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MicroRNA profiling of human hepatocytes induced by HBx in hepatocarcinogenesis.January 2009 (has links)
Yip, Wing Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 100-119). / Abstract also in Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgments --- p.v / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma --- p.1 / Chapter 1.1.1 --- Epidermiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.1 / Chapter 1.2 --- Hepatitis B Virus --- p.3 / Chapter 1.2.1 --- The Epidermiology of Hepatitis B Virus Infection --- p.3 / Chapter 1.2.2 --- The Morphology and Genome of Hepatitis B Virus --- p.4 / Chapter 1.2.3 --- HBV Genotypes and Their Significance --- p.8 / Chapter 1.3 --- Hepatitis B Virus X Protein --- p.9 / Chapter 1.3.1 --- HBx Alters Various Signal Transduction Pathways --- p.10 / Chapter 1.3.2 --- HBx Interacts with Various Transcription Factors and Co-activators --- p.12 / Chapter 1.3.3 --- HBx Induces Epigenetic Alterations --- p.14 / Chapter 1.3.4 --- Identification of COOH-terminal Truncated HBx in Liver Tumors --- p.15 / Chapter 1.4 --- MicroRNAs --- p.17 / Chapter 1.4.1 --- Transcriptional Regulation and Biogenesis of MicroRNAs --- p.18 / Chapter 1.4.2 --- MicroRNAs and Cancer --- p.21 / Chapter 1.4.3 --- MicroRNAs and HCC --- p.25 / Chapter 1.5 --- Hypothesis and Aims of the Study --- p.29 / Chapter CHAPTER 2 --- MATERIALS and METHODS --- p.30 / Chapter 2.1 --- Patients --- p.30 / Chapter 2.2 --- Cell Lines --- p.30 / Chapter 2.3 --- Cloning of Various HBx Constructs --- p.32 / Chapter 2.3.1 --- PCR Amplification of HBx Fragments --- p.32 / Chapter 2.3.2 --- Cloning of HBx Fragments into TA-vectos --- p.33 / Chapter 2.3.3 --- Heat Shock Transformation --- p.33 / Chapter 2.3.4 --- Sub-cloning of HBx Fragments into Lentiviral Vectors --- p.34 / Chapter 2.4 --- Generation of Lentivirus --- p.37 / Chapter 2.4.1 --- Lentivirus Infection --- p.37 / Chapter 2.5 --- RNA Extraction --- p.38 / Chapter 2.6 --- Western Blot Analysis --- p.39 / Chapter 2.7 --- MiRNA Microarray --- p.40 / Chapter 2.7.1 --- Cyanine3-pCp Labeling of RNA Samples --- p.40 / Chapter 2.7.2 --- Sample Hybridization --- p.41 / Chapter 2.7.3 --- Microarray Wash --- p.41 / Chapter 2.7.4 --- Array Slide Scanning and Processing --- p.41 / Chapter 2.8 --- Detection of HBx Gene Deletion by PCR --- p.43 / Chapter 2.9 --- Immunohistochemistry --- p.44 / Chapter 2.10 --- Quantitative Real-time PCR --- p.45 / Chapter 2.11 --- Proliferation Assay --- p.47 / Chapter 2.12 --- Cell Cycle Analysis --- p.48 / Chapter 2.13 --- Annexin V Apoptosis Assay --- p.49 / Chapter 2.14 --- Colony Formation Assay --- p.50 / Chapter 2.15 --- Statistical Analysis --- p.51 / Chapter CHAPTER 3 --- RESULTS --- p.52 / Chapter 3.1 --- Detection of Full-length and COOH-terminal Truncated HBx in HCC Tissues --- p.52 / Chapter 3.2 --- Confirmation of HBx Expression in HCC Tissues --- p.55 / Chapter 3.3 --- Comparison of HBx from Different HBV Genotypes for Study --- p.61 / Chapter 3.4 --- Functional Characterization of COOH-tterminal Truncated HBx --- p.64 / Chapter 3.4.1 --- Selection of COOH-terminal Truncated HBx --- p.64 / Chapter 3.4.2 --- Generation of Various HBx-expressing Hepatocyte Cell Lines --- p.66 / Chapter 3.4.3 --- Effect of Full-length and COOH-terminal Truncated HBx on Cell Proliferation --- p.69 / Chapter 3.4.4 --- Effect of Full-length and COOH-terminal Truncated HBx Cell Cycle --- p.34 / Chapter 3.4.5 --- Effect of Full-length and COOH-terminal Truncated HBx on Apoptosis --- p.45 / Chapter 3.5 --- MicroRNA Profiling of Various HBx-expressing Hepatocyte Cell Lines --- p.76 / Chapter 3.5.1 --- Identification of Deregulated MicroRNAs by Microarray --- p.76 / Chapter 3.5.2 --- Validation of Deregulated MicroRNAs by Real-time PCR Analysis --- p.80 / Chapter 3.5.3 --- Confirmation of Deregulated MiRNAs in HCC and Adjacent Non-tumor Tissues --- p.84 / Chapter 3.5.4 --- Potential Downstream Targets of the HBx-deregulated MiRNAs --- p.87 / Chapter CHAPTER 4 --- DISCUSSION --- p.91 / Chapter 4.1 --- The Impact of COOH-terminal Truncated HBx in HCC --- p.91 / Chapter 4.2 --- The Biological Significance of COOH-terminal Truncated HBx Induced MiRNAs --- p.94 / Chapter 4.3 --- Limitations of the Present Study --- p.97 / Chapter 4.4 --- Future Studies --- p.98 / Chapter CHAPTER 5 --- CONCLUSION --- p.99 / REFERENCES --- p.100
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Development of a Non-Invasive Electrode for Intradermal Electrically Mediated DNA VaccinationDonate, Amy Lynn 01 January 2011 (has links)
Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use against a wide variety of diseases. Alone, this vaccine technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity against the expressed antigen. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. This study evaluated the use of the MEA for the development of DNA vaccines. We assessed the appropriate delivery conditions for gene expression and the development of humoral immunity. We used both B. anthracis and HBV as infectious models for our experiments. Our results indicated that the MEA can enhance gene expression in a mouse model with minimal to no tissue damage. Optimal delivery conditions, based on generation of antibodies, were determined to be 125-175V/cm and 150ms with 200ug and a prime boost protocol administered on Day 0 and 14. Under these conditions, end-point titers of 20,000-25,000 were generated. Neutralizing antibodies were noted in 40-60% of animals.
Additionally, we utilized a guinea pig model to assess the translation potential of this electrode. The plasmid encoding HBsAg, pHBsAg, was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in the experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to the injection only groups. Results from both experimental models demonstrate that protective levels of humoral immunity can be generated and that this electrode should translate well to the clinic.
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Development of molecular diagnostic system for detection of hepatitis B virus in blood donationsFun, Sze-tat., 范思達. January 2003 (has links)
published_or_final_version / Medical Sciences / Master / Master of Medical Sciences
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Perfil das mutações de resistência do vírus da Hepatite B aos análogos de nucleos(t)ídeos entre pacientes com hepatite B crônicaSantos, Maria Isabel Magalhães Andrade dos January 2014 (has links)
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Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Introdução: A doença causada pelo vírus da hepatite B (HBV) é um problema de saúde pública mundial. No Brasil, o sistema único de saúde (SUS) tem disponibilizado drogas antivirais para o tratamento de hepatite B crônica há mais de 10 anos, mas um sistema para o monitoramento e avaliação de resistência a estas drogas ainda não está disponível. Objetivo: Este estudo teve como objetivo determinar o perfil de mutações do HBV associadas com a resistência aos análogos de nucleos(t)ídeos entre 81 pacientes com infecção crônica pelo HBV: virgens de tratamento para hepatite B e tratados com diferentes análogos de nucleosídeos e nucleotídeos, no Hospital Professor Edgar Santos (HUPES-UFBA)- Salvador-BA. Metodologia: O HBV-DNA foi isolado de amostras de soro, amplificado por nested-PCR, utilizando-se primers deduzidos da região flaqueadora da domínio rt do gene P e sequenciados (ABI Prism 3730, Applied Biosystems, EUA). Duas a seis sequências de cada isolado foram alinhados e os sítios conflitantes foram resolvidos usando o software CLC Main Workbench v. 5.0 por inspeção visual dos eletroferogramas. As sequências consenso tinham um tamanho de 1032 pb (compreendendo os aminoácido 1-344 da rt). Estas sequências foram submetidas ao banco de dados HBVrt DB (Stanford University, EUA) para a análise de cada mutação de acordo com o genótipo e tratamento. Resultado: O genótipo A1 foi o mais prevalente (85,2%) seguido pelo genótipo A2 (4,9%) F (6,2%) e C1, D2 e D4 (1,2% cada). Seis pacientes (7 %) apresentaram mutações de resistência para LAM, ETV, TDF: dois com o padrão L180M + M204V e quatro com padrões diversos (L80I + L180M + M204I ;L80V + L180M + M204V; M204I; A194T). Todas estas mutações foram associadas ao genótipo A (quatro A1 e dois A2). Além disso, foi encontrado um paciente com HBV genótipo C típico do leste da Ásia. Destes pacientes, dois foram virgens de tratamento e quatro tinham histórico de tratamento para HIV ou HBV. Foram detectadas quatro mutações no gene S (três casos com a mutação sI195M e um a mutação sW196L) associadas às mutações do domínio rt do gene P, correspondendo à uma taxa de 6% de mutações de escape vacinal. A prevalência das mutações de resistência às drogas antivirais variou de acordo com a duração do tratamento e com o nível da barreira genética da droga utilizada. Neste estudo, foi encontrada uma forte associação entre a ocorrência de mutações de resistência do HBV e positividade para o AgHBe, co-infecção com o HIV e histórico de tratamento para HBV e/ou HIV. Conclusão: Antes da terapia ser iniciada é extremamente importante o monitoramento da carga viral e a identificação destas mutações para suportar decisões clinicas sobre o manejo dos pacientes e prevenir a emergência de vírus multi- resistentes. / Introduction: Hepatitis B virus (HBV) infection is a public health issue. The Brazilian public health system (SUS) has provided antiviral drugs for chronic hepatitis B treatment for over 10 years, but a system for monitoring for drug-related resistance mutations is not available. Objective: This study aims to determine the presence of HBV mutations associated with resistance to nucleos(t)ide analogs among 81 patients with chronic HBV infection-naïve and treated from University Hospital Professor Edgard Santos, Salvador-BA (HUPES-UFBA). Methods: Briefly, HBV-DNA was PCR amplified with primers deduced from the flanking of the rt domain at the HBV P gene and sequenced using ABI Prism 3730 (Applied Biosystems, USA). From two to six forward and reverse sequences of each isolate were assembled and conflicting sites were resolved using software CLC Main Workbench v. 5.0 by visual inspection of the electropherograms. Consensus sequence extended 1032 bp and encompassed the entire rt domain (from amino acid 1 to 344). Those sequences were submitted to the HBV drug resistance database (HBVrt DB, Stanford University, USA) to retrieve each mutation according to genotype and treatment. Results: HBV genotype A1 (85.2%) was the most prevalent followed by genotype A2 (4.9%), F (6.2%), and C1, D2 and D4 (1.2% each). Six patients (7%) exhibited resistance mutations to LAM, ETV and TDF: two with patterns L180M + M204V and four with other different patterns: L80I + L180M + M204I; L80V + L180M + M204V; M204I; A194T. All of these mutations were present in patients with genotype A (four A1 and two A2). Furthermore, this study found one patient with genotype C, common in East Asian. Of these patients, two were naïve and four had a history of treatment for HIV or HBV. In addition, four mutations in gene S (sI195M three cases with the mutation and one with the mutations W196L) associated with mutations in the rt domain of the P gene were detected, corresponding to a rate of 6% of vaccine escape mutations. Prevalence of drug-related resistance mutations varied according to treatment duration and the level of genetic barrier for the drugs used. Conclusion: In this study a strong association was found between the occurrence of HBV resistance mutations and HBeAg positivity, co-infection with HIV and a history of treatment for HBV and / or HIV. Once the drug therapy is initiated it is extremely important to monitor viral load and identify those mutations in order to support clinical decisions about patient management and also to prevent the emergence of multidrug-resistant viruses.
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Avaliação de mutações de resistência ao tratamento com análogos de nucleos(t)ídeos e de escape vacinal do vírus da hepatite B (VHB) em pacientes com hepatite crônica.Pacheco, Sidelcina Rugieri January 2016 (has links)
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Previous issue date: 2016 / CAPES / CNPq / Fundação Gonçalo Moniz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / INTRODUÇÃO: A hepatite B (VHB) é uma infecção dinâmica crônica, que apesar de existir programas de imunização e tratamento antiviral disponível, existe o risco de emergência de mutações de resistência aos análogos de núcleos(t)ídeos (AN) que devem ser rastreadas, devido as suas implicações clínicas. O Brasil disponibiliza pelo SUS cinco drogas para o tratamento antiviral: IFN, LAM, ADF, ETV e TDF e um guia de conduta clínica para orientar o tratamento no território nacional, o Protocolo de Diretrizes Terapêuticas para Hepatite B e co-infecções. OBJETIVO: O objetivo do presente estudo foi avaliar as mutações de resistência aos AN, mutações de escape vacinal e genótipos circulantes em pacientes com hepatite B crônica em dois centros de referencia em Hepatites, na Bahia (região Nordeste) e no Acre (região Norte) do Brasil. MATERIAL E MÉTODOS: Foi utilizadas ferramentas de biologia molecular e bioinformática, através de nested PCR e sequenciamento direto das amostras, para rastrear as mutações de resistência, a região alvo foi a transcriptase reversa (RT) do gene P e as mutações de escape vacinal foi a região do gene S do VHB, como também os genótipos e subgenotipos do VHB. RESULTADOS: Foram incluídos 527 pacientes durante o período de 2011-2015, sendo 320 pacientes do HUPES/BA e 207 do FUNDHACRE/AC. Os pacientes que representam a região Nordeste foram 59,3 % do sexo masculino e uma média de idade de 44,75±12,4 DP, os pacientes da região Norte 42% foram do sexo masculino e a média de idade foi de 40,36±13,9 DP. Todos os pacientes incluídos apresentaram AgHBs persistente por mais de seis meses e 86,1% apresentaram AgHBe negativo. Foram sequenciadas 296 amostras dos pacientes com VHB crônica. Foram encontradas mutações de resistência aos AN na Região Norte 1,2% (2), Região Nordeste 7,4%(8) e no global 3,8%(20). Os padrões de mutações de resistência primária encontrados foram: rtA194T, (3) rtL180M+M204V, rtL180M+M204I, rtS202I, rtM204I, rtA181S, rtA181E e rtA184S. Em relação ao escape vacinal a frequencia para a Região Norte foi de 7,1% (11), Região Nordeste 8,4% (9) e no global 7,6% (20). Nos pacientes virgens de tratamento (n=189), a frequência de mutações de resistência foi de 6%, somente nas amostras da região Nordeste. Não houve diferença estatisticamente significante entre o grupo com ou sem mutação dos pacientes virgens de tratamento. Não foram encontradas mutações de resistência nas amostras da região Norte. Os genótipos circulantes nas duas regiões foram A, D e F, e a região Nordeste foi encontrada o genótipo C (C2). CONCLUSÃO: Os resultados demonstram a importância de rastrear e monitorar as mutações de resistência aos AN e de escape vacinal devido a importância epidemiológica e clínica na conduta terapêutica. / INTRODUTION: Hepatitis B virus (HBV) is a chronic dynamic infection, which although there immunization programs and antiviral therapy available, there is a risk of emergence of resistance mutations cores analogs (t) ide to be screened, because of their implications clinics. The Brazil offers the SUS five drugs for antiviral treatment: IFN, LAM, ADF, ETV and TDF and clinical guide of conduct to guide treatment in the country, the Therapeutic Guidelines Protocol for Hepatitis B and co-infections. AIM: The aim of this study was to evaluate the resistance mutations core analogues (t) ide, vaccine escape mutations and circulating genotypes in patients with chronic hepatitis B in two reference centers in Hepatitis, Bahia (Northeast) and Acre (Northern region) of Brazil. MATERIAL AND METHODS: Was used tools of molecular biology and bioinformatics by nested PCR and direct sequencing of samples to track resistance changes, the target region is the reverse transcriptase (RT) P gene and vaccine escape mutations was region of the gene S of HBV, as well as the HBV genotypes and subgenotipos. RESULTS: 527 patients were included during the period 2011-2015, with 320 patients HUPES / BA and 207 FUNDHACRE / AC. Patients representing the Northeast were 59.3% male and an average age of 44.75 ± 12.4 PD patients in the northern region 42% were male and the average age was 40, 36 ± 13.9 DP. All patients had persistent HBsAg for more than six months and 86.1% were HBeAg negative. We were sequenced 296 samples from patients with chronic HBV. the cores of similar resistance mutations were found (t) ide in the North 1.2% (2), Northeast 7.4% (8) and 3.8% overall (20). The patterns of primary resistance mutations were: rtA194T (3) rtL180M + M204V, M204I + rtL180M, rtS202I, rtM204I, rtA181S, and rtA181E rtA184S. Regarding vaccine escape the frequency for the Northern Region was 7.1% (11), Northeast 8.4% (9) and the global 7.6% (20). In treatment-naïve patients (n = 189), the frequency of resistance mutations was 6%, only the samples in the Northeast. There was no statistically significant difference between the groups with or without mutation of naive patients. There were no resistance mutations in samples from the North. Circulating genotypes in the two regions A, D and F, and the Northeast found the C genotype (C2). CONCLUSION: The results demonstrate the importance of tracking and monitoring the resistance mutations similar cores (t) ide and vaccine escape due to epidemiological and clinical importance in the therapeutic approach.
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The histone chaperone HIRA is crucial for the early establishment of hepatitis B virus minichromosome / La chaperone d'histones, HIRA, est essentielle dans l'établissement précoce du minichromosome du virus de l'hépatite BLocatelli, Maëlle 18 September 2018 (has links)
Le virus de l'hépatite B (HBV) infecte de manière chronique 240 millions de personnes dans le monde, et est la principale cause de carcinome hépatocellulaire. Actuellement, les traitements standards permettent une suppression virale à long terme, mais ne sont pas capables d'éliminer complètement le virus, en raison de la persistance de l'ADN circulaire et clos de façon covalente (ADNccc). Ce minichromosome viral réside dans le noyau des hépatocytes infectés, grâce à sa structure chromatinienne. En effet, lors de l'infection d'un hépatocyte, l'ADN viral partiellement double brin (ADN relâché circulaire (rc)) est libéré dans le noyau, où il est réparé et enveloppé par des protéines histones, afin de former une structure d'épisome chromatinisé. Les mécanismes conduisant à la formation ainsi qu'à la chromatinisation de l'ADNccc sont encore largement inconnus, et leur élucidation constituerait une première étape vers l'identification de nouvelles cibles thérapeutiques, susceptibles d'altérer la persistance de l'ADNccc. Dans ce but, nous avons étudié le rôle des facteurs hôtes de réparation de l'ADN, et des voies d'assemblage des nucléosomes, dans la formation de l'ADNccc, à des stades précoces (entre 30 minutes et 72 heures) de l'infection, dans des lignées cellulaires d'HepG2-NTCP, ainsi que dans des hépatocytes primaires humains. Nous nous sommes particulièrement concentrés sur la protéine chaperone d'histones, HIRA, qui est connue pour déposer le variant d'histone 3.3 (H3.3) sur l'ADN cellulaire d'une manière indépendante de la réplication et en association avec le remaniement des nucléosomes pendant la transcription et la réparation de l'ADN. Nous avons été capables de détecter l'ADNccc dans la fraction nucléaire des hépatocytes dès 30 minutes et 24 heures post-infection, par qPCR et Southern Blotting (SB), respectivement. L'extinction de HIRA par ARN interférent (siARN) avant l'inoculation du virus, a conduit à une forte diminution de l'accumulation de l'ADNccc (à la fois par qPCR et Southern Blot), qui était indépendante de la protéine HBx (en utilisant un virus HBx-défectueux). Les niveaux d'ADNrc sont restés stables, indiquant soit une éventuelle transition de l'ADNrc en ADNccc incomplète, ou retardée. L'analyse par immunoprécipitation de la chromatine a montré que HIRA était liée à l'ADNccc dès 30 minutes après infection, et que son recrutement était concomitant avec le dépôt de l'histone H3.3, ainsi que la liaison de la protéine de capside du HBV (HBc). Après 24 heures d'infection, une augmentation de la liaison de H3.3 et de l'ARN polymérase II sur l'ADNccc a été observée, en corrélation avec l'initiation de la transcription de l'ARN viral de 3.5 kb. Par des expériences de co-immunoprécipitation et de test de proximité entre protéines (PLA), nous avons montré que HIRA était capable d'interagir avec HBc dans des hépatocytes infectés et dans une lignée cellulaire HepaRG exprimant HBc de manière inductible. En conclusion, nos résultats suggèrent que la chromatinisation de l'ADN viral entrant est un événement très précoce, nécessitant l'histone chaperone HIRA. Bien que HBx ne soit pas requis pour ce processus, HBc pourrait jouer un rôle majeur, suggérant que l'interaction entre HIRA et HBc pourrait représenter une nouvelle cible thérapeutique à étudier / Hepatitis B virus (HBV) chronically infects 240 million people worldwide and is the major cause of hepatocellular carcinoma (HCC). Currently standard-of-care treatments can achieve longterm viral suppression, but are not able to completely eliminate the virus, due to the persistence of the covalently closed circular DNA (cccDNA). cccDNA, the viral minichromosome, resides in the nucleus of infected hepatocytes by virtue of its chromatin structure. Indeed, upon entry into hepatocytes, the partially double stranded viral DNA (relaxed circular (rc)DNA) is released into the nucleus, where it is repaired and wrapped by histones to form an episomal chromatinized structure. The mechanisms leading to cccDNA formation and chromatinization are still largely unknown and their elucidation would be a first step toward the identification of new therapeutic targets to impair cccDNA persistence. To this aim, we investigated the role of host factors belonging to DNA repair and nucleosome assembly pathways in cccDNA formation at early time points (i.e. between 30 minutes and 72 hours) post-infection in both HepG2-NTCP cell line and Primary Human Hepatocytes (PHH). We particularly focused on the histone chaperone Hira, which is known to deposit histone variant 3.3 (H3.3) onto cellular DNA in a replication-independent manner and in association to nucleosome reshuffling during transcription and DNA repair. We were able to detect cccDNA in the nuclear fraction of hepatocytes as early as 30 minutes and 24h post-infection, by qPCR and Southern Blotting (SB), respectively. Knock-down of Hira by RNA interference before virus inoculation led to a strong decrease in cccDNA accumulation (both in qPCR and SB) which was independent from HBx protein expression (using an HBx defective virus). rcDNA levels remained stable, indicating either a possible incomplete or delayed rcDNA to cccDNA transition. Chromatin Immunoprecipitation analysis showed that Hira was bound to cccDNA already at 2 hours post-infection and that its recruitment was concomitant with the deposition of histone H3.3 and the binding of HBV capsid protein (HBc). After 24 hours of infection, an increase of H3.3 and Pol2 binding on cccDNA was observed, correlating with the initiation of the transcription of the 3.5 kb RNA. By Co-Immunoprecipitation and Proximity Ligation Assay experiments, we showed that Hira was able to interact with HBc in infected hepatocytes and in a HepaRG cell line expressing HBc in an inducible manner. Altogether, our results suggest that chromatinization of incoming viral DNA is a very early event, requiring the histone chaperone Hira. While HBx is not required for this process, HBc could play a major role, suggesting that the interaction between Hira and HBc could represent a new therapeutic target to be investigated
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A multipathogen vaccine for rabies, hepatitis B, Japanese encephalitis and enterovirus 71Lauer, Katharina January 2016 (has links)
To enhance the global control of encephalitis and hepatitis caused by rabies virus (RABV), Japanese encephalitis virus (JEV), enterovirus 71 (EV71) and hepatitis B virus (HBV), novel immunisation strategies are needed. All four diseases particularly affect low income countries with marginal health services – an affordable combined vaccine strategy could alleviate the large burden of disease. Therefore, we aimed to construct a multipathogen vaccine assessing the immunising activity of a recombinant modified vaccinia Ankara (MVA), expressing key antigens (RABV-glycoprotein, JEV pre-membrane & envelope protein, EV71-P1 protein and large hepatitis B surface antigen) from the various pathogens. Successful delivery of the pathogen sequences into non-essential sites (deletion site I, II, VI) of MVA via homologous recombination with a transfer plasmid, was demonstrated by transient color selection (LacZ-marker) in vitro. The stable insertion of the expression cassettes was validated over ten virus passages by PCR with specific primer sets, targeting the pathogen sequence. Two recombinants, one carrying the EV71 and JEV pathogen sequence and one carrying the RABV-HBV pathogen sequence were generated and validated by PCR.To ensure similar expression of the key antigens, a T7-promoter was linked to the expression cassettes of all pathogen sequences. Direct regulation of this promoter was achieved through co-infection with a second T7-polymerase expressing MVA under the control of a vaccinia p7.5 promoter. Protein expression from recombinant MVA using the co-infection model of expression in vitro, was further characterised by Western blot, dot blot and immunocytochemistry. All inserted transgenes were expressed using an avian (chicken embryo fibroblasts) or mammalian (human fetal lung fibroblasts) cell culture system. To investigate the co-infection model of antigen delivery in vivo, a pilot murine immunogenicity study was performed in six Balb/c mice using the MVA-RABV-HBV recombinant in a homologous prime-boost regimen two weeks apart. To detect antibodies against the expressed pathogen sequences in the mouse serum an antibody-capture assay was performed (Western blot, dot blot). The antigen (used to capture murine antibodies) was purified RABV-glycoprotein or large hepatitis B surface antigen expressed from a baculovirus. The murine antibodies were detected by a secondary anti-mouse antibody, conjugated with horseradish peroxidase for a chemiluminescent reporter assay. Although, serum antibodies against MVA were induced in all mice, no serum antibodies against RABV or HBV could be detected. In summary, we were able to demonstrate that two transgenes, when inserted into one or two different loci in the MVA genome, can be expressed in vitro when using the co-infection model of gene expression with a T7-expression system. This project has provided new insights into a novel group of vaccines, the multipathogen viral vector vaccines, employing MVA as a vector. Future studies will be needed to further explore this vaccine-group, as well as the co-infection model of expression.
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Impact des facteurs de restriction sur la réplication du virus de l'hépatite B / Impact of restriction factors on hepatitis B virus replicationHallez, Camille 25 September 2017 (has links)
Le Virus de l'Hépatite B (VHB) infecte 350 millions d'individus à l'échelle mondiale. Il est responsable d'hépatites aigües pouvant évoluer vers la chronicité puis le carcinome hépatocellulaire. Le génome du VHB est constitué d'un ADN partiellement bicaténaire. De par sa nature, il pourrait être sensible à l'action de certaines nucléases cellulaires qui hydrolysent l'ADN double brin. Nous avons ainsi mis en évidence la capacité de la Désoxyribonuclase I (DNase I) à être incorporée dans les virions du VHB, ce qui permet la dégradation du génome viral et la diminution de son infectivité. La DNAse I est particulièrement surexprimée en hypoxie et pourrait contribuer à l'élimination du virus chez les individus cirrhotiques. Par ailleurs, nous avons montré que la cytidine désaminase APOBEC3DE appartenant à une famille de facteurs de restriction viraux possède un rôle proviral. En effet, son association avec APOBEC3F et APOBEC3G mène à une diminution de l'activité de ces dernières et ceci favorise la réplication du VHB. La formation d'hétérodimères APOBEC3DE/APOBEC3F et APOBEC3DE/APOBEC3G semble génèrer un encombrement stérique ne permettant pas l'encaspidation d'APOBEC3F et APOBEC3G, raison pour laquelle le génome du VHB est moins muté lorsqu'APOBEC3DE est exprimée. / Hepatitis B Virus (HBV) infects 350 millions people worldwilde. It triggers accute hepatitis that can turn into cirrhosis then hepatocellular carcinoma. HBV genome is composed of a partially double-stranded DNA.Thus, it could be targeted by some cellular nucleases that hydrolyze double-stranded DNA. We have highlighted that Deoxyribunuclease I (DNase I) can be incorporated into HBV virions and degrade its genome, leading to a loss of viral infectivity. Moreover, DNase I is upregulated under hypoxia which is a caracteristic of liver cirrhosis. DNase I could be involved in HBV elimination in cirrhotic patients. In an other study, we found that APOBECDE, a cytidine deaminase of the same family than some restriction factors, has a proviral activity. Indeed, association of APOBEC3DE with APOBEC3F or APOBEC3G leads to a loss of cytidine deaminase activity and a better viral replication. When APOBEC3DE is associated with those two proteins, APOBEC3F and APOBEC3G cannot be incorporated into HBV virions. This is the reason why HBV is more infectious when APOBEC3DE is expressed.
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Prevalência de marcadores sorológicos dos vírus das Hepatites B (VHB) e C (VHC) em indivíduos infectados por Schistosoma mansoni no bairro Santa Maria, Aracaju/SE / PREVALENCE OF SEROLOGICAL MARKERS OF HEPATITIS B VIRUS (HBV) AND C (HCV) IN INDIVIDUALS INFECTED WITH SCHISTOSOMA MANSONI IN THE NEIGHBORHOOD SANTA MARIA, ARACAJU, STATE OF SERGIPE.Santos, Márcio Bezerra 19 July 2012 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Schistosomiasis is a parasitic disease, severe, chronic waterborne and development, whose etiologic agent is Schistosoma mansoni. It occurs in 74 countries with 207
million people infected and 700 million in risky areas. In Brazil, data indicate a prevalence of eight million. The Schistosomiasis can be exacerbated when patients are
carriers of the Hepatitis B Virus (HBV) and C (HCV), resulting in simultaneous evolution of both pathologies. In Brazil, the prevalence of co-infection with HBV/HCV
and S. mansoni found in studies ranged from 13.6% to 40% for HBV and 0.5% to 19.66% for the HCV. On this basis, this research aimed to identify the prevalence of
serological markers of HBV and HCV and risky factors in individuals carrying the S. mansoni in the Santa Maria neighborhood, Aracaju, state of Sergipe. We conducted a
cross-sectional epidemiological study. Data were collected for each patient by means of a questionnaire investigation. This questionnaire included variables identifying the
subject of the research participants and variables that included the epidemiological risky factors for infection with HBV or HCV. We collected serum samples of research
participants and forwarded to the laboratory testing to identify serological markers of hepatitis B and C: Total Anti-HBc IgG, Anti-HBs, HBsAg and Anti-HCV. All
procedures performed using the technique of chemiluminescence immunoassay using the ARCHITECT assay for each serological marker following the protocols established by the laboratory equipment manufacturer SYSTEM ARCHITECT. In addition, we carried out the analysis of spatial distribution of co-infection in the district through the program using the TerraView Kernel intensity estimation. It was found that 16 individuals had contact with HBV (9.41%), one of these was positive for HBsAg. Only Thirty-two samples (18.82%) were positive for the marker Anti-HBs. Three samples were positive for anti-HCV (1.76%), and also a positive for Anti-HBc. The main risks of HBV and HCV infection were related to parenteral interventions of health services, as well as sexual activity without condom use in the case of HBV. Spatial analysis of
cases of co-infection (Schistosomiasis and Hepatitis) allowed the visualization of areas of higher concentration of these infections, as well as those that are exposed to different degrees of risk of transmission. The survey results allow to offer, the municipal health services, a tool to facilitate the understanding of the spatial distribution of
schistosomiasis and hepatitis (B and C) in Santa Maria neighborhood. Although our values are above the estimated prevalence for the Brazilian population and the
Northeast, we can t infer that the individuals with Schistosomiasis are more susceptible to infection with HBV or HCV, since the risky factors were the means of risky transmission of causative agents of hepatitis and not infected with S. mansoni. / A Esquistossomose Mansônica é uma doença parasitária grave, de veiculação hídrica e evolução crônica, cujo agente etiológico é o Schistosoma mansoni. Ocorre em 74 países, com 207 milhões de pessoas infectadas e 700 milhões em áreas de risco. No Brasil, dados indicam uma prevalência de oito milhões. A Esquistossomose pode ser agravada quando os pacientes são portadores dos Vírus das Hepatites B (VHB) e C (VHC), resultando na evolução simultânea de ambas as patologias. No Brasil, As prevalências da co-infecção VHB/VHC e S. mansoni encontradas nos estudos realizados variaram de 13,6% a 40% para o HBV e de 0,5% a 19,66% para o HCV. Com base nisso, esta pesquisa objetivou identificar a prevalência de marcadores sorológicos do VHB e VHC
e os fatores de risco em indivíduos portadores do S. mansoni no Bairro Santa Maria, Aracaju/SE. Foi realizado um estudo epidemiológico do tipo transversal. Coletaram-se dados de cada paciente por meio de um questionário investigativo. Este questionário contemplou variáveis de identificação do sujeito participante da pesquisa e variáveis
epidemiológicas que incluíram os fatores de risco para infecção pelo HBV ou HCV. Foram coletadas alíquotas de soro dos participantes da pesquisa e encaminhadas para a
realização das análises laboratoriais para identificação de marcadores sorológicos dos Vírus das Hepatites B e C: Anti-HBc Total IgG, Anti-HBs, HBsAg e Anti-HCV. Todos
os procedimentos realizados utilizaram a técnica de Imunoensaio Quimioluminescente através do Ensaio ARCHITECT para cada marcador sorológico seguindo os protocolos de análises laboratoriais estabelecidos pelo fabricante do equipamento ARCHITECT SYSTEM. Além disso, realizou-se a análise espacial da distribuição da co-infecção no bairro através do programa TerraView utilizando o estimador de intensidade Kernel. Constatou-se que 16 indivíduos tiveram contato com o HBV (9,41%), desses um foi positivo para HBsAg. Apenas Trinta e duas amostras (18,82%) foram positivas para o marcador Anti-HBs. Três amostras foram positivas para Anti-HCV (1,76%), sendo uma também positiva para Anti-HBc. Os principais riscos de infecção pelo HBV e HCV foram relacionados às intervenções parenterais dos serviços de saúde, assim como à atividade sexual sem uso de preservativo, no caso do HBV. A análise espacial dos casos
de co-infecção (Esquistossomose e Hepatite) permitiu a visualização de áreas de maior concentração dessas infecções, assim como as que são expostas a diferentes graus de risco de transmissão. Os resultados da pesquisa possibilitam oferecer, aos serviços municipais de saúde, um instrumento que facilite a compreensão da distribuição
espacial da Esquistossomose e Hepatites (B e C) no bairro Santa Maria. Embora nossos valores sejam acima da prevalência estimada para a população brasileira e da região
nordeste, não podemos inferir que os indivíduos portadores da Esquistossomose são mais susceptíveis à infecção pelo HBV ou HCV, uma vez que, os fatores de risco foram as vias de risco de transmissão de agentes causadores de hepatite e não a infecção pelo S. mansoni.
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Infecção oculta pelo virus da hepatite B em pacientes hemodialisados e em pacientes infectados pelo virus da imunodeficiencia humana / Occult hepatitis B infection in dialysis patients and in HIV infected patientsJardim, Ruth Nogueira Cordeiro de Moraes 23 August 2006 (has links)
Orientadores: Fernando Lopes Gonçales Junior, Neiva Sellan Lopes Gonçales / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T15:36:18Z (GMT). No. of bitstreams: 1
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Previous issue date: 2006 / Resumo: A infecção oculta pelo VHB é caracterizada pela presença do DNA-VHB em indivíduos com o antígeno de superfície (HBsAg) indetectável. A prevalência e significado clínico desta infecção ainda não são totalmente conhecidos. Este trabalho teve por objetivo determinar a prevalência de infecção oculta pelo VHB em dois grupos de pacientes imunossuprimidos (pacientes em tratamento por hemodiálise e pacientes HIV positivos) com anti-HBc positivo co-infectados ou não pelo vírus da hepatite C (VHC). Foi investigada uma possível correlação entre prevalência do DNA-VHB e carga viral, níveis de CD4, fatores de risco e administração de lamivudina no grupo de HIV positivos. O primeiro grupo (G1) foi formado por 34 pacientes hemodialisados que eram HBsAg negativo/anti-HIV negativo. O segundo grupo (G2) formado por 159 pacientes infectados pelo vírus da imunodeficiência humana (anti-HIV positivo) e HBsAg negativo. Foi utilizado como grupo controle (G3), 150 doadores de sangue com marcadores sorológicos negativos para o HBsAg e para o HIV, mas reagentes para o anti-HBc. A pesquisa do DNA do VHB foi realizada pela PCR ?in house? , segundo a técnica de Kaneko et al. (1989), utilizando-se ?primers? específicos da região do core do VHB (limite de detecção 100 cópias/ml). Entre os pacientes hemodialisados, a infecção oculta não esteve presente, talvez devido ao controle da infecção pelo VHB nestes centros, pela adoção das medidas de precauções universais e imunização específica por vacinação contra o VHB que possivelmente, foram eficazes para evitar a presença de infecção oculta pelo VHB. A prevalência de positividade do VHB-DNA entre os pacientes HIV positivos foi de 5,03% (8/159). Este dado mostrou que não houve diferença significativa entre o grupo HIV positivo e o grupo controle (5,03% x 4%). De acordo com estes resultados, provavelmente, a imunossupressão nestes pacientes HIV positivos não foi fator determinante na prevalência de infecção oculta. O DNA-VHB foi observado independente da presença do anti-HBs nos pacientes HIV positivos coinfectados ou não pelo VHC. Esta observação sugere que o anti-HBs não foi capaz de proteger, na totalidade, os pacientes. A prevalência de infecção oculta pelo vírus da hepatite B não foi diferente entre os grupos estudados, HIV positivos e doadores de sangue. Não foi observada associação entre contagem de CD4, carga viral, fator de risco e nem à utilização da lamivudina como parte do tratamento anti-retroviral e ocorrência de hepatite B oculta / Abstract: Occult hepatitis B virus infection is characterized by presence of HBV-DNA in individuals with undetectable hepatitis B surface antigen (HBsAg). The prevalence and clinical significance of this infection remain incompletely defined. The aim of this study was to determinate the prevalence of occult HBV infection between two immunosuppressed populations (maintenance haemodialysis patients and HIV positive patients) with anti-HBc positive, co-infected or not with hepatitis C virus (HCV). Possible correlations were investigated between prevalence of HBV-DNA, viral load, CD4 levels, risk factors and lamivudina administration in the HIV group. The first group (G1) was formed by 34 hemodialysed patients that were HBsAg negative/anti-HIV negative. The second group (G2) formed by 159 human immunodeficiency virus infected patients (anti- HIV positive) and HBsAg negative. Used as a control group (G3), 150 blood donors with serologic markers negative to HBsAg and HIV, but positive for anti-HBc. HBV-DNA testing was performed using ?in house? nested PCR as described by Kaneko et al. and was detected using specific primers derivated from core regions of HBV genome (detection limit 100 copies / ml). Occult hepatitis B virus infection was absent in hemodialysis patients may be due the control of HBV infection in these centers, by adoption universal precautions measures and specific immunization by hepatitis B vaccination perhaps were effective to avoid occult HBV infection. The prevalence of HBV-DNA in HIV positive patients was 5,03% (8/159). This data showed that there was not significant difference between group HIV positive and control (5,03% x 4%). According these results probably the Immunosuppression in this HIV positive patients were not a determinant factor in prevalence of occult infection. The HBV-DNA was observed independent the presence of anti-HBs in HIV positive patients co-infected or not by HCV. This observation suggest that anti-HBs was not able to protect in the totality this patents. The prevalence of occult hepatitis B virus infection was not different among the group studied HIV positive patients and blood donors. It was not observed association between CD4 cell count, viral load, risk factor or treatment antiretroviral with lamivudine and occurrence of occult hepatitis B / Mestrado / Ciencias Basicas / Mestre em Clinica Medica
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