Introdução: A evolução dos marcadores sorológicos da hepatite B em pacientes com hepatite B crônica coinfectados com o vírus da imunodeficiência humana (HIV) tem sido pouco documentada. Objetivos: O objetivo deste estudo foi descrever a evolução dos marcadores sorológicos AgHBe e AgHBs, com ênfase na avaliação da frequência de perda definitiva ou transitória desses marcadores, neste grupo de pacientes. Buscamos, também, comparar as variáveis clínicas e demográficas desses pacientes segundo a evolução desses marcadores sorológicos. Pacientes e métodos: A população de estudo foi composta por pacientes atendidos em um ambulatório de referência para atendimento a pacientes infectados pelo HIV em São Paulo, Brasil. Todos os pacientes selecionados eram portadores de HIV e de hepatite B crônica. Foram incluídos nesse estudo pacientes AgHBs positivos, com confirmação da presença desse marcador em, no mínimo, duas sorologias consecutivas, com intervalo mínimo de seis meses entre elas. Variáveis clínicas foram coletadas: idade, sexo, fator de exposição ao HIV/VHB, contagem de células T CD4+, carga viral do HIV, níveis de alaninoaminotransferase (ALT), uso de terapia antirretroviral, incluindo lamivudina, tenofovir ou outras drogas com ação anti-VHB. Resultados: Entre 2.242 pacientes HIV positivos encontrados, foram identificados 105 (4,68%) pacientes com hepatite B crônica. O tempo de seguimento variou de 06 meses a 20,5 anos e o número de coletas variou de 2 a 18 por paciente no período. A maioria dos pacientes era do sexo masculino (97%) e 43,9% (46/105) tinha história de uma ou mais infecções oportunistas. Todos os pacientes tiveram terapia antirretroviral iniciada durante o seguimento. Entre os pacientes com hepatite B crônica, 58% (61/105) eram AgHBe positivos na primeira avaliação. Entre eles, 15% (16/105) apresentaram clareamento de AgHBs e 50% (8/16) dos que clarearam AgHBs apresentaram posterior reativação desse marcador durante a evolução clínica. Dentre os pacientes AgHBe positivos na primeira sorologia, 57% (35/61) apresentaram clareamento desse marcador, sendo que 28,5% (10/35) dos que clarearam AgHBe voltaram a apresentar este marcador durante a evolução clínica. Conclusão: Observamos uma significativa taxa de clareamento e posterior reativação dos marcadores AgHBs e AgHBe no grupo de pacientes avaliados. Estes resultados sugerem que o monitoramento frequente desses marcadores sorológicos deveria ser recomendado / Introduction: Evolution of hepatitis B serological markers among HIV co-infected patients has rarely been documented. Objectives: The aim of this study was to describe the evolution of HBsAg and HBeAg serological markers, with emphasis on the frequency of transient or permanent loss of these markers, among this group of patients. It was also our objective to compare patients\' demographic and clinical variables according to the evolution of these serological markers. Patients and methods: The enrolled patients were selected from those registered at a HIV-Outpatient Clinic in Sao Paulo, Brazil. All included patients were diagnosed with HIV infection and chronic hepatitis B. HBsAg patients who underwent at least two repeated HBV serological testing during clinical follow up , with tests taken at least six months apart, were included in the analysis. Clinical information was collected: age, sex, patient history regarding HIV/HBV transmission, CD4 T+ cell count, HIV viral load, alanine amino transferase (ALT) level, and use of antiretroviral drugs including lamivudine, tenofovir or other anti-HBV drugs. Results: Among 2,242 HIV-positive patients 105 (4.68%) patients were identified with chronic hepatitis B. Follow-up time for these patients varied from 06 months to 20.5 years and the number of serological testing for each patient varied from 2 to 18 along this period. Most patients were male (97%) and 43.9% (46/105) had a history of one or more opportunistic infections. All patients had initiated antiretroviral medication during follow-up. Among patients with chronic hepatitis B, 58% (61/105) were HBeAg reagent at the first assessment. Also, fifteen percent of them (16/105) underwent HBsAg clearance and 50% (8/16) of those who initially lost HBsAg underwent HBsAg reactivation during clinical follow up. Among HBeAg positive patients in the first serology, 57% (35/61) lost this marker during clinical follow up, whereas 28.5% (10/35) of those who initially cleared this serological marker underwent HBeAg reactivation. Conclusion: A significant rate of changes of HBsAg and HBeAg was observed, during clinical follow up among this group of patients. These results suggest that periodic monitoring of HBV serological markers should be recommended

A Região Amazônica é considerada área de alta endemicidade para hepatite B. No Estado do Acre e na capital Rio Branco caracteriza-se um quadro em que jovens e adultos jovens, em pleno período reprodutivo e produtivo para o trabalho e para o estudo, constituem população gravemente acometida pela doença. Assim, procurouse neste estudo a compreensão do modo de vida de pessoas portadoras do HBV, de ambos os sexos, entre 15 e 30 anos, para contribuir no enfrentamento da complexidade desse quadro, através de ações de saúde mais condizentes com as necessidades desses sujeitos. Os métodos qualitativos se mostraram mais apropriados, pois possibilitam a compreensão dos acontecimentos e da forma como os sujeitos vivenciam as experiências. Reconheceu-se no cotidiano, históricooriginal- significativo, uma oportunidade de se descobrir novos caminhos de entendimento para a realidade. Buscou-se os participantes a partir do Serviço de Assistência Especializada, valendo-se de instrumentos de pesquisa exclusivos, utilizados para obtenção de informações socioeconômicas e para nortear as entrevistas com os sujeitos. Foram realizadas doze entrevistas, duas das quais com familiares. Os depoimentos prestados, juntamente com as anotações de campo e os apontamentos da observação participante, além das informações colhidas pelos questionários iniciais, constituíram os registros analisados. A análise do material possibilitou as seguintes categorias: descoberta da doença, contemplando as situações que precipitaram o diagnóstico nos casos assintomáticos e sintomáticos, além das principais informações acerca da enfermidade; doença no cotidiano, abrangendo as percepções quanto aos efeitos adversos do tratamento, as restrições procedentes da doença e/ou do tratamento farmacológico e os cuidados profiláticos; modo de vida, apresentando as estratégias de enfrentamento à doença, modalidades através das quais os sujeitos confrontam-se com as implicações da hepatite B, quais sejam, a religiosidade, o apoio espiritual e familiar, e o planejamento para o futuro, além das relações afetivas no cotidiano, e por último, a categoria preconceito e estigma, realidade ainda presente no contexto de doenças infecciosas. Assinalam-se assim, as compreensões do modo de vida, a partir do cotidiano, de pessoas que vivem com o vírus B, para colaborar no maior entendimento de suas necessidades de saúde, podendo subsidiar estratégias de assistência mais equânimes, vislumbrando a atenção integral desses sujeitos / The Amazon region is considered an area of high endemicity for hep B. In the State of Acre, and in the city of Rio Branco, the reality of teens and young adults, on the heights of their reproductive and work/study productive period, constituting a population severely affected by the disease, is characteristic. Therefore, it\'s been aimed in this study, the comprehension of the way of life of the HBV affected people, both sexes, between 15 and 30 years, to contribute on the understanding of the complexity of this situation., through health actions more appropriate to the needs of these individuals. The qualitative methods were shown more suitable, because they allow the understanding of the events and the way those individuals lived their experiences. It\'s been recognized on the routine, \"historical-original-meaningful\", an opportunity of discovering new paths of understanding for reality. The participants were found on the Specialized Assistance Service, by the use of exclusive research tools, handled for obtaining socioeconomic informations and guiding the interviews with the individuals. Eleven interviews were made, two of them with relatives. The testimonies given, along with the field notes and the pointers of the participant observation, as well as the information gathered by the initial questionnaires, constituted the analyzed records. The material analysis permitted the following categories: disease discovery, covering the situations that hastened the diagnosis on the asymptomatic and symptomatic cases, as well as the main information about the disorder; everyday disease, covering the perceptions around the adverse effects of the treatment, the restrictions derived of the disease and/or farmacologic treatment and the prophylactic care; way of life, presenting the strategies of coping the disease, arrangements which the individuals confront with the implications of Hep B, whichever they are, religiosity, familiar and spiritual support, and planning for the future, as well as the affective relations on routine, and at last, the discrimination and stigma, reality still present on the context of infectious diseases. So, the comprehension of the way of life of people who live with the B virus, based on the routine, is pointed out to colaborate on the greater understanding of their health needs, enabling the allowance of fairer assistance strategies, glimpsing the full attention of these individuals

Orientador: Artênio José Isper Garbin / Banca: Renato Moreira Arcieri / Banca: Paula Caetano Araújo / Resumo: A hepatite B e a AIDS são doenças virais de grandes destaques na saúde pública, devido seus elevados índices epidemiológicos, de morbidade e mortalidade. O estigma criado às doenças virais, principalmente pelo vírus da hepatite B (VHB) e vírus da imunodeficiência humana (HIV), repercute de forma negativa e impactante à vida da pessoa infectada, gerando atitudes discriminatórias e preconceituosas, principalmente no acesso aos serviços de saúde. Entre as duas doenças, a hepatite B é a única que tem a imunoprevenção, a qual é realizada através da vacinação. A imunização contra VHB é a principal forma de prevenção da doença, além de ser uma medida de autocuidado que deve ser preconizado no exercício da odontologia. O objetivo deste estudo foi avaliar o conhecimento e a existência da discriminação, através das atitudes dos graduandos em odontologia, frente à representação da Hepatite B e HIV/AIDS; e verificar a imunização dos graduandos, contra o vírus da hepatite B utilizando o teste imunocromatográfico, para o rastreamento de anticorpos anti-HBs. No primeiro capítulo, o universo amostral do estudo foi composto de todos os alunos de graduação regularmente matriculados (n=525). Foi criado um inquérito semiestruturado e autoaplicável, exclusivamente para este estudo, que versava sobre HIV/AIDS e Hepatite B. No segundo capítulo, foram convidados a participar da pesquisa, todos os alunos que desenvolviam atividades clínicas (n=263). Como instrumento de pesquisa, foi utilizado ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: To assess the knowledge, and the existence of discrimination by the attitudes of academic dentistry when facing HIV / AIDS and hepatitis B. Method ology: This is an exploratory cross - sectional study conducted in a public college of dentistry. The sample consisted of 462 (88%) academics who agreed to participate. In data collection, we used a semi - structured questionnaire, created exclusively for this study, addressing the theme HIV / AIDS and hepatitis B. In the data analysis, we used the chi - square test of proportions, in order to verify the associations between the study variabilities and the academic level. The significance level was 5% (0.050). Re sults: It was found that although 85.5% and 88.7% of the students affirm that they knew or have had some information about AIDS and hepatitis B, only 58.9% and 55.8% respectively, had some knowledge on etiological agents. The statistical differences found in relation to the knowledge of the disease and the success of the etiological agent of AIDS, was evident as in undergraduate students it's been noticed a relevant raise on statistical data but the knowledge of the etiology of hepatitis B was significant at the beginnin g of the course. On the attitudes of students in dental treatment of patients infected with HIV and HBV, 85.3% and 91.8% respectively they said that would accept performing it, however, a considerable part of the students believed that there are difference s in clinical procedures to be followed, with statistical values significant for those who attend the first year of graduation. When asked to participants if they would accept to be treated by a dental surgeon with AIDS or hepatitis B, only 31.4% and 38.7% would consent to do the treatment. Conclusion: We conclude that there are gaps in knowledge of students in relation to AIDS and Hepatitis B. The attitudes of students, compared ... (Complete abstract electronic access below) / Mestre

Yu Chi Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 124-139). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Table of Contents --- p.ii / Abbreviations --- p.iv / Abstract --- p.v / 論文摘要 --- p.vi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction / Chapter 1.2 --- HBV and its potential oncogenic properties / Chapter 1.3 --- Aim of the present study / Chapter 1.4 --- Expressed sequence tag (EST) analysis: an approach to reveal gene expression pattern in a specific tissue / Chapter 1.5 --- cDNA subtraction / Chapter Chapter 2 --- Materials and Methods --- p.17 / Chapter 2.1 --- Plating out the adult human normal liver cDNA library / Chapter 2.2 --- PCR amplification of cloned human normal liver cDNA inserts / Chapter 2.3 --- Cycle sequencing of cloned human normal liver cDNA inserts / Chapter 2.4 --- mRNA preparation from the HCC tissue and its surrounding normal counterpart / Chapter 2.5 --- PCR-Select cDNA subtraction / Chapter 2.6 --- Construction of HCC subtracted cDNA library by T/A cloning method / Chapter 2.7 --- PCR amplification of cloned subtracted cDNA / Chapter 2.8 --- Cycle sequencing of cloned subtracted cDNA / Chapter 2.9 --- Sequence analysis / Chapter 2.10 --- Differential hybridization of HCC subtracted clones / Chapter Chapter 3 --- Results --- p.46 / Chapter 3.1 --- The sequencing results of adult human normal liver cDNA clones / Chapter 3.2 --- Categorization of ESTs sequenced from the adult normal liver / Chapter 3.3 --- Adaptor ligation efficiency analysis / Chapter 3.4 --- Primary and secondary PCR Amplification / Chapter 3.5 --- PCR analysis of subtraction efficiency / Chapter 3.6 --- The sequencing results of subtracted HCC cDNA clones / Chapter 3.7 --- Categorization of ESTs sequenced from the subtracted HCC cDNA library / Chapter 3.8 --- Differential hybridization of subtracted cDNA clones / Chapter Chapter 4 --- Discussions --- p.90 / Chapter 4.1 --- Characterization of the ESTs generated from human normal liver cDNA library / Chapter 4.2 --- EST analysis on subtracted HCC cDNA clones / Chapter 4.3 --- Candidate genes differentially expressed in HCC / Appendix A The coordinates of dot blots (in numerical order according to clone numbers) / Appendix B The coordinates of dot blots (in alphabetical order according to putative identity) / References --- p.124

by Cheung Yuk Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves xx-xxviii). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iii / Table of Content --- p.iv / Abbreviations / for Amino Acids --- p.viii / for Standard Genetic Code --- p.ix / for Units --- p.x / for Prefixes --- p.xi / for Terms commonly used in the report --- p.xii / List of Figures --- p.xiii / List of Tables --- p.xiv / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Epidemiology of Hepatitis B Virus (HBV) --- p.1 / Chapter 1.2 --- Relationship between Hepatitis B Virus and Hepatocellular Carcinoma --- p.2 / Chapter 1.3 --- Brief Description of HBV Genome --- p.2 / Chapter 1.4 --- Possible Roles of HBx in Hepatocellular Carcinoma --- p.4 / Chapter 1.5 --- Novel Interacting Partner of HBx - HBx-lnteracting Protein (XIP) --- p.6 / Chapter 1.6 --- Objective --- p.6 / Chapter Chapter 2 --- Methodology / Chapter 2.1 --- Information of the HBx and XIP Clones --- p.7 / Chapter 2.2 --- "Information of the Expression Vectors (pRSETA, 6xHis-pRSETA and pET8C)" --- p.7 / Chapter 2.3 --- Sub-Cloning of HBx and XIP into Different Vectors --- p.9 / Chapter 2.3.1 --- Design of Primers for Cloning of HBx and XIP into Different Vectors --- p.9 / Chapter 2.3.2 --- Polymerase Chain Reaction (PCR) Protocol --- p.12 / Chapter 2.3.3 --- Enzyme Digestion Reaction Protocol --- p.14 / Chapter 2.3.4 --- Ligation Protocol --- p.16 / Chapter 2.3.5 --- Preparation of Competent Cells --- p.17 / Chapter 2.3.6 --- Transformation --- p.18 / Chapter 2.3.7 --- Gel Extraction Protocol --- p.19 / Chapter 2.3.7.1 --- Life Technologies CONCERT´ёØ Rapid Gel Extraction System --- p.19 / Chapter 2.3.7.2 --- QIAGEN Gel Extraction Kit --- p.20 / Chapter 2.3.8 --- Plasmid Preparation Protocol --- p.22 / Chapter 2.3.8.1 --- Life Technologies CONCERT´ёØ Rapid Plasmid Minipreps --- p.22 / Chapter 2.3.8.2 --- QIAGEN Plasmid Maxi Kit --- p.23 / Chapter 2.4 --- Expression of HBx and XIP in E. coli Strain C41 (DE3) --- p.25 / Chapter 2.4.1 --- Transformation --- p.25 / Chapter 2.4.2 --- Expression of HBx and 6xHis-HBx in E. coli Strain C41 (DE3) --- p.26 / Chapter 2.4.3 --- Expression of XIP in E. coli Strain C41 (DE3) --- p.27 / Chapter 2.5 --- Preparation of Buffers for Chromatography and Circular Dichroism Spectrum Measurement --- p.28 / Chapter 2.6 --- Purification and Refolding of HBx and His-Tagged HBx --- p.28 / Chapter 2.6.1 --- Washing of HBx and His-Tagged HBx Inclusion Bodies --- p.28 / Chapter 2.6.2 --- His-Tagged HBx Purification by Affinity Chromatography --- p.29 / Chapter 2.6.3 --- HBx Purification by Size Exclusion Chromatography --- p.30 / Chapter 2.6.4 --- Refolding of HBx and His-Tagged HBx by Oxidative Dialysis --- p.30 / Chapter 2.7 --- Purification of XIP --- p.33 / Chapter 2.7.1 --- Screening of Chromatographic Conditions for the Purification of XIP --- p.33 / Chapter 2.7.2 --- XIP 1st Step of Purification by Hydrophobic Interaction Chromatography --- p.34 / Chapter 2.7.3 --- XIP 2nd step of Purification by Size Exclusion Chromatography --- p.34 / Chapter 2.8 --- Chemical Denaturation Experiment of HBx and XIP --- p.36 / Chapter 2.8.1 --- Preparation of Urea Buffers for the Chemical Denaturation of HBx --- p.37 / Chapter 2.8.2 --- Preparation of Different GdnHCI Buffer for the Chemical Denaturation of XIP --- p.38 / Chapter 2.8.3 --- Calculation for Chemical Denaturation Experiment --- p.39 / Chapter 2.8.3.1 --- Protein Concentration Calculation --- p.39 / Chapter 2.8.3.2 --- Residual Molar Elipticity Calculation --- p.39 / Chapter 2.8.3.3 --- Free Energy Change (ΔGu) Calculation --- p.40 / Chapter 2.9 --- Two-dimensional Heteronuclear Nuclear Magnetic Resonance (NMR) Experiment --- p.41 / Chapter 2.10 --- Interaction Confirmation between HBx and XIP --- p.42 / Chapter 2.10.1 --- "Transfection of pEGFP, pEGFP-HBx and pEGFP-XIP into HepG2" --- p.42 / Chapter 2.10.2 --- Yeast Two Hybrid System for Confirmation of HBx and XIP Interaction --- p.44 / Chapter 2.10.2.1 --- Preparation of Y187 Competent Cells --- p.44 / Chapter 2.10.2.2 --- Transformation of pGBKT7-HBx and pACT2-XIP into Y187 --- p.45 / Chapter 2.10.2.3 --- β-galactosidase Colony Lift Assay --- p.46 / Chapter Chapter 3 --- "Expression, Purification and Characterization of Hepatitis B Virus X Protein (HBx)" / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.2 --- Construction of Recombinant HBx-pRSETA and 6xHis-HBx-pRSETA Plasmids --- p.48 / Chapter 3.3 --- Expression of 6xHis-HBx in E. coli C41 (DE3) using M9ZB Medium --- p.52 / Chapter 3.4 --- Expression of HBx in E. coli C41 (DE3) using M9ZB Medium --- p.54 / Chapter 3.5 --- Purification and Refolding of 6xHis-HBx Fusion Proteins --- p.56 / Chapter 3.6 --- Purification and Refolding of HBx Proteins --- p.60 / Chapter 3.7 --- Structural Characterization of Refolded HBx --- p.65 / Chapter 3.7.1 --- Introduction --- p.55 / Chapter 3.7.2 --- Experimental Analysis of HBx Secondary Structure --- p.66 / Chapter 3.7.3 --- Chemical Unfolding Experiment of HBx --- p.68 / Chapter 3.8 --- Discussion --- p.70 / Chapter 3.8.1 --- "HBx was Expressed, Purified and Characterized instead of 6xHis-HBx" --- p.71 / Chapter 3.8.2 --- High Concentration of DTT was used to Minimize Formation of HBx Aggregates --- p.72 / Chapter 3.8.3 --- Oxidative Refolding to Ensure Proper Disulfide Bond Formation --- p.73 / Chapter 3.8.4 --- Computational Prediction and Experimental Prediction of Secondary Structure of HBx --- p.75 / Chapter 3.9 --- Concluding Remarks --- p.77 / Chapter Chapter 4 --- "Expression, Purification and Characterization of HBx-lnteracting Protein (XIP)" / Chapter 4.1 --- Introduction --- p.78 / Chapter 4.2 --- Construction of Recombinant XIP-pET8C --- p.78 / Chapter 4.3 --- Expression of XIP in E. coli C41 (DE3) using M9ZB and M9 Mediums --- p.82 / Chapter 4.4 --- Screening of Chromatographic Conditions for the Purification of XIP --- p.83 / Chapter 4.4.1 --- Introduction --- p.83 / Chapter 4.4.2 --- Purification Details --- p.83 / Chapter 4.5 --- Purification of XIP by HiTrap Phenyl HP 5-ml Column --- p.87 / Chapter 4.6 --- Purification of XIP by HiLoad 26/60 Superdex 75 Prep Grade --- p.89 / Chapter 4.7 --- Structural Characterization of XIP --- p.92 / Chapter 4.7.1 --- CD Spectrum --- p.92 / Chapter 4.7.2 --- Chemical Denaturation Experiment of XIP --- p.93 / Chapter 4.7.3 --- Two-Dimensional Heteronuclear Nuclear Magnetic Resonance (NMR) Spectrum of 15N Labeled XIP --- p.95 / Chapter 4.8 --- Discussion --- p.97 / Chapter 4.8.1 --- Purification Method Development --- p.97 / Chapter 4.8.2 --- "Do Different Protein Cosolutes, Protein Stabilizers and Detergents Help XIP to Adopt a Stable Conformation?" --- p.99 / Chapter 4.9 --- Concluding Remarks --- p.101 / Chapter Chapter 5 --- In vivo Studies of HBx and XIP Interactions / Chapter 5.1 --- Investigation of Sub-Cellular Localization of HBx and XIP in Liver Cells --- p.102 / Chapter 5.1.1 --- Introduction --- p.102 / Chapter 5.1.2 --- "Construction of Recombinant HBx-pECFP-C1, HBx-pEGFP-C1, HBx-pEYFP-C1 and XIP-pECFP-C1, XIP-pEGFP-C1, XIP-pEYFP-C1" --- p.103 / Chapter 5.1.3 --- Transfection of pEGFP-C1 HBx and pEGFP-C1 XIP into HepG2 to Find Out HBx and XIP Sub-Cellular Localization --- p.106 / Chapter 5.1.3.1 --- Introduction --- p.107 / Chapter 5.1.3.2 --- Investigation of EGFP Proteins Expression using the Confocal Microscope and the Leica TCS Software --- p.108 / Chapter 5.1.4 --- Discussion and Future Prospects --- p.111 / Chapter 5.2 --- Interaction of HBx and XIP Studied by Yeast Two-Hybrid System --- p.113 / Chapter 5.2.1 --- Introduction --- p.113 / Chapter 5.2.2 --- Construction of Recombinant HBx-pGBKT7 and XIP-pACT2 Plasmids --- p.114 / Chapter 5.2.3 --- Confirmation of HBx and XIP Interaction by Yeast Two-Hybrid System --- p.117 / Chapter 5.2.4 --- Discussion --- p.121 / Chapter Chapter 6 --- Conclusion --- p.123 / Appendix I Sequence of HBx and XIP --- p.I / Chapter II --- Vector Sequences --- p.II / Chapter III --- Vector Maps --- p.VI / Chapter IV --- Electrophoresis Markers --- p.XI / Chapter V --- Agarose Gel Electrophoresis --- p.XII / Chapter VI --- SDS-PAGE Eectrophoresis --- p.XIII / Chapter VII --- Medium for Bacterial Culture --- p.XV / Chapter VIII --- Medium for Cell Culture --- p.XVII / Chapter IX --- Medium for Yeast Culture --- p.XVIII / Chapter X --- Buffers for Yeast Transformation --- p.XIX / Reference --- p.XX

by Ray, Kit Ng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 112-121). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgments --- p.iv / Abbreviations --- p.x / List of Figures --- p.xii / List of Tables --- p.xiv / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatitis B Virus (HBV) --- p.1 / Chapter 1.2 --- Hepatitis B Virus X Protein (HBx) --- p.5 / Chapter 1.2.1 --- The Genomic Structure of HBx --- p.5 / Chapter 1.2.2 --- The HBx Protein Structure --- p.6 / Chapter 1.2.3 --- Subcellular Localization of HBx --- p.7 / Chapter 1.2.4 --- Possible Functions of HBx --- p.8 / Chapter 1.3 --- Etiology of Hepatocellular Carcinoma (HCC) --- p.12 / Chapter 1.4 --- Relationship between HCC and HBx --- p.13 / Chapter 1.5 --- Aims of Study --- p.14 / Chapter 1.6 --- The Basis of Tet-On System --- p.15 / Chapter 1.7 --- The Basis of DNA Microarray --- p.18 / Chapter 1.8 --- The Basis of Two-Dimensional Electrophoresis --- p.20 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of a Tet-On HBx Expressing Cell Model --- p.22 / Chapter 2.1.1 --- Cloning of HBx Gene into pTRE2 Vector --- p.22 / Chapter 2.1.1.1 --- PCR of HBx Gene --- p.22 / Chapter 2.1.1.2 --- Purification of the PCR Product --- p.23 / Chapter 2.1.1.3 --- Restriction Enzyme Digestion --- p.23 / Chapter 2.1.1.4 --- Ligation of HBx into pTRE Vector --- p.24 / Chapter 2.1.1.5 --- Transformation of the Ligation Product into Competent Cells --- p.24 / Chapter 2.1.2 --- Preparation of the Plasmid DNA --- p.24 / Chapter 2.1.2.1 --- DNA Sequencing of the Cloned Plasmid DNA --- p.25 / Chapter 2.1.3 --- Cell Culture of AML12 Cell Line --- p.26 / Chapter 2.1.4 --- Transfection of pTet-On Vector into AML12 Cells --- p.26 / Chapter 2.1.5 --- Selection of the Transfected AML12 Cells by G418 --- p.27 / Chapter 2.1.6 --- Single Clone Isolation --- p.27 / Chapter 2.1.6.1 --- Luciferase Assay for Selection of Highly Inducible Clones --- p.28 / Chapter 2.1.7 --- Second Transfection of pTRE-HBx Plasmid --- p.28 / Chapter 2.1.8 --- Selection of the Transfected Cells by Hygromycin --- p.29 / Chapter 2.1.9 --- Second Single Clone Isolation --- p.29 / Chapter 2.1.10 --- Total RNA Isolation --- p.29 / Chapter 2.1.11 --- DNase I Digestion --- p.30 / Chapter 2.1.12 --- First-Strand cDNA Synthesis --- p.31 / Chapter 2.1.13 --- RT-PCR of HBx Gene --- p.31 / Chapter 2.1.14 --- Northern Blotting --- p.32 / Chapter 2.1.15 --- Preparation of the Probe --- p.33 / Chapter 2.1.16 --- Northern Blot Hybridization --- p.33 / Chapter 2.1.17 --- 3H-Thymidine Incorporation Assay --- p.34 / Chapter 2.1.18 --- Analysis of Cell Cycle by Flow Cytometry --- p.35 / Chapter 2.2 --- Microarray Analysis of Differential Gene Expression upon HBx Induction --- p.35 / Chapter 2.2.1 --- Sample Preparation for Microarray Analysis --- p.35 / Chapter 2.2.2 --- Probe Labelling --- p.36 / Chapter 2.2.3 --- Microarray Hybridization --- p.37 / Chapter 2.2.4 --- RT-PCR of the Candidate Genes --- p.38 / Chapter 2.2.5 --- Northern Blot Analysis of the Candidate Genes --- p.39 / Chapter 2.3 --- Two-Dimensional (2D) Gel Electrophoretic Analysis --- p.40 / Chapter 2.3.1 --- Protein Sample Preparation for 2D Gel Electrophoresis --- p.40 / Chapter 2.3.2 --- First-Dimension Isoelectric Focusing (IEF) --- p.40 / Chapter 2.3.3 --- Second-Dimension SDS-PAGE --- p.41 / Chapter 2.3.4 --- Silver Stain of 2D Gel --- p.42 / Chapter 2.3.5 --- Mass Spectroscopic Analysis --- p.43 / Chapter 2.4 --- Subcellular Localization of HBx --- p.44 / Chapter 2.4.1 --- Cloning of HBx into Green Fluorescent Protein (GFP) Expression Vector --- p.44 / Chapter 2.4.2 --- Transfection of GFP-HBx --- p.44 / Chapter 2.4.3 --- Propidium Iodide (PI) Staining --- p.45 / Chapter 2.4.4 --- Mitochondria Staining --- p.45 / Chapter 2.4.5 --- Subcellular Localization Study using Epi-Fluorescent Microscopy --- p.45 / Chapter 2.5 --- Analysis of Mitochondrial Transmembrane Potential --- p.46 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Construction of Tet-On AML12 Cell Line of HBx Gene --- p.47 / Chapter 3.2 --- Characterization of the HBx-Expressing Cell Model --- p.53 / Chapter 3.2.1 --- 3H-Thymidine Proliferation Assay --- p.53 / Chapter 3.2.2 --- Cell Cycle Analysis --- p.55 / Chapter 3.3 --- Microarray Analysis of Differential Gene Expression Pattern upon HBx Induction --- p.57 / Chapter 3.4 --- Northern Blot Analysis and RT-PCR of the Candidate Genes --- p.65 / Chapter 3.5 --- Differential Protein Expression Pattern under HBx Induction --- p.70 / Chapter 3.6 --- Subcellular Localization of HBx --- p.77 / Chapter 3.7 --- Analysis of Mitochondrial Transmembrane Potential --- p.83 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Conditional HBx-Expressing Cell Model --- p.84 / Chapter 4.2 --- The Effects of HBx in Clone X18 --- p.86 / Chapter 4.2.1 --- Proliferative Effect of HBx --- p.86 / Chapter 4.2.2 --- Deregulation of G2/M Checkpoint by HBx --- p.86 / Chapter 4.3 --- Early Differential Gene Expression due to HBx Induction --- p.88 / Chapter 4.4 --- The Relationship of the Potential Candidate Genes and Cancer Development --- p.90 / Chapter 4.5 --- The Protein Expression Pattern due to HBx Induction --- p.93 / Chapter 4.6 --- The Subcellular Localization of HBx --- p.96 / Chapter 4.7 --- The Possible Involvement of HBx in Mitochondrial Transmembrane Potential --- p.98 / Chapter 4.8 --- Conclusions --- p.101 / Chapter 4.9 --- Future Prospects --- p.104 / Appendix --- p.107 / References --- p.112

Au Chi Chuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 157-169). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Table of Contents --- p.ii / Abstract --- p.v / 論文摘要 --- p.vii / Abbreviations --- p.viii / List of Figures --- p.ix / List of Tables --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Hepatitis B virus and Hepatocellular carcinoma --- p.3 / Chapter 1.3 --- Pathogenesis of HBV related HCC --- p.6 / Chapter 1.4 --- Current screening test and tumor markers --- p.10 / Chapter 1.5 --- Expressed sequence tag (EST) sequencing --- p.13 / Chapter 1.6 --- Aim of the present study --- p.15 / Chapter 1.7 --- Characterization of KIAA0022 --- p.16 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of liver HCC and normal counterpart libraries --- p.19 / Chapter 2.2 --- Plating out the human liver cDNA libraries --- p.19 / Chapter 2.3 --- PCR amplification of clones human liver cancer and the normal counterpart cDNA libraries --- p.21 / Chapter 2.4 --- Cycle sequencing of cloned human liver cancer and the normal counterpart cDNA libraries --- p.21 / Chapter 2.4.1 --- Dye-primer cycle sequencing (Pharmacia) --- p.21 / Chapter 2.4.1.1 --- Using Pharmacia LBKA.L.F. DNA sequencer --- p.21 / Chapter 2.4.1.2 --- Using Li-Cor 4200 Automated DNA sequencer --- p.22 / Chapter 2.4.2 --- Dye-terminator cycle sequencing (Pharmacia) --- p.22 / Chapter 2.5 --- Sequences analysis --- p.23 / Chapter 2.6 --- Cloning of full-length cDNA of KIAA0022 --- p.24 / Chapter 2.6.1 --- Amplification of KIAA0022 gene using PCR --- p.24 / Chapter 2.6.2 --- Purification of the PCR product --- p.25 / Chapter 2.6.3 --- Ligation --- p.25 / Chapter 2.6.4 --- One Shot® TOP 10 Chemical Transformation --- p.25 / Chapter 2.6.5 --- Small-scale preparation of the plasmid DNA --- p.26 / Chapter 2.6.6 --- Large-scale preparation of the plasmid DNA Table of Contents (continued) --- p.26 / Chapter 2.6.7 --- DNA sequencing of the full-length cDNA of KIAA0022 --- p.28 / Chapter 2.7 --- Northern Hybridization --- p.29 / Chapter 2.7.1 --- The Human multiple tissue Northern Blot --- p.29 / Chapter 2.7.2 --- Synthesis of the radiolabeled DNA probe --- p.29 / Chapter 2.7.3 --- Hybridization of the Northern blot --- p.30 / Chapter 2.8 --- Subcellular localization of KIAA0022 by tagging with green fluorescence protein (GFP) --- p.30 / Chapter 2.8.1 --- Amplification and purification of the KIAA0022 gene product --- p.30 / Chapter 2.8.2 --- Restriction enzymes digestion --- p.31 / Chapter 2.8.3 --- DNA ligation --- p.31 / Chapter 2.8.4 --- Preparation of the Escherichia coli competent cells for transformation --- p.31 / Chapter 2.8.5 --- Transformation of the plasmid DNA into competent Escherichia coli cells --- p.32 / Chapter 2.8.6 --- Small-scale preparation of the plasmid DNA --- p.32 / Chapter 2.8.7 --- Large-scale preparation of the plasmid DNA --- p.32 / Chapter 2.8.8 --- DNA sequencing of the cloned plasmid DNA --- p.33 / Chapter 2.8.9 --- Transfection --- p.33 / Chapter 2.8.10 --- Fluorescence microscopy examination --- p.33 / Chapter 2.9 --- Yeast two-hybrid screening assay --- p.34 / Chapter 2.9.1 --- "Cloning of the KIAA0022 gene into the yeast two-hybrid DNA-BD vector, pGBKT7" --- p.34 / Chapter 2.9.2 --- Small-scale transformation of pGBKT7-KIAA0022 plasmid --- p.34 / Chapter 2.9.2.1 --- Preparation of yeast competent cells --- p.34 / Chapter 2.9.2.2 --- Transformation of the pGBKT7-KIAA 0022 plasmid into the yeast strain PJ69-2A --- p.35 / Chapter 2.9.3 --- Screening a pretransformed library by yeast mating --- p.35 / Chapter 2.9.4 --- β -Galactosidase analysis - colony lift filter assay --- p.36 / Chapter 2.9.5 --- Analysis of yeast plasmid inserts using PCR and DNA sequencing --- p.37 / Chapter 2.9.5.1 --- PCR --- p.37 / Chapter 2.9.5.2 --- DNA sequencing --- p.37 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Results of ESTs sequencing in normal counterpart and HCC libraries --- p.38 / Chapter 3.1.1 --- The sequencing results of the normal counterpart cDNA clones --- p.38 / Chapter 3.1.2 --- Sequencing results of the human liver cancer cDNA clones --- p.41 / Chapter 3.1.3 --- The accuracy of the automated sequencing technique --- p.41 / Chapter 3.1.4 --- Catalogue of normal counterpart ESTs --- p.45 / Chapter 3.1.5 --- Catalogue of liver cancer ESTs --- p.47 / Chapter 3.2 --- Identification of genes differentially expressed in HCC using in silico method --- p.115 / Chapter 3.3 --- Sequence analysis of KIAA0022 --- p.121 / Chapter 3.3.1 --- Structural analysis of KIAA0022 --- p.121 / Chapter 3.3.2 --- Homology alignment --- p.122 / Chapter 3.4 --- Tissue distribution and expression profile of KIAA0022 using Northern blot analysis --- p.132 / Chapter 3.5 --- Subcellular localization of the KIAA0022 tagging by green fluorescence protein --- p.134 / Chapter 3.6 --- Yeast two-hybrid screening assay --- p.136 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Large-scale partial cDNA sequencing --- p.138 / Chapter 4.2 --- Characterization of ESTs --- p.139 / Chapter 4.3 --- Identification of genes differentially expressed in liver cancer using Poisson probability --- p.143 / Chapter 4.4 --- Characterization of KIAA0022 --- p.154 / Reference --- p.157 / Appendix --- p.170

Le virus de l'hépatite B (VHB) reste un problème de santé majeur dans le monde malgré la disponibilité du vaccin. Le VHB n’est pas éradiqué par les thérapies actuelles et 240 millions de personnes infectées chroniquement restent à risque de développer une cirrhose du foie et un carcinome hépatocellulaire (CHC).Le VHB est un petit virus hépatotrope doté d'un génome à ADN double brin partiel (ADNrc). Après infection l'ADNrc est converti en ADN épisomal (ADNccc) qui est ensuite organisé en minichromosome viral, qui est le modèle pour la transcription et qui initie la réplication. La protéine de l'hépatite B x (HBx) est recrutée sur l'ADNccc pour initier et maintenir la transcription de l'ADN ccc. HBx cible aussi directement des gènes cellulaires impliqué dans le développement du CHC.Nous avons utilisé une approche ChIP-Seq pour identifier toutes les cibles génomiques de HBx dans les cellules qui répliquent le VHB. Les cibles HBx sont à la fois des gènes codant les protéines et des ARNnc (75 miARN et 34 lncRNA). Nous avons montré que HBx réprimait un sous-ensemble de miARNs qui réguleraient négativement la réplication virale (ex : miR-24) et des miARNs impliqués dans le développement du CHC (ex : miR-21). Parmi les lncARNs ciblés pour HBx, nous avons étudié DLEU2, qui est fortement surexprimé dans l’infection par le VHB et le CHC. Nous avons en outre montré que DLEU2 lie à la fois HBx et l’histone méthyltransférase Ezh2, la sous-unité catalytique du complexe répressif PRC2. L'interaction avec DLEU2 et HBx relie les fonctions Ezh2/PRC2 conduisant à l'activation constitutive d'un sous-ensemble de gènes cibles d'Ezh2 qui sont normalement conservés dans un état réprimé. Nous avons également montré que l’interaction de HBx avec DLEU2 se produisait sur le minichromosome de l’ADNccc où elle stimulait la transcription/réplication du virus. Enfin, nous avons caractérisé par ATAC-Seq les changements d'accessibilité de la chromatine imposés par HBV dans les hépatocytes humains primaires / Hepatitis B virus (HBV) remains a major health problem worldwide despite the availability of the vaccine. No cure is available for the 240 million peoples chronically infected with HBV that are at risk to develop liver cirrhosis and hepatocellular carcinoma (HCC). Viral suppression, achieved by long term treatment with nucleotides analogues (NUCs), impacts on liver fibrosis and prevents liver decompensation but HCC risk is not reduced in the first 5 years of treatment. HBV is a small hepatotropic virus with a partially double strand DNA (rcDNA) genome. After hepatocyte infection the rcDNA is converted into the cccDNA episome that is then organized into a viral minichromosome that is the template for all viral transcripts and initiates replication. The hepatitis B x protein (HBx) is recruited on the cccDNA and is required to launch and maintain cccDNA transcription. HBx has also been shown to directly target cellular genes and this has been related to HCC development.We used a ChIP-Seq approach to determine the full repertoire of HBx genomic targets in HBV replicating cells. HBx targets include both protein coding genes and ncRNA (75 miRNAs and 34 lncRNAs). We showed that HBx represses a subset of miRNAs that would negatively regulate viral replication (i.e. miR-24) and miRNAs involved in HCC development (i.e. miR-21). Among the HBx targeted lncRNAs we focused DLEU2, which is strongly upregulated in HBV infection and HCC. We further showed that DLEU2 binds both HBx the Ezh2 histone methyltransferase, the catalytic subunit of the repressive PRC2 complex. The interaction with DLEU2 and HBx re-wires Ezh2/PRC2 functions leading to the constitutive activation of a subset of Ezh2 target genes that are normally kept in a repressed state. We also showed that HBx interaction with DLEU2 occurs on the cccDNA minichromosome where it boosts HBV transcription/replication. Finally, we characterized by ATAC-Seq HBV imposed changes of chromatin accessibility in primary human hepatocytes

Made available in DSpace on 2015-02-04T21:42:14Z (GMT). No. of bitstreams: 1 Celina Soares.pdf: 585127 bytes, checksum: dbb978516e57361340a9d4e0b3953a00 (MD5) Previous issue date: 2006-04-27 / Esta pesquisa é um estudo transversal, com método quantitativo e análise de prevalência dos dados. Apresenta como objetivo estimar a soroprevalência das hepatites virais B e C em pacientes infectados pelo HIV. A justificativa deste estudo evidencia-se nas atuais doenças sexualmente transmissíveis (HIV e HBV) e de transmissão sanguínea (HIV, HBV e HCV), por protagonizar substancialmente o panorama do enfrentamento mundial em saúde pública. Santos/SP, cidade litorânea e turística, onde está localizado o maior porto marítimo da América Latina, com um grande número de homens que fazem sexo com homens, mulheres trabalhadores do sexo e usuários de drogas injetáveis, além de apresentar população flutuante de turistas, trabalhadores diretos e indiretos na atividade portuária. Neste sentido constrói um perfil humano relevante que caracteriza a elaboração do atual estudo. A coleta de dados foi realizada na população de pacientes matriculados no Centro de Referência em Aids CRAIDS, Santos, SP. A amostra foi constituída por 1438 pessoas, no período de fevereiro de 2004 a fevereiro de 2005. A análise nos prontuários foi realizada selecionando resultados de marcadores sorológicos para as hepatite B (HBsAg e anti-HBs) e hepatite C (anti-HCV), pela técnica de ELISA, além dos exames sorológicos de menor contagem de linfócitos T CD4+ e maior contagem de carga viral para o HIV, considerando as variáveis: sexo, idade, escolaridade, estado civil, forma de contaminação. Os resultados demonstraram que na população dos 1438 indivíduos HIV+, 54.6% eram do sexo masculino e 45.4% do feminino. Em relação à idade, 71% estavam entre 30 e 49 anos e 54.3% com nível de escolaridade no ensino fundamental. A contagem de linfócitos CD4+ revelou 43.7% com níveis abaixo de 200 células /mm³. Considerando a soroprevalência dos marcadores HBsAg, anti-HBs, Anti-HCV e fatores de risco para essas doenças obtivemos uma prevalência global de HBsAg de 7.4% e 23.5% para o anti-HCV. As variáveis que apresentaram associação com o HBV foram: idade, baixos níveis de células T CD4+, homossexualidade masculina e UDI; para HCV: idade, sexo, escolaridade de nível superior e UDI.

mormaeche@dge.gob.pe / Objective: To assess the seroprevalence of hepatitis B virus (HBV), syphilis, and HIV and associated risk factors in pregnant women and their male partners from six indigenous populations of the Peruvian Amazon Basin. Methods: A cross-sectional study was performed in six indigenous populations from the Peruvian Amazon Basin. Blood samples were obtained and tested for HBV (antibodies to the hepatitis B core antigen (anti-HBc) and hepatitis B surface antigen (HBsAg)), for syphilis (rapid plasma reagin and microhemagglutination assay for Treponema pallidum antibodies), and for HIV (ELISA and indirect immunofluorescence test). A survey was also performed to identify associated risk factors. Results: One thousand two hundred and fifty-one pregnant women and 778 male partners were enrolled in the study. The seroprevalence of anti-HBc in pregnant women was 42.06% (95% confidence interval (CI) 39.28–44.85%) and in their male partners was 54.09% (95% CI 50.32–57.86%). The seroprevalence of HBsAg in pregnant women was 2.11% (95% CI 0.78–3.44%) and in their male partners was 3.98% (95% CI 1.87–6.08%). The seroprevalence of syphilis in pregnant women was 1.60% (95% CI 0.86–2.33%) and in their male partners was 2.44% (95% CI 1.22–3.66%). HIV seroprevalence in pregnant women was 0.16% (95% CI 0.02–0.58%) and in their male partners was 0.29% (95% CI 0.04–1.03%). Sexual risk factors were strongly related to blood markers of syphilis and HBV. Conclusions: Hepatitis B was found to be hyperendemic and strongly related to sexual factors, suggesting an important sexual component in the transmission of the disease in the populations studied. Syphilis was found to have an endemicity in pregnant women above the national level and this may be indicative of high mother-to-child transmission. HIV has started to show its presence in indigenous populations of the Amazon Basin and the results suggest the epidemic is concentrated. / Revisión por pares