Hepatitis C Virus (HCV) is one of the most important causes of chronic liver disease, affecting more than 170 million people worldwide. The mechanisms of hepatitis C pathogenesis are unknown. Viral cytotoxicity and immune mediated mechanisms might play an important role in its pathogenesis. HCV infection and alcohol abuse frequently coexist and together lead to more rapid progression of liver disease, increasing the incidence and prevalence of cirrhosis and hepatocellular carcinoma. The cytopathic effect of HCV proteins, especially the core, E1 and E2 structural proteins, which induce liver steatosis, oxidative stress and cell transformation may be amplified by alcohol abuse. The purpose of this study was to characterize the liver damage mechanisms induced by HCV structural proteins and alcohol and to determine the potential molecular mechanism(s) that may promote chronic, progressive liver damage. A transgenic mouse model expressing HCV core, E1 and E2 was used to investigate whether alcohol increased HCV RNA expression. Real-time RT-PCR analysis of genes involved in lipid metabolism and transport confirmed their abnormal expression in the alcohol-fed transgenic mice. In addition, light and electron microscopy analysis were performed on liver tissues of transgenic mice on an alcoholic diet versus those on a normal diet, in order to identify histological changes. The severe hepatopathy in HCV transgenic mice was exacerbated by alcohol. Mitochondria and endoplasmic reticulum had severe abnormalities in the electron microscopy analysis. The second part of this study focused on adaptive immune responses, which may also play an important role in HCV pathogenesis. I focused my analysis on dendritic cells (DC), which have been the main suspects to explain immune impairment in HCV infection. Their powerful antigen-presenting function allows them to stimulate the antiviral response of CD4+ and CD8+ T cells, the effector cells of the immune system. This unique function of the DC makes them possible targets for immune evasion by the Hepatitis C virus. In this study, DCs were generated from mouse bone marrow cells. I investigated their maturation capacity in the presence of structural proteins of HCV. The impact of HCV core/E1/E2 polyprotein on DCs cytokine expression and ability to activate T-cell lymphocytes was also analyzed. A dysfunctional CD4 T cell response was observed after exposure of DCs to core/E1/E2 polyprotein, indicating inefficient CD4 priming, which might lead to chronic HCV infection in humans. The presence of the core/E1/E2 polyprotein reduced the DC maturation capacity and the expression of certain cytokines (IL-12, IFNg, IL-6, MCP-1) important for stimulation and chemotaxis of T cells and other immune cells. My studies contribute to the understanding of HCV pathogenesis and may have implications to the development of better therapies for HCV infection.

Blood-borne infectious diseases such as Human Immunodeficiency Virus (HIV)and Hepatitis C Virus (HCV) spread when the addicts share needles and injecting equipmentwithin contact networks. A quantitative cross-sectional study was conducted in Winnipeg,Canada in December 2003 - September 2004. Through a special questionnaire, respondentswere asked to answer questions about themselves and about their contacts, about relationshipsand how they shared syringes and other injection supplies. Blood tests were used to identifywhether they carried any blood-borne infectious diseases. We selected some variables from theoriginal data set. The purpose was to explain who shared syringes and why these particularindividuals shared syringes. The key finding was that injection drug users were more concernedabout the health of others than their own health; this was shown particularly when it came tosharing of needles. Respondents chose to use their contacts needles despite knowing that thecontact was HCV positive, they were more cautious regarding HIV-infected contacts. Womenshared syringes to a greater extent with their sexual partners, while men more frequently sharedsyringes with his drug connections. It was also shown that ethnicity played a role regarding thesharing of needles.

Hepatitis C virus (HCV) is a serious global health problem with an estimate of 170 million carriers worldwide. Most individuals exposed to this blood-borne pathogen develop chronic infection, which may result in severe liver complications as well as end-stage liver diseases including cirrhosis and hepatocellular carcinoma. Current treatment options are suboptimal with no effective vaccines available to date. Development of a readily accessible mouse model that is permissive to natural HCV infection is important to facilitate drug and vaccine discovery, and also to better understand the viral pathogenesis. The inherent difficulty is that HCV displays very limited tropism, infecting only livers from humans or chimpanzees. An attempt was made to elucidate the key determinants in rendering the murine intracellular environment permissive to HCV replication. The results revealed that deletion of the interferon regulatory factor-3 and overexpression of microRNA-122 can independently enhance viral subgenomic replication in murine fibroblasts, with microRNA-122 being the stronger determinant. Interestingly, the phenotype established by these genetic manipulations was insufficient to support full-length HCV genome replication. Murine hepatic cell lines, with or without microRNA-122 expression, were also non-permissive to genomic HCV replication, despite the fact that translation of viral RNA was observed. These results suggest that additional host-specific factor(s) are required to support replication of full-length HCV RNA. These studies provide insight on the essential factors capable of influencing permissiveness of rodent cells to HCV replication, and also suggest genetic modifications to be considered when modeling the complete viral life cycle in a rodent animal model.

Studien syftar till att belysa faktorer som kan påverka de möten som sker inom svenska sprutbytesprogram. Utöver målet med att minska smittspridning av blodsjukdomar ska sprutbytesprogrammen vara en kontaktskapande arena där deltagarna motiveras till vård och behandling. Den metodologiska utgångspunkten är en hermeneutisk kvalitativ ansats med semistrukturerade intervjuer. Tre sprutbytesprogram har besökts och två stycken respondenter på varje verksamhet har intervjuats. Resultatet undersöks tillsammans med Wrights teorier om punktuella och relationella pedagogiska möten. Studien visar att deltagarna återvänder mer frekvent då vårdpersonalen genom sitt bemötande skapar förtroendefulla relationer. Vårdpersonalen kan då påbörja ett motivationsarbete som mynnar ut i ett drogfritt liv. En varierad arbetsgrupp är att föredra inom sprutbytesprogrammen för att kunna kombinera betydande faktorer för motivationsarbetet. Två av tre verksamheter samarbetade med övrig beroendevård i regionen och en verksamhet hade en kurator knuten till sig. En av våra slutsatser är att ramarna för det pedagogiska arbetet bör ses över.

This dissertation is composed of three studies that examined illness-related stigma, depressive symptoms and attachment style among patients living with HIV and Hepatitis C (HCV). The first study examined the psychometric properties of a brief HIV Stigma Scale (B-HSS) in a sample of adult patients living with HIV (PHA) (n=94). The second study developed and explored the psychometric properties of the HCV Stigma Scale in a sample of adult patients living with HCV (PHC) (n =92). Psychometric properties were evaluated with classical test theory and item response theory methodology. The third study explored whether illness-related stigma mediated the relationship between insecure attachment styles (anxious attachment or avoidant attachment) and depressive symptoms among PHA (n =72) and PHC (n=83). From June to December 2008, patients were recruited to participate in a questionnaire study at the outpatient clinics in The Ottawa Hospital. Findings indicated that the 9-item B-HSS is a reliable and valid measure of HIV stigma with items that are highly discriminatory, which indicates that items are highly effective at discriminating patients with different levels of stigma. The 9-item HCV Stigma Scale was also found to be reliable and valid with highly discriminatory items that effectively differentiate PHC. Construct validity for both scales was supported by relationships with theoretically related constructs: depression and quality of life. Among PHA, when HIV stigma was controlled the relationship between anxious attachment style and depression was not significant. However, the relationship between avoidant attachment style and depressive symptoms decreased but remained significant. Among PHC when HCV stigma was controlled the relationship between insecure attachment styles and depressive symptoms was not significant. Dissertation results emphasize the importance of identifying patients experiencing illness-related stigma and the relevance of addressing stigma and attachment style when treating depressive symptoms among PHA and PHC.

Hepatitis C Virus (HCV) is one of the most important causes of chronic liver disease, affecting more than 170 million people worldwide. The mechanisms of hepatitis C pathogenesis are unknown. Viral cytotoxicity and immune mediated mechanisms might play an important role in its pathogenesis. HCV infection and alcohol abuse frequently coexist and together lead to more rapid progression of liver disease, increasing the incidence and prevalence of cirrhosis and hepatocellular carcinoma. The cytopathic effect of HCV proteins, especially the core, E1 and E2 structural proteins, which induce liver steatosis, oxidative stress and cell transformation may be amplified by alcohol abuse. The purpose of this study was to characterize the liver damage mechanisms induced by HCV structural proteins and alcohol and to determine the potential molecular mechanism(s) that may promote chronic, progressive liver damage. A transgenic mouse model expressing HCV core, E1 and E2 was used to investigate whether alcohol increased HCV RNA expression. Real-time RT-PCR analysis of genes involved in lipid metabolism and transport confirmed their abnormal expression in the alcohol-fed transgenic mice. In addition, light and electron microscopy analysis were performed on liver tissues of transgenic mice on an alcoholic diet versus those on a normal diet, in order to identify histological changes. The severe hepatopathy in HCV transgenic mice was exacerbated by alcohol. Mitochondria and endoplasmic reticulum had severe abnormalities in the electron microscopy analysis. The second part of this study focused on adaptive immune responses, which may also play an important role in HCV pathogenesis. I focused my analysis on dendritic cells (DC), which have been the main suspects to explain immune impairment in HCV infection. Their powerful antigen-presenting function allows them to stimulate the antiviral response of CD4+ and CD8+ T cells, the effector cells of the immune system. This unique function of the DC makes them possible targets for immune evasion by the Hepatitis C virus. In this study, DCs were generated from mouse bone marrow cells. I investigated their maturation capacity in the presence of structural proteins of HCV. The impact of HCV core/E1/E2 polyprotein on DCs cytokine expression and ability to activate T-cell lymphocytes was also analyzed. A dysfunctional CD4 T cell response was observed after exposure of DCs to core/E1/E2 polyprotein, indicating inefficient CD4 priming, which might lead to chronic HCV infection in humans. The presence of the core/E1/E2 polyprotein reduced the DC maturation capacity and the expression of certain cytokines (IL-12, IFNg, IL-6, MCP-1) important for stimulation and chemotaxis of T cells and other immune cells. My studies contribute to the understanding of HCV pathogenesis and may have implications to the development of better therapies for HCV infection.

Hepatitis C virus (HCV) replication is directed by NS5b, the viral RNA dependent RNA polymerase (RdRp). To date, our understanding of the HCV polymerase has come almost entirely from genotype 1. The aim of this study was to examine the influence of sequence variation in the polymerase region by characterising a polymerase derived from genotype 3a. The genotype 3a CB strain polymerase was cloned into the bacterial expression vector pTrcHis2C incorporating a hexahistidine tag to facilitate purification. An optimised process produced 2.5 mg of highly purified recombinant protein per litre of bacterial culture. The 3a preparation possessed an RdRp activity and could utilise both homopolymeric and heteropolymeric RNA templates. Optimal activity was seen at 30oC at pH 8 in reactions containing 160nM enzyme, 10??g/ml RNA template and 2.5mM MnCl2. Subsequently, three genotype 1b polymerases including the HCV-A, Con1 and JK1 strains were cloned for the comparison of activity under identical conditions. Steady state kinetic parameters for GMP incorporation revealed the 3a polymerase exhibited the highest activity, with an almost two fold higher catalytic efficiency (Kcat/Km) than HCVA-1b, primarily due to differences in Km for GTP (2.984??M vs 5.134??M). Furthermore, the 3a polymerase was 3.5 fold and 15 fold more active than JK1-1b and Con1-1b respectively. Improving our understanding of the influence of sequence difference on polymerase activity, particularly in the context of replication will be crucial to developing effective antiviral therapies.

Hepatitis C is a public health crisis in both developing and developed countries. Direct acting antiviral therapies have revolutionized the fight against Hepatitis C, making the worldwide eradication of the disease feasible. However, screening and access to care for vulnerable patients – especially for patients experiencing homelessness – are lacking. Homelessness exacerbates the effects of Hepatitis C, leading to poor health outcomes for individual patients and high costs for health providers and taxpayers. One potential solution is investing in affordable housing and the housing first model that provide the stability needed to address both acute and chronic health conditions, including Hepatitis C. Partnerships between patients and providers facilitated by supportive housing can benefit individual outcomes and decrease the financial and social costs to communities.

Submitted by Hiolanda Rêgo (hiolandar@gmail.com) on 2013-08-14T20:32:07Z No. of bitstreams: 1 Dissertação_ICS_ Luciana Bastianelli Knop.pdf: 1536859 bytes, checksum: 7cfedb077faecccffed02fda3ab313f1 (MD5) / Made available in DSpace on 2013-08-14T20:32:07Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ Luciana Bastianelli Knop.pdf: 1536859 bytes, checksum: 7cfedb077faecccffed02fda3ab313f1 (MD5) / O avanço tecnológico das últimas décadas nas técnicas dos imunodiagnósticos permitiu o desenvolvimento de métodos capazes de detectar o complexo antígeno-anticorpo com elevada eficiência e confiabilidade. Contudo, essas ténicas ainda não alcançaram um patamar de baixo custo, fácil manuseio, de resultado imediato para amostras múltiplas e de pessoal sem qualificação técnica para a aplicação dos testes. A Precision Photonic Corporation (PPC), juntamente com a mBio Inc. e em parceria com a Universidade de San Diego (UCSD), EUA, criaram um leitor de circuito integrado (Biochip Reader), baseado em arranjos multiplex para a detecção de multimarcadores biológicos a um só tempo, com a utilização de sistemas ópticos de baixo custo e fluorescência, rápido e de fácil manuseio. O objetivo deste estudo piloto, realizado na Bahia, em parceria com a Universidade Federal da Bahia (UFBA), foi o de avaliar a operacionalidade do sistema de leitor de circuito integrado (Biochip Reader), dos protocolos encaminhados e dos resultados dos testes multiplex para detecção de anticorpos contra HIV e HCV. Foram testadas 65 amostras que apresentaram uma sensibilidade e especificidade de 100% quando comparadas com os resultados realizados por ELISA para HIV e HCV. Apesar disto, a análise sobre a operacionalidade do sistema, dos protocolos e dos resultados obtidos na Bahia apresentaram instabilidade das lâminas devido à suceptibilidade excessiva à umidade, formação de cristais e resíduos de trealose, excesso de etapas e manipulação das lâminas, e levarem a alterações dos protocolos. Portanto, apesar da similaridade dos resultados encontrados nos testes deste estudo-piloto quaondo comparado com os testes realizados por ELISA nos pacientes testados, o protótipo necessita de aprimoramento tecnológico, ampliação dos biomarcadores e mais experimentos de validação, a fim de que o sistema de biochip como uma ferramenta eficaz para o diagnóstico de doenças infecciosas seja inserido no mercado. / Salvador

Made available in DSpace on 2014-06-11T19:23:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-30Bitstream added on 2014-06-13T18:50:11Z : No. of bitstreams: 1 neo_ta_me_arafcf.pdf: 1125295 bytes, checksum: 23377b72364333a1ac1bf48473dfab17 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O vírus da Hepatite C (VHC) é o principal agente etiológico das hepatites não-A e não-B, infectando aproximadamente 170 milhões de pessoas no mundo (3% da população mundial). O vírus da hepatite C (HCV; hepatitis C-virus) é envelopado tem de 50 a 70nm de diâmetro, possui uma única fita positiva de RNA e pertence ao gênero do Hepacivirus e à família Flaviridae. Seu genoma é constituído por cerca de 9.500 nucleotídeos com regiões curtas não codificadoras e hiperconservadas nas extremidades 5’ e 3’UTR, flanqueando uma única ORF. A região estrutural do vírus é constituída por 3 genes: core, E1 e E2. As proteínas do envelope, E1 e E2 do VHC, são altamente glicosiladas e apresentam 30 e 70 kDa, respectivamente. Estudos demonstram que ambas apresentam funções específicas em diferentes etapas do ciclo de replicação do vírus, atuando de forma essencial para entrada, ligação ao receptor e fusão com a membrana da célula hospedeira. A glicoproteína E2 do VHC liga-se com alta afinidade a uma alça do receptor CD81, também denominado de TAPA-1, uma tetraespanina encontrada na superfície de muitas células, incluindo hepatócitos. No entanto, o CD81 isoladamente não é suficiente para mediar a entrada celular do vírus, e vários outros co-fatores podem atuar nessa interação. Os receptores de lipoproteína de baixa densidade (LDL-r) e receptor scavenger tipo B classe I apresentam grande importância nessa relação com o VHC. Estudos sobre as glicoproteínas E1 e E2 têm mostrado que estas se associam com os LDL-r, sugerindo que o VHC use estes receptores para invadir a célula hospedeira. Além disso, estudos anteriores relatam o fato de que, as lipoproteínas poderiam proporcionar acréscimos da infectividade ao VHC. Desta forma, neste trabalho foram desenvolvidas estratégias de clonagem e expressão heteróloga da proteína E2, e avaliou-se sua imunogenicidade... / Hepatitis C is currently recognized as the primary cause of hepatitis non A - non B associated to the blood transfusion. The hepatitis C virus (HCV) is enveloped in about 50 to 70nm in diameter, presenting a positive single strand RNA and belongs to the genus Hepacivirus and the family Flaviridae. Its genome consists of 9,500 nucleotides with short non-coding regions and hiperconservadas ends 5´ and 3'UTR flanking a single ORF. The virus structural region is based on three core genes, E1 and E2. HCV E1 and E2 are highly glycosylated and have 30 and 70 kDa, respectively. Studies show that both have key role in different stages of the cycle of virus replication, acting as essential for entry, receptor binding and fusion with host cell membrane. Glycoprotein E2 of HCV binds with high affinity to a loop of CD81, a tetraspanin, also named TAPA-1, found on the surface of many cells, including hepatocytes. However, the CD81 alone is not sufficient to mediate the cellular entry of the virus, and several other co-factors may be operating in this interaction. Recipients of low density lipoprotein (LDL-r) and scavenger receptor class B type I (SR-BI) would present great importance in relation to HCV. The LDL-r plays an important role in infection for virus of the hepatitis C. Studies on the glycoproteins E1 and E2 have shown that these are associated with the LDL-r suggesting that HCV uses the LDL-r to invade the host cell. Besides, previous studies showed that lipoprotein could improve HCV infectivity. Thus, in this work the capacity of recognition of the antibodies present anti-HCV was evaluated in the positive human serum for HCV of recognizing the protein E2 recombinant produced in bacteria of the lineage Rosetta and also the capacity of connection of the protein E2 of HCV in bind LDL-r present in the surface of human cells with characteristics endoteliais (ECV 304), and such capacity was... (Complete abstract click electronic access below)