Introdução e Objetivos: A Hepatite pelo Vírus E (HEV) é uma infecção conhecida mundialmente por ter seu curso assintomático e autolimitado na maioria dos casos. Nos últimos anos, casos de evolução para cronicidade têm sido descritos envolvendo população de pacientes imunocomprometidos, particularmente os transplantados de órgãos sólidos. A prevalência da infecção pelo HEV na população de transplantados de fígado é ainda desconhecida em nosso meio. Pacientes e Métodos: Nós avaliamos parâmetros de infecção pelo HEV (HEV RNA, anti-HEV IgM e anti-HEV IgG) em uma coorte de 294 adultos transplantados de fígado seguidos no HCFMUSP. Para pesquisa dos anticorpos foi utilizada a metodologia ELISA (RecomWell HEV IgM/ IgG da Mikrogen®), sendo os resultados indeterminados e com IgM isoladamente positivo confirmados por Immunoblotting (RecomLine IgM/ IgG da Mikrogen®). Para pesquisa do HEV RNA utilizou-se a metodologia de PCR em tempo real One-Step com primers e sondas que amplificam um fragmento da ORF3 do genoma viral. Foram avaliados dados demográficos e laboratoriais de toda população de transplantados. Posteriormente, foram analisadas apenas as biópsias hepáticas de 122 pacientes transplantados devido à Hepatite pelo Vírus C (VHC) possuidores ou não de marcadores sorológicos ou moleculares do HEV de acordo com a classificação METAVIR. Resultados: Amostras de 8,2% (24/294) dos pacientes foram positivas para anti-HEV IgG, 2% (6/294%) positivas para anti-HEV IgM e 5,8% (17/274) mostraram positividade para o HEV RNA. Apenas um paciente mostrou positividade tanto para anti-HEV IgM quanto para o anti-HEV IgG. Setenta e sete vírgula oito por cento (95/122) dos pacientes transplantados pelo VHC foram tratados com esquema que incluía ribavirina por um período mínimo de 6 meses antes da coleta do sangue. Dentre os transplantados pela cirrose por VHC que apresentaram positividade para o anti-HEV IgG apenas 37,5% (3/122) mostraram níveis de fibrose maior que 2, enquanto que 41,7% (5/122) dos positivos para o HEV RNA demonstraram níveis de fibrose maior que 2. Em geral, a prevalência do HEV no cenário pós transplante hepático parece ser baixa podendo não trazer um dano histológico significativo no cenário pós transplante. Conclusão: Nós concluímos que apesar de alguns estudos demonstrarem riscos de cronificação pelo HEV, esse vírus tem baixa prevalência em nosso meio e os pacientes transplantados de fígado pelo VHC que apresentaram marcadores sorológicos ou moleculares para o HEV não tiveram níveis mais acentuados de fibrose quando comparados com os pacientes que não apresentaram indícios de infecção pelo HEV no momento da análise / Background and Aims: Hepatitis E Virus (HEV) is an infection known worldwide for its asymptomatic and self-limited course in most of the cases. In the last five years, cases evolving to chronicity have been described involving immunosuppressed patients, especially in recipients of solid organs transplants. The HEV infection prevalence in liver transplanted patients has yet to be fully assessed by our community. Patients and Methods: We evaluated laboratory parameters for the HEV infection (HEV RNA, anti-HEV IgM and anti-HEV IgG) in a cohort of 294 adult patients who had liver transplants on the HCFMUSP (Clinical Hospital of University of São Paulo School of Medicine). In order to investigate the antibodies, we used Elisa methodology (RecomWell HEV IgM/ IgG by Mikrogen®) and the indeterminate results and isolates positive anti-HEV IgM were confirmed by immunoblotting (RecomLine IgM/ IgG by Mikrogen®). For screening of HEV RNA, One-Step PCR in real time was used with primers and probes that amplify a fragment of the ORF3 from the viral genome. Laboratory and demographic data were collected in the entirety of the transplanted population. Following that, the hepatic biopsies of 122 patients transplanted due to Hepatitis C (HCV) were analysed with or without serological or molecular markers of HEV according to METAVIR score. Results: Serological samples of 24 (8.2%) of the patients tested positive for anti-HEV IgG, 06 (2%) were positive for anti-HEV IgM and 17 (5.8%) showed positive results for HEV RNA. Only one patient showed positive results for both anti- HEV IgM and anti-HEV IgG. Also, 95 (77.8%) of the patients transplanted because of the HCV infection received treatment including Ribavirin for at least 6 months before the blood sample collection. Among the transplanted patients due to HCV cirrhosis who presented positive results for anti-HEV IgG, only 3 (37.5%) showed fibrosis beyond stage 2, while 5 (41.7%) of the HEV RNA positive patients had liver fibrosis higher than 2. Conclusion: Overall, the prevalence of HEV in the post-hepatic transplant scenario appears to be low, seemingly not harmful histologically in the post-transplant cases. We have concluded that although some studies showed risks of HEV chronification, patients who had their livers transplanted due to HCV who showed serological or molecular markers for HEV did not have higher levels of fibrosis when compared to patients who showed no indications of HEV infection at the time of the analysis

A infecção pelo vírus da hepatite C (HCV) pode evoluir gradualmente para hepatite crônica, cirrose e carcinoma hepatocelular (CHC) ao longo de 20 a 30 anos [1-3]. O carcinoma hepatocelular é a quinta neoplasia mais comum em todo o mundo, sendo responsável por mais de 600.000 mortes por ano. Atualmente, cerca de 170 milhões de indivíduos estão infectados pelo HCV, o que corresponde a aproximadamente 3% da população do mundo. A hepatocarcinogênese é um processo complexo, com várias etapas que envolvem alterações genéticas e epigenéticas. Estudos relatam que substituições de aminoácidos (aa) na posição 70 e 91 da região core do HCV podem estar relacionados ao desenvolvimento de CHC. O conhecimento sobre os mecanismos da carcinogênese que envolvem o HCV são importantes para a descoberta de biomarcadores e potenciais alvos terapêuticos do CHC. Neste estudo, foram analisados os genótipos virais e a presença de mutações na região core do HCV, em 94 pacientes com CHC e em 79 pacientes cirróticos (sem CHC). As sequências da região core do HCV foram obtidas pelo método de sequenciamento populacional baseado na metodologia de Sanger. Características demográficas, bioquímicas e sorológicas também foram avaliadas. A idade dos pacientes com CHC foi significativamente maior do que a dos pacientes sem CHC (63 vs 60,5 anos, P=0,025). Uma proporção maior de homens foi observada no grupo CHC (64,4% vs 54%, P=0,329), qual apresentou nível de alfafetoproteína significativamente mais elevado (P=0,003) e menores níveis de albumina em relação ao grupo sem CHC (P=0,012). Elevada variabilidade genética do HCV foi observada. Ao todo, quatro genótipos e sete subtipos foram encontrados. O subtipo 1 b foi o mais frequente em ambos os grupos. Os subtipos encontrados no grupo CHC e cirróticos foram, 1a (13,6%), 1 b (45,7%), 3a (28,8%), 2b (6,8%), 2a (1,7%), 2c (1,7%), 5a (1,7%); e 1a (30%), 1 b (44%), 3a (22%), 2b (2%) e 5a (2%). As mutações R70Q e UC91 M foram observadas principalmente no HCV genótipo 1 b. Não houve associação entre mutações nas posições 70 e 91 na região core do HCV e o desenvolvimento de CHC / Hepatitis C virus (HCV) infection is often persistent and gradually advances from chronic hepatitis (CH) to liver cirrhosis, and hepatocellular carcinoma (HCC) over 20 to 30 years [1-3]. Worldwide, hepatocellular carcinoma is the fifth most common neoplasm and is responsible for more than 600,000 deaths annually due to very poor prognosis. There are about 170 million individuais infected with HCV, corresponding to approximately 3% of world population. Hepatocarcinogenesis is a complex process involving genetic and epigenetic modifications. Studies have reported that amino acid substitutions (a a) at position 70 and 91 of HCV core region may be related to development of HCC. Understanding the pathogenesis of HCV-induced hepatocarcinogenesis is important to identify novel biomarkers and potential therapeutic targets. In this study, the viral genotypes and the presence of mutations in HCV core region were analyzed in 94 patients with HCC, and also in 79 cirrhotic patients (without HCC). HCV core sequences were obtained using population sequencing based on Sanger method. Demographic, biochemical and serological characteristics were also evaluated. The age of patients with HCC were significantly higher than in patients without HCC (63 vs. 60.5 years, P=0.025). High proportion of men was observed in HCC group (64.4% vs 54%, P=0.329). Alpha-fetoprotein levei was significantly higher in HCC group compared to cirrhotic group (P=0.003), and low rates of albumin was observed in cirrhotic group (P=0.012). High genetic variability of HCV was observed, in HCC group, however genotype 1 b was the most common in both groups. Other genotypes were found in HCC group: 1a (13.6%), 1 b (45.7%), 3a (28.8%) 2b (6.8%), 2a (1.7%), 2c (1.7%) and 5a (1.7%). In cirrhotic group was found genotypes 1 a (30%), 1 b (44%), 3a (22%), 2b (2%) and 5a (2%). Mutations R70Q and LlC91 M were mainly observed in individuais infected with HCV genotype 1 b. In the present study, no association between mutations at positions 70 and 91 of HCV

As hepatites B e C constituem ainda importante problema de saúde pública no mundo, com cifras de portadores crônicos estimados em cerca de 350 milhões e de 170 milhões, respectivamente, fazendo com que número elevado de pessoas se encontrem sob risco de cronificação e evolução para cirrose hepática e hepatocarcinoma. Entre os fatores de risco mais conhecidos para hepatite B estão às transfusões de sangue e derivados, o contato com sangue e/ou com secreções, por relações sexuais desprotegidas, compartilhamento de seringas e/ou agulhas para uso de drogas injetáveis, tatuagem e piercing com material contaminado. A hepatite C é transmitida primordialmente por via parenteral e a sua prevenção faz-se de modo semelhante à hepatite B. Diversos fatores de risco para ambas parecem ser fazer presentes na população surda, a qual é historicamente marginalizada em termos de acesso a informações e serviços de saúde e sobre a qual não existem referências de investigações sobre hepatites virais. Esse trabalho teve como objetivos estimar a prevalência de sorologia positiva para hepatite B e C e investigar possíveis fatores de risco entre a população surda de Ribeirão Preto. Foram estudados 88 surdos, aos quais foi apresentado DVD explicativo sinalizado em Libras sobre características e riscos das hepatites B e C. Aos que concordaram em participar foi aplicado questionário padronizado e coletada uma amostra de sangue para realização de testes imunoenzimáticos para detecção dos marcadores HBsAg, anti-HBs, anti-HBc e anti-HCV, realizados no Laboratório de Sorologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto. A análise sorológica revelou presença de infecção atual ou pregressa por hepatite B em sete participantes, correspondendo a 8% de prevalência total de marcadores (IC95%: 2,3 13,7). Ao se analisar as possíveis variáveis de risco, encontrou-se associação entre a infecção e as variáveis: ser nascido em outra Unidade Federada que não São Paulo e antecedente de encarceramento. Os participantes mostraram grande desconhecimento sobre aspectos básicos relacionados à transmissão das hepatites virais, indicando necessidade de políticas de saúde pública voltadas para esta população, e que levem em consideração suas particularidades linguística e cultural. / Hepatitis B and C are still an important public health problem in the world, with chronic carriers estimated at around 350 million and 170 million, respectively, causing a large number of people to be at risk of chronic forms and progression to liver cirrhosis and hepatocellular carcinoma. Among the most well-known risk factors for hepatitis B are blood and derivatives transfusions, contact with blood and/or secretions by unprotected sex, sharing needles and/or syringes in injectable drug use, tattooing and piercing with contaminated material. Hepatitis C is transmitted primarily by parenteral way and its prevention is similar to hepatitis B. Several risk factors for both seem to be present in the deaf population, which is historically marginalized in terms of access to health information and services and on which there are no references to research on viral hepatitis. This work aimed to estimate the prevalence of positive serology for hepatitis B and C and to investigate possible risk factors among the deaf population of Ribeirão Preto. An explanatory DVD about the features and risks of hepatitis B and C was presented to them in Brazilian Sign Language. Eighty eight deaf agreed to participate, signed a free and informed consent form and were included in the investigation. A standardized questionnaire was applied and a sample of blood was collected. Immunoenzymatic tests for detection of HBsAg, anti-HBS, anti-HBC and anti-HCV markers were carried out at the Serum Laboratory of the University Hospital of the Ribeirão Preto Medical School, University of São Paulo. The serological analysis revealed the presence of current or previous infection by hepatitis B in seven participants, representing 8% of total prevalence of markers (CI95%: 2,3 13,7). When analyzing the possible risk factors, it was found association between infection and the variables being born in another State other than São Paulo and past history of imprisonment. No positive samples for hepatitis C were found. The participants showed great ignorance about basic aspects related to the transmission of viral hepatitis, indicating need for public health policies directed to this population that takes into account their linguistic and cultural singularities.

O transplante de fígado é definido como a terapêutica de escolha para as doenças hepáticas em estágio terminal. Por muitos anos, o sucesso deste procedimento foi mensurado pelas taxas de mortalidade e a freqüência de complicações nas anastomoses biliares e complicações infecciosas. No entanto, mais recentemente um novo foco de preocupação voltou-se para a avaliação da qualidade de vida no grupo de pacientes transplantados. Os benefícios do transplante hepático sobre a qualidade de vida dos pacientes é um evento já demonstrado previamente em alguns estudos que utilizaram questionários genéricos de avaliação de qualidade de vida. A qualidade de vida em saúde também pode ser acessada através de questionários específicos, como o LDQOL (Liver Disease Quality of Life). Este é um instrumento voltado especificamente para sintomas relacionados às doenças hepáticas, desenvolvido nos Estados Unidos e recentemente traduzido para o Português e adaptado culturalmente para à população brasileira. Objetivo Aplicar este novo instrumento na população de pacientes em lista de espera de transplante de fígado e naqueles submetidos a transplante, reavaliando diferentes aspectos da qualidade de vida destes pacientes e avaliando o impacto da recidiva da doença de base após o transplante hepático. Método: Foram aplicados os questionários LDQOL e SF?36 a 126 pacientes em acompanhamento ambulatorial regular no Serviço de Transplante e Cirurgia do Fígado do Hospital das Clínicas da Universidade de São Paulo, dos quais 65 eram pacientes cirróticos em lista de espera de transplante de fígado e 61 deles eram pacientes submetidos a transplante hepático há pelo menos 6 meses e no máximo há 60 meses. Também foi aplicado o questionário SF-36 a um grupo de doadores de sangue sadios, pareados por sexo e idade e que funcionou como grupo controle. As avaliações, realizadas a partir da formação destes 3 grupos, incluiram a comparação da qualidade de vida entre cirróticos e transplantados, a comparação entre indivíduos saudáveis e portadores de hepatopatia e a influência de outros aspectos como etiologia da doença hepática, escore do MELD, classificação de Child-Pugh, presença de co-morbidades, tempo decorrido do transplante e efeito da recidiva da doença hepática sobre a qualidade de vida do paciente transplantado. O método estatístico aplicado foi o teste de Mann- Whitney. Resultados: As pontuações atingidas para os pacientes em lista de transplante hepático para mensuração da qualidade de vida foram significantemente mais baixas do que aquelas atingidas pelo grupo controle. Na comparação entre cirróticos com MELD <= 15 e cirróticos com MELD > 15, tanto o SF-36 como o LDQOL mostraram pior qualidade de vida nos pacientes com MELD mais elevado. A comparação entre indivíduos antes e depois do transplante evidenciou melhor qualidade de vida no grupo de pacientes transplantados. Isto ficou evidenciado tanto pelo LDQOL como SF-36. No grupo de pacientes pré-transplante, o questionário LDQOL mostrou maior comprometimento da qualidade de vida naqueles pacientes com cirrose por VHC, quando comparado a outras etiologias, enquanto o SF-36 não teve a mesma acurácia em demonstrar esta diferença. Nos pacientes transplantados, a recidiva da hepatite C determina comprometimento da qualidade de vida, quando avaliados pelo LDQOL, mas não pelo SF-36. Conclusões: O uso de um instrumento de medida de qualidade de vida - o LDQOL - pôde demonstrar, com maior acurácia, o seu comprometimento nos pacientes cirróticos, a melhora observada nos pacientes transplantados e o impacto negativo da recidiva da hepatite C no pós-transplante. / Liver transplantation has been established as the standard treatment for patients with end stage liver disease and for many years the outcomes of its procedure has been measured as mortality rates and biliary and infectious complications. More recently a new issue has been raised and many studies have been changing the focus to analyze the health related quality of life among the population of transplanted patients. A positive effect of liver transplantation on health-related quality of life (HRQOL) has been documented in many studies using generic instruments. Health-related quality of life can also be assessed with specific instruments, as the Liver Disease Quality of Life(LDQOL), a questionnaire targeted to symptoms of hepatic disease, that has been recently translated to Portuguese and culturally adapted to Brazilian population. Our aim was to reevaluate different aspects of HRQOL before and after liver transplantation with this new instrument, and determinate the impact of liver disease recurrence after the surgical procedure. Methods: The LDQOL and SF-36 questionnaires were applied to 126 patients at the Service of Transplant and Liver Surgery of Clinical Hospital of University of São Paulo, 65 of them on the transplant waiting list and 61 of them after 6 to 60 months of liver transplantation. It was also analyzed the quality of life, by using the SF-36 questionnaire, of health blood donors, paired by sex and age, to serve as control group. Multiple comparisons were made concerning etiology of cirrhosis, co-morbidities, MELD scores, time elapsed of transplant, recurrence of liver disease after liver transplantation, using the Mann-Whitney test. Results: HRQOL scores for patients waiting for transplantation were significantly lower than those for the control group. Patients with MELD scores > 15 showed worse healthrelated quality of life than patients with MELD scores <= 15, both with SF-36 and LDQOL. When the group of transplanted patients was compared with patients before transplant, an improvement of HRQOL was found in the transplanted group with both SF-36 and LDQOL. Quality of life in pretransplant patients was found to be worse in those with cirrhosis due to hepatitis C than in those with cirrhosis due to other etiologies; the reduction in quality of life was found to be greater using LDQOL than using SF-36. Transplanted patients with recurrence of hepatitis C had worse HRQOL when measured with LDQOL, but not with SF-36. Conclusions: LDQOL, a specific instrument for measuring quality of life is more reliable than SF-36 in showing quality of life impairment. A deterioration in health-related quality of life after recurrence of hepatitis C could be observed with LDQOL.

Abstract not available. / by Wong Lai-hung, Grace. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 218-252). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.

Li, Sai Kam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 162-179). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.ii / Acknowledgements --- p.iii / Table of Content --- p.iv / Abbreviations --- p.xi / List of Figures --- p.xiv / List of Tables --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Hepatitis B Virus / Chapter 1.1.1 --- General information --- p.1 / Chapter 1.1.2 --- Classification --- p.2 / Chapter 1.1.3 --- Virus life cycle and genome --- p.3 / Chapter 1.1.4 --- Hepatitis B virus X protein (HBx) --- p.7 / Chapter 1.2 --- Enigmatic functions of HB --- p.x / Chapter 1.2.1 --- HBx as a transactivator --- p.10 / Chapter 1.2.2 --- HBx as a cell cycle regulator --- p.12 / Chapter 1.2.3 --- HBx as an apoptosis modulator --- p.13 / Chapter 1.3 --- Etiology of HBV-mediated hepatocarcinogenesis --- p.14 / Chapter 1.4 --- Clinical mutants of HBV --- p.16 / Chapter 1.5 --- Hypothesis and aims of the research --- p.16 / Chapter 1.6 --- Basis of Tet-On system --- p.18 / Chapter CHPATER 2 --- EXPERIMENT MATERIALS / Chapter 2.1 --- Cell culture / Chapter 2.1.1 --- Cell-lines --- p.21 / Chapter 2.1.2 --- Culture medium --- p.22 / Chapter 2.1.3 --- Culture medium supplements --- p.23 / Chapter 2.2 --- Reagents for subcloning / Chapter 2.2.1 --- Reagents for polymerase chain reaction (PCR) --- p.24 / Chapter 2.2.2 --- Reagents for restriction enzyme digestion --- p.24 / Chapter 2.2.3 --- Reagents for ligation --- p.25 / Chapter 2.2.4 --- Reagents for electrophoresis --- p.25 / Chapter 2.2.5 --- Reagents for E. coli DH5a preparation --- p.25 / Chapter 2.2.6 --- Materials for bacterial culture work --- p.27 / Chapter 2.3 --- Reagents for subcellular localization study / Chapter 2.3.1 --- Reagents for cell staining --- p.28 / Chapter 2.3.2 --- Reagents for mounting slides --- p.29 / Chapter 2.3.3 --- Materials for site-directed mutagenesis --- p.29 / Chapter 2.4 --- Reagents for cell cycle analysis and cellular proliferation / Chapter 2.4.1 --- Reagents for cell cycle analysis --- p.29 / Chapter 2.4.2 --- Reagents for cellular proliferation study --- p.30 / Chapter 2.5 --- Reagents for protein expression study / Chapter 2.5.1 --- Cell lysis buffer --- p.30 / Chapter 2.5.2 --- Reagents for SDS-PAGE --- p.30 / Chapter 2.5.3 --- Reagents for Western blot --- p.33 / Chapter 2.5.4 --- Antibodies --- p.34 / Chapter 2.6 --- Reagents for gene expression study / Chapter 2.6.1 --- Reagents for RNA extraction --- p.36 / Chapter 2.6.2 --- Reagents for first strand cDNA synthesis --- p.37 / Chapter 2.6.3 --- Reagents for real-time PCR --- p.37 / Chapter 2.7 --- Reagents for establishment of Tet-On inducible stable cell-lines / Chapter 2.7.1 --- Reagents for MTT assay --- p.38 / Chapter 2.7.2 --- Reagents for selection of stable clones --- p.38 / Chapter 2.8 --- Vectors used in the project / Chapter 2.8.1 --- Vectors for subcellular localization study --- p.39 / Chapter 2.8.2 --- Vectors for establishment of Tet-on inducible cell-lines --- p.39 / Chapter 2.9 --- Primers used in the project / Chapter 2.9.1 --- Primers used for subcloning --- p.42 / Chapter 2.9.2 --- Primers used for site-directed mutagenesis --- p.43 / Chapter 2.9.3 --- Primers used in real-time chain polymerase reaction --- p.43 / Chapter CHAPTER 3 --- RESEARCH METHODS / Chapter 3.1 --- Subcloning of HBx and mutant genes into a green fluorescence protein (GFP) expression vector / Chapter 3.1.1 --- Amplification of HBxWt，HBxΔC44 and HBxAN60 genes --- p.45 / Chapter 3.1.2 --- Purification of PCR products --- p.46 / Chapter 3.1.3 --- Restriction enzyme digestion --- p.47 / Chapter 3.1.4 --- Ligation of gene products with pEGFP-C 1 vector --- p.47 / Chapter 3.1.5 --- Preparation of chemically competent bacterial cells E. coli strain DH5α --- p.47 / Chapter 3.1.6 --- Transformation of the ligation product into competent cells --- p.48 / Chapter 3.1.7 --- PCR confirmation of successful ligation --- p.48 / Chapter 3.1.8 --- Small scale preparation of bacterial plasmid DNA --- p.49 / Chapter 3.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.50 / Chapter 3.1.10 --- Large scale preparation of target recombinant plasmid DNA --- p.50 / Chapter 3.2 --- Subcellular localization pattern study / Chapter 3.2.1 --- Cell transfection --- p.51 / Chapter 3.2.2 --- Mitochondria and nucleus staining --- p.52 / Chapter 3.2.3 --- Epi-fluorescence microscopy --- p.53 / Chapter 3.2.4 --- Analysis of fluorescence images --- p.53 / Chapter 3.2.5 --- In vitro site-directed mutagenesis --- p.53 / Chapter 3.3 --- Cell cycle phase analysis with flow cytometry / Chapter 3.3.1 --- Cell transfection --- p.55 / Chapter 3.3.2 --- Cell staining --- p.55 / Chapter 3.3.3 --- Flow cytometry --- p.55 / Chapter 3.4 --- Cellular proliferation quantification by BrdU proliferation assay / Chapter 3.4.1 --- Cell transfection --- p.57 / Chapter 3.4.2 --- BrdU ELISA measurement --- p.57 / Chapter 3.5 --- Protein expression / Chapter 3.5.1 --- Cell lysate collection --- p.58 / Chapter 3.5.2 --- Quantification of protein samples --- p.59 / Chapter 3.5.3 --- SDS-PAGE --- p.59 / Chapter 3.5.4 --- Western blot --- p.60 / Chapter 3.5.5 --- Western blot luminal detection --- p.60 / Chapter 3.6 --- Gene expression / Chapter 3.6.1 --- Primer design --- p.61 / Chapter 3.6.2 --- Cell transfection --- p.61 / Chapter 3.6.3 --- RNA extraction --- p.61 / Chapter 3.6.4 --- Reverse transcription for first strand complementary DNA (cDNA) --- p.63 / Chapter 3.6.5 --- Quantitative real-time PCR --- p.63 / Chapter 3.7 --- Establishment of Tet-On inducible stable cell-lines / Chapter 3.7.1 --- Subcloning of HBx gene into pTRE2 vector --- p.64 / Chapter 3.7.2 --- Construction of WRL68/Tet-On stable cell-lines --- p.64 / Chapter 3.7.3 --- Construction of WRL68/Tet-On HBx and mutants expression cell-lines --- p.68 / Chapter 3.7.4 --- Characterization of Tet-On gene expression monoclones --- p.69 / Chapter 3.8 --- Statistical analyses --- p.70 / Chapter CHPATER 4 --- STUDY ON MITOCHONDRIA TARGETING / Chapter 4.1 --- Establishment of pEGFP-Cl-HBx and mutants constructs --- p.71 / Chapter 4.2 --- Transactivation C-terminus domain is essential for granular localization --- p.73 / Chapter 4.3 --- Wild-type HBx localizes in mitochondria --- p.76 / Chapter 4.4 --- C-terminal transactivation domain is sufficient for mitochondria targeting --- p.79 / Chapter 4.5 --- Mapping of the HBx region crucial for mitochondria targeting --- p.81 / Chapter 4.6 --- The 111-117 amino acids in HBx do not work as a signal peptide --- p.83 / Chapter 4.7 --- Site-directed mutagenesis identifies the key amino acid at 115 in HBx for mitochondrial targeting --- p.85 / Chapter CHAPTER 5 --- CELL PROLIFERATION AND REGULATION / Chapter 5.1 --- Alteration of S-phase distribution in cell cycle --- p.88 / Chapter 5.2 --- Analysis of DNA synthesis using BrdU proliferation ELISA --- p.92 / Chapter 5.3 --- Differential molecular regulation of cell cycle --- p.94 / Chapter 5.4 --- Regulation of the mRNA expression levels of cyclin-dependent kinases inhibitors p2raf/cipl and p27kipl --- p.98 / Chapter CHAPTER 6 --- TRANSACTIVATION AND RAS/RAF/MAPK PHOSPHORYLATION / Chapter 6.1 --- Determination of p53-dependency of p21、vaf/cipl expression --- p.101 / Chapter 6.2 --- Ras/Raf/MAPK pathway activation by HBx variants / Chapter 6.2.1 --- ERK1/2 phophorylation by HBx variants --- p.104 / Chapter 6.2.2 --- ERK inhibition blocks the regulation effect on p53Wt and p21waf/cipl --- p.107 / Chapter 6.3 --- Transactivation activity on oncogenes/ proto-oncogenes / Chapter 6.3.1 --- Effect on c-myc (NM´ؤ002467) mRNA expression --- p.109 / Chapter 6.3.2 --- Effect on RhoC (NM_017744) and Rabl4 (NM´ؤ016322) mRNA expression --- p.112 / Chapter CHAPTER 7 --- CONSTRUCTION OF TET-ON INDUCIBLE CELL-LINES / Chapter 7.1 --- Establishment of WRL/Tet-On monoclonal cell-lines Page / Chapter 7.1.1 --- Determination of geneticin selection dosage --- p.116 / Chapter 7.1.2 --- Selection of the best WRL/TOn clone using luciferase assay --- p.118 / Chapter 7.2 --- Establishment of inducible WRL/TOn/Gene monoclonal cell-lines / Chapter 7.2.1 --- Determination of hygromycin selection dosage --- p.120 / Chapter 7.2.2 --- Selection of positive WRL/TOn/Gene clones with viral genes --- p.122 / Chapter 7.3 --- Characterization of TOXDC1 cell-line / Chapter 7.3.1 --- Cell morphology --- p.125 / Chapter 7.3.2 --- Growth pattern of TOXDC1 --- p.126 / Chapter 7.3.3 --- HBxAC44 induced p21waf/cipl mRNA expression --- p.127 / Chapter 7.3.4 --- Doxycycline concentration dependent HBxAC44 expression in TOXDC1 --- p.129 / Chapter CHAPTER 8 --- DISCUSSION / Chapter 8.1 --- Selection of cell model / Chapter 8.1.1 --- Selection of cell models --- p.130 / Chapter 8.1.2 --- Selection of truncation mutant --- p.131 / Chapter 8.2 --- Differential sub-cellular localization of HBx and its variants / Chapter 8.2.1 --- Mechanisms of mitochondria targeting --- p.132 / Chapter 8.2.2 --- Mitochondria as site of HBx-induced apoptosis --- p.134 / Chapter 8.2.3 --- Stimulation of calcium release from mitochondria by wild-type HBx --- p.135 / Chapter 8.3 --- Cell cycle distribution profiling and its regulations / Chapter 8.3.1 --- Cell cycle pattern and cell proliferation --- p.136 / Chapter 8.3.2 --- Differential cell cycle molecular pathway activation --- p.138 / Chapter 8.4 --- Ras/Raf/MAPK mediated transactivation by HBxWt and its mutants / Chapter 8.4.1 --- p53-mediated p21waf/cipl expression --- p.142 / Chapter 8.4.2 --- ERK-mediated p21waf/cipl and wild-type p53 mRNA expression --- p.143 / Chapter 8.4.3 --- Regulation of oncogenes/ proto-oncogenes expression --- p.147 / Chapter 8.5 --- General discussions on differential effects of HBxWt and HBxAC44 --- p.149 / Chapter 8.6 --- Establishment of Tet-On/HBxAC44 cell-line TOXDC1 --- p.153 / Chapter 8.7 --- Conclusions --- p.154 / Chapter 8.8 --- Future Prospects / Chapter 8.8.1 --- From mitochondria targeting to calcium signaling --- p.157 / Chapter 8.8.2 --- Construction of a complete cell cycle regulation pathway --- p.158 / Chapter 8.8.3 --- Elucidation of the transcriptional transactivation regulation --- p.159 / Chapter 8.8.4 --- To make the best use of the Tet-on stable cell-line TOXDC1 --- p.159 / Chapter 8.8.5 --- Study with other carboxy-terminal truncation mutants --- p.160 / Chapter 8.8.6 --- In vivo study --- p.160 / REFERENCES --- p.162

Leung, Chung Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 100-113). / Abstracts in English and Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgements --- p.V / List of figures --- p.VI / List of tables --- p.VIII / Abbreviations --- p.IX / Table of Contents --- p.XII / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Epidemiology of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.2 --- Etiology of heptocellular carcinoma (HCC) --- p.1 / Chapter 1.3 --- HBV genome structure --- p.2 / Chapter 1.4 --- HBV pathogenesis --- p.2 / Chapter 1.5 --- Hepatitis B virus X protein (HBx) --- p.3 / Chapter 1.6 --- Oxidative stress and antioxidant --- p.5 / Chapter 1.6.1 --- Glutathione (GSH) --- p.5 / Chapter 1.6.2 --- Superoxide dismutase (SOD) --- p.7 / Chapter 1.6.3 --- Oxidative stress in HBV-related liver disease and HCC --- p.8 / Chapter 1.7 --- Apoptosis and necrosis --- p.9 / Chapter 1.7.1 --- Apoptotic pathways --- p.9 / Chapter 1.8 --- Role of HBx in apoptosis --- p.10 / Chapter 1.9 --- Transcriptional activity by HBx --- p.12 / Chapter 1.10 --- Chemotherapy drug resistance --- p.13 / Chapter 1.11 --- Objectives of study --- p.14 / Chapter Chapter 2: --- Methods and materials / Chapter 2.1 --- Construction of plasmid --- p.23 / Chapter 2.1.1 --- PCR amplification of wild-type and mutant HBx --- p.23 / Chapter 2.1.2 --- Agarose gel extraction --- p.25 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.26 / Chapter 2.1.4 --- Ligation of vectors and gene of interest --- p.26 / Chapter 2.1.5 --- Preparation of competent cells for transformation --- p.27 / Chapter 2.1.6 --- Transformation of plasmid in competent cells --- p.27 / Chapter 2.1.7 --- Plasmid extraction by mini-prep --- p.28 / Chapter 2.1.8 --- DNA sequencing of the inserted genes --- p.29 / Chapter 2.2 --- Transfection --- p.30 / Chapter 2.2.1 --- Cell line --- p.30 / Chapter 2.2.2 --- Lipofectamine transfection --- p.31 / Chapter 2.2.3 --- Construction of stably-transfected cell lines --- p.31 / Chapter 2.3 --- Detection of expression of transfected gene in mRNA level by RT-PCR --- p.32 / Chapter 2.3.1 --- RNA extraction --- p.32 / Chapter 2.3.2 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.33 / Chapter 2.3.3 --- Agarose gel electrophoresis --- p.36 / Chapter 2.4 --- Detection of expression of transfected gene in protein level by Western blot --- p.36 / Chapter 2.4.1 --- Sample preparation --- p.36 / Chapter 2.4.2 --- Measurement of protein concentration --- p.36 / Chapter 2.4.3 --- Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) --- p.37 / Chapter 2.4.4 --- Transfer of proteins to nitrocellulose membrane --- p.38 / Chapter 2.4.5 --- Immunoblotting of protein --- p.38 / Chapter 2.5 --- Measurement of reduced glutathione (GSH) concentration in cell lines --- p.39 / Chapter 2.5.1 --- Sample preparation --- p.39 / Chapter 2.5.2 --- Measurement of GSH concentration --- p.39 / Chapter 2.6 --- Superoxide dismutase (SOD) activity in cell lines --- p.40 / Chapter 2.6.1 --- Sample preparation --- p.40 / Chapter 2.6.2 --- Measurement of total SOD activity --- p.41 / Chapter 2.6.3 --- Measurement of Cu/ZnSOD and MnSOD by Western blot --- p.42 / Chapter 2.7 --- Cell proliferation assay --- p.43 / Chapter 2.7.1 --- Drugs and concentration --- p.43 / Chapter 2.7.2 --- "MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay" --- p.43 / Chapter 2.7.3 --- Cell proliferation and cytotoxicity of the drugs --- p.44 / Chapter 2.8 --- Detection of apoptosis by flow-cytometry --- p.44 / Chapter 2.8.1 --- Cell culture --- p.44 / Chapter 2.8.2 --- Cell fixation --- p.45 / Chapter 2.8.3 --- Cell staining --- p.45 / Chapter 2.8.4 --- Flow cytometry analysis --- p.46 / Chapter 2.9 --- Detection of protein involved in apoptotic pathway --- p.46 / Chapter 2.9.1 --- Antibodies --- p.46 / Chapter 2.9.2 --- Sample Preparation --- p.47 / Chapter 2.9.3 --- Measurement of protein concentration --- p.48 / Chapter 2.9.4 --- Western blotting --- p.49 / Chapter Chapter 3: --- Establishment of HBx transfected stable cell lines / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Results --- p.56 / Chapter 3.2.1 --- Plasmid construction --- p.56 / Chapter 3.2.2 --- Stable transfection of cell lines --- p.57 / Chapter 3.2.3 --- Morphology of wild type and mutant HBx-transfected cell lines --- p.58 / Chapter 3.3 --- Discussion --- p.58 / Chapter Chapter 4: --- Antioxidant level in HBx transfected cell lines / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Results --- p.70 / Chapter 4.2.1 --- Glutathione (GSH) concentration in different cell lines --- p.70 / Chapter 4.2.2 --- Superoxide dismutase (SOD) activity in different cell lines --- p.71 / Chapter 4.2.2.1 --- Total SOD activity --- p.71 / Chapter 4.2.2.2 --- Cu/ZnSOD --- p.71 / Chapter 4.2.2.3 --- MnSOD --- p.72 / Chapter 4.3 --- Discussion --- p.72 / Chapter Chapter 5: --- Involvement of HBx in apoptotic pathway / Chapter 5.1 --- Introduction --- p.81 / Chapter 5.2 --- Results --- p.82 / Chapter 5.2.1 --- Cell proliferation of HBx transfected cells --- p.82 / Chapter 5.2.2 --- Apoptosis of HBx transfected cells --- p.83 / Chapter 5.2.3 --- Cytotoxicity of fluorouracil (5FU) and doxorubicin (DOX) in HBx transfected cells --- p.84 / Chapter 5.2.4 --- Detection of anti-apoptotic proteins cIAP2 and Bcl-2 in HBx-transient and stably transfected cells --- p.84 / Chapter 5.3 --- Discussion --- p.85 / Chapter Chapter 6: --- Concluding remarks and general discussion / Chapter 6.1 --- General discussion --- p.93 / Chapter 6.2 --- Future work --- p.97 / Chapter 6.3 --- Summary --- p.99 / References --- p.100 / Appendix 1 --- p.114

This thesis aimed to describe the epidemiology of HCV in South Australian prisons - prevalence, transmission and risk factors. This thesis also aimed to determine the impact of incarceration on reported risk behaviours. A related objective was to evaluate the epidemiological effectiveness of the ELISA - 3 HCV antibody test using PCR as the gold standard. Finally, this thesis aimed to explore the potential for minimising HCV risk in the South Australian prison population. Methods: Two case note audits were conducted at each of eight publicly operated SA prisons ( in summer and winter ) to identify any documented HCV - antibody test results. Prisoners recruited at entry to prison were offered tests for HCV - antibody and completed a pre - entry risk factor survey. Participants completed additional risk factor surveys and ( if HCV - negative at last test ) underwent further antibody tests at three - monthly intervals for up to 15 months. A sample of participants also provided blood specimens for HCV - RNA testing. Limited stakeholder consultations with prison officers and nurses were also conducted. Quantitative data were analysed using univariate and multivariate techniques. Results: 1347 case notes were audited in summer, and 1347 in winter and an overall HCV prevalence of 42 % was estimated. In both univariate and multivariate analyses, HCV prevalence was significantly higher in female prisoners ( 65 % ), those aged above 28 years ( 48 % ), and in Indigenous prisoners originating from metropolitan areas ( 56 % ). Indigenous prisoners originating from remote areas had significantly lower HCV prevalence ( 20 % ). 666 prisoners were recruited at entry, and 42 % were estimated to be HCV - antibody positive. Three seroconversions were noted in 151 initially HCV - seronegative negative individuals followed up for a median time of 121 days - a rate 4.6 per 100 person years - but community exposure could not be ruled out. Overall agreement between HCV - antibody and HCV - RNA assays was 86 % ( 100% in the HCV negative samples ) - kappa = 0.71. Injecting history was highly prevalent in prison entrants ( 70 % ) and both community and prison injecting ( but not tattooing ) were independent predictors of entry HCV status. Prison history was also independently associated with entry HCV status. Injecting in prison during the study was infrequently reported, but significantly more likely in those testing HCV - antibody positive at prison entry ( risk ratio = 2.48, P = 0.046 ). Stakeholders were most supportive of strategies to increase education and to minimise risks associated with hair clippers, but did not support most other suggested preventive strategies. Other issues related to communicable diseases and infection control were explored in the stakeholder interviews. Conclusions: HCV prevalence in South Australian prisoners is extremely high and may have contributed to a ' ceiling effect ' , minimising the seroconversion rate observed in this population. Injecting is relatively infrequently reported in prison, but more likely in those already infected with HCV. Thus, contaminated injecting equipment represents a significant threat to other prisoners and prison staff. Strategies aimed at reducing HCV risk in prisons, which address the concerns of those expected to implement them, are proposed in this thesis. / Thesis (Ph.D.)--School of Population Health and Clinical Practice, 2006.

I) Hintergrund Die chronische Hepatitis-C-Infektion stellt global ein wesentliches Gesundheitsproblem dar. Diese Virusinfektion kann bei unbehandelten Patienten zur Leberzirrhose und im weiteren Verlauf bis hin zur Entwicklung eines hepatozellulären Karzinoms führen. Die einzige Behandlungsoption mit der Aussicht auf dauerhafte Viruselimination besteht in modernen Kombinationstherapien, die das Zytokin Interferon alfa enthalten. Wesentliche Merkmale sind – neben inzwischen sehr hohen Ansprechraten – eine Behandlungsdauer zwischen 24 und 48 Wochen, hohe Therapiekosten und ein Nebenwirkungsprofil, das sowohl somatische als auch psychopathologische Symptome umfassen kann. II) Untersuchungsgegenstand und Fragestellungen Sowohl die chronische Virusinfektion an sich als auch die aktuell verfügbaren Therapieverfahren bergen ein erhebliches psychisches Belastungspotential. Hauptgegenstand dieser Dissertation ist die Erfassung der psychologischen Aspekte der Erkrankung und der psychischen und psychopathologischen Nebenwirkungen einer Interferonbehandlung. Wesentliche bearbeitete Fragestellungen sind: - Welchen Belastungsfaktoren sind Hepatitis-C-Patienten bereits ohne aktuelle antivirale Interferontherapie ausgesetzt bzw. welche psychopathologischen Symptome zeigen diese Patienten? - Wie ist der zeitliche Verlauf psychopathologischer Symptome bei Hepatitis-C-Patienten vor, während und nach einer antiviralen Therapie? - Wie wirksam und wie sicher ist eine medikamentöse Behandlung der Interferon-induzierten Depression mit selektiven Serotonin-Wiederaufnahmehemmern (SSRI) unter Fortführung der antiviralen Therapie? III) Patienten und Methoden Studienteilnehmer waren Hepatitis-C-Patienten, die sich ambulant vorstellten bzw. in unsere Ambulanz überwiesen wurden und die jeweiligen Einschlusskriterien erfüllten. Zu den wichtigsten verwendeten psychometrischen Selbstbeurteilungsskalen zählen: HADS (Depressivität, Angst), SCL-90-R (psychopathologische Symptome), SF-36 (Lebensqualität) und FKV (Krankheitsverarbeitung). IV) Wesentliche Forschungsergebnisse Bereits ohne Einfluss des Zytokins Interferon bestehen starke Krankheits-assoziierte psychische bzw. psychosoziale Belastungen der Patienten, die sich in einem erhöhten Depressionsrisiko ausdrücken. Die erhobenen Depressionsscores stehen in signifikantem Zusammenhang mit der Erkrankungsdauer und den individuell bestehenden Optionen und Erfolgsaussichten einer antiviralen Interferontherapie. Prospektive Erfassungen der Auftretenshäufigkeit klinisch relevanter Interferon-assoziierter Depressionen ergeben Raten von ca. 30 %. Diese Größenordnung wurde sowohl in einer eigenen prospektiven Studie als auch im Rahmen einer vorgestellten Übersichtsarbeit bestätigt. Die Umstellung der verwendeten Formulierung des Medikaments von herkömmlichem Interferon alfa auf die pegylierte Variante brachte keine Verbesserung der Verträglichkeit z.B. im Hinblick auf die interferonassoziierte Depression. Ein rechtzeitiges Erkennen der entsprechenden Symptome vorausgesetzt, ist die antidepressive Behandlung der Interferon-assoziierten Depression mit Hilfe von selektiven Serotonin-Reuptake-Inhibitoren auch ohne generelle Prophylaxe sehr effektiv und sicher möglich. V) Diskussion Empfohlen wird ein engmaschiges psychometrisches Monitoring aller Hepatitis-C-Patienten im Therapieverlauf. Ausführliche Aufklärung, enger Arzt-Patienten-Kontakt während der Therapie, sowie die Betreuung durch einen festen Ansprechpartner während der bis zu einem Jahr dauernden Therapie sind wichtige Rahmenbedingungen für eine solche Behandlung. Für die medikamentöse Behandlung der Interferon-induzierten Depression gilt: Bei besonderer Indikation (z.B. Interferon-assoziierte Depression bei früheren Therapieversuchen) sollte eine SSRI-Sekundärprophylaxe in Betracht gezogen werden. Ansonsten ist eine entsprechende SSRI-Intervention beginnend mit dem Einsetzen einer klinisch relevanten Depression ausreichend.

Die Virushepatitis gehört weltweit zu einer der häufigsten viralen Erkrankungen. Doch durch die Entwicklung von immer effizienteren Impfstoffen kann bei einer frühzeitigen Immunisierung eine Infektion verhindert werden. Ziel dieser Arbeit war es, ein Patientenkollektiv zu untersuchen, welches eine Impfung mit einem Hepatitis B-Impfstoff erhalten hatte, und dieses mit Probanden zu vergleichen, die nicht immunisiert waren. Diese Proben wurden auf ihre serologische und zelluläre Reaktivität mittels ELISA und ELISpot untersucht. Im ELISA zeigte sich bei 95 % der geimpften Personen eine positive Serokonversion nach zurückliegender Hepatitis B-Impfung. Um den Impfstatus genauer zu analysieren und bei seronegativen geimpften Probanden den zellulären Arm des Immun-systems zu verifizieren, wurde mittels ELISpot die IFN-γ-Sekretion von HBs-reaktiven T-Zellen untersucht und mit den Ergebnissen, welche in der Serologie gewonnen wurden, verglichen. Dabei zeigte sich, dass die zelluläre Untersuchung bei 43 von 94 untersuchten Patientenproben (46 %) ein positives Ergebnis aufwies. Zweiundzwanzig der 48 geimpften Patienten (46 %) hatten eine antigenspezifische IFN-γ-Sekretion und 21 der 46 Proben der aktuell nicht geimpften Patienten fielen im ELISpot positiv aus. Bei zwei seronegativ geimpften Patienten konnte jeweils ein positives Ergebnis im ELISpot gezeigt werden. Ein direkter Zusammenhang zwischen der Höhe des anti-HBs-Titers im ELISA und Anzahl der spot-bildenden Zellen im ELISpot konnte nicht gezeigt werden. Die Ergebnisse dieser Arbeit zeigen, dass die serologische Untersuchung mittels ELISA weiterhin als Goldstandard verwendet werden soll, um den aktuellen Schutz gegenüber einer Hepatitis B-Infektion zu verifizieren. Durch die zelluläre Untersuchung mit dem ELISpot-Verfahren kann bei weiterer Testoptimierung in Zukunft eine Nachweismethode für seronegative Geimpfte entwickelt werden. Vor allem sollten diese mit Hilfe des ELISpots genauer analysiert werden, um gegebenenfalls bei nicht vorhandener humoralen Immunität und einer ebenfalls fehlenden zellulären Immunität prophylaktische Maßnahmen einzuleiten.