Li Ching-wan. / Thesis submitted in: November 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 104-130). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACTS (ENGLISH/CHINESE) --- p.iii / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xiii / LIST OF ABBREVIATIONS --- p.xiv / Chapter CHAPTER1 --- INTRODUCTION / Chapter 1.1. --- Liver Cancer --- p.1 / Chapter 1.2. --- Hepatocellular Carcinoma --- p.1 / Chapter 1.2.1. --- Types of Liver Cancer --- p.1 / Chapter 1.2.2. --- Epidemiology --- p.4 / Chapter 1.2.2.1. --- Geographical Distribution --- p.4 / Chapter 1.2.2.2. --- Age and Gender Distribution --- p.8 / Chapter 1.2.3. --- Etiologic Factors --- p.9 / Chapter 1.2.3.1. --- Chronic Infection with Hepatitis B (HBV) and C (HCV) Viruses --- p.9 / Chapter 1.2.3.2. --- Aflatoxin B1 --- p.11 / Chapter 1.2.3.3. --- Alcohol --- p.12 / Chapter 1.2.3.4. --- Summary --- p.12 / Chapter 1.3. --- HCC in Hong Kong --- p.14 / Chapter 1.4. --- Role of Viral Hepatitis B in HCC --- p.17 / Chapter 1.4.1. --- HBV Genome --- p.17 / Chapter 1.4.2. --- Consequences of HBV DNA Integration --- p.17 / Chapter 1.4.2.1. --- HBV Integration --- p.17 / Chapter 1.4.2.2. --- Transactivation of Cellular Genes by HBV DNA --- p.19 / Chapter 1.4.2.3. --- Chromosomal DNA Instability --- p.20 / Chapter 1.5. --- Genetic Alterations in HCC --- p.21 / Chapter 1.5.1. --- Tumor Suppressor Gene --- p.21 / Chapter 1.5.2. --- Proto-oncogene --- p.23 / Chapter 1.5.3. --- Genetic Studies in HCC --- p.23 / Chapter 1.5.3.1. --- Loss of Heterozygosity (LOH) --- p.25 / Chapter 1.5.3.2. --- Comparative Genomic Hybridization (CGH) --- p.26 / Chapter 1.5.3.3. --- Array CGH --- p.26 / Chapter 1.5.4. --- Large-Scale Genetic Analysis in HCC --- p.27 / Chapter CHAPTER2 --- RATIONALE IN THIS STUDY --- p.35 / Chapter CHAPTER3 --- MATERIALS AND METHODS / Chapter 3.1. --- Patients and Materials --- p.38 / Chapter 3.1.1. --- DNA Extraction --- p.40 / Chapter 3.2. --- Loss of Heterozygosity Analysis on Chromosome 4q --- p.40 / Chapter 3.2.1. --- Microsatellite Markers --- p.41 / Chapter 3.2.2. --- Amplification of Target Sequences by PCR --- p.42 / Chapter 3.2.2.1. --- 5-end Labeling Primers --- p.42 / Chapter 3.2.2.2. --- Amplification of Target Sequences --- p.42 / Chapter 3.2.3. --- Denaturing Polyacrylamide Gel --- p.44 / Chapter 3.2.3.1. --- Electrophoresis --- p.44 / Chapter 3.2.4. --- Detection of Loss of Heterozygosity (LOH) --- p.45 / Chapter 3.2.5. --- Duplex PCR Analysis of Homozygous Deletion --- p.45 / Chapter 3.3. --- Amplification Analysis by Array-CGH --- p.46 / Chapter 3.3.1. --- Nick-Translation --- p.49 / Chapter 3.3.2. --- Hybridization --- p.49 / Chapter 3.3.3. --- Imaging and Data Analysis --- p.50 / Chapter 3.3.4. --- Determination of Normal Range for All Cases --- p.51 / Chapter 3.3.5. --- Assessment of Data Quality --- p.51 / Chapter 3.4. --- Statistical Analysis --- p.52 / Chapter CHAPTER4 --- RESULTS / Chapter 4.1. --- Loss of Heterozygosity Analysis on Chromosome 4q --- p.53 / Chapter 4.1.1. --- Region I of Smallest Common Deletion Region --- p.54 / Chapter 4.1.2. --- Region II of Smallest Common Deletion Region --- p.54 / Chapter 4.2. --- Amplification Analysis by Array-CGH --- p.62 / Chapter CHAPTER5 --- DISCUSSION / Chapter 5.1. --- LOH Analysis on Chromosome 4q --- p.73 / Chapter 5.1.1. --- LOH of Chromosome 4q in Various Cancers --- p.74 / Chapter 5.1.1.1. --- Hepatocellular Carcinomas --- p.74 / Chapter 5.1.1.2. --- Other Neoplasia --- p.76 / Chapter 5.1.2. --- Functional Studies on Chromosome 4 --- p.76 / Chapter 5.1.3. --- Putative Tumor Suppressors on Chromosome 4q --- p.80 / Chapter 5.1.3.1. --- Region I (4q27-q28.1) --- p.80 / Chapter 5.1.3.1.1. --- MAD2L1 (4q27) --- p.80 / Chapter 5.1.3.2. --- Region II (4q35.2) --- p.81 / Chapter 5.1.3.2.1. --- INGlL(4q35.1) --- p.81 / Chapter 5.1.3.2.2. --- FAT (4q34-q35) --- p.81 / Chapter 5.1.3.2.3. --- Caspase 3 (4q35) --- p.82 / Chapter 5.1.4. --- Limitation of this Study --- p.83 / Chapter 5.1.4.1. --- Markers --- p.83 / Chapter 5.1.4.1.1. --- Limitation of the Markers --- p.83 / Chapter 5.1.4.1.2. --- Location of the Microsatellite Markers --- p.83 / Chapter 5.1.4.2. --- Tissue Samples --- p.84 / Chapter 5.1.4.2.1. --- Normal Reference --- p.84 / Chapter 5.1.4.2.2. --- Pathologic Characterization --- p.85 / Chapter 5.1.5. --- Future Studies --- p.85 / Chapter 5.1.5.1. --- Improvement of the Experiment --- p.85 / Chapter 5.1.5.2. --- Extension of the Present Study --- p.86 / Chapter 5.2. --- Amplification Analysis by Array-CGH --- p.88 / Chapter 5.2.1. --- Amplicons Showing Amplification in HCC --- p.89 / Chapter 5.2.1.1. --- Locus of 17q23 --- p.89 / Chapter 5.2.1.1.1. --- D17S1670 --- p.89 / Chapter 5.2.1.1.2. --- RPS6KB1 --- p.91 / Chapter 5.2.1.2. --- Locus of 1q25-q31 --- p.92 / Chapter 5.2.1.2.1. --- LAMC2 --- p.92 / Chapter 5.2.1.3. --- Locus of 3q26.3 --- p.93 / Chapter 5.2.1.3.1. --- PIK3CA --- p.93 / Chapter 5.2.1.4. --- Locus of 8p22 --- p.94 / Chapter 5.2.1.4.1. --- CTSB --- p.94 / Chapter 5.2.1.5. --- Locus of 6q22 --- p.95 / Chapter 5.2.1.5.1. --- MYB --- p.95 / Chapter 5.2.1.6. --- Locus of 20ql3 --- p.96 / Chapter 5.2.1.6.1. --- CSE1L --- p.96 / Chapter 5.2.1.7. --- Locus of Ip36.2-p35.1 --- p.97 / Chapter 5.2.1.7.1. --- FGR --- p.97 / Chapter 5.2.1.8. --- Locus of 7q21.1 --- p.98 / Chapter 5.2.1.8.1. --- PGY1 --- p.98 / Chapter 5.2.2. --- Amplicons Showing Deletion in HCC --- p.99 / Chapter 5.2.2.1. --- Loss at 11ql3 and 14q32.3 --- p.99 / Chapter 5.2.3. --- Limitation of the Study --- p.100 / Chapter 5.2.3.1. --- Samples and Materials --- p.100 / Chapter 5.2.4. --- Further Study --- p.101 / Chapter 5.2.4.1. --- Confirmation of the Result in Various Levels --- p.101 / Chapter 5.2.4.2. --- Assessment of the Significant Losses on Chromosomes 11ql3 and 14ql3 --- p.101 / Chapter 5.2.5. --- Application of Microarray in Genetic Studies --- p.102 / Chapter 5.2.5.1. --- Deletion Analysis --- p.102 / Chapter 5.2.5.2 --- Tissue Microarray --- p.103 / Chapter 5.2.5.3. --- cDNA Microarray --- p.103 / Chapter chapter6 --- references --- p.104

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