L'hépatite B chronique reste un problème majeur de santé publique. Sous traitement par analogues nucléos(t)idiques (NUCs), l'objectif thérapeutique ultime est la clairance de l'antigène (Ag) HBs. Nous avons étudié l'influence de la variabilité des protéines d'enveloppe, impliquées dans l'entrée cellulaire du virus et cibles de la réponse immune, sur la clairance de l'Ag HBs. Des patients traités par NUCs ayant obtenu une clairance de l'Ag HBs (resolvers) ont été appariés à des non-resolver. Deux mutations combinées sT125M/sP127T, caractéristiques des non-resolver, étaient associées à une baisse de l'antigénicité prédite. L'analyse par séquençage haut débit montrait une plus grande variabilité du gène S chez les non resolver. Des tests fonctionnels portant sur des particules virales mutées en sT125M et sP127T sont en cours. Ces données moléculaires sont en faveur de l'existence de "motifs" spécifiques dans le gène S associés à la persistance de l'Ag HBs sous traitement par NUCs / Hepatitis B virus (HBV)-related chronic infection remains difficult to eradicate. On treatment by nucleos(t)ide analogues (NUCs), HBs Antigen (Ag) clearance is the ultimate but difficult therapeutic goal. Our aim was to investigate how variability of HBV envelope protein, crucial in viral cellular entry and targeted by host immune response, could play a role in HBsAg clearance. HBV chronically infected patients, treated by NUCs with HBsAg clearance (resolver) were matched with patients without HBsAg clearance (non resolver). Combined mutations sT125M/sP127T, associated with HBsAg persistence, displayed a lower predicted antigenicity. Ultra Deep Sequencing of S gene showed a higher variability in non resolver. Functional assays on viral particles including sT125M and sP127T mutations versus reference particles are in progress. As a conclusion, molecular features observed in non NR argue in favor of a different pattern in HBV S characteristics according to variable NUCs efficiency

L’hépatite B chronique touche environ 257 millions de personnes dans le monde. La perte de l’antigène HBs (AgHBs), marqueur de guérison fonctionnelle, n’est que très rarement observée, même sous traitement antiviral (3-16 %). Les protéines d’enveloppe du virus de l’hépatite B (VHB), formant l’AgHBs, sont très variables et cruciales pour le pouvoir infectieux du virus de l’hépatite B (VHB) et la physiopathologie. Nous avons émis l’hypothèse que cette variabilité pourrait expliquer, au moins partiellement, l’évolution de l’infection par le VHB, évaluée par la clairance de l’AgHBs, chez des patients traités ou non par analogues nucléos(t)idiques anti-VHB. Chez 29 patients infectés par différents génotypes du VHB (A, C et D), présentant différents profils cliniques (infection aigüe ou chronique, co-infection VHB/VIH) et thérapeutiques, une très grande variabilité des protéines d’enveloppe du VHB a été mise en évidence. Chez ces patients, la persistance de l’AgHBs était corrélée avec la présence de mutations et délétions localisées dans des régions des protéines d’enveloppe virale jouant un rôle important dans la reconnaissance du virus par le système immunitaire. Ces résultats renforcent l’hypothèse que l’étude des protéines d’enveloppe du VHB pourrait mettre en évidence des signatures moléculaires influençant le fitness du VHB et par conséquent l’évolution clinique de la maladie liée à l’infection par le VHB / Chronic hepatitis B affects about 257 million people worldwide. The loss of HBS antigen (HBsAg), a marker of the functional cure, is very rarely observed, even on anti-HBV treatment (3-16%). The hepatitis B virus (HBV) envelope proteins (HBsAg) are highly variable and crucial for the viral infectivity and pathogeny. We hypothesized that the HBV variability in the envelope proteins could explain, at least partially, the evolution of HBV infection, evaluated by HBsAg clearance, in patients treated or not by anti-HBV nucleos(t)idic analogues. For 29 patients infected with different HBV genotypes (A, C and D), presenting different clinical profiles (acute or chronic infection, HBV/HIV co-infection) and therapies, a very high variability of HBV envelope proteins was observed. In these patients, the persistence of HBsAg was correlated with the presence of mutations and deletions located in areas that play a key role in the viral recognition by the immune system. These results reinforce the hypothesis that the study of HBV envelope proteins could highlight molecular signatures influencing HBV fitness which would subsequently modify the clinical evolution of HBV-related disease

Made available in DSpace on 2016-03-22T17:27:14Z (GMT). No. of bitstreams: 1 Elza Bueno.pdf: 2003941 bytes, checksum: 176576d54fbdbc371386c7215923debb (MD5) Previous issue date: 2013-12-02 / Depressive symptoms have been frequently observed in association with immune activation. To prospectively evaluate, depressive symptoms and risk factors for major depression in patients with hepatitis C virus (HCV) treated with antiviral combined therapy. This study is a convenience cohort that evaluated 50 patients with HCV by the structured diagnostic interview - Mini International Neuropsychiatric Interview (MINI) to screen for depressive symptoms before antiviral combined therapy, and in the follow-up visits (4 and 12th week). Laboratorial analysis were performed during the follow-up. The study was approved by the University s Ethics Committee (151.642). We evaluated 50 patients, in which prevalence of genotype 1 was 42%. Pegylated interferon alpha (IFN-α) and ribavirin was the most prevalent treatment used for HCV (86%). During the follow-up of patients, treatment for HCV increases the risk of depression in the 4th week (43.5.9%), but not at 12th week (30.7%) treatment compared with the baseline (25.6%) (p=0.04). We found differences between the prevalences of depression and genotypes of the virus in regard to time of the follow-up with higher odds ratio in the 4th week (OR=2.2) compared to baseline and 12th week (OR=1.8) using pairwise comparisons with Bonferroni adjustment (p=0.03). Also, patients with genotype 2 and 3 had significantly lower odds of presenting depression compared genotype 1 (p≤0.05). However, the average score on the BDI-II did not differ in the follow-up.This study provide evidence of an association between HCV genotype and major depression. During the follow-up, depressive symptoms increase in 4th week, corresponding to conditions of immune activation. Major depression in HCV patients influence their health-related quality of life and their adherence to antiviral treatment 8 being important screening programmes, for early recognition and treatment of interferon-induced depression / Introdução:Os sintomas depressivos têm sido freqüentemente observados em associação com ativação imune .Objetivos: Avaliar prospectivamente , sintomas depressivos e fatores de risco para depressão maior em pacientes com o vírus da hepatite C (HCV ) tratados com terapia combinada antiviral . Metodologia: Este estudo é uma coorte de conveniência , que avaliou 50 pacientes com HCV por entrevista diagnóstica estruturada - Mini International Neuropsychiatric Interview ( MINI ) - para triagem de sintomas depressivos antes da terapia antiviral combinada , e nas visitas de acompanhamento (4 e 12 semanas ) . A análise laboratorial foi realizada durante o follow-up. O estudo foi aprovado pelo Comitê de Ética da Universidade (151,642). Resultados: Foram avaliados 50 pacientes, nos quais a prevalência do genótipo 1 foi de 42% . Interferon peguilado alfa (IFN - α) e ribavirina era o tratamento mais prevalente utilizado para HCV ( 86 % ) . Durante o seguimento de pacientes, o tratamento para HCV aumenta o risco de depressão na 4 ª semana ( 43.5 %) , mas não a 12 ª semana (30,7%) em comparação com o tratamento inicial ( 25,6% ) (p = 0,04) . Foram encontradas diferenças entre as prevalências de depressão e genótipos do vírus em relação ao tempo de seguimento com maior razão de odds na 4 ª semana (OR = 2,2 ) em relação à linha de base e 12 ª semana (OR = 1,8 ), utilizando comparações pareadas com ajuste de Bonferroni (p = 0,03). Além disso, os pacientes com genótipo 2 e 3 tiveram chances significativamente menores de apresentar depressão, em comparação genótipo 1 (p ≤ 0,05). No entanto, a pontuação média no BDI- II não foi diferente no estudo follow- up. Conclusão: Este estudo fornece evidências de uma associação entre o genótipo HCV e depressão maior. Durante o seguimento, os sintomas depressivos aumentam na 4 ª semana, o que corresponde a condições de ativação imune . A depressão maior em pacientes HCV influencia a qualidade de vida e sua adesão ao tratamento antiviral, sendo importantes programas de rastreio, para a identificação precoce e tratamento da depressão induzida por interferon.

Le VHC infecte les hépatocytes, cellules polarisées du foie. Le cycle de réplication du VHC est dépendant du métabolisme lipidique de la cellule hôte. Mais la relation entre VHC, polarité cellulaire et métabolisme lipidique est mal connue. Nous avons démontré que SHIP2 joue un rȏle important dans l’établissement de la polarité apicobasale des cellules épithéliales. La protéine core du HCV induit la perte de polarité cellulaire et diminue l’expression de la phosphatase SHIP2. La réintroduction de SHIP2 dans les cellules exprimant core restitue la polarité cellulaire et diminue l’expression de core. SHIP2 agit aussi sur l’accumulation et l’organisation des gouttelettes lipidiques qui sont des éléments cellulaires nécessaires à la réplication du VHC. Ces résultats montrent le rôle de SHIP2 dans la polarité cellulaire et le désigne comme une cible intéressante pour des recherches dans la lutte contre les infections du VHC. / HCV infects hepatocytes, polarized cells of the liver. HCV replication cycle is dependent on lipid metabolism of the host cell. But the relationship between HCV cell polarity and lipid metabolism is unknown. We demonstrated that SHIP2 plays an important role in establishment of the apicobasal epithelial cell polarity. The HCV core protein induces loss of cell polarity and decreases the expression of the phosphatase SHIP2. The reintroduction of SHIP2 in cells expressing core restores cell polarity and decreases the expression of core protein. SHIP2 also negatively affect lipid droplets, which are important for HCV replication. These results show the role of SHIP2 in cell polarity and designate it as an attractive target for research in the fight against HCV infection.

Introdução: Pacientes com infecção crônica pelo VHC e superinfecção pelo vírus da hepatite A (VHA) ou o vírus da hepatite B (VHB), têm maior morbi-mortalidade quando comparados com pacientes que apresentam infecção aguda somente pelo VHA ou VHB. A mortalidade associada à hepatite A aguda pode estar particularmente elevada em pacientes com pré-existência de hepatite crônica causada pelo VHC. Por esta razão, a imunização ativa com vacinas contra o VHA e o VHB vem a ser obrigatória nesta população, e consequentemente esta sorologia deve ser determinada. Objetivos: O objetivo deste trabalho foi avaliar a prevalência de marcadores sorológicos da hepatite A e hepatite B em 1.000 pacientes com infecção crônica pelo VHC atendidos no Ambulatório de Hepatites da Divisão de Gastroenterologia e Hepatologia Clínica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Resultados: O anti-VHA IgG foi positivo em 923 de 1000 pacientes (92,3%). Quando estratificados por idade, o anti-VHA IgG foi encontrado em 61% dos pacientes entre 20 e 29 anos, 70% entre 30 e 39 anos, 85% entre 40 e 49 anos, 94% entre 50 e 59 anos e 99% nos pacientes com mais de 60 anos . O anti-HBc total foi positivo em 244 pacientes (24%). Estratificados por idade, em 4,3% dos pacientes entre 20 e 29 anos, 17% entre 30e 39 anos, 21% entre 40 e 49 anos, 24% entre 50 e 59 anos, e 28% dos pacientes com mais de 60 anos. Dos 244 pacientes anti-HBc IgG positivos, 0,8% são HBsAg positivo, 8,5% anti-HBc IgG isolado e 16% anti-HBs positivo. Conclusões: A prevalência de anti-VHA IgG nod nossos pacientes com hepatite C crônica foi semelhante à da população geral no município de São Paulo. No entanto, o anti-HBc totaI foi maior em nossos pacientes, quando comparada historicamente à população geral dos países ocidentais, sugerindo fatores de risco semelhantes para as hepatites B e C, o que enfatiza a importância dos programas de imunização nesta população / Background and Aims: Patients with chronic HCV and superinfection by hepatitis A virus (HAV) or hepatitis B virus (HBV) have higher morbidity and mortality when compared with those without HCV. For this reason, HAV and HBV active immunization has become mandatory in this population and hence their serological markers must be determined. The aim of this study was to evaluate the prevalence of serological markers of HAV and HBV infection in patients with chronic HCV. Methods: 1.000 chronic HCV infected patients at the University of Sao Paulo School of Medicine outpatient Liver Clinic were evaluated for the prevalence of serological markers of HAV and HBV infection. Results: Anti-HAV IgG was positive in 923 of 1000 patients (92.3%). When stratified by age, the anti-HAV IgG was found in 61% of patients between 20-29 years, 70% between 30-39 years, 85% between 40-49 years, 94% between 50-59 years, and 99% over 60 years of age. Anti-HBc IgG was positive in 244 patients (24%). Stratified by age, anti-HBc IgG was found in 4.3% of patients between 20-29 years, 17% between 30-39 years, 21% between 40 -49 years, 24% between 50-59 years, and 28% of patients over 60 years of age. Of the 244 anti-HBc IgG positive patients, 0.8% were also HBsAg positive, 8.5% were anti-HBc IgG isolated and 16% were also anti-HBs positive. Conclusions: The prevalence of anti-HAV IgG was similar to the general population in the city of São Paulo. However, anti-HBc IgG was higher in our chronic HCV patients, when compared historically to the general population of western countries, suggesting similar risk factors for HBV and HCV acquisition, so emphasizing the importance of immunization programs in this population. Keywords: Hepatitis C, Chronic; Hepatitis C; Hepacivirus, Prevalence; Hepatitis A; Hepatitis B Título: Prevalência de Marcadores Sorológicos das Hepatites A e B em Pacientes com Hepatite C Crônica atendidos no Ambulatório de Hepatites do Serviço de Gastroenterologia Clínica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo - HCFMUSP Background and Aims: Patients with chronic HCV and superinfection by hepatitis A virus (HAV) or hepatitis B virus (HBV) have higher morbidity and mortality when compared with those without HCV. For this reason, HAV and HBV active immunization has become mandatory in this population and hence their serological markers must be determined. The aim of this study was to evaluate the prevalence of serological markers of HAV and HBV infection in patients with chronic HCV. Methods: 1.000 chronic HCV infected patients at the University of Sao Paulo School of Medicine outpatient Liver Clinic were evaluated for the prevalence of serological markers of HAV and HBV infection. Results: Anti-HAV IgG was positive in 923 of 1000 patients (92.3%). When stratified by age, the anti-HAV IgG was found in 61% of patients between 20-29 years, 70% between 30-39 years, 85% between 40-49 years, 94% between 50-59 years, and 99% over 60 years of age. Anti-HBc IgG was positive in 244 patients (24%). Stratified by age, anti-HBc IgG was found in 4.3% of patients between 20-29 years, 17% between 30-39 years, 21% between 40 -49 years, 24% between 50-59 years, and 28% of patients over 60 years of age. Of the 244 anti-HBc IgG positive patients, 0.8% were also HBsAg positive, 8.5% were anti-HBc IgG isolated and 16% were also anti-HBs positive. Conclusions: The prevalence of anti-HAV IgG was similar to the general population in the city of São Paulo. However, anti-HBc IgG was higher in our chronic HCV patients, when compared historically to the general population of western countries, suggesting similar risk factors for HBV and HCV acquisition, so emphasizing the importance of immunization programs in this population

by Sung Ying-Man, Mandy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 112-125). / Abstracts in English and Chinese. / Acknowledgments --- p.I / List of Abbreviations --- p.II / Abstract --- p.IV / 摘要 --- p.VII / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Epidemiology --- p.1 / Chapter 1.2 --- Aetiological factors --- p.4 / Chapter 1.2.1 --- Hepatitis B infection --- p.4 / Chapter 1.2.2 --- Aflatoxin exposure --- p.5 / Chapter 1.2.3 --- Alcohol consumption --- p.5 / Chapter 1.2.4 --- Genetic risk factors --- p.6 / Chapter 1.3 --- Aims of the study --- p.7 / Chapter Chapter 2 --- Polymorphisms of candidate genes in Interleukin- signalling pathway in HCC --- p.6 / Chapter 2.1 --- Introduction --- p.9 / Chapter 2.2 --- Materials and Methods --- p.15 / Samples and Genomic DNA isolation --- p.15 / PCR-PFLP --- p.15 / dHPLC --- p.16 / Direct Sequencing --- p.17 / Stattistical Analysis --- p.17 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Known IL6 polymorphisms --- p.25 / Chapter 2.3.2 --- IL-6R and gp130 polymorphisms --- p.28 / Chapter 2.3.3 --- Stat-3 polymorphisms --- p.29 / Chapter 2.3.4 --- SOCS-1 polymophisms --- p.32 / Chapter 2.3.5 --- Mutation screening of IL-6 gene --- p.34 / Chapter 2.3.6 --- Mutation screening of SOCS-1 gene --- p.39 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.4.1 --- Interleukin-6 --- p.40 / Chapter 2.4.2 --- Gp130 and IL6-R --- p.43 / Chapter 2.4.3 --- STAT-3 --- p.44 / Chapter 2.4.4 --- SOCS-l --- p.46 / Chapter Chapter 3 --- Methylation status of SOCS-1 gene in Chinese HCC patients / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Methods and Materials --- p.53 / Tissue Sampling --- p.53 / Methylation specific PCR (MSP) --- p.53 / Chapter 3.3 --- Results --- p.55 / Chapter 3.4 --- Discussion --- p.56 / Chapter Chapter 4 --- Polymorphisms of enzyme encoding genesin steroidogenesis in Chinese / Chapter 4.1 --- Introduction --- p.59 / Chapter 4.1.1 --- Steroid 5a reductases (SRD5A) --- p.62 / Chapter 4.1.1a --- Steroid 5a reductase type II (SRD5A2) --- p.63 / Chapter 4.1.1b --- Steroid 5a reductase type I (SRD5A1) --- p.65 / Chapter 4.1.2 --- Cytochrome P450al7 (CYP17) --- p.67 / Chapter 4.1.3 --- "Cytochrome P450, family 1，subfamily A polypeptide1 (CYP1A1)" --- p.69 / Chapter 4.1.4 --- "Cytochrome P450, subfamily IIIA (niphedipine oxidase) polypeptide 4 (CYP3A4)" --- p.71 / Chapter 4.2 --- Materials and Methods --- p.74 / Samples and Genomic DNA isolation --- p.74 / PCR-PFLP --- p.74 / Direct Sequencing --- p.74 / Statistical Analysis --- p.74 / Chapter 4.3 --- Results --- p.77 / Chapter 4.3.1 --- SRD5A2 --- p.77 / Chapter 4.3.2 --- Linkage Disequilibrium in SRD5A2 gene --- p.83 / Chapter 4.3.2 --- SRD5A1 --- p.84 / Chapter 4.3.3 --- CYP17 --- p.87 / Chapter 4.3.4 --- CYP1A1 --- p.89 / Chapter 4.3.5 --- CYP3A4 --- p.92 / Chapter 4.3.6 --- Logistic regression --- p.95 / Chapter 4.4 --- Discussion --- p.96 / Chapter 4.4.1 --- SRD5A2 --- p.96 / Chapter 4.4.2 --- SRD5A1 --- p.99 / Chapter 4.4.3 --- CYP17 --- p.101 / Chapter 4.4.4 --- CYP1A1 --- p.103 / Chapter 4.4.5 --- CYP3A4 --- p.106 / Chapter 4.4.6 --- Logistic Regression --- p.107 / Chapter Chapter 5 --- Conclusions and Future Prospect --- p.108 / Chapter 5.1 --- Conclusions --- p.108 / Chapter 5.2 --- Future works and prospect --- p.111 / References --- p.113

GBF1 a émergé autant que facteur cellulaire nécessaire pour la réplication de plusieurs virus à ARN. Au cours de l’infection par le virus de l’hépatite C (VHC), GBF1 est essentiel pour les étapes précoces de la réplication, bien qu’il soit dispensable lorsque celle-ci est établie. Afin de mieux comprendre la fonction de GBF1 dans la régulation de l'infection par le VHC, nous avons tenté d’explorer les interactions entre GBF1 et les protéines du VHC. Ainsi, grâce à l’approche du double hybride en levure et par co-immunoprécipitation et par PLA (proximity ligation assay), nous avons pu montrer que NS3 interagit avec GBF1. De plus, NS3 semble interférer avec la localisation subcellulaire de GBF1 dans des cellules exprimant NS3. Cette interaction a été retrouvée entre le domaine protéase de NS3 et Sec7, le domaine catalytique de GBF1. Un crible sur des mutations altérant l’interaction GBF1-NS3, par double hybride en levure, a permis révéler un mutant NS3 (N77D de la souche Con1) qui est non-réplicatif malgré une activité protéase bien conservée. De plus, le résidu muté est exposé à la surface, ce qui suggère qu’il pourrait appartenir à la zone d’interaction de NS3 avec GBF1. La mutation correspondante dans la souche JFH1 produit le même phénotype que la souche Con1 du VHC. L’ensemble des résultats révèlent l’existence d’une interaction entre GBF1 et NS3 et suggèrent qu’une altération de cette interaction est délétère pour la réplication du VHC. / GBF1 has emerged as a host factor required for the replication of RNA viruses of different families. During the hepatitis C virus (HCV) life cycle, GBF1 performs a critical function at the onset of replication, but is dispensable when the replication is established. To better understand how GBF1 regulates HCV infection, we have looked for interactions between GBF1 and HCV proteins. NS3 was found to interact with GBF1 in yeast two-hybrid, in co-immunoprecipitation and in proximity ligation assays, and to interfere with GBF1 function and alter GBF1 intracellular localization in cells expressing NS3. The interaction was mapped to the Sec7 domain of GBF1 and the protease domain of NS3. A yeast two-hybrid screen for mutations altering NS3-GBF1 interaction yielded an NS3 mutant (N77D, Con1 strain) that is non-replicative despite conserved protease activity. The mutated residue is exposed at the surface of NS3, suggesting it could be part of the domain of NS3 that interacts with GBF1. The corresponding mutation in JFH-1 strain (S77D) produces the same phenotype. Our results provide evidence for an interaction between NS3 and GBF1 and suggest that an alteration of this interaction is detrimental to HCV replication.

Orientador: Paula Rahal / Banca: Hamilton Cabral / Banca: Nelson José Freitas da Silveira / Banca: Maria Tercília Vilela de Azeredo Oliveira / Banca: José Osmar Gaspar / Resumo: A proteína NS3 apresenta dois domínios e é bifuncional. Apresenta três funções enzimáticas que são; 1) atividade de protease; 2) NTPase e 3) helicase. A função protease relaciona-se a tradução da proteína precursora e as funções NTPase e helicase tem grande participação na replicação do material genético viral. Trata-se de uma molécula essencial para o processamento da poliproteína precursora e também para a replicação viral e portanto, um dos principais alvos para o desenvolvimento de drogas antivirais. No domínio Protease foram evidenciadas substituições na tríade catalítica e na região de ligação ao íon zinco nos pacientes avaliados. Estas substituições, quando somadas podem explicar a resposta ao tratamento. Também foram visualizadas alterações na porção Helicase da NS3. As substituições ocorreram nos sítios de ligação ao ATP e ao RNA. Outros resíduos da Helicase relevantes para o desenvolvimento de inibidores, como R2133 e F258 e F264 não apresentaram substituições, evidenciando tratarem-se de aminoácidos conservados nessa região. Os resultados obtidos nesse trabalho fornecem informações sobre o perfil genético do vírus HCV do genótipo 3 especificamente da região codificadora da proteína NS3, permitindo o conhecimento do genoma viral e a identificação de regiões para ligação de possíveis inibidores. Este projeto certifica que a modelagem é uma ferramenta útil para a biologia estrutural e funcional, e que os modelos obtidos aqui contribuem para o desenho de novas drogas anti-virais específicas para o genótipo 3 do vírus HCV / Abstract: The NS3 protein has two domains and is bifuntional. It presents three functions: 1) protease activity, 2) NTPase and 3) helicase. The protease function is related to the translation of the poliprotein precursor and functions NTPase and helicase has great participation in the replication of the viral genetic material. So. The NS3 is considered the major target for the development of antiviral drugs. In the Protease portion substitutions were evidenced in catalytic triad and the zinc ion binding sites, in the patients evaluated. These substitutions, when added up can explain the response to treatment. Also were observed changes in Helicase portion of NS3. The substitutions took place on ATP and RNA binding sites. Other residues of Helicase relevant to the development of inhibitors, as R2133 and F258 and F264, showed no substitutions, highlighting the great conservation of amino acids in this region. The results obtained in this work provide information on the genetic profile of the HCV virus genotype 3, specifically the region of NS3 protein, allowing the knowledge of the viral genome and the identification of regions for possible connection of inhibitors. This project certifies that the modeling is a useful tool for structural biology and functional, and that the models obtained here contribute to the design of new anti-viral drugs specific to the genotype 3 of HCV virus / Doutor

O objetivo deste estudo foi avaliar a qualidade virológica da água de reúso produzida em uma das estações de tratamento de esgoto da cidade de São Paulo. Para tanto, foram coletadas concomitantemente 177 amostras de esgoto tratado (100L) e bruto (15L) e os vírus concentrados utilizando método Viradel-ultracentrifugação. Em seguida as amostras foram tratadas com Vertrel XF e os ácidos nucléicos extraídos para a detecção de adenovírus (HAdV), rotavírus (RV-A), norovírus e vírus da hepatite A (VHA). A detecção por PCR e/ou RT-PCR evidenciou RV-A (G1-G5), VHA e HAdV incluindo os da espécie F tanto no esgoto bruto quanto no tratado, no entanto norovírus não foram detectados em ambos os efluentes. A infectividade de RV-A e HAdV foi avaliada por cultivo celular e os rotavírus RV-A foram também quantificados por reação de imunoperoxidase direta. PCR em tempo real foi padronizada para quantificação de vírus não cultiváveis ou de difícil cultivo como os VHA. Com base nos resultados obtidos foi verificada a ocorrência e a distribuição anual de cada vírus nas águas de reúso. / The aim of the study was to evaluate the virological quality from one Sewage Treatment Plant in the state of São Paulo. From January/2005 to November/2006, 177 samples (15L) of raw sewage were collected at the entrance and another 177 (100L) at the end of treatment, twice a week. Viruses were concentrated by filtration through positively charged microporous filters, followed by ultracentrifugation. Virus concentrates were treated by using Vertrel-XF and the viral genomes extracted for detection of adenovirus (HAdV), rotavirus (RV-A), norovirus and hepatitis A virus (HAV). PCR and RT-PCR revealed RV-A (G1-G5), HAV and HAdV, including the enteric ones (species F) in sewage and treated wastewater samples. Norovirus was not detected in any samples. The infectivity of HAdV and RV-A was assayed by inoculating onto suitable cell line. Immunoperoxidase assay was used to calculate the rotavirus FFU/L in the samples. Real time-PCR was standardized for enumeration of non-cultivable virus. The occurrence and annual distribution of each virus in reuse water were analyzed.

Introdução: A hepatite C é um grande problema de saúde pública e sua prevalência global está estimada em torno de 3%. O risco de transmissão do VHC via fluído seminal é muito discutida tanto na área de reprodução assistida como em estudos sobre o fator de risco da hepatite C ser ou não uma DST. Foram investigados e analisados 23 pacientes sabidamente infectados pelo VHC. Objetivos: 1.Estabelecer uma técnica para detectar a ausência ou presença do vírus da Hepatite C no sêmen de pacientes infectados; 2.Comparar técnicas de manuseio das amostras de sêmen, procurando diminuir a quantidade de inibidores presentes nas amostras; 3.Comparar técnicas de PCR e detecção do vírus da Hepatite C nas amostras de sêmen, procurando aumentar a sensibilidade dos testes. Métodos: Na primeira fase do estudo foram recrutados 20 pacientes (13 preencheram os critérios de inclusão). Amostras de sêmen e soro foram coletadas. As amostras de sêmen foram processadas com o auxílio do Percoll 90% e 45%. Foi analisada a presença do HCVRNA em soro pelo método Amplicor Roche, teste qualitativo. Se positivas, as amostras de sangue foram genotipadas e as amostras de sêmen foram extraídas, pelo mesmo método, e a PCR executada. Na segunda fase do estudo 23 pacientes foram selecionados, sendo alguns reconvocados da primeira fase (20 preencheram os critérios de inclusão). Amostras de sêmen e soro foram coletadas. As amostras de sêmen foram processadas através de diluições seriadas. Foi analisada a presença do HCV-RNA em soro e sêmen pelo método Amplicor Roche, teste qualitativo e por PCR em Tempo-real. Os dados epidemiológicos e os genótipos foram analisados, assim como os resultados da detecção do soro. Sêmen e frações realizadas pelas 2 (duas) técnicas de processamento estabelecidas foram comparados e analisados. Resultados: Dos 23 pacientes selecionados, a média de idade foi de 40,7 anos, com mediana de 45 anos. O tempo médio de descoberta da infecção pelo VHC foi de 7,15 anos. Dez pacientes (37,1%) não possuíam epidemiologia aparente, oito pacientes (29,6%) adquiriram a infecção pelo VHC através da utilização de drogas injetáveis e/ou inalatórias; seis (22,2%) por transfusão sanguínea; dois (7,4%) apresentaram histórico de transfusão sanguínea e uso de drogas e um (3,7%) relatou ser profissional da saúde. O genótipo 3a foi encontrado em 40,7% dos pacientes, seguido pelo 1a com 26%, 1b com 14,8%, 2b em 11,1% e 1a/1b em 7,4%. Das amostras processadas pelo Percoll, 86,5% apresentaram resultados inibidos, enquanto que nas amostras processadas pela diluição seriada e amplificadas através do PCR convencional, apenas 25,62% das amostras apresentou inibição, 65% não foram detectadas e 9,38% das amostras apresentou positividade. Nas amostras processadas pela diluição seriada na PCR em Tempo-real, 95% das amostras não foram detectadas e somente 5% apresentou positividade. Conclusão: Na tentativa de driblar os inibidores presentes nas amostras de sêmen, o procedimento de diluições seriadas mostrou maior eficácia quando comparado com o processamento através do gradiente descontínuo de concentração. Contudo, a grande quantidade de não detectados mostrou que a carga viral pode ter sido diluída, gerando a necessidade da utilização de técnicas mais sensíveis. Não foi observada diferença significativa entre os resultados da PCR convencional e Tempo-real. Porém o aumento na quantidade dos resultados negativos pode ser conseqüência da ausência de um controle interno nas reações da PCR em Tempo-real. / Introduction: Hepatitis C vírus is a huge problem for public health, and its global prevalence is estimated around 3%. Its transmission by seminal fluid is still in discussion in several fields, such as assisted reproduction and in studies about risk factors, whether the hepatitis C virus is an STD (sexually transmitted disease) or not. Twenty-three patients were investigated. Objectives: 1.Establish a technique to detect the presence or absence of the HCV in semen from chronically infected patients; 2.Compare semen samples handling techniques, in order to decrease the amount of inhibitors on the samples; 3.Compare different PCR and detection techniques for the HCV in semen samples, in order to increase the sensibility of the test. Methods: On the first phase 20 patients were selected (13 filled the inclusion criterion). Semen and serum samples were collected. The semen samples were processed with the help of Percoll® 90% and 45%. The presence of the RNA-HCV were analyzed in serum with Amplicor Roche method, qualitative test. When positive, the serum samples were genotyped and the semen samples were extracted, by the same method, and the PCR was done. On the second phase 23 patients were selected, some of them were old patients from the first phase (20 filled the inclusion criterion). Semen and serum samples were collected. The semen samples were processed through a dilution series. The presence of HCV-RNA was analised by Amplicor Roche, qualitative test and by PCR in Real-time. The epidemiological data and genotypes were analised. Resultados: From the 23 patients selected the mean age was 40,7 years, mean 45 years. The mean time of Discovery was 7,15 years. Ten patients (37,1%) didn´t present any apparent epidemiology, eight patients (29,6%) contracted HCV through injection and inhalatory drug use; six patients (22,2%) through blood transfusion; two patients (7,4%) had history of drug use and blood transfusion and one patient (3,7%) who was a health professional. Genotype 3a was found in 40,7% of the patients, followed by 1a with 26% of the patients, 1b with 14,8%, 2b with 11,1% e 1a/1b in 7,4% of the patients. The samples processed with Percoll, 86,5% presented inhibited results. Whereas on the samples that were processed with dilution series and amplified on the conventional PCR only 25,62% presented inhibited results, 65% were undetected and 9,38% were positive. On the samples processed with dilution series on the Real-time PCR 95% were undetected and only 5% were positive. Conclusion: On the attempt of decreasing the amount of inhibitors found on the semen samples, the procedure of dilution series showed us more efficient results when compared to the Percoll procedure. However, the great amount of undetected showed that the viral load might have being diluted, leading us to the necessity of a more sensitive technique. There was no significant difference between the results of the conventional PCR and the Real-time. These increase on the undetected results may be a consequence of the absence of a internal control on the PCR reactions.