Thesis (Ph. D.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Also available online.

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.

Renal cell carcinoma (RCC) is the commonest urogenital tumor, characterized by increased expression of hypoxia inducible factors (HIFs). During normoxia, HIFα subunits are targeted for proteasomal degradation by the product of the von Hippel Lindau gene (pVHL). In RCC, mutations in the VHL gene allow the HIFα subunits to escape degradation and translocate to the nucleus where they activate transcription of their target genes. Although both HIF1α and HIF2α are upregulated in RCC, it has been suggested that HIF2α plays the dominant role. To further elucidate the function of HIF2α in RCC, we generated a transgenic mouse model that permits temporal stabilization of HIF2α in renal tubular cells. Induction of HIF2α results in the rapid development of renal cysts - a feature observed in RCC. Taken together, these results suggest that HIF2α is a key player in development of RCC and an excellent candidate target for therapy in this disorder.

Medulloblastoma is the most common malignant paediatric brain tumour. Similar to other tumours, medulloblastoma pathogenesis involves abnormal regulation of several developmental growth pathways. As my thesis project, I studied the effects of aberrant HGF/MET signalling on medulloblastoma formation in two ways. In my first objective, I investigated the role that mutations play in activated HGF/MET signalling in medulloblastoma by searching for mutations in HGF/MET pathway genes, SPINT1, SPINT2, and MET, within primary medulloblastoma specimens. This screen identified several single nucleotide polymorphisms (SNPs) and two novel variations, one in each SPINT1 and SPINT2 genes. In my second objective, I generated a transgenic mouse model with cerebellar-specific aberrant MET signalling. These mice developed extensive cerebellar abnormalities but formed no tumours. These results indicate that mutations in the HGF/MET pathway components alone are not sufficient to initiate medulloblastoma formation and must coincide with additional genetic insults to promote tumour formation, maintenance, and progression.

Medulloblastoma is the most common malignant paediatric brain tumour. Similar to other tumours, medulloblastoma pathogenesis involves abnormal regulation of several developmental growth pathways. As my thesis project, I studied the effects of aberrant HGF/MET signalling on medulloblastoma formation in two ways. In my first objective, I investigated the role that mutations play in activated HGF/MET signalling in medulloblastoma by searching for mutations in HGF/MET pathway genes, SPINT1, SPINT2, and MET, within primary medulloblastoma specimens. This screen identified several single nucleotide polymorphisms (SNPs) and two novel variations, one in each SPINT1 and SPINT2 genes. In my second objective, I generated a transgenic mouse model with cerebellar-specific aberrant MET signalling. These mice developed extensive cerebellar abnormalities but formed no tumours. These results indicate that mutations in the HGF/MET pathway components alone are not sufficient to initiate medulloblastoma formation and must coincide with additional genetic insults to promote tumour formation, maintenance, and 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.

Title from PDF of title page (University of Missouri--Columbia, viewed on March 8, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Nicholas Kalaitzandonakes. Vita. Includes bibliographical references.