Thesis (M.Sc. (Microbiology))--Mahidol University, 1979. / Financial support from University Development Committee and National Cancer Institute.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 157-186). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 1995. / Includes bibliographical references.

Stuttgart, Univ., Diss., 2004.

Thesis (doctoral)--Utrecht, 1979.

München, Techn. Univ., Diss., 2002.

Um estudo de caso-controle para investigar a associação entre a hanseníase e infecção pelo vírus da hepatite B (VHB) foi conduzido no período de 1992/93, na cidade de Goiânia e municípios contíguos - Estado de Goiás. Avaliou-se, também, a distribuição espacial da hanseníase neste aglomerado urbano. Inicialmente, os indivíduos com suspeita clínica de hanseníase foram submetidos a exames baciloscópicos e histopatológicos, independentemente da rotina do Programa de Controle de Hanseníase. Do total de 855 pacientes recémdiagnosticados de hanseníase, 600 eram residentes em área urbana, e foram categorizados em casos multibacilares (31,3 por cento ), paucibacilares (51,8 por cento ) e prováveis (16,8 por cento ). Foi realizada análise descritiva desta casuística, havendo nítida predominância do sexo masculino na forma multibacilar de hanseníase. A distribuição espacial dos pacientes possibilitou, através da análise exploratória das taxas de detecção, discriminar estratos de risco intra-urbano. Para o estudo de caso-controle, 552 pacientes de hanseníase de 1 O a 70 anos foram incluídos. Os controles (N =552) foram selecionados de indivíduos com ausência de sinais e sintomas sugestivos de hanseníase oriundos da demanda espontânea de ambulatórios de 7 unidades de saúde, localizadas na região de procedência dos casos. Os participantes - casos e controles - foram entrevistados para avaliar fatores de risco para hanseníase e infecção pelo vírus da hepatite B. Foram coletadas amostras de sangue para detecção de marcadores ao vírus da hepatite B pela técnica de ELISA. Comparou-se a prevalência de marcadores de exposição (anti-HBc), de imunidade (anti-HBs) e de portador (AgHBs) entre casos e controles. Foram avaliados como potenciais fatores de confusão: sexo, idade, condições sócio-econômicas, estado nutricional, cicatriz vacinal de BCG e utilização dos serviços de saúde. Casos e controles foram similares quanto às características sócio-econômicas e nutricionais indicando que o princípio de selecionar controles da mesma base populacional que os casos parece ter sido adequado. Cicatriz vacinal de BCG esteve estatisticamente associada aos diferentes tipos de hanseníase. Houve maior proporção de indivíduos hospitalizados nos útimos 5 anos entre casos que em controles indicando que o emparelhamento por local de residência não eliminou completamente as diferenças entre os grupos em relação ao uso dos serviços de saúde. Entre os participantes do estudo, 18,1 por cento dos casos e 19,6 por cento dos controles foram soropositivos ao anti-HBc. Em análise multivariada, utilizando-se o modelo de regressão logística politômica, a associação da hanseníase e anti-HBc entre casos e controles apresentou odds ratio de 0,9 (IC95 por cento O, 7-1 ,3) para a categoria de multibacilar; 1,0 (IC 95 por cento 0,7-1,3) para a de paucibacilar e 1,1 (IC95 por cento 0,8-1,5) para a de provável. Estes resultados mostraram que subgrupos de casos e os controles estiveram igualmente expostos ao vírus da hepatite B. As proporções de indivíduos imunes foram semelhantes nos grupos de casos (9,2 por cento ) e controles (10,2 por cento ). Casos multibacilares responderam à exposição viral com formação de anticorpos protetores, qualitativa e quantitativamente de maneira semelhante aos pacientes paucibacilares e grupo controle. Os resultados dos índices de persistência de infecção (PPI) indicaram não haver diferença quanto ao clearance do antígeno viral nos subgrupos de casos e controles. Os resultados obtidos nesta investigação mostraram nos subgrupos de casos e controles: (i) prevalências semelhantes dos marcadores de exposição, de imunidade e de estado de portador; (ii) capacidade similar para produção de anticorpos protetores, avaliada através dos percentuais do marcador anti-HBs e, quantitativamente, através do Índice de Elisa e (iii) baixa probabilidade de persistência da antigenemia mensurada pelo PPI. Em conclusão, não houve evidências epidemiológicas de uma associação entre hanseníase e infecção pelo vírus da hepatite B, avaliada através de estudo de caso-controle, conduzido em área de baixa endemicidade ao VHB e alta endemicidade de hanseníase. / A case-control study was conducted in Goiânia, Central Brazil, a highly endemic area for leprosy and Iow endemic region for hepatitis B virus (HBV) infection. The purpose was to investigate the association between leprosy types and hepatitis B infection. The spatial distribution of leprosy in urban area was assessed. Between 1992 and 1993, newly detected leprosy cases (N=855) were investigated and 600 cases lived in the urban area. They were classified in multibacillary (31.3 per cent ), paucibacillary (51.8 per cent ) and probable cases (16.8 per cent ) according to histopathological and baciloscopic exams, independently of the leprosy control routine. The majority of multibacillary cases was males. Detection rates of leprosy were calculated by mapping cases and several risk strata were identified by using exploratory data analysis. This methodology seems to be particularly useful for targeting control activities in urban areas. Cases were 552 leprosy patients from the urban area and adjacent counties, between the ages of 1O and 70 years who self-referred or were referred to the main outpatient clinic for treatment in the region. 552 controls were selected from among self-referred outpatients from 7 health centers geographically located in areas where the cases came from. The main criteria for eligibility for control subjects was that they must not have any signs or symptoms indicative of leprosy. Blood samples were collected for all participants to determine serological markers of HBV infection and tested by enzyme immunoabsorbent assay technique (ELISA). Cases and controls were interviewed in order to evaluate risk factors for leprosy and hepatitis B vírus (HBV) infection. Prevalence of HBV exposure (anti-HBc), immunity (anti-HBs) and carrier status (AgHBs) were compared among cases and controls. Cases and controls were also compared for age, sex, socio-economic conditions, nutritional status, BCG scars and previous hospitalization. The participants had similar socio-economic pattern and also nutrition status, suggesting that the source of control selection was adequate for controlling for the most common confounding variables. BCG vaccine appeared to provide protection against multibacillary and paucibacillary types of leprosy and percentage of hospitalization was higher among cases. Prevalence of anti-HBc was similar among leprosy cases (18.1 per cent ) compared to controls (19.6 per cent ). An analysis of association between anti-HBc infection and leprosy types in terms of odds ratio, calculated by polytomous logistic regression, showed no positive association: multibacillary (OR=0.9 CI95 per cent 0.7-1.3); paucibacillary (OR= 1.0 CI95 per cent 0.7-1.3) and probable (OR= 1.1 CI95 per cent 0.8-1.5). The main findings of the case-control study were: (i) cases and controls had similar leveis of viral exposure, immune and carrier status (íi) the persistence of antigen response (PPI) was low among cases and controls respectively; (iii) ELISA índices were similar among multibacillary, paucibacillary and control group indicating that all participants mount antibody response to viral infection. In conclusion, there was no association between multibacillary leprosy and HBV infection in this setting.

Mathematical models have long been used to describe complex biological interactions with the aim of predicting mechanistic interactions hard to distinguish from data. This dissertation uses modeling, mathematical analyses, and data fitting techniques to provide hypotheses on the mechanisms of immune response formation and function. The immune system, comprised of the innate and adaptive immune responses, is responsible for protecting the body against invading pathogens, with disease or vaccine induced immune memory leading to fast responses to subsequent infections. While there is some agreement about the underlying mechanisms of adaptive immune memory, innate immune memory is poorly understood. Stimulation with lipopolysaccharide induces differential phenotypes in innate immune cells depending on the strength of the stimulus, such that a secondary lipopolysaccharide encounter of a constant dose results in either strong or weak inflammatory cytokine expression. We model the biochemical kinetics of three molecules involved in macrophages responses to lipopolysaccharide and find that once a macrophage is programed to show a weak inflammatory response this cannot be reverted. Contrarily, a secondary lipopolysaccharide stimulus of a very high dose or applied prior to waning of the effects of the primary stimulus can induce a phenotype switch in macrophages initially programed to show strong inflammatory responses. Some pathogens, such as the hepatitis B virus, have developed strategies that hinder an efficient innate immune response. Hepatitis B virus infection is a worldwide pandemic with approximately 257 million chronically infected people. One beneficial event in disease progression is the seroclearance of hepatitis B e antigen often in combination with hepatitis B antibody formation. We propose mathematical models of within-host interactions and use them to predict that hepatitis B e antibody formation causes hepatitis B e antigen seroclearance and the subsequent reactivation of cytotoxic T cell immune responses. We use the model to quantify the time between antibody formation and antigen clearance and the average monthly hepatocyte turnover during that time. We further expand the study of hepatitis B infection, by investigating the kinetics of the virus under an experimental drug administered during a clinical trial. Available drugs usually fail to induce hepatitis B s antigen clearance, defined as the functional cure point of chronic hepatitis B infections. Drug therapy clinical trials that combined RNA interference drug ARC-520 with entecavir have shown promising results in reducing hepatitis B s antigen titers. We develop pharmacokinetic-pharmacodynamic models describing the mechanistic interactions of the drugs, hepatitis B virus DNA, and virus proteins. We fit the model to clinical trial data and predict that ARC-520 alone is responsible for the reduction of hepatitis B s and e antigens, while entecavir is the driving force behind viral reduction. This work was supported by Simons Foundation, Grant No. 427115, and National Science Foundation, Grant No. 1813011. / Doctor of Philosophy / Mathematical models have long been used to describe complex biological interactions with the aim of predicting interactions that explain observed data and informing new experiments. This dissertation uses modeling, mathematical analyses, and data fitting techniques to provide hypotheses on the mechanisms of immune response formation and function. The immune system, comprised of the innate and adaptive immune responses, is responsible for protecting the body against invading pathogens, such as viruses, bacteria, or fungi. If an immune response to a secondary pathogen encounter differs from the response when the body first encounters the specific pathogen, this is called immune memory. The mechanisms underlying the memory of immune responses are well understood in the context of adaptive immune responses, but less so for innate immune responses. Stimulation with lipopolysaccharide, a cell wall component of many bacteria, programs innate immune cells, such as macrophages, to be in one of two states, called phenotypes, depending on the strength of the stimulus. Based on their phenotype the macrophages show either a weak or strong inflammatory response upon a secondary lipopolysaccharide encounter of a constant dose. We model the biochemical kinetics of three molecules involved in macrophages responses to lipopolysaccharide. We find that once a macrophage is programed to show a weak inflammatory response this cannot be reverted. Contrarily, a secondary lipopolysaccharide stimulus that is either of a very high dose or applied before the effects of the primary stimulus have waned, can induce a phenotype switch in macrophages initially programed to show strong inflammatory responses. Some pathogens, such as the hepatitis B virus, have developed strategies that hinder an efficient innate immune response. Hepatitis B virus infection is a worldwide pandemic with approximately 257 million chronically infected people. Hepatitis B e antigen is a protein that infected liver cells release into blood and that impairs adaptive immune responses. It is considered a beneficial event in disease progression, and called hepatitis B e antigen clearance, when hepatitis B e antigen becomes indetectable in a patient's blood. We propose mathematical models of interactions between liver cells, the virus, hepatitis B e antigens and hepatitis B e antibodies, which neutralize the antigens. We predict that antibody formation causes antigen clearance and a reactivation of immune responses. We furthermore use the model to quantify the time between antibody formation and antigen clearance and the average number of liver cells killed during that time. We further expand the study of hepatitis B infection, by investigating the kinetics of the virus under an experimental drug administered during a clinical trial. Available drugs rarely induce hepatitis B s antigen clearance, but clinical trials that combined a novel drug, called ARC-520, with the commonly used drug entecavir have shown promising results in reducing hepatitis B s antigen titers in the blood of infected patients. Following the clearance of hepatitis B s antigen, a protein that is released by infected cells and impairs adaptive immunity, the body usually has the capability to control the infection without medication. We develop mathematical models describing the interactions of the drugs, hepatitis B virus, and virus proteins. We fit the model to clinical trial data and predict that ARC-520 alone is responsible for the reduction of hepatitis B s and e antigens, while entecavir is the driving force behind viral reduction.

Student Number : 9906382M MSc (Med) dissertation - Faculty of Health Sciences / The G1862T mutation in the bulge of the RNA encapsidation signal, in the precore region of hepatitis B virus, results in reduced expression of HBeAg and accumulation of the HBeAg precursor in the endoplasmic reticulum (ER)/Golgi apparatus of the cell. This accumulation can disturb the functioning of the ER and lead to the ER stress response that can affect various cellular pathways, in turn affecting cell viability. The aim of this study was to determine whether apoptosis or necrosis occurred when cultured Huh7 cells were transfected with a plasmid expressing the G1862T mutation. Plasmid constructs, with and without the G1862T mutation, were used to transfect cells. To differentiate between necrosis and apoptosis cells were stained with propidium iodide or YO-PRO-1®, respectively. These were analyzed quantitatively using flow cytometry and qualitatively using confocal microscopy. Confocal microscopy, using monoclonal anti- HBe and the Hoechst stain, was performed to ensure that apoptosis was present as a result of the accumulation of the G1862T mutant HBeAg precursor. Caspase profiling was carried out using a fluorogenic-based assay. When cells were transfected with wild-type plasmid, necrosis predominated over apoptosis. Apoptosis predominated when the cells were transfected with the G1862T mutant plasmid. The highest levels of apoptosis occurred at 72 hours post-transfection. Confocal microscopy revealed the co-localization of aggregates of mutant HBeAg precursor with apoptotic nuclei. Transfection with G1862T mutant plasmids resulted in significant differences in the expression of caspase 3, 8, and 9 relative to the wild-type, at 48 and 72 hours post-transfection. The accumulation of the G1862T mutant HBeAg precursor, in the ER/ Golgi compartment, leads to apoptosis and affects the levels of caspase expression.

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