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.

Introdução: A infecção crônica pelo vírus da hepatite C (VHC) é uma epidemia que atinge mais de 170 milhões de pessoas em todo o mundo e freqüentemente associa-se a fenômenos de auto-imunidade. O papel dos alelos de HLA de classe II vem sendo estudado em diversas condições autoimunes. Objetivos: investigar a presença de auto-imunidade em portadores de VHC; realizar análise de HLA de classe II em portadores de VHC com e sem marcadores de auto-imunidade. Casuística e Métodos: obtiveram-se retrospectivamente os dados clínicos, laboratoriais, histológicos hepáticos de 1312 indivíduos com VHC, e definiu-se a presença de HAI associada segundo critérios adotados pelo Grupo Internacional de Estudos da HAI (escore >= 10). Constituíram-se os subgrupos: VHC + HAI (n = 44); VHC + anticorpo antimicrossoma de fígado e rim tipo 1 (AAMFR-1) (n = 7); VHC+ anticorpo antimúsculo liso padrão tubular/antiactina (AAML-T/AAA) (n = 5) e controle de pacientes com VHC sem características de auto-imunidade (n = 29). A tipagem do HLA foi realizada em DNA leucócitário de sangue periférico, extraído pela técnica de DTAB/CTAB, seguido de SSCP com o kit Micro SSPTM HLA DNA Typing (One Lambda Inc., CA, USA). A análise estatística foi realizada com o teste de X2 de Pearson com correção de Yates ou teste exato de Fisher quando apropriado e nos casos de existência de associação foi calculado o coeficiente de Yule para quantificá-la. Resultados: observou-se no grupo VHC + HAI, em comparação com a casuística geral predominância de idade > 40 anos e níveis de ALT acima de três vezes o limite superior da normalidade, associação positiva com o HLADR4 (45,1% vs 3,4%, p = 0,0006, coeficiente de Yule = 0,92) e DQ3 (67,7% vs 37,9%, p = 0,04, coeficiente de Yule = 0,54) e associação negativa com o HLADR51 (9,6% vs 34,4%, p = 0,04, coeficiente de Yule = -0,66) e DR2 (9,6% vs 34,4%, p = 0,04, coeficiente de Yule = -0,66). Pacientes do grupo VHC + AAMFR-1 situaram-se em faixa etária inferior a 40 anos em relação ao grupo controle (p = 0,001). Conclusão: Idade superior a 40 anos, níveis de aminotransferases elevados caracterizam os pacientes com VHC e marcadores de auto-imunidade; níveis elevados de gamaglobulinas não são observados em portadores de VHC + HAI; o grupo VHC + AAMFR-1 manifesta a doença em faixa etária inferior a 40 anos; há associação positiva dos alelos HLA-DR4 e DQ3 e associação negativa dos alelos HLA-DR51 e DR2 com o grupo VHC + HAI; não foi possível estabelecer na população estudada semelhanças de marcadores imunogenéticos com os da HAI tipo 1 e 2, devido à baixa freqüência do AAA e AAMFR-1 na presente série. / Introduction: HCV chronic infection is an epidemic condition that affects more than 170 million of people around the world, and often is associated with autoimmunity phenomena. The role of HLA class II alleles has been studied in many autoimmune diseases. Aim: To investigate the presence of laboratory markers of autoimmunity and compatible anatomo pathologic histology for autoimmune hepatitis (AIH) in HCV patients; to perform HLA class II typing in HCV patients with and without autoimmunity markers. Material and Methods: Clinical, laboratory and liver histology data from 1312 HCV patients were obtained retrospectively. AIH was considered in association based on the International Group for the Study of AIH criteria score system (>= 10). The following groups were constituted: HCV + AIH (n = 44); HCV + anti-liver-kidney-microsome type 1 (LKM-1) (n = 7); HCV + antismooth muscle/anti-actin antibodies (SMA/AAA) (n = 5); control (HCV without autoimmunity) (n = 29). HLA typing was performed DNA extracted from leucocyte from periferic blood by DTAB/CTAB techniques, followed by SSCP with Micro SSPTM HLA DNA Typing kit (One Lambda Inc., CA, USA). Statistical analysis was performed with Pearson\'s X2 test, with Yates correction or Fisher exact test, when appropriated; when significant association was detected, Yule coefficient was calculated. Results: Age > 40 years old and ALT three times above upper normal limit predominated in the HCV + AIH group, when compared with the whole cohort (n = 1312). HLA typing in VHC+HAI group (n = 31) revealed positive association with HLA-DR4 (45.1% vs 3.4%, p = 0.0006, Yule = 0.92) and DQ3 (67.7% vs 37.9%, p = 0.04, Yule = 0.54) and negative association with HLA-DR51 (9.6% vs 34.4%, p = 0.04, Yule = -0.66) and DR2 (9.6% vs 34.4%, p = 0.04, Yule = -0.66), when compared with VHC control (n = 29). HCV + LKM-1 patients were younger than 40 years old at the time of initial disease manifestations (p = 0,001). Conclusion: Age above 40 years old, ALT levels three times upper the normal limit, but not gamma globulin levels, characterize HCV + AIH; HCV + LKM-1 patients manifest disease before 40 years old; HLA-DR4 and DQ3 allele have a positive association and HLA-DR51 and DR2 have a negative association with the HCV + AIH group. It was not possible to establish immunogenetic markers of AIH type 1 and 2 because of low frequency of SMA and AAA in this series.

by Shuk-kei Lau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 141-148). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Table of Contents --- p.ii / Abstract --- p.vi / 論文摘要 --- p.viii / Abbreviations --- p.ix / List of Figures --- p.x / List of Tables --- p.xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- HBV and its role in hepatocarcinogenesis --- p.3 / Chapter 1.2.1 --- Current situation of HBV infection and the HCC incidencein the world --- p.3 / Chapter 1.2.2 --- Current situation of HBV infection and the HCC incidencein Hong Kong --- p.4 / Chapter 1.2.3 --- Genetic organization of HBV --- p.4 / Chapter 1.2.4 --- Principle of hepatocarcinogenesis induced by HBV --- p.5 / Chapter 1.2.4.1 --- Role of chronic hepatitis in hepatocarcinogenesis --- p.5 / Chapter 1.2.4.2 --- Role of HBV in hepatocarcinogenesis --- p.6 / Chapter 1.2.5 --- Current screening tests for HCC --- p.7 / Chapter 1.2.6 --- Current therapies for HCC --- p.9 / Chapter 1.3 --- Aim of the present study --- p.13 / Chapter 1.4 --- "Combining Expressed Sequence Tag (EST), Suppression Subtractive Hybridization and cDNA microarray for rapid differentially by expressed genes screening" --- p.14 / Chapter 1.4.1 --- Expressed Sequence Tag (EST) --- p.14 / Chapter 1.4.2 --- cDNA subtraction --- p.15 / Chapter 1.4.3 --- cDNA microarray --- p.16 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- PCR-select cDNA subtraction --- p.17 / Chapter 2.1.1 --- Amplification of subtracted cDNA clones by PCR --- p.17 / Chapter 2.1.2 --- Cycle sequencing of subtracted cDNA clones --- p.18 / Chapter 2.1.3 --- Sequence analysis using BLAST server and Stanford Online Universal Resource for Clones and ESTs (SOURCE) --- p.19 / Chapter 2.2 --- cDNA microarray analysis --- p.20 / Chapter 2.2.1 --- Array fabrication --- p.20 / Chapter 2.2.1.1 --- Amplification of cDNA clones by PCR --- p.20 / Chapter 2.2.1.2 --- Purification of PCR products --- p.21 / Chapter 2.2.1.3 --- Cycle sequencing for clones checking --- p.22 / Chapter 2.2.2 --- Microarray printing --- p.22 / Chapter 2.2.2.1 --- Preparation of cDNA target --- p.22 / Chapter 2.2.2.2 --- Arraying --- p.22 / Chapter 2.2.3 --- Screening of differentially expressed genes in hepatocellular carcinoma and its surrounding normal counterpart by cDNA microarray --- p.23 / Chapter 2.2.3.1 --- Extraction of RNA --- p.23 / Chapter 2.2.3.2 --- RNA labeling --- p.24 / Chapter 2.2.3.3 --- Microarray hybridization --- p.26 / Chapter 2.2.3.4 --- Collection of data --- p.27 / Chapter 2.2.3.5 --- Data normalization and analysis --- p.28 / Chapter 2.3 --- Molecular cloning and characterization of a novel cDNA clone differentially expressed in HCC --- p.30 / Chapter 2.3.1 --- Tissue distribution of T2L522 gene --- p.30 / Chapter 2.3.1.1 --- Northern hybridization --- p.30 / Chapter 2.3.1.2 --- Reverse-transcriptase polymerase chain reaction (RT-PCR) --- p.33 / Chapter 2.3.2 --- Expression level of T2L522 in HCC and its surrounding normal counterpart --- p.33 / Chapter 2.3.3 --- Identification of interacting partner of T2L522 using yeast two-hybrid assay --- p.35 / Chapter 2.3.3.1 --- "Cloning of T2L522 gene into the yeast two-hybrid DNA-BD vector, pGBKT7" --- p.35 / Chapter 2.3.3.2 --- Transformation of yeast competent cells --- p.39 / Chapter 2.3.3.3 --- Mating of T2L522-BD with pretransformed human liver cDNA library --- p.40 / Chapter 2.3.3.4 --- Colony lift p-galactosidase filter assay --- p.42 / Chapter 2.3.4 --- Subcellular localization of T2L522 gene by tagging with green fluorescence protein (GFP) --- p.43 / Chapter 2.3.4.1 --- "Cloning of T2L522 gene into the eukaryotic GFP expression vector, pEGFP-Cl" --- p.43 / Chapter 2.3.4.2 --- Transfection of pEGFP-T2L522 into HepG2 cell --- p.43 / Chapter Chapter 3 --- Results / Chapter 3.1 --- PCR-select cDNA subtraction --- p.45 / Chapter 3.1.1 --- The sequencing results of subtracted-HCC cDNA clones --- p.45 / Chapter 3.1.2 --- Categorization of ESTs sequenced from subtracted-HCC library --- p.45 / Chapter 3.2 --- Microarray analysis --- p.49 / Chapter 3.2.1 --- Array fabrication --- p.49 / Chapter 3.2.1.1 --- Amplification of cDNA microarray targets --- p.49 / Chapter 3.2.2 --- Microarray printing --- p.52 / Chapter 3.2.3 --- Microarray analysis of differentially expressed genesin hepatocellular carcinoma and its surrounding normal counterpart --- p.55 / Chapter 3.2.4 --- Data collection --- p.57 / Chapter 3.2.5 --- Image processing: spots finding and quantitation --- p.61 / Chapter 3.2.6 --- Data normalization and analysis --- p.61 / Chapter 3.3 --- Molecular cloning and characterization of a novel cDNA clone differentially expressed in HCC --- p.73 / Chapter 3.3.1 --- Tissue distribution of T2L522 --- p.77 / Chapter 3.3.1.1 --- Northern hybridization --- p.77 / Chapter 3.3.1.2 --- Reverse-transcriptase polymerase chain reaction (RT-PCR) --- p.79 / Chapter 3.3.2 --- Expression level of T2L522 in hepatocellular carcinoma and its surrounding normal counterpart --- p.81 / Chapter 3.3.3 --- Identification of interacting partner of T2L522 using yeast two-hybrid assay --- p.85 / Chapter 3.3.4 --- Subcellular localization of GFP tagged T2L522 --- p.87 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- EST analysis on subtracted-HCC cDNA library --- p.89 / Chapter 4.2 --- cDNA microarray analysis --- p.92 / Chapter 4.2.1 --- Generation of reliable data using cDNA microarray --- p.92 / Chapter 4.2.1.1 --- Reproducibility of signal and normalized ratio --- p.92 / Chapter 4.2.2 --- Comparison of data between multiple slides --- p.96 / Chapter 4.2.2.1 --- Assession of data quality and statistical significance --- p.96 / Chapter 4.2.2.2 --- Interpretation of gene expression data from single and multiple hybridizarion --- p.97 / Chapter 4.3 --- Candidate genes differentially expressed in HCC and its surrounding normal counterpart --- p.99 / Chapter 4.3.1 --- Protein up-regulated in HCC --- p.99 / Chapter 4.3.1.1 --- Extracellular matrix protein --- p.99 / Chapter 4.3.1.2 --- Protein involved in other metabolism --- p.100 / Chapter 4.3.1.3 --- Protein involved in transcription and translation --- p.100 / Chapter 4.3.2 --- Protein down-regulated in HCC --- p.101 / Chapter 4.3.2.1 --- Membrane associated protein --- p.101 / Chapter 4.3.2.2 --- Protein involved in other metabolism --- p.102 / Chapter 4.3.2.2 --- Secretory protein --- p.104 / Chapter 4.3.3 --- Novel protein differentially expressed in HCC --- p.107 / Chapter 4.4 --- "TBC1 domain containing protein, T2L522" --- p.108 / Chapter 4.4.1 --- Possible involvement of T2L522 gene in HCC --- p.109 / Chapter 4.4.2 --- Tissue distribution and expression pattern of T2L522 --- p.110 / Chapter 4.4.3 --- Potential interacting partner of T2L522 --- p.110 / Chapter 4.4.4 --- Subcellular localization of T2L522 --- p.112 / Chapter 4.5 --- Summary --- p.113 / Appendix --- p.114 / References --- p.141

Chan Ka Ping Sophie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 140-144). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Acknowledgements --- p.ii / Abstract --- p.viii / 摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xii / List of Tables --- p.xiv / Chapter 1 --- Introduction / Chapter 1.1 --- Pathogenesis of HBV Infection --- p.1 / Chapter 1.2 --- Classification and Structure --- p.2 / Chapter 1.3 --- HBV Genome --- p.4 / Chapter 1.4 --- Replication Cycle --- p.7 / Chapter 1.5 --- HBV Genotypes and Nomenclature --- p.9 / Chapter 1.5.1 --- Asian prevalent genotypes --- p.9 / Chapter 1.5.2 --- Numbering system --- p.9 / Chapter 1.6 --- Identification of Markers in HBV Genome for HCC Development --- p.11 / Chapter 1.7 --- Project Objective --- p.13 / Chapter 1.8 --- Promoters of HBV --- p.14 / Chapter 1.8.1 --- Pre-S1 promoter --- p.14 / Chapter 1.8.2 --- X promoter and enhancer I --- p.14 / Chapter 1.8.3 --- Core promoter and enhancer II --- p.15 / Chapter 1.8.4 --- Pair of mutations at BCP --- p.17 / Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of pGL3-promoter Plasmids --- p.18 / Chapter 2.1.1 --- Templates selection --- p.18 / Chapter 2.1.2 --- Amplification of promoters --- p.19 / Chapter 2.1.3 --- Cloning into pGL3-basic vector --- p.21 / Chapter 2.1.4 --- Screening and plasmid preparation --- p.21 / Chapter 2.2 --- Construction of Mutant Promoter Clones --- p.23 / Chapter 2.2.1 --- Site-directed mutagenesis --- p.23 / Chapter 2.2.2 --- pPreS 1 /2712C mutant clone --- p.24 / Chapter 2.3 --- Cloning of Full-length HBV Genomes --- p.26 / Chapter 2.3.1 --- Replication-competent HBV clone --- p.26 / Chapter 2.3.2 --- Amplification of full-length HBV genome --- p.28 / Chapter 2.3.3 --- Cloning into pUC19 vector --- p.28 / Chapter 2.3.4 --- Screening for insert and sequence confirmation --- p.29 / Chapter 2.3.5 --- Excision of full-length HBV from plasmid --- p.29 / Chapter 2.4 --- Re-construction into a 1.3-fold HBV Clone --- p.32 / Chapter 2.4.1 --- Cloning of HBV fragment nucleotide 979-2617 (nt 979-2617) --- p.32 / Chapter 2.4.2 --- Screening for insert and sequence confirmation --- p.33 / Chapter 2.4.3 --- Cloning of HBV fragment (nt 905-2000) --- p.33 / Chapter 2.4.4 --- Construction of a 1.3-fold HBV genotype Cs clone --- p.34 / Chapter 2.5 --- Cell Culture --- p.37 / Chapter 2.5.1 --- Cell culture maintenance --- p.37 / Chapter 2.5.2 --- Transient transfection of promoter clones --- p.37 / Chapter 2.5.3 --- Transient transfection of HBV genomes --- p.38 / Chapter 2.6 --- Dual-Luciferase® Reporter Assay System --- p.40 / Chapter 2.6.1 --- Principle of the assay --- p.40 / Chapter 2.6.2 --- Cell harvest --- p.43 / Chapter 2.6.3 --- Luciferase assay --- p.43 / Chapter 2.7 --- Data Analysis --- p.44 / Chapter 2.8 --- Extraction of HBV DNA from Intracellular Cores --- p.45 / Chapter 2.8.1 --- Harvest of intracellular cores --- p.45 / Chapter 2.8.2 --- Phenol/chloroform extraction --- p.45 / Chapter 2.9 --- Southern Blotting --- p.47 / Chapter 2.9.1 --- Transfer of DNA to membrane --- p.47 / Chapter 2.9.2 --- Preparation of probes --- p.47 / Chapter 2.9.3 --- Hybridization with radiolabeled probes --- p.48 / Chapter 2.10 --- Detection of HBeAg and HBsAg --- p.50 / Chapter 2.10.1 --- HBsAg assays --- p.50 / Chapter 2.10.2 --- HBeAg assays --- p.51 / Chapter 2.11 --- SEAP Reporter Gene Assay --- p.52 / Chapter 3 --- Results / Chapter 3.1 --- Templates Selected --- p.53 / Chapter 3.2 --- Results of Luciferase Assays --- p.58 / Chapter 3.2.1. --- BCP mutation of genotype A as control --- p.58 / Chapter 3.2.2. --- Effect of C1165T mutation on Xpro/enhI activity of HBV genotype B --- p.60 / Chapter 3.2.3. --- Effect ofT2712C mutation on pre-S1 promoter activity of HBV Genotype B --- p.60 / Chapter 3.2.4. --- Effect of G1613A mutation on core pro/enhII activity of HBV Genotype Cs --- p.64 / Chapter 3.2.5. --- G1613A and BCP mutation --- p.67 / Chapter 3.3 --- Full-length HBV Genome Clones --- p.70 / Chapter 3.3.1. --- Construction of replication-competent full-length HBV genome clones --- p.70 / Chapter 3.3.2. --- Drawbacks of the system --- p.78 / Chapter 3.4 --- Construction of a Replication-competent 1.3-fold HBV Clone --- p.82 / Chapter 3.4.1. --- Construction of the HBV (nt 979-2617) clone --- p.82 / Chapter 3.4.2. --- Construction of the HBV (nt 905-2000) clone --- p.86 / Chapter 3.4.3. --- Construction of 1.3-fold genotype Cs HB V clone --- p.89 / Chapter 3.4.4. --- Test for replication competency --- p.92 / Chapter 4 --- Discussion / Chapter 4.1 --- BCP Mutation as Control of the Luciferase Assay --- p.94 / Chapter 4.2 --- Promoter Activities Not Altered by T2712C and C1165T --- p.96 / Chapter 4.3 --- Mutation G1613A of Core pro/enhll --- p.98 / Chapter 4.3.1 --- Mutation resides in negative regulatory element of core promoter --- p.98 / Chapter 4.3.2 --- NRE and NRE-binding protein --- p.98 / Chapter 4.3.3 --- Relationship with BCP mutation --- p.101 / Chapter 4.4 --- HBV Constructs --- p.103 / Chapter 4.4.1 --- Rationale in re-construction of 1.3-fold HB V clone --- p.103 / Chapter 4.4.2 --- Replication competency --- p.104 / Chapter 4.5 --- Conclusion --- p.106 / Chapter 4.6 --- Future Work --- p.107 / Appendix --- p.108 / References --- p.140

O café uma é das bebidas mais consumidas no mundo e seus efeitos benéficos são objetivo do estudo durante anos. O café, por ser uma bebida antioxidante, pode inibir as enzimas hepáticas diminuindo a lesão de hepatócitos e com isso temos um efeito hepatoprotetor. Esta melhora no fígado é relacionado diretamente à ingestão de café. Portanto, este estudo tem como objetivo avaliar o efeito do consumo de café em grupos de portadores de hepatite B crônica e de hepatite C crônica supondo que o café pode retardar a progressão da lesão hepática. Métodos: Um total de 1169 pacientes com doenças hepáticas crônicas foram selecionados do banco de dados do ambulatório de hepatologia do Hospital das Clínicas de São Paulo, sendo 514 (44%) com o vírus da hepatite B (HBV) e 655 (56%) com hepatite C (HCV). Foram consideradas as variáveis como tabagismo, etilismo, consumo de café, exames laboratoriais (ALT, AST, GGT, INR, plaquetas, bilirrubina total, bilirrubina direta e bilirrubina indireta, albumina e creatinina), APRI e FIB4 para avaliar fibrose e o grau de lesão hepática. Resultados: Através da análise descritiva dos dados observamos que 758/1169 (65%) pacientes consumiam café. Pacientes que consumem café apresentam menores índices de AST (p=0,004), APRI (p=0,002) e FIB4 (p=0,003). Ao se analisar por etiologia observou-se que pacientes portadores de hepatite crônica C que consumem café apresentam menores índices de ALT (p=0,021), AST (p=0,005), APRI (p=0,013) e FIB4 (p=0,013) e maiores níveis de albumina (p=0,006). O mesmo não foi observado para os portadores de hepatite crônica B. Conclusões: A ingestão de café está associada com a redução das enzimas do fígado e parece estar diretamente ligada a diminuição dos valores de APRI e FIB4 em pacientes portadores de hepatite crônica C. O mesmo não é observado para hepatite crônica B / Coffee is one of the most consumed beverages in the world and its beneficial effects are objective of study for years. Coffee is considered an antioxidant drink and can inhibit injury of hepatocytes decreasing liver enzymes, thus having a hepatoprotective effect. This improvement in the liver is directly related to coffee intake. Therefore, this study aims to evaluate the consumption of coffee in groups of chronic hepatitis B and C patients assuming that coffee can slow the progression of liver damage. Methods: 1169 patients with chronic liver disease were consecutively selected in our clinic hepatology database of the Hospital das Clinicas in Sao Paulo. There were 514 (44%) patients with hepatitis B virus (HBV) and 655 (56%) with hepatitis C virus (HCV). Variables such as smoking, alcohol consumption, coffee consumption, laboratory tests (ALT, AST, GGT, INR, platelet, total bilirubin, direct bilirubin and indirect bilirubin, albumin and creatinine), APRI and FIB4 were analyzed to assess fibrosis and degree of liver injury. Results: Through descriptive analysis, we found that 758/1169 (65%) patients consumed coffee. Patients who consume coffee have lower levels of AST (p = 0.004), APRI (p = 0.002) and FIB4 (p = 0.003). When analyzed by etiology it was observed that patients with chronic hepatitis C who consume coffee have lower levels of ALT (p = 0.021), AST (p = 0.005), APRI (p = 0.013) and FIB4 (p = 0.013) and higher albumin level (p = 0.006). The same was not observed for patients with chronic hepatitis B. Conclusions: Coffee intake is associated with reduced liver enzymes and appears to be directly linked to the reduction of APRI and FIB4 values in patients with chronic hepatitis C. The same is not observed for chronic hepatitis B

Viral hepatitis - knowledge of upper grade high school students This thesis is focused on the issue of all types of viral hepatitis. The work is divided into two large units. The first part is theoretical and provides a comprehensive overview of all types of viral hepatitis in general. It deals in detail with each type separately; it describes its characteristics, what causes it, its epidemiology, course, diagnosis, treatment and prevention. The empirical part investigates the level of knowledge of students of third and fourth grade of high schools from a quantitative survey. It includes pedagogical research, whose task was to determine what knowledge students had acquired during compulsory education, and it also includes evaluation of this research.

Estudo observacional transversal que teve como objetivos caracterizar os pacientes com diabetes mellitus segundo as variáveis demográficas e clínicas; estimar a prevalência de infecção pelo vírus da hepatite B e C e a cobertura vacinal contra hepatite B em pacientes com diabetes mellitus e identificar potenciais fatores de risco relacionados. A amostra de conveniência foi constituída de 255 pacientes com diabetes mellitus que compareceram à consulta médica nos ambulatórios de Endocrinologia e de Clínica Médica Integrado de Unidade Distrital de Saúde de Ribeirão Preto-SP, no período de julho a dezembro de 2014. As variáveis demográficas selecionadas foram sexo, idade e escolaridade e, as clínicas o uso de insulina, monitoramento da glicemia capilar, história de intervenções médicas, cirúrgicas, diagnósticas e terapêuticas e situações e comportamentos de risco para hepatite B e C. Os marcadores sorológicos investigados foram HBsAg, Anti-HBc IgG, Anti-HBc IgM, Anti-HBs e Anti-HCV. Considerou-se vacinação completa três ou mais doses da vacina contra hepatite B registrada. Para a coleta de dados utilizou-se um questionário, consulta ao registro de vacinação no Sistema Informatizado de Gestão em Saúde da Secretaria Municipal de Saúde e coleta de sangue venoso. Os dados foram apresentados por meio de estatística descritiva. A análise univariada das possíveis associações entre as variáveis demográficas e clínicas e os desfechos infecção pelo vírus da hepatite B e C e cobertura vacinal contra hepatite B, foi determinada pelos testes de Qui-quadrado corrigido por Pearson ou Teste exato de Fisher bicaudal e Wilcoxon para amostras não pareadas. Para a análise multivariada, foi construído um modelo de regressão logística onde foram incluídas as variáveis que exibiram p<0,2 na análise univariada. Valores de \'p\' inferiores a 5% foram considerados significativos. Os resultados mostraram que 16,8% dos pacientes apresentaram marcador Anti-HBc total reagente, 8,2% Anti-HBs isolado e 75% foram não reagentes para todos os marcadores de hepatite B. Nenhum caso de HBsAg reagente foi encontrado na amostra estudada. Quanto à infecção pelo vírus da hepatite C, 3,3% dos pacientes apresentaram marcador anti-HCV reagente. A prevalência de infecção pelo vírus da hepatite B (Anti-HBc reagente) em pacientes com diabetes mellitus foi de 16,8%, superior à nacional e mostrou-se diretamente associado ao tempo da doença. A prevalência de infecção pelo vírus da hepatite C (Anti-HCV reagente) foi de 3,3%, superior à nacional, mas não teve associação com as variáveis demográficas e clínicas investigadas. A cobertura vacinal contra hepatite B mostrou-se baixa (15%) em pacientes com diabetes mellitus evidenciando a sua vulnerabilidade a essa doença grave e potencialmente fatal. Maior escolaridade e o trabalho na área da saúde foram associados à melhor cobertura vacinal. Estes resultados fornecem subsídios importantes para a avaliação da prática clínica dos enfermeiros na atenção primaria à saúde para a prestação de cuidados relacionados à cobertura vacinal a pessoas com diabetes mellitus. Assim, esforços devem ser empenhados pelos profissionais e serviços de saúde para enfrentar o desafio de prover ampla cobertura vacinal a essa população, garantindo a prevenção da infecção pelo vírus da hepatite B e C / A cross-sectional observational study aimed at characterizing patients with diabetes mellitus according to demographic and clinical variables; to estimate the prevalence of hepatitis B and C virus infection and vaccine coverage in patients with diabetes mellitus and to identify potential related risk factors. The convenience sample consisted of 255 patients with diabetes mellitus who attended the medical consultation in the outpatient clinics of Endocrinology and Integrated Medical Clinic of the District Health Unit of Ribeirão Preto-SP, from July to December 2014. The selected demographic variables the use of insulin, capillary blood glucose monitoring, history of medical, surgical, diagnostic and therapeutic interventions and risky situations and behaviors for hepatitis B and C. The serological markers investigated were HBsAg, Anti -HBc IgG, Anti-HBc IgM, Anti-HBs and Anti-HCV. Three or more doses of the registered hepatitis B vaccine were considered complete vaccination. To collect data, a questionnaire was used, consulting the vaccination record of the Health Management Computerized System of Health of the Municipal Health Department and venous blood collection. The data were presented by means of descriptive statistics. Univariate analysis of possible associations between demographic and clinical variables and outcomes of hepatitis B and C virus infection and vaccine coverage against hepatitis B was determined by the Pearson-corrected chi-square test or Wilcoxon\'s exact Fisher\'s test for Unpaired samples. For the multivariate analysis, a logistic regression model was constructed in which variables that exhibited p <0.2 were included in the univariate analysis. Values of \'p\' below 5% were considered significant. The results showed that 16.8% of the patients presented total anti-HBc marker reagent, 8.2% Anti-HBs alone and 75% were non-reactive for all hepatitis B markers. No case of HBsAg reagent was found in the sample studied. Regarding hepatitis C virus infection, 3.3% of the patients presented anti-HCV marker reagent. The prevalence of hepatitis B virus (Anti-HBc reagent) in patients with diabetes mellitus was 16.8% higher than the national level and was directly associated with the time of diabetes mellitus. The prevalence of hepatitis C virus infection (Anti-HCV reagent) was 3.3%, higher than the national level, but had no association with the demographic and clinical variables investigated. Hepatitis B vaccination coverage was shown to be low (15%) in patients with diabetes mellitus evidencing their vulnerability to this serious and potentially fatal disease. Higher schooling and work in the health area were associated with better vaccine coverage. These results provide important insights for the evaluation of nurses\' clinical practice in primary health care for care related to vaccination coverage for people with diabetes mellitus. Thus, efforts must be made by health professionals and services to meet the challenge of providing ample vaccination coverage to this population, ensuring the prevention of hepatitis B and C virus infection

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2015-02-04T09:24:45Z No. of bitstreams: 2 Dissertação - Leandro Nascimento da Silva - 2014.pdf: 6086146 bytes, checksum: 75f020e70d97640ac095871c4913b275 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-02-05T14:16:04Z (GMT) No. of bitstreams: 2 Dissertação - Leandro Nascimento da Silva - 2014.pdf: 6086146 bytes, checksum: 75f020e70d97640ac095871c4913b275 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-05T14:16:04Z (GMT). No. of bitstreams: 2 Dissertação - Leandro Nascimento da Silva - 2014.pdf: 6086146 bytes, checksum: 75f020e70d97640ac095871c4913b275 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-09-09 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Crack is considered a public health problem in Brazil and in the world because of its impact on social relationships, physical and mental integrity of the user, and the risk associated with infections, such as those caused by the hepatitis B virus (HBV). This study investigated the epidemiology of infection with the hepatitis B virus and immunization status among users of crack institutionalized in Goiania, Brazil. During August 2012 to April 2013, a total of 600 individuals were interviewed, and blood samples collected for the detection of serological markers of HBV (HBsAg, total anti HBc and anti-HBs) by enzyme-linked immune sorbent assay (ELISA). Subsequently a cohort of individuals susceptible to hepatitis B was formed to assess compliance, completion of the vaccination series, and vaccine response against hepatitis B, using an accelerated scheme. Prior exposure to HBV (anti-HBc) was 7.0% (95% CI: 5.22 to 9.32), and 17.7% (95% CI: 14.8 to 20.9) were anti -HBs isolated, suggesting previous vaccination against hepatitis B. The use of crack cocaine through improvised pipes, history of sexually transmitted disease, and exchanging sex for drugs or money were significantly associated with exposure to HBV (p < 0.05). Of the total of individuals who received the first dose of hepatitis B vaccine and eligible to complete the full vaccine scheme (n = 406), 229 (56.4%) and 96 (26.6%) received the second and third doses, respectively. It was possible to evaluate the vaccine response in only 23/96 subjects, and 78% responded with protective titers. The high frequency of risk behaviors, the low frequency of vaccinations, and improper compliance with the vaccination schedule, even using the accelerated scheme, highlights the need for strategies for health education and prevention to reach this population so vulnerable to sexually transmitted infections and parenteral transmission of hepatitis B. / O crack é considerado um problema de saúde pública no Brasil e no mundo devido ao seu impacto nas relações sociais, na integridade física e mental do usuário e no risco associado às infecções, como a causada pelo vírus da hepatite B (HBV). Este estudo investigou a epidemiologia da infecção pelo vírus da Hepatite B e situação vacinal em usuários de crack institucionalizados em Goiânia – Goiás. Durante agosto de 2012 a abril de 2013 um total de 600 indivíduos foram entrevistados e amostras sanguíneas coletadas para detecção dos marcadores sorológicos do HBV (HBsAg, anti-HBc total e anti-HBs) pelo ensaio imunoenzimático (ELISA). Posteriormente foi formada uma coorte de indivíduos suscetíveis a hepatite B para avaliação da adesão, completude do esquema e resposta vacinal contra hepatite B, utilizando-se um esquema super acelerado. A exposição prévia ao HBV (anti-HBc) foi de 7,0% (IC 95%: 5,22-9,32), e 17,7% (IC 95%: 14,8-20,9) apresentaram positividade isolada para o anti-HBs, sugerindo vacinação prévia contra hepatite B. O consumo de crack por meio de lata improvisada como cachimbo, história de doença sexualmente transmissível e troca de sexo por droga ou dinheiro foram significativamente associados à exposição ao HBV (p< 0,05). Do total de indivíduos que recebeu a primeira dose da vacina contra hepatite B e elegíveis para completar o esquema (n=406), 229 (56,4%) e 96 (26,6%) receberam a segunda e terceira doses, respectivamente. Em somente 23/96 indivíduos foi possível avaliar a resposta vacinal, sendo que 78% responderam com títulos protetores. A frequência elevada de comportamentos de risco, a baixa frequência de indivíduos vacinados e adesão ao esquema vacinal, mesmo com esquema super acelerado, evidencia a necessidade de estratégias de educação em saúde e prevenção que alcancem essa população vulnerável as doenças de transmissão sexual e parenteral como a hepatite B.