by Mo Flora. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references (leaves 168-184). / ACKNOWLEDGEMENT --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1 --- Stroke as a major disabling disease --- p.1 / Chapter 1.2 --- Classification of stroke --- p.4 / Chapter 1.3 --- Risk factors attributing to stroke --- p.15 / Chapter 1.4 --- Experimental methods to induce cerebral ischemia --- p.19 / Chapter 1.4.1 --- The establishment of animal models for stroke --- p.21 / Chapter 1.4.2 --- Gerbil as a putative model --- p.25 / Chapter 1.5 --- Mechanisms of focal ischemia damage --- p.30 / Chapter 1.6 --- Potential biochemical markers for cerebral ischemia --- p.38 / Chapter 1.7 --- Aim of investigation --- p.48 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Common chemicals --- p.49 / Chapter 2.2 --- Common bench solutions --- p.52 / Chapter 2.3 --- Animals / Chapter 2.3.1 --- Gerbils --- p.52 / Chapter 2.3.2 --- Rabbit --- p.53 / Chapter 2.4 --- Establishment of an animal model / Chapter 2.4.1 --- Surgical methods for common carotid artery (CCA) ligation --- p.54 / Chapter 2.5 --- Methods to determine stroke conditions of gerbils / Chapter 2.5.1 --- Ocular fundus examination --- p.56 / Chapter 2.5.2 --- Stroke index --- p.56 / Chapter 2.5.3 --- Inclined plane method --- p.59 / Chapter 2.6 --- Preparation of gerbil brain for subsequent analysis / Chapter 2.6.1 --- Preparation of gerbil brain slices --- p.61 / Chapter 2.6.2 --- "2,3,5-triphenytetrazolium chloride (TTC) for quantitative staining of brain slices" --- p.61 / Chapter 2.6.3 --- Preparation of normal and stroke gerbil brain extract --- p.62 / Chapter 2.7 --- Polyacrylamide gel electrophoresis (PAGE) using a discontinuous buffer system / Chapter 2.7.1 --- Stock reagents --- p.63 / Chapter 2.7.2 --- Separation gel preparation --- p.65 / Chapter 2.7.3 --- Stacking gel preparation --- p.66 / Chapter 2.7.4 --- Electrophoresis conditions --- p.67 / Chapter 2.7.5 --- Staining and destaining --- p.67 / Chapter 2.8 --- Two dimensional slab gel electrophoresis / Chapter 2.8.1 --- Equipment --- p.70 / Chapter 2.8.2 --- Chemical --- p.70 / Chapter 2.8.3 --- Procedure --- p.74 / Chapter 2.9 --- Production of rabbit polyclonal antibodies against isolated stroke protein / Chapter 2.9.1 --- Isolation of stroke protein band from SDS-PAGE slab gel --- p.78 / Chapter 2.9.2 --- Production of anti-stroke protein serum in rabbits --- p.79 / Chapter 2.10 --- Western blotting method / Chapter 2.10.1 --- Reagents --- p.80 / Chapter 2.10.2 --- Procedures --- p.81 / Chapter 2.11 --- Extraction of total cellular RNA by lithium chloride method / Chapter 2.11.1 --- Reagents --- p.83 / Chapter 2.11.2 --- Procedures --- p.84 / Chapter 2.11.3 --- Checking the purity of the extracted RNA --- p.85 / Chapter 2.12 --- Purification of mRNA / Chapter 2.12.1 --- Reagents --- p.85 / Chapter 2.12.2 --- Procedure --- p.86 / Chapter 2.13 --- Verification of purity of mRNA / Chapter 2.13.1 --- Reagents --- p.87 / Chapter 2.13.2 --- Procedure --- p.88 / Chapter 2.14 --- Translation of gerbil brain mRNA in reticulocyte lysates and analysis of its product by SDS PAGE / Chapter 2.14.1 --- Reagents --- p.89 / Chapter 2.14.2 --- Procedures --- p.89 / Chapter CHAPTER THREE --- ESTABLISHMENT OF AN ANIMAL STROKE MODEL / Chapter 3.1 --- Foreword --- p.92 / Chapter 3.2 --- Preliminary studies / Chapter 3.2.1 --- Introduction --- p.92 / Chapter 3.2.2 --- Results --- p.93 / Chapter 3.2.3 --- Discussion --- p.96 / Chapter 3.3 --- Survival rate analysis / Chapter 3.3.1 --- Introduction --- p.97 / Chapter 3.3.2 --- Result --- p.98 / Chapter 3.3.3 --- Discussion --- p.102 / Chapter 3.4 --- Neurologic signs of ischemia / Chapter 3.4.1 --- Introduction --- p.103 / Chapter 3.4.2 --- Result --- p.105 / Chapter 3.4.3 --- Discussion --- p.111 / Chapter 3.5 --- Ocular fundus examination / Chapter 3.5.1 --- Introduction --- p.112 / Chapter 3.5.2 --- Result --- p.114 / Chapter 3.5.3 --- Discussion --- p.116 / Chapter 3.6 --- Inclined plane method / Chapter 3.6.1 --- Introduction --- p.117 / Chapter 3.6.2 --- Result --- p.118 / Chapter 3.6.3 --- Discussion --- p.121 / Chapter 3.7 --- Histologic examination using TTC as staining agent / Chapter 3.7.1 --- Introduction --- p.122 / Chapter 3.7.2 --- Result --- p.124 / Chapter 3.7.3 --- Discussion --- p.129 / Chapter CHAPTER FOUR --- IDENTIFICATION OF ALTERED PROTEIN PATTERN IN THE - BRAINS OF STROKE GERBILS BY ELECTROPHORETIC METHODS / Chapter 4.1 --- Separation of soluble brain extracts by SDS-PAGE analysis / Chapter 4.1.1 --- Introduction --- p.130 / Chapter 4.1.2 --- Result --- p.132 / Chapter 4.1.3 --- Discussion --- p.140 / Chapter 4.2 --- Two dimensional electrophoretic analysis of soluble brain extracts from stroke gerbils / Chapter 4.2.1 --- Introduction --- p.142 / Chapter 4.2.2 --- Result --- p.143 / Chapter 4.2.3 --- Discussion --- p.148 / Chapter CHAPTER FIVE --- ISOLATION OF STROKE-ASSOCIATED PROTEIN FROM BRAINS OF STROKE GERBILS BY IMMUNOCHEMICAL METHOD / Chapter 5.1 --- Introduction --- p.149 / Chapter 5.2 --- Result --- p.151 / Chapter 5.3 --- Discussion --- p.153 / Chapter CHAPTER SIX --- DETECTION OF NEW PROTEIN TRANSLATED FROM MESSENGER RIBONUCLEIC ACID FROM BRAINS OF STROKE GERBIL / Chapter 6.1 --- Introduction / Chapter 6.1.1 --- Extraction of stroke gerbil brain messenger ribonucleic acid --- p.154 / Chapter 6.1.2 --- Translation of mRNA --- p.154 / Chapter 6.2 --- Results / Chapter 6.2.1 --- Yield of total cellular RNA --- p.157 / Chapter 6.2.2 --- Verification of purity of mRNA --- p.157 / Chapter 6.2.3 --- Autoradiographic patterns of translated proteins --- p.159 / Chapter 6.3 --- Discussion --- p.163 / Chapter CHAPTER SEVEN --- GENERAL DISCUSSION --- p.165 / BIBLIOGRAPHY --- p.168

by Mo-Lung Chen. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 70-75). / Abstract also in Chinese. / ABBREVIATIONS --- p.iv / LIST OF TABLES --- p.v / LIST OF FIGURES --- p.vii / ACKNOWLEDGEMENT --- p.ix / ABSTRACT --- p.xi / Chapter CHAPTER 1. --- INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- BACKGROUND --- p.4 / Chapter 2.1 --- Production of pleural fluid --- p.4 / Chapter 2.2 --- Pathophysiology of pleural effusion --- p.5 / Chapter 2.3 --- Separating exudate from transudate --- p.8 / Chapter 2.4 --- Receiver operating characteristic curve --- p.9 / Chapter CHAPTER 3. --- ADENOSINE DEAMINASE --- p.12 / Chapter 3.1 --- Background --- p.12 / Chapter 3.2 --- Differentiation of tuberculous and non-tuberculous pleural effusion --- p.12 / Chapter CHAPTER 4. --- MATERIALS AND METHODS --- p.17 / Chapter 4.1 --- Patients --- p.17 / Chapter 4.2 --- Collection and handling of specimens --- p.17 / Chapter 4.3 --- Diagnostic criteria --- p.18 / Chapter 4.4 --- Methods --- p.19 / Chapter 4.4.1 --- Routine chemistries --- p.19 / Chapter 4.4.2 --- Protein zone electrophoresis --- p.19 / Chapter 4.4.3 --- Adenosine deaminase --- p.19 / Chapter 4.4.3.1 --- Instrumentation --- p.22 / Chapter 4.4.3.2 --- Optimization of reaction time --- p.24 / Chapter 4.4.4 --- Analytical performance --- p.24 / Chapter 4.4.4.1 --- Imprecision --- p.24 / Chapter 4.4.4.2 --- Recovery --- p.26 / Chapter 4.4.4.3 --- Lowest detection limit --- p.26 / Chapter 4.4.4.4 --- Linearity --- p.26 / Chapter 4.4.4.5 --- Interference by ammonia --- p.26 / Chapter 4.4.4.6 --- Interference by turbidity --- p.28 / Chapter 4.4.4.7 --- Interference by haemoglobin --- p.28 / Chapter 4.4.4.8 --- Interference by bilirubin --- p.29 / Chapter 4.4.4.9 --- Storage stability of ADA at -80°C --- p.29 / Chapter 4.4.5 --- Statistical analysis --- p.30 / Chapter CHAPTER 5. --- RESULTS OF OPTIMIZATION AND EVALUATION EXPERIMENTS --- p.31 / Chapter 5.1 --- Optimization of reaction time --- p.31 / Chapter 5.2 --- Analytical performance --- p.31 / Chapter 5.2.1 --- Imprecision --- p.31 / Chapter 5.2.1.1 --- Within-run --- p.31 / Chapter 5.2.1.2 --- Between-run --- p.31 / Chapter 5.2.2 --- Recovery --- p.31 / Chapter 5.2.3 --- Lowest detection limit --- p.34 / Chapter 5.2.4 --- Linearity --- p.34 / Chapter 5.2.5 --- Interference by / Chapter 5.2.5.1 --- ammonia --- p.34 / Chapter 5.2.5.2 --- turbidity --- p.34 / Chapter 5.2.5.3 --- haemoglobin --- p.37 / Chapter 5.2.5.4 --- bilirubin --- p.37 / Chapter 5.2.6 --- Storage stability of ADA at -80°C --- p.37 / Chapter CHAPTER 6. --- TRANSUDATIVE AND EXUDATIVE PLEURAL EFFUSION --- p.39 / Chapter 6.1 --- Results of routine chemistries --- p.39 / Chapter 6.2 --- Decision thresholds by ROC curve --- p.39 / Chapter 6.3 --- Discussion --- p.39 / Chapter 6.4 --- Results of protein zone electrophoresis --- p.49 / Chapter 6.5 --- Discussion --- p.51 / Chapter 6.6 --- Comparison of protein zone electrophoresis and Light's criteria --- p.55 / Chapter 6.7 --- Discussion --- p.55 / Chapter CHAPTER 7. --- TUBERCULOUS AND NON-TUBERCULOUS EXUDATIVE PLEURAL EFFUSION --- p.59 / Chapter 7.1 --- Results of adenosine deaminase assay --- p.59 / Chapter 7.2 --- Combinations of analysis --- p.59 / Chapter 7.3 --- Decision thresholds by ROC curve --- p.64 / Chapter 7.4 --- Discussion --- p.64 / Chapter CHAPTER8. --- GENERAL DISCUSSION --- p.69 / REFERENCES --- p.70

Chen, Yu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 75-78). / Abstracts in English and Chinese. / Isolation and Identification of Differentially Expressed Protein in Serum of Patients with Sleep Disorders --- p.I / Abstract --- p.IV / 論文摘要 --- p.VII / Acknowledgements --- p.IX / Table of Contents --- p.X / List of Figures --- p.XII / List of Tables --- p.XII / List of Abbreviations --- p.XIII / Chapter Chapter 1: --- Introduction --- p.2 / Chapter 1.1 --- Definition of narcolepsy --- p.2 / Chapter 1.2 --- Symptoms of narcolepsy --- p.2 / Chapter 1.2.1 --- Excessive Daytime Sleepiness (EDS) --- p.2 / Chapter 1.2.2 --- Cataplexy --- p.2 / Chapter 1.2.3 --- Associated features --- p.3 / Chapter 1.3 --- Prevalence of narcolepsy --- p.4 / Chapter 1.4 --- Pathophysiology and molecular genetics of narcolepsy --- p.7 / Chapter 1.4.1 --- Pathophysiology of narcolepsy --- p.7 / Chapter 1.4.2 --- Molecular genetics research --- p.8 / Chapter 1.5 --- Diagnostic criteria for narcolepsy --- p.12 / Chapter 1.6 --- Treatment of narcolepsy --- p.16 / Chapter 1.7 --- The Burden of narcolepsy --- p.18 / Chapter 1.8 --- Human blood serum/plasma --- p.19 / Chapter 1.9 --- Cerebrospinal fluid (CSF) --- p.23 / Chapter 1.10 --- Aims of study --- p.26 / Chapter Chapter 2: --- Materials and Methods --- p.28 / Chapter 2.1 --- Participants and measurements --- p.28 / Chapter 2.1.1 --- Participants --- p.28 / Chapter 2.1.2 --- Diagnosis measurements --- p.28 / Chapter 2.2 --- "Serum extraction, albumin and IgG depletion" --- p.30 / Chapter 2.2.1 --- Albumin and IgG Depletion Kit --- p.30 / Chapter 2.2.2 --- Chemicals and reagents --- p.30 / Chapter 2.2.3 --- Preparation of solutions --- p.30 / Chapter 2.2.4 --- Procedure --- p.30 / Chapter 2.3 --- Reversed Phase High Performance Liquid Chromatography (RP-HPLC) --- p.32 / Chapter 2.3.1 --- RP-HPLC method --- p.32 / Chapter 2.3.2 --- Chemicals and reagents --- p.33 / Chapter 2.3.3 --- Preparation of mobile phases --- p.33 / Chapter 2.3.4 --- Procedure --- p.33 / Chapter 2.4 --- MALDI-TOF/TOF Mass Spectrometry --- p.35 / Chapter 2.4.1 --- Chemicals and reagents --- p.35 / Chapter 2.4.2 --- Preparation of solutions --- p.35 / Chapter 2.4.3 --- Procedure --- p.35 / Chapter 2.5 --- SDS-PAGE and double staining --- p.37 / Chapter 2.5.1 --- Chemicals and reagents --- p.37 / Chapter 2.5.2 --- Preparation of solutions --- p.37 / Chapter 2.5.3 --- Procedure --- p.39 / Chapter 2.6 --- N-terminal amino acid analysis --- p.42 / Chapter 2.6.1 --- Procedure --- p.42 / Chapter 2.6.2 --- Sequence analysis --- p.42 / Chapter 2.7 --- CSF analysis --- p.43 / Chapter Chapter 3: --- Results --- p.45 / Chapter 3.1 --- Albumin and IgG depletion of human serum samples --- p.45 / Chapter 3.2 --- Peak identification --- p.47 / Chapter 3.2.1 --- Peak identification on HPLC profiles --- p.47 / Chapter 3.2.2 --- Statistical results --- p.51 / Chapter 3.2.3 --- Family cases analysis --- p.54 / Chapter 3.3 --- MALDI-TOF/TOF Mass Spectrometry --- p.56 / Chapter 3.4 --- SDS-PAGE and double staining --- p.58 / Chapter 3.5 --- Protein sequence analysis --- p.60 / Chapter 3.6 --- Cerebrospinal fluid (CSF) analysis --- p.62 / Chapter Chapter 4: --- Discussion --- p.65 / Chapter 4.1 --- RP-HPLC methods --- p.65 / Chapter 4.2 --- The detected peptide fragment and Hlark --- p.66 / Chapter 4.2.1 --- "Human Lark protein (Hlark, hlark)" --- p.66 / Chapter 4.2.2 --- Circadian clocks --- p.67 / Chapter 4.2.3 --- "Hlark, circadian rhythm and narcolepsy" --- p.71 / Chapter 4.3 --- Familial and genetic analysis --- p.72 / Chapter 4.4 --- Clinical implications --- p.73 / Chapter 4.5 --- Conclusion --- p.74 / References --- p.75

多聚谷氨酰胺疾病 (Polyglutamine diseases) 是一類在各自的致病基因編碼區的CAG重複編碼擴張造成的顯性遺傳神經退退化疾病。已擴大的CAG訊息核醣核酸 (Expanded CAG transcripts) 在多聚谷氨酰胺蛋白疾病作出細胞毒性作用。從基因減弱篩查中，我發現U2AF50能修飾已擴大的CAG訊息核醣核酸的毒性。並發現U2AF50能與已擴大的CAG訊息核醣核酸作實體互動，能參與已擴大的CAG訊息核醣核酸的核出口 (Nuclear export)。U2AF50的基因減弱增強已擴大CAG訊息核醣核酸在細胞核的累積和毒性。這突出核醣核酸的核出口在多聚谷氨酰胺疾病的重要性，並暗示細胞核是已擴大的CAG訊息核醣核酸毒性的起源地。此外，我鑑定已擴大的CAG訊息核醣核酸在亞細胞的分佈，並發現它們特別累積在核仁 (Nucleolus) 內。核仁是核糖體核醣核酸（rRNA）的轉錄場所。我發現已擴大的CAG訊息核醣核酸減弱rRNA基因啟動子 (rRNA promoter) 的活性並且抑制核糖體核醣核酸的轉錄。 核糖體核醣核酸基因轉錄的抑制，促進核糖體蛋白RpL23和E3連接酶MDM2蛋白作實體互動，從而增強p53的穩定性導。穩定的p53能夠轉移至線粒體 (Mitochondria)。我還發現，線粒體內的p53能打亂Bcl-xL與 Bak的實體互動，導致細胞色素C釋放到細胞質，這導致凋亡蛋白酶 (Caspases) 的活化和細胞凋亡。我的研究，首次證明核仁參與在多聚谷氨酰胺疾病的發病機制中，揭示了在多聚谷氨酰胺疾病中的新致病機制。 / Polyglutamine (polyQ) diseases are a class of dominantly inherited neurodegenerative disorders caused by the expansion of CAG-repeat encoding glutamine within the coding region of the respective disease genes. Expanded CAG transcripts have been reported to contribute to cytotoxicity in polyQ diseases. From a candidate gene knockdown screen, I identified U2AF50 as a modifier of RNA toxicity. U2AF50 has been reported to be involved in RNA nuclear export, and I showed that it interacted specifically with expanded CAG transcripts. Knockdown of U2AF50 expression enhanced nuclear accumulation of expanded CAG transcripts and neurotoxicity. This part of my work highlights the role of RNA nuclear export in polyQ degeneration and implies that the nucleus is a major site for RNA toxicity. In addition, I determined the subcellular distribution of expanded CAG transcripts and found that they particularly localized in the nucleolus. The nucleolus is a critical sub-nuclear compartment for ribosomal RNA (rRNA) transcription. I discovered that expanded CAG transcripts in nucleolus inhibited rRNA transcription by inactivating the rRNA gene promoter activity. Inhibition of rRNA transcription promoted the interaction between ribosomal protein L23 and the ubiquitin E3 ligase MDM2, which led to the stabilization of p53 and its accumulation in mitochondria. I also found that mitochondrial p53 disrupted the interaction between the anti-apoptotic protein, Bcl-xL, and pro-apoptotic protein, Bak, subsequently causing Cytochrome c release, caspase activation, and apoptosis. In summary, my study first describes the involvement of nucleolar function in polyQ pathogenesis and uncovers a new pathogenic mechanism in polyQ diseases. / Detailed summary in vernacular field only. / Tsoi, Ho. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 220-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Thesis Committee --- p.ii / Declaration --- p.iii / Acknowledgement --- p.iv / Abstract --- p.v / Abstract in Chinese --- p.vii / List of Abbreviations --- p.viii / List of Figures --- p.x / List of Tables --- p.xvi / Table of Contents --- p.xvi / Chapter 1 --- Introduction / Chapter 1.1 --- Introduction to Polyglutamine Diseases --- p.1 / Chapter 1.1.1 --- Etiology of Polyglutamine Diseases --- p.1 / Chapter 1.1.2 --- Common Features of Different Types of Polyglutamine Disease --- p.1 / Chapter 1.2 --- Pathogenic Mechanisms of Expanded Polyglutamine Proteins --- p.4 / Chapter 1.2.1 --- Pathogenesis of Polyglutamine Diseases --- p.4 / Chapter 1.2.1.1 --- Loss-of-function toxicity --- p.4 / Chapter 1.2.1.2 --- Gain-of-function toxicity --- p.4 / Chapter 1.3 --- Expanded CAG Transcript-mediated Pathogenic Mechanism --- p.6 / Chapter 1.3.1 --- Identification of the Toxic Role of Expanded CAG Transcripts --- p.6 / Chapter 1.3.2 --- Nuclear Foci Formation of Expanded CAG Transcripts and Polyglutamine Pathogenesis --- p.8 / Chapter 1.4 --- Receptor-mediated RNA nuclear export Transport --- p.9 / Chapter 1.4.1 --- Introduction to RNA Nuclear Export --- p.9 / Chapter 1.4.2 --- Regulation of RNA Nucleocytoplasmic Transport and Human Diseases --- p.11 / Chapter 1.5 --- Function of Nucleolus --- p.12 / Chapter 1.5.1 --- Ribosomal RNA Transcription --- p.12 / Chapter 1.5.2 --- Nucleolar Stress and Apoptosis --- p.15 / Chapter 1.6 --- Research Plan --- p.17 / Chapter 1.6.1 --- Project Objective --- p.17 / Chapter 1.6.2 --- Experimental Model --- p.17 / Chapter 1.6.2.1 --- In vivo Drosophila Model --- p.17 / Chapter 1.6.2.2 --- In vitro Cell Culture Model --- p.19 / Chapter 1.6.2.3 --- Transgenic Mouse Model --- p.20 / Chapter 1.6.3 --- Significance of the Present Study --- p.21 / Chapter 2 --- Materials and Methods / Chapter 2.1 --- Molecular Cloning --- p.22 / Chapter 2.1.1 --- Polymerase Chain Reaction (PCR) --- p.22 / Chapter 2.1.2 --- Primers Used for PCR --- p.29 / Chapter 2.1.3 --- Restriction Digestion --- p.31 / Chapter 2.1.4 --- Agarose Gel Electrophoresis --- p.32 / Chapter 2.1.5 --- Preparation of genomic DNA from A Single Adult Fly --- p.34 / Chapter 2.1.6 --- Removal of 5' Phosphate Groups on Linearized Plasmids --- p.35 / Chapter 2.1.7 --- Addition of 5' Phosphate Group to Linker Oligonucleotides --- p.35 / Chapter 2.1.8 --- Ligation Reaction --- p.37 / Chapter 2.1.9 --- Bacterial Transformation --- p.37 / Chapter 2.2 --- Mammalian Cell Culture --- p.40 / Chapter 2.3 --- Drosophila Culture --- p.44 / Chapter 2.4 --- Semi-quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.48 / Chapter 2.5 --- Microscopy --- p.51 / Chapter 2.6 --- Protein Sample Preparation and Concentration Measurement --- p.53 / Chapter 2.7 --- Co-immunoprecipitation --- p.57 / Chapter 2.8 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting --- p.62 / Chapter 2.9 --- Bacterial Protein Purification --- p.65 / Chapter 2.1 --- DNA Methylation Assay --- p.68 / Chapter 2.11 --- Mitochondrial Fraction Isolation --- p.79 / Chapter 3 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50 Modulates Polyglutamine Diseases Toxicity by Altering the Subcellular Localization of Expanded CAG Transcripts in vivo / Chapter 3.1 --- The Nuclear Accumulation of Expanded CAG Transcripts Correlates with the Neurodegeneration in vivo --- p.72 / Chapter 3.1.1 --- Expanded CAG Transcripts Predominantly Localize in the Nucleus in Drosophila Model of Machado-Joseph Disease --- p.72 / Chapter 3.1.2 --- Nuclear Accumulation of Expanded CAG Transcripts Correlates with the Neurodegeneration in an Inducible Model of Machado-Joseph Disease --- p.73 / Chapter 3.1.3 --- Nuclear Accumulation of Expanded CAG Transcripts Correlates with the Neurodegeneration in Inducible DsRed[subscript CAG100] Model. --- p.76 / Chapter 3.1.3.1 --- Expanded CAG Transcripts Induce the Expression of Pro-apoptotic Genes --- p.77 / Chapter 3.1.3.2 --- Co-expression of p35 Suppresses the Toxicity Induced by the Expanded CAG Transcripts --- p.80 / Chapter 3.2 --- A Candidate-gene RNA Interference Approach was Employed to Identify Genetic Factors Involved in Nuclear Export of Expanded CAG Transcripts --- p.80 / Chapter 3.3 --- Confirmation of the Modulatory Effect of U2 Small Nuclear Riboprotein Auxiliary Factor 50 on Machado-Joseph Disease in vivo --- p.84 / Chapter 3.4 --- The Modulatory Effect of U2 Small Nuclear Riboprotein Auxiliary Factor 50 on Different Drosophila Models of Polygultamine Diseases --- p.84 / Chapter 3.5 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50 Specifically Modulates Expanded CAG Transcript-induced Toxicity in vivo --- p.87 / Chapter 3.5.1 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Enhances Expanded CAG Transcript-induced Toxicity --- p.87 / Chapter 3.5.2 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Does Not Modulate Expanded PolyQ Protein-induced Toxicity --- p.89 / Chapter 3.5.3 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Does Not Alter the Expression Level of Expanded CAG Transcripts in vivo --- p.89 / Chapter 3.5.4 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Does Not Modulate the Toxicity in Fragile X syndrome in vivo --- p.91 / Chapter 3.6 --- Over-expression of Human U2 Small Nuclear Riboprotein Auxiliary Factor 65 Does Not Modulate Expanded CAG Transcript-induced Toxicity in Drosophila --- p.91 / Chapter 3.7 --- Expanded CAG Transcripts Does Not Compromise Endogenous Function of U2 Small Nuclear Riboprotein Auxiliary Factor 50 --- p.94 / Chapter 3.8 --- A Correlation between Nucleocytoplasmic Localization of Expanded CAG Transcripts and Its Induced Toxicity --- p.97 / Chapter 3.8.1 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Enriched DsRedCAG100 Transcripts in the Nucleus in vivo --- p.99 / Chapter 3.8.2 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Enriched MJDCAG78 Transcripts in the Nucleus in vivo --- p.99 / Chapter 3.9 --- Expanded CAG-repeat on the Transcripts Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 50/65 in vivo and in vitro --- p.102 / Chapter 3.9.1 --- Expanded CAG Transcripts Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 50 in vivo --- p.102 / Chapter 3.9.2 --- Expanded CAG Transcripts Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 65 in vitro --- p.103 / Chapter 3.9.3 --- Expanded CAG Transcripts Directly Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 65 in vitro --- p.103 / Chapter 3.10 --- Identification of Expanded CAG Transcripts Interacting Domain on U2 Small Nuclear Riboprotein Auxiliary Factor 65 --- p.107 / Chapter 3.10.1 --- Generation of Different Myc-tagged U2 Small Nuclear Riboprotein Auxiliary Factor 65 Expression Constructs --- p.107 / Chapter 3.10.2 --- RNA Recognition Motif 3 on U2 Small Nuclear Riboprotein Auxiliary Factor 65 Is Essential for the Interaction with Expanded CAG Transcripts --- p.109 / Chapter 3.11 --- Nuclear RNA Export Factor 1 is Involved in U2 Small Nuclear Riboprotein Auxiliary Factor 65-mediated Nuclear Export of Expanded CAG Transcripts --- p.113 / Chapter 3.11.1 --- The Effect of Full Length U2 Small Nuclear Riboprotein Auxiliary Factor 65 and its Corresponding Deletion Mutants on Nuclear Export of Expanded CAG Transcripts --- p.113 / Chapter 3.11.2 --- Formation of Complexes Composed of Nuclear RNA Export Factor 1/U2 Small Nuclear Riboprotein Auxiliary Factor 65/Expanded CAG Transcripts in HEK293 Cells --- p.115 / Chapter 3.12 --- The Nuclear Export of Expanded CAG Transcripts is Mediated by U2 Small Nuclear Riboprotein Auxiliary Factor 65 and Nuclear RNA Export Factor 1 --- p.120 / Chapter 3.13 --- Aging Compromises the Nuclear Export of Expanded CAG Transcripts in Polyglutamine Disease Mouse Model --- p.123 / Chapter 3.13.1 --- Expanded CAG Transcripts Accumulate in the Nucleus of Polyglutamine Disease Mouse Model --- p.123 / Chapter 3.13.2 --- Expression Level of U2 Small Nuclear Riboprotein Auxiliary Factor 65 Declines with Age in Mice --- p.124 / Chapter 3.14 --- Discussion --- p.127 / Chapter 3.14.1 --- Expanded CAG Transcripts Induce Nuclear Toxicity through a Mechanism Independent on Pathogenic Mechanism Mediated by Other Trinucleotide Repeats Expansion --- p.127 / Chapter 3.14.2 --- Nuclear Accumulation of Expanded CAG Transcripts Leads to Neurodegeneration --- p.128 / Chapter 3.14.3 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50 Modulates Expanded CAG Transcript-induced Toxicity by Mediating the Subcellular Localization of Expanded CAG Transcripts --- p.129 / Chapter 3.14.4 --- U2 Small Nuclear Riboprotein Auxiliary Factor 65 and Nuclear RNA Export Factor 1 Regulate the Nuclear Export of Expanded CAG Transcripts --- p.130 / Chapter 3.14.4.1 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50/65 Interacts with Expanded CAG Transcripts and Mediates the Subcellular localization of Expanded CAG Transcripts --- p.130 / Chapter 3.14.4.2 --- U2 Small Nuclear Riboprotein Auxiliary Factor 65 Requires Nuclear RNA Export Factor 1 to Mediate the Nuclear Export of Expanded CAG Transcripts --- p.131 / Chapter 3.14.4.3 --- Developmental Decline of U2 Small Nuclear Riboprotein Auxiliary Factor 65 Protein Level is a Factor That Leads to Progressive Neurodegeneration in Polyglutamine Diseases --- p.134 / Chapter 4 --- Expanded CAG Transcripts Induce Nucleolar Stress / Chapter 4.1 --- Expanded CAG-repeat Sequence Mediates the Nucleolar Localization of RNA Transcripts in vitro --- p.135 / Chapter 4.1.1 --- Machado-Joseph Disease Cell Model --- p.135 / Chapter 4.1.2 --- EGFPCAG Cell Model --- p.137 / Chapter 4.2 --- Expanded CAG Transcripts Suppress Nucleolar Function in vitro and in vivo --- p.140 / Chapter 4.2.1 --- Expanded CAG Transcripts Suppress Ribosomal RNA Transcription in vivo --- p.140 / Chapter 4.2.1.1 --- Drosophila Model of Machado-Joseph Disease --- p.140 / Chapter 4.2.1.2 --- Drosophila Model of DsRedCAG --- p.142 / Chapter 4.2.1.3 --- Transgenic Mouse Model of PolyQ Disease --- p.142 / Chapter 4.2.2 --- Expanded CAG Transcripts Suppress rRNA Transcription in vitro --- p.145 / Chapter 4.2.2.1 --- Machado-Joseph Disease Patient-derived Fibroblast Cell Lines --- p.145 / Chapter 4.2.2.2 --- Expanded CAG Transcript-expressing HEK293 Cells --- p.145 / Chapter 4.3 --- Expanded CAG Transcripts Disrupt the Interaction between RNA Polymerase I and rRNA Promoter in vitro --- p.148 / Chapter 4.4 --- Expanded CAG Transcripts Disrupt the Interaction between Upstream Binding Factor and Upstream Control Element in vitro and in vivo --- p.149 / Chapter 4.4.1 --- Expanded CAG Transcripts Compromise the Interaction between Upstream Binding Factor and Upstream Control Element in vitro --- p.149 / Chapter 4.4.2 --- Expanded CAG Transcripts Compromise the Interaction between Upstream Binding Factor and Upstream Control Element in vivo --- p.151 / Chapter 4.5 --- Expanded CAG Transcripts Induce DNA Hyper-methylation on Upstream Control Element in vitro and in vivo --- p.151 / Chapter 4.5.1 --- The HpaII-PCR Assay for DNA Methylation --- p.154 / Chapter 4.5.2 --- Expanded CAG Transcripts Lead to DNA Hyper-methylation of Upstream Control Element in vitro --- p.154 / Chapter 4.5.2.1 --- Expanded CAG Transcript-expressing HEK293 Cells --- p.154 / Chapter 4.5.2.2 --- Machado-Joseph Disease Patient-derived Fibroblast Cell Lines --- p.156 / Chapter 4.5.3 --- Expanded CAG Transcripts Lead to DNA Hyper-methylation of Upstream Control Element in vivo --- p.156 / Chapter 4.5.4 --- Expanded CAG Transcripts Disrupt the Regulatory Mechanism of Upstream Control Element Methylation in vitro --- p.159 / Chapter 4.6 --- Expanded CAG Transcripts Induce Nucleolar Stress and Apoptosis --- p.161 / Chapter 4.6.1 --- Expanded CAG Transcripts Induce Nucleolar Stress in vitro and in vivo --- p.162 / Chapter 4.6.1.1 --- Expanded CAG Transcript-expressing HEK293 Cells --- p.162 / Chapter 4.6.1.2 --- Transgenic Mouse Model of PolyQ Disease --- p.162 / Chapter 4.6.2 --- Expanded CAG Transcripts Lead to Stabilization of p53 in vitro and in vivo --- p.165 / Chapter 4.6.2.1 --- Expanded CAG Transcripts Lead to Stabilization of p53 in vitro --- p.165 / Chapter 4.6.2.2 --- Expanded CAG Transcripts Lead to Stabilization of p53 in vivo --- p.167 / Chapter 4.6.3 --- Expanded CAG Transcripts Enrich p53 in Mitochondria in vitro --- p.167 / Chapter 4.6.4 --- Expanded CAG Transcripts Lead to Disruption of interaction between Bcl-xL and Bak by p53 in mitochondria in vitro --- p.169 / Chapter 4.6.5 --- Expanded CAG Transcripts Lead to Release of Cytochrome c in vitro --- p.171 / Chapter 4.6.6 --- Expanded CAG Transcripts Lead to Activation of Caspase 3 in vitro --- p.173 / Chapter 4.7 --- Discussion --- p.176 / Chapter 4.7.1 --- Expanded CAG Transcripts Compromise Nucleolar Function --- p.176 / Chapter 4.7.2 --- Expanded CAG Transcripts Induce Apoptosis via Nucleolar Stress --- p.176 / Chapter 4.7.3 --- The Origin of Nucleolar Stress Induced by Expanded CAG Transcripts --- p.178 / Chapter 5 --- Expanded CAG Transcripts Interact with Nucleolin and Deplete It from Upstream Control Element to Suppress Ribosomal RNA Transcription / Chapter 5.1 --- Nucleolin is an Interacting Partner of Expanded CAG Transcripts --- p.180 / Chapter 5.1.1 --- Nucleolin is Pulled down by S1-tagged Expanded CAG Transcripts in vitro --- p.180 / Chapter 5.1.2 --- Expanded CAG Transcripts Interact with Endogenous Nucleolin in vitro --- p.181 / Chapter 5.1.3 --- Expanded CAG Transcripts Directly Interact with Nucleolin in vitro --- p.184 / Chapter 5.2 --- RNA Recognition Motifs 2 and 3 on Nucleolin Interact with Expanded CAG Transcripts --- p.184 / Chapter 5.2.1 --- Generation of Expression Constructs Carrying Full Length Nucleolin and its Deletion Mutants --- p.184 / Chapter 5.2.2 --- Mapping of Domains on Nucleolin Required for Interacting with Expanded CAG Transcripts --- p.187 / Chapter 5.3 --- Nucleolin Regulates Ribosomal RNA Transcription by Mediating the DNA Methylation of Upstream Control Element in HEK293 Cells --- p.187 / Chapter 5.3.1 --- Nucleolin is involved in Regulating the Interaction between Upstream Binding Factor and Upstream Control Element in vitro --- p.191 / Chapter 5.3.2 --- Nucleolin is Involved in Regulating DNA Methylation Level of Upstream Control Element in vitro --- p.191 / Chapter 5.3.3 --- Nucleolin Associates with Upstream Control Element in vitro --- p.194 / Chapter 5.4 --- Expanded CAG Transcripts Deplete Nucleolin from Upstream Control Element in vitro and in vivo --- p.194 / Chapter 5.4.1 --- Expanded CAG Transcripts Compete Nucleolin with Upstream Control Element in vitro --- p.197 / Chapter 5.4.2 --- Expanded CAG Transcripts Compete Nucleolin with Upstream Control Element in vivo --- p.197 / Chapter 5.4.3 --- Expanded Polyglutamine Proteins does not Interact with Nucleolin in vitro --- p.200 / Chapter 5.5 --- Over-expression of Nucleolin Counteracts the Effect of Expanded CAG Transcripts on Ribosomal RNA Transcription in vitro --- p.200 / Chapter 5.5.1 --- Over-expression of Nucleolin Restores the Methylation Level of Upstream Control Element in Dose-dependent Manner in vitro --- p.200 / Chapter 5.5.1.1 --- The Dosage Effect of Nucleolin on DNA Hyper-methylation of Upstream Control Element Induced by Expanded CAG Transcripts in vitro --- p.202 / Chapter 5.5.1.2 --- Does-dependent Expression of Nucleolin in vitro --- p.202 / Chapter 5.5.1.3 --- The Effect of Nucleolin Over-expression on DNA Hyper-methylation of Upstream Control Element Induced by Expanded CAG Transcripts is Dose-dependent in HEK293 cells --- p.205 / Chapter 5.5.2 --- Over-expression of Nucleolin Does Not Alter the Expression Level of Expanded CAG Transcripts in vitro --- p.205 / Chapter 5.5.3 --- Over-expression of Nucleolin Relieves the Nucleolar Stress induced by Expanded CAG Transcripts in vitro --- p.208 / Chapter 5.6 --- Discussion --- p.212 / Chapter 5.6.1 --- The Physical Interaction between Expanded CAG Transcripts and Nucleolin Leads to Suppression of Ribosomal RNA Transcription --- p.212 / Chapter 5.6.2 --- Expanded CAG Transcripts Deprive Upstream Control Element of Nucleolin to Induce Toxicity --- p.212 / Chapter 5.6.3 --- Nucleolin Suppresses Expanded CAG Transcript-induced Cell Death --- p.213 / Chapter 5.6.4 --- Expanded CAG Transcripts Employ both p53-dependent and p53-independent pathways to Induce Cell Death --- p.214 / Chapter 6 --- Concluding Remarks --- p.216 / References --- p.220

Alan George Miller. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 204-230). / 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.

腎細胞癌是一種成人惡性腫瘤，治療效果不理想且常發生腫瘤轉移。目前對腎細胞癌的研究主要集中於鑒定並驗證可用於癌癥早期診斷和預後判斷的新型潛在生物標誌物。擬遺傳學變化尤其是啟動子CpG二核苷酸甲基化所導致的抑癌基因功能喪失已被廣泛認為是腫瘤發生的一個主要機理。迄今為止，已有許多抑癌基因在腎細胞癌中被報道出現啟動子甲基化。這些發現為腎癌發生的分子機制及潛在生物標誌物提供了新的思路。本課題旨在探索ZNF382和BCL6B這兩個鋅指蛋白抑癌基因在腎細胞癌中的啟動子甲基化情況，及其與腫瘤抑制有關的生物學功能和可能的分子機制。 / 鋅指蛋白轉錄抑制子ZNF382已在多種癌癥中被報道為功能性抑癌基因， 且常伴隨有啟動子甲基化導致的基因沈默，但其在腎細胞癌中尚未被報道。我們發現ZNF382在腎癌細胞系中由於啟動子CpG甲基化而致表達下調或基因沈默，並且其表達下調或沈默可被去甲基化藥物逆轉，在正常細胞系中則觀察不到這一現象。腎癌原發腫瘤組織中也廣泛檢測到ZNF382異常甲基化。在ZNF382沈默的腎細胞癌細胞系中，外源表達的ZNF382顯著地抑制了腫瘤細胞集落形成和細胞遷移，並且誘導細胞發生雕亡。而且，我們發現ZNF382在腎癌細胞系中可抑制多種致瘤基因和幹細胞標誌基因的表達。因此，本研究證明ZNF382通過抑制下遊致癌基因和幹細胞標誌基因的表達從而發揮抑制腫瘤的功能，並且其在腎細胞癌中常因啟動子高度甲基化而導致基因失活。 / 另一個鋅指蛋白基因BCL6B（ZNF62）已被證實可通過招募組蛋白去乙酰化酶抑制靶基因的轉錄，但其在腎細胞癌中的表達情況和生物學功能尚不清楚。我們發現，BCL6B基因在正常腎組織和正常細胞系中穩定表達， 但在腎癌細胞系中由於啟動子甲基化其表達下調或沈默。去甲基化藥物可以重新激活BCL6B的表達，同時伴隨其啟動子的去甲基化。BCL6B甲基化在腎癌原發腫瘤組織中也被頻繁檢測到。在腎癌細胞系中，外源表達BCL6B顯著抑制了腫瘤細胞集落形成和細胞遷移，並且誘導腫瘤細胞雕亡。我們進一步發現，BCL6B作為功能性轉錄抑制子在腎癌細胞系中抑制多種致癌基因和幹細胞標誌基因的表達。這些結果表明BCL6B是腎細胞癌的一個抑癌基因且其在腎癌中常被甲基化。 / 綜上所述，本課題從擬遺傳學和生物學功能兩個方面分別鑒定了腎癌中的兩個鋅指蛋白抑癌基因，ZNF382 和BCL6B。此研究可以幫助更好地了解腎癌發生的分子機理，並且為發展新的腎癌標誌物提供了更多思路。 / Renal cell carcinoma (RCC) is a malignant cancer in adults, often with poor outcome and frequent metastasis. Recent studies on this disease focus on the identification and verification of novel potential biomarkers for early detection and prognostic prediction of cancer. Epigenetic alterations, especially promoter CpG methylation, leading to the loss of tumor suppressor gene (TSG) function have been widely recognized as a major cause for tumor pathogenesis. To date, a number of TSGs with aberrant promoter methylation have been reported in RCC, which provides new insights into the molecular mechanism of renal cancer and the potential as biomarkers. The aim of this study is to characterize promoter methylation of two zinc finger tumor suppressors, ZNF382 and BCL6B, their biological functions and underlying molecular mechanisms in RCC. / Transcription repressor ZNF382 (zinc finger protein 382) was reported as a functional TSG with frequent inactivation by promoter methylation in multiple carcinomas, but not studied in RCC yet. I found that ZNF382 was silenced or downregulated in RCC cell lines due to promoter CpG methylation which could be reversed by pharmacologic demethylation treatment, but not in normal renal cell lines. Aberrant methylation of ZNF382 was also frequently detected in the RCC primary tumors. Ectopic expression of ZNF382 in the silenced RCC cells strongly inhibited their clonogenicity and migration, as well as promoted cell apoptosis. Moreover, I found that ZNF382 repressed the expression of multiple oncogenes and stem cell markers in RCC cells. Therefore, my results demonstrate ZNF382 exerts the tumor suppressive function through repressing the downstream target oncogenes and stem cell markers, and is often epigenetically inactivated by promoter methylation in RCC. / Another zinc finger protein, B cell CLL/lymphoma 6 member B (BCL6B, ZNF62) has been identified to repress transcription of its target genes by recruiting histone deacetylases, but its expression and biological function in RCC remain largely unknown. BCL6B was readily expressed in normal kidney tissue and renal cell line. BCL6B was silenced or downregulated by promoter CpG methylation in RCC cell lines. Pharmacologic demethylation reactivated BCL6B expression along with concomitant promoter demethylation. BCL6B methylation was also frequently detected in RCC primary tumors. Ectopic expression of BCL6B in RCC cells significantly inhibited tumor clonogenicity and migration of RCC cells, and induced tumor cell apoptosis. We further found that BCL6B as functional repressor suppressed the expression of multiple oncogenes and stem cell markers. These data indicated BCL6B was a functional tumor suppressor frequently methylated in RCC. / In summary, my study identified two zinc finger tumor suppressors, ZNF382 and BCL6B, in RCC from both epigenetical and functional aspects. This work may contribute to a better understanding of the molecular mechanisms of renal cancer pathogenesis and also give more clues to the discovery of novel biomarkers for RCC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Rong, Rong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 110-128). / Abstracts also in Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / List of abbreviations --- p.xi / List of Figures --- p.xv / List of Tables --- p.xvii / List of Publications --- p.xviii / Chapter Chapter 1 --- Literature Reviews --- p.1 / Chapter 1.1 --- Molecular basis of cancer --- p.1 / Chapter 1.1.1 --- Oncogenes and TSGs --- p.2 / Chapter 1.1.2 --- Cancer genetics --- p.3 / Chapter 1.1.3 --- Cancer epigenetics --- p.4 / Chapter 1.1.4 --- DNA methylation --- p.4 / Chapter 1.1.4.1 --- Mechanism of DNA methylation --- p.5 / Chapter 1.1.4.2 --- DNA methylation and gene transcription --- p.6 / Chapter 1.1.4.3 --- Types of DNA methylation in human cancers --- p.7 / Chapter 1.1.4.3.1 --- Hypomethylation in cancer genome --- p.8 / Chapter 1.1.4.3.2 --- Hypermethylation of TSGs in cancer --- p.8 / Chapter 1.1.5 --- The link between cancer genetics and cancer epigenetics --- p.9 / Chapter 1.2 --- Renal cell carcinoma (RCC) --- p.10 / Chapter 1.2.1 --- Epidemiology of RCC --- p.10 / Chapter 1.2.2 --- Histopathology of RCC --- p.14 / Chapter 1.2.3 --- Genetic and epigenetic alterations in RCC --- p.16 / Chapter 1.2.3.1 --- Genetic alterations --- p.17 / Chapter 1.2.3.2 --- Epigenetic alterations --- p.22 / Chapter 1.2.3.2.1 --- Aberrant DNA hypermethylation in RCC --- p.22 / Chapter 1.2.3.2.2 --- Histone and chromatin regulations in RCC --- p.24 / Chapter 1.2.4 --- Signaling pathways associated with RCC --- p.25 / Chapter 1.2.4.1 --- VHL/HIF signaling in RCC --- p.26 / Chapter 1.2.4.2 --- PI3K/AKT/mTOR signaling in RCC --- p.27 / Chapter 1.2.4.3 --- Wnt/β-catenin signaling in RCC --- p.28 / Chapter 1.2.4.4 --- HGF/MET signaling in RCC --- p.31 / Chapter 1.3 --- Transcription factor family of zinc finger proteins --- p.32 / Chapter 1.3.1 --- Zinc Finger Protein 382 (ZNF382) --- p.33 / Chapter 1.3.2 --- B cell CLL/lymphoma 6, member B (BCL6B) --- p.34 / Chapter Chapter 2 --- Aim of Study --- p.36 / Chapter 2.1 --- Identify two zinc finger repressors as TSGs for RCC --- p.37 / Chapter 2.2 --- Study their tumor suppressor roles in RCC --- p.37 / Chapter 2.3 --- Explore the mechanisms of their tumor suppressor function --- p.38 / Chapter Chapter 3 --- Materials and Methods --- p.39 / Chapter 3.1 --- Cell lines and tissue samples --- p.39 / Chapter 3.1.1 --- Cell lines, tumors and normal tissue samples --- p.39 / Chapter 3.1.2 --- Maintenance of cell lines --- p.39 / Chapter 3.1.3 --- Drug treatment of cell lines --- p.40 / Chapter 3.1.4 --- Total RNA extraction --- p.40 / Chapter 3.1.5 --- Genomic DNA extraction --- p.41 / Chapter 3.2 --- General techniques --- p.42 / Chapter 3.2.1 --- Agarose gel electrophoresis --- p.42 / Chapter 3.2.2 --- TA cloning --- p.43 / Chapter 3.2.3 --- Transformation of cloning vectors into E. coli competent cells --- p.43 / Chapter 3.2.4 --- Plasmid DNA extraction --- p.44 / Chapter 3.2.4.1 --- Mini-prep of plasmid DNA --- p.44 / Chapter 3.2.4.2 --- Midi-prep of plasmid DNA --- p.45 / Chapter 3.2.5 --- Measurement of DNA and RNA concentrations --- p.45 / Chapter 3.2.6 --- Preparation of reagents and medium --- p.46 / Chapter 3.2.6.1 --- Reagents for agarose gel electrophoresis --- p.46 / Chapter 3.2.6.2 --- Reagents for mini-prep of plasmid DNA --- p.46 / Chapter 3.2.6.3 --- LB medium and LB plates --- p.46 / Chapter 3.3 --- Semi-quantitative Reverse transcription (RT)-PCR --- p.47 / Chapter 3.3.1 --- Reverse Transcription --- p.47 / Chapter 3.3.2 --- Semi-quantitative PCR --- p.48 / Chapter 3.3.2.1 --- Primer design --- p.48 / Chapter 3.3.2.2 --- PCR reaction --- p.49 / Chapter 3.4 --- Real-time PCR --- p.49 / Chapter 3.5 --- Methylation analysis --- p.50 / Chapter 3.5.1 --- Bisulfite treatment of genomic DNA --- p.50 / Chapter 3.5.2 --- Bioinformatical analysis of CpG island --- p.51 / Chapter 3.5.3 --- Methylation-specific PCR (MSP) --- p.51 / Chapter 3.5.3.1 --- Primers design --- p.51 / Chapter 3.5.3.2 --- PCR reaction --- p.53 / Chapter 3.5.4 --- Bisulfite genomic sequencing (BGS) --- p.53 / Chapter 3.5.4.1 --- Primers design --- p.53 / Chapter 3.5.4.2 --- PCR amplification and TA-cloning --- p.54 / Chapter 3.6 --- Construction of expression plasmids for studied genes --- p.54 / Chapter 3.6.1 --- Construction of the ZNF382-expressing vector --- p.54 / Chapter 3.6.2 --- Construction of the BCL6B-expressing vector --- p.55 / Chapter 3.7 --- Functional Study --- p.56 / Chapter 3.7.1 --- Colony formation assay on monolayer culture --- p.56 / Chapter 3.7.2 --- Wound healing assay --- p.57 / Chapter 3.7.3 --- TUNEL assay --- p.58 / Chapter 3.8 --- Western blot --- p.58 / Chapter 3.9 --- Statistical analysis --- p.58 / Chapter Chapter 4 --- Results --- p.60 / Chapter 4.1 --- Epigenetic and Functional study of ZNF382 in RCC --- p.60 / Chapter 4.1.1 --- Expression profiling of ZNF382 in human adult tissues --- p.60 / Chapter 4.1.2 --- Expression profiling of ZNF382 in RCC cell lines --- p.61 / Chapter 4.1.3 --- Dense promoter CpG methylation of ZNF382 correlated with its reduced expression in RCC --- p.62 / Chapter 4.1.4 --- Restoration of ZNF382 expression by pharmacologic demethylation --- p.65 / Chapter 4.1.5 --- Frequent methylation of ZNF382 in RCC primary tumors --- p.67 / Chapter 4.1.6 --- Functional study of ZNF382 in RCC --- p.68 / Chapter 4.1.6.1 --- Ectopic expression of ZNF382 inhibits clonogencity of RCC cells --- p.68 / Chapter 4.1.6.2 --- Ectopic expression of ZNF382 inhibits migration of RCC cells --- p.71 / Chapter 4.1.7 --- ZNF382 induces apoptosis of RCC cells --- p.72 / Chapter 4.1.8 --- ZNF382 represses the expression of multiple oncogenes and stem cell markers in RCC --- p.73 / Chapter 4.1.9 --- Discussion --- p.76 / Chapter 4.2 --- Epigenetic and Functional study of BCL6B in RCC --- p.82 / Chapter 4.2.1 --- Expression profiling of BCL6B in human adult tissues --- p.82 / Chapter 4.2.2 --- Expression profiling of BCL6B in RCC cell lines --- p.83 / Chapter 4.2.3 --- Correlation of the methylation status of ZNF382 promoter CpG island with its aborted expression in RCC --- p.84 / Chapter 4.2.4 --- Restoration of BCL6B expression by pharmacological demethylation --- p.87 / Chapter 4.2.5 --- Frequent BCL6B methylation in RCC primary tumors --- p.88 / Chapter 4.2.6 --- Functional study of BCL6B in RCC --- p.90 / Chapter 4.2.6.1 --- Ectopic expression of BCL6B inhibits clonogencity of RCC cells --- p.90 / Chapter 4.2.6.2 --- Ectopic expression of ZNF382 inhibits migration of RCC cells --- p.92 / Chapter 4.2.7 --- BCL6B induces apoptosis of RCC cells --- p.93 / Chapter 4.2.8 --- BCL6B represses the expression of multiple oncogenes and stem cell markers in RCC --- p.94 / Chapter 4.2.9 --- Discussion --- p.97 / Chapter Chapter 5 --- General discussion --- p.103 / Chapter Chapter 6 --- Summary --- p.106 / Chapter Chapter 7 --- Future Study --- p.108 / Chapter 7.1 --- Identification of key responsive elements in gene promoter --- p.108 / Chapter 7.2 --- Study of genetic alterations leading to gene inactivation --- p.109 / Chapter 7.3 --- Elucidation of the transcription-repressor activity in RCC --- p.109 / Reference List --- p.110

Background: Asthma is characterized by chronic airway inflammation in which leukocytes are attracted into the inflamed airway under the influence of chemokines. Molecular studies and allergen bronchoprovocation suggested that chemokines such as thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), eotaxin and regulated upon activation normal T-cell expressed and secreted (RANTES) were involved in the airway responses to allergen exposure. / Conclusions: Chemokines are important mediators in the pathophysiology of asthma and atopy. TARC in plasma and MDC in EBC appear to be useful biomarkers for assessing childhood asthma. Besides, MDC concentrations in UCB may predict the susceptibility to develop wheezing during infancy. / Methods: Asthmatic patients, non-allergic controls and healthy singleton newborns were recruited from attendants of a university teaching hospital. Atopy-related chemokines in peripheral blood and EBC were measured by enzyme-linked immunosorbent assays. Genomic DNA from asthmatics and controls was genotyped by restriction fragment length polymorphism to characterize single nucleotide polymorphisms (SNPs) in the genes encoding TARC, RANTES, interleukin-13 and CD14. / Objectives: This thesis investigated the relation between chemokines and asthma and atopy by: (a) measuring their concentrations in peripheral blood and exhaled breath condensate (EBC); (b) performing case-control association studies for genes encoding atopy-related chemokines and related molecules; and (c) analyzing chemokines in umbilical cord blood (UCB) in relation to wheezing phenotypes during infancy. / Results: Plasma TARC concentrations were higher in children with chronic asthma than controls, and also correlated with plasma total IgE. Among children with asthmatic exacerbation, plasma TARC concentrations showed inverse correlation with peak expiratory flow rates at presentation. When measured in EBC, MDC but not TARC or eotaxin was higher in asthmatics than controls. In our genetic association studies, SNPs in IL13, RANTES and TARC were associated with serum total and/or allergen-specific IgE. TARC C-431T was also linked to peripheral eosinophilia. However, none of these polymorphisms was associated with physician-diagnosed asthma. Interestingly, C-159T in CD14 was also associated with serum total IgE, but only among atopic asthmatic children. In the last part involving 124 singleton healthy newborns, MDC concentrations in UCB were significantly increased in newborns who wheezed during infancy. / Leung Ting-fan. / Adviser: Gary W.K. Wong. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 196-231). / 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.

Mast cells are pivotal effector cells in the pathogenesis of allergic and inflammatory diseases. Activation of FcepsilonRI in mast cells by antigen initiates a complex series of biochemical events leading to the release and synthesis of myriads of chemical mediators and cytokines. Adenosine is an endogenous nucleoside formed from cleavage of AMP by the enzyme 5'-nucleotidase. It exerts modulating effects in a large number of cellular systems by acting through four distinct subtypes of adenosine receptors (A1, A 2A, A2B and A3) which belong to the G-protein-coupled receptor (GPCR) family. Increasing evidence have been provided to show that adenosine plays a role in the pathophysiology of asthma through a mast cell dependent mechanism. / Pharmacological studies using specific adenosine agonists and antagonists revealed that A1 receptor was responsible for the potentiating effect of adenosine with the involvement of the pertussis toxin-sensitive Galphai-protein. Conversely, inhibition of HCMC activation was mediated by A2B receptor and was accompanied by the elevation of cAMP level suggesting the participation of Galphas-protein. / Taken together, the current studies explored the dual effect of adenosine on human mast cells activation which enhanced our understanding of adenosine receptor biology. The effectiveness of adenosine in modulating the important mast cell activation pathways definitely facilitates the rational exploitation of these receptors as therapeutic targets that could be converted into clinical benefits for asthmatic patients. / To better characterize the effect of adenosine on human mast cell under asthmatic environment, we incubated HCMC under different inflammatory condition found in asthmatic, including toll-like receptor (TLR) ligands and inflammatory cytokines. Functional studies on mediator release from HCMC indicated that out of all tested substances, Peptidoglycan (PGN) pre-incubation enhanced the IL-8 synthesis from HCMC in response to low concentration of adenosine (10-9--10-7 M). / We also investigated the action of adenosine on key signal transduction pathways involved in mast cells activation. Study on intracellular calcium concentration ([Ca2+]i) revealed that low concentration of adenosine (10-8 M) through activation of PI3Kgamma significantly enhanced Ca2+ influx. In contrast, high concentration of adenosine at 10-4 M substantially inhibited [Ca2+] i in response to anti-IgE. Furthermore, investigation on intracellular signaling molecules provided evidence that adenosine at concentrations over 10-6 M does-dependently inhibited the immunoglobulin (IgE)-dependent activation of ERK, JNK or NF-kappaB pathways, whereas enhancement of IkappaBalpha was found on low concentration of adenosine. The above observation help to justify the dual action of adenosine on anti-IgE-induced mediators release from HCMC. Our investigation further suggested that adenosine may inhibit HCMC activation through a novel cAMP-dependent, but PKA- and EPAC-independent, signaling pathway. / We generated human cultured mast cells (HCMC) from human buffy coat and confirmed the expression of all adenosine receptor subtypes in them. We showed that adenosine alone did not induce HCMC degranulation and cytokine release. However, adenosine and the non-selective agonist, 5'-N-Ethylcarbox-amidoadenosine (NECA), produced a biphasic response on anti-IgE induced mast cell activation. An enhancement of HCMC activation was observed with low concentrations of adenosine and NECA (10-9--10-7 M), whereas a predominant inhibitory action was observed at concentrations higher than 10-6 M. / Yip, Kwok Ho. / Adviser: Alaster H.Y. Lau. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 237-263). / 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.

As CFTR plays an important role in HCO3- transport, and HCO3- sensitive soluble adenylyl cyclase (sAC) has been shown to be largely responsible for the cAMP production in spermatogenetic cells, we hypothesized that CFTR-mediated HCO3- transport was important to spermatogenesis via sAC pathway in spermatogenetic and Sertoli cells. Using intracellular pH measurement, we demonstrated that CFTR is involved in HCO3- transport in Sertoli cells. RT-PCR results showed that increased HCO3- concentrations in the culture medium resulted in upregulation of CFTR expression. The results also showed that the intracellular cAMP level in Sertoli cells increased as the extracellular HCO3- concentration increased. HCO3- also caused phosphorylation of the cAMP response element binding (pCREB) proteins transcription factor on serine 133, a modification known to be required by Sertoli cells to support spermatogenesis. This phosphorylation could be inhibited by CFTR inhibitor, further lending support to the notion that CFTR is important for HCO3- transport in Sertoli cells, leading to HCO3- dependent events that are important for spermatogenesis. / CFTR is known to be widely expressed in epithelial cells of male reproductive tracts, but its expression in spermatogenic cells is less well known. We first confirmed the expression of CFTR in spermatogenic cells and mature sperm in rodents. Our study thus focused on the important role of CFTR in the processes related to male fertility including spermatogenesis and sperm capacitation. / Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel, mutations of which cause cystic fibrosis, a disease characterized by defective Cl- and HCO3- transport. While over 95% of CF male patients are infertile because of congenital bilateral absence of the vas deferens (CBAVD), the question whether CFTR mutations are involved in other forms of male infertility is under intense debates. / In conclusion, our study has demonstrated the role of CFTR in male reproductive system. We have further elucidated its possible physiological role and the underlying molecular mechanisms. These studies may pave the way for the development of method strategies for diagnosis and treatment of CFTR related infertility in male. / Our study also detected CFTR in both human and mouse sperm. CFTR inhibitor or antibody significantly reduced sperm capacitation, and the associated HCO 3--dependent events including increases in intracellular pH, cAMP production and membrane hyperpolarization. The fertilizing capacity of the sperm obtained from heterozygous CFTR mutant mice is also significantly lower as compared to that of the wild type. These results suggest that CFTR in sperm may be involved in the transport of HCO3- important for sperm capacitation and that CFTR mutations with impaired CFTR function may lead to reduced sperm fertilizing capacity and male infertility other than CBAVD. / We further demonstrated the physiological role of CFTR in spermatogenesis using CFTR knockout mice as an in vivo model. Although TUNNEL staining showed normal percentage of apoptotic cells in seminiferous tubules, Cftr -/- mice had spermatogenetic defects in histology section and fewer number of mature sperm compared with wild type (WT) mice. Consistent with the proposed role of CFTR in spermatogenesis, RT-PCR and Western blot results showed reduced expression of spennatids specific gene, Protamine 1, Protamine 2, and CREM, which have been known to be involved in the process of spermatogenesis, in Cftr-/- mice. / Xu, Wenming. / "January 2008." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4506. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 121-138). / 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.

Zhao Hailu. / "June 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 192-210). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.