. The analysis of cytokine production by
CD4+ and CD8+ T cells showed a mixed Th1/Th2 pattern after vaccination. Two weeks
after the challenge, we observed an increase in the production of antibodies and IL-6,
IFN gamma and TNF alpha. The control group (saline) showed more clinical signs of
disease (lethargy, weight loss and huddling) than vaccinated animals and more Evans
HEPARIN MODULATION OF PRION SEEDING ACTIVITY
1- VIEIRA, T.C.R.G.; 2- CAUGHEY, B.;1- SILVA, J.L.
1-Inst de Bioquímica Médica, IBqM-UFRJ, RJ, Brazil; 2-LPVD, RML, NIAID, NIH, MT, USA.
"INTRODUCTION. The conversion of PrP into scrapie PrP is the central event of prion
diseases (TSEs). Such conversion and propagation is seeded or templated by a
polymerization mechanism. Some authors have suggested that glycosaminoglycans
(GAGs) directly convert PrP into a protease resistant form, while others have proposed
that these molecules have a protective activity. Our group recently reported that low
molecular weight heparin (LMWHep) does not induce recombinant mouse prion protein
(rPrP23-231) conversion, protecting rPrP23-231 from RNA-induced aggregation (1).
MATERIAL AND METHODS: Real-time quaking-induced conversion (RT-QuIC) is an assay
in which disease-associated PrP initiates a rapid conformational transition in
recombinant PrP, resulting in the formation of amyloid fibrils that can be monitored in
real time using the dye thioflavin T. We used rPrP from mouse and hamster as substrate
(23-231 and 90-231), and TSE-associated forms were from mouse and hamster brain
homogenates (RML and 263K strain respectively). LMWHep was used in order to
determine the effect of this GAG on PrP fibrillization. RESULTS AND DISCUSSION: In the
present work, we show that LMWHep delays and decrease fibril formation. It also
inhibits fibrilization depending on the seed used. There is no effect when rPrP 90-231 is
used, or with high salt concentration. Moreover, it is effective when added at the lag
phase of the polymerization process. When a soluble LMWHep-rPrP complex is added to
the reaction, no fibrils are detected. On the contrary, the addition of a LMWHep-rPrP
aggregated complex results in conversion. CONCLUSIONS: Through electrostatic
interactions, LMWHep interaction with PrP N-terminal domain, modulates PrP
fibrilization. It affects the nucleation processes and the formation of oligomers, the first
step of fibrilization. Our findings may explain the protective effect of these molecules in
different models.
References: Vieira TCRG, et AL. J Amer Chem Soc 2011; 133:334-344.
Keywords: Prion, Gycosaminoglycan, Aggregation, Neurodegeneration"
NIAID (NIH), CNPq, INBEB and FAPERJ.
PD24.
STRUCTURAL BASIS FOR THE INTERACTION OF HUMAN Β-DEFENSINS 1 AND
6 AND ITS PUTATIVE CHEMOKINE RECEPTOR CCR2 AND BREAST CANCER
MICROVESICLES
De Paula, V.S.1, Gomes, N.S.F.1, Lima, L.G.1, Monteiro, R.Q.1, Almeida, F.C.L. 1, Valente,
A.P.
1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, RJ 21941-902, Brasil
Human β-defensins (hBD) are believed to function as alarm molecules that stimulate the
adaptive immune system when a threat is present. In addition to its antimicrobial
activity, defensins present other activities such as chemoattraction of a range of
different cell types to the sites of inflammation. We have solved the structure of the
human β-defensins 6 (hBD6) by NMR spectroscopy that contains a conserved β-defensin
domain followed by an extended C-terminus. We also investigated the interaction of βdefensin 1 and 6 with microvesicles shed by breast cancer cell lines using NMR.
Página 64
Chemical shift mapping of the interaction showed that both defensins interact with
microvesicles but in slightly different way, suggesting an inverse correlation with the
aggressiveness potential of the cell. Furthermore, molecular docking using restraints
derived from the NMR chemical shift data produced a model of the complex between
hBD6 and a peptide derived from the extracellular domain of CC chemokine receptor 2
(Nt-CCR2) that reveals a contiguous binding surface on hBD6, which comprises amino
acid residues of the α-helix, loop between β2-β3 and C-terminal. The microvesicles
binding surface partially overlaps with the chemokine receptor interface. These data
offer new insights into the structure–function relation of the hBD6–CCR2 interaction
and may be helpful for the design of novel anti-cancer agents.
INBEB, CAPES, FAPERJ,CNPq
Página 65
Pesquisadores
P1.
MICE DEFICIENT IN BRADYKININ B1 RECEPTOR, A GPCR UPREGULATED IN
INFLAMED TISSUES, ARE PROTECTED FROM CHRONIC MYOCARDITIS AND ADVERSE
CARDIAC REMODELLING
OLIVEIRA AC1, ANDRADE, D1, CORDOVIL, T1, RAMOS-JUNIOR E.S1, ALMEIDA, LN1;
CARVALHO-PINTO, C.E2, MONTEIRO, RQ3, SVENSJO, E1, SIROIS, P.4, SCHARFSTEIN, J1
1Instituto de Biofísica Carlos Chagas Filho, UFRJ; 2Instituto de Biologia, Departamento
de Imunologia, UFF; 3 Departamento de Bioquímica Médica, UFRJ; 4CHUL Research
Center, Laval University, Quebec, Canada
Chronic chagasic myocarditis (CCM) depends on Trypanosoma cruzi persistence in the
myocardium. In a recent article (Scharfstein et al., Frontiers in Immunology, 2013) we
have suggested that extracellular trypomastigotes released from heavily parasitized
heart cells evoke intermittent “flares” of plasma-leakage through the activation of the
kallikrein-kinin cascade. Based on multiple evidences, we hypothesized that T. cruzi may
exploit this window of opportunity (formation of a transient intramyocardial edema) to
proteolytically generate infection-promoting signals, such as bradykinin (BK) and DesArg-BK, respectively the agonists of BK2R (constitutive) and BK1R, a NFk-B-inducible
GPCR upregulated in inflamed tissues. Consistent with this hypothesis, here we show
evidence that R954 (BK1R antagonist) reduced the parasite tissue load (skeletal muscle)
in LPS-treated infected (3 d pi) mice, but not PBS-treated infected. Extending this
analysis to the classical intraperitoneal model of infection, we found that BK1R-/- mice
displayed reduced heart parasitism as compared to WT B6 mice (14 d pi). Immunological
studies revealed that adaptive responses were fairly similar in both strains.
Interestingly, analysis of innate response profiles revealed a significant reduction
(p<0.05) of intracardiac frequencies of Ly6C low monocytes, a subset implicated in
tissue repair and collagen deposition. Reminiscent of the cardioprotective phenotype of
BK1R-/- mice in models of diabetic cardiomyopathy, phenotypic analysis of chronic
chagasic mice (90 d) revealed that myocarditis and heart fibrosis were profoundly
reduced in BK1R-/- mice. Combined with the in vivo pharmacological data, these studies
support the hypothesis that BK1R, GPCR upregulated in inflamed tissues, might serve a
preferential gateway for T. cruzi infection.
INBEB, CNPq, FAPERJ
P2.
CARACTERIZAÇÃO ESTRUTURAL DA TXNIP, UMA PROTEINA CHAVE NO
METABOLISMO CELULAR
1-AGUIAR, R.P.; 1-AMORIM, G.C.; 1- VALENTE, A.P.; ALMEIDA, F.C.L.
1- Centro Nacional de Ressonância Magnética Nuclear, Instituto de Bioquimica Médica,
UFRJ
"A Txnip (Thioredoxin interacting protein) é um importante regulador multifuncional do
metabolismo celular, o que vem atraindo muita atenção para esta proteína atualmente.
Entre suas funções está a regulação do crescimento, diferenciação, sinalização e morte
celular. Ela atua ainda no metabolismo da glicose e de lipidios. A Txnip faz parte da
família das α-arrestinas, que inclui outras cinco proteínas, Arrdc1-5. No entanto, a Txnip
é a única proteína desta família capaz de interagir com a Tioredoxina (Trx), uma enzima
antioxidante ubíqua que regula a homeostasia oxido-redutora das células. A Trx
desempenha papel importante em diversos processos celulares relacionados ao
equilíbrio saúde-doença. Pouca informação estrutural e funcional está disponível para a
família das α-arrestinas e, especialmente, para a Txnip e sua interação com a Trx. Com o
objetivo de compreender estes mecanismos, estamos fazendo a caracterização
estrutural e funcional da Txnip e sua interação com a Trx utilizando RMN, SAXS e
fluorescência. Por se tratar de uma proteína grande (390 resíduos), estudamos seus
domínios estruturais isolados. Além disso, também utilizamos peptídeos
correspondentes a prováveis regiões de interação. Esta abordagem nos permitiu
caracterizar a participação de cada domínio na interação com a Trx. Os primeiros
experimentos de RMN e SAXS para o cálculo da estrutura tridimensional já foram
realizados e os peptídeos sintetizados. Estes dados fornecerão ferramentas para a
melhor compreensão dos mecanismos de ação e das funções desempenhadas por essa
classe de proteínas, pouco conhecidas, mas de grande importância para a regulação das
funções celulares."
FAPERJ, CNPq
P3.
NEW THIAZOLIDINEDIONE DERIVATIVE RA-4 DECREASES THE EXPRESSION
OF INFLAMMATORY MARKERS IN MURINE MODEL OF CARRAGEENAN-INDUCED
PLEURISY
1- BARBOSA,K.P.S.; 1- ROCHA, S.W.S.; 1- SANTOS, L.A.M.; 1- SANTANA, A.K.; 1FRAGOSO, I.T.; 1- FRANÇA, M.E.R.; 1- GOMES, F.O.S.; 1- RIBEIRO, E.L; 1- SILVA, B.S.; 1SILVA, A.K.S.; 1- DONATO, M.M.A.; 3- SILVA, T.G; 3- LIMA, M.C.A.; 3- PITTA, I.R.;
GALDINO, S.L.; 1,2- PEIXOTO, C.A.
1- Centro de Pesquisas Aggeu Magalhães - CPqAM/FIOCRUZ; 2- Centro de Tecnologias
Estratégicas do Nordeste - CETENE/MCT, Universidade Federal de Pernambuco; 3Departamento de Antibióticos, Universidade Federal de Pernambuco
Background: Many studies have demonstrated several biological activities of
peroxisome proliferator-activated receptor (PPAR), however, few studies have been
conducted on the effects of agonist these receptors on lung diseases, which pose a
serious public health problem. Objective: The present study evaluated the antiinflammatory action of a new synthetic thiazolidinedione derivative RA-4 on acute lung
inflammation (pleurisy) induced by carrageenan. Methodology: Forty mice were
randomly allocated into the following groups: Control group (sham) (n=10); carrageenan
group (CAR) (n=10); CAR + LPSF/RA-4 group (n=10); which was treated with LPSF/RA-4
(60 µMol/kg); CAR + INDO group (5 mg/kg) (n=10). The mice were anaesthetized and
submitted to injection into the pleural cavity with Saline (0.1ml) or saline containing 1%
carrageenan (0.1ml) injected into the pleural cavity. After 4h, the animals were
sacrificed under CO2 vapors. The levels of nitric oxide (NO) quantified on pleural
exudates and lung fragments were processed for light microscopy, transmission
electron microscopy, immunohistochemistry and Western blotting assays. Results: The
influx of leukocytes and nitric oxide levels were significantly reduced after treatment
with RA-4 and INDO, compared with the CAR group. Histopathological and
ultrastructural analysis showed that tissue injury was significantly reduced in the groups
treated with RA-4 or INDO. Immunohistochemistry showed an increase of inflammatory
markers such as COX-2, iNOS, TNF-α, IL-1β in lung tissue CAR group, whereas the groups
treated with RA-4 and INDO presented significant reduction of these immunomarkers.
Analysis by Western blotting revealed increased expression of COX-2 and IL-1 in the CAR
group, which were significantly reduced by treatment with RA-4. Conclusions: These
results demonstrate the potent anti-inflammatory action of the new derivative
thiazolidinedione RA-4 (60µMol/kg) in acute lung injury induced by carrageenan.
Therefore, assays are in development in our laboratory in order to clarify the role of RA4 on the molecular mechanisms involved in the inflammation.
INBEB, FACEPE
Página 66
FAPERJ
P4.
ESTUDO DA IMOBILIZAÇÃO DA PROTEÍNA PRÍON EM NANOPARTÍCULAS
MAGNÉTICAS
1 - de MORAES, M.C.; 1- dos ANJOS, D.M.; 1- BOSCO, J.; 1- da SILVA, J.L.
Encefalopatias espongiformes transmissíveis são desencadeadas quando a proteína
príon nativa é convertida em sua isoforma infecciosa, o que provavelmente requer um
fator celular. Assim, diversos compostos têm sido avaliados como inibidores desta
conversão. Considerando a importância das interações ligante-proteína nos sistemas
biológicos, estudos de afinidade podem contribuir para a compreensão do mecanismo
de conversão e da avaliação de possíveis inibidores. Neste trabalho, foi selecionado
como modelo o estudo de afinidade por "fishing" de ligantes. Para o desenvolvimento
desta metodologia, a proteína príon deve ser imobilizada covalentemente em
nanopartículas magnéticas, sem afetar significativamente a estrutura da proteína.
Assim, foram preparadas nanopartículas magnéticas (NPMs) de Fe3O4 pelo método da
co-precipitação de íons Fe3+ e Fe2+. Posteriormente, as MNPs foram derivadas com
tetraetoxisilano, aminopropriltrietoxisilano e glutaraldeído, com a posterior ligação
covalente da proteína através da formação de bases de Schiff. Cada etapa de derivação
foi confirmada pela análise do potencial zeta de amostras coletadas após cada reação.
As NPMs foram caracterizadas também por infravermelho, morfologicamente por
microscopia eletrônica de transmissão, e a distribuição do tamanho das NPMs foi
investigada por DLS (tamanho médio de 51,12nm). A ligação da proteína príon foi
estimada medindo-se a absorbância a 280nm da solução protéica antes e após a reação
com as NPMs. Com o estudo de otimização, observou-se que aproximadamente 0,6mg
de proteína príon se liga a 2,5mg de NPMs. A próxima etapa do trabalho consiste em
imergir as NPMs com o príon ligado em soluções contendo compostos com conhecida
afinidade pela proteína e, após incubação, realizar magneticamente uma extração
líquido-sólido. Após algumas lavagens, pode-se identificar, por espectrometria de
massas, os compostos com maior afinidade pela proteína, objetivo dos ensaios de
“fishing” de ligantes, obtendo-se assim um método de triagem rápida para ligantes do
príon.
P5.
SILDENAFIL
ESTIMULA
A
PROLIFERAÇÃO
ENDOMETRIAL
DE
CAMUNDONGOS C57BL/6
1- DONATO, M. A. M.; 1- RIBEIRO, E. L.; 1- GOMES, F. O. S.; 1- OLIVEIRA, W. B.; 1FRAGOSO, I. T.; 1- SANTOS, L. A. M.; 1,2- PEIXOTO, C. A.
1 - Centro de Pesquisas Aggeu Magalhães, FIOCRUZ, Recife-PE; 2 - CETENE, Recife-PE
Recentemente, o sildenafil tem sido utilizado como estratégia terapêutica no
tratamento de mulheres com problemas de fertilidade, sugerindo uma possível ação no
trato reprodutor feminino. Vários autores sugerem que o tratamento com o Sildenafil
estimula o crescimento endometrial ou aumenta a possibilidade de implantação. No
presente trabalho, camundongos C57BL/6 selvagens e knocauteados para o gene da
iNOS (iNOS-/-) foram tratados com Sildenafil na concentração de 25 mg/kg/dia por 60
dias. Após esse período, os animais foram eutanasiados, os úteros dissecados e
submetidos a análises de microscopia óptica e eletrônica, assim como a análise
imunohistoquímica para avaliar se houve alteração na expressão de proteínas essenciais
para a receptividade endometrial. Nossos resultados confirmam que o Sildenafil induziu
a proliferação celular no endométrio e miométrio no útero em camundongos C57Bl/6
wild type assim como aumentou a expressão de enzimas angiogênicas VEGF e eNOS no
tecido estromal, além de reduzir a expressão de TGF-β. O grupo controle knockout para
a iNOS apresentou miométrio e endométrio com maior espessura em relação ao grupo
controle selvagem, apresentando glândulas endometriais com lúmen reduzido e células
cilíndricas, indicando que o gene da iNOS apresenta um possível papel na preparação
endometrial pré-implantacional e na implantação embrionária. Após o tratamento dos
camundongos knockout para a iNOS com sildenafil, foram obtidos resultados similares
em relação à expressão de VEGF, eNOS e TGF-β, indicando que a ação do sildenafil no
endométrio independe da presença do gene iNOS.
INBEB
Página 67

Instituto Nacional de Ciência e tecnologia de Biologia Estrutural e

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