After alignment of 300 HBV sequences randomly downloaded from GenBank, we found that the frequency of A1762T and G1764A mutations in genotype C was found as high as 64%, while 34% was found for other genotypes (A, B, D to H). Besides, recent clinical studies have also shown that A1762T/G1764A mutations occur frequently in HCC patients with genotype B infection (81%, 30 of 37 patients), but were relatively lower in asymptomatic carriers (43%, 22 of 51 patients). These indicate that the contribution of A1762T/G1764A mutations to liver cancer might not be limited to genotype C. As the double mutations are present within the region of HBV Enhancer II/Basal core promoter (BCP) and cause residue substitution of HBx (Lys130Met and Val131Ile); therefore, their effects on the promoter and HBx activities were examined. / Chronic infection of hepatitis B virus (HBV) increases the risk of hepatocellular carcinoma (HCC) by more than 100-fold. However, the underlying molecular mechanism of this process is not fully understood. Several recent studies have shown that A1762T and G1764A mutations of HBV were associated with the aggressiveness of liver disease, in which inactive carriers would develop active hepatitis, and eventually liver cirrhosis and HCC. In Asia, genotypes B and C are the predominant genotypes of HBV infections. Our longitudinal five-year follow-up study of 426 chronic hepatitis B patients in Hong Kong found that the genotype C HBV (normally with A1762T/G1764A mutations) was closely associated with higher risk of HCC than genotype B HBV (non-frequent mutations with A1762T/G1764A). / In this study, systemic site-directed mutagenesis studies, promoter assays, replication capacity assays and overexpression of HBx assays were carried out to demonstrate the molecular mechanisms of these mutations for the increases risk of HCC. Three conclusions were drawn from this study. (1) A1762T and/or G1764A mutations of HBV could reduce BCP activities in a synergistic manner with 1764A contributing more. Reversed T1762A and/or A1764G mutations increase the BCP activities also in a synergistic manner with 1764G contributing more; (2) HBx could increase HBV BCP activity, HBV replication and HBsAg expression. The Lys130Met and Val131Ile mutations of HBx could further increase the above abilities while the A1762T/G1764A double mutations in the BCP region could not affect the interaction of HBx and HBV BCP; (3) The G1677T/A1679C and T1706C mutations could increase the BCP activity; The ectopic expression of HBx could further increase the BCP activity while the mutated HBx (130Met and 131Ile) has less effect on these mutated promoters. / Dong, Qingming. / Adviser: Ming-Liang He. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: December 2008. / Thesis submitted in: December 2008. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 132-154). / 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. / Abstracts 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.

Song Jingzheng. / "June 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 170-186). / 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.

研究背景和目的 / 肝癌是非常高死亡率的恶性肿瘤之一。由于传统化疗方式的局限性，表观遗传治疗方法可能成为肝癌治疗的替代方法。研究报道ZBP-89诱导肝癌细胞Bak的表达，表观调控是否参与该诱导作用，目前仍然不清楚。 / HDAC3被认为是化疗靶点和肝癌复发的肿瘤标记物。它常常在肝癌组织中高表达，对HDAC3的抑制作用可以增加肝癌的化疗效果。我们的研究表明ZBP-89可以降低肝癌细胞HDAC3的表达，但机制未明。蛋白的翻译后调控是细胞生化过程的重要调节因素。所以，研究调节HDAC3的降低途径对肝癌的发生和复发具有非常重要的研究意义。 / 本研究旨在研究ZBP-89调控Bak表达的表观遗传机制。同时，弄清楚DNA甲基化转移酶和组蛋白去乙酰化酶是否参与ZBP-89对Bak的调控作用，进一步阐明ZBP-89对HDAC3降低通路的机制。 / 方法和结果 / 肝癌病人组织蛋白分析表明，相对于癌旁组织，肝癌组织Bak和ZBP-89蛋白表达降低，而DNMT1和HDAC3表达升高。免疫共沉淀技术显示ZBP-89与HDAC3、 DNMT1结合，但不与HDAC4， DNMT3a和DNMT3b结合。相应地，HDAC3和 DNMT1免疫沉淀分析也显示三者形成免疫复合物。我们在肝癌细胞中过表达ZBP-89，验证它会不会影响HDACs和DNMTs的活性。实验结果表明过表达的ZBP-89抑制HDACs和DNMTs的活性。进一步发现ZBP-89调节的Bak表达可能是通过抑制HDACs活性和维持组蛋白H3和H4乙酰化水平实现的。另一方面，我们同样证明HDAC的抑制剂（HDACi）VPA和TSA可以诱导肝癌细胞Bak表达，此外，siRNA干扰HDAC3的表达同样可以诱导Bak表达。 / 对DNMT1表达的抑制和使用DNMT抑制剂（DNMTi）Zebularine也可以诱导Bak的表达。染色质免疫沉淀结果显示ZBP-89结合于Bak的启动子区域，从-3188bp到-3183bp，从-275到-49。 ZBP-89可以抑制DNMT的活性，那么ZBP-89是否会影响DNA中CpG岛甲基化状态和甲基化结合蛋白（MeCP2）的结合能力，这一点仍需要进一步证实。结果表明ZBP-89可以抑制MeCP2结合基因组DNA。为进一步揭示MeCP2是否由于启动子区域CpG岛去甲基化影响其结合能力，我们采用亚硫酸盐测序方法。测序结果显示ZBP-89过表达可以影响Bak启动子CpG岛的甲基化状态，并促进其去甲基化。 / 腺病毒介导的ZBP-89过表达降低HDAC3表达呈现剂量依赖性，然而HDAC3 的mRNA水平并没有受到ZBP-89的表达。免疫共沉淀方法和蛋白免疫印迹实验用于分析Pin1和HDAC3复合物，磷酸化IκB和HDAC3复合物的结合情况。结果表明Pin1结合HDAC3并促进HDAC3的减少。同时，HDAC3与磷酸的IκB结合并进入蛋白减少途径。 / 构建的mU6-siPin1表达质粒用于敲除肝癌细胞Pin1的表达，方法检测基因表达水平。Pin1的缺失表达阻碍ZBP-89介导的HDAC3降低。在Pin1 敲除细胞系 JB6 C141 Pin1⁻/⁻ 和Pin1过表达细胞系的研究，ZBP-89更加能促进Pin1⁺/⁺细胞中HDAC3减少，而对Pin1⁺/⁺的细胞则没那么明显。由此肯定了Pin1在ZBP-89介导的HDAC3降低中的重要作用。进一步研究发现， IκB激酶 (IKK)抑制剂，CAY10576，能抑制 ZBP-89介导的HDAC3的降低；而SN50, p65/p50人核抑制多肽，则不影响HDAC3的降低。研究结果证明HDAC3的降低依赖IκB通路，而不是NF- κB活性。 / 我们用人肝癌细胞的裸鼠移植瘤模型研究ZBP-89调控Bak表达的表观遗传机制，及其对肝癌的治疗效果。研究结果表明ZBP-89蛋白和组蛋白抑制剂VPA和DNA甲基化抑制zebularine都能抑制肿瘤的生长，并诱导肿瘤组织Bak表达及细胞凋亡。VPA和zebularine联合治疗的效果更好。研究也表明ZBP-89可以在体内降低HDAC3蛋白水平。 / 结论 / 本研究揭示了ZBP-89调节Bak蛋白表达和肝癌细胞凋亡的表观遗传机制。同时，进一步揭示ZBP-89联合Pin1经由IκB通路调节HDAC3降低的机制. 本研究为肝癌表观遗传学的治疗提供研究基础和科学依据。 / Background / Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide with a very high mortality. Because the success of the conventional therapies is limited, epigenetic therapy may represent an alternative for HCC management. ZBP-89 is known to induce Bak in HCC. However, it is unclear whether epigenetic mechanisms contribute to ZBP-89-mediated Bak. / Histone acetylase 3 (HDAC3) is realized as a chemotherapy target and a biomarker of recurrence in HCC. HDAC3 is frequently overexpressed in HCC and its inhibition enhances the efficacy of anti-HCC chemotherapy. The pilot data have indicated that ZBP-89 reduced HDAC3 in HCC but the mechanism responsible was unknown. The post-translational modification of proteins functions as a key regulatory factor in cellular physiological procedures, such as ubiquitinoylation degradation. As a biomarker of HCC development and recurrence, it is important to understand how ZBP-89 mediates the reduction of HDAC3. / This study focuses on if ZBP-89 regulates Bak expression through epigenetic mechanisms. It is designed to investigate whether DNA methyltransferases (DNMTs), histone acetylases (HDACs) are involved in regulation of ZBP-89-induced Bak expression. The study also elucidates the mechanism how ZBP-89 reduces the level of HDAC3 protein. / Methods and Results / The levels of Bak and ZBP-89 as shown on western blots were reduced but DNMT1 and HDAC3 were increased in HCC cancer tissues compared to the corresponding non-cancer tissues. Co-immunoprecipitation experiments showed that ZBP-89 bound to HDAC3 and DNMT1 but not other epigenetic enzymes, such as HDAC4, DNMT3a and DNMT3b. To clarify if ZBP-89 affects the activities of HDACs and DNMTs, ZBP-89 was overexpressed in HCC cells. Enzyme activities of HDACs and DNMTs were determined using relevant assay kits. Results showed that overexpressed ZBP-89 inhibited the activities of HDACs and DNMTs. Further experiments indicated that ZBP-89-mediated Bak up-regulation might contribute to maintenance of histone H3 and H4 acetylation through inhibition of HDACs activity. In another set of experiments, we also found an increased Bak expression in HCC cells when the cells were treated with HDAC inhibitors (HDACi) VPA and TSA. HDAC3 siRNA also increased Bak expression. / Both knockdown of DNMT1 expression and administration of DNMTs inhibitors (zebularine) induced Bak expression. Chromatin immunoprecipitation (ChIP) showed that ZBP-89 bound to Bak promoter at the region from -3188bp to -3183bp and from -275 to -49. As ZBP-89 inhibits DNMT activity, it is essential to know whether its inhibition affectes DNA CpG methylation status and methyl-CpG binding protein (MeCP) binding. The results showed that ZBP-89 overexpression inhibited MeCP2 binding to genomic DNA. The finding indicated that decreased MeCP2 binding to DNA might be due to decreased methyl-CpG number in Bak promoter, suggesting that ZBP-89 might affect CpG island methylation status. Therefore, the bisulfite modified DNA sequencing method was used to clarify if Bak promoter CpG island methylation status was altered after ZBP-89 overexpression. Results revealed that ZBP-89 overexpression could demethylate the CpG islands in Bak promoter. / ZBP-89 overexpression dose-dependently reduced the expression of HDAC3 at protein level but not at mRNA level. Co-immunoprecipitation and western blot methods were used to analyze Peptidyl-prolyl cis/trans isomerase 1 (Pin1) and HDAC3, phospho-I kappa B (pIκB), and the result revealed that HDAC3 could bound with either Pin1 or pIκB to promote the reduced expression of HDAC3. / Constructed mU6-siPin1 vector was used to knockdown Pin1 expression in HCC cells. We found that knockdown of Pin1 expression blocked ZBP-89-mediated HDAC3 reduction. Experiments performed in Pin1 allele-knockdown JB6 C141 Pin1⁻/⁻ and Pin1⁺/⁺ cells showed that the reduction of HDAC3 by ZBP-89 was greater in Pin1⁺/⁺ cells than in Pin1⁻/⁻ cells, confirming the role of Pin1 in ZBP-89-mediated HDAC3 reduction. Furthermore, the ZBP-89-mediated HDAC3 reduction was suppressed by CAY10576, an IκB kinase (IKK) activation inhibitor but not by SN50, a p65/p50 translocation inhibitor, suggesting that HDAC reduction may depend on IκB kinase rather than NF-κB activity. / HCC xenograft mouse model was used to support the involvement of epigenetic mechanism in ZBP-89-induced Bak expression and its therapeutic effects against HCC. Results showed that ZBP-89 as well as HDAC inhibitor valproic acid (VPA) or/and DNMT inhibitor zebularine stimulated Bak expression and induced apoptosis of tumor cells in an HCC xenograft mouse model, arresting tumor growth. In HCC xenografe model, treatment by injection of Ad-ZBP-89 viral expression vector mediated ZBP-89 expression decreased HDAC3 expression, but not HDAC4. / Conclusions / In conclusion, the study demonstrates a novel mechanism through which ZBP-89 mediates an epigenetic pathway to promote Bak expression, and induce apoptosis in HCC cells. It also reveals the mechanism of HDAC3 reduction by ZBP-89 is dependent on IκB, which requires the presence of Pin1. This pathway may help develop future epigenetic therapy against HCC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ye, Caiguo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 123-140). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.v / Publications --- p.viii / Acknowledgements --- p.ix / Abbreviations --- p.xi / List of Tables --- p.xiii / List of figures --- p.xiv / Chapter Chapter One: --- General Introduction --- p.1 / Chapter 1.1 --- Background --- p.2 / Chapter 1.2 --- The complexity of HDAC family and functions --- p.3 / Chapter 1.2.1 --- HDAC family --- p.4 / Chapter 1.2.2 --- Multifunction of HDACs --- p.6 / Chapter 1.3 --- HDACs and apoptosis --- p.6 / Chapter 1.3.1 --- HDAC regulates apoptotic-related gene expression --- p.9 / Chapter 1.3.2 --- HDACs regulate apoptosis through protein complexes --- p.18 / Chapter 1.3.3 --- HDACs mediates non-histone deacetylation and apoptosis --- p.21 / Chapter 1.3.4 --- HDACs degradation deficiency and apoptosis --- p.24 / Chapter 1.4 --- DNMTs and epigenetic modification --- p.25 / Chapter 1.4.1 --- DNMT family --- p.25 / Chapter 1.4.2 --- CpG islands methylation and HCC --- p.26 / Chapter 1.5 --- Perspectives --- p.28 / Chapter Chapter Two: --- ZBP-89 up-regulates Bak expression through inhibition the activity of HDACs and DNMTs --- p.30 / Chapter 2.1 --- Introduction --- p.31 / Chapter 2.2 --- Materials and Methods --- p.33 / Chapter 2.2.1 --- Hepatocellular carcinoma patient samples and cell lines --- p.33 / Chapter 2.2.2 --- Chemicals and reagents --- p.34 / Chapter 2.2.3 --- Cell proliferation --- p.34 / Chapter 2.2.4 --- Adenovirus infection of cells --- p.35 / Chapter 2.2.5 --- Apoptosis detection --- p.36 / Chapter 2.2.6 --- Transfection of siRNA and plasmid --- p.36 / Chapter 2.2.7 --- Co-immunoprecipitation (co-IP) --- p.37 / Chapter 2.2.8 --- Western blotting --- p.37 / Chapter 2.2.9 --- Immunohistochemistry and Immunofluorescence --- p.38 / Chapter 2.2.10 --- Chromatin immunoprecipitation --- p.38 / Chapter 2.2.11 --- Sodium bisulfite modified sequencing of Bak promoter --- p.40 / Chapter 2.2.12 --- Histone deacetylase activity assay --- p.41 / Chapter 2.2.13 --- DNA methyltransferases enzyme activity --- p.42 / Chapter 2.2.14 --- Xenograft animal model --- p.43 / Chapter 2.2.15 --- Statistical analysis --- p.43 / Chapter 2.3 --- Results --- p.45 / Chapter 2.3.1 --- ZBP-89 interacts with DNMT1 and HDAC3 --- p.45 / Chapter 2.3.2 --- DNA methyltransferase-1 and histone deacetylase 3 are overexpressed in cancer tissues --- p.48 / Chapter 2.3.3 --- Inhibition of HDACs and DNMTs induces Bak expression and apoptosis --- p.58 / Chapter 2.3.4 --- Adenovirus mediated ZBP-89 expression inhibits HDACs activity --- p.65 / Chapter 2.3.5 --- ZBP-89 suppresses DNMTs activity --- p.67 / Chapter 2.3.6 --- Overexpressed ZBP-89 demethylates methyl-CpG islands --- p.69 / Chapter 2.3.7 --- Downregulation of HDAC3 and DNMT1 enhances Bak expression --- p.74 / Chapter 2.3.8 --- Xenograft nude mouse model reveals that Ad-ZBP-89 adenovirus diminishes tumor volume and induces Bak expression and apoptosis --- p.75 / Chapter 2.4 --- Discussion --- p.81 / Chapter Chapter Three: --- ZBP-89 targets IkappaB to reduce HDAC3 via a Pin1-dependent pathway --- p.86 / Chapter 3.1 --- Introduction --- p.87 / Chapter 3.2 --- Materials and Methods --- p.89 / Chapter 3.2.1 --- Cell lines, chemicals and reagents --- p.89 / Chapter 3.2.2 --- Transfection of siRNA plasmid --- p.89 / Chapter 3.2.3 --- Plasmid extraction by mini-prep --- p.90 / Chapter 3.2.4 --- Co-immunoprecipitation (co-IP) and Western blotting --- p.91 / Chapter 3.2.5 --- Total RNA extraction --- p.92 / Chapter 3.2.6 --- Reverse transcription and real-time PCR --- p.93 / Chapter 3.2.7 --- Immunohistochemistry and Immunofluorescence --- p.94 / Chapter 3.2.8 --- Xenograft animal model --- p.95 / Chapter 3.2.9 --- Statistical analysis --- p.95 / Chapter 3.3 --- Results --- p.97 / Chapter 3.3.1 --- ZBP-89 overexpression diminishes HDAC3 expression but not HDAC4 --- p.97 / Chapter 3.3.2 --- Knockdown of Pin1 blocks ZBP-89-mediated HDAC3 reduction --- p.99 / Chapter 3.3.3 --- ZBP-89 reduces the level of IκB --- p.103 / Chapter 3.3.4 --- IκB degradation inhibitors suppresses ZBP-89-meditaed HDAC3 reduction --- p.105 / Chapter 3.3.5 --- ZBP-89 decreases HDAC3 but increases Bak in xenograft tumor tissues --- p.111 / Chapter 3.4 --- Discussion --- p.115 / Chapter Chapter Four: --- Conclusions and Future Perspectives --- p.119 / Chapter 4.1 --- Summary of results --- p.120 / Chapter 4.2 --- Conclusions --- p.121 / Chapter 4.3 --- Future Perspectives --- p.121 / References --- p.123

Pang, Pei Shin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 96-109). / Abstract also in Chinese. / Acknowledgement --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iv / Table of Content --- p.vi / List of Figures --- p.x / List of Tables --- p.xiii / Abbreviations --- p.xiv / Chapter CHAPTER ONE: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Incidence --- p.2 / Chapter 1.2 --- Etiological Factors --- p.7 / Chapter 1.2.1 --- Viral Hepatitis Infection --- p.7 / Chapter 1.2.1.1 --- Hepatitis B Virus --- p.10 / Chapter 1.2.1.2 --- Hepatitis C Virus --- p.11 / Chapter 1.2.2 --- Cirrhosis and Chronic Inflammation --- p.14 / Chapter 1.2.3 --- Dietary Aflatoxin Contamination --- p.16 / Chapter 1.2.4 --- Obesity --- p.16 / Chapter 1.3 --- Genomic Aberrations of HCC --- p.17 / Chapter 1.3.1 --- Chromosomal Imbalances --- p.17 / Chapter 1.3.2 --- Candidate Tumour Suppressor Genes and Oncogenes in HCC --- p.18 / Chapter 1.3.2.1 --- Chr 1q21-q22 gain --- p.18 / Chapter 1.3.2.2 --- Chr 8p21-p23 loss and 8q21-q24 gain --- p.18 / Chapter 1.3.2.3 --- Chr 13ql2-ql4 loss --- p.19 / Chapter 1.3.2.4 --- Chr 17pl3 loss --- p.20 / Chapter 1.3.3 --- Chromosomal Rearrangement --- p.21 / Chapter 1.4 --- Cell Lines as In-vitro Study Models --- p.22 / Chapter 1.5 --- Aim of study --- p.23 / Chapter 1.5.1 --- Objectives --- p.24 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS --- p.25 / Chapter 2.1 --- Materials --- p.26 / Chapter 2.2 --- Cell Lines and Cell Culture --- p.29 / Chapter 2.2.1 --- Cell Lines --- p.29 / Chapter 2.2.2 --- Cell Culture --- p.29 / Chapter 2.3 --- Comparative Genomic Hybridization (CGH) --- p.30 / Chapter 2.4 --- Spectral Karyotyping (SKY) --- p.31 / Chapter 2.5 --- Expression Profiling --- p.34 / Chapter 2.6 --- Functional Investigations --- p.37 / Chapter 2.6.1 --- Growth Kinetics --- p.37 / Chapter 2.6.2 --- Cytotoxic Assay --- p.38 / Chapter CHAPTER THREE: --- RESULTS --- p.39 / Chapter 3.1 --- Molecular Cytogenetic Analysis --- p.40 / Chapter 3.1.1 --- Comparative Genomic Hybridization (CGH) --- p.40 / Chapter 3.1.1.1 --- Introduction --- p.40 / Chapter 3.1.1.2 --- CGH Results --- p.41 / Chapter 3.1.1.3 --- Clustering Analysis of CGH Data --- p.49 / Chapter 3.1.2 --- Spectral Karyotyping (SKY) --- p.51 / Chapter 3.1.2.1 --- Introduction --- p.51 / Chapter 3.1.2.2 --- Ploidy Status --- p.51 / Chapter 3.1.2.3 --- Structural Rearrangements --- p.54 / Chapter 3.1.2.4 --- Chromosomes Susceptible to Further Rearrangements --- p.63 / Chapter 3.1.2.5 --- Maintained Common HCC Translocations --- p.65 / Chapter 3.2 --- Expression Profiling --- p.67 / Chapter 3.2.1 --- Introduction --- p.67 / Chapter 3.2.2 --- Gene Expression Profiling --- p.69 / Chapter 3.2.3 --- The Ontologies of Deregulated Genes --- p.71 / Chapter 3.2.4 --- Maintained Biological Pathways --- p.74 / Chapter 3.3 --- Functional Investigation --- p.76 / Chapter 3.3.1 --- Introduction --- p.76 / Chapter 3.3.2 --- Cell Morphology --- p.76 / Chapter 3.3.3 --- Growth Kinetics --- p.79 / Chapter 3.3.4 --- Cytotoxic Assay --- p.83 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.86 / Chapter 4.1 --- Introduction --- p.87 / Chapter 4.2 --- Molecular Cytogenetic Analysis --- p.87 / Chapter 4.3 --- Expression Profiling --- p.91 / Chapter 4.4 --- Conclusion --- p.94 / REFERENCES --- p.95

To, Ka Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (p. 115-120). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 中文摘要 --- p.vi / List of abbreviations --- p.ix / List of tables --- p.xii / List of figures --- p.xiii / Contents --- p.xvi / Chapter Chapter One: --- General introduction --- p.1 / Chapter 1.1 --- Background of Hepatocellular carcinoma (HCC) --- p.2 / Chapter 1.2.1 --- ZBP-89 structure and its expression in cancers --- p.3 / Chapter 1.2.2 --- Transcriptional regulation of ZBP-89 --- p.5 / Chapter 1.3.1 --- Apoptosis and necrosis --- p.6 / Chapter 1.3.2 --- Mechanisms of Apoptosis --- p.7 / Chapter 1.4 --- Bcl-2 family --- p.11 / Chapter 1.5 --- Regulation of p53 cancer cells --- p.12 / Chapter 1.6 --- The aim of the study --- p.13 / Chapter Chapter Two: --- Up-regulation of Bak expression by Ad-ZBP-89 induce apoptosis in human liver cancer cells --- p.15 / Chapter 2.1. --- Introduction --- p.16 / Chapter 2.2. --- Materials and methods --- p.18 / Chapter 2.2.1. --- Cell culture --- p.18 / Chapter 2.2.2. --- RT-PCR --- p.19 / Chapter 2.2.3. --- Western blotting --- p.21 / Chapter 2.2.4. --- Adenovirus infection and Cell viability assay --- p.24 / Chapter 2.2.5. --- Detection of apoptosis --- p.26 / Chapter 2.2.6. --- RNA interference --- p.27 / Chapter 2.2.7. --- Statistical analysis --- p.29 / Chapter 2.3. --- Results --- p.30 / Chapter 2.3.1. --- Endogenous expression of ZBP-89 and Bak of human liver cancer cells --- p.30 / Chapter 2.3.2. --- Effects of Ad-ZBP-89 on proliferation in HCC cell lines --- p.31 / Chapter 2.3.3. --- Effects of Ad-ZBP-89 on the expression of Bcl-2 family members --- p.34 / Chapter 2.3.4. --- ZBP-89 induced Bak expression and release of cytochrome c --- p.39 / Chapter 2.3.5. --- Effects of Ad-ZBP-89 on apoptosis rate in HCC cell lines --- p.41 / Chapter 2.3.6. --- Effects of ZBP-89 siRNA on expression of Bcl-2 family members and proliferation in HCC cell lines --- p.43 / Chapter 2.3.7. --- Effects of Bak siRNA and its combined effect with Ad-ZBP-89 on the expression of Bak and reduced apoptosis in HCC cell lines --- p.50 / Chapter 2.4. --- Discussion --- p.55 / Chapter Chapter Three: --- Identification of ZBP-89 protein as an apoptosis activator for a pro-apoptotic Bak gene promoter --- p.60 / Chapter 3.1. --- Introduction --- p.61 / Chapter 3.2. --- Materials and methods --- p.64 / Chapter 3.2.1. --- Cell lines and tissues --- p.64 / Chapter 3.2.2. --- Transient transfection and Luciferase activity assay --- p.64 / Chapter 3.2.3. --- pGL3-Bak-promoter vector construction --- p.67 / Chapter 3.2.4. --- "Preparation of mitochondrial, cytosolic and nuclear fractions" --- p.74 / Chapter 3.2.5. --- Electrophoretic mobility shift assay --- p.75 / Chapter 3.2.6. --- Overexpression of Bak --- p.76 / Chapter 3.2.7. --- RT-PCR and Western blot analysis on HCC tissues samples --- p.80 / Chapter 3.2.8. --- Statistical Analysis --- p.80 / Chapter 3.3. --- Results --- p.81 / Chapter 3.3.1. --- ZBP-89 activates Bak-luciferase promoter genes in HCC cells --- p.81 / Chapter 3.3.2. --- ZBP-89 activates shortened Bak-luc-promoter in PLC/PRF/5 and SK-Hep-1 cells --- p.82 / Chapter 3.3.3. --- ZBP-89 is a potential binding protein to the Bak promoter gene region -457/-407 --- p.85 / Chapter 3.3.4. --- The combined effects of Bak overexpression and Ad-ZBP-89 induce apoptosis in HCC cells --- p.89 / Chapter 3.3.5. --- The combined effects on Bak protein expression --- p.94 / Chapter 3.3.6. --- Bak expression in HCC tissues --- p.98 / Chapter 3.4. --- Discussion --- p.99 / Chapter Chapter Four: --- Conclusion and Future Perspectives --- p.104 / Chapter 4.1. --- Conclusion --- p.104 / Chapter 4.2. --- Future Perspectives --- p.112 / Reference --- p.113

Chen, Shuyan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 91-98). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ACKNOWLEGGMENTS --- p.v / LIST OF FIGURES --- p.vi / LIST OF TABLES --- p.vii / ABBREVIATIONS --- p.viii / CONTENTS --- p.ix / Chapter Chapter I --- Introduction / Chapter 1.1 --- Introduction of BRE / Chapter 1.1.1 --- Discovery of BRE --- p.1 / Chapter 1.1.2 --- Isoforms of BRE --- p.2 / Chapter 1.1.3 --- Homology and orthologs of BRE --- p.3 / Chapter 1.1.4 --- Expression studies of BRE mRNA --- p.4 / Chapter 1.1.5 --- Expression and cellular localization of BRE protein --- p.5 / Chapter 1.1.6 --- Interaction between BRE and death receptor --- p.6 / Chapter 1.1.7 --- Anti-apoptotic effect of BRE in cell line studies --- p.9 / Chapter 1.1.8 --- Anti-apoptotic effect of BRE in vivo --- p.11 / Chapter 1.1.9 --- BRE's role in DNA repair and ubiquitination --- p.12 / Chapter 1.1.10 --- BRE's role in regulation of Prohibitin and p53 expression --- p.13 / Chapter 1.2 --- Hepatocellular carcinoma / Chapter 1.2.1 --- Carcinogenesis --- p.15 / Chapter 1.2.2 --- Diethylnitrosamine -induced HCC --- p.15 / Chapter 1.2.3 --- Mouse model for HCC studies --- p.17 / Chapter 1.2.4 --- BRE in human HCC --- p.18 / Chapter 1.3 --- Green Fluorescent Protein / Chapter 1.3.1 --- Application of GFP in biological research --- p.19 / Chapter 1.3.2 --- Advantage of GFP applied in protein localization --- p.19 / Chapter Chapter II --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Primer used for cloning --- p.20 / Chapter 2.1.2 --- DNA clones used in the studies --- p.21 / Chapter 2.1.3 --- Materials for DNA manipulation --- p.24 / Chapter 2.1.4 --- Materials for protein manipulation --- p.24 / Chapter 2.1.5 --- Antibodies --- p.25 / Chapter 2.1.6 --- Chemicals --- p.25 / Chapter 2.1.7 --- Kits --- p.26 / Chapter 2.1.8 --- Culture media and reagents --- p.26 / Chapter 2.1.9 --- Bacterial strain used for transformation and cloning --- p.26 / Chapter 2.1.10 --- Instrumentation --- p.27 / Chapter 2.1.11 --- Animals --- p.27 / Chapter 2.1.12 --- Slides --- p.27 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Construction of Plasmids / Chapter 2.2.1.1 --- Polymerase chain reaction (PCR) --- p.28 / Chapter 2.2.1.2 --- Enzyme Digestion and Ligation --- p.29 / Chapter 2.2.1.3 --- Transformaion / Chapter 2.2.1.3.1 --- Preparation of competent cells --- p.29 / Chapter 2.2.1.3.2 --- Heat-shock Transformation --- p.29 / Chapter 2.2.1.4 --- Midi Prep of plasmids --- p.30 / Chapter 2.2.2 --- Cell Culture --- p.30 / Chapter 2.2.3 --- Transfection --- p.30 / Chapter 2.2.4 --- MG-132 treatment --- p.31 / Chapter 2.2.5 --- Flow Cytometry --- p.32 / Chapter 2.2.6 --- Western blotting / Chapter 2.2.6.1 --- SDS-PAGE --- p.32 / Chapter 2.2.6.2 --- Immunoblotting --- p.32 / Chapter 2.2.7 --- Production of Monoclonal Antibody --- p.33 / Chapter 2.2.8 --- Mice --- p.34 / Chapter 2.2.9 --- Tissue Processing --- p.35 / Chapter 2.2.10 --- Tissue Section --- p.35 / Chapter 2.2.11 --- Immunostaining --- p.36 / Chapter 2.2.12 --- H&E staining --- p.36 / Chapter 2.2.13 --- Picture Capture --- p.37 / Chapter 2.2.14 --- Confocal imaging --- p.37 / Chapter 2.2.14 --- Statistical Analysis --- p.37 / Chapter Chapter III --- BRE promotes growth of chemically-induced hepatocellular carcinoma / Chapter 3.1 --- DEN induced HCC in male mice --- p.38 / Chapter 3.2 --- BRE facilitates HCC in female mice --- p.44 / Chapter 3.3 --- Over-expression of BRE in tumor portion --- p.45 / Chapter 3.4 --- Direct effect of DEN on BRE expression --- p.47 / Chapter 3.5 --- Contribution of infiltrating cells in up-regulation of BRE --- p.50 / Chapter Chaper IV --- Subcellular localization of BRE / Chapter 4.1 --- GFP-BRE fusion constructs --- p.55 / Chapter 4.1.1 --- Transfection of GFP-BRE fusions --- p.58 / Chapter 4.1.2 --- Flow cytometry analysis of GFP-BRE fusions --- p.59 / Chapter 4.1.3 --- Western blot analysis of GFP-BRE fusions --- p.62 / Chapter 4.1.4 --- Stabilities of GFP-BRE fusions --- p.64 / Chapter 4.2 --- Fusions between GFP and the deletion mutants of BRE --- p.66 / Chapter 4.2.1 --- Transfection of mutants --- p.68 / Chapter 4.2.2 --- Low expression of mutants --- p.69 / Chapter 4.3 --- MG-132 treatments / Chapter 4.3.1 --- Increased expression of fusion proteins --- p.74 / Chapter 4.3.2 --- Subcellular localization of GFP-BRE fusions --- p.77 / Chapter Chapter V --- Discussion / Chapter 5.1 --- Functional role of BRE in HCC / Chapter 5.1.1 --- Stage model of carcinogenesis --- p.81 / Chapter 5.1.2 --- Anti-apoptotic genes in cancer --- p.84 / Chapter 5.1.3 --- Limitation of the study --- p.85 / Chapter 5.1.4 --- Conclusion --- p.85 / Chapter 5.2 --- Subcellular localization of BRE / Chapter 5.2.1 --- Low expression of GFP-BRE fusions --- p.86 / Chapter 5.2.2 --- Additional study --- p.90 / Chapter 5.2.3 --- Conclusion --- p.90 / Reference --- p.91 / Appendix --- p.99

Kan, Hoi Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 103-124). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.viii / LIST OF TABLES --- p.xii / LIST OF FIGURES --- p.xiii / LIST OF ABBREVIATIONS --- p.xiv / PUBLICATION --- p.xvi / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter Chapter 1: --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.1. --- Epidemiology of HCC --- p.3 / Chapter 1.2. --- Etiology of HCC --- p.3 / Chapter 1.2.1. --- Cirrhosis --- p.4 / Chapter 1.2.2. --- Hepatitis virus --- p.4 / Chapter 1.2.3. --- Plant carcinogens --- p.5 / Chapter 1.2.4. --- Miscellaneous factors --- p.6 / Chapter 1.3. --- Clinical presentation of HCC --- p.6 / Chapter 1.4. --- Existing diagnostic tests for HCC --- p.6 / Chapter 1.4.1. --- Alpha-fetoprotein (AFP) --- p.7 / Chapter 1.4.2. --- Imaging --- p.7 / Chapter 1.5. --- Treatment of HCC --- p.8 / Chapter 1.5.1. --- Surgical Resection and Transplantation --- p.8 / Chapter 1.5.2. --- Tumor Ablation or Embolization --- p.8 / Chapter 1.5.3. --- Chemotherapy and Radiotherapy --- p.9 / Chapter 1.6. --- Tumor marker development for HCC detection --- p.10 / Chapter 1.6.1. --- Oncofetal antigens and glycoprotein antigens --- p.11 / Chapter 1.6.2. --- Enzymes and isoenzymes --- p.12 / Chapter 1.6.3. --- Growth factors --- p.12 / Chapter 1.6.4. --- Genetics and epigenetics - mRNA and methylation --- p.13 / Chapter Chapter 2: --- Hypermethylation of tumor suppressor genes in cancer --- p.14 / Chapter 2.1. --- Cancer epigenetics --- p.14 / Chapter 2.2. --- DNA methylation in normal cells --- p.15 / Chapter 2.3. --- Physiological role of DNA methylation in normal cells --- p.18 / Chapter 2.4. --- Aberrant DNA methylation in cancer --- p.19 / Chapter 2.4.1. --- DNA hypomethylation in cancer --- p.20 / Chapter 2.4.2. --- DNA hypermethylation in cancer --- p.20 / Chapter 2.5. --- Development of methylation markers in tumor diagnosis --- p.21 / Chapter 2.5.1. --- Methods for the analysis of DNA methylation markers --- p.22 / Chapter 2.5.2. --- Detection of tumor-associated methylated DNA in the circulation of cancer patients / Chapter 2.6. --- Aim of thesis --- p.27 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.28 / Chapter Chapter 3: --- Methods for detecting DNA methylation --- p.29 / Chapter 3.1. --- Subject recruitment --- p.29 / Chapter 3.2. --- Sample collection and processing --- p.29 / Chapter 3.2.1. --- Tumor tissue samples --- p.29 / Chapter 3.2.2. --- Peripheral blood samples --- p.29 / Chapter 3.3. --- DNA extraction --- p.30 / Chapter 3.3.1. --- Plasma samples --- p.30 / Chapter 3.3.2. --- Blood cells --- p.33 / Chapter 3.3.3. --- Tumor tissue --- p.33 / Chapter 3.4. --- Quantitative analysis of methylated DNA using methylation-sensitive restriction enzyme-mediated real-time quantitative PCR (MSRE-qPCR) --- p.34 / Chapter 3.4.1. --- Methylation-sensitive restriction enzyme-mediated real-time quantitative PCR --- p.34 / Chapter 3.4.3. --- Real-time PCR primer design --- p.36 / Chapter 3.4.4. --- Duplex real-time PCR --- p.40 / Chapter 3.4.5. --- "Real-time detection of GSTP1, SOCS1, A PC, pl6 and ACTB sequences" --- p.41 / Chapter 3.4.6. --- Statistical analysis of real-time PCR results --- p.41 / Chapter 3.5. --- "Methylation study of GSTP1, SOCS1, APC, pl6 and ACTB in tumor tissues and blood cells using bisulfite sequencing" --- p.46 / Chapter 3.5.1. --- Principle of bisulfite modification --- p.46 / Chapter 3.5.2. --- Bisulfite conversion --- p.47 / Chapter 3.5.3. --- Sequencing primer design --- p.47 / Chapter 3.5.4. --- Conventional PCR after bisulfite treatment --- p.49 / Chapter 3.5.5. --- Cloning and bisulfite genomic sequencing --- p.53 / Chapter 3.5.6. --- Data acquisition and interpretation --- p.54 / Chapter SECTION III: --- DEVELOPMENT OF METHYLATION MARKERS IN HCC DETECTION / Chapter Chapter 4: --- Evaluation of the real-time PCR assay for quantification of methylated tumor suppressor genes --- p.57 / Chapter 4.1. --- Development of real-time PCR assays --- p.57 / Chapter 4.2. --- Methylation analyses by bisulfite sequencing were concordant with the real-time quantification results --- p.61 / Chapter Chapter 5: --- Clinical application of methylated markers in the detection of hepatocellular carcinoma --- p.69 / Chapter 5.1. --- Demographics of HCC patients and HB V carriers --- p.69 / Chapter 5.2. --- Quantitative analysis of hypermethylated tumor suppressor genes in tumor and plasma samples --- p.71 / Chapter 5.3. --- Effect of cirrhosis on the plasma methylated tumor suppressor gene concentrations --- p.77 / Chapter 5.4. --- Changes in the concentration of the tumor suppressor genes one month after surgical resection of the cancer --- p.81 / Chapter 5.5. --- Concurrent use of serum AFP level and plasma methylated markers for HCC diagnosis --- p.84 / Chapter 5.6. --- Prognostic value of plasma methylated TSGs --- p.86 / Chapter SECTION IV: --- DISCUSSION --- p.90 / Chapter Chapter 6: --- Discussion --- p.91 / Chapter 6.1. --- Tumor and plasma detection of hypermethylated tumor suppressor genes --- p.92 / Chapter 6.2. --- No effect of cirrhosis on plasma methylated DNA level --- p.94 / Chapter 6.3. --- Clearance of methylated TSG sequences after tumor resection --- p.95 / Chapter 6.4. --- Concurrent use of serum AFP level and the presence of methylated markers in the plasma in HCC diagnosis --- p.95 / Chapter 6.5. --- Prognostic significance of circulating methylated tumor markers --- p.96 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.98 / Chapter Chapter 7: --- Conclusions and future perspectives --- p.99 / REFERENCES --- p.103

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.

Leung, Kin Chung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 148-164). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.