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

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.

In conclusion, two new methods of cccDNA quantitation were developed and validated. The two assays are complementary to each other and may be used in patients with extreme HBV DNA levels. These cccDNA assays should be further validated in larger studies and may become important tests for diagnostic, prognostic and treatment monitoring purposes. / Over 350 million people worldwide suffer from chronic hepatitis B virus (HBV) infection, which leads to many cases of cirrhosis and hepatocellular carcinoma. HBV covalently closed circular DNA (cccDNA) is a critical intracellular replicative intermediate and cannot be eliminated during antiviral therapy. Current methods for cccDNA detection are limited by false positive detection due to the interference by HBV relaxed circular DNA (rcDNA). The tests also have limited sensitivity to detect cccDNA at low concentrations. Hence, we aimed to develop a highly sensitive and highly specific assay for cccDNA detection with wide linear range. / The modified Bowden's assay had the highest intrahepatic cccDNA detection rate (60 positive results out of 61 cases). The detection rate of the modified Bowden's assay is significantly higher than that of the Bowden's assay. On the other hand, the cccDNA detection rate in serum samples was low at 20--27% by all 3 assays. In 5 samples in which cccDNA was undetectable by the Bowden's assay but detectable by the other two assays, a point mutation in the HBV genome was found in the forward primer binding site of the Bowden's assay. This partly explained the false negative results. / The quantification result of cccDNA by the bisulfite conversion assay was significantly lower than that by the Bowden's assay assay (P=0.001) and the modified Bowden's assay (P=0.003). When the total HBV DNA was higher than 107 copies/ml, the serum cccDNA level detected by the bisulfite conversion assay was significantly lower than that detected by the Bowden's assay (P=0.008) and the modified Bowden's assay (P=0.046). When the total HBV DNA is less than 107 copies/ml, there were no significant differences. This suggests that the bisulfite conversion assay was less affected by rcDNA even in samples containing a high viral load. / With this background, two new cccDNA assays were developed and optimized. Bowden's assay was used as a standard to evaluate the performance of new assays. The first new assay (modified Bowden's assay) involved the use of new primers and probes that targeted more conserved regions in the HBV genome. The second assay adopted the bisulfite conversion method, which introduced gene sequence changes into the HBV genome and thereby enhance the specificity of the assay. Capillary sequencing was performed to find mutations in primers and probe range of different assays. / Yu, Ling. / Advisers: Vincent Wai-Sun Wang; Joseph Jao-Yiu Sung. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 105-111). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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

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

Ng Wah. / Thesis submitted in: August 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 101-113). / Abstracts in English and Chinese. / ABSTRACT --- p.II / 摘要 --- p.IV / ACKNOWLEDGEMENT --- p.VI / TABLE OF CONTENTS --- p.VII / LIST OF TABLES --- p.X / LIST OF FIGURES --- p.XI / ABBREVIATIONS --- p.XII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Etiological Factors of Hepatocellualr Carcinoma (HCC) --- p.4 / Chapter 1.2.1 --- Dietary Aflatoxins --- p.4 / Chapter 1.2.2 --- Liver Cirrhosis --- p.5 / Chapter 1.2.3 --- Alcohol Abuse --- p.6 / Chapter 1.2.4 --- Viral Hepatitis Infection --- p.6 / Chapter 1.3 --- Literature Review on the Investigations of HBV Integrants in HCC --- p.16 / Chapter 1.3.1 --- Affected Host Junctions --- p.17 / Chapter 1.3.2 --- Viral Junctions --- p.18 / Chapter 1.4 --- Restriction Site Polymerase Chain Reaction (RS-PCR) --- p.19 / Chapter 1.5 --- Aims of Thesis --- p.21 / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Chemicals --- p.23 / Chapter 2.1.2 --- Buffers --- p.24 / Chapter 2.1.3 --- Cell Cultures --- p.24 / Chapter 2.1.4 --- Nucleic Acids --- p.24 / Chapter 2.1.5 --- Enzymes --- p.25 / Chapter 2.1.6 --- Equipment --- p.25 / Chapter 2.1.7 --- Software and Web Resources --- p.26 / Chapter 2.2 --- Methods --- p.27 / Chapter 2.2.1 --- DNA Extraction --- p.27 / Chapter 2.2.2 --- RS-PCR --- p.31 / Chapter 2.2.3 --- Sequencing --- p.37 / Chapter 2.2.4 --- Spectral Karyotyping (SKY) --- p.38 / Chapter 2.2.5 --- Fluorescence In situ hybridization --- p.39 / Chapter Chapter 3 --- Investigation of HBV Integration Sites in HCC Cell lines --- p.45 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Materials and Methods --- p.47 / Chapter 3.2.1 --- Cell Lines --- p.47 / Chapter 3.2.2 --- RS-PCR --- p.47 / Chapter 3.2.3 --- Spectral Karyotyping --- p.48 / Chapter 3.2.4 --- Tyramide Signal Amplification for HBV in FISH Analysis --- p.48 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Identification of HBV Integration Sites in Cell Lines --- p.51 / Chapter 3.3.2 --- Evaluation of RSO Primer Efficiency --- p.52 / Chapter 3.3.3 --- SKY and FISH Analysis --- p.53 / Chapter 3.4 --- Discussion --- p.64 / Chapter 3.4.1 --- HBV Insertions in HCC Cell Lines --- p.64 / Chapter 3.4.2 --- Efficacy of RSO Primers --- p.65 / Chapter 3.4.3 --- Investigation of HBV Integration on Chromosomal Rearrangement --- p.65 / Chapter Chapter 4 --- Investigation of Hepatitis B Virus Integration Sites in Primary HCC --- p.67 / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Materials and Methods --- p.69 / Chapter 4.2.1 --- Patients --- p.69 / Chapter 4.2.2 --- RS-PCR --- p.70 / Chapter 4.3 --- Results --- p.72 / Chapter 4.3.1 --- HBV Integration Sites in Primary HCC Tumors and Adjacent Non- malignant Liver --- p.72 / Chapter 4.4 --- Discussion --- p.88 / Chapter 4.4.1 --- HBV integration Sites in Primary HCC Tumors and Adjacent Non- malignant Liver --- p.88 / Chapter 4.4.2 --- Summary on HBV Integrants Identified --- p.91 / Chapter Chapter 5 --- Proposed Future Studies --- p.98 / Chapter 5.1 --- Correlation of Structural Aberrations with HBV Integrations --- p.99 / Chapter 5.2 --- Transcriptional Expression Study on the Genes Interrupted by or Located near the Virus Host Junctions --- p.100 / Chapter Chapter 6 --- References --- p.101

Hepatocellular carcinoma (HCC) is a highly malignant tumor that is prevalent in Southeast Asia and China, where hepatitis B viral (HBV) infection is the main etiologic factor. Despite a high incidence of HCC developing in patients with HBV-induced liver cirrhosis, the molecular events underlying the malignant liver progression remain largely unclear. In an effort to characterize the genetic abnormalities involved in the HBV-related liver carcinogenesis, genome-wide exploration by metaphase comparative genomic hybridization (CGH) was performed on 100 cirrhotic HCC tumors that were derived from chronic hepatitis B carriers. CGH analysis indicated chromosomal instability in both early and advanced stage tumors where common genomic copy gains on 1q, 8q and 17q, and deletions on 4q, 8p, 13q, 16q and 17p found in both groups are suggestive of early events in hepatocarcinogenesis. Nevertheless, a combined univariate and multivariate statistical analyses highlighted for the first time preferential regional 3q26-q28, 7q21-q22 and 7q34-q36 gains in association with advanced stage HCC. The novel aberrant gains identified here thus formed basis for further mapping analysis for causative genes related to HCC progression in this thesis. / Near 50% of the advanced stage HCC manifested copy gains of chr 7q21-q22. High resolution mapping analysis by cDNA microarray-based CGH nominated 13 amplified candidates within the region 7q21-q22 Analysis on the mRNA expresson levels of these genes in a cohort of primary HCC compared to paired adjacent non-tumorous liver tissues by quantitative RT-PCR (qRT-PCR) indicated the up-regulation of the PFTK1 (PFTAIRE protein kinase 1) gene as the only candidate that demonstrated a close association with advanced metastatic tumors. The effects of PFTK1 on cell proliferation, migration and invasive phenotypes were further studied to substantiate its role in HCC progression. Upon gene suppression of PFTK1 in vitro by RNA interference (RNAi), a significant reduction in chemotactic migration, cellular invasion and an inhibition on cell motility were indicated, albeit cell proliferation remained unaffected. / Sub-cellular localization study of translated PFTK1 protein indicated protein localization in both the nucleus and cytoplasm. This has led to the further investigations of potential PFTK1 function at both the transcriptional and protein levels. (Abstract shortened by UMI.) / Sy Ming Hui. / "July 2005." / Advisers: Winnie Yeo; Nathalie Wong. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3571. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 124-139). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

A case-control study was previously carried out to identify HCC-associated genomic markers on HBV. Some of them are clustered at the preS1 and X promoter regions of HBV genotype B and core promoter of HBV subgenotype Cs. The functional significance of these markers to the virus was investigated in our study. Our result showed that one of those markers, the G1613A mutation on core promoter, can significantly increase the promoter activity in a genotype-dependent manner and the effect is reversible by the A-to-G back mutation. We have established an in vitro full-length HBV genome transfection system and the result suggested that the G1613A mutation suppressed the e antigen (HBeAg) secretion and enhanced virus DNA production by downregulating the precore (preC) mRNA transcription. In consistence to the clinical study, the mutation was associated to serum HBV DNA level higher than 6 log copies/1M in female HBV carriers in a univariate analysis. In addition, we demonstrated that the G1613A mutation is a hot spot mutation situated on the negative regulatory element (NRE) on the core promoter in an alignment analysis. To further investigate the molecular mechanism of the mutation, two unknown protein complexes had been shown to bind on the NRE. They showed different binding affinity to the G1613-wild-type and A1613-mutant NRE sequence. Moreover, we showed that in vitro synthesized RFX1 protein could bind to the mutated NRE probe at a higher affinity than that to wild-type NRE probe. Overall, our result suggests that the G1613A mutation exerts its effect by differential binding to some proteins via the NRE region. Studying the mechanism of the mutations may provide insights to the viral pathogenesis and HBV-associated HCC, which has long been a health burden in Asia-Pacific countries. / Infection of hepatitis B virus (HBV) causes acute and chronic hepatitis and is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC). Approximately 60-80% of world's HCC is related to HBV, and it is the third most common cause of cancer death in Asia-Pacific region. Almost 400 million people are chronically infected with HBV and one-third was likely to die of complications of cirrhosis, including liver failure and HCC. As there is a shortage of effective curative treatments, detection and prognosis of the risk of cancer development will be essential to improve survival of patients with chronic HBV infection. / Li, Man Shan. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 198-210). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Further functional investigation revealed that knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells, concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium Conversely, overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-beta 1 (IFN-beta1). In this connection, IFN-beta1-mediated 2', 5'-oligoadenylate synthetase (OAS) and ribonuclease L (RNase L) signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover, GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p= 0.019) as compared with their counterpart pre-treatment liver biopsies. / Hepatitis B virus (HBV) infection is a global public health problem, which plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis and hepatocellular carcinoma. Although considerable progress has been made over the past decade, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. / In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via IFN-beta1-OAS-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection. / In this study, we applied a two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic approach to globally analyze the host early response to HBV by using an inducible HBV-producing cell line HepAD38. Twenty-three proteins were identified as differentially expressed, with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, as well as in HBV-infected human liver biopsies by immunohistochemistry. / Ma, Yan. / Adviser: Ming-Liang He. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 111-129). / 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. / Abstract also in Chinese.

Lau, Chi Chiu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 133-142). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.