by Chan Ka Lee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 94-112). / Abstracts in English and Chinese. / Acknowledgements --- p.i / abstract(english/chinese) --- p.iii / Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / Chapter I --- Introduction --- p.1 / Chapter I.1 --- Gastric Lymphoma --- p.1 / Chapter I.1.1 --- Background --- p.1 / Chapter I.1.2 --- Mucosa-Associated Lymphoid Tissue --- p.2 / Chapter I.1.3 --- Classification of Primary Gastric Lymphomas --- p.3 / Chapter I.1.3.1 --- Mucosa-Associated Lymphooid Tissue Type Lymphomas --- p.3 / Chapter I.1.3.2 --- High Grade Primary Gastric Lymphomas --- p.5 / Chapter I.1.3.3 --- Other Gastric Lymphomas --- p.7 / Chapter I.2 --- Helicobater Pylori --- p.8 / Chapter I.3 --- Epstein-Barr Virus --- p.9 / Chapter I.3.1 --- Epidemiology --- p.9 / Chapter I.3.2 --- Virus and Genome Structure --- p.9 / Chapter I.3.3 --- Latent Infection --- p.11 / Chapter I.3.4 --- Latent Membrane Protein-1 --- p.12 / Chapter I.3.5 --- "EBV-Encoded, Small Non-polydenylated RNAs (EBERs)" --- p.13 / Chapter I.3.6 --- Disease Associated with EBV --- p.13 / Chapter I.3.7 --- EBV and PGL --- p.14 / Chapter I.4 --- Genetic Alterations --- p.15 / Chapter I.4.1 --- Background --- p.15 / Chapter I.4.2 --- Tumor Suppressor Genes (TSGs) --- p.16 / Chapter I.4.2.1 --- Origin and Structure of p53 Gene and Protein --- p.16 / Chapter I.4.2.2 --- Functions of p53 Gene --- p.18 / Chapter I.4.2.3 --- Inactivation Mechanisms of p53 --- p.21 / Chapter I.4.2.4 --- p53 Mutations and Protein Expression in NHLs --- p.24 / Chapter I.4.3 --- Oncogene --- p.25 / Chapter I.4.3.1 --- Bcl-2 --- p.25 / Chapter I.4.3.2 --- Other Oncogenes --- p.27 / Chapter II --- OBJECTIVES OF STUD Y --- p.30 / Chapter III --- ma terials and methods --- p.31 / Chapter III.1 --- Materials --- p.31 / Chapter III.2 --- Detection of EB V Latent Gene Product by In-situ Hybridization --- p.33 / Chapter III.2.1 --- Pretreatment of Paraffin-embedded Tissues and Apparatus --- p.33 / Chapter III.2.2 --- In-situ Hybridization of EBERs --- p.34 / Chapter III.3 --- Detection of p53 and bcl-2 and LMP-1 Protein Expression by Immunohisiochemistry --- p.35 / Chapter III.4 --- Microdissection of Formalin-fixed Paraffin-embedded Tissues --- p.37 / Chapter III.5 --- Extraction of Genomic DNA from Formalin-fixed Paraffin-embedded Tissues --- p.38 / Chapter III.5.1 --- Phenol / Chloroform Extraction --- p.38 / Chapter III.5.2 --- Commercially Available DNA Extraction Kit --- p.40 / Chapter III.6 --- Mutational Analysis p53 --- p.41 / Chapter III.6.1 --- Polymerase Chain Reaction - Single Strand Conformation Polymorphism (PCR-SSCP) Analysis --- p.41 / Chapter III.6.1.1 --- PCR primers --- p.41 / Chapter III.6.1.2 --- PCR Amplification ofp53 gene --- p.42 / Chapter III.6.1.3 --- Non-denaturing Polyacrylamide Gel Electrophoresis --- p.42 / Chapter III.6.2 --- DNA Sequencing Analysis --- p.44 / Chapter III.6.2.1 --- Purification of DNA from Shifts on Non-denaturing Gels --- p.44 / Chapter III.6.2.2 --- 5' end Labeling of Primer --- p.45 / Chapter III.6.2.3 --- Cycle Sequencing --- p.45 / Chapter III.6.2.4 --- Denaturing Gel Electrophoresis --- p.46 / Chapter III.7 --- Loss of Heterozygosity (LOH) Analysis on Chromosome 17p --- p.47 / Chapter III.7.1 --- Microsatellite Markers --- p.49 / Chapter III.7.2 --- PCR Amplification of DNA Fragments Containing Polymorphic Microsatellites --- p.49 / Chapter III.7.3 --- Denaturing Polyacrylamide Gel Electrophoresis --- p.50 / Chapter III.7.4 --- Determination of Allelic Abnormalities --- p.51 / Chapter III. 8 --- Statistical Analysis --- p.52 / Chapter IV --- results --- p.53 / Chapter IV.1 --- Association with Helicobactor Pylori (HP) --- p.53 / Chapter IV.2 --- Detection of EBERs by ISH --- p.53 / Chapter IV.3 --- Immunohistochemical Analysis --- p.54 / Chapter IV.3.1 --- Protein Expression of EBV LMP-1 --- p.54 / Chapter IV.3.2 --- Protein Expression of p53 --- p.54 / Chapter IV.3.3 --- Protein Expression of bcl-2 --- p.55 / Chapter IV.3.4 --- Correlation between p53 and bcl-2 Protein Expression --- p.55 / Chapter IV.4 --- Mutational Analysis of p53 --- p.56 / Chapter IV.5 --- LOH Analysis on Chromosome 17p --- p.57 / Chapter V --- DISCUSSION --- p.58 / Chapter V.1 --- Helicobactor Pylori Association --- p.58 / Chapter V.2 --- Association with EE V --- p.60 / Chapter V.3 --- Protein Expression of p53 and bcl-2 --- p.61 / Chapter V.3.1 --- p53 --- p.61 / Chapter V.3.2 --- Bcl-2 --- p.62 / Chapter V.3.3 --- Correlation between p53 and bcl-2 Expression --- p.63 / Chapter V.4 --- p53 Gene Molecular Analysis --- p.65 / Chapter V.5 --- Distribution of Mutations and Molecular Fingerprinting --- p.67 / Chapter V.6 --- Possible Role of p53 Mutation in EBV+ Gastric Lymphomas --- p.69 / ILLUSTRATIONS --- p.71 / references --- p.94

by Wan Yu Kwan. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 112-125). / Abstracts in English and Chinese. / Acknowledgement --- p.2 / Abstract --- p.3 / Table of Contents --- p.5 / Chapter Chapter 1 - --- Introduction --- p.7 / Chapter 1.1 --- Contribution of lifting techniques on risk assessment and training on manual materials handling --- p.7 / Chapter 1.2 --- Objectives --- p.12 / Chapter 1.3 --- Hypotheses --- p.12 / Chapter 1.4 --- Significance of Study --- p.13 / Chapter Chapter 2 - --- Literature Review --- p.14 / Chapter 2.1 --- Societal concerns on employee compensation --- p.14 / Chapter 2.2 --- Etiology of low back injury --- p.15 / Chapter 2.2.1 --- Compression forces on vertebral joints --- p.16 / Chapter 2.2.2 --- Shear forces on vertebral joints --- p.17 / Chapter 2.3 --- Lifting techniques --- p.18 / Chapter 2.3.1 --- Effect of lifting techniques on curvature of the spine --- p.22 / Chapter 2.3.2 --- Importance of leg muscles in manual lifting --- p.22 / Chapter 2.4 --- Prediction of low back injury in manual lifting --- p.24 / Chapter 2.4.1 --- Compression forces and moments --- p.26 / Chapter 2.4.2 --- Balance control --- p.29 / Chapter 2.4.3 --- Application of Surface electromyography in ergonomics --- p.31 / Chapter Chapter 3 - --- Method --- p.34 / Chapter 3.1 --- Subject recruitment --- p.34 / Chapter 3.2 --- Equipment --- p.35 / Chapter 3.2.1 --- Force platform --- p.35 / Chapter 3.2.2 --- Motion analysis system --- p.37 / Chapter 3.2.3 --- Surface electromyography --- p.38 / Chapter 3.3 --- Maximum voluntary contraction test --- p.40 / Chapter 3.3.1 --- MVC of vastus medialis --- p.41 / Chapter 3.3.2 --- MVC of medial gastrocnemius --- p.42 / Chapter 3.3.3 --- MVC of erector spinae --- p.43 / Chapter 3.4 --- Lifting techniques --- p.44 / Chapter 3.5 --- Experimental procedures --- p.48 / Chapter 3.6 --- Statistical analysis --- p.53 / Chapter Chapter 4 - --- Results and Dicussion --- p.54 / Chapter 4.1 --- No. of cases in the study --- p.54 / Chapter 4.2 --- Phases of lifting --- p.55 / Chapter 4.3 --- Process time --- p.56 / Chapter 4.4 --- Trunk inclination angles --- p.62 / Chapter 4.5 --- Hip joint angles --- p.67 / Chapter 4.6 --- Knee joint angles --- p.73 / Chapter 4.7 --- Center of mass of box --- p.79 / Chapter 4.8 --- Muscle activities --- p.84 / Chapter Chapter 5 - --- Conclusion --- p.109 / Chapter Chapter 6 - --- References --- p.112

Yau, Ka Long. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 97-113). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Ng Wah. / Thesis submitted in: August 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 101-113). / Abstracts in English and Chinese. / ABSTRACT --- p.II / 摘要 --- p.IV / ACKNOWLEDGEMENT --- p.VI / TABLE OF CONTENTS --- p.VII / LIST OF TABLES --- p.X / LIST OF FIGURES --- p.XI / ABBREVIATIONS --- p.XII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Etiological Factors of Hepatocellualr Carcinoma (HCC) --- p.4 / Chapter 1.2.1 --- Dietary Aflatoxins --- p.4 / Chapter 1.2.2 --- Liver Cirrhosis --- p.5 / Chapter 1.2.3 --- Alcohol Abuse --- p.6 / Chapter 1.2.4 --- Viral Hepatitis Infection --- p.6 / Chapter 1.3 --- Literature Review on the Investigations of HBV Integrants in HCC --- p.16 / Chapter 1.3.1 --- Affected Host Junctions --- p.17 / Chapter 1.3.2 --- Viral Junctions --- p.18 / Chapter 1.4 --- Restriction Site Polymerase Chain Reaction (RS-PCR) --- p.19 / Chapter 1.5 --- Aims of Thesis --- p.21 / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Chemicals --- p.23 / Chapter 2.1.2 --- Buffers --- p.24 / Chapter 2.1.3 --- Cell Cultures --- p.24 / Chapter 2.1.4 --- Nucleic Acids --- p.24 / Chapter 2.1.5 --- Enzymes --- p.25 / Chapter 2.1.6 --- Equipment --- p.25 / Chapter 2.1.7 --- Software and Web Resources --- p.26 / Chapter 2.2 --- Methods --- p.27 / Chapter 2.2.1 --- DNA Extraction --- p.27 / Chapter 2.2.2 --- RS-PCR --- p.31 / Chapter 2.2.3 --- Sequencing --- p.37 / Chapter 2.2.4 --- Spectral Karyotyping (SKY) --- p.38 / Chapter 2.2.5 --- Fluorescence In situ hybridization --- p.39 / Chapter Chapter 3 --- Investigation of HBV Integration Sites in HCC Cell lines --- p.45 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Materials and Methods --- p.47 / Chapter 3.2.1 --- Cell Lines --- p.47 / Chapter 3.2.2 --- RS-PCR --- p.47 / Chapter 3.2.3 --- Spectral Karyotyping --- p.48 / Chapter 3.2.4 --- Tyramide Signal Amplification for HBV in FISH Analysis --- p.48 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Identification of HBV Integration Sites in Cell Lines --- p.51 / Chapter 3.3.2 --- Evaluation of RSO Primer Efficiency --- p.52 / Chapter 3.3.3 --- SKY and FISH Analysis --- p.53 / Chapter 3.4 --- Discussion --- p.64 / Chapter 3.4.1 --- HBV Insertions in HCC Cell Lines --- p.64 / Chapter 3.4.2 --- Efficacy of RSO Primers --- p.65 / Chapter 3.4.3 --- Investigation of HBV Integration on Chromosomal Rearrangement --- p.65 / Chapter Chapter 4 --- Investigation of Hepatitis B Virus Integration Sites in Primary HCC --- p.67 / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Materials and Methods --- p.69 / Chapter 4.2.1 --- Patients --- p.69 / Chapter 4.2.2 --- RS-PCR --- p.70 / Chapter 4.3 --- Results --- p.72 / Chapter 4.3.1 --- HBV Integration Sites in Primary HCC Tumors and Adjacent Non- malignant Liver --- p.72 / Chapter 4.4 --- Discussion --- p.88 / Chapter 4.4.1 --- HBV integration Sites in Primary HCC Tumors and Adjacent Non- malignant Liver --- p.88 / Chapter 4.4.2 --- Summary on HBV Integrants Identified --- p.91 / Chapter Chapter 5 --- Proposed Future Studies --- p.98 / Chapter 5.1 --- Correlation of Structural Aberrations with HBV Integrations --- p.99 / Chapter 5.2 --- Transcriptional Expression Study on the Genes Interrupted by or Located near the Virus Host Junctions --- p.100 / Chapter Chapter 6 --- References --- p.101

研究背景 / 系統性紅斑狼瘡（SLE）是一種較常見的累及多系統多器官的自身免疫性疾病，由於細胞和體液免疫功能障礙，產生多種自身抗體，確切病因不明。研究一般認為是在遺傳、環境等諸多因素的共同作用下導致了機體固有免疫及獲得性免疫系統功能紊亂，從而發病。作為機體抵抗病原體入侵的第一道防線，固有免疫系統通過模式識別受體（PRRs），不僅可以識別結合外源性的病原體相關模式分子（PAMPs），也可識別機體自身細胞所釋放的內源性危險信號又稱破壞相關模式分子（DAMPs），從而啟動信號傳導途徑，激活天然免疫細胞，從而導致一系列免疫應答的發生。本文將初步探討三類PRRs在SLE發病機制及病毒感染中的作用：（i）胞質中識別細胞壁肽聚糖的NOD樣受體（NLR），（ii）識別危險信號分子（DAMP）的膜結合型晚期糖基化終末產物受體（RAGE）及（iii）識別核酸的胞內Toll樣受體（TLR）。 / NLR是一種新發現的PAMP識別受體，在配體識別及信號傳導方面有別於膜型PRRs，在固有免疫應答中發揮重要作用。目前報道中NLR至少有23個成員，其中最有代表性的是NOD1和NOD2，通過特異性識別細菌胞壁肽聚糖產物從而參與固有免疫應答並誘導炎症反應和細胞凋亡。前期研究多集中於NODs與SLE基因易感性的探討，而SLE患者體內免疫細胞是否功能性表達NOD2及其下遊 的效應如何，仍有待進一步探討。 / RAGE是一種多配體受體，廣泛分布於上皮細胞、血管及炎症細胞表面，低表達於正常組織細胞，但與其配體結合後可啟動激活細胞內部各種信號傳導機制從而產生相應的生物學效應。HMGB1作為RAGE的重要配體在細胞和組織中分佈十分廣泛，近年研究證實，胞外高表達的HMGB1為一種重要的內源性危險信號分子，通過RAGE受體通路，可促進趨化作用，並通過激活NF-κB途徑誘導炎症反應。越來越多的證據表明HMGB1在自身免疫性疾病中起積極作用， RAGE-HMGB1軸在SLE的炎症反應及組織損傷中的重要作用值得研究。 / RAGE基因可因RNA的選擇性剪接而分為：全長RAGE（flRAGE）、截去N端的RAGE及截去C端的可溶性RAGE（sRAGE）。sRAGE有兩種來源，其中由細胞分泌而來的又稱為內分泌性RAGE（esRAGE）。sRAGE通過與flRAGE競爭性與結合配體從可抑制RAGE誘導的細胞信號傳導途徑，故又稱為作為“誘餌受體，作為潛在的治療靶點，對疾病的進展有保護作用。因此，評估sRAGE/esRAGE與flRAGE及配體HMGB1在SLE患者體內的動態平衡具有顯著臨床意義。 / 人類乳頭狀病毒（HPV）感染是子宮頸癌的致病因素，高危型HPV感染的持續存在是子宮頸癌的重要風險因素之一。早期病例對照研究已提示伴高炎症狀態及長期多重高危型HPV感染的SLE患者其子宮頸巴氏塗片異常和宮頸癌的發病率顯著高於對照人群，但在前瞻性隊列研究中其致高危致病因素及預測因子並未得到證實。TLR家族在早期固有免疫中對入侵病原微生物的識別發揮重要作用，胞內TLR3、TLR7、TLR8、TLR9通過識別病毒核酸成分通過介導下遊信號轉導誘導免疫反應。是否固有免疫系統異常參與SLE患者體內HPV持續及高危感染，仍有待進一步探討。 / 研究目的 / 1.研究NOD2受體通路及RAGE-HMGB1軸在SLE發病機制中的作用； / 2.闡明sRAGE/esRAGE作為“保護因子與flRAGE及其配體HMGB1在SLE患者體內的動態平衡及評估與疾病活動相關性； / 3.探討TLR受體通路在宿主SLE抗 HPV感染中的作用。 / 研究方法 / 本文通過三項臨床病例對照研究分別探討NLR、RAGE、TLR在SLE發病機制及病毒感染中的作用。 / 研究結果 / 1.SLE外周高表達於單核細胞內的NOD2可通過特異性識別配體誘導外周血單個核細胞的異常活化及促炎細胞因子的產生；而免疫抑制治療可下調CD8+ T細胞及抗原體提呈細胞內NOD2表達及抗炎細胞因子的產生； / 2.FlRAG高表達於SLE患者外周血單核細胞；血漿sRAGE作為獨立風險因素，與SLE疾病活動指數呈負相關；HMGB1單獨或與TLR9配體協同作用可刺激單核細胞分泌促炎細胞因子並激活信號轉導通路； / 3.TLR拮抗劑（羥氯喹）及強的松治療作為獨立風險因素可下調SLE患者子宮頸上皮細胞中TLR7和TLR9的表達；腫瘤相關的高危型HPV細胞株內核酸識別受體TLR及幹擾素刺激基因（ISGs）表達明顯下調伴功能異常。 / 研究結論 / 一方面，異常活化的PRRs通過識別結合外源及內源性病原體相關分子從而啟動固有免疫應答，激活一系列信號轉導通路，參與SLE的自身免疫反應： / 1.胞內受體NOD2可能通過特異性識別細菌胞壁酰二肽及誘導炎症反應，參與固有免疫應答反應抵禦外源性病原體侵襲，為感染因素導致SLE發病的假說尋找進一步理論依據； / 2.膜表面受體RAGE與配體HMGB1結合可激活細胞內多信號轉導機制，參與固有免疫應答反應抵禦內源性病原體侵襲，為胞內危險信號分子释放導致SLE無菌性炎症的假說提供初步理論依據； / 3.可溶性“誘餌受體RAGE作為潛在治療靶點可抑制SLE體內高炎症反應。 / 另一方面，多重因素交叉作用可引起SLE患者體內PRR轉錄及表達下調，從而抑制固有免疫應答，導致病毒逃避宿主免疫系統的監視及清除而長期潛伏： / 1.TLR拮抗劑（羥氯喹）和強的松治療可能引起SLE患者體內TLR7和TLR9表達下調，從而抑制固有免疫系統對外源侵入性病原體HPV的識別； / 2.腫瘤相關的高危型HPV細胞株亦可通過抑制TLR7和TLR9轉錄、下調受體表達及功能致 HPV逃避宿主免疫防禦而長期潛伏。 / Introduction / The pathogenesis of systemic lupus erythematosus (SLE) is a complicated process caused by genetic and environmental factors resulting in abnormalities of both the innate and the adaptive immune system. Sensing the presence of a pathogen is the first step for the immune system to mount an effective response to eliminate invading microorganisms and establish protective immunity. The innate immune system constitutes an important defense system to respond rapidly to both endogenous and exogenous molecules, in which the pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs) can interact with the pattern recognition receptors (PRRs) and then activate the antigen presenting cells (APCs), T, B cells. / Effective sensing of endogenous and exogenous molecules promotes autoreactivity via immune activation and antigen presentation. In lupus, these molecules may have a special role in the pathogenesis since they can serve as targets of autoreactivity as well as inducers. In this series of experiments, we focused on the roles of various PRRs including nucleic acid sensing toll-like receptors (TLRs), bacterial peptidoglycans sensing NOD-like receptors (NLRs) and dangerous signals sensing receptor for advanced glycation end products (RAGE), which are involved in the recognition of PAMPs and DAMPs sharing between microbes and the host in the pathogenesis of SLE. / In contrast to the well elucidated membrane-bound TLRs, cytoplasmic NLRs are a new family of PRRs for the recognition of extracellular PAMPs. NLRs can participate in the signaling events triggered by host recognition of specific motifs of bacterial peptidoglycans and, upon activation, induce the production of proinflammatory mediators. Apart from the putative link between genetic mutations of NOD2 and SLE, little is known regarding the expression and function of NOD2 in SLE. / RAGE is a transmembrane cell-surface receptor on a variety of immune effector cells, which is expressed at low levels in normal tissues and vasculature, but is upregulated wherever the accumulation of its proinflammatory ligands, especially the key ligand, high mobility group box protein 1 (HMGB1). Both endogenous secretory RAGE (esRAGE) as well as soluble RAGE (sRAGE) can be detected in blood serum and are able to bind the circulating ligands, neutralizing their actions. In those conditions characterized by high concentrations of the circulating ligands, the decoy receptors are reduced drastically, revealing the system function. Therefore, the relationship between the upregulation of full-length (fl) RAGE/RAGE ligands and the levels of “protective“ esRAGE/sRAGE in SLE is of obvious clinical interest. / Apart from inducing and perpatating autoreactivity, abnormal innate response may also be responsible for the increased risk of infection in patients with lupus. The prevalence of abnormal Papanicolaou (Pap) smear was significantly increased in lupus patients in cross-sectional studies, associated with a higher prevalence of high-risk and multiple human papillomavirous (HPV) infections. However, none of the clinical, lifestyle, gynecological and treatment parameters was predictive of persistent HPV infection. Innate immune recognition of viral infection triggers antiviral immune responses. Whether the abnormal host innate immune response in lupus patients may play a role in enhancing HPV persistence remained unknown. / Hypothesis / 1.Aberrant activation of NLR and RAGE pathways by endogenous or exogenous ligands lead to the initation and/or perpetuation of autoimmune responses in SLE; / 2.HPV infection suppresses the host immune response by deregulating the TLRs transcript, leading to increased viral persistence in SLE. / Aims / 1.To evaluate the role of NOD2 pathway in the pathogenesis of SLE; / 2.To elucidate the relationship and regulatory mechanisms among members of the RAGE axis in the pathogenesis of SLE; / 3.To investigate the role of TLR in the defense against HPV infection in SLE. / Methods / The present thesis comprised of three cross-sectional studies in Chinese patients with SLE and controls in Hong Kong. Clinical assessments and review of medical records were performed to obtain information regarding disease status. / Results / 1.Over-expression of NOD2 in monocytes was observed in immunosuppressant naive SLE patients, and was positively associated with longer disease duration. Immunosuppressive therapy was an independent explanatory variable for downregulating NOD2 expression in CD8⁺ T, monocytes and dendritic cells (DCs). Ex vivo basal productions of cytokines [Interleukin (IL)-6, IL-8 and IL-10] were significantly increased in immunosuppressant naive patients and patients with active disease despite immunosuppressants compared with healthy controls. Upon muramyl dipeptide (MDP) stimulaiton, relative induction (%) of cytokines (IL-1β) from peripheral blood mononuclear cells (PBMCs) was significantly increased in immunosuppressant naive patients with inactive disease, and patients with active disease despite immunosuppressant treatment compared with healthy controls. Immunosuppressant usage was associated with a decreased basal production and MDP induced relative induction (%) of IL-10 in patients with inactive disease compared with immunosuppressant naive patients and healthy controls. / 2.Plasma sRAGE level was negatively correlated with SLE disease activities. The reduction in sRAGE levels in SLE patients with flare indicates that sRAGE may play a regulatory role on disease activity. HMGB1 alone could only mildly induce IL-6 production, which resulted in a transient phosporylation of intracellular p38 mitogen activated protein kinase (MAPK), c-Jun NH2- terminal protein kinase (JNK) and nuclear factor (NF)-κB. On the other hand, CpG-oligodeoxynucleotides (ODN) (TLR9 ligand) together with HMGB1 not only had a additive effect on IL-6 and IL-12p70 secretions compared with each agent alone, but also activated the phosphorylation of p38 MAPK and NF-κB. / 3.TLR inhibitor (hydroxychloroquine) and prednisolone may down-regulate protein levels of TLRs 7 and 9 in cervical epithelial cells of lupus patients. In the cervical cell lines, TLRs 3, 7, 8, 9 protein levels and antiviral interferon-stimulated genes (ISG) 15 and myxovirus resistance (Mx) 1 gene expressions were inhibited in two oncogenic HPV types. Functional data showed that the induction of pro-inflammatory cytokines by TLR ligands [R837, single stranded (ss) RNA and CpG-ODN] was greatly impaired in CaSki and HeLa than C33A cells. / Conclusions / Aberrant activation of PRR pathways by endogenous or exogenous molecules triggers the initation and/or perpetuation of autoimmune responses as follows: / 1.NOD2 may participate in the pathogenesis of lupus via the recognition of MDP and induction of proinflammatory effects, implicating the innate immune response for endogenous pathogens in the immunopathological mechanisms in SLE; / 2.Over-expression of RAGE may amplify the pro-inflammatory effects of DAMP such as HMGB1, while soluble RAGE may serve as a decoy receptor to suppress inflammation in patients with lupus nephritis. / 3.Upregulated HMGB1 may act alone or in combine with TLR9 ligand through the phosphorylation of p38 MAPK and NF-κB to promote inflammation in lupus. / On the other hand, the immune evasion strategy via avoidance of stimulation and downregulation of PRRs may promote establishment of persistent infection as listed below: / 1.TLR inhibitor (hydroxychloroquine) and prednisolone may down-regulate protein levels of TLRs 7 and 9 in lupus patients, thereby decreasing the innate immune response against HPV infection. / 2.Upon infection, HPV further down-regulate TLRs 7 and 9 levels for viral persistence. / 3.Reduction of TLRs 7, 8 and 9 in carcinogenic HPVs ensures that the expression of inducible pro-inflammatory cytokines is minimized to prevent the expression of antiviral ISGs on a biologically relevant antiviral response. / 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. / 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. / Yu, Shuilian. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 114-141). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.v / ACKNOWLEDGEMENTS --- p.viii / LIST OF PUBLICATIONS --- p.ix / LIST OF AWARDS AND GRANDT RECORD --- p.x / CONTENTS --- p.viii / LIST OF TABLES --- p.xiii / LIST OF FIGURES --- p.xv / LIST OF APPENDIXS --- p.xvi / ABBREVIATIONS --- p.xvii / Chapter CHAPTER 1 --- REVIEW OF THE LITERATURE --- p.1 / Chapter 1.1 --- What is systemic lupus erythematosus (SLE)? --- p.1 / Chapter 1.2 --- Epidemiology of SLE --- p.1 / Chapter 1.3 --- Etiology and pathogenesis of SLE --- p.3 / Chapter 1.3.1 --- Genetic factors --- p.5 / Chapter 1.3.2 --- Environmental triggers --- p.5 / Chapter 1.3.3 --- Cellular abnormalities --- p.6 / Chapter 1.3.3.1 --- APCs and cell debris clearance --- p.6 / Chapter 1.3.3.2 --- B cell and the production of autoantibodies --- p.8 / Chapter 1.3.3.3 --- T cell and the regulation of the immune responses --- p.9 / Chapter 1.3.4 --- Disturbance of the innate immune response --- p.10 / Chapter 1.3.4.1 --- PAMPs and DAPMs: all we need to know about danger in SLE --- p.10 / Chapter 1.3.4.2 --- Sensors of innate immunity --- p.12 / Chapter 1.3.4.2.1 --- TLRs sensing nucleic acids --- p.14 / Chapter 1.3.4.2.2 --- NLRs sensing bacterial peptidoglycans --- p.16 / Chapter 1.3.4.2.3 --- RAGE sensing dangerous signals --- p.16 / Chapter 1.3.5 --- Dysregulation of cytokine networks --- p.19 / Chapter 1.3.5.1 --- Anti-inflammatory cytokines --- p.20 / Chapter 1.3.5.2 --- Proinflammatory cytokines --- p.20 / Chapter 1.3.6 --- Abnormal signaling transduction --- p.22 / Chapter 1.4 --- Clinical features of SLE --- p.22 / Chapter 1.5 --- Laboratory features of SLE --- p.26 / Chapter 1.6 --- Assessing disease activity and damage of SLE --- p.28 / Chapter 1.7 --- Treatment of SLE --- p.28 / Chapter 1.7.1 --- Current immunosuppressive therapy --- p.28 / Chapter 1.7.2 --- Novel biologic therapies --- p.30 / Chapter 1.8 --- Human papillomavirus (HPV) infection in SLE --- p.32 / Chapter 1.8.1 --- Are women with lupus at higher risk of HPV infection? --- p.32 / Chapter 1.8.2 --- Abnormalities of TLR-IFN axis potentially increases HPV risk --- p.32 / Chapter 1.8.3 --- TLR suppressing mediciation potentially increases HPV risk --- p.34 / Chapter CHAPTER 2 --- HYPOTHESISS AND AIMS --- p.35 / Chapter CHAPTER 3 --- METHODOLOGIES --- p.36 / Chapter 3.1 --- Materials --- p.36 / Chapter 3.1.1 --- Selection of patients and controls --- p.36 / Chapter 3.1.2 --- Blood and cervical samples --- p.36 / Chapter 3.1.3 --- Cervical epithelial cell lines --- p.37 / Chapter 3.1.4 --- Culture medium and serum supplement --- p.37 / Chapter 3.1.5 --- Culture ligands --- p.37 / Chapter 3.1.6 --- Reagents for flow cytometric analysis (FCM) --- p.37 / Chapter 3.1.7 --- Antibodies for FCM --- p.38 / Chapter 3.1.8 --- Quantative assay kits --- p.39 / Chapter 3.1.9 --- Membrane array of phosphorylated intracellular kinases --- p.39 / Chapter 3.1.10 --- Primers for qPCR --- p.40 / Chapter 3.2 --- Methods --- p.40 / Chapter 3.2.1 --- Study design and patient assessment --- p.40 / Chapter 3.2.2 --- Isolation of PBMCs --- p.41 / Chapter 3.2.3 --- Isolation of monocytes --- p.42 / Chapter 3.2.4 --- Cell culture --- p.42 / Chapter 3.2.5 --- Sampling procedure of cervical epithelial cells --- p.42 / Chapter 3.2.6 --- HPV identification --- p.43 / Chapter 3.2.7 --- Flow cytometry gating of target cells --- p.43 / Chapter 3.2.8 --- FCM of target molecules and phosphorylated signaling molecules --- p.45 / Chapter 3.2.9 --- Membrane array of phosphorylated intracellular kinases --- p.46 / Chapter 3.2.10 --- Cytokine cytometric bead array --- p.46 / Chapter 3.2.11 --- Enzyme-linked immunosorbent assay --- p.46 / Chapter 3.2.12 --- Real-time qPCR --- p.46 / Chapter 3.2.13 --- Statistical analysis --- p.47 / Chapter CHAPTER 4 --- DOWN-REGULATED NOD2 BY IMMUNOSUPPRESSANTS IN PERIPHERAL BLOOD CELLS IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS REDUCES THE MURAMYL DIPEPTIDE-INDUCED IL-10 PRODUCTION --- p.48 / Chapter 4.1 --- INTRODUCTION --- p.48 / Chapter 4.2 --- METHODS --- p.49 / Chapter 4.2.1 --- Patient selection and assessment --- p.49 / Chapter 4.2.2 --- FCM of NOD2 expression in T, B cells, monocytes and DCs --- p.49 / Chapter 4.2.3 --- Cell culture --- p.49 / Chapter 4.2.4 --- Quantitative assay --- p.49 / Chapter 4.2.5 --- Statistical analyses --- p.49 / Chapter 4.3 --- RESULTS --- p.50 / Chapter 4.3.1 --- Characteristics of lupus patients and control subjects --- p.50 / Chapter 4.3.2 --- Identification of DCs, T, B lymphocytes and monocytes --- p.50 / Chapter 4.3.3 --- Protein level of NOD2 in DCs, T, B lymphocytes and monocytes --- p.53 / Chapter 4.3.4 --- Potential explanatory variables associated with NOD2 levels in lupus patients --- p.55 / Chapter 4.3.5 --- Induction of inflammatory cytokines by NOD2 ligand --- p.61 / Chapter 4.4 --- DISCUSSION --- p.63 / Chapter 4. --- 5 CONCLUSIONS --- p.67 / Chapter CHAPTER 5 --- MEMBERS OF THE RECEPTOR FOR ADVANCED GLYCATION ENDPRODUCTS AXIS AS POTENTIAL THERAPEUTIC TARGETS IN SYSTEMIC LUPUS ERYTHEMATOSUS --- p.68 / Chapter 5.1 --- INTRODUCTION --- p.68 / Chapter 5.2 --- METHODS --- p.68 / Chapter 5.2.1 --- Patients selection and assessment --- p.68 / Chapter 5.2.2 --- FCM of monocyte-surface flRAGE --- p.69 / Chapter 5.2.3 --- Cell culture --- p.69 / Chapter 5.2.4 --- Quantitative assay --- p.69 / Chapter 5.2.5 --- Membrane array of phosphorylated of intracellular kinases --- p.69 / Chapter 5.2.6 --- FCM of activated intracellular signaling molecules --- p.69 / Chapter 5.2.7 --- Statistical analysis --- p.69 / Chapter 5.3 --- RESULTS --- p.69 / Chapter 5.3.1 --- Characteristics of SLE patients --- p.69 / Chapter 5.3.3 --- Relationships between RAGE isoforms and HMGB1 --- p.75 / Chapter 5.3.4 --- Potential explanatory variables associated with levels of RAGE isoforms and HMGB1 in LN patients --- p.77 / Chapter 5.3.5 --- Activity of HMGB1 alone or in combine with TLR9 ligand --- p.81 / Chapter 5.4 --- DISCUSSION --- p.84 / Chapter 5.5 --- CONCLUSIONS --- p.88 / Chapter CHAPTER 6 --- ANTAGONIST-MEDIATED DOWN-REGULATION OF TOLL-LIKE RECEPTOR INCREASES THE PREVALENCE OF HUMAN PAPILLOMAVIRUS INFECTION IN SYSTEMIC LUPUS ERYTHEMATOSUS --- p.89 / Chapter 6.1 --- INTRODUCTION --- p.89 / Chapter 6.2 --- METHODS --- p.90 / Chapter 6.2.1 --- Patient selection and assessment --- p.90 / Chapter 6.2.2 --- HPV sampling procedure and identification --- p.90 / Chapter 6.2.3 --- FCM of TLRs 3, 7, 8 and 9 in cervical epithelial cells --- p.90 / Chapter 6.2.4 --- Cell culture --- p.90 / Chapter 6.2.5 --- Quantitative assay --- p.90 / Chapter 6.2.6 --- Real-time qPCR of Interferon-stimulated genes (ISGs) --- p.90 / Chapter 6.2.7 --- Statistical analysis --- p.91 / Chapter 6.3 --- RESULTS --- p.91 / Chapter 6.3.1 --- Pap smear findings, socio-demographic and clinical characteristics --- p.91 / Chapter 6.3.2 --- Identification of cervical epithelial cells --- p.95 / Chapter 6.3.3 --- Protein level of TLRs 3, 7, 8 and 9 in cervical epithelial cells --- p.96 / Chapter 6.3.4 --- Potential explanatory variables associated with TLR levels in lupus patients --- p.98 / Chapter 6.3.5 --- TLRs and ISGs expressions are inhibited by oncogenic HPVs --- p.102 / Chapter 6.3.6 --- Induction of inflammatory cytokines by TLR agonists was impaired in oncogenic HPVs --- p.103 / Chapter 6.4 --- DISCUSSION --- p.105 / Chapter 6.5 --- CONCLUSIONS --- p.107 / Chapter CHAPTER 7 --- CONCLUSIONS OF THE THESIS --- p.108 / Chapter 7.1 --- Answers to the hypotheses --- p.108 / Chapter 7.2 --- Conclusions and implications --- p.109 / Chapter 7.3 --- Liminations and future plan --- p.110 / Chapter 7.3.1 --- Liminations of study design --- p.110 / Chapter 7.3.2 --- Liminations of methodology --- p.111 / Chapter 7.3.3 --- Liminations of CHAPTER 4 and future plan --- p.111 / Chapter 7.3.4 --- Liminations of CHAPTER 5 and future plan --- p.112 / Chapter 7.3.5 --- Liminations of CHAPTER 6 and future plan --- p.112 / Chapter CHAPTER 8 --- REFERENCES --- p.114 / Chapter CHAPTER 9 --- APPENDIX --- p.142

Yip Yin Bing. / "July 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 163-177). / 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. / Yip Yin Bing.

Du Jing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 70-89). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGMENTS --- p.IV / PUBLICATIONS --- p.V / LIST OF FIGURES --- p.VI / LIST OF TABLES --- p.VII / ABBREVIATIONS --- p.VIII / CONTENTS --- p.X / Chapter CHAPTER ONE: --- INTRODUCTION AND LITERATURE / Chapter 1.1 --- Laryngeal carcinoma and HPV --- p.1 / Chapter 1.2 --- HPV --- p.2 / Chapter 1.3 --- Human papillomavirus E6 protein --- p.6 / Chapter 1.3.1 --- Transformation by HPV E6 --- p.7 / Chapter 1.3.2 --- Inhibition of apoptosis by E6 --- p.8 / Chapter 1.3.3 --- Alteration of gene transcription --- p.11 / Chapter 1.3.4 --- E6 interation with other proteins --- p.12 / Chapter 1.3.5 --- E6 as a therapeutic target --- p.14 / Chapter 1.4 --- HPV E7 protein --- p.15 / Chapter 1.4.1 --- Regulation of viral life cycle by HPV E7 --- p.16 / Chapter 1.4.2 --- Degradation of retinoblastoma tumor suppressor by HPV E7 --- p.18 / Chapter 1.4.3 --- Inhibition of p53 by HPV E7 --- p.22 / Chapter 1.4.4 --- Interaction with other proteins by HPV E7 --- p.24 / Chapter 1.5 --- Objective --- p.26 / Chapter CHAPTER TWO: --- GENERAL MATERIALS AND METHODS --- p.28 / Chapter 2.1 --- Materials --- p.28 / Chapter 2.1.1 --- Materials for cDNA and RNA manipulation --- p.28 / Chapter 2.1.2 --- Culture media and transfection reagents --- p.28 / Chapter 2.1.3 --- Antibodies --- p.29 / Chapter 2.1.4 --- Materials for protein manipulation --- p.29 / Chapter 2.1.5 --- Kits --- p.30 / Chapter 2.1.6 --- Instrumentation --- p.31 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Plasmid construction --- p.32 / Chapter 2.2.1.1 --- DNA preparation --- p.34 / Chapter 2.2.1.2 --- DNA ligation --- p.34 / Chapter 2.2.1.3 --- Transformation of competent E. coli --- p.35 / Chapter 2.2.2 --- Mini preparation --- p.35 / Chapter 2.2.3 --- Clone selection and confirmation --- p.37 / Chapter 2.2.4 --- Sequencing gel electrophoresis --- p.37 / Chapter 2.2.5 --- Cell culture and cytokine treatment --- p.39 / Chapter 2.2.6 --- Plasmid transfection --- p.39 / Chapter 2.2.7 --- Confirming construction of stable cell lines by RT-PCR --- p.40 / Chapter 2.2.7.1 --- Total cellular RNA extraction --- p.40 / Chapter 2.2.7.2 --- First strand cDNA synthesis --- p.41 / Chapter 2.2.7.3 --- Polymerase chain reaction (PCR) --- p.41 / Chapter 2.2.8 --- Fluorescence microscopy and imaging --- p.43 / Chapter 2.2.9 --- DNA fragmentation assay --- p.44 / Chapter 2.2.10 --- Protein detection --- p.46 / Chapter 2.2.10.1 --- Preparation of protein extract --- p.46 / Chapter 2.2.10.2 --- SDS-PAGE electrophoresis and protein transfer --- p.47 / Chapter 2.2.10.3 --- Immunoblotting analysis --- p.47 / Chapter 2.2.11 --- Statistical analysis --- p.48 / Chapter CHAPTER THREE: --- RESULTS --- p.49 / Chapter 3.1 --- Plasmid construction --- p.49 / Chapter 3.2 --- Expression of HPV16 viral oncogenes in transfected UMSCC12 --- p.51 / Chapter 3.3 --- HPV16 E6 and E7 protect apoptosis induced by TNF-alpha and CHX --- p.53 / Chapter 3.4 --- Detection of apoptosis with fluorescence staining --- p.55 / Chapter 3.5 --- Regulation of the expression of apoptosis-associated proteins by E6 and E7 oncoproteins --- p.57 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.59 / Chapter CHAPTER FIVE: --- CONCLUSION AND FUTURE PERSPECTIVE --- p.68 / REFERENCES --- p.70 / APPENDIX DNA SEQUENCING RESULTS --- p.90

血管內膜的進行性增厚是動脈粥樣硬化的重要標誌，並最終導致閉塞性血管病。血管內膜增生的一個主要因素是血管中膜的平滑肌細胞遷移至內膜層並增殖。大量研究證實，在動脈粥樣硬化的發生發展中，過量產生的活性氧簇（ROS）參與了血管壁的增厚。M型瞬時受體電位通道亞家族的成員TRPM2在血管平滑肌細胞中有表達，它能被ROS激活並對Ca²⁺通透，但其在血管平滑肌中的功能以及與心血管疾病的聯繫尚未見報道。 / 本論文著眼於探討TRPM2在鼠和人血管內膜增生中的作用。用血管外周套管法建立在體齧齒類動脈內膜增生模型。套管放置2周後，大鼠股動脈可見明顯的內膜增厚。免疫染色顯示新生內膜及其鄰近中膜區域內有大量增殖細胞核抗原陽性細胞，提示在增生的動脈中，細胞週期活動增強。動脈內膜和中膜内二氫乙錠螢光信號顯著增強，提示了ROS的過量生成。免疫染色和免疫印跡法均顯示，套管損傷導致TRPM2表達上調。免疫螢光雙標TRPM2與α-平滑肌肌動蛋白顯示內膜區域有大量TRPM2陽性的平滑肌細胞。與正常股動脈中膜平滑肌細胞相比，次黃嘌呤和黃嘌呤氧化酶在套管損傷的動脈來源的新生內膜平滑肌細胞中引起更大幅度的細胞內鈣離子濃度升高，而TRPM2抑制性抗體TM2E3預處理可消除這種差異。套管放置3周可引起小鼠頸動脈新生內膜形成，並伴隨著TRPM2表達上調。敲除TRPM2基因可顯著抑制內膜增生。取冠狀動脈搭橋術後殘餘的大隱靜脈，離體培養2周誘導內膜增生。免疫螢光雙標TRPM2與α-平滑肌肌動蛋白顯示新生內膜內含有大量TRPM2陽性的平滑肌細胞。TM2E3和另一TRPM2抑制劑2-氨乙氧基二苯酯硼酸處理均可有效降低內膜的增生。培養齧齒類主動脈平滑肌細胞，用劃痕試驗和MTT法檢測TRPM2阻斷劑和TRPM2基因敲除對過氧化氫誘導的細胞遷移和增殖的影響。結果顯示，暴露於過氧化氫48小時，細胞的遷移和增殖均明顯加快。TM2E3和2-氨乙氧基二苯酯硼酸處理有效抑制過氧化氫誘導的大鼠主動脈平滑肌細胞遷移和增殖；類似地，TRPM2基因敲除可顯著抑制過氧化氫誘導的小鼠主動脈平滑肌細胞遷移和增殖。 / 以上結果表明，血管內膜增生伴隨著TRPM2表達的上調；TRPM2參與了血管內膜增生以及血管平滑肌細胞的遷移、增殖；抑制TRPM2可能是對抗血管內膜增厚的潛在治療手段。 / A hallmark in atherosclerosis is progressive intimal thickening, which leads to occlusive vascular diseases. A causation of neointimal hyperplasia is the migration of medial smooth muscle cells (SMCs) to the intima where they proliferate. It is well recognized that excessive production of reactive oxide species (ROS) contributes to vascular wall thickening during arteriosclerotic development. TRPM2, a member of the melastatin-like transient receptor potential channel subfamily, is a Ca²⁺-permeable cation channel activated by ROS and is expressed in vascular smooth muscle cells (VSMCs). The functional properties of TRPM2 in vascular smooth muscle remain to be identified and an association between TRPM2 and cardiovascular diseases has not been reported. / In the present study, I investigated the involvement of TRPM2 in rodent and human neointimal hyperplasia. In vivo neointimal hyperplasia in rodent arteries was induced by perivascular cuff placement. After the cuff placement for 2 weeks, rat femoral arteries showed distinct intimal thickening. Immunostaining showed a great number of PCNA-positive proliferating cells in the neointima and its adjacent media region, indicating the enhanced cell cycle activity in the hyperplasic arteries. Dihydroethidium signal was markedly increased in the neointima and media of the cuffed arteries, suggesting that ROS is over-produced. Interestingly, both immunostaining and immunoblot showed that cuff-injury also led to an up-regulated expression of TRPM2. Double immunofluorescent labeling of TRPM2 and α-smooth muscle actin showed a large amount of TRPM2-positive SMCs in the neointimal region. Compared with the normal medial SMCs isolated from non-cuffed arteries, the neointimal SMCs from cuff-injured arteries displayed a greater [Ca²⁺] rise in response to hypoxanthine-xanthine oxidase, which was inhibited by pre-treatment with a TRPM2-specific blocking antibody TM2E3. In mouse carotid arteries, cuff placement for 3 weeks caused clear neointimal formation, accompanied by up-regulated expression of TRPM2. Trpm2 disruption dramatically reduced the neointimal growth. Human saphenous vein samples obtained during CABG surgery were organ-cultured for 2 weeks to allow growth of neointima. Double immunofluorescent labeling of TRPM2 and α-smooth muscle actin showed that the neointima contained numerous TRPM2-positive SMCs. Neointimal hyperplasia in the veins was effectively suppressed by in vitro treatment with TM2E3 or a chemical blocker 2-aminoethoxydiphenyl borate. Furthermore, the effect of TRPM2 blockers and Trpm2 disruption on hydrogen peroxide-induced migration and proliferation of cultured rodent aortic SMCs were evaluated by scratch wound healing assay and MTT assay, respectively. It was found that exposure to hydrogen peroxide for 48 hour substantially enhanced the migration and proliferation of rodent aortic SMCs. In rat aortic SMCs, both TM2E3 and 2-aminoethoxydiphenyl borate significantly inhibited the hydrogen peroxide-induced cell migration and proliferation. The hydrogen peroxide-induced cell migration and proliferation of SMCs was also reduced in Trpm2 knockout mice. / Taking together, these results provide strong evidences that in vivo neointimal hyperplasia is accompanied by an up-regulated expression of TRPM2 and that TRPM2 plays a key role in neointimal hyperplasia, VSMCs migration and proliferation. Blocking TRPM2 can be a potential therapeutic approach for protecting blood vessels against intimal thickening. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ru, Xiaochen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 125-151). / Abstracts also in Chinese. / Declaration of Originality --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Abbreviations and Units --- p.vii / Table of Content --- p.x / List of Figures --- p.xvi / List of Tables --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Neointimal hyperplasia --- p.1 / Chapter 1.1.1 --- Definition of neointimal hyperplasia --- p.2 / Chapter 1.1.2 --- Medical significance of coronary neointimal hyperplasia --- p.3 / Chapter 1.1.3 --- Pathogenesis of neointimal hyperplasia --- p.5 / Chapter 1.1.3.1 --- “Response to injury“ hypothesis --- p.6 / Chapter 1.1.3.2 --- Role of VSMCs --- p.7 / Chapter 1.1.3.2.1 --- VSMC phenotypic switch --- p.7 / Chapter 1.1.3.2.2 --- Ca²⁺ channel modulation in VSMCs --- p.8 / Chapter 1.1.3.2.3 --- VSMC migration --- p.9 / Chapter 1.1.3.2.4 --- VSMC proliferation --- p.10 / Chapter 1.1.3.2.5 --- Extracellular matrix production by VSMCs --- p.11 / Chapter 1.1.3.3 --- Endothelial dysfunction --- p.11 / Chapter 1.1.3.4 --- Platelet adhesion --- p.12 / Chapter 1.1.3.5 --- Inflammation --- p.13 / Chapter 1.1.4 --- Role of ROS in neointimal hyperplasia --- p.14 / Chapter 1.1.4.1 --- Types of ROS --- p.15 / Chapter 1.1.4.1.1 --- Superoxide anion --- p.16 / Chapter 1.1.4.1.2 --- Hydroxyl radical --- p.16 / Chapter 1.1.4.1.3 --- Hydrogen peroxide --- p.16 / Chapter 1.1.4.1.4 --- Nitric oxide --- p.17 / Chapter 1.1.4.2 --- Sources of ROS in vessel wall --- p.17 / Chapter 1.1.4.3 --- ROS signaling in endothelial cells --- p.19 / Chapter 1.1.4.4 --- ROS signaling in VSMCs --- p.20 / Chapter 1.1.4.5 --- ROS and atherosclerosis --- p.21 / Chapter 1.1.5 --- Current therapeutic approaches to neointimal hyperplasia --- p.23 / Chapter 1.1.5.1 --- Pharmacological approaches --- p.23 / Chapter 1.1.5.2 --- Technical Approaches --- p.25 / Chapter 1.2 --- Transient receptor potential melastatin 2 (TRPM2) channel --- p.27 / Chapter 1.2.1 --- TRP Channels --- p.27 / Chapter 1.2.2 --- TRPM2 structure and expression --- p.29 / Chapter 1.2.2.1 --- Structure --- p.29 / Chapter 1.2.2.2 --- Alternative splicing isoforms --- p.30 / Chapter 1.2.2.3 --- Expression pattern --- p.32 / Chapter 1.2.3 --- TRPM2 channel properties --- p.32 / Chapter 1.2.4 --- TRPM2 activators and inhibitors --- p.32 / Chapter 1.2.4.1 --- Activators --- p.33 / Chapter 1.2.4.1.1 --- ADPR --- p.33 / Chapter 1.2.4.1.2 --- NAD, cADPR and NAADP --- p.33 / Chapter 1.2.4.1.3 --- H₂O₂ and oxidative stress --- p.34 / Chapter 1.2.4.1.4 --- Ca²⁺ --- p.34 / Chapter 1.2.4.1.5 --- Other regulators --- p.35 / Chapter 1.2.4.2 --- Inhibitors --- p.35 / Chapter 1.2.5 --- Biological relevance of TRPM2 --- p.36 / Chapter 1.2.5.1 --- TRPM2 in insulin release --- p.36 / Chapter 1.2.5.2 --- TRPM2 in inflammation --- p.36 / Chapter 1.2.5.3 --- TRPM2 in cell death --- p.37 / Chapter 1.2.5.4 --- TRPM2-mediated lysosomal Ca²⁺ release --- p.38 / Chapter 1.2.5.5 --- TRPM2 and cardiovascular diseases --- p.39 / Chapter Chapter 2 --- Objectives of the Present Study --- p.40 / Chapter Chapter 3 --- Materials and Methods --- p.42 / Chapter 3.1 --- Materials --- p.42 / Chapter 3.1.1 --- Chemicals --- p.42 / Chapter 3.1.2 --- Media, supplements and other reagents for cell/tissue culture --- p.44 / Chapter 3.1.3 --- Antibodies --- p.45 / Chapter 3.1.4 --- Solutions --- p.46 / Chapter 3.1.4.1 --- Solutions for immunohistochemical and immunocytochemical staining --- p.46 / Chapter 3.1.4.2 --- solutions for immunoblotting --- p.47 / Chapter 3.1.4.3 --- Solutions for Genotyping --- p.49 / Chapter 3.1.4.4 --- Solutions for hematoxylin and eosin (HE) staining --- p.50 / Chapter 3.1.4.5 --- Solutions for [Ca²⁺]i measurement --- p.51 / Chapter 3.1.4.6 --- Solutions for IgG purification --- p.51 / Chapter 3.1.5 --- Animals --- p.51 / Chapter 3.1.5.1 --- Rat --- p.51 / Chapter 3.1.5.2 --- Trpm2 knockout mice --- p.52 / Chapter 3.1.5.3 --- Rabbit --- p.52 / Chapter 3.1.5.4 --- Ethics --- p.52 / Chapter 3.1.6 --- Human Tissue --- p.52 / Chapter 3.2 --- Methods --- p.54 / Chapter 3.2.1 --- Rodent models of neointimal hyperplasia --- p.54 / Chapter 3.2.1.1 --- Cuff-induced vascular injury in rat femoral artery --- p.54 / Chapter 3.2.1.2 --- Cuff-induced vascular injury in mouse carotid artery --- p.54 / Chapter 3.2.2 --- Genotyping for Trpm2 knockout mice --- p.55 / Chapter 3.2.2.1 --- Genomic DNA extraction from tail --- p.55 / Chapter 3.2.2.2 --- Polymerase Chain Reaction (PCR) --- p.55 / Chapter 3.2.2.3 --- Agarose gel electrophoresis of DNA --- p.56 / Chapter 3.2.3 --- Human saphenous vein culture and treatment --- p.56 / Chapter 3.2.4 --- Generation of anti-TRPM2 antibody, TRPM2-specific blocking antibody TM2E3 and preimmune IgG --- p.57 / Chapter 3.2.5 --- Histological analysis and immunohistochemistry --- p.58 / Chapter 3.2.6 --- Western blotting --- p.59 / Chapter 3.2.7 --- Detection of ROS production by dihydroethidium fluorescence --- p.60 / Chapter 3.2.8 --- Isolation of rodent neointimal and medial smooth muscle cells --- p.60 / Chapter 3.2.9 --- Culture of rodent aortic smooth muscle cells --- p.61 / Chapter 3.2.9.1 --- Cell culture --- p.61 / Chapter 3.2.9.2 --- Cell identification --- p.61 / Chapter 3.2.10 --- [Ca²⁺]i measurement --- p.62 / Chapter 3.2.11 --- Cell proliferation assay --- p.63 / Chapter 3.2.12 --- Cell migration assay --- p.63 / Chapter 3.2.13 --- Statistical analysis --- p.64 / Chapter Chapter 4 --- ROS over-production and TRPM2 up-regulation in cuff-induced rodent neointimal hyperplasia --- p.65 / Chapter 4.1 --- Introduction --- p.65 / Chapter 4.2 --- Materials and Methods --- p.66 / Chapter 4.2.1 --- Cuff-induced vascular injury in rat femoral artery --- p.66 / Chapter 4.2.2 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.66 / Chapter 4.2.3 --- Histological analysis and immunohistochemistry --- p.66 / Chapter 4.2.4 --- Western blotting --- p.67 / Chapter 4.2.5 --- Detection of ROS production --- p.67 / Chapter 4.2.6 --- Isolation of rat neointimal and medial smooth muscle cells --- p.68 / Chapter 4.2.7 --- [Ca²⁺]i measurement --- p.68 / Chapter 4.2.8 --- Statistical analysis --- p.68 / Chapter 4.3 --- Results --- p.69 / Chapter 4.3.1 --- Cuff-induced neointimal hyperplasia in rat femoral arteries --- p.69 / Chapter 4.3.2 --- ROS over-production in neointimal region of cuff-injured rat femoral arteries --- p.69 / Chapter 4.3.3 --- TRPM2 up-regulation in neointimal region of cuff-injured rat femoral arteries --- p.69 / Chapter 4.3.4 --- Enhanced [Ca²⁺]i response to HX-XO in rat neointimal smooth muscle cells --- p.70 / Chapter 4.4 --- Discussion --- p.81 / Chapter Chapter 5 --- TRPM2 contributes to human and rodent neointimal hyperplasia --- p.86 / Chapter 5.1 --- Introduction --- p.86 / Chapter 5.2 --- Materials and Methods --- p.87 / Chapter 5.2.1 --- Cuff-induced vascular injury in mouse carotid artery --- p.87 / Chapter 5.2.2 --- Genotyping for Trpm2 knockout mice --- p.87 / Chapter 5.2.3 --- Organ culture of human saphenous vein --- p.87 / Chapter 5.2.4 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.88 / Chapter 5.2.5 --- Histological analysis and immunohistochemistry --- p.88 / Chapter 5.2.6 --- Western blotting --- p.88 / Chapter 5.2.7 --- Isolation of mouse neointimal and medial smooth muscle cells --- p.89 / Chapter 5.2.8 --- [Ca²⁺]i measurement --- p.89 / Chapter 5.2.9 --- Statistical analysis --- p.90 / Chapter 5.3 --- Results --- p.90 / Chapter 5.3.1 --- Cuff-induced neointimal hyperplasia was reduced in Trpm2 knockout mice --- p.90 / Chapter 5.3.2 --- [Ca²⁺]i response to HX-XO in mouse neointimal smooth muscle cells --- p.90 / Chapter 5.3.3 --- Inhibiting TRPM2 reduced the neointimal hyperplasia in in vitro cultured human saphenous vein --- p.91 / Chapter 5.4 --- Discussion --- p.99 / Chapter Chapter 6 --- Role of TRPM2 in H₂O₂-stimulated migration and proliferation of vascular smooth muscle cells --- p.103 / Chapter 6.1 --- Introduction --- p.103 / Chapter 6.2 --- Materials and Methods --- p.104 / Chapter 6.2.1 --- Culture of rodent aortic smooth muscle cells --- p.104 / Chapter 6.2.2 --- Immunocytochemistry --- p.104 / Chapter 6.2.3 --- Genotyping for Trpm2 knockout mice --- p.104 / Chapter 6.2.4 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.104 / Chapter 6.2.5 --- [Ca²⁺]i measurement --- p.105 / Chapter 6.2.6 --- Cell proliferation assay --- p.105 / Chapter 6.2.7 --- Western blotting --- p.105 / Chapter 6.2.8 --- Cell migration assay --- p.106 / Chapter 6.2.9 --- Statistical analysis --- p.106 / Chapter 6.3 --- Results --- p.106 / Chapter 6.3.1 --- H₂O₂-induced [Ca²⁺]i rises in rodent aortic smooth muscle cells --- p.106 / Chapter 6.3.2 --- Role of TRPM2 in H₂O₂-stimulated smooth muscle cell proliferation --- p.107 / Chapter 6.3.3 --- Role of TRPM2 in H₂O₂-stimulated smooth muscle cell migration --- p.108 / Chapter 6.4 --- Discussion --- p.118 / Chapter Chapter 7 --- General Conclusion and Future Work --- p.121 / Chapter 7.1 --- Concluding remarks --- p.121 / Chapter 7.2 --- Future work --- p.123 / Chapter 7.2.1 --- Specific downstream signaling pathway of TRPM2 that mediates ROS-induced VSMC proliferation and migration --- p.123 / Chapter 7.2.2 --- Involvement of TRPM2 in leukocyte infiltration and inflammation in vascular wall --- p.124 / References --- p.125 / List of Publications --- p.152

廣泛的區域內和跨民族的轉錄變化反映了人類的適應和自然選擇。基因表達是轉化基因組信息為功能基因產品 - 蛋白質的主要機制。異常基因的表達和疾病的發病機制有關。基因組革命提供了獨特的機會為複雜的人類轉錄組進行全面的研究。轉錄分析需要複雜的生物信息學方法。在技術角度，一個實證模型用了哺乳動物基因組中內含子長度幾何尾分佈的定律準確地確定剪接交界處和非唯一映射讀取的位置。這種方法在處理非唯一映射讀取比BWA更好。這方法還比其他工具檢測出更多已經實驗證實的剪接交界處。核糖核酸測序首先用於北京漢人和西歐之間的表達表型與的轉錄變化的詳盡研究。民族的具體剪接交界處被發現。此外，民族的具體特點體現在相對異構體的豐度差。最後，這分子表型剪接頻譜的變化在不同種族之間的不同表明了另一個描繪種族多樣性的方法，核糖核酸測序還被用於探索的一種複雜的疾病：二型糖尿病的分子異常。二型糖尿病表現在廣泛不同的基因表達。（1）這研究證實先前公佈的全基因組關聯研究;（2）改善策劃不佳的位點和（3）發現新型2型糖尿病相關的基因。本研究通過整合各種改變的信號，並在一個高度可信的基因 - 基因相互作用網絡進行解釋，增強表達異常在2型糖尿病的認識。在更廣泛的69×79的情況下，對照組的結果進行了驗證。本研究增強表達異常在2型糖尿病的認識。 / Extensive intra- and inter- ethnic transcriptome variation reflects human adaptation and natural selection. Gene expression is the primary mechanism that translates genome information into functional gene product that lead to physiological phenotypes. Aberrant gene expression has been associated to the pathogenesis of diseases. The genome revolution has offered unique opportunity for a comprehensive interrogation of the complexity of human transcriptome. Analysis of transcriptome using RNA-Seq requires sophisticated bioinformatics approach. In a technical perspective, an empirical model based on the geometric-tail distribution of intron lengths in mammalian genome was developed to accurately determine splice junctions from junction reads and locations of non-uniquely mapped reads. Such method handles non-uniquely mapped reads better than BWA. The method can also detect more experimentally confirmed splice junction than other tools. Expressional phenotyping was employed to explore global transcriptomic variation between Beijing Han Chinese and Western European. In addition to inter-ethnic variations in gene expression, ethnic specific splice juctions were found. Further, ethnic specific trait manifests in differential relative isoform abundance. Lastly, such spectrum of variations was different between different ethnic groups, suggesting alternative splicing as another molecular phenotype that delineates ethnic diversity. Expressional phenotyping was then used in a case-control study to explore the molecular abnormalities of a complex disease: Type 2 Diabetes (T2DM). T2DM manifested in wide-spread repression of gene expression. The study (1) confirmed previously reported Genome-wide Association Study (GWAS) loci; (2) curated poorly characteriezed GWAS loci and (3) discovered novel T2DM associated genes. By integrating various alteration signals and interpretation performed in a highly confident gene-gene interaction network, this study augmented the understanding of expressed abnormalities in T2DM. The results were validated in a broader 69 x 79 case-control group. / Detailed summary in vernacular field only. / Li, Jing Woei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 118-130). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.v / 中文擇要 --- p.vi / Thesis/Assessment Committee --- p.ix / Acknowledgement --- p.ix / List of figures --- p.x / List of tables --- p.xii / List of Abbreviations --- p.xiii / Scientific contributions --- p.xv / List of Publication(s) related to this thesis --- p.xvi / Conference presentations --- p.xvii / Chapter Chapter 1: --- Introduction and Literature Reviews --- p.1 / Chapter 1.1 --- The variable human transcriptome --- p.1 / Chapter 1.2 --- Significance of variation in gene expression and transcript variants --- p.2 / Chapter 1.3 --- Transcriptomic study in a technological perspective --- p.8 / Chapter 1.3.1 --- Microarray: Probing what was designed to be probed --- p.8 / Chapter 1.3.2 --- RNA-Seq: the ab initio decoder of biological sequences --- p.9 / Chapter 1.4 --- Analysis of RNA-Seq data --- p.10 / Chapter 1.4.1 --- The bioinformatics challenges prevail --- p.10 / Chapter 1.4.2 --- Identifying changes in gene expression --- p.16 / Chapter 1.4.3 --- Identifying splice site, quantification of isoform level expression --- p.17 / Chapter 1.5 --- Conclusion --- p.19 / Chapter 1.6 --- Aims of this study --- p.20 / Chapter 1.6.1 --- Splice junction determination --- p.20 / Chapter 1.6.2 --- Expressional phenotyping in ethnical context --- p.20 / Chapter 1.6.3 --- Expressional phenotyping in a disease context --- p.20 / Chapter Chapter 2: --- Detection of splicing events --- p.21 / Chapter 2.1 --- Abstract --- p.21 / Chapter 2.2 --- Introduction --- p.22 / Chapter 2.3 --- Methods and workflow --- p.25 / Chapter 2.4 --- Algorithm --- p.29 / Chapter 2.5 --- Geometric-tail distribution --- p.32 / Chapter 2.6 --- Insert-size distribution --- p.33 / Chapter 2.7 --- Multiread analysis --- p.34 / Chapter 2.7.1 --- GT model probably places multiread more accurately than BWA --- p.35 / Chapter 2.8 --- Splice-site comparison --- p.37 / Chapter 2.8.1 --- GT model discovers more experimentally confirmed splice junction --- p.37 / Chapter 2.8.2 --- GT model is highly accurate --- p.39 / Chapter 2.9 --- Discussion --- p.40 / Chapter 2.10 --- Limitation --- p.40 / Chapter Chapter 3: --- Transcriptomic variation in a ethnicity context --- p.41 / Chapter 3.1 --- Abstract --- p.41 / Chapter 3.2 --- Introduction --- p.42 / Chapter 3.3 --- Materials and Methods --- p.46 / Chapter 3.3.1 --- HapMap lymphoblastoid cell-lines --- p.46 / Chapter 3.3.2 --- Sequenced samples --- p.48 / Chapter 3.3.3 --- Paired-end RNA-Seq, dataset and reads processing --- p.48 / Chapter 3.3.4 --- Genome reference and annotation --- p.49 / Chapter 3.3.5 --- Strategies for reads mapping --- p.49 / Chapter 3.3.6 --- Pathway and Gene Ontology analysis --- p.50 / Chapter 3.3.7 --- Differential gene expression analysis --- p.50 / Chapter 3.3.8 --- Ethnic specific splice junction --- p.51 / Chapter 3.3.9 --- Junction sites saturation analysis --- p.51 / Chapter 3.3.10 --- Ethnical novel transcribed regions --- p.52 / Chapter 3.3.11 --- Isoform dynamics and meta-analysis --- p.53 / Chapter 3.4 --- Result --- p.54 / Chapter 3.4.1 --- Paired-end RNA-Seq --- p.54 / Chapter 3.4.2 --- Differential gene expression and meta-analysis --- p.56 / Chapter 3.4.3 --- Ethnic specific splice junction is rare --- p.58 / Chapter 3.4.4 --- Saturation of discovery of highly confident annotated junctions --- p.59 / Chapter 3.4.5 --- Novel transcribed regions --- p.62 / Chapter 3.4.6 --- Isoform dynamics and meta-analysis --- p.63 / Chapter 3.5 --- Discussion --- p.66 / Chapter 3.6 --- Limitations --- p.67 / Chapter 3.6.1 --- HapMap LCLs may not reflect the entire spectrum of natural variation --- p.67 / Chapter 3.6.2 --- Sequencing depth and the usefulness of published dataset --- p.67 / Chapter 3.6.3 --- Knowledge gap in understanding of the human genome --- p.69 / Chapter Chapter 4: --- Transcriptomic investigation of complex disease: Type 2 Diabetes --- p.70 / Chapter 4.1 --- Abstract --- p.70 / Chapter 4.2 --- Introduction --- p.72 / Chapter 4.3 --- Materials and Methods --- p.75 / Chapter 4.3.1 --- Subjects --- p.75 / Chapter 4.3.2 --- Strand-specific RNA-Seq Library Construction --- p.77 / Chapter 4.3.3 --- Genome annotation sequencing reads processing --- p.81 / Chapter 4.3.4 --- Reads mapping for expression analysis --- p.82 / Chapter 4.3.5 --- Differential Gene expression analysis --- p.82 / Chapter 4.3.6 --- GWAS candidate genes --- p.83 / Chapter 4.3.7 --- Individual network, pathway and Gene Ontology analysis --- p.83 / Chapter 4.3.8 --- Alternative Splicing Variation --- p.83 / Chapter 4.3.9 --- Reads mapping and processing for expressed genomic variants discovery --- p.84 / Chapter 4.3.10 --- Expressed and functional genomic variants --- p.85 / Chapter 4.3.11 --- Screening for gene fusion --- p.86 / Chapter 4.3.12 --- Sense and Antisense analysis --- p.86 / Chapter 4.3.13 --- Integrated multi-level T2DM alternations gene interaction network --- p.87 / Chapter 4.3.14 --- Validation of selected genes --- p.87 / Chapter 4.4 --- Results --- p.88 / Chapter 4.4.1 --- High quality strand-specific pair-ended RNA-Seq facilitated downstream analyses --- p.88 / Chapter 4.4.2 --- Definition of significance --- p.91 / Chapter 4.4.3 --- Wide-spread repressed gene expression in T2DM --- p.91 / Chapter 4.4.4 --- Confirmation and curation of T2DM GWAS loci by RNA-Seq --- p.92 / Chapter 4.4.5 --- Global expression alteration on T2DM associated genes --- p.97 / Chapter 4.4.6 --- Alteration of relative splicing isoforms variations and T2DM specific isoforms --- p.100 / Chapter 4.4.7 --- Rare and deleterious SNPs --- p.100 / Chapter 4.4.8 --- Absence of alteration in Sense/Antisense ratio and expressed fusion gene --- p.101 / Chapter 4.4.9 --- T2DM manifests a broad spectrum of expressed abnormalities --- p.101 / Chapter 4.4.10 --- Pathway-based integration of multiple levels of alteration expanded the T2DM network --- p.103 / Chapter 4.4.11 --- Validation of selected genes --- p.107 / Chapter 4.5 --- Discussion --- p.108 / Chapter Chapter 5: --- Conclusions and future perspectives --- p.115 / Chapter 5.1 --- Conclusions --- p.115 / Chapter 5.2 --- Future perspective --- p.115 / Chapter 5.2.1 --- Splicing detection --- p.115 / Chapter 5.2.2 --- Studies related to ethnicity --- p.116 / Chapter 5.2.3 --- Complex diseases --- p.116 / References --- p.118 / Appendix --- p.131

Hepatocellular carcinoma (HCC) is a highly malignant tumor that is prevalent in Southeast Asia and China, where hepatitis B viral (HBV) infection is the main etiologic factor. Despite a high incidence of HCC developing in patients with HBV-induced liver cirrhosis, the molecular events underlying the malignant liver progression remain largely unclear. In an effort to characterize the genetic abnormalities involved in the HBV-related liver carcinogenesis, genome-wide exploration by metaphase comparative genomic hybridization (CGH) was performed on 100 cirrhotic HCC tumors that were derived from chronic hepatitis B carriers. CGH analysis indicated chromosomal instability in both early and advanced stage tumors where common genomic copy gains on 1q, 8q and 17q, and deletions on 4q, 8p, 13q, 16q and 17p found in both groups are suggestive of early events in hepatocarcinogenesis. Nevertheless, a combined univariate and multivariate statistical analyses highlighted for the first time preferential regional 3q26-q28, 7q21-q22 and 7q34-q36 gains in association with advanced stage HCC. The novel aberrant gains identified here thus formed basis for further mapping analysis for causative genes related to HCC progression in this thesis. / Near 50% of the advanced stage HCC manifested copy gains of chr 7q21-q22. High resolution mapping analysis by cDNA microarray-based CGH nominated 13 amplified candidates within the region 7q21-q22 Analysis on the mRNA expresson levels of these genes in a cohort of primary HCC compared to paired adjacent non-tumorous liver tissues by quantitative RT-PCR (qRT-PCR) indicated the up-regulation of the PFTK1 (PFTAIRE protein kinase 1) gene as the only candidate that demonstrated a close association with advanced metastatic tumors. The effects of PFTK1 on cell proliferation, migration and invasive phenotypes were further studied to substantiate its role in HCC progression. Upon gene suppression of PFTK1 in vitro by RNA interference (RNAi), a significant reduction in chemotactic migration, cellular invasion and an inhibition on cell motility were indicated, albeit cell proliferation remained unaffected. / Sub-cellular localization study of translated PFTK1 protein indicated protein localization in both the nucleus and cytoplasm. This has led to the further investigations of potential PFTK1 function at both the transcriptional and protein levels. (Abstract shortened by UMI.) / Sy Ming Hui. / "July 2005." / Advisers: Winnie Yeo; Nathalie Wong. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3571. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 124-139). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.