by Hiu-Ming Li. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 176-198). / 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. / Abstracts in English and Chinese.

by Ng Chor Yin Maggie. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 358-403). / 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.

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.

Lee Siu-wah. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves [106]-116). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Figures --- p.ix / List of Tables --- p.x / Abbreviations --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.2 --- Etiology of Hepatocellular Carcinoma --- p.5 / Chapter 1.2.1 --- Viral Infection --- p.5 / Chapter 1.2.1.1 --- Hepatitis B Virus --- p.5 / Chapter 1.2.1.2 --- Hepatitis C Virus --- p.7 / Chapter 1.2.2 --- Cirrhosis and Chronic Inflammation --- p.8 / Chapter 1.2.3 --- Aflatoxin --- p.9 / Chapter 1.3 --- Genetic Studies of Hepatocellular Carcinoma --- p.9 / Chapter 1.3.1 --- Conventional Cytogenetics --- p.9 / Chapter 1.3.2 --- Molecular Cytogenetics --- p.12 / Chapter 1.3.3 --- Molecular Genetic Studies --- p.12 / Chapter 1.3.3.1 --- Proto-oncogenes --- p.12 / Chapter 1.3.3.2 --- Tumour Suppressor Genes --- p.13 / Chapter 1.3.3.3 --- Cell Cycle Genes --- p.14 / Chapter 1.4 --- Background of Study --- p.16 / Chapter 1.5 --- Objectives of Study --- p.17 / Chapter Chapter 2 --- Material and Methods --- p.18 / Chapter 2.1 --- Materials --- p.19 / Chapter 2.2 --- Analysis of Chromosomal Imbalances by Comparative Genomic Hybridization --- p.23 / Chapter 2.2.1 --- Comparative Genomic Hybridization --- p.23 / Chapter 2.2.2 --- Methods / Chapter 2.2.2.1 --- Preparation of Normal Metaphases --- p.30 / Chapter 2.2.2.2 --- Extraction of High Molecular Weight DNA --- p.30 / Chapter 2.2.2.3 --- Labeling of DNA by Nick Translation --- p.31 / Chapter 2.2.2.4 --- Labeling Efficiency --- p.31 / Chapter 2.2.2.5 --- Preparation of Probe --- p.33 / Chapter 2.2.2.6 --- Hybridization --- p.33 / Chapter 2.2.2.7 --- Washing and Detection of Signals --- p.35 / Chapter 2.2.2.8 --- Image Acquisition and Analysis --- p.35 / Chapter 2.2.2.9 --- Control Experiments --- p.36 / Chapter 2.3 --- Positional Mapping of Novel Amplicon by Interphase Cytogenetics --- p.39 / Chapter 2.3.1 --- Fluorescence in situ Hybridization --- p.39 / Chapter 2.3.2 --- Using Yeast Artificial Chromosomes (YAC) as Probe --- p.41 / Chapter 2.3.3 --- Methods --- p.48 / Chapter 2.3.3.1 --- Culture of Yeast Artificial Chromosomes --- p.48 / Chapter 2.3.3.2 --- Extraction of Total YAC DNA --- p.48 / Chapter 2.3.3.3 --- Verification of YAC Clones for Chimerism by FISH --- p.49 / Chapter 2.3.3.4 --- Inter-Alu-PCR --- p.49 / Chapter Chapter 3 --- Assessment of Genetic Changes in HCC by Comparative Genomic Hybridization (CGH) --- p.57 / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and Methods --- p.58 / Chapter 3.2.1 --- Patients and Specimens --- p.58 / Chapter 3.2.2 --- Comparative Genomic Hybridization --- p.60 / Chapter 3.2.3 --- Statistical Analysis --- p.60 / Chapter 3.3 --- Results --- p.61 / Chapter 3.3.1 --- Overall Copy Number Aberrations in 67 HCC and Surrounding Cirrhotic Tissues --- p.61 / Chapter 3.3.2 --- TNM Staging --- p.61 / Chapter 3.3.3 --- Tumour Size --- p.72 / Chapter 3.3.4 --- Serum AFP Elevation --- p.72 / Chapter 3.3.5 --- Chromosomal Aberrations in HCC arising from Cirrhotic and Non- cirrhotic Livers --- p.72 / Chapter 3.4 --- Discussion --- p.73 / Chapter 3.4.1 --- Recurrent Gains --- p.73 / Chapter 3.4.2 --- Recurrent Losses --- p.75 / Chapter 3.4.3 --- Tumour Progression --- p.76 / Chapter 3.5 --- Conclusion --- p.78 / Chapter Chapter 4 --- Positional Mapping of a Novel Amplicon on Chromosome 1q21-q25 by Interphase Cytogenetics --- p.79 / Chapter 4.1 --- Introduction --- p.80 / Chapter 4.2 --- Materials --- p.82 / Chapter 4.3 --- Methods --- p.82 / Chapter 4.3.1 --- Preparation of Paraffin-embedded Tissue Sections --- p.82 / Chapter 4.3.2 --- Verification of YAC Probes for Chimerism --- p.83 / Chapter 4.3.3 --- Hybridization Efficiency of Test and Reference Probes --- p.83 / Chapter 4.3.4 --- Slide Pretreatment and FISH with YAC Probes --- p.88 / Chapter 4.3.5 --- Scoring of FISH Signals --- p.88 / Chapter 4.4 --- Results --- p.89 / Chapter 4.4.1 --- Relative Copy Number Gain --- p.89 / Chapter 4.4.2 --- Intratumour Heterogeneity --- p.90 / Chapter 4.5 --- Discussion --- p.90 / Chapter 4.6 --- Further Studies --- p.104 / Chapter 4.6.1 --- Fine Mapping of Chromosomal Region 1 q21 --- p.104 / Chapter 4.6.2 --- Isolation of Novel Genes in the Amplicon --- p.105 / Chapter 4.6.3 --- Expression Status of the EDC Genes --- p.105 / References --- p.106

Lam Wai-Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves [64]-[73]). / Abstracts in English and Chinese. / Abstract (in English) --- p.i-ii / Abstract (in Chinese) --- p.iii -iv / Acknowledgements --- p.v / Table of contents --- p.vi -ix / List of Figures --- p.x / List of Tables --- p.x / Abbreviations --- p.xi -xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatocellular Carcinoma (HCC) --- p.1-2 / Chapter 1.2 --- Major risk factors of HCC / Chapter (1) --- Hepatitis B Virus (HBV) --- p.2-4 / Chapter (2) --- Hepatitis C Virus (HCV) --- p.5-6 / Chapter (3) --- Cirrhosis --- p.6 / Chapter (4) --- Dietary alfatoxin B1 (AFB1) --- p.6 -7 / Chapter (5) --- Alcoholic consumption --- p.7 / Chapter (6) --- Iron overload --- p.8 / Chapter 1.3 --- Genetic aberrations in HCC --- p.8-9 / Chapter (1) --- Chromosomal loss --- p.10-13 / Chapter (2) --- Chromosomal gains --- p.13-15 / Chapter 1.4 --- roposed study --- p.15 / Chapter (1) --- Hypomethylation of heterochromatin in chromosome 1q copy number gain. --- p.16 / Chapter (2) --- ositional mapping on 1q21 - q22 by interphase cytogenetics. --- p.16-17 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Southern Blot Analysis for Satellite DNA Hypomethylation. --- p.18-19 / Chapter 2.1.2 --- ositional Mapping by Interphase Cytogenetics. --- p.19 -24 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Southern Blot Analysis for Satellite DNA Hypomethylation / Chapter (1) --- Extraction of high molecular weight DNA --- p.25 / Chapter (2) --- DNA digestion with methyl-sensitive restriction enzyme --- p.25 -26 / Chapter (3) --- Control for the complete DNA digestion. --- p.26 / Chapter (4) --- Southern Blotting. --- p.26 -27 / Chapter 2.2.2 --- ositional Mapping by Interphase Cytogenetics / Chapter (1) --- Yeast Artificial Chromosome (YAC) --- p.28 -29 / Chapter (i) --- YAC culturing --- p.29 -30 / Chapter (ii) --- YAC DNA extraction --- p.30 -31 / Chapter (iii) --- Inter-Alu-Polymerase Chain Reaction --- p.32 -33 / Chapter (2) --- -1 derived Bacterial Artificial Chromosome (PAC) --- p.34 / Chapter (i) --- AC culturing and DNA extraction --- p.34 -35 / Chapter (3) --- FISHrobe labeling by nick translation. --- p.35 / Chapter (4) --- FISHrobereparation --- p.36 / Chapter (5) --- Dot-blot analysis. --- p.36 -37 / Chapter (6) --- Verification of the YAC andACrobes by metaphase FISH --- p.37 / Chapter (7) --- Hybridization efficiency test --- p.38 / Chapter Chapter 3 --- Southern Blot Analysis for Satellite DNA Hypomethylation / Chapter 3.1 --- Introduction --- p.39 -40 / Chapter 3.2 --- Materials and Methods / Chapter (1) --- atients --- p.41 / Chapter (2) --- Mathyl-sensitive restriction enzyme digestion. --- p.42 / Chapter (3) --- Classical satellite 2 DNArobe labeling and hybridization. --- p.42 -43 / Chapter (4) --- Membrane washing and signal detection. --- p.43 / Chapter (5) --- Signal detection and reference ratio determination. --- p.43 -44 / Chapter (6) --- Comparative Genomic Hybridization (CGH) --- p.44 -45 / Chapter 3.3 --- Results / Chapter (1) --- Heterochromatin hypomethylation and 1q12 breakpoint. --- p.45 / Chapter (2) --- Heterochromatin hypomethylation in adjacent hepatitis Infected liver tissue. --- p.46 / Chapter 3.4 --- Discussion --- p.47-51 / Chapter Chapter4 --- ositional Mapping of 1q21 - q22 by Interphase Cytogenetics / Chapter 4.1 --- Introduction --- p.52-53 / Chapter 4.2 --- Materials and Methods / Chapter (1) --- atients --- p.53 / Chapter (2) --- YAC clones --- p.53 -54 / Chapter (3) --- AC clones --- p.55 / Chapter (4) --- Formalin-fixedaraffin-embedded tissue sections pretreatment. --- p.55 / Chapter (5) --- Hybridization --- p.56 / Chapter (6) --- Signal detection --- p.56 -57 / Chapter 4.3 --- Results / Chapter (1) --- Relative copy number gain on YAC examined. --- p.57 -59 / Chapter (2) --- AC findings --- p.60 / Chapter 4.4 --- Discussion --- p.60 -63 / References

by Ray, Kit Ng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 112-121). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgments --- p.iv / Abbreviations --- p.x / List of Figures --- p.xii / List of Tables --- p.xiv / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatitis B Virus (HBV) --- p.1 / Chapter 1.2 --- Hepatitis B Virus X Protein (HBx) --- p.5 / Chapter 1.2.1 --- The Genomic Structure of HBx --- p.5 / Chapter 1.2.2 --- The HBx Protein Structure --- p.6 / Chapter 1.2.3 --- Subcellular Localization of HBx --- p.7 / Chapter 1.2.4 --- Possible Functions of HBx --- p.8 / Chapter 1.3 --- Etiology of Hepatocellular Carcinoma (HCC) --- p.12 / Chapter 1.4 --- Relationship between HCC and HBx --- p.13 / Chapter 1.5 --- Aims of Study --- p.14 / Chapter 1.6 --- The Basis of Tet-On System --- p.15 / Chapter 1.7 --- The Basis of DNA Microarray --- p.18 / Chapter 1.8 --- The Basis of Two-Dimensional Electrophoresis --- p.20 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of a Tet-On HBx Expressing Cell Model --- p.22 / Chapter 2.1.1 --- Cloning of HBx Gene into pTRE2 Vector --- p.22 / Chapter 2.1.1.1 --- PCR of HBx Gene --- p.22 / Chapter 2.1.1.2 --- Purification of the PCR Product --- p.23 / Chapter 2.1.1.3 --- Restriction Enzyme Digestion --- p.23 / Chapter 2.1.1.4 --- Ligation of HBx into pTRE Vector --- p.24 / Chapter 2.1.1.5 --- Transformation of the Ligation Product into Competent Cells --- p.24 / Chapter 2.1.2 --- Preparation of the Plasmid DNA --- p.24 / Chapter 2.1.2.1 --- DNA Sequencing of the Cloned Plasmid DNA --- p.25 / Chapter 2.1.3 --- Cell Culture of AML12 Cell Line --- p.26 / Chapter 2.1.4 --- Transfection of pTet-On Vector into AML12 Cells --- p.26 / Chapter 2.1.5 --- Selection of the Transfected AML12 Cells by G418 --- p.27 / Chapter 2.1.6 --- Single Clone Isolation --- p.27 / Chapter 2.1.6.1 --- Luciferase Assay for Selection of Highly Inducible Clones --- p.28 / Chapter 2.1.7 --- Second Transfection of pTRE-HBx Plasmid --- p.28 / Chapter 2.1.8 --- Selection of the Transfected Cells by Hygromycin --- p.29 / Chapter 2.1.9 --- Second Single Clone Isolation --- p.29 / Chapter 2.1.10 --- Total RNA Isolation --- p.29 / Chapter 2.1.11 --- DNase I Digestion --- p.30 / Chapter 2.1.12 --- First-Strand cDNA Synthesis --- p.31 / Chapter 2.1.13 --- RT-PCR of HBx Gene --- p.31 / Chapter 2.1.14 --- Northern Blotting --- p.32 / Chapter 2.1.15 --- Preparation of the Probe --- p.33 / Chapter 2.1.16 --- Northern Blot Hybridization --- p.33 / Chapter 2.1.17 --- 3H-Thymidine Incorporation Assay --- p.34 / Chapter 2.1.18 --- Analysis of Cell Cycle by Flow Cytometry --- p.35 / Chapter 2.2 --- Microarray Analysis of Differential Gene Expression upon HBx Induction --- p.35 / Chapter 2.2.1 --- Sample Preparation for Microarray Analysis --- p.35 / Chapter 2.2.2 --- Probe Labelling --- p.36 / Chapter 2.2.3 --- Microarray Hybridization --- p.37 / Chapter 2.2.4 --- RT-PCR of the Candidate Genes --- p.38 / Chapter 2.2.5 --- Northern Blot Analysis of the Candidate Genes --- p.39 / Chapter 2.3 --- Two-Dimensional (2D) Gel Electrophoretic Analysis --- p.40 / Chapter 2.3.1 --- Protein Sample Preparation for 2D Gel Electrophoresis --- p.40 / Chapter 2.3.2 --- First-Dimension Isoelectric Focusing (IEF) --- p.40 / Chapter 2.3.3 --- Second-Dimension SDS-PAGE --- p.41 / Chapter 2.3.4 --- Silver Stain of 2D Gel --- p.42 / Chapter 2.3.5 --- Mass Spectroscopic Analysis --- p.43 / Chapter 2.4 --- Subcellular Localization of HBx --- p.44 / Chapter 2.4.1 --- Cloning of HBx into Green Fluorescent Protein (GFP) Expression Vector --- p.44 / Chapter 2.4.2 --- Transfection of GFP-HBx --- p.44 / Chapter 2.4.3 --- Propidium Iodide (PI) Staining --- p.45 / Chapter 2.4.4 --- Mitochondria Staining --- p.45 / Chapter 2.4.5 --- Subcellular Localization Study using Epi-Fluorescent Microscopy --- p.45 / Chapter 2.5 --- Analysis of Mitochondrial Transmembrane Potential --- p.46 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Construction of Tet-On AML12 Cell Line of HBx Gene --- p.47 / Chapter 3.2 --- Characterization of the HBx-Expressing Cell Model --- p.53 / Chapter 3.2.1 --- 3H-Thymidine Proliferation Assay --- p.53 / Chapter 3.2.2 --- Cell Cycle Analysis --- p.55 / Chapter 3.3 --- Microarray Analysis of Differential Gene Expression Pattern upon HBx Induction --- p.57 / Chapter 3.4 --- Northern Blot Analysis and RT-PCR of the Candidate Genes --- p.65 / Chapter 3.5 --- Differential Protein Expression Pattern under HBx Induction --- p.70 / Chapter 3.6 --- Subcellular Localization of HBx --- p.77 / Chapter 3.7 --- Analysis of Mitochondrial Transmembrane Potential --- p.83 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Conditional HBx-Expressing Cell Model --- p.84 / Chapter 4.2 --- The Effects of HBx in Clone X18 --- p.86 / Chapter 4.2.1 --- Proliferative Effect of HBx --- p.86 / Chapter 4.2.2 --- Deregulation of G2/M Checkpoint by HBx --- p.86 / Chapter 4.3 --- Early Differential Gene Expression due to HBx Induction --- p.88 / Chapter 4.4 --- The Relationship of the Potential Candidate Genes and Cancer Development --- p.90 / Chapter 4.5 --- The Protein Expression Pattern due to HBx Induction --- p.93 / Chapter 4.6 --- The Subcellular Localization of HBx --- p.96 / Chapter 4.7 --- The Possible Involvement of HBx in Mitochondrial Transmembrane Potential --- p.98 / Chapter 4.8 --- Conclusions --- p.101 / Chapter 4.9 --- Future Prospects --- p.104 / Appendix --- p.107 / References --- p.112

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.