Wang, Yajun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 118-126). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Figures --- p.xi / List of Tables --- p.xii / Abbreviations and Symbols --- p.xiii / List of Publications and Sequence Submissions during the Study --- p.xv / Chapter Chapter One: --- General Introduction --- p.1 / Chapter Chapter Two: --- Literature Review --- p.8 / Chapter 2.1 --- DNA methylation --- p.8 / Chapter 2.1.1 --- Epigenetic changes --- p.8 / Chapter 2.1.2 --- Differential methylation pattern in normal and tumor cells --- p.10 / Chapter 2.2 --- TSGs --- p.13 / Chapter 2.2.1 --- "Cancer initiation, progression and cancer genes" --- p.13 / Chapter 2.2.2 --- TSGs could be inactivated through promoter hypermethylation --- p.14 / Chapter 2.3 --- NPC --- p.17 / Chapter 2.3.1 --- Epidemiology ofNPC --- p.18 / Chapter 2.3.2 --- Molecular genetic and epigenetic studies ofNPC --- p.19 / Chapter 2.3.3 --- NPC and chromosome 11q --- p.21 / Chapter 2.4 --- ESCC --- p.21 / Chapter 2.4.1 --- Epidemiology of ESCC --- p.22 / Chapter 2.4.2 --- Genetic and epigenetic studies of ESCC --- p.23 / Chapter 2.4.3 --- ESCC and chromosome 11q --- p.24 / Chapter 2.5 --- Chromosome 11q and other carcinomas --- p.24 / Chapter 2.5.1 --- Breast cancer --- p.24 / Chapter 2.5.2 --- Ovarian cancer --- p.25 / Chapter 2.5.3 --- Neuroblastoma --- p.26 / Chapter 2.5.4 --- Melanoma --- p.27 / Chapter 2.5.5 --- Multiple myeloma --- p.27 / Chapter 2.5.6 --- Lung Cancer --- p.27 / Chapter 2.6 --- Important candidate genes located at the project study 1 lq region --- p.28 / Chapter 2.6.1 --- ETS1 --- p.28 / Chapter 2.6.2 --- FLI1 --- p.29 / Chapter 2.6.3 --- P53AIP1 --- p.30 / Chapter 2.6.4 --- RICS --- p.30 / Chapter 2.6.5 --- BARX2 --- p.30 / Chapter 2.6.6 --- ST14 --- p.32 / Chapter 2.6.7 --- ADAMTS8 --- p.33 / Chapter 2.6.8 --- ADAMTS15 --- p.35 / Chapter 2.6.9 --- HNT --- p.36 / Chapter 2.6.10 --- OPCML --- p.36 / Chapter Chapter Three: --- Materials and Methods --- p.37 / Chapter 3.1 --- Cell lines and primary tumor samples --- p.37 / Chapter 3.2 --- Cell line demethylation treatment --- p.38 / Chapter 3.3 --- DNA and RNA extraction from cell lines and tissues --- p.39 / Chapter 3.4 --- Semiquantitative RT-PCR --- p.41 / Chapter 3.5 --- DNA bisulfite treatment --- p.42 / Chapter 3.6 --- Promoter analysis and identification of 5' CpG islands of target genes --- p.45 / Chapter 3.7 --- Methylation-Specific PCR (MSP) --- p.45 / Chapter 3.8 --- Bisulfite Genomic Sequencing (BGS) --- p.46 / Chapter 3.8.1 --- BGS PCR reaction --- p.46 / Chapter 3.8.2 --- TA cloning of the PCR products into the sequencing vector --- p.47 / Chapter 3.8.3 --- Plasmid mini-preparation on 96-well plate --- p.48 / Chapter 3.8.4 --- Plasmid sequencing --- p.49 / Chapter 3.9 --- Homozygous deletion detection --- p.50 / Chapter 3.10 --- Construction of expression plasmids --- p.51 / Chapter 3.10.1 --- The strategy of full length cDNA cloning --- p.51 / Chapter 3.10.2 --- Obtaining of full length covered cDNA by cloning PCR --- p.53 / Chapter 3.10.3 --- Ligation and transformation --- p.54 / Chapter 3.10.4 --- Mini preparation of plasmid in Eppendorf tubes --- p.54 / Chapter 3.10.5 --- Verification of correct inserts in the plasmid --- p.55 / Chapter 3.10.6 --- Subcloning --- p.55 / Chapter 3.10.7 --- Bacteria storage --- p.57 / Chapter 3.11 --- Colony formation assays (CFA) --- p.57 / Chapter 3.11.1 --- Midiprep of the transfection grade plasmid --- p.57 / Chapter 3.11.2 --- Transfection --- p.58 / Chapter 3.11.3 --- Selection of the transfected cells with G418 --- p.59 / Chapter 3.11.4 --- Colony staining --- p.60 / Chapter 3.12 --- Statistical analysis --- p.60 / Chapter Chapter Four: --- Results --- p.61 / Chapter 4.1 --- Narrow down the candidate genes for further study --- p.61 / Chapter 4.1.1 --- Define the study chromosome region --- p.61 / Chapter 4.1.2 --- Database search of all candidate genes --- p.61 / Chapter 4.1.3 --- Transcriptional expression analysis of the candidate genes --- p.63 / Chapter 4.1.4 --- Selection of the genes with tumor specific expression downregulation for further intensive study --- p.64 / Chapter 4.2 --- Further characterization of ADAMTS8 --- p.69 / Chapter 4.2.1 --- Tissue transcriptional expression panel --- p.69 / Chapter 4.2.2 --- Semiquantitative RT-PCR results in tumor cell lines --- p.70 / Chapter 4.2.3 --- Promoter CpG island identification and promoter methylation study --- p.70 / Chapter 4.2.4 --- Transcription reactivation by demethylation treatment --- p.72 / Chapter 4.2.5 --- High resolution promoter methylation analysis by BGS --- p.72 / Chapter 4.2.6 --- Detection of homozygous deletion --- p.73 / Chapter 4.2.7 --- Analysis of ADAMTS8 promoter methylation in clinical samples --- p.74 / Chapter 4.2.8 --- ADAMTS8 full length cDNA cloning --- p.74 / Chapter 4.2.9 --- Colony formation assay --- p.75 / Chapter 4.3 --- Further characterization of HNT --- p.80 / Chapter 4.3.1 --- Tissue transcriptional expression panel --- p.80 / Chapter 4.3.2 --- Semiquantitative RT-PCR results in tumor cell lines --- p.80 / Chapter 4.3.3 --- Promoter CpG island identification and promoter methylation study --- p.81 / Chapter 4.3.4 --- Transcription reactivation by demethylation treatment --- p.82 / Chapter 4.3.5 --- HNT full length cDNA cloning --- p.82 / Chapter 4.4 --- Further characterization of BARX2 --- p.87 / Chapter 4.4.1 --- Tissue transcriptional expression panel --- p.87 / Chapter 4.4.2 --- Semiquantitative RT-PCR results in tumor cell lines --- p.87 / Chapter 4.4.3 --- Promoter CpG island identification and promoter methylation study --- p.88 / Chapter 4.4.4 --- Transcription reactivation by demethylation treatment --- p.89 / Chapter 4.4.5 --- BARX2 full length cDNA cloning --- p.89 / Chapter 4.5 --- Further study of other downregulated genes --- p.92 / Chapter 4.5.1 --- FLII --- p.92 / Chapter 4.5.2 --- ADAMTS15 --- p.94 / Chapter 4.5.3 --- P53AIP1 --- p.97 / Chapter Chapter Five: --- Discussion --- p.100 / Reference List --- p.118 / Appendix I: Reagents Preparation Recipe --- p.127 / Appendix II: PCR Primers for cDNA Cloning --- p.129

Lou, Pak Kin. / Thesis (M.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 136-170). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xiii / List of Publications --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Aims of Study --- p.1 / Chapter 1.2. --- Literature Review --- p.2 / Chapter 1.2.1. --- Nasopharyngeal Carcinoma --- p.2 / Chapter 1.2.1.1. --- Overview --- p.2 / Chapter 1.2.1.2. --- Histopathology --- p.2 / Chapter 1.2.1.3. --- Epidemiology --- p.3 / Chapter 1.2.1.4. --- Etiology --- p.5 / Chapter 1.2.1.4.1. --- Epstein-Barr Virus (EBV) Latent Infection --- p.5 / Chapter 1.2.1.4.2. --- Environmental Factors --- p.5 / Chapter 1.2.1.4.3. --- Genetic Factors --- p.6 / Chapter 1.2.1.5. --- Molecular Pathogenesis --- p.7 / Chapter 1.2.1.5.1. --- Chromosomal Alterations --- p.7 / Chapter 1.2.1.5.2. --- NPC-associated Tumour Suppressor Genes --- p.7 / Chapter 1.2.1.5.3. --- NPC-associated Oncogenes --- p.8 / Chapter 1.2.2. --- Epstein-Barr Virus --- p.9 / Chapter 1.2.2.1. --- Overview --- p.9 / Chapter 1.2.2.2. --- Lytic and Latent Infection of EBV --- p.9 / Chapter 1.2.2.3. --- EBV Latency Programs and Associated --- p.10 / Malignancies --- p.11 / Chapter 1.2.2.4. --- The Role of EBV in NPC --- p.12 / Chapter 1.2.3. --- NF-kB Signalling Pathways --- p.12 / Chapter 1.2.3.1. --- Overview --- p.12 / Chapter 1.2.3.2. --- Pathway Components --- p.12 / Chapter 1.2.3.2.1. --- NF-kB Subunits --- p.16 / Chapter 1.2.3.2.2. --- Inhibitors of kB (IkBs) --- p.16 / Chapter 1.2.3.2.3. --- IkB Kinases (IKKs) --- p.17 / Chapter 1.2.3.3. --- NF-kB Activation and Signalling --- p.17 / Chapter 1.2.3.3.1. --- The Canonical Pathway --- p.18 / Chapter 1.2.3.3.2. --- The Non-canonical Pathway --- p.18 / Chapter 1.2.3.3.3. --- Physiological Functions of NF-kB --- p.19 / Chapter 1.2.3.4. --- NF-kB Signalling and Tumourigenesis --- p.20 / Chapter 1.2.3.4.1. --- Oncogenic Activation of NF-kB in Hematological Malignancies --- p.20 / Chapter 1.2.3.4.2. --- Oncogenic Activation of NF-kB in Solid and Epithelial Tumours --- p.22 / Chapter Chapter 2 --- Material and Methods --- p.22 / Chapter 2.1. --- Tumour Specimens --- p.24 / Chapter 2.2. --- NPC Tumour Lines and Immortalized NP Cell Lines --- p.24 / Chapter 2.2.1. --- Cell Lines --- p.24 / Chapter 2.2.2. --- Xenografts --- p.27 / Chapter 2.3. --- DNA Sequence Analysis --- p.27 / Chapter 2.3.1. --- Genomic DNA Extraction --- p.27 / Chapter 2.3.2. --- Polymerase Chain Reaction (PCR) --- p.28 / Chapter 2.3.3. --- DNA Sequencing --- p.32 / Chapter 2.4. --- RNA Expression Analysis --- p.32 / Chapter 2.4.1. --- Total RNA Extraction and Reverse Transcription --- p.33 / Chapter 2.4.2. --- Quantitative Real-time Polymerase Chain Reaction (QRT-PCR) --- p.35 / Chapter 2.5. --- Protein Expression Analysis --- p.35 / Chapter 2.5.1. --- Total Protein Extraction --- p.35 / Chapter 2.5.2. --- Nuclear and Cytoplasmic Protein Isolation --- p.36 / Chapter 2.5.3. --- Western Blotting --- p.39 / Chapter 2.6. --- Immunohistochemical Staining --- p.41 / Chapter 2.7. --- Statistical Analysis --- p.41 / Chapter 2.8. --- Immunoprecipitation --- p.43 / Chapter 2.9. --- Electrophoretic Mobility Shift Assay (EMSA) and Supershift Assay --- p.44 / Chapter 2.10. --- Enzyme-Linked Immunosorbent Assay (ELISA) --- p.45 / Chapter 2.11. --- Plasmid Preparation --- p.45 / Chapter 2.11.1. --- Plasmids --- p.45 / Chapter 2.11.2. --- Bacterial Transformation and Plasmid DNA Extraction --- p.46 / Chapter 2.12. --- Transfections --- p.46 / Chapter 2.12.1. --- Transient Transfection --- p.46 / Chapter 2.12.2. --- Stable Transfection --- p.47 / Chapter 2.13. --- Immunofluorescence --- p.47 / Chapter 2.14. --- Cell Proliferation and Viability Analysis --- p.47 / Chapter 2.15. --- Small Interfering RNA (siRNA) Knockdown --- p.49 / Chapter 2.16. --- Expression Microarray --- p.49 / Chapter 2.16.1. --- Agilent Oligonucleotide Microarray --- p.50 / Chapter 2.16.2. --- Data Analysis --- p.51 / Chapter Chapter 3 --- Activation of NF-kB Signals in NPC --- p.51 / Chapter 3.1. --- Introduction --- p.52 / Chapter 3.2. --- Results --- p.52 / Chapter 3.2.1. --- Expression Pattern of NF-kB Subunits in NPC Tumour Lines --- p.55 / Chapter 3.2.2. --- Distinct NF-kB Complexes in NPC Tumour Lines --- p.60 / Chapter 3.2.3. --- Expression of NF-kB Subunits in NPC Primary Tumours --- p.67 / Chapter 3.3. --- Discussion / Chapter Chapter 4 --- Alterations of NF-kB Components in NPC --- p.71 / Chapter 4.1. --- Introduction --- p.72 / Chapter 4.2. --- Results --- p.72 / Chapter 4.2.1. --- Homozygous Deletion of IicBa and TRAF3 in NPC Tumour Lines --- p.76 / Chapter 4.2.2. --- Mutation of TRAF2 and A20 in NPC Tumour Lines / Chapter 4.2.3. --- Aberrant Expression of Multiple NF-kB Signalling Components in NPC Tumour Lines --- p.80 / Chapter 4.2.4. --- Expression of NF-kB Signalling Components in NPC --- p.85 / Primary Tumour --- p.92 / Chapter 4.3. --- Discussion --- p.99 / Chapter Chapter 5 --- Identification of Downstream Targets for NPC-associated NF-kB Signalling --- p.99 / Chapter 0.1. --- Introduction --- p.99 / Chapter 0.2. --- Results --- p.100 / Chapter 0.2.1. --- Target Genes Modulated by p50 --- p.100 / Chapter 0.2.2. --- Functional Annotation of p50 Target Genes --- p.105 / Chapter 0.2.3. --- Target Genes Modulated by RelB --- p.105 / Chapter 0.2.4. --- Functional Annotation of RelB Target Genes --- p.105 / Chapter 0.2.5. --- Functional Annotation of Genes Modulated by both p50 and RelB --- p.111 / Chapter 0.3. --- Discussion --- p.118 / Chapter Chapter 6 --- Functional Role of TRAF3 Inactivation in NPC --- p.118 / Chapter 0.1. --- Introduction --- p.118 / Chapter 0.2. --- Results --- p.118 / Chapter 0.2.1. --- Effect of TRAF3 Restoration on NF-kB Activity --- p.119 / Chapter 0.2.2. --- Effect of TRAF3 Expression on Cell Proliferation --- p.123 / Chapter 0.2.3. --- TRAF3 Expression Modulates Interferon Transcription in NPC Cells --- p.128 / Chapter 0.3. --- Discussion / Chapter Chapter 7 --- General Discussion --- p.132 / Chapter Chapter 8 --- Conclusion / Chapter Chapter 9 --- References / Appendix --- p.136

Tong Hung Man Joanna. / "June 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 137-149). / 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.

Based on matching analysis between different EBV strains, we found two nucleotide variations in miR-BART21 and four nucleotide changes in miR-BART22. Interestingly, two nucleotide variations upstream of mature miR-BART22 likely favor its biogenesis by Drosha/DGCR8 processing and we experimentally confirmed this augmentation by in-vitro Drosha digestion, and thus may underline the high and consistent expression of miR-BART22 in NPC tumors. / Infection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Many viral gene products including EBNA1, LMP1 and LMP2 have been implicated in NPC tumorigenesis, although the de novo control of these viral oncoproteins remain largely unclear. / MicroRNAs (miRNAs) are a class of small, non-coding RNAs with a size around 18--24 nucleotides with significant roles in regulating gene expression by either transcriptional silencing or translational suppression. As gene regulators, recent miRNA studies have emphasized the contribution of aberrant miRNA expression in cancer development. The recent discovery of EBV encoded viral miRNAs (ebv-miRNAs) in lymphoid malignancies has prompted us to examine the NPC-associated EBV miRNAs. In this study, we have systematically examined the NPC associated EBV genome for viral-encoded miRNA expression. By constructing small cDNA libraries from a native EBV positive NPC cell line (C666-1) and a xenograft (X2117), we screened about 3000 clones and detected several small EBV fragments, within which two novel ebv-miRNAs in the BARTs region were identified. These two newly identified miRNAs, now named miR-BART21 and miR-BART22, were proven to be abundantly expressed in most NPC samples by both Northern blot and QRT-PCR analysis. / Taken together, this thesis shows that two newly identified EBV-encoded miRNAs are highly expressed in latent EBV infection in NPC. Frequent expression of miR-BART22 can be explained partially by a specific EBV strain that is associated with NPC in our locality. Our findings emphasize the role of miR-BART22 in modulating LMP-2A expression. Because LMP-2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells (CTLs), down-modulation of LMP-2A expression by mir-BART22 may permit escape of EBV-infected cells from host immune surveillance. / We attempted to predict the potential viral and cellular targets of miR-BART21 and miR-BART22 by public available computer programs, miRanda and RNAhybrid. A number of potential cellular mRNA targets were suggested, although many failed to be validated by luciferase reporter assay. However, we found a putative miR-BART22 binding site in the LMP2A-3'UTR. Although the LMP-2A transcript is consistently detected in NPC, only 6 out of 26 (23%) primary NPC tumors show weak LMP-2A expression by immunohistochemistry (IHC). The expression levels of miR-BART22 and LMP-2A mRNA have also been determined in eleven of these tumors. Interestingly, the LMP-2A mRNA expression level did not directly correlate with protein expression, and relatively low expression levels of miR-BART22 miRNA were observed in all 3 LMP-2A positive-primary tumors. The suppressive effect of miR-BART22 on LMP-2A was also experimentally validated by a series of dual luciferase reporter assays using reporter constructs containing the putative or mutated recognition site at the LMP-2A 3'UTR. By co-transfection of different amounts of miR-BART22 with the LMP-2A-3'UTR expression vector in reporter assay, we confirmed that miR-BART22 suppressed the LMP-2A protein level in a dose-dependent manner. Furthermore, transfection of miR-BART22 into HEK293 cells that had been stably transfected with pcDNA3.1-LMP-2A, which contains a complete LMP-2A ORF and 3'UTR, readily suppressed levels of the LMP-2A protein. / Lung, Wai Ming Raymond. / Adviser: To Ka Fai. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 197-226). / 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.

In brief, mRNA expression profiling of candidate genes in each locus was performed using semi-quantitative RT-PCR in a panel of ESCC and NPC cell lines, normal tissues and immortalized epithelial cell lines. Genes downregulated in cancer cells but with high expression in normal tissues and immortalized epithelial cells were subjected to promoter methylation analysis using methylation-specific PCR (MSP), bisulfite genomic sequencing (BGS) and pharmacological demethylation treatment. Genes with tumor-specific downregulation and methylation were further selected as candidates and their tumor suppressive roles were verified via functional studies. / In conclusion, RAB39 and WDRX, epigenetically silenced in multiple cancer cell lines, were identified as novel TSG candidates in this study. Meanwhile, the tumor suppressive functions of ADAMTS8 were further validated, proving the efficiency of this integrative approach. Further study on these novel TSG candidates may help to elucidate the detailed molecular mechanisms for ESCC and NPC, and provide novel therapeutic targets and biomarkers. / In this study, RAB39 and WDRX were identified as candidate TSGs in 11q22.3 and 15q21.3, respectively. Both genes were broadly expressed in normal tissues and immortalized epithelial cell lines, but significantly downregulated and methylated in multiple cancer cell lines. Demethylation treatment with 5-Aza-2'-deoxycytidine restored their mRNA expression, indicating that CpG methylation directly contributed to their transcriptional inactivation. Methylation of RAB39 and WDRX was detected in primary ESCC and NPC, but rarely observed in normal tissues, implicating that their tumor-specific methylation might be used as biomarkers. Ectopic expression of both genes significantly inhibited the clonogenicity of multiple cancer cell lines, supporting their potential roles as functional TSGs. Moreover, WDRX repressed WNT/beta-catenin signaling, underscoring a possible anti-tumorigenic mechanism for it. In addition, ADAMTS8 was revealed to inhibit clonogenicity of NPC and ESCC cell lines, acting as a negative modulator for ERK pathway and a potential pro-apoptotic metalloprotease. / Inactivation of tumor suppressor genes (TSGs) contributes to the genesis of cancers including esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC), two prevalent causes of death in Hong Kong. Apart from genetic abnormalities, epigenetic disruptions including CpG methylation represent another major mechanism for TSG inactivation. Promoter methylation of multiple TSGs was detected in different cancer types, suggesting that it could be utilized as therapeutic target or biomarker for disease diagnosis and prognosis. / TSGs are often located at frequently deleted chromosomal regions and subjected to tumor-specific methylation, making it possible to use an integrative epigenetic and genomic approach combining array comparative genomic hybridization (aCGH) with epigenetic profiling to screen for novel TSGs. Previous aCGH revealed that several loci in 11822.3, 15q14, 15q21.1 and 15q21.3 underwent frequent copy number loss in ESCC cell lines. Loss of heterozygosity (LOH) of these regions was also reported in other cancers, indicating that TSGs might reside within them. The aim of this study was thus to identify the candidate TSGs in these loci and study their anti-tumorigenic roles. In addition, the tumor suppressive function of ADAMTS8, a silenced 11q25 candidate TSG previously identified in our lab via this approach, was also studied. / Li, Jisheng. / Adviser: Qian Tao. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 136-159). / 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.

Wong, Ho Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 66-77). / Abstracts in English and Chinese. / Acknowledgement --- p.i / List of abbreviations --- p.ii / List of tables and figures --- p.iv / Abstract in English --- p.vi / Abstract in Chinese --- p.viii / Table of content --- p.x / Chapter Chapter 1 --- Literature review / Chapter 1.1 --- Nasopharyngeal carcinoma (NPC) and treatments --- p.1 / Chapter 1.2 --- EBV and NF-kB signaling in NPC / Chapter 1.2.1 --- Role of EBV and NF-kB in NPC --- p.2 / Chapter 1.2.2 --- NF-kB signaling in cancer --- p.4 / Chapter 1.2.3 --- NF-kB activation in NPC --- p.7 / Chapter 1.2.3.1 --- NF-kB activation by LMP1 --- p.8 / Chapter 1.2.3.2 --- NF-kB and LMP2A --- p.10 / Chapter 1.2.3.3 --- NF-kB activation by non-viral factors --- p.10 / Chapter 1.2.4 --- NF-kB target genes in NPC --- p.11 / Chapter 1.3 --- NF-kB targeting / Chapter 1.3.1 --- NF-kB targeting agents --- p.14 / Chapter 1.3.2 --- "DHMEQ, a novel blocker of NF-kB Transactivation" --- p.15 / Chapter Chapter 2 --- Aim of study and Research plan --- p.18 / Chapter Chapter 3 --- Materials and Methods / Chapter 3.1 --- Cell lines and Reagents --- p.20 / Chapter 3.2 --- Cell viability assay --- p.21 / Chapter 3.3 --- Cell apoptosis detection / Chapter 3.3.1 --- PARP cleavage --- p.22 / Chapter 3.3.2 --- DNA fragmentation --- p.22 / Chapter 3.4 --- Cell cycle analysis --- p.22 / Chapter 3.5 --- Transwell migration or Matrigel invasion assay --- p.23 / Chapter 3.6 --- Soft agar colony formation assay --- p.24 / Chapter 3.7 --- Drug treatment for western blotting --- p.25 / Chapter 3.8 --- "Protein extraction and quantification, SDS-PAGE and western blotting" / Chapter 3.8.1 --- Protein extraction and quantification --- p.25 / Chapter 3.8.2 --- SDS-PAGE and western blotting --- p.26 / Chapter 3.9 --- Fractionation --- p.28 / Chapter 3.10 --- NF-kB transcriptional activity assay / Chapter 3.10.1 --- Construction of NF-kB reporter system --- p.29 / Chapter 3.10.2 --- Luciferase assay --- p.29 / Chapter 3.11 --- Statistical Analysis --- p.30 / Chapter Chapter 4 --- Results / Chapter 4.1 --- Anti-tumor activity of DHMEQ in NPC / Chapter 4.1.1 --- Growth inhibition in NPC cell lines --- p.31 / Chapter 4.1.2 --- Apoptotic induction in NPC cell lines --- p.35 / Chapter 4.1.3 --- Cell cycle arrest in NPC cell lines --- p.38 / Chapter 4.1.4 --- Inhibition of migration and invasive behavior of NPC cell lines --- p.38 / Chapter 4.1.5 --- Abrogation of soft agar colony formation ability of NPC cell lines --- p.43 / Chapter 4.2 --- Mechanistic study of DHMEQ in NPC / Chapter 4.2.1 --- Blockade of p65 nuclear translocation --- p.48 / Chapter 4.2.2 --- Attenuation of NF-kB transcriptional activity --- p.48 / Chapter 4.2.3 --- Downregulation of NF-kB target genes --- p.53 / Chapter Chapter 5 --- Discussion --- p.54 / Chapter Chapter 6 --- Summary --- p.60 / Chapter Chapter 7 --- Future Study --- p.63 / Reference List --- p.66 / Appendix / Chapter Appendix 1 --- Construction of NF-kb report plasm id --- p.78 / Chapter Appendix 2 --- Wound healing assay --- p.86 / Chapter Appendix 3 --- Reverse-phase protein Array --- p.88

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

First, expression profiling of ZNFs with CpG islands at 10 clusters of Chr19 was examined in a panel of NPC and ESCC cell lines by semi-quantitative RT-PCR, with adult normal tissues - larynx and esophagus as controls. Several down-regulated genes were identified, and I further focused on 5 candidates: ZNF382, ZNF545, ZFP30, ZNFT1 and ZNFT2. These genes were frequently downregulated in NPC, ESCC, lung, gastric, colon and breast carcinomas. Their promoters were frequently methylated in multiple downregulated cell lines but less in non-tumor cell lines as revealed by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS). Their expression could be restored by pharmacologic or genetic demethylation, suggesting that DNA methylation was directly involved in their silencing. The frequent methylation of these genes indicated they could act as potential biomarkers. / In conclusion, several novel candidate TSGs epigenetically silenced in tumor cells were identified in this study. Their downregulation by promoter methylation was tumor-specific, which could be use as epigenetic biomarkers for diagnosis. / More functional studies were done for ZNF382 and ZNF545, I found that ectopic expression of ZNF382 and ZNF545 in tumor cells lacking endogenous expression could inhibit tumor cell clonogenicity, proliferation and induce apoptosis. I found that ZNF382 suppressed tumorigenesis through mediating heterochromatin formation, as ZNF382 was revealed to be co-localized and interacts with heterochromatin protein. For ZNF545, I found that it is a transcriptional repressor. I further showed that ZNF545 was located in the nucleus and sequestered in the nucleolus. ZNF545 could inhibit tumorigenesis at least partially through downregulating the transcription of target genes or regulating nucleolus function such as ribosome biogenesis. / The development of a tumor from a normal cell is a complex and multi-step process. A large number of oncogenes, tumor suppressor genes (TSGs) and signal transduction pathways are involved in this process. Tumor-specific methylation of TSGs in multiple tumors indicated that it could be used as epigenetic biomarker for molecular diagnosis and therapeutics. / The functions of KRAB-containing proteins are critical to cell differentiation, proliferation, apoptosis and neoplastic transformation. A large number of ZNF genes are located in 10 clusters at chromosome 19. Some of the KRAB-ZNF may function as potential TSGs with epigenetic alterations. Thus, I try to identify silenced novel KRAB-ZNF candidate TSGs through screening chromosome 19. / Cheng, yingduan. / Adviser: Tao Qian. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 110-136). / 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.

Fong, Kwan Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 90-103). / Abstracts in English and Chinese. / Chapter Chapter 1: --- "Introduction, problems and objectives" --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Problems --- p.3 / Chapter 1.3 --- Objectives --- p.3 / Chapter Chapter 2: --- Background --- p.4 / Chapter 2.1. --- Head and Neck Cancer --- p.4 / Chapter 2.2 --- Diagnostic Imaging of Head and Neck Cancer --- p.5 / Chapter 2.3. --- Magnetic Resonance Imaging- Physics --- p.8 / Chapter 2.3.1 --- Nuclear Magnetic Resonance Principle --- p.8 / Chapter 2.3.2 --- Proton Magnetic Resonance Imaging --- p.8 / Chapter 2.3.3 --- Relaxation --- p.12 / Chapter 2.3.4 --- Tl- and T2-weighted Imaging --- p.12 / Chapter 2.3.5 --- Diffusion Weighted Imaging (DWI) --- p.13 / Chapter 2.3.6 --- Magnetic Resonance Spectroscopy- Single Voxel Spectroscopy and Chemical Shift Imaging --- p.15 / Chapter Chapter 3: --- Diffusion-weighted imaging in the evaluation head of and neck cancer --- p.21 / Chapter 3.1 --- Introduction - Diffusion-Weighted Imaging in Tumors --- p.21 / Chapter 3.2 --- DWI of Nasopharyngeal Carcinoma --- p.22 / Chapter 3.2.1 --- Introduction and Objectives --- p.22 / Chapter 3.2.2. --- Methods --- p.23 / Chapter 3.2.3. --- Results --- p.27 / Chapter 3.2.4 --- Discussion --- p.31 / Chapter 3.3 --- DWI of Primary Tumors: Comparison of NPC with Squamous Cell Carcinoma and Extra-nodal Non-Hodgkin Lymphoma --- p.33 / Chapter 3.3.1 --- Introduction and Objectives --- p.33 / Chapter 3.3.2. --- Methods --- p.34 / Chapter 3.3.3. --- Results --- p.35 / Chapter 3.3.4 --- Discussion --- p.42 / Chapter 3.3.5 --- Summary of DWI in Head and Neck Cancer --- p.44 / Chapter Chapter 4: --- Chemical shift imaging of head and neck tumors --- p.45 / Chapter 4.1 --- Introduction - Single Voxel Spectroscopy and Chemical Shift Imaging --- p.45 / Chapter 4.2 --- CSI - Methods Used to Reduce Magnetic Field Inhomogeneity --- p.48 / Chapter 4.3 --- Phantom studies - CSI Experiments Using Phantoms --- p.51 / Chapter 4.3.1 --- Introduction and Objectives --- p.51 / Chapter 4.3.2. --- Methods --- p.51 / Chapter 4.3.3 --- Experiment and MR Protocol --- p.54 / Chapter 4.3.4 --- Data Analysis --- p.58 / Chapter 4.3.5 --- Phantom Experimental Results --- p.59 / Chapter 4.3.6 --- Discussion and Conclusion on Phantom Experiments --- p.69 / Chapter 4.4 --- In vivo CSI Study of Human Head and Neck Tumors --- p.72 / Chapter 4.4.1 --- Introduction and Objectives --- p.72 / Chapter 4.4.2 --- Patient Selection --- p.73 / Chapter 4.4.3 --- MRI and CSI Protocol --- p.73 / Chapter 4.4.4 --- Data Analysis --- p.74 / Chapter 4.4.5 --- Results from CSI on Patients --- p.74 / Chapter 4.4.6 --- Discussion and Conclusion of CSI on Patients --- p.81 / Chapter Chapter 5: --- "Summary, conclusion and future studies" --- p.87 / Chapter 5.1 --- Summary --- p.87 / Chapter 5.2 --- Conclusion --- p.89 / Chapter 5.3 --- Future Studies --- p.89 / References --- p.90 / Publications --- p.104