Contexto: A suplementação de ácido fólico (AF) melhora a função endotelial de indivíduos infectados pelo HIV em uso contínuo de terapia antirretroviral (TARV). A literatura não demonstra com clareza esse benefício em indivíduos coinfectados HIV-HCV. Introdução: Indivíduos infectados pelo HIV ou em coinfecção pelo HIV-HCV apresentam um conjunto de fatores de risco que podem levar a disfunção endotelial. Estudos demonstram que a administração de AF possui efeitos benéficos sobre a função endotelial de diferentes populações com risco cardiovascular, inclusive em HIV monoinfectados. Objetivo: Determinar o efeito da suplementação de AF por quatro semanas sobre a dilatação mediada (FMD) pelo fluxo da artéria braquial em indivíduos infectados pelo HIV ou HIV-HCV em uso contínuo de TARV. Delineamento: Ensaio clínico randomizado (ECR), controlado por placebo. Local: Ambulatório de doenças infecciosas do Hospital Universitário de Santa Maria. População: Foram avaliados 69 indivíduos com idade entre 18-50 anos, de ambos os sexos, infectados pelo HIV com ou sem coinfecção pelo HCV, em TARV e com carga viral indetectável há mais de seis meses. Excluíram-se participantes com diabetes mellitus, infarto agudo do miocárdio, revascularização miocárdica, ou acidente vascular encefálico prévios, com creatinina >1,5 mg/dL, diagnóstico clínico, ecográfico, endoscópico ou laboratorial de cirrose hepática, em uso de estatinas, fibratos, terapia de reposição hormonal, sulfonamidas, suplementos vitamínicos, ou AF nos últimos 30 dias. Adicionalmente foram excluídas mulheres grávidas, e participantes de outro ECR. Intervenção: Indivíduos alocados para o grupo intervenção receberam AF, 5 mg, via oral, em dose única diária, pela manhã, durante quatro semanas. Os participantes alocados para grupo placebo receberam orientação para seguir a mesma posologia, sendo os comprimidos indistinguíveis em cor, aroma, sabor, forma e tamanho. Desfechos: Utilizaram-se as variações nos níveis de homocisteína, vitamina B12 e na FMD, aferida por Doppler, na artéria braquial, obtidos na randomização e ao final do seguimento. Resultados: Realizou-se o rastreamento de 239 participantes, sendo 72 elegíveis, 69 randomizados e 68 acompanhados, entre outubro de 2012 e julho de 2013. Em indivíduos infectados pelo HIV houve aumento significativo nos níveis de AF (12,8 ng/ml) no grupo intervenção em comparação ao placebo (P<0,001), acompanhada de variação negativa de homocisteína (-1,9 umol/l) no grupo intervenção e aumento mínimo no grupo placebo (P<0,001). Não houve variação significativa na FMD (P=0,9). Entre indivíduos coinfectados por HIV-HCV, a variação nos níveis de AF foi decorrente da elevação no grupo intervenção (12,6 ng/ml) e redução no grupo placebo (-0,7 ng/ml) (P<0,001). Os níveis de homocisteína aumentaram no grupo placebo (4,6 umol/l) e diminuíram no grupo intervenção (-1,0 umol/l) (P<0,0003). Em relação ao FMD, houve tendência à redução percentual no grupo intervenção e aumento no grupo placebo (P=0,007). As variações de vitamina B12 não foram significativas, independente do status de infecção para HCV. Conclusão: Esse estudo demonstrou que a suplementação de AF por um curto período de tempo, esteve associada com redução de homocisteína sérica, mas não modificou a FMD da artéria braquial, aferida por Doppler, em indivíduos adultos infectados pelo HIV ou HIV-HCV em uso de TARV. / Context: The supplementation of folic acid (FA) improves endothelial function in HIV-infected individuals in continuous use of highly active antiretroviral therapy (HAART). The literature does not clearly show this benefit in coinfected HIV-HCV. Introduction: Individuals infected with HIV or HIV-HCV coinfected presents a set of risk factors that can lead to endothelial dysfunction. Studies show that FA management has beneficial effects on endothelial function in different populations with cardiovascular risk, including HIV monoinfected. Objective: To determine the effect of FA supplementation for four weeks on the mediated dilation (FMD) by brachial artery flow in patients infected with HIV or HIV-HCV continuous HAART. Design: Randomized clinical trial (RCT), placebo-controlled study. Location: Division of Infectious Diseases, University Hospital of Santa Maria. Population: A total of 69 subjects aged 18-50 years, of both sex, HIV or HIV-HCV infected, on HAART, with undetectable viral load for more than six months. Patients presenting with diabetes mellitus, acute myocardial infarction, coronary revascularization, or stroke prior, with creatinine >1,5 mg/dL, clinical diagnosis, ultrasound, endoscopic or laboratory evidence of liver cirrhosis, on statins, fibrates, hormone replacement therapy, sulfonamides, vitamin supplements, or FA in the last 30 days. In addition pregnant women were excluded, and participants in a RCT. Intervention: Subjects allocated to the intervention group received FA, 5 mg orally once daily in the morning for four weeks. Participants allocated to placebo group were instructed to follow the same dosage, being indistinguishable tablets in color, aroma, taste, shape and size. Outcomes: changes were used in the levels of homocysteine, vitamin B12 and FMD, measured by Doppler, in the brachial artery obtained at randomization and at the end of follow-up. Results: We carried out the screening of 239 participants, 72 eligible, 69 randomized and 68 accompanied, between October 2012 and July 2013. In HIV-infected patients there was a significant increase in the levels of FA (12.8 ng/ml) in the intervention group compared to placebo (P <0.001), accompanied by negative variation of homocysteine (1.9 umol/L) in the group intervention and minimal increase in the placebo group (P <0.001). There was no significant change in FMD (P = 0.9). Between individuals coinfected with HIV-HCV, the variation in FA levels was due to the increase in the intervention group (12.6 ng/ml) and reduction in the placebo group (-0.7 ng / ml) (P <0.001). Homocysteine levels increased in the placebo group (4.6 umol/L) and decreased in the intervention group (-1.0 umol/L) (P <0.0003). Regarding the FMD, there was a tendency to percentage reduction in the intervention group and increased in the placebo group (P = 0.007). Variations of B12 were not significant, independent of HCV infection status. Conclusion: This study showed that AF supplementation for a short term, was associated with reduced serum homocysteine, but did not change the FMD of the brachial artery, measured by Doppler in adults infected with HIV or HIV-HCV in HAART.

Contexto: A suplementação de ácido fólico (AF) melhora a função endotelial de indivíduos infectados pelo HIV em uso contínuo de terapia antirretroviral (TARV). A literatura não demonstra com clareza esse benefício em indivíduos coinfectados HIV-HCV. Introdução: Indivíduos infectados pelo HIV ou em coinfecção pelo HIV-HCV apresentam um conjunto de fatores de risco que podem levar a disfunção endotelial. Estudos demonstram que a administração de AF possui efeitos benéficos sobre a função endotelial de diferentes populações com risco cardiovascular, inclusive em HIV monoinfectados. Objetivo: Determinar o efeito da suplementação de AF por quatro semanas sobre a dilatação mediada (FMD) pelo fluxo da artéria braquial em indivíduos infectados pelo HIV ou HIV-HCV em uso contínuo de TARV. Delineamento: Ensaio clínico randomizado (ECR), controlado por placebo. Local: Ambulatório de doenças infecciosas do Hospital Universitário de Santa Maria. População: Foram avaliados 69 indivíduos com idade entre 18-50 anos, de ambos os sexos, infectados pelo HIV com ou sem coinfecção pelo HCV, em TARV e com carga viral indetectável há mais de seis meses. Excluíram-se participantes com diabetes mellitus, infarto agudo do miocárdio, revascularização miocárdica, ou acidente vascular encefálico prévios, com creatinina >1,5 mg/dL, diagnóstico clínico, ecográfico, endoscópico ou laboratorial de cirrose hepática, em uso de estatinas, fibratos, terapia de reposição hormonal, sulfonamidas, suplementos vitamínicos, ou AF nos últimos 30 dias. Adicionalmente foram excluídas mulheres grávidas, e participantes de outro ECR. Intervenção: Indivíduos alocados para o grupo intervenção receberam AF, 5 mg, via oral, em dose única diária, pela manhã, durante quatro semanas. Os participantes alocados para grupo placebo receberam orientação para seguir a mesma posologia, sendo os comprimidos indistinguíveis em cor, aroma, sabor, forma e tamanho. Desfechos: Utilizaram-se as variações nos níveis de homocisteína, vitamina B12 e na FMD, aferida por Doppler, na artéria braquial, obtidos na randomização e ao final do seguimento. Resultados: Realizou-se o rastreamento de 239 participantes, sendo 72 elegíveis, 69 randomizados e 68 acompanhados, entre outubro de 2012 e julho de 2013. Em indivíduos infectados pelo HIV houve aumento significativo nos níveis de AF (12,8 ng/ml) no grupo intervenção em comparação ao placebo (P<0,001), acompanhada de variação negativa de homocisteína (-1,9 umol/l) no grupo intervenção e aumento mínimo no grupo placebo (P<0,001). Não houve variação significativa na FMD (P=0,9). Entre indivíduos coinfectados por HIV-HCV, a variação nos níveis de AF foi decorrente da elevação no grupo intervenção (12,6 ng/ml) e redução no grupo placebo (-0,7 ng/ml) (P<0,001). Os níveis de homocisteína aumentaram no grupo placebo (4,6 umol/l) e diminuíram no grupo intervenção (-1,0 umol/l) (P<0,0003). Em relação ao FMD, houve tendência à redução percentual no grupo intervenção e aumento no grupo placebo (P=0,007). As variações de vitamina B12 não foram significativas, independente do status de infecção para HCV. Conclusão: Esse estudo demonstrou que a suplementação de AF por um curto período de tempo, esteve associada com redução de homocisteína sérica, mas não modificou a FMD da artéria braquial, aferida por Doppler, em indivíduos adultos infectados pelo HIV ou HIV-HCV em uso de TARV. / Context: The supplementation of folic acid (FA) improves endothelial function in HIV-infected individuals in continuous use of highly active antiretroviral therapy (HAART). The literature does not clearly show this benefit in coinfected HIV-HCV. Introduction: Individuals infected with HIV or HIV-HCV coinfected presents a set of risk factors that can lead to endothelial dysfunction. Studies show that FA management has beneficial effects on endothelial function in different populations with cardiovascular risk, including HIV monoinfected. Objective: To determine the effect of FA supplementation for four weeks on the mediated dilation (FMD) by brachial artery flow in patients infected with HIV or HIV-HCV continuous HAART. Design: Randomized clinical trial (RCT), placebo-controlled study. Location: Division of Infectious Diseases, University Hospital of Santa Maria. Population: A total of 69 subjects aged 18-50 years, of both sex, HIV or HIV-HCV infected, on HAART, with undetectable viral load for more than six months. Patients presenting with diabetes mellitus, acute myocardial infarction, coronary revascularization, or stroke prior, with creatinine >1,5 mg/dL, clinical diagnosis, ultrasound, endoscopic or laboratory evidence of liver cirrhosis, on statins, fibrates, hormone replacement therapy, sulfonamides, vitamin supplements, or FA in the last 30 days. In addition pregnant women were excluded, and participants in a RCT. Intervention: Subjects allocated to the intervention group received FA, 5 mg orally once daily in the morning for four weeks. Participants allocated to placebo group were instructed to follow the same dosage, being indistinguishable tablets in color, aroma, taste, shape and size. Outcomes: changes were used in the levels of homocysteine, vitamin B12 and FMD, measured by Doppler, in the brachial artery obtained at randomization and at the end of follow-up. Results: We carried out the screening of 239 participants, 72 eligible, 69 randomized and 68 accompanied, between October 2012 and July 2013. In HIV-infected patients there was a significant increase in the levels of FA (12.8 ng/ml) in the intervention group compared to placebo (P <0.001), accompanied by negative variation of homocysteine (1.9 umol/L) in the group intervention and minimal increase in the placebo group (P <0.001). There was no significant change in FMD (P = 0.9). Between individuals coinfected with HIV-HCV, the variation in FA levels was due to the increase in the intervention group (12.6 ng/ml) and reduction in the placebo group (-0.7 ng / ml) (P <0.001). Homocysteine levels increased in the placebo group (4.6 umol/L) and decreased in the intervention group (-1.0 umol/L) (P <0.0003). Regarding the FMD, there was a tendency to percentage reduction in the intervention group and increased in the placebo group (P = 0.007). Variations of B12 were not significant, independent of HCV infection status. Conclusion: This study showed that AF supplementation for a short term, was associated with reduced serum homocysteine, but did not change the FMD of the brachial artery, measured by Doppler in adults infected with HIV or HIV-HCV in HAART.

Contexto: A suplementação de ácido fólico (AF) melhora a função endotelial de indivíduos infectados pelo HIV em uso contínuo de terapia antirretroviral (TARV). A literatura não demonstra com clareza esse benefício em indivíduos coinfectados HIV-HCV. Introdução: Indivíduos infectados pelo HIV ou em coinfecção pelo HIV-HCV apresentam um conjunto de fatores de risco que podem levar a disfunção endotelial. Estudos demonstram que a administração de AF possui efeitos benéficos sobre a função endotelial de diferentes populações com risco cardiovascular, inclusive em HIV monoinfectados. Objetivo: Determinar o efeito da suplementação de AF por quatro semanas sobre a dilatação mediada (FMD) pelo fluxo da artéria braquial em indivíduos infectados pelo HIV ou HIV-HCV em uso contínuo de TARV. Delineamento: Ensaio clínico randomizado (ECR), controlado por placebo. Local: Ambulatório de doenças infecciosas do Hospital Universitário de Santa Maria. População: Foram avaliados 69 indivíduos com idade entre 18-50 anos, de ambos os sexos, infectados pelo HIV com ou sem coinfecção pelo HCV, em TARV e com carga viral indetectável há mais de seis meses. Excluíram-se participantes com diabetes mellitus, infarto agudo do miocárdio, revascularização miocárdica, ou acidente vascular encefálico prévios, com creatinina >1,5 mg/dL, diagnóstico clínico, ecográfico, endoscópico ou laboratorial de cirrose hepática, em uso de estatinas, fibratos, terapia de reposição hormonal, sulfonamidas, suplementos vitamínicos, ou AF nos últimos 30 dias. Adicionalmente foram excluídas mulheres grávidas, e participantes de outro ECR. Intervenção: Indivíduos alocados para o grupo intervenção receberam AF, 5 mg, via oral, em dose única diária, pela manhã, durante quatro semanas. Os participantes alocados para grupo placebo receberam orientação para seguir a mesma posologia, sendo os comprimidos indistinguíveis em cor, aroma, sabor, forma e tamanho. Desfechos: Utilizaram-se as variações nos níveis de homocisteína, vitamina B12 e na FMD, aferida por Doppler, na artéria braquial, obtidos na randomização e ao final do seguimento. Resultados: Realizou-se o rastreamento de 239 participantes, sendo 72 elegíveis, 69 randomizados e 68 acompanhados, entre outubro de 2012 e julho de 2013. Em indivíduos infectados pelo HIV houve aumento significativo nos níveis de AF (12,8 ng/ml) no grupo intervenção em comparação ao placebo (P<0,001), acompanhada de variação negativa de homocisteína (-1,9 umol/l) no grupo intervenção e aumento mínimo no grupo placebo (P<0,001). Não houve variação significativa na FMD (P=0,9). Entre indivíduos coinfectados por HIV-HCV, a variação nos níveis de AF foi decorrente da elevação no grupo intervenção (12,6 ng/ml) e redução no grupo placebo (-0,7 ng/ml) (P<0,001). Os níveis de homocisteína aumentaram no grupo placebo (4,6 umol/l) e diminuíram no grupo intervenção (-1,0 umol/l) (P<0,0003). Em relação ao FMD, houve tendência à redução percentual no grupo intervenção e aumento no grupo placebo (P=0,007). As variações de vitamina B12 não foram significativas, independente do status de infecção para HCV. Conclusão: Esse estudo demonstrou que a suplementação de AF por um curto período de tempo, esteve associada com redução de homocisteína sérica, mas não modificou a FMD da artéria braquial, aferida por Doppler, em indivíduos adultos infectados pelo HIV ou HIV-HCV em uso de TARV. / Context: The supplementation of folic acid (FA) improves endothelial function in HIV-infected individuals in continuous use of highly active antiretroviral therapy (HAART). The literature does not clearly show this benefit in coinfected HIV-HCV. Introduction: Individuals infected with HIV or HIV-HCV coinfected presents a set of risk factors that can lead to endothelial dysfunction. Studies show that FA management has beneficial effects on endothelial function in different populations with cardiovascular risk, including HIV monoinfected. Objective: To determine the effect of FA supplementation for four weeks on the mediated dilation (FMD) by brachial artery flow in patients infected with HIV or HIV-HCV continuous HAART. Design: Randomized clinical trial (RCT), placebo-controlled study. Location: Division of Infectious Diseases, University Hospital of Santa Maria. Population: A total of 69 subjects aged 18-50 years, of both sex, HIV or HIV-HCV infected, on HAART, with undetectable viral load for more than six months. Patients presenting with diabetes mellitus, acute myocardial infarction, coronary revascularization, or stroke prior, with creatinine >1,5 mg/dL, clinical diagnosis, ultrasound, endoscopic or laboratory evidence of liver cirrhosis, on statins, fibrates, hormone replacement therapy, sulfonamides, vitamin supplements, or FA in the last 30 days. In addition pregnant women were excluded, and participants in a RCT. Intervention: Subjects allocated to the intervention group received FA, 5 mg orally once daily in the morning for four weeks. Participants allocated to placebo group were instructed to follow the same dosage, being indistinguishable tablets in color, aroma, taste, shape and size. Outcomes: changes were used in the levels of homocysteine, vitamin B12 and FMD, measured by Doppler, in the brachial artery obtained at randomization and at the end of follow-up. Results: We carried out the screening of 239 participants, 72 eligible, 69 randomized and 68 accompanied, between October 2012 and July 2013. In HIV-infected patients there was a significant increase in the levels of FA (12.8 ng/ml) in the intervention group compared to placebo (P <0.001), accompanied by negative variation of homocysteine (1.9 umol/L) in the group intervention and minimal increase in the placebo group (P <0.001). There was no significant change in FMD (P = 0.9). Between individuals coinfected with HIV-HCV, the variation in FA levels was due to the increase in the intervention group (12.6 ng/ml) and reduction in the placebo group (-0.7 ng / ml) (P <0.001). Homocysteine levels increased in the placebo group (4.6 umol/L) and decreased in the intervention group (-1.0 umol/L) (P <0.0003). Regarding the FMD, there was a tendency to percentage reduction in the intervention group and increased in the placebo group (P = 0.007). Variations of B12 were not significant, independent of HCV infection status. Conclusion: This study showed that AF supplementation for a short term, was associated with reduced serum homocysteine, but did not change the FMD of the brachial artery, measured by Doppler in adults infected with HIV or HIV-HCV in HAART.

The Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs) have become a mainstay of newer all-oral combination therapies. Despite improvements in potency of this inhibitor class, drug resistance remains a problem with the rapid emergence of resistance-associated substitutions (RASs). In this thesis I elucidate the molecular mechanisms of drug resistance for PIs against a resistant variant and apply insights toward the design of inhibitors with improved resistance profiles using structural, biochemical and computational techniques. Newer generation PIs retain high potency against most single substitutions in the protease active site by stacking on the catalytic triad. I investigated the molecular mechanisms of resistance against the Y56H/D168A variant. My analysis revealed that the Y56H substitution disrupts these inhibitors’ favorable stacking interactions with the catalytic residue His57. To further address the impact of drug resistance, I designed new inhibitors that minimize contact with known drug resistance residues that are unessential in substrate recognition. The initially designed inhibitors exhibited flatter resistance profiles than the newer generation PIs but lost potency against the D168A variant. Finally, I designed inhibitors to extend into the substrate envelope (SE) and successfully regained potency against RAS variants maintaining a flat profile. These inhibitors both pack well in the enzyme and fit within the SE. Together these studies elucidate the molecular mechanisms of PI resistance and highlight the importance of substrate recognition in inhibitor design. The insights from this thesis provide strategies toward the development of diverse NS3/4A PIs that may one day lead to the eradication of HCV.

Introdução e objetivos: A infecção crônica pelo VHC é considerada um grave problema de saúde pública mundial. O perfil epidemiológico vem mudando desde 1992 com a obrigatoriedade da pesquisa sorológica em doadores de sangue. Atualmente o uso de drogas ilícitas injetáveis é o fator de risco mais importante. Em muitos casos o mecanismo de contaminação não é identificado sendo definido como forma esporádica. O presente trabalho avaliou aspectos demográficos e epidemiológicos de pacientes com infecção crônica pelo VHC, em acompanhamento no ambulatório de Hepatologia do Serviço de Gastroenterologia da Divisão de Clínica Médica II do Hospital das Clínicas da FMUSP. Pacientes e métodos: Foram entrevistados 700 de um total de 1.112 pacientes adultos, no período de outubro de 2001 a novembro de 2003 (49% homens, 51% mulheres). Após a assinatura do termo de consentimento esclarecido, todos os pacientes foram submetidos à entrevista com questionário elaborado para este estudo, abrangendo aspectos demográficos, fatores de risco e uso de bebida alcoólica. O diagnóstico da infecção pelo VHC foi realizado através de teste sorológico Elisa de terceira geração e pesquisa do RNA viral através da reação em cadeia da polimerase. A determinação do genótipo do VHC foi realizada em 540 amostras de soro através do sequenciamento da região 5\' UTR. A biópsia hepática foi analisada em 470 pacientes e estadiada segundo critérios das Sociedades Brasileiras de Patologia e Hepatologia. Resultados: Não houve diferença significante entre a média de idade de homens e mulheres (49 ± 12,2 anos e 51 ± 12,5, respectivamente). 60% cursaram até o ensino fundamental, 19,7% o ensino médio e 12,4% o superior. 1% dos pacientes tem ocupações ligadas ao setor primário da economia, 8% ao setor secundário e 52% ao setor terciário. 62% foram classificados como brancos e 67% como católicos. 60% referiram relacionamento estável monogâmico. Em 68,5% dos casos o diagnóstico foi estabelecido através de exames de rotina e em 19,4% durante doação de sangue. O principal fator de risco para infecção foi a realização de hemotransfusão antes de 1993 (46,4%). 10% dos pacientes referiram uso de drogas injetáveis e 3%, apresentavam ambos os fatores. Em 42% dos casos o mecanismo de infecção foi considerado como forma esporádica. O genótipo 1 foi responsável por 70% das infecções seguidas pelo genótipo 3 (25%). Grau leve de fibrose classificado como 0 ou 1 foi encontrado em 48,7% dos pacientes, estádio 2 em 18,5%, estádio 3 em 12,3% e estádio 4 (cirrose) em 20,4% dos casos. A análise multivariada mostrou que os fatores de risco para desenvolvimento de cirrose foram: uso de álcool(> 60g/dia, odds ratio 2.01), raça branca (odds ratio 2,2) e idade acima de 55,8 anos (odds ratio 2,2). Conclusões: A infecção crônica pelo VHC foi caracterizada por alta freqüência de formas esporádicas e predominância dos genótipos 1 e 3. Na maioria dos casos o diagnóstico da infecção foi realizado através de exames de rotina. Consumo de álcool acima de 60 g/dia, raça branca e idade acima de 55,8 anos foram fatores de risco para a progressão para a cirrose / Background and aims: Hepatitis C virus infection is considered a world-wide serious public health problem. Since 1992, the epidemiological profile of the infection has changed with the systematic serological testing in blood donors. Nowadays the use of illicit intravenous drugs remains one of the most important risk factor. Nevertheless, in a variable percentage of cases, the mechanisms of contamination can not be identified and those cases are referred as sporadic forms. The current work was aimed at evaluating the demographic and epidemiological profile of HCV infection in outpatients attending the Hepatology branch of the Division of Gastroenterology of University of São Paulo School of Medicine teaching hospital. Patients and methods: From October 2001 and November 2003, 700 out of 1.112 adult patients were enrolled in this study (49% men, 51% women). After the written informed consent was obtained, all patients were interviewed, using standardized questionnaire for collecting data about demographic data, risk factors and alcohol use. Routine serological testing for HCV was performed using third-generation ELISA assays and circulating HCV-RNA was detected by polymerase chain reaction. HCV genotyping was performed in 540 subjects by sequencing of the 5\' UTR region. Liver biopsy slides from 470 patients were available for the assessment of the degree of fibrosis, which was performed according to the criteria of the Brazilian Societies of Pathology and Hepatology Results: There was no significant difference between the mean age of men and women (49 ± 12.2 years and 51 ± 12.5, respectively). 60% had completed no more than basic education, 19.7% had finished high school and 12.4% had graduated from university. 1% worked in activities of the primary sector of economy, 8% in the secondary and 52% in the tertiary one. 62% were caucasian descendants. 67% were catholic. 70% were born in the Southeastern region of Brazil and 97% lived in the State of São Paulo. 60% declared to have a monogamic relationship in the last 6 months. In 68.5% the diagnosis of HCV infection was established by routine check-up tests and in 19.4% during blood donation. The major risk factor for HCV infection was blood transfusion before 1993 (46.4%). 10% of the patients were intravenous drug users, and 3% had both risk factors. In 42% of the cases, the mechanism of infection was considered sporadic. Genotype 1 was found in 70% of the cases, followed by genotype 3 (25%) and genotype 2 (2.7%). Liver fibrosis stage 0 or 1 was found in 48.7%, stage 2 in 18.5%, stage 3 in 12.3% and stage 4 (liver cirrhosis) in 20.4% of cases. Linear regression multivariate analysis showed that risk factors for developing liver cirrhosis were: alcohol abuse (> 60g/day, odds ratio 2.01), caucasian origin (OR 2.2) and age > 55.8 year old (OR 2.2). Conclusions: HCV infection profile in this cohort was characterized by a high frequency of sporadic forms and predominance of genotypes 1 and 3. The infection was diagnosed in most of the cases by routine check-up tests. Heavy alcohol use, caucasian origin and older age were risk factors for progression to cirrhosis

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-03-18T21:03:29Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InfeccaoHCVUnidade.pdf: 506575 bytes, checksum: 7ec90063c7175dc2b178b996d08ad0d0 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-03-21T16:39:44Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InfeccaoHCVUnidade.pdf: 506575 bytes, checksum: 7ec90063c7175dc2b178b996d08ad0d0 (MD5) / Made available in DSpace on 2013-03-21T16:39:44Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InfeccaoHCVUnidade.pdf: 506575 bytes, checksum: 7ec90063c7175dc2b178b996d08ad0d0 (MD5) Previous issue date: 1998 / O presente estudo objetivou comparar o desempenho de quatro kits para detecção do anti-HCV, por enzimaimunoensaio, frente à detecção do HCV-RNA por meio de reação em cadeia pela polimerase (PCR), em 19 funcionários (FC) e 66 pacientes, sendo 54 submetidos à hemodiálise (HD) e 12 a diálise peritoneal (DP), em uma unidade de diálise em Belém, Pará. A pesquisa do anti-HCV foi realizada com kits de 3ª geração do Ortho, Ubi, BioChem e Abbott. A amplificação e quantificação de HCV-RNA foram realizadas utilizando-se kits do Roche. A concordância de resultados entre os quatro kits imunoenzimáticos foi de 89,5% entre os funcionários e 81,8% entre os pacientes. A prevalência do anti-HCV variou de 34,1 a 37,5% entre a população estudada, e de 42,4 a 47% entre os pacientes. O HCV-RNA foi detectado em 25 (37,9%) dos pacientes, dos quais 24 eram submetidos à hemodiálise. Não foi detectada infecção pelo HCV entre os funcionários. Não houve diferença estatística à análise dos índices de sensibilidade, especificidade e valores preditivos, entre os quatro kits em relação ao PCR, contudo, deixou-se de identificar, na dependência do kit utilizado, de 12,5 a 20% de indivíduos infectados pelo HCV. Não se observou correlação entre infecção pelo HCV e aumento nas aminotransferases. Foi possível mostrar correlação estatística entre a infecção pelo HCV e o tratamento por hemodiálise, tempo de tratamento superior a seis meses e idade dos pacientes superior a cinqüenta anos. Além disso, houve alta prevalência de portadores do HBV (13%) entre os hemodiálisados. / The main objective of this study was to make a comparative evaluation of four 3rd generation enzyme-linked immunosorbent assay (ELISA) kits (Ortho, Ubi, BioChem and Abbott), for the detection of HCV antibody and Roche polymerase chain reaction (PCR) kit. Serum samples were obtained from 19 members of the staff (grupo A) and 66 patients (grupo B), who were undergoing either hemodialysis (n=54) or peritoneal dialisys (n=12) in a dialysis unit in Belém, Pará. Altogether, the four ELISA kits agreed in 89,5% and 81,8% when results of groups A and B were compared, respectively. Overall rates of HCV antibody ranged from 34,1% to 37,5%. These rates among the patients ranged from 42,4% to 47%. While HCV-RNA was detected by PCR in 25 (37,9%) of the patients (of whom 24 in the hemodialysis subgroup), no PCR-positive results could be yielded among the staff members. When compared with the PCR, the four ELISA kits showed sensitivity, specificity and predictive values with differences that were not statistically significant. Of interest, serum samples from 12,5% to 20% of HCV-infected patients – as diagnosed by PCR – yielded negative results when tested by the four ELISA kits. An additional finding was that increase in the aminotransferasis levels does not seem occur as a result of HCV infection, it is notable that the risk of HCV infection increases following hemodialysis procedure during six months or more, among those patients who are older than fifty years. In addition, among patients subjected to hemodialysis, a high rate (13%) of HBV carriers was found.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-04-03T17:35:16Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_UsoTecnicaReacao.pdf: 38039015 bytes, checksum: 7f428bfd06dc0f92b66c083c3dbef073 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-04-17T17:01:33Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_UsoTecnicaReacao.pdf: 38039015 bytes, checksum: 7f428bfd06dc0f92b66c083c3dbef073 (MD5) / Made available in DSpace on 2013-04-17T17:01:34Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_UsoTecnicaReacao.pdf: 38039015 bytes, checksum: 7f428bfd06dc0f92b66c083c3dbef073 (MD5) Previous issue date: 2003 / O HCV é um vírus esférico, que apresenta um genoma de RNA com polaridade positiva. Atualmente está classificado na família Flaviviridae num gênero separado que é o Hepacivirus, apresenta cerca de 9,4 Kb constituído por uma única e longa fase de leitura aberta (ORF) que compreende quase todo o genoma. Apresenta duas regiões não traduzidas nas extremidades 5' e 3' denominadas 5' UTR e 3' UTR. A poli proteína precursora é clivada em dez proteínas, resultando em proteínas virais estruturais e proteínas nãoestruturais. É um vírus de transmissão preferencialmente parenteral, com distribuição universal, cujo diagnóstico é feito na grande maioria de maneira acidental, sendo atualmente utilizado os testes sorológico e molecular. Este trabalho tem como objetivo comparar o teste sorológico de imunoensaio enzimático (ELISA) e a reação em cadeia da polimerase (PCR) na ocasião da seleção de pré-doadores de sangue. Foram feitos testes de detecção do vírus C por PCR em 290 amostras com resultado positivo ou indeterminado para o teste ELISA. A análise dos resultados revelou que as amostras com testes ELISA positivo/PCR positivo e ELISA positivo/PCR negativo são duas amostras diferentes e independentes (p=0,0006). Esta diferença pode ser supostamente devido a resposta imune diferenciada nas amostras que apresentaram resultado no teste PCR positivas. Esperava-se que houvesse correlação entre os resultados do DO/Cutoff (ELISA) e carga viral (PCR) como o que ocorre em outros vírus como o HIV, no entanto os resultados apresentaram-se totalmente dispersos (R2=0,025), confirmando a não correlação entre os dois testes: ELISA e PCR para o vírus C. / The HCV is a spherical virus that presents a RNA genome with a positive polarity. Actually classified into the flaviridae family, and Hepacivirus genre, presenting a 9.4 Kb constituted by a unique and long open reading frame (ORF) which comprises almost all the genome. It presents two untranslated regions (UTR) at the 5' and 3' extremities. The major polyprotein is clivated in ten minors proteins, resulting in structural and non-structural proteins. This virus shows preferentially the blood transmission and is distributed around the world. The diagnosis has been done accidentally in the most of cases while sorological and molecular screening is done. This work has as the main objective, to compare the imuno-enzematic assay (ELISA) with polimerase chain reaction test (PCR) in the occasion of pre-selection of blood donors. Detection screening by PCR was done in 290 samples that were positive or indeterminate for ELISA assay. The result analysis showed that the samples positive-ELISA/positive- PCR and positive-ELISA/negative-PCR are two different and independent samples (p=0,0006). This difference is supposed to be due a differential immunologic response of the samples that presented positive PCR result. We attended a correlation between DO/cut-off (ELISA) and viral load in PCR as we see in other virus like HIV, however the results appears totally disperse (R2=0,025), confirming the non-correlation between the two testes, ELISA and PCR for detection of virus C.

Introdução e objetivos: A infecção crônica pelo VHC é considerada um grave problema de saúde pública mundial. O perfil epidemiológico vem mudando desde 1992 com a obrigatoriedade da pesquisa sorológica em doadores de sangue. Atualmente o uso de drogas ilícitas injetáveis é o fator de risco mais importante. Em muitos casos o mecanismo de contaminação não é identificado sendo definido como forma esporádica. O presente trabalho avaliou aspectos demográficos e epidemiológicos de pacientes com infecção crônica pelo VHC, em acompanhamento no ambulatório de Hepatologia do Serviço de Gastroenterologia da Divisão de Clínica Médica II do Hospital das Clínicas da FMUSP. Pacientes e métodos: Foram entrevistados 700 de um total de 1.112 pacientes adultos, no período de outubro de 2001 a novembro de 2003 (49% homens, 51% mulheres). Após a assinatura do termo de consentimento esclarecido, todos os pacientes foram submetidos à entrevista com questionário elaborado para este estudo, abrangendo aspectos demográficos, fatores de risco e uso de bebida alcoólica. O diagnóstico da infecção pelo VHC foi realizado através de teste sorológico Elisa de terceira geração e pesquisa do RNA viral através da reação em cadeia da polimerase. A determinação do genótipo do VHC foi realizada em 540 amostras de soro através do sequenciamento da região 5\' UTR. A biópsia hepática foi analisada em 470 pacientes e estadiada segundo critérios das Sociedades Brasileiras de Patologia e Hepatologia. Resultados: Não houve diferença significante entre a média de idade de homens e mulheres (49 ± 12,2 anos e 51 ± 12,5, respectivamente). 60% cursaram até o ensino fundamental, 19,7% o ensino médio e 12,4% o superior. 1% dos pacientes tem ocupações ligadas ao setor primário da economia, 8% ao setor secundário e 52% ao setor terciário. 62% foram classificados como brancos e 67% como católicos. 60% referiram relacionamento estável monogâmico. Em 68,5% dos casos o diagnóstico foi estabelecido através de exames de rotina e em 19,4% durante doação de sangue. O principal fator de risco para infecção foi a realização de hemotransfusão antes de 1993 (46,4%). 10% dos pacientes referiram uso de drogas injetáveis e 3%, apresentavam ambos os fatores. Em 42% dos casos o mecanismo de infecção foi considerado como forma esporádica. O genótipo 1 foi responsável por 70% das infecções seguidas pelo genótipo 3 (25%). Grau leve de fibrose classificado como 0 ou 1 foi encontrado em 48,7% dos pacientes, estádio 2 em 18,5%, estádio 3 em 12,3% e estádio 4 (cirrose) em 20,4% dos casos. A análise multivariada mostrou que os fatores de risco para desenvolvimento de cirrose foram: uso de álcool(> 60g/dia, odds ratio 2.01), raça branca (odds ratio 2,2) e idade acima de 55,8 anos (odds ratio 2,2). Conclusões: A infecção crônica pelo VHC foi caracterizada por alta freqüência de formas esporádicas e predominância dos genótipos 1 e 3. Na maioria dos casos o diagnóstico da infecção foi realizado através de exames de rotina. Consumo de álcool acima de 60 g/dia, raça branca e idade acima de 55,8 anos foram fatores de risco para a progressão para a cirrose / Background and aims: Hepatitis C virus infection is considered a world-wide serious public health problem. Since 1992, the epidemiological profile of the infection has changed with the systematic serological testing in blood donors. Nowadays the use of illicit intravenous drugs remains one of the most important risk factor. Nevertheless, in a variable percentage of cases, the mechanisms of contamination can not be identified and those cases are referred as sporadic forms. The current work was aimed at evaluating the demographic and epidemiological profile of HCV infection in outpatients attending the Hepatology branch of the Division of Gastroenterology of University of São Paulo School of Medicine teaching hospital. Patients and methods: From October 2001 and November 2003, 700 out of 1.112 adult patients were enrolled in this study (49% men, 51% women). After the written informed consent was obtained, all patients were interviewed, using standardized questionnaire for collecting data about demographic data, risk factors and alcohol use. Routine serological testing for HCV was performed using third-generation ELISA assays and circulating HCV-RNA was detected by polymerase chain reaction. HCV genotyping was performed in 540 subjects by sequencing of the 5\' UTR region. Liver biopsy slides from 470 patients were available for the assessment of the degree of fibrosis, which was performed according to the criteria of the Brazilian Societies of Pathology and Hepatology Results: There was no significant difference between the mean age of men and women (49 ± 12.2 years and 51 ± 12.5, respectively). 60% had completed no more than basic education, 19.7% had finished high school and 12.4% had graduated from university. 1% worked in activities of the primary sector of economy, 8% in the secondary and 52% in the tertiary one. 62% were caucasian descendants. 67% were catholic. 70% were born in the Southeastern region of Brazil and 97% lived in the State of São Paulo. 60% declared to have a monogamic relationship in the last 6 months. In 68.5% the diagnosis of HCV infection was established by routine check-up tests and in 19.4% during blood donation. The major risk factor for HCV infection was blood transfusion before 1993 (46.4%). 10% of the patients were intravenous drug users, and 3% had both risk factors. In 42% of the cases, the mechanism of infection was considered sporadic. Genotype 1 was found in 70% of the cases, followed by genotype 3 (25%) and genotype 2 (2.7%). Liver fibrosis stage 0 or 1 was found in 48.7%, stage 2 in 18.5%, stage 3 in 12.3% and stage 4 (liver cirrhosis) in 20.4% of cases. Linear regression multivariate analysis showed that risk factors for developing liver cirrhosis were: alcohol abuse (> 60g/day, odds ratio 2.01), caucasian origin (OR 2.2) and age > 55.8 year old (OR 2.2). Conclusions: HCV infection profile in this cohort was characterized by a high frequency of sporadic forms and predominance of genotypes 1 and 3. The infection was diagnosed in most of the cases by routine check-up tests. Heavy alcohol use, caucasian origin and older age were risk factors for progression to cirrhosis

Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors.

Two decades after the discovery of the Hepatitis C Virus (HCV), Hepatitis C infection still persists to be a global health problem. With the recent approval of the first set of directly acting antivirals (DAAs), the rate of sustained viral response for HCV-infected patients increased significantly. However, a complete cure has not been found yet. Drug development efforts primarily target NS3/4A protease, bifunctional serine protease-RNA helicase of HCV. HCV NS3/4A is critical in viral function; protease domain processes the viral polyprotein and helicase domain aids replication of HCV genome by unwinding double stranded RNA transcripts produced by NS5B, RNA-dependent RNA polymerase of HCV. Protease and helicase domains can be isolated, expressed and purified separately while retaining function. Isolated domains of HCV NS3/4A have been extensively used in biochemical and biophysical studies for scientific and therapeutic purposes to evaluate functional capability and mechanism. However, these domains are highly interdependent and modulate the activities of each other bidirectionally. Interdomain dependence was demonstrated in comparative studies where activities of isolated domains versus the full length protein were evaluated. Nevertheless, specific factors affecting interdependence have not been thoroughly studied. Chapter II investigates the domain-domain interface formed between protease and helicase domains as a determinant in interdependence. Molecular dynamics simulations performed on single chain NS3/4A constructs demonstrated the importance of interface in the coupled dynamics of the two domains. The role of the interface in interdomain communication was experimentally probed by disrupting the domain-domain interface through Ala-scanning mutations in selected residues in the interface with significant buried surface areas. These interface mutants were assayed for both helicase and protease related activities. Instead of downregulating the activities of either domain, interface mutants caused enhancement of protease and helicase activities. In addition, the interface had minimal effect in RNA unwinding activity of the helicase domain, the mere presence of the protease domain was the main protagonist in elevated RNA unwinding activity. In conclusion, I suspect that the interface formed between the domains is transient in nature and plays a regulatory role more than a functional role. In addition, I found results supporting the suggestion that an alternate domain-domain arrangement other than what is observed in crystal structures is the active, biologically relevant conformation for both the helicase and the protease. Chapter III investigates structural features of HCV NS3/4A protease inhibitors in relation to effects on inhibitor potency, susceptibility to drug resistance and modulation of potency by the helicase domain. Nearly all NS3/4A protease inhibitors share common features, with major differences only in bulky P2 extension groups and macrocyclization statuses. Enzymatic inhibition profiles of different drugs were analyzed for wildtype isolated protease domain and single chain NS3/4A helicase-protease construct, their multi drug resistant variants, and additional helicase mutants. Inhibitor potency was mainly influenced by macrocyclization, where macrocyclic drugs were significantly more potent compared to acyclic variants. Potency loss with respect to resistance mutations primarily depended on the P2 extension, while macrocyclization had minimal effect except for P2-P4 macrocyclic compounds which were up to an order of magnitude more susceptible to mutations A156T and, in lesser extent, D168A. Modulation by helicase domain was also dependent on P2 extension, although opposite trends were observed for danoprevir analogs versus others. In conclusion, this study provides a basis for future inhibitor development in both avoiding drug resistance and exploitation of the helicase domain for additional efficacy. In this thesis, I have provided evidence further supporting and revealing the details of domain-domain dependency in HCV NS3/4A. Lessons learned here will aid future research for dissecting the interdependency to gain a better understanding of HCV NS3/4A function, which can possibly be extended to all Flaviviridae NS3 protease-helicase complexes. In addition, interdomain dependence can be exploited in future drug development efforts to create better drugs that will pave the way to an effective cure.