published_or_final_version / Medicine / Master / Doctor of Medicine

Ho Kar Fai, William. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 121-129). / Abstracts in English and Chinese. / Abstract --- p.I / Acknowledgement --- p.V / Table of Contents --- p.VI / Abbreviations --- p.VIII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The recent situation of hepatitis B infection and HBV-induced HCC in Hong Kong / Chapter 1.2 --- Natural history of HBV infection in human / Chapter 1.3 --- The genomic organization of HBV / Chapter 1.4 --- Potential oncogenic mechanism of HBV-induced hepatocarcinogenesis / Chapter 1.5 --- Aim of the present study / Chapter Chapter 2 --- Materials and methods --- p.16 / Chapter 2.1 --- Transformation in E.coli for subtracted normal-counterpart library / Chapter 2.2 --- PCR amplification of subtracted clones / Chapter 2.3 --- Sequencing of subtracted clones with dye-terminator cycle sequencing technology / Chapter 2.4 --- Sequence analysis and database construction / Chapter 2.5 --- Molecular cloning and characterization of novel gene / Chapter 2.6 --- In silico structural and functional analysis of Z313 / Chapter 2.7 --- Cloning and sequencing analysis of zinc finger protein 313 (Z313) / Chapter 2.7.1 --- PCR amplification of target gene -Z313 / Chapter 2.7.2 --- Mini-preparation of plasmid DNA / Chapter 2.7.3 --- Cycle sequencing of cloned cDNA -Z313 with dye-primer technology / Chapter 2.8 --- Multiple Tissue Northern (MTN) blot hybridisation / Chapter 2.9 --- RT-PCR analysis of Z313 / Chapter 2.10 --- Subcellular localization study of Z313 by Green Fluorescent Protein (GFP) / Chapter 2.10.1 --- Directional cloning of Z313 into pEGFP-Cl / Chapter 2.10.2 --- Mini-preparation of plasmid DNA / Chapter 2.10.3 --- Transient transfection of plasmids in different cell lines / Chapter 2.10.4 --- Microscope observation of GFP transfected cells / Chapter Chapter 3 --- Results --- p.49 / Chapter 3.1 --- PCR selection of subtracted clones for sequencing analysis / Chapter 3.2 --- Partial sequencing of selected subtracted clones / Chapter 3.3 --- DNA homology searching using program - BLASTN / Chapter 3.4 --- Catalogue of the 467 ESTs from the subtracted normal-counterpart library / Chapter 3.5 --- Classification and frequency of the subtracted normal-counterpart cDNA clones / Chapter 3.6 --- Identification of putative differentially expressed genes in HCC surrounding normal liver / Chapter 3.7 --- Categorization of ESTs exclusively appeared in the subtracted normal- counterpart library / Chapter 3.8 --- In silico structural and functional analysis of zinc finger protein313 (Z313) / Chapter 3.9 --- Molecular cloning of zinc finger protein 313 (Z313) / Chapter 3.10 --- Northern analysis of zinc finger protein 313 (Z313) / Chapter 3.11 --- RT-PCR analysis of zinc finger protein 313 (Z313) / Chapter 3.12 --- Subcellular localization study of zinc finger protein 313 (Z313) / Chapter Chapter 4 --- Discussion --- p.104 / Chapter 4.1 --- EST analysis on the subtracted normal-counterpart cDNA clones / Chapter 4.1.1 --- Characterization of ESTs generated from the subtracted normal-counterpart library / Chapter 4.1.2 --- Putative differentially expressed genes in HCC surrounding normal liver related to hepatocellular carcinoma / Chapter 4.2 --- Molecular cloning and characterization of zinc finger protein313 (Z313) / Chapter 4.3 --- Future aspects / References --- p.121

The human hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae family of viruses. Various other hepadnaviruses are used as models to study human HBV infections as all Hepadnaviridae family members have similar virus structure and replication strategies. The studies performed and described in this thesis were carried out using duck hepatitis B virus (DHBV) infection of Pekin ducks as a model system. Hepadnavirus infections can have either an acute or a chronic outcome. The factors that contribute to these outcomes include the immune response, the age of the host at the time of infection as well as size of viral inoculum. The overall aim of this project was to gain a detailed understanding of the mechanisms involved in clearance of virus and resolution of acute DHBV infections. As a first step, molecular and immunohistochemical detection methods for a range of cellular markers in ducks had to be developed as assays were not readily available. Quantitative reverse transcription PCR assays (qRT-PCR) were developed for the detection of mRNA of the duck T-lymphocyte markers, CD3, CD4, CD8, duck cytokines, IFN-α, IFN-γ, TNF-α and the duck housekeeping genes, β-actin and GAPDH. Immunohistochemistry was developed for the detection of duck CD4 + and CD8 + on T cells and for the detection of proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation. These methods were then widely used throughout the project. The innate immune response during HBV infections is not well understood. Toll-like receptors (TLR) are a family of pattern recognition receptors that form part of the innate immune response and are involved in the recognition of bacterial, fungal and viral pathogens. The only TLR that have been reported to recognise viral pathogens are TLR- 2, TLR-3, TLR-4, TLR-7 and TLR-9. The possible role of TLR during hepadnavirus infections had not been well characterized to date. In this project cDNA sequences for duck TLR-2, TLR-4 and TLR-7 were identified and characterised and qRT-PCR assays were developed for their detection. Changes in duck TLR-2, TLR-4 and TLR-7 mRNA expression during hepadnavirus infection were identified following DHBV infection of primary duck hepatocytes (PDH) in vitro. The results showed increased levels of expression of duck TLR early during infection indicating an involvement of TLR and the innate immune response during DHBV infection. During the in vivo DHBV infection studies performed to date TLR mRNA expression remained unchanged. As previously mentioned hepadnavirus infection can have an acute or chronic outcome. We aimed to understand the mechanisms involved during the resolution of acute DHBV infection and to elucidate specific factors contributing to the successful resolution of infection. During acute infections immune markers were monitored by qRT-PCR and histological analysis of fixed liver sections was performed. Liver sections were analysed to detect liver inflammation, the number and size of Kupffer cells, hepatocyte apoptosis and changes in hepatocyte proliferation throughout the course of acute DHBV infection in 6-week-old ducks. By determining the percentage of DHBV-positive hepatocytes two patterns of clearance of acute DHBV infection were observed; early clearance of infected hepatocytes occurring before day 14 post infection (p.i.), and late clearance occurring after day 14, but before or on day 31 p.i. This viral clearance was seen to occur in a cell by cell pattern. Higher levels of hepatocyte proliferation and apoptotic hepatocytes were detected during the clearance phase (on day 14 p.i.) of the late clearance group. Periodic acid schiff-diastase (PAS-D) staining was used to show significant increases in both cell number and size of Kupffer cells. Levels of IFN-γ mRNA increased significantly over the uninfected age-matched control ducks on day 14 p.i. Levels of CD3, CD4 and CD8 mRNA expression also increased over the uninfected controls on days 14 and 31 p.i. In summary, we found that resolution of acute DHBV infection occurred on a cell by cell pattern of clearance, it was accompanied by increases in hepatocyte proliferation, apoptotic hepatocytes and activated Kupffer cells, indicating that T lymphocytes and cell death play important roles in the rapid clearance of DHBV infection. Following resolution of acute hepadnaviral infections residual viral DNA has been found to persist. Residual HBV DNA in humans can result in reactivation of HBV infection following liver transplantation or immunosuppressive drug treatment. This leads to possible pathogenic outcomes thus the need for further investigations. Previous studies performed in the duck model have shown that the major form of residual DNA is present as covalently closed circular DNA (cccDNA). We aimed to understand how this residual cccDNA was being maintained and if replication was involved in the process. Following resolution of infection in ducks, levels of residual DHBV DNA were monitored by quantitative PCR (qPCR). Ducks were treated with the Bristol-Myers Squibb nucleoside analogue Entecavir (ETV) in order to suppress any possible replication that might be maintaining levels of residual cccDNA. In DHBV-infected but non-ETV treated ducks, the levels of residual DHBV DNA decreased gradually when measured on days 60, 221 and 316 p.i. The observed decrease in residual DHBV DNA occurred in parallel with decreases in the rate of hepatocyte proliferation measured by PCNA staining. This finding suggests that levels of residual DHBV DNA and hepatocyte proliferation are linked and we hypothesise that hepatocyte turnover is involved in the clearance of residual DHBV DNA. ETV treatment did not have an effect on the levels of residual DHBV DNA which suggests that it is present in a subset of long-lived hepatocytes that do not support virus replication. Mathematical modelling was performed to predict the rate of hepatocyte proliferation required for the elimination of residual cccDNA. The mathematical modelling showed that the predicted rate of hepatocyte proliferation was consistent with the rate of hepatocyte proliferation measured by PCNA. Further mathematical modelling showed that residual cccDNA is most likely to survive mitosis and it decreases due to several rounds of hepatocyte proliferation required for its elimination. Altogether, this project has elucidated mechanisms involved during the resolution of acute DHBV infection and also possible mechanisms by which residual DHBV DNA is maintained following resolution of infection. Detailed understanding of the virological and immunological events that occur during the resolution of an acute hepadnavirus infection would assist in the development of new therapeutic treatments for the cure of chronic HBV infections. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345121 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 1997. / Typescript. Bibliography: leaves 189-204.

published_or_final_version / Surgery / Master / Master of Philosophy

Hepatitis B virus is a member of the hepadnavirus family. The hepatitis B virus core gene codes for two proteins viz. core protein and pre-core protein. These proteins assemble to form particles viz. HBcAg and HBeAg respectively. The structure of the HBcAg has been widely studied but very little is known about the structure of HBeAg. Therefore, the aim of this study was to identify the disulfide bonding patterns in HBeAg. Recombinant HBeAg was isolated from E.coli and used for this study along with various mutants of HBeAg. There are four cysteines present in HBeAg each at position -7, 48, 61 and 107. From this study it can be inferred that the cysteine at 61 and 48 were found to be involved in inter-molecular disulfide bonds between the cysteine at 61 and 48 of other identical monomers. These di-mers were further inter-molecularly linked with cysteine at -7 to form chains. Moreover, the cysteine at -7 and cysteine at 107 were sometimes involved in intra-molecular disulfide bond formation. Thus, the HBeAg in a solution was found be particulate with a heterogeneous pattern of inter chain disulfide bonds.

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