This thesis explores the impact of hepatitis C on women’s childbearing decisions and experiences of motherhood. A partial grounded theory approach was used, in which 34 women living with hepatitis C participated in semi-structured interviews to determine the direct and indirect effects of hepatitis C on their own personal decisions regarding childbearing and to describe their lived experiences of motherhood. The qualitative interview data were analysed thematically, in which common themes were identified and explored.¶ Three key areas are explored: women’s social experience of hepatitis C; hepatitis C and childbearing decisions; and the meaning of motherhood for women with hepatitis C.¶ The interviews revealed that living with hepatitis C had direct effects on the childbearing decisions of women. The direct effects of the virus which impacted on women’s childbearing decisions included poor physical and emotional hepatitis C related health, the perceived risk of vertical transmission of hepatitis C, concerns their future hepatitis C related health might impact upon their mothering abilities, and childbearing can conflict with treatment for hepatitis C. However, of greater importance to these women, appeared to be the indirect effects of living with a virus which is so highly stigmatised within our society. In particular, hepatitis C is closely associated with injecting drug use, which means these women are often assumed to possess the stereotypical characteristics associated with injecting drug users. As a result, they experience widespread medical discrimination and social rejection. Hepatitis C also impacts indirectly upon a wide range of factors that most women in contemporary society take into consideration in their childbearing decisions, for example, available social support, financial security and age.¶ The experiences reported by these women are discussed in terms of their concordance or discord with prevailing theories of deviance, stigma and the social construction of motherhood. The interview data, considered in light of such theories reveal that possibly the greatest impact that hepatitis C can have upon women is to prevent them from achieving a legitimate adult female status through childbearing and becoming a ‘good mother’.¶ The implications of these findings are discussed in terms of public health and social policy.

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, a life threatening disease, caused by the hepatitis C virus (HCV) currently affects over 170-200 million people worldwide (~3% of global human population), more than five times the percentage of total HIV infections. HCV infection has been shown to be a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma and is the leading cause of liver transplantation in the U.S. HCV has escaped every therapeutic target to date by means of its error-prone RNA polymerase, which allows it to mutate prolifically. The current standard anti-HCV therapy, which is pegylated interferon a combined with ribavirin, is difficult to tolerate, and more than 50% of HCV patients are refractory to it. No protective vaccine or therapeutic antibody is available, making the need for the development of an efficacious immunoprophylactic and therapeutic agent imperative. HCV is an enveloped virus with a positive sense RNA genome of ~9.6 kilobases (kb), which carries a large open reading frame (ORF), flanked by 5'- and 3'- untranslated regions (UTRs). Interestingly, within the highly mutational HCV RNA, there are a limited number of 100% conserved and functionally vital motifs, located in the 5' UTR, coding region and in the 3' UTR. Within the HCV genome, these motifs have been proposed to be involved in multiple exclusive interactions with each other and furthermore, these interactions have been demonstrated to be essential for HCV replication and/or translation of the viral proteins. / Bayer School of Natural and Environmental Sciences; / Chemistry and Biochemistry / PhD; / Dissertation;

Cellular immune responses to Hepatitis C (HCV) epitopes are crucial for successful host response to HCV infection. We investigated a platform to assess specific and global immune responses in HCV infection. We identified 57 HCV peptides from literature (24 of CD4+, 33 of CD8+ specificity) and tested them in two peptide pools to assess specific response in non-transplanted and post-liver transplant (LT) patients. Robust interferon-gamma (IFN) response to CD4+ peptide and mitogen stimulation was seen in sustained virological clearance. IFN response to the CD4+ peptide pool could differentiate between SVR and NR with 82% accuracy. In patients with recurrent HCV post-LT, HCV-specific responses were attenuated, but global immune responses were preserved. Significantly lower specific (CD4+) and global immune responses (mitogen response) were observed in patients with advanced allograft disease (fibrosis score>2). Quantiferon-HCV may identify patients likely to respond to anti-HCV treatment, as well as post-LT patients with aggressive HCV recurrence. / Experimental Surgery

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.

Context & Rationale: Individuals with similar CD4 cell counts and RNA levels can vary considerably with regards to clinical progression. This variation is likely the result of a complex interplay between viral, host and environmental factors. This study aimed to characterize and identify predictors associated with disease progression to AIDS or death in Saskatoon, Saskatchewan. Methods: This is a retrospective cohort study of 343 seroprevalent HIV positive patients diagnosed from Jan 2005 to Dec 2010. Of these, 73 had an estimated seroconversion date. Data was extracted from medical charts at two clinics specialized in HIV/AIDS care. Disease progression was measured as time from HIV diagnosis (or seroconversion) to immunological AIDS and death. The Cox hazard model was used. Results: The 3-year and 5-year immunological AIDS free probability was 53% and 33%, respectively. The 3-year and 5-year survival probability was 89% and 77%, respectively. Among the seroconversion cohort, the 3-year immunological AIDS free probability was 76%. Due to multicollinearity, separate models were built for IDU, hepatitis C and ethnicity. A history of IDU (HR, 3.0; 95%CI, 1.2-7.1), hepatitis C coinfection (HR, 2.9; 95%CI, 1.2-6.9), baseline CD4 counts (HR, 0.95; 95%CI, 0.92-0.98, per ever 10 unit increase), ever on ART, and year of diagnosis were significant predictors of progression to immunological AIDS among the seroprevalent cohort. Age at diagnosis, sex and ethnicity were not. For survival, only treatment use was a significant predictor (HR, 0.34; 95%CI, 0.1-0.8). Hepatitis C coinfection was marginally significant (p=0.067), while a history of IDU, ethnicity, gender, age at diagnosis, and year of diagnosis were not. Among the seroconversion cohort, no predictors of progression to immunological AIDS were identified. Ethnicity, hepatitis C coinfection and history of IDU could not be assessed. Conclusion: Our study found that IDU, HCV coinfections, baseline CD4 counts, and ART use were significant predictors of disease progression. This highlights the need for increased testing and early detection and for targeted interventions for these particularly vulnerable populations to slow disease progression.

Context & Rationale: Individuals with similar CD4 cell counts and RNA levels can vary considerably with regards to clinical progression. This variation is likely the result of a complex interplay between viral, host and environmental factors. This study aimed to characterize and identify predictors associated with disease progression to AIDS or death in Saskatoon, Saskatchewan. Methods: This is a retrospective cohort study of 343 seroprevalent HIV positive patients diagnosed from Jan 2005 to Dec 2010. Of these, 73 had an estimated seroconversion date. Data was extracted from medical charts at two clinics specialized in HIV/AIDS care. Disease progression was measured as time from HIV diagnosis (or seroconversion) to immunological AIDS and death. The Cox hazard model was used. Results: The 3-year and 5-year immunological AIDS free probability was 53% and 33%, respectively. The 3-year and 5-year survival probability was 89% and 77%, respectively. Among the seroconversion cohort, the 3-year immunological AIDS free probability was 76%. Due to multicollinearity, separate models were built for IDU, hepatitis C and ethnicity. A history of IDU (HR, 3.0; 95%CI, 1.2-7.1), hepatitis C coinfection (HR, 2.9; 95%CI, 1.2-6.9), baseline CD4 counts (HR, 0.95; 95%CI, 0.92-0.98, per ever 10 unit increase), ever on ART, and year of diagnosis were significant predictors of progression to immunological AIDS among the seroprevalent cohort. Age at diagnosis, sex and ethnicity were not. For survival, only treatment use was a significant predictor (HR, 0.34; 95%CI, 0.1-0.8). Hepatitis C coinfection was marginally significant (p=0.067), while a history of IDU, ethnicity, gender, age at diagnosis, and year of diagnosis were not. Among the seroconversion cohort, no predictors of progression to immunological AIDS were identified. Ethnicity, hepatitis C coinfection and history of IDU could not be assessed. Conclusion: Our study found that IDU, HCV coinfections, baseline CD4 counts, and ART use were significant predictors of disease progression. This highlights the need for increased testing and early detection and for targeted interventions for these particularly vulnerable populations to slow disease progression.

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.

Existen cada vez más evidencias de que el virus de la hepatitis C favorece el desarrollo de diabetes. Objetivos: 1.- Analizar la prevalencia de las alteraciones del metabolismo hidrocarbonado (diabetes y glucemia anómala en ayunas) en los pacientes con infección crónica por el VHC (n=498) y compararla con la que presentan los pacientes con otras enfermedades hepáticas (n=144), considerando el grado de lesión hepática (hepatitis crónica o cirrosis) y los genotipos del VHC en el análisis de los resultados. 2.- Evaluar los mecanismos etiopatogénicos por los que el VHC puede favorecer el desarrollo de diabetes (citocinas proinflamatorias, resistencia a la insulina, disfunción célula beta) mediante un estudio caso control en pacientes con hepatitis crónica no diabéticos (14 VHC (-) y 28 (+)) estrictamente equiparados por los factores relacionados con la aparición de diabetes.3.- Analizar si el aumento de ferritina que existe en los pacientes infectados por el VHC es debido a la propia infección o, si por el contrario, puede atribuirse a la mayor prevalencia de diabetes (enfermedad que también cursa con ferritina elevada) en estos pacientes.4.- Complementando al último objetivo, determinar mediante el análisis de los receptores solubles de la transferrina (sTfR), si la concentración de ferritina en los pacientes diabéticos tipo 2 es un buen indicador de los depósitos de hierro.Conclusiones:1.- Los pacientes con infección por el VHC presentan una elevada prevalencia tanto de glucemia anómala en ayunas como de diabetes en comparación con los pacientes con otras enfermedades hepáticas no atribuibles al VHC (17% vs. 7% y 15% vs. 5%, respectivamente). Este aumento de prevalencia se produce fundamentalmente en los pacientes con hepatitis crónica e incluso se objetiva en sujetos con transaminasas normales.2.- El análisis del genotipo no parece útil para identificar una población de pacientes infectados por el VHC con mayor riesgo para desarrollar alteraciones hidrocarbonadas.3.- La infección por el VHC debe consideradarse un factor de riesgo para el desarrollo de diabetes. Por tanto, es recomendable realizar un cribado sistemático de las alteraciones hidrocarbonadas en los pacientes infectados por el VHC.4.- En los pacientes infectados por el VHC la prueba de la sobrecarga oral de glucosa es el método de elección para realizar el diagnóstico de la diabetes.5.- En los pacientes infectados por el VHC que aún no han desarrollado una diabetes existe un hiperinsulinismo, tanto en situación basal como tras la estimulación con glucagón endovenoso o la ingesta de una comida estándar, lo que traduce un estado de insulinresistencia. 6.- El aumento de resistencia a la insulina en los pacientes infectados por el VHC se asocia a un incremento de citocinas proinflamatorias.7.- Los pacientes infectados por el VHC no diabéticos presentan concentraciones similares de ferritina que la población general. De ello puede deducirse que el aumento de los depósitos de hierro, evaluados mediante los niveles séricos de ferritina, no parece un elemento fundamental en la etiopatogenia de la diabetes asociada al VHC.8.- La diabetes mellitus es el principal factor relacionado con el aumento de las concentraciones séricas de ferritina observado en los pacientes con infección crónica por el VHC. Por tanto, la diabetes mellitus debe ser tenida en cuenta al evaluar el metabolismo del hierro en los pacientes infectados por el VHC.9.- El aumento de la ferritina en los pacientes diabéticos no se acompaña de un descenso recíproco de los niveles de sTfR, lo que sugiere que es un reflejo del estado inflamatorio crónico de baja intensidad característico de la DM tipo 2.10.- La determinación de la concentración sérica de ferritina no es un marcador útil de los depósitos de hierro en los pacientes con DM tipo 2. / There is growing evidence to suggest an association between hepatitis C virus infection and type 2 diabetes mellitus. Objetives: 1.- To compare the prevalence of both diabetes and impaired fasting glucose between hepatitis C virus (HCV)-infected patients (n=498) and patients with other liver diseases but not HCV (n=144), taking into account the degree of liver damage (chronic hepatitis and cirrhosis) and the different HCV genotypes. 2.- To explore the specific mechanisms responsible for the development of diabetes in HCV infected patients (proinflammatory cytokines, insulin resistance and beta-cell function) in a case-control study with nondiabetic noncirrhotic patients (14 anti-HCV negative and 28 anti-HCV positive) carefully matched for the main items related with diabetes development.3.- To investigate if the high ferritin levels observed in HCV-infected patients are related with HCV infection itself or could be associated with the higher prevalence of diabetes (also related with hyperferritinemia) in HCV infected patients.4.- To determine circulating transferrin receptor levels (sTfR) to evaluate if serum ferritin reflects iron body stores in type 2 diabetic patients.Conclusions:1.- Both impaired fasting glucose and diabetes were more prevalent among patients with HCV infection than among anti-HCV negative patients. This finding was mainly due to the group of patients with chronic hepatitis, and it was also present among patients with normal transaminases.2.- No differences in the prevalence of either impaired fasting glucose or diabetes among HCV genotypes were observed.3.- HCV infected patients must be considered as a high risk group for type 2 diabetes development, and testing for glucose abnormalities should be mandatory in these patients.4.- The high percentage of new cases of diabetes detected using postload hyperglycemia in the subset of patients with chronic hepatitis suggests that the oral glucose tolerance test (OGTT) should be recommended as the primary screening test for diabetes in these patients.5.- In HCV nondiabetic noncirrhotic patients an hyperinsulinemia is detected, both basal and after stimulation tests (intravenous administration of 1 mg of glucagon and the standard food intake test), suggesting an insulin resistance state. 6.- This state of insulin resistance, before the development of glucose abnormalities, is accompanied with a marked increase of proinflammatory cytokines.7.- Serum ferritin levels in anti-HCV positive nondiabetic patients were similar to those in the control group, suggesting that iron deposition is not one of the main mechanisms linking HCV infection and diabetes. 8.- The increase in ferritin levels detected in HCV patients was closely related to the presence of diabetes. So, diabetes should be taken into consideration when evaluating iron metabolism in HCV-infected patients.9.- Serum ferritin levels are increased in Type 2 diabetic patients in the absence of a reciprocal decrease of sTfR. This finding suggest that elevated ferritin levels in Type 2 diabetic patients are mainly as a result of inflammatory mechanisms rather than iron overload.10.- Serum ferritin levels may not be a reliable tool for evaluating iron deficiency anaemia in Type 2 diabetes.

En aquesta tesi s'estudia una població de pacients seropositius al VHC i en ells se'ls ha investigat els factors de risc que poden afavorir el patir aquesta infecció i a un grup se'ls ha practicat biòpsia hepàtica per via transjugular amb estidi posterior dels paràmetres histològics i factors que es relacionen amb un major mal hepàtic.S'estudia també l'evolució i pronòstic de la infecció pel VHC en aquests pacients, així com l'evolucióde la malaltia hepàtica en el posttrasplantament. / En esta tesis se estudia una población de pacientes seropositivos al VHC y en ellos se los ha investigado los factores de riesgo que pueden favorecer el padecer esta infección y a un grupo se los ha practicado biopsia hepática por vía transjugular con estidio posterior de los parámetros histológicos y factores que se relacionan con uno mayor mal hepático. Se estudia también la evolución y pronóstico de la infección por el VHC en estos pacientes, así como la evolución de la enfermedad hepática en el posttrasplante.