Flavonoide sind weitverbreitete sekundäre Pflanzeninhaltsstoffe. Ihr Beitrag zur Prävention von chronischen Erkrankungen wird zu großen Teilen auf immunmodulatorische und neuroprotektive Effekte zurückgeführt. Eine Voraussetzung für die Nutzung dieser Eigenschaften der Flavonoide stellt die Erfassung cytotoxischer Effekte dar. Mit Ausnahme von Xanthohumol und Quercetin ist für alle im Rahmen der vorliegenden Arbeit untersuchten Flavonoide, Hispidulin, Baicalein, Scutellarein, Hesperetin, Chrysin, Apigenin, Naringenin, Catechin, Pelargonidinchlorid und EMD 21388, sowohl in T-Zellen (Jurkat) als auch in neuronalen (SK-N-SH)-Zellen nach 24-stündiger Inkubation eine geringgradige Cytotoxizität festzuhalten. Für Xanthohumol bzw. Quercetin wird ein halbmaximaler Verlust der Zellvitalität je nach Modell in Konzentrationen von 33-45 µM bzw. 118-208 µM erreicht. Der weiterführenden Charakterisierung (zVAD, DNA-Laddering) ist zu entnehmen, dass die zellulären Veränderungen substanzabhängig differieren und sowohl nekrotische Mechanismen (Xanthohumol) als auch apoptotische Vorgänge (Quercetin) einschließen. Eine erhöhte Lipidperoxidation im oberen Dosisbereich lässt darüber hinaus auf eine Beteiligung von oxidativem Stress an den von Xanthohumol-induzierten nekrotischen Prozessen schließen. Eine positive Einflussnahme auf die Zellvitalität durch Antioxidantien wie GSH und NAC lässt des Weiteren vermuten, dass die erfassten Flavonoid-induzierten Prozesse jeweils sensitiv zum Redoxzustand der Zelle sind. Während die Effekte von Xanthohumol auch in anderen Zellmodellen (HL-60) nachweisbar bleiben, verhält sich Quercetin nicht durchgehend vitalitätsmindernd. Unterschiede zwischen den Testsubstanzen bestehen auch hinsichtlich antioxidativer Effekte. Das Eliminieren freier Radikale zählt zu den wichtigsten Mechanismen, die bei Flavonoid-vermittelter Neuroprotektion eine Rolle spielen. Insgesamt sind alle diesbezüglich untersuchten Substanzen als starke Superoxidanionen-Radikalfänger einzustufen. Im Co-Inkubationsversuch zeigt Scutellarein den stärksten Effekt, gefolgt von Quercetin, Hispidulin und Xanthohumol. Im Prä-Inkubations-Versuchsmodell liegen in der Reihenfolge ihrer Effektstärken Xanthohumol vor Quercetin, Hispidulin und schließlich Scutellarein. Die modellabhängigen Konstanten können, unter Beteiligung einer passiven Diffusion der hydrophoben Flavonoidaglykone, auf eine substanzgebundene Membranpermeabilität zurückzuführen sein. Das antioxidative Potential der Flavonoide resultiert u.a. aus einer komplexen Einflußnahme auf die Genexpression in der Zelle. In der vorliegenden Arbeit sind anhand von cDNA-Arrays für mehrere Vertreter übereinstimmend Wechselwirkungen mit Genen der zellulären Abwehr dargestellt. Demnach führen Scutellarein, Hispidulin, Quercetin und Xanthohumol zu einer deutlich reduzierten Expressionsstärke von STK4, CHD4, ARHGDIB, IL16, ISG20, PFN1 und SOD2. Unter den Flavonoid-induzierten Veränderungen ragen die Effekte auf ADAR1 heraus, dessen Genexpression von Scutellarein bis auf ein 0,1-faches der Referenzwerte reduziert wird. Gleichsinnige Auswirkungen von Scutellarein auf die Expression von ADAR1-Protein in Western Blots unterstreichen diese Interaktion und legen nahe, dass ADAR-vermittelte enzymatische Deaminierungen durch Flavonoide moduliert werden können. Diese Beobachtung wird ergänzt durch den nachgewiesenen Effekt von Flavonoiden auf die Expression einer Reihe weiterer Gene (ADAR2, APOBEC3B, APOBEC3C, APOBEC3F und APOBEC3G), die analoge posttranskriptionale Mechanismen steuern und gleichermaßen in Immunabwehr und Neuroprotektion eingebunden sind. Zu den wichtigsten Substraten von ADAR zählen Glutamatrezeptoren. Erwartungsgemäß ist nach der Einwirkung von Scutellarein auf humane Zellen, die Glutamatrezeptoren exprimieren, ein Rückgang der Deaminierung im Bereich der Glutamatrezeptoruntereinheit GluR 2 zu verzeichnen (Q/R-Position). Dem entspricht in elektrophysiologischen Modellen eine gesteigerte Ca2+-Permeabilität der jeweiligen Ionenkanäle und eine veränderte neuronale Exzitabilität. Hieraus ergibt sich ein breites Spektrum zusätzlicher Optionen für die Induktion von gesundheitsrelevanten Flavonoidfunktionen in der Zelle. So spielt die Modulation von Deaminierungen zugleich eine entscheidende Rolle im Vermehrungszyklus viraler Erreger. Die Annahme einer möglichen antiviralen Qualität von Scutellarein wird durch ein HBV-Infektionsmodell anhand drei Parameter der Virusreplikation (Virus-DNA-Konzentration, HBs- bzw. HBe-Antigenproduktion) bestätigt. Offen bleibt auch nach ausführlicher Prüfung, ob der deutliche antivirale Effekt als das Produkt von Flavonoid-induzierten Veränderungen der Deaminierungsraten oder als Folge eines Effekts auf die virale Polymerase zu interpretieren ist. Die hier dargestellten Wirkmechanismen leisten einen Beitrag zum Verständnis der Bedeutung von Flavonoiden für neue Anwendungen in Neuroprotektion und Immunabwehr. / Flavonoids are common secondary plant metabolites that confer numerous nutritional health effects. Their role in preventing chronic diseases is attributed to immunmodulatory and neuroprotective effects among others. In order to fully exploit these properties the limitations imposed by the compounds cytotoxic profiles must be addressed. For the majority of compounds investigated, hispidulin, baicalein, scutellarein, hesperetin, chrysin, apigenin, naringenin, catechin, pelargonidinchloride and EMD 21388, the present study confirms minimal cytotoxicity in T-cells (Jurkat) and in neuronal cells (SK-N-SH). As for xanthohumol and quercetin a 50% decline in cell-vitality is observed at concentrations of 33-45 µM and 118-208 µM, respectively. Further characterization using zVAD and DNA-laddering indicate that cell-vitality may be compromised both by necrotic mechanisms (xanthohumol) and by apoptotic effects (quercetin). An increase in lipidperoxidation in the upper dose range suggests that oxidative stress may be involved in xanthohumol toxicity. As this is counteracted by antioxidants such as GSH and NAC, these flavonoids impact on cell-vitality is likely codetermined by the cells redox state. While the effects of xanthohumol extend to other cell models, quercetin toxicity in HL-60 cells is less pronounced. Test compounds are also found to differ with regard to antioxidative profiles. The elimination of free radicals is a key mechanism in flavonoid-induced neuroprotection and is shown to vary with different incubation protocols. In short incubation experiments (5 min; co-incubation) scutellarein is identified as the most powerful scavenger, followed by quercetin, hispidulin and xanthohumol. In prolonged incubations (24 hrs; prä-incubation) xanthohumol and quercetin are followed by hispidulin and scutellarein. Model-specific constants suggest that passive diffusion of the hydrophobic flavonoid-aglyca may occur across cell membranes, alongside with other modes of permeation. Flavonoids antioxidative potential is mediated by complex effects on gene expression. The present work uses data from cDNA-arrays to highlight interactions with genes involved in cellular defense. Specifically, scutellarein, hispidulin, quercetin and xanthohumol downregulate expression for STK4, CHD4, ARHGDIB, IL16, ISG20, PFN1 and SOD2. In addition, flavonoids consistently downregulate ADAR1-expression, which drops to 0,1-fold of reference values and is paralleled by scutellarein-effects on ADAR1-protein-expression. Together, these findings indicate, that ADAR-mediated enzymatic deamination may be modulated by flavonoids. Similar effects are noted on related genes (ADAR2, APOBEC3B, APOBEC3C, APOBEC3F and APOBEC3G), relevant to posttranscriptional processing underlying immune defense and neuroprotection. Glutamate receptors count among the most important neuronal substrates of ADAR. Following exposure to scutellarein a decrease in deamination rates is confirmed with respect to the glutamate receptor subunit GluR 2 (Q/R-site). As a result, an enhanced Ca2+-permeability of the respective ion channels is anticipated, and modified neuronal excitability. Overall, the regulation of enzymatic deamination by flavonoids offers opportunities for multilevel balancing of cell homeostasis. Thus deaminations may interfere with the replication cycle of viral pathogens. Using an ex-vivo HBV-infection model and three parameters of viral replication (viral load, HBs and HBe indices), antiviral properties of scutellarein are illustrated. Despite extensive investigation, it remains to be seen whether these effects can be ascribed to deaminations of viral DNA or to an interaction with other substrates, e.g. the viral polymerase. In summary, the present observations serve to foster our understanding of flavonoids roles in neuroprotection and immune defense.

Ph.D. thesis, Faculty of Health Sciences, University of the Witwatersrand, 2009

Thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Science (Biochemistry), University of the Witwatersrand, Johannesburg, 1992 / An investigation has been made into aspects of the expression of oncogenes in normally dividing cells and in hepatoceilular carcinoma (Hee). HOC occurs commonly in Southern Africs, and thf1aetiology ·ofthis tumour lsaseccieted with hepatitis a virus (HBV) infection. c·erbA, c..mva and e-tos but not c~Ha..res mANA were elevatad in tumours and adjacent hepatic tissue from the same petiEJ;htswhen compared to normal liver. Amounts of Fos and MYQ prot~in in the liver tumour specimens were else raised. The"e was some correlation between the patients' serum a..fetoproteirt concentretlons, histological features of tumour differentiatic)t"l, c..mvc and c40s r.ixpression. expression of e-tas and c..myc has been reportec to be elevated after stimulation of cells to alvlde, ,'1$ occurs during liver r19ganeration. This was corroborated by the findin~ that c-mvc, c·fo~· and c-jun mRNA concentratlona "Jere increased it"! cultured 3T6 mouse fibroblasts following treatment with alkaline medium aa a mitogenlo stimulus. The time course of the expression of these oncogenes was similar to that reported after gro\l'l/th factor sttmulation, The H[~V X..gene ma\' be responsible for increased oncogene expression it' YCC as a result of its documented trans activating properties. This vi!'a~ gene is unusual in that it has a codon preferanc";which is similar to that of eukarvotic ceU genes. Also HBV may ha'V& evolved from ti similar ancestral virus to that giving rise to retroviruses. These ideas suggest that the HBV X·gene is a viral oncogene derived from a host homologue. Low stringency Northern brot hybridisation using a X-gene probe denlonstrated a murine transcrlpt in heart and thymus. Attempts to isolate the sequence from mouse heart and thymus eDNA libraries ware unsuccessful despite ext,~n$jve screening with sensitive probes (SP6 palymerfjsa and peR fab(':.lUed X~gen~~fragments). Conserved X~gene \ . I sequences were also used fot the desigr:Jof primers in .~.peR bas£'d method " . II aimed at isolating a mammalian sequence. No sinnificant sequsnce \\ homology was found bet\lveen the HBVI\X..gene and Ol\A ampllfle'd from \1 l! gen(llmic and eDNA I1br'srytemplate sou~\pes.The peR preducts ttppeared to have been artef.,ots of arnplWaation. ~~n'IJreto detect the hQrtll.)logous gene may have resu~ted from poo' complS,JIlentarity between the VIral ant! \\ mammalian secuencec, 1\ \\ Non..~pecific amplification is commonly enct~unter&d when u$1110 PCli'. A qtJick asvmmatrlc re·ampW~catj(ii1 method I,?ssed on eXUOSilin of an " interm.uly' hybrfdising X·gelllapfimar we! davisQ\j to confirm FICRprOdu(,ts. The l"n1ithodwas specific irlthat "ver~ single bas~ mlsmatohe$ betwsen the internal primer and tem1>late re;.,ultad in fatJut~ of dete(;tabla \tUim$f extension. / GR 2016

Chan Ming Lok. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 93-108). / Abstracts in English and Chinese. / Ackowledgments --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iii / List of Abbreviations --- p.iv / List of Tables --- p.v / List of Figures --- p.vi / Chapter Chapter 1 --- Introduction and Objectives / Chapter 1.1 --- Hepatocellular Carcinoma --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Geographical Distribution --- p.1 / Chapter 1.1.3 --- Sex and Age --- p.1 / Chapter 1.1.4 --- Etiology --- p.2 / Chapter 1.1.5 --- Molecular Basis of HCC --- p.3 / Chapter 1.1.6 --- Situation in China and Hong Kong --- p.4 / Chapter 1.2 --- The Hepatitis B Virus --- p.5 / Chapter 1.2.1 --- Morphology --- p.5 / Chapter 1.2.2 --- Structure of the HBV Genome --- p.6 / Chapter 1.2.3 --- Functional Domains of the HBV Genome --- p.9 / Chapter 1.2.4 --- Pathogenesis of HBV Infection --- p.11 / Chapter 1.3 --- HBx --- p.12 / Chapter 1.3.1 --- The HBV x Gene --- p.12 / Chapter 1.3.2 --- The HBX Protein --- p.13 / Chapter 1.3.3 --- "Preferential HBX Expression in Sera, Hepatitis, Cirrhosis and HCC" --- p.13 / Chapter 1.3.4 --- Cellular Localization of HBX --- p.14 / Chapter 1.3.5 --- Animal Studies --- p.15 / Chapter 1.3.6 --- Functional Studies on HBX --- p.15 / Chapter 1.3.7 --- Variations in the HBx Gene --- p.21 / Chapter 1.4 --- Objectives of this Study --- p.24 / Chapter Chapter 2 --- Methods and Materials Methods / Chapter 2.1 --- Paraffin Embedding of Patient Tissue Samples --- p.26 / Chapter 2.1.1 --- Tissue Processing --- p.26 / Chapter 2.1.2 --- Paraffin Embedding of Tissue Samples --- p.26 / Chapter 2.2 --- Sectioning of Paraffin Embedded Tissue Sections --- p.26 / Chapter 2.3 --- Immunohistochemical Staining of Paraffin Embedded Tissue Sections --- p.26 / Chapter 2.3.1 --- Dewaxing of Paraffin-Embedded Tissue Sections --- p.26 / Chapter 2.3.2 --- Rehydration of Tissue Sections --- p.27 / Chapter 2.3.3 --- Antigen Retrieval --- p.27 / Chapter 2.3.4 --- Quenching of Endogenous Hydrogen Peroxidase --- p.27 / Chapter 2.3.5 --- Blocking of Endogenous Biotin and Non-Specific Protein Binding --- p.27 / Chapter 2.3.6 --- Antibody Incubation and Color Development --- p.27 / Chapter 2.3.7 --- Counterstaining and Coverslip Mounting --- p.28 / Chapter 2.3.8 --- Interpretation of Immunostaining Results --- p.28 / Chapter 2.4 --- DNA Extraction from HCC Tissues --- p.28 / Chapter 2.4.1 --- Sectioning of Frozen HCC Specimens --- p.28 / Chapter 2.4.2 --- Proteinase K Digestion and Phenol Chloroform Extraction --- p.29 / Chapter 2.4.3 --- Ethanol Precipitation and Re-suspension in Tris-EDTA (TE) Buffer --- p.29 / Chapter 2.5 --- Quantitation and Purity Check of Extracted DNA --- p.29 / Chapter 2.6 --- Quality Check for Extracted Genomic DNA --- p.30 / Chapter 2.6.1 --- Agarose Gel Electrophoresis --- p.30 / Chapter 2.6.2 --- Polymerase Chain Reaction (PCR) of the β-globin Gene --- p.30 / Chapter 2.6.3 --- Analysis of PCR Fragments by Agarose Gel Electrophoresis --- p.30 / Chapter 2.7 --- Polymerase Chain Reaction Amplification of HBs and HBx Genes of the Hepatitis B Virus --- p.31 / Chapter 2.8 --- Southern Blot of HBx PCR Fragments --- p.31 / Chapter 2.8.1 --- Immobilization of DNA onto a Positively Charged Nylon Membrane and Pre-hybridization --- p.31 / Chapter 2.8.2 --- Radio-labeling of an HBV Probe --- p.32 / Chapter 2.8.3 --- Hybridization of a 32P-labeled HBV Probe and Film Exposure --- p.32 / Chapter 2.9 --- Cloning of PCR Fragments into pGEM®-T Vector for Sequencing --- p.33 / Chapter 2.9.1 --- Gel Extraction and Purification --- p.33 / Chapter 2.9.2 --- Ligation --- p.33 / Chapter 2.10 --- Transformation of Competent DH5a cells --- p.34 / Chapter 2.10.1 --- Preparation of Competent DH5α Using Calcium Chloride --- p.34 / Chapter 2.10.2 --- Heat Shock of Competent DH5α Cells --- p.34 / Chapter 2.10.3 --- Plating of Transformed Cells onto LB Agar Plates --- p.34 / Chapter 2.10.4 --- Screening of Transformants for Inserts --- p.35 / Chapter 2.11 --- Miniprep of Plasmid DNA --- p.35 / Chapter 2.11.1 --- Inoculation of Bacterial Clones --- p.35 / Chapter 2.11.2 --- DNA Extraction by Alkaline Lysis and Phenol/Chloroform --- p.35 / Chapter 2.11.3 --- Ethanol Precipitation and Re-suspension in TE Buffer --- p.35 / Chapter 2.11.4 --- Confirmation of Positive Clones --- p.36 / Chapter 2.12 --- Sequencing of pGEM®-T Cloned HBx PCR Fragments --- p.36 / Chapter 2.13 --- Construction of the HBx-GFP Plasmid --- p.36 / Chapter 2.13.1 --- PCR Amplification of HBx Gene Inserts --- p.36 / Chapter 2.13.2 --- Confirmation of HBx Insert Sequence by DNA Sequencing --- p.37 / Chapter 2.13.3 --- Restriction Digest of HBx-pGEM®-T Plasmids to Obtain HBx Inserts --- p.37 / Chapter 2.13.4 --- Restriction Digest of pEGFP-Nl Cloning Vector for Cloning --- p.37 / Chapter 2.13.5 --- Ligation of HBx Inserts into the pEGFP Cloning Vector --- p.37 / Chapter 2.14 --- Large Scale Plasmid DNA Preparation --- p.38 / Chapter 2.15 --- Cell Culture --- p.39 / Chapter 2.16 --- Transfection using LipofectAminéёØ --- p.39 / Chapter 2.16.1 --- Seeding of Cells for Coverslip Growth --- p.39 / Chapter 2.16.2 --- Transfection using LipofecAminéёØ --- p.39 / Chapter 2.17 --- Cell Fixation and DAPI Staining Materials --- p.40 / Chapter 2.18 --- Chemicals --- p.41 / Chapter 2.19 --- Antibodies --- p.41 / Chapter 2.20 --- "Formalin-fixed, Paraffin Embedded Tissues of HCC Tissues from Xiamen" --- p.41 / Chapter 2.21 --- Frozen Liver Tissues --- p.41 / Chapter 2.22 --- PCR Reagents --- p.43 / Chapter 2.23 --- Primers --- p.43 / Chapter 2.24 --- Plasmid --- p.43 / Chapter 2.25 --- Enzymes --- p.43 / Chapter 2.26 --- Ligation Reagents --- p.43 / Chapter 2.27 --- Cloning Vectors --- p.45 / Chapter 2.28 --- Competent Cell --- p.45 / Chapter 2.29 --- Hela and HepG2 Cell Line --- p.45 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Hepatitis B Virus Status of HCC Patients from Hong Kong and Xiamen --- p.46 / Chapter 3.2 --- Immunohistochemical Studies of the HBx Protein in Hong Kong and Xiamen HCC --- p.46 / Chapter 3.2.1 --- Cross Reaction of Anti-99 with Cytokeratin 18 (CK18) --- p.46 / Chapter 3.2.2 --- HBx Expression in HCC Patient Tissue Samples from Hong Kong --- p.50 / Chapter 3.2.3 --- HBxAg Staining in HCC Tissue Samples from Xiamen --- p.50 / Chapter 3.3 --- Agarose Gel Electrophoresis of DNA Extracted from Frozen Liver Tissues --- p.50 / Chapter 3.4 --- PCR Amplification of the β-globin Gene --- p.55 / Chapter 3.5 --- PCR Amplification of the HBs Gene from Liver Samples of HCC Patients from Hong Kong --- p.55 / Chapter 3.6 --- PCR Amplification of the HBx Gene from Liver Samples of HCC Patients from Hong Kong --- p.55 / Chapter 3.7 --- Amplification of the HBx Gene from Serum Samples of Chronic Hepatitis B Virus from Hong Kong Using Nested PCR --- p.61 / Chapter 3.8 --- Southern Blot of HBx PCR Fragments --- p.61 / Chapter 3.9 --- Cloning and Sequencing of the HBx Gene in HCC and Chronic Hepatitis B Patient Samples from Hong Kong --- p.61 / Chapter 3.10 --- Expression Pattern of Wild-type HBx-GFP Fusion Protein in Transiently Transfected HeLa and HepG2 Cells --- p.73 / Chapter 3.11 --- Expression Patterns of HBx-GFP with and without Mutations at Codons 130 and 131 in HeLa and HepG2 Cell Line --- p.78 / Chapter 3.12 --- Growth Kinetics of HeLa Cells Transfected with GFP and Wild-type HBx-GFP with and without Mutations in Codons 130 and131 --- p.81 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- HBxAg Expression in Tumorous and Surrounding Non-tumorous Tissues --- p.83 / Chapter 4.2 --- "Detection of the HBx Gene in Sera, Non-tumorous and Tumorous Tissues" --- p.84 / Chapter 4.3 --- HBx Gene Mutations in Chronic Hepatitis and HCC --- p.85 / Chapter 4.3.1 --- Codon 127 (HBV nt 1752-1754) --- p.85 / Chapter 4.3.2 --- Codons 130 and 131 (HBV nt 1761-1766) --- p.86 / Chapter 4.3.3 --- Lack of Correlation between HBx Gene Mutations and Lack of HBxAg Expression --- p.87 / Chapter 4.4 --- Cellular Localization of HBxAg in Transiently Transfected Cells Lines --- p.88 / Chapter 4.5 --- Functional Difference Between Wild-type and Mutant HBX Protein --- p.89 / Chapter Chapter 5 --- Conclusions and Directions for Further Studies / Chapter 5.1 --- Conclusions --- p.91 / Chapter 5.2 --- Directions for Further Studies --- p.92 / References --- p.93 / Appendix / Chapter A1 --- Recipes of Reagents Used in this Study --- p.109 / Chapter A2 --- Schematic Setup of Downward Capillary Transfer of DNA --- p.112 / Chapter A3 --- Circle Map of the pGEM®-T Cloning Vector and Construct of the HBx-pGEM®-T Plasmid --- p.113 / Chapter A4 --- Circle Map of the pEGFP-Nl Cloning Vector and Construct of the HBx-GFP Plasmid --- p.114

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.

by Zhong Sheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (p. 186-203). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

by Winnie Yeo. / Thesis (M.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 213-256). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Zhu, Lin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-118). / Abstracts in English and Chinese. / Contents --- p.I / List of Abbreviations --- p.IV / List of Tables and Figures --- p.V / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Viral Hepatitis --- p.2 / Chapter 1.2 --- Global Epidemiology of HBV --- p.3 / Chapter 1.3 --- Modes of Transmission --- p.4 / Chapter 1.4 --- Diagnostic Tests --- p.5 / Chapter 1.4.1 --- HBeAg and Anti-HBe --- p.7 / Chapter 1.4.2 --- Serum Enzymes --- p.8 / Chapter 1.4.3 --- HBV DNA Assays --- p.9 / Chapter 1.4.3.1 --- HBV DNA Assays --- p.9 / Chapter 1.4.3.2 --- Clinical Applications of DNA Assays --- p.10 / Chapter 1.4.4 --- Histology --- p.13 / Chapter 1.5 --- Natural Course of Chronic Hepatitis infection --- p.18 / Chapter 1.5.1 --- Phases of chronic hepatitis B --- p.18 / Chapter 1.5.2 --- HBeAg-negative chronic hepatitis B --- p.21 / Chapter 1.6 --- Molecular biology of HBV --- p.23 / Chapter 1.6.1 --- Overview --- p.23 / Chapter 1.6.2 --- Genomic structure and organization --- p.24 / Chapter 1.6.2.1 --- Surface ORF --- p.24 / Chapter 1.6.2.2 --- Precore/Core ORF --- p.25 / Chapter 1.6.2.3 --- Polymerase ORF --- p.25 / Chapter 1.6.2.4 --- X ORF --- p.26 / Chapter 1.7 --- Genetic Variation of HBV --- p.31 / Chapter 1.7.1 --- HBV genotypes --- p.31 / Chapter 1.7.2 --- Predominant genotypes and their subgroups in Asia --- p.33 / Chapter 1.7.3 --- HBV mutations --- p.36 / Chapter 1.7.3.1 --- Precore mutations --- p.37 / Chapter 1.7.3.2 --- Core promoter mutations --- p.38 / Chapter 1.7.3.3 --- Other Mutations associated with clinical outcome --- p.40 / Chapter Chapter Two: --- Methodology --- p.44 / Chapter 2.1 --- Aims and Hypothesis --- p.45 / Chapter 2.1.1 --- Aims --- p.46 / Chapter 2.1.2 --- Hypothesis --- p.47 / Chapter 2.2 --- Patient Recruitment --- p.48 / Chapter 2.3 --- Laboratory Assays --- p.49 / Chapter 2.3.1 --- Preparation of serum HBV DNA --- p.49 / Chapter 2.3.2 --- Quantification of serum HBV DNA --- p.51 / Chapter 2.4 --- Full-genome Amplification of HBV DNA --- p.53 / Chapter 2.5 --- Full-genome Sequencing of HBV DNA --- p.55 / Chapter 2.6 --- Assembly of HBV Full-genome Sequence --- p.58 / Chapter 2.7 --- Phylogenetic Analysis --- p.59 / Chapter 2.7.1 --- Construction of phylogenetic tree --- p.59 / Chapter 2.7.2 --- Genotype and subgenotype determination --- p.60 / Chapter 2.8 --- HBV Mutations --- p.62 / Chapter 2.9 --- Info-gain program --- p.64 / Chapter 2.10 --- Statistical Analysis --- p.65 / Chapter Chapter Three: --- Results --- p.67 / Chapter 3.1 --- Patient Information --- p.68 / Chapter 3.2 --- Phylogenetic Analysis --- p.69 / Chapter 3.3 --- HBV genotypes/subgenotypes --- p.76 / Chapter 3.4 --- “Hot-spo´tح HBV Mutants --- p.79 / Chapter 3.5 --- HBV Mutation Associated with Liver Fibrosis --- p.82 / Chapter 3.5.1 --- Mutant selection --- p.82 / Chapter 3.5.2 --- Clinical significance of novel mutants --- p.84 / Chapter Chapter Four: --- Discussion --- p.88 / Chapter 4.1 --- Full-genome Sequencing Strategy --- p.89 / Chapter 4.2 --- HBV genotypes/subgenotypes Distribution and Disease Activity --- p.90 / Chapter 4.2.1 --- HBV genotypes/subgenotypes distribution --- p.90 / Chapter 4.2.2 --- Clinical significance of genotypes/subgenotypes --- p.91 / Chapter 4.3 --- HBV Hotspot Mutants and Disease Activity --- p.93 / Chapter 4.4 --- HBV Novel Mutants --- p.96 / Chapter 4.5 --- Limitation of the Study and Future Work --- p.97 / Chapter 4.5.1 --- Limitation --- p.97 / Chapter 4.5.2 --- Future Direction --- p.98 / Chapter Chapter Five: --- Conclusions --- p.99 / References --- p.102

Thesis ( M Med (Virological Pathology))--University of Limpopo, 2010. / Introduction: Hepatitis B virus (HBV) is a serious problem worldwide causing various liver diseases such as chronic hepatitis and hepatocellular carcinoma (HCC). The pathogenesis of HBV related HCC is not well established. Hepatitis B X protein (HBx) plays an important role in the pathogenesis of HCC. HBx coded by HBV X gene enhances several cellular pathways in hepatocytes which may lead to HCC. The genetic variability of other HBV genomic regions plays a significant role in diagnosis, vaccine development and drug resistance. However, the genetic variability of HBV X gene is not well understood. In addition the dual basal core promoter mutations found within the X gene have been implicated in the inhibition of hepatitis B e antigen (HBeAg) expression. Studies focusing on HBV X gene are scarce in South Africa. Consequently HBV X gene variability may reveal interesting mutations and substitutions that are important in chronic liver diseases or HCC. This study aimed at characterising HBV X gene at a molecular level isolated from patients with different serological profiles. Methods: This was an exploratory study which used 20 stored sera (-70°C) collected from adult patients at Dr George Mukhari hospital, Pretoria. The samples were already tested for HBsAg, anti-HBs, anti-HBc and HBeAg serological markers (Elecsys, Roche Diagnostics, Penzburg, Germany). HBV DNA extraction was performed from serum using High Pure Viral Nucleic Acid Assay (Roche Diagnostics, Penzburg, Germany). Nested PCR assay was used for the amplification of 465 nucleotide HBV X gene. Sequencing of PCR positive samples was done using spectruMedix SCE2410 genetic analysis system. Six samples selected, were cloned into the pGEM®-T Easy vector system (Promega, Madison, USA). Three clones of each sample were selected and their plasmids purified using Pure Yield™ Plasmid Miniprep System (Promega, Madison, USA). The plasmid DNA was recovered using optimised nested PCR assay and sequenced. A total of 38 sequences were generated from the study and compared with reference strains retrieved from GenBank. Phylogenetic analysis based on HBV X gene sequences was done using MEGA 4 software to determine different genotype clusters. vi Results: HBV X gene was successfully detected and amplified in 20 study samples. The sequenced HBV X gene products revealed mutations and insertions. Particularly a six nucleotide insertion, GCATGG between nucleotides 1611 and 1618 which was detected in five samples. In addition, the six cloned samples confirmed the six nucleotide insertion and other mutations associated with inhibition of hepatitis B e antigen (HBeAg) detected in the study. The substitutions within HBx were detected in the N (1-50 amino acids) and C (51-154) terminals by comparing our sequences with archival sequences from GenBank. Important substitutions found within the N and C terminals were S31A, P38S, A42P, F73L, H94Y, P101S, K118T, D119N, I127T/N, K130M and V131I. These substitutions are associated with various biological functions and pathogenesis. Other substitutions with unknown functions detected in the study include A2G, A3G, A4G, C6W, P42S and V116L. Further mutations of T1753M, A1762T and G1764A associated with inhibition of HBeAg expression were detected in most samples and only one sample had C1766T mutation. Phylogenetic analysis resulted in A, C and D HBV genotypes. Five samples and 11 clones clustered with genotype D, two samples and four clones clustered with genotype C and finally 13 samples and 3 clones clustered with genotype A. Conclusion: HBV X gene was successfully characterised using various molecular methods. HBx substitutions detected are involved in various pathogenic effects and may present a risk of HCC for patients infected with HBV. Genotype D samples displayed most mutations/substitutions and this can be regarded as an important genotype with high risk of HCC. The detection of a six nucleotide insertion (GCATGG) in 5 samples may emerge as a new variant of genotype D. Furthermore triple mutations of T1753M/A1762T/G1764A within basal core promoter region were detected mostly in HBeAg negative samples. However further analysis of HBV X gene variability is needed.

This thesis investigates the epidemiology of hepatitis B virus infection (HBV) and estimates HBV-related liver disease burden in Vietnam and Australia using a cross-sectional study design and mathematical modelling. A population-based seroprevalence survey was undertaken in rural Northern Vietnam. In a sample of 870 study participants, prevalence of anti-HBV core antibody (anti-HBc) and hepatitis B virus surface antigen (HBsAg) was 68.2% and 19.0%, respectively, and hepatitis B e antigen (HBeAg) was detected in 16.4% of the HBsAg-positive group. Factors associated with HBV infection (anti-HBc and/or HBsAg-positive) were age 60 years or older (adjusted odds ratio (AOR), 3.82; 95% CI, 1.35??10.80; P = 0.01), residence in Vu Thu district (AOR, 3.00; 95% CI, 2.16??4.17; P <0.001), hospital admission (AOR, 2.34; 95% CI, 1.33??4.13; P = 0.003) and history of acupuncture (AOR, 2.01; 95% CI, 1.29??3.13; P = 0.002). Household contact with a person with liver disease (AOR, 2.13; 95% CI, 1.29??3.52; P = 0.003), reuse of syringes (AOR, 1.81; 95% CI, 1.25??2.62; P = 0.002) and sharing of razors (AOR, 1.69; 95% CI, 1.03??2.79; P = 0.04) were independent predictors of HBsAg positivity. Alanine aminotransferase (ALT) level was elevated (>40 IU/L) in 43% of the HBsAg-positive group; the proportion of elevated ALT was higher in HBeAg-positive (65%) compared with HBeAg-negative (39%) (P = 0.02). Based on data from the seroprevalence study, other prevalence estimates and HBV natural history parameters, a mathematical model was used to estimate HBV-related liver disease burden in Vietnam. Estimated chronic HBV prevalence increased from 6.4 million cases in 1990 to around 8.4 million cases in 2005 and was projected to decrease to 8.0 million by 2025. Estimated HBV-related liver cirrhosis and hepatocellular carcinoma (HCC) incidence increased linearly from 21 900 and 9400 in 1990 to 58 650 and 25 000 in 2025. Estimated HBV-related mortality increased from 12 600 in 1990 to 40 000 in 2025. To estimate HBV-related HCC incidence among Australians born in the Asia-Pacific region (APR), a mathematical modelling was developed utilising HBV natural history parameters, HBV prevalence estimates in APR countries and immigration data. Chronic HBV cases among the APR-born population increased rapidly from the late 1970s, reaching a peak of 4182 in 1990. Chronic HBV prevalence increased to more than 53 000 in 2005. Estimates of HBV-related HCC increased linearly from one in 1960 to 140 in 2005, with a projected increase to 250 in 2025. Universal HBV vaccination programs in countries of origin had limited impact on projected HBV-related HCC to 2025. HBV-related HCC survival was analysed in a population-based linkage study in New South Wales (NSW), Australia. Between 1994 and 2002, 278 HCC cases notified to the NSW Cancer Registry were linked to chronic HBV infection notifications to the NSW Health Department. The majority of cases were male (83.5%) and overseas born (93.6%); Asian-born cases accounted for 72.1%. Median survival following HCC diagnosis was 15 months. HCC survival was poorer among older age groups (P <0.001), and among cases with regional spread (HR 3.23; 95% CI, 1.83??5.69; P <0.001) and distant metastases (HR 3.85; 95% CI, 2.44??6.08; P <0.001). Sex, region of birth, and study period (1994??1997 versus 1998??2002) were not associated with HCC survival. The results of these studies show that HBV infection remains a major public health challenge in highly endemic countries such as Vietnam. HBV-related liver disease burden in Vietnam was estimated to increase for at least two decades despite the introduction of a universal infant HBV-vaccination program. Similarly, HBV-related HCC among Australians born in the APR was estimated to continue to increase over the next two decades. Survival for HBV-related HCC even in settings such as Australia continues to be extremely poor. Strategies are required to expand HBV treatment to individuals with chronic HBV infection who are at greatest risk of progression to advanced liver disease.