Tam On Yi Ann. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 138-152). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGMENTS --- p.VI / ABBREVIATIONS --- p.VIII / LIST OF FIGURES --- p.X / LIST OF TABLES --- p.XI / CONTENTS --- p.XII / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- History of human papillomavirus --- p.2 / Chapter 1.2 --- Biology of human papillomavirus --- p.4 / Chapter 1.2.1 --- Classification --- p.4 / Chapter 1.2.2 --- Genome structure --- p.5 / Chapter 1.2.3 --- Properties of gene products --- p.6 / Chapter 1.2.3.1 --- El gene --- p.6 / Chapter 1.2.3.2 --- E2 gene --- p.7 / Chapter 1.2.3.3 --- E4 gene --- p.7 / Chapter 1.2.3.4 --- E5 gene --- p.7 / Chapter 1.2.3.5 --- E6 gene --- p.7 / Chapter 1.2.3.6 --- E7 gene --- p.8 / Chapter 1.2.3.7 --- LI and L2 genes --- p.9 / Chapter 1.2.4 --- Latent and lytic life cycle --- p.9 / Chapter 1.2.5 --- Host specificity --- p.10 / Chapter 1.2.6 --- Site of infection --- p.11 / Chapter 1.2.7 --- Clinical manifestations --- p.11 / Chapter 1.2.8 --- Mode of infection --- p.12 / Chapter 1.2.9 --- Detection method --- p.13 / Chapter 1.2.9.1 --- DNA hybridization --- p.13 / Chapter 1.2.9.2 --- DNA amplification methods --- p.15 / Chapter 1.2.9.3 --- Hybrid capture assay --- p.16 / Chapter 1.2.9.4 --- Other DNA detection methods --- p.17 / Chapter 1.2.9.5 --- Serology --- p.18 / Chapter 1.3 --- Biology of cervical intraepithelial neoplasia and cervical cancer --- p.19 / Chapter 1.3.1 --- Grading of severity of cervical neoplasia --- p.20 / Chapter 1.3.2 --- Treatment of cervical intraepithelial lesions --- p.22 / Chapter 1.3.3 --- Prognosis after treatment --- p.22 / Chapter 1.4 --- Epidemiology of cervical cancer --- p.23 / Chapter 1.4.1 --- Global burden of disease --- p.23 / Chapter 1.4.2 --- Local burden of disease --- p.23 / Chapter 1.4.2.1 --- Incidence --- p.23 / Chapter 1.4.2.2 --- Mortality --- p.24 / Chapter 1.4.2.3 --- Age distribution --- p.24 / Chapter 1.4.2.4 --- Trends of incidence and mortality --- p.25 / Chapter 1.4.2.5 --- Morbidity --- p.25 / Chapter 1.4.2.6 --- International comparison --- p.25 / Chapter 1.5 --- Aetiology and risk factors --- p.26 / Chapter 1.5.1 --- Human papillomavirus infection --- p.26 / Chapter 1.5.2 --- Number of sexual partners --- p.26 / Chapter 1.5.3 --- Age of first sexual intercourse --- p.27 / Chapter 1.5.4 --- Presence of other sexually-transmitted diseases --- p.28 / Chapter 1.5.5 --- Cigarette smoking --- p.29 / Chapter 1.5.6 --- Diet --- p.30 / Chapter 1.5.7 --- Oral contraceptives --- p.30 / Chapter 1.5.8 --- Parity --- p.31 / Chapter 1.5.9 --- Age --- p.32 / Chapter 1.5.10 --- Socio-economic status --- p.32 / Chapter 1.6 --- Malignant transformation of human papillomavirus infection --- p.33 / Chapter 1.7 --- Primary prevention of cervical cancer - vaccine for human papillomavirus --- p.38 / Chapter 1.7.1 --- Classification of vaccine for human papillomavirus --- p.38 / Chapter 1.7.2 --- Human papillomavirus vaccination combined with human papillomavirus screening --- p.39 / Chapter 1.8 --- Secondary prevention of cervical cancer --- p.40 / Chapter 1.8.1 --- Cytology screening --- p.40 / Chapter 1.8.2 --- Detection of human papillomavirus --- p.41 / Chapter 1.9 --- Human papillomavirus and cervical cancer --- p.43 / Chapter 1.9.1 --- Risk association between cervical cancer and human papillomavirus infection --- p.43 / Chapter 1.9.2 --- World-wide prevalence of human papillomavirus types in cervical cancer --- p.43 / Chapter 1.9.3 --- Human papillomavirus prevalence in China and Hong Kong --- p.44 / Chapter Chapter Two: --- Materials and Methods --- p.49 / Chapter 2.1 --- Ethics approval --- p.50 / Chapter 2.2 --- Sample management --- p.50 / Chapter 2.2.1 --- Sample collection --- p.50 / Chapter 2.2.2 --- Sample storage and labelling --- p.50 / Chapter 2.3 --- DNA extraction --- p.51 / Chapter 2.3.1 --- Physical extraction 226}0ؤ heating --- p.51 / Chapter 2.3.2 --- Chemical extraction - Qiagen kit extraction --- p.51 / Chapter 2.4 --- Polymerase chain reaction --- p.53 / Chapter 2.4.1 --- Controls for polymerase chain reaction --- p.53 / Chapter 2.4.2 --- Beta-globin polymerase chain reaction --- p.53 / Chapter 2.4.3 --- HPV 52-specific human papillomavirus polymerase chain reaction --- p.56 / Chapter 2.4.4 --- Consensus human papillomavirus L1 open-reading frame polymerase chain reaction --- p.57 / Chapter 2.4.4.1 --- GP5+/6+ polymerase chain reaction --- p.57 / Chapter 2.4.4.2 --- MY09/11 polymerase chain reaction --- p.60 / Chapter 2.4.4.3 --- PGMY09/11 polymerase chain reaction --- p.63 / Chapter 2.5 --- Genotyping of human papillomavirus --- p.65 / Chapter 2.5.1 --- Restriction fragment length polymorphism --- p.65 / Chapter 2.5.2 --- Reverse line-blot hybridization --- p.67 / Chapter 2.6 --- Sequencing --- p.69 / Chapter 2.6.1 --- Sequencing for HPV genotyping --- p.69 / Chapter 2.6.2 --- Sequencing of HPV 52 E6 and E7 genes --- p.69 / Chapter 2.7 --- Statistical analysis --- p.70 / Chapter Chapter Three --- Study I 226}0ؤ Comparison of Three HPV DNA Detection Methods --- p.71 / Chapter 3.1 --- Objective --- p.72 / Chapter 3.2 --- Study plan --- p.72 / Chapter 3.3 --- Results --- p.74 / Chapter 3.3.1 --- Study population --- p.74 / Chapter 3.3.2 --- Optimisation of polymerase chain reactions --- p.74 / Chapter 3.3.3 --- Method 1: GP5+/6+ PCR followed by cycle sequencing --- p.76 / Chapter 3.3.4 --- Method 2: MY09/11 PCR followed by restriction fragment length polymorphism --- p.76 / Chapter 3.3.5 --- Method 3: PGMY09/11 PCR followed by reverse line-blot hybridization --- p.77 / Chapter 3.3.6 --- Prevalence and genotype distribution of human papillomavirus infection in cervical cancer patients --- p.81 / Chapter 3.3.7 --- Detection of multiple infections --- p.81 / Chapter 3.3.8 --- Sensitivity of the detection methods --- p.82 / Chapter 3.3.9 --- Comparison of prevalence rates of human papillomavirus genotypes --- p.82 / Chapter 3.3.10 --- Comparison of genotype distribution in Hong Kong cervical cancer patients with other geographic regions --- p.83 / Chapter 3.3.11 --- Follow-up investigation of GP5+/6+ primer binding site in HPV 52 --- p.84 / Chapter 3.4 --- Discussion --- p.91 / Chapter Chapter Four --- Study II - Post-treatment Follow-up Study on Patients with High-grade Cervical Lesions --- p.95 / Chapter 4.1 --- Objective --- p.96 / Chapter 4.2 --- Study plan --- p.96 / Chapter 4.3 --- Results --- p.97 / Chapter 4.3.1 --- Study population --- p.97 / Chapter 4.3.2 --- The prevalence and genotype distribution of human papillomavirus infection before treatment --- p.98 / Chapter 4.3.3 --- Persistent human papillomavirus infection --- p.99 / Chapter 4.3.4 --- Risk-factors associated with persistent human papillomavirus infection --- p.99 / Chapter 4.3.4.1 --- Excision margin status --- p.99 / Chapter 4.3.4.2 --- Multiple human papillomavirus infections --- p.99 / Chapter 4.4 --- Discussion --- p.108 / Chapter 4.4.1 --- Prevalence and genotype distribution of human papillomavirus in high-grade cervical neoplasia --- p.108 / Chapter 4.4.2 --- Risk factors for cervical intraepithelial neoplasia recurrence --- p.110 / Chapter Chapter Five --- Study III - Investigation of Human Papillomavirus 52 Sequence Variation --- p.115 / Chapter 5.1 --- Objective --- p.116 / Chapter 5.2 --- Study plan --- p.116 / Chapter 5.3 --- Results --- p.117 / Chapter 5.3.1 --- Study population --- p.117 / Chapter 5.3.2 --- Nucleotide sequence variations --- p.119 / Chapter 5.3.2.1 --- Human papillomavirus 52 E6 open-reading frame --- p.119 / Chapter 5.3.2.2 --- Human papillomavirus 52 E7 open-reading frame --- p.123 / Chapter 5.3.2.3 --- Comparison of nucleotide sequence variations in HPV 52 E6 and E7 open-reading frame --- p.128 / Chapter 5.4 --- Discussion --- p.134 / References --- p.137

目的： 转化生长因子－1（TGF-β1）通过与II型受体结合而引起I型受体活化，进一步激活其下游信号分子蛋白Smad2 和Smad3，它们与Smad4（Co-Smad）结合后形成Smad复合体并发生核转移，从而发挥广泛的生物学效应。同时，整个TGF-β信号通路又受到其抑制因子Smad7的负反馈调节。研究结果显示Smad3是肾脏炎症和纤维化中重要的致病分子，相反，Smad7在多种肾脏疾病中起保护作用。然而，由于转化生长因子II型受体（TβRII），Smad2 或Smad4基因敲除的小鼠无法存活，这些分子在TGF-β1介导的肾脏炎症和纤维化中的功能尚未见报道。因此，本研究旨在剖析TβRII、Smad2 和Smad4 在肾脏疾病发生发展中的作用及机制。 / 方法：本研究利用Cre/LoxP系统分别靶向敲除小鼠肾小管上皮细胞的TβRII、Smad2 或者Smad4，通过结扎小鼠单侧输尿管建立梗阻性肾病模型，观察这些分子对肾脏炎症和纤维化的影响，并用体外实验进行验证。具体实验结果请参见本论文第III,IV, V章。 / 结果：通过分析，本论文取得以下新的发现： / (1) TβRII在TGF-β1介导的肾脏炎症和纤维化的双向调节中起到了决定性的作用：研究结果显示条件性敲除TβRII明显抑制TGF-β/Smad3介导的肾脏纤维化，同时增强NF-κB引起的肾脏炎症反应。由此可见，TRII不仅仅是TGF-β/Smad信号通路的启动因子，更决定了TGF-β1对肾脏炎症和纤维化的双向性调节。(参见第III章) / (2)尽管Smad2和Smad3结构相似并共同介导了TGF-β1的生物学效应，本研究意外发现Smad2可反向调节Smad3引起的纤维化。体内和体外实验共同证实，敲除Smad2基因增强了Smad3的磷酸化，核转位及其转录子活性，并能促进Smad3与I型胶原转录子的结合，进而加重肾脏纤维化（参见第IV章）。 / (3)我们还发现Smad4不仅作为TGF-β/Smad信号通路的共有蛋白，它在TGF-β1介导肾脏炎症和纤维化中起到了重要的双向性调节作用：条件敲除Smad4显著降低了Smad7对NF-κB介导肾脏炎症的抑制作用，同时在转录水平（而非磷酸化水平）抑制Smad3的功能，从而减轻纤维化。（参见第V章） / 结论：TβRII和Smad4 在TGF-β1介导肾脏炎症和纤维化中起到了重要的双向性作用；Smad2通过抑制Smad3信号传导和功能，在肾脏纤维化中起保护作用。 / Objectives: TGF-β1 binds its receptor II (TβRII) and then activates receptor I to initiate the downstream Smad signaling, called Smad2 and Smad3 which bind a common Smad4 to form the Smad complex and then translocate to nucleus to exert its biological activities. This process is negatively regulated by an inhibitory Smad7. While the pathogenic role of Smad3 and the protective role of Smad7 in renal fibrosis and inflammation are clearly understood, the functional role of TβRII, Smad2 and Smad4 in kidney diseases remains largely unexplored due to the lethality of these knockout mice. Therefore, the aim of present study is to dissect the functional role of these TGF-β/Smad signaling molecules in renal inflammation and fibrosis. / Methods: Kidney conditional knockout (KO) mice for TβRII, Smad2 and Smad4 were generated by crossing the FloxFlox mice with the kidney specific promoter driven Cre (KspCre) mice, in which TβRII, Smad2 or Smad4 were specifically deleted from the kidney tubular epithelial cells (TEC) respectively. Then, a well-characterized progressive renal inflammation and fibrosis mouse model of Unilateral ureteral obstructive (UUO) nephropathy was induced in these conditional KO mice and the specific roles for TβRII, Smad2, and Smad4 in renal inflammation and fibrosis were investigated in vivo and in vitro as described in the Chapter III, IV and V of this thesis. / Results: There were several novel findings through this thesis: / 1. TGF-β1 signals through its TβRII to diversely regulate renal fibrosis and inflammation. We found that disrupted TRII suppressed Smad3-dependent renal fibrosis while enhancing NF-κB-driven renal inflammation. Thus, TβRII not only acts as a binding receptor for initiating the TGF-β signaling, but also determines the diverse role of TGF-β1 in inflammation and fibrosis, which was described in the Chapter III. / 2. As shown in the Chapter IV, an unexpected finding from this thesis was that although Smad2 and Smad3 were homologically similar and bound together in response to TGF-β1 stimulation, Smad2 counter-regulated Smad3-mediated renal fibrosis. This was evidenced by the findings that conditional deletion of Smad2 enhanced Smad3 signaling including phosphorylation, nuclear translocation, the Smad3 responsive promoter activity, and the binding of Smad3 to Col1A2 promoter. Thus, disrupted Smad2 from the kidney significantly enhanced Smad3-mediated renal fibrosis in the UUO kidney and in cultured TEC. / 3. Finally, we also showed that that Smad4 acted not only as a common Smad in TGF-β signaling, but exerted its regulatory role in determining the diverse role of TGF-β1 in renal inflammation and fibrosis. Disruption of Smad4 significantly enhanced renal inflammation by impairing inhibitory effect of Smad7 on NF-κB-driven renal inflammation. In contrast, disrupted Smad4 inhibited renal fibrosis by blocking Smad3 functional activity without influencing Smad3 signaling. Because deletion of Smad4 inhibited TGF-β1-induced Smad3 responsive promoter activity and the binding of Smad3 to the Col1A2 promoter without altering the phosphorylation and nuclear translocation of Smad3 (Chapter V). / Conclusions: TβRII and Smad4 may function as key regulators of TGF-β signaling and diversely regulate the renal inflammation and fibrosis. Smad2 plays a protective role in renal fibrosis by counter-regulating Smad3 signaling. / 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. / Meng, Xiaoming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 202-231). / 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 / Declaration --- p.viii / Acknowledgement --- p.ix / Table of Contents --- p.xii / List of Abbreviations --- p.xxvii / List of Figures/Tables --- p.xxix / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter 1.1 --- TGF-β signaling pathway --- p.2 / Chapter 1.1.1 --- TGF-β superfamily --- p.2 / Chapter 1.1.2 --- TGF-β signaling transduction --- p.3 / Chapter 1.1.2.1 --- Smad-dependent TGF-β signaling --- p.4 / Chapter 1.1.2.2 --- Smad-independent TGF-β signaling --- p.10 / Chapter 1.2 --- Chronic Kideny disease (CKD) --- p.12 / Chapter 1.2.1 --- Epidemiology of CKD --- p.12 / Chapter 1.2.2 --- Pathophysiology of CKD --- p.12 / Chapter 1.3 --- TGF-β signaling in renal diseases --- p.13 / Chapter 1.3.1 --- Role of TGF-β1 in renal diseases --- p.13 / Chapter 1.3.2 --- Potential role of TβRII in renal diseases --- p.15 / Chapter 1.3.3 --- Potential role of Smad2 in renal diseases --- p.17 / Chapter 1.3.4 --- Potential role of Smad4 in renal diseases --- p.20 / Chapter 1.3.5 --- Role of Smad7 in renal diseases --- p.23 / Chapter 1.3.6 --- Role of Smad-independent TGF-β signaling in renal disease --- p.24 / Chapter CHAPTER II --- MATERIALS AND METHODS --- p.26 / Chapter 2.1 --- MATERIALS --- p.27 / Chapter 2.1.1 --- Reagents and Equipments --- p.27 / Chapter 2.1.1.1 --- General reagents and equipments for cell culture --- p.27 / Chapter 2.1.1.2 --- General reagents and equipments for real-time RT-PCR --- p.28 / Chapter 2.1.1.3 --- General reagents and equipments for Masson Trichrome Staining --- p.28 / Chapter 2.1.1.4 --- General reagents and equipments for Immunohistochemistry --- p.29 / Chapter 2.1.1.5 --- General reagents and equipments for Immunofluorescence --- p.29 / Chapter 2.1.1.6 --- General reagents and equipments for Western Blot --- p.29 / Chapter 2.1.1.7 --- General reagents and equipments for Promoter assay --- p.31 / Chapter 2.1.1.8 --- General reagents and equipments for ChIP assay --- p.32 / Chapter 2.1.2 --- Buffers --- p.32 / Chapter 2.1.2.1 --- Buffers for Immunohistochemistry --- p.32 / Chapter 2.1.2.2 --- Buffers for Western blot --- p.35 / Chapter 2.1.3 --- Sequences of Primers and siRNAs --- p.40 / Chapter 2.1.4 --- Antibodies --- p.42 / Chapter 2.2 --- METHODS --- p.44 / Chapter 2.2.1 --- Animal model of Unilateral Ureteral Obstruction (UUO) --- p.44 / Chapter 2.2.2 --- Cell culture --- p.44 / Chapter 2.2.2.1 --- NRK52E cell line --- p.44 / Chapter 2.2.2.2 --- Smad2 WT/KO mouse embryonic fibroblasts (MEFs) --- p.45 / Chapter 2.2.2.3 --- Primary culture of kidney fibroblasts --- p.45 / Chapter 2.2.2.4 --- Primary culture of peritoneal macrophages --- p.46 / Chapter 2.2.3 --- PAS staining --- p.47 / Chapter 2.2.3.1 --- Tissue Handling and Fixation --- p.47 / Chapter 2.2.3.2 --- Tissue embedding and sectioning --- p.47 / Chapter 2.2.3.3 --- Preparation of Paraffin Tissue Sections for PAS staining --- p.48 / Chapter 2.2.3.4 --- PAS staining --- p.48 / Chapter 2.2.4 --- Real-time RT-PCR --- p.48 / Chapter 2.2.4.1 --- Total RNA isolation --- p.48 / Chapter 2.2.4.2 --- Reverse Transcription --- p.49 / Chapter 2.2.4.3 --- Real-time PCR --- p.50 / Chapter 2.2.4.4 --- Analysis of Real-time PCR --- p.50 / Chapter 2.2.5 --- Masson Trichrome Staining --- p.51 / Chapter 2.2.6 --- Immunohistochemistry --- p.52 / Chapter 2.2.6.1 --- Preparation of Paraffin Tissue Sections for IHC --- p.52 / Chapter 2.2.6.2 --- Antigen-Antibody Reaction --- p.52 / Chapter 2.2.6.3 --- Signal Detection --- p.53 / Chapter 2.2.6.4 --- Semi-quantification of Immunohistochemistry --- p.53 / Chapter 2.2.7 --- Immunofluorescence --- p.54 / Chapter 2.2.8 --- Western blot analysis --- p.54 / Chapter 2.2.8.1 --- Protein preparation --- p.55 / Chapter 2.2.8.2 --- SDS-PAGE --- p.56 / Chapter 2.2.8.3 --- Transmembrane of protein --- p.56 / Chapter 2.2.8.4 --- Incubation of first and second antibody --- p.57 / Chapter 2.2.8.5 --- Signal capture and analysis --- p.57 / Chapter 2.2.8.6 --- Stripping --- p.57 / Chapter 2.2.9 --- Promoter assay --- p.58 / Chapter 2.2.10 --- ChIP assay --- p.61 / Chapter 2.2.11 --- Statistical analysis --- p.62 / Chapter CHAPTER III --- THE DIVERSE ROLE OF TGF-BETA RECEPTOR II IN RENAL INFLAMMATION AND FIBROSIS --- p.63 / Chapter 3.1 --- INTRODUCTION --- p.64 / Chapter 3.2 --- AIMS --- p.64 / Chapter 3.3 --- MATERIALS AND METHODS --- p.66 / Chapter 3.3.1 --- Generation and characterization of TβRII conditional Knockout mice --- p.66 / Chapter 3.3.2 --- Generation and characterization of TβRII disrupted tubular epithelial cell line (NRK52E) and kidney interstitial fibroblasts --- p.67 / Chapter 3.3.3 --- Animal model of Unilateral Ureteral Obstruction --- p.67 / Chapter 3.3.4 --- Cell culture --- p.67 / Chapter 3.3.5 --- Real-time RT-PCR --- p.68 / Chapter 3.3.6 --- Masson Trichrome Staining --- p.68 / Chapter 3.3.7 --- Immunohistochemistry --- p.68 / Chapter 3.3.8 --- PAS staining --- p.69 / Chapter 3.3.9 --- Immunofluorescence --- p.69 / Chapter 3.3.10 --- Western blot analysis --- p.70 / Chapter 3.3.11 --- Promoter assay --- p.70 / Chapter 3.3.12 --- Statistical analysis --- p.70 / Chapter 3.4 --- RESULTS --- p.71 / Chapter 3.4.1 --- Characterization of TβRII conditional Knockout mice and TβRII disrupted cells --- p.71 / Chapter 3.4.2 --- Disruption of TβRII suppresses renal interstitial damage in the UUO kidney --- p.72 / Chapter 3.4.3 --- Disruption of TβRII suppresses renal fibrosis in UUO kidney and TGF-β1-induced fibrotic response in vitro --- p.76 / Chapter 3.4.3.1 --- Conditional knockout of TβRII from the kidney decreases the collagen I level in UUO kidney --- p.76 / Chapter 3.4.3.2 --- Disruption of TβRII inhibits TGF-β1 induced collagen I level in vitro --- p.79 / Chapter 3.4.3.3 --- Conditional knockout of TβRII from the kidney decreases the α-SMA positive cells infiltration in vivo --- p.81 / Chapter 3.4.3.4 --- Disruption of TβRII inhibits TGF-β1-induced α-SMA expression in vitro --- p.83 / Chapter 3.4.3.5 --- Conditional knockout of TβRII from the kidney decreases the FN level in UUO nephropathy --- p.85 / Chapter 3.4.3.6 --- Disruption of TβRII decreases TGF-β1-induced FN expression in vitro --- p.87 / Chapter 3.4.4 --- Disruption of TβRII impairs the TGF-β/Smad signaling in vivo in the UUO kidney and in vitro in TGF-β1 treated tubular epithelial cells and kidney fibroblasts --- p.89 / Chapter 3.4.4.1 --- Conditional knockout of TβRII decreases the UUO induced TGF-β1 expression in vivo and the TGF-β1 auto-induction in vitro --- p.89 / Chapter 3.4.4.2 --- Disrupted TβRII decreases CTGF level in the UUO nephropathy in vivo and the TGF-β1 induced CTGF mRNA level in vitro --- p.91 / Chapter 3.4.4.3 --- Conditional knockout of TβRII impairs the Smad3 signaling in the injured kidney --- p.93 / Chapter 3.4.4.4 --- Disrupted TβRII inhibits TGF-β1-induced Smad3 phosphorylation, P-Smad3 nuclear translocation and Smad3 responsive promoter activity in vitro --- p.95 / Chapter 3.4.4.5 --- Conditional knockout of TβRII doesn’t alter the activation of ERK and P38 signaling in the UUO kidney --- p.97 / Chapter 3.4.4.6 --- Disrupted TβRII inhibits TGF-β1-induced ERK and P38 phosphorylation in vitro --- p.99 / Chapter 3.4.5 --- Disruption of TβRII enhances inflammatory cytokines expression in the UUO kidney and impairs the anti-inflammatory effect of TGF-β1 in response to IL-1β triggered inflammatory response in the TEC cells --- p.101 / Chapter 3.4.5.1 --- Conditional knockout of TβRII increases the TNF-α expression in the UUO nephropathy --- p.101 / Chapter 3.4.5.2 --- Conditional knockout of TβRII increases the IL-1β expression in the UUO nephropathy --- p.103 / Chapter 3.4.5.3 --- Conditional knockout of TβRII doesn’t enhance the MCP-1 expression and macrophages infiltration in the UUO nephropathy --- p.104 / Chapter 3.4.5.4 --- Disruption of TβRII in TECs decreases the anti-inflammatory effect of TGF-β1 in response to IL-1β --- p.106 / Chapter 3.4.6 --- Disruption of TβRII enhances NFκB activation in vivo and in vitro --- p.108 / Chapter 3.5 --- DISCUSSION --- p.110 / Chapter 3.6 --- CONCLUSION --- p.114 / Chapter CHAPTER IV --- Smad2 protects against TGF-β/Smad3 mediated renal fibrosis --- p.115 / Chapter 4.1 --- INTRODUCTION --- p.116 / Chapter 4.2 --- AIMS --- p.117 / Chapter 4.3 --- MATERIALS AND METHODS --- p.117 / Chapter 4.3.1 --- Generation and characterization of Smad2 conditional Knockout mice --- p.117 / Chapter 4.3.2 --- Generation and characterization of Smad2 KO MEFs and Smad2 knockdown/overexpression tubular epithelial cell line (NRK52E) --- p.118 / Chapter 4.3.3 --- Animal model of Unilateral Ureteral Obstruction --- p.118 / Chapter 4.3.4 --- Cell culture --- p.118 / Chapter 4.3.5 --- Real-time RT-PCR --- p.119 / Chapter 4.3.6 --- Western blot analysis --- p.119 / Chapter 4.3.7 --- Immunohistochemistry --- p.119 / Chapter 4.3.8 --- Masson Trichrome Staining --- p.119 / Chapter 4.3.9 --- Immunofluorescence --- p.120 / Chapter 4.3.10 --- Promoter assay --- p.120 / Chapter 4.3.11 --- ChIP assay --- p.120 / Chapter 4.3.12 --- Statistical analysis --- p.120 / Chapter 4.4 --- RESULTS --- p.121 / Chapter 4.4.1 --- Characterization of Smad2 disrupted mice and cells --- p.121 / Chapter 4.4.1.1 --- Characterization of Smad2 conditional Knockout mice --- p.121 / Chapter 4.4.1.2 --- Characterization of Smad2 knockout MEFs, Smad2 knockdown/overexpression TECs --- p.123 / Chapter 4.4.2 --- Disruption of Smad2 further enhances renal fibrosis in vivo and in vitro --- p.124 / Chapter 4.4.2.1 --- Conditional knockout of Smad2 increases total collagen deposition and Col.I level in the UUO kidney --- p.124 / Chapter 4.4.2.2 --- Disruption of Smad2 in MEFs and TECs increases Col.I production in a time- and dosage-dependent manner in response to TGF-β1 --- p.126 / Chapter 4.4.2.3 --- Conditional knockout of Smad2 increases Col.III level in the UUO kidney --- p.128 / Chapter 4.4.2.4 --- Disruption of Smad2 in MEFs and TECs increases Col.III production in a time- and dosage-dependent manner in response to TGF-β1 --- p.130 / Chapter 4.4.3 --- Disruption of Smad2 further enhances renal fibrosis by suppressing the collagen degradation system in vivo and in vitro --- p.132 / Chapter 4.4.3.1 --- Conditional knockout of Smad2 inhibits the MMP2 mRNA while enhances TIMP-1 production in UUO kidney --- p.132 / Chapter 4.4.3.2 --- Disruption of Smad2 in MEFs and TECs decreases the MMP2 level while enhances TIMP-1 production in response to TGF-β1 --- p.133 / Chapter 4.4.4 --- Disruption of Smad2 further increases renal fibrosis by increasing TGF-β1 auto-induction and CTGF level in vivo and in vitro --- p.135 / Chapter 4.4.4.1 --- Disruption of Smad2 increases TGF-β1 auto-induction in vivo and in vitro --- p.135 / Chapter 4.4.4.2 --- Disruption of Smad2 increases CTGF synthesis in vivo and in vitro --- p.137 / Chapter 4.4.5 --- Disruption of Smad2 further increases renal fibrosis by enhancing Smad3 signaling in vivo and in vitro --- p.139 / Chapter 4.4.5.1 --- Conditional knockout of Smad2 further enhances Smad3 phosphorylation and nuclear translocation --- p.139 / Chapter 4.4.5.2 --- Disruption of Smad2 in MEFs and TECs further enhances Smad3 phosphorylation, nuclear translocation, Smad3 responsive promoter activity and the binding to the Col1A2 promoter --- p.141 / Chapter 4.4.6 --- Overexpression of Smad2 suppresses Smad3 signaling therefore ameliorates the TGF-β1-induced fibrotic response in TECs --- p.144 / Chapter 4.4.6.1 --- Overexpression of Smad2 ameliorates the TGF-β1- induced fibrotic response in TECs --- p.144 / Chapter 4.4.6.2 --- Overexpression of Smad2 suppresses Smad3 phosphorylation --- p.146 / Chapter 4.5 --- DISCUSSION --- p.147 / Chapter 4.6 --- CONCLUSION --- p.150 / Chapter CHAPTER V --- THE DISTINCT ROLE OF SMAD4 IN RENAL INFLAMMATION AND FIBROSIS --- p.151 / Chapter 5.1 --- INTRODUCTION --- p.152 / Chapter 5.2 --- AIMS --- p.152 / Chapter 5.3 --- MATERIALS AND METHODS --- p.153 / Chapter 5.3.1 --- Generation and characterization of Smad4 conditional Knockout mice --- p.153 / Chapter 5.3.2 --- Generation and characterization of Smad4 disrupted kidney interstitial fibroblasts and peritoneal macrophages --- p.153 / Chapter 5.3.3 --- Animal model of Unilateral Ureteral Obstruction (UUO) --- p.154 / Chapter 5.3.4 --- Cell culture --- p.154 / Chapter 5.3.5 --- Real-time RT-PCR --- p.155 / Chapter 5.3.6 --- Western blot analysis --- p.155 / Chapter 5.3.7 --- Immunohistochemistry --- p.155 / Chapter 5.3.8 --- Masson Trichrome Staining --- p.155 / Chapter 5.3.9 --- Promoter assay --- p.156 / Chapter 5.3.10 --- ChIP assay --- p.156 / Chapter 5.3.11 --- Statistical analysis --- p.156 / Chapter 5.4 --- RESULTS --- p.157 / Chapter 5.4.1 --- Characterization of Smad4 conditional Knockout mice and Smad4 disrupted cells --- p.157 / Chapter 5.4.2 --- Disruption of Smad4 suppresses renal fibrosis in the UUO nephropathy in vivo and TGF-β1-induced fibrotic response in vitro --- p.160 / Chapter 5.4.2.1 --- Conditional knockout of Smad4 from the kidney decreases the total collagen deposition in the UUO nephropathy --- p.160 / Chapter 5.4.2.2 --- Conditional knockout of Smad4 from the kidney decreases the Col.I production in the UUO nephropathy --- p.161 / Chapter 5.4.2.3 --- Disruption of Smad4 inhibits TGF-β1-induced Col.I production in vitro --- p.163 / Chapter 5.4.3 --- Disruption of Smad4 impairs the Smad3 function in vivo and in vitro --- p.164 / Chapter 5.4.3.1 --- Conditional knockout of Smad4 doesn’t decrease Smad3 phosphorylation and P-Smad3 nuclear translocation in vivo and in vitro --- p.164 / Chapter 5.4.3.2 --- Disruption of Smad4 inhibits TGF-β1 induced Smad3 promoter activity and the Smad3 binding to Col1A2 promoter --- p.166 / Chapter 5.4.3.3 --- Disruption of Smad4 has minimal effect on the activation of ERK signaling in vivo and in vitro --- p.167 / Chapter 5.4.4 --- Disruption of Smad4 enhances renal inflammation and impairs the anti-inflammatory effect of TGF-β1 in response to IL-1β triggered inflammatory response in vitro --- p.169 / Chapter 5.4.4.1 --- Conditional knockout of Smad4 increases the inflammatory cells infiltration --- p.169 / Chapter 5.4.4.2 --- Conditional knockout of Smad4 increases the TNFα expression in the UUO nephropathy --- p.171 / Chapter 5.4.4.3 --- Conditional knockout of Smad4 increases the IL-1β expression in the UUO nephropathy --- p.172 / Chapter 5.4.4.4 --- Conditional knockout of Smad4 increases the MCP-1 expression in the UUO nephropathy --- p.173 / Chapter 5.4.4.5 --- Conditional knockout of Smad4 increases the ICAM-1 level in the UUO nephropathy --- p.174 / Chapter 5.4.4.6 --- Time and dosage dependent experiments in response to IL-1β in macrophages --- p.175 / Chapter 5.4.4.7 --- Disruption of Smad4 in macrophages decreases the anti-inflammatory effect of TGF-β1 in response to IL-1β --- p.176 / Chapter 5.4.5 --- Disruption of Smad4 impairs the inhibitory effect of Smad7 on NFκB activation in vivo and in vitro --- p.178 / Chapter 5.4.5.1 --- Conditional knockout of Smad4 largely inhibits Smad7 level in UUO kidney --- p.178 / Chapter 5.4.5.2 --- Conditional knockout of Smad4 suppresses IκBα and further increases NF-κB p65 activation in UUO kidney --- p.180 / Chapter 5.4.5.3 --- Disruption of Smad4 inhibits Smad7 synthesis in macrophages --- p.182 / Chapter 5.4.5.4 --- Conditional knockout of Smad4 impair the inhibition effect of TGF-β1 on the activation of NFκB p65 in macrophages --- p.184 / Chapter 5.5 --- DISCUSSION --- p.186 / Chapter 5.6 --- CONCLUSION --- p.189 / Chapter CHAPTER VI --- SUMMARY AND DISCUSSION OF THE MAJOR FINDINGS --- p.190 / Chapter 6.1 --- SUMMARY AND DISCUSSION --- p.192 / Chapter 6.1.1 --- The diverse role of TβRII in renal inflammation and fibrosis both in vivo and in vitro --- p.192 / Chapter 6.1.2 --- Smad2 protects renal fibrosis by counter-regulating Smad3 signaling --- p.192 / Chapter 6.1.3 --- Disruption of Smad4 increased renal inflammation while suppressed the renal fibrosis in vivo and in vitro --- p.194 / Chapter 6.1.4 --- Comparative analysis of functions and related mechanisms between TβRII and Smad4 in renal disease --- p.195 / Chapter 6.1.5 --- Inadequacies of current work and future plan --- p.197 / Chapter 6.1.6 --- Perspectives (1) : The balance within the TGF-b/Smad signaling may determine the fate of renal diseases --- p.197 / Chapter 6.1.7 --- Perspectives(2)：The balance within the TGF-β/Smad signaling may determine the fate of renal diseases --- p.198 / Chapter 6.2 --- CONCLUSION --- p.201 / REFERENCES --- p.202 / PUBLICATION LIST --- p.232 / HONORS AND AWARDS --- p.237

青少年特發性脊柱側凸(Adolescent idiopathic scoliosis , AIS)是一種複雜的脊柱三維畸形，常見於10-16 歲處於生長發育高峰期的青少年女性。儘管AIS 發生率較高並且臨床影響較大，但是到目前為止其病因未明。在眾多關於AIS 病因學的假設和理論研究中，普遍認為低骨密度是AIS 的一個重要影響因素。然而近年來對於AIS 患者低骨密度研究不足，其潛在的機制尚不明確。我們之前初步的組織學研究發現，AIS 患者的松質骨中成骨細胞功能下降，此研究為AIS中存在骨礦化異常提供了初步依據。 / 骨橋蛋白是骨組織中一種重要的非膠原細胞外基質蛋白，其在骨礦化過程中起著重要作用。近期的研究報導AIS 患者血漿中骨橋蛋白水準高於年齡匹配的正常對照。因此本研究假設AIS 患者血漿及骨組織中骨橋蛋白高於正常對照，并可能影響了骨基質的礦化，從而導致低骨密度。 / 本系列研究的第一部分旨在通過外周定量電腦斷層掃描（pQCT）明確AIS患者中皮質骨密度及松質骨密度是否均低於正常對照。pQCT 可以準確地三維評估皮質骨密度，松質骨密度及其他骨品質的相關參數。採用雙能X 線骨密度儀（DXA）測量受試者的非優勢側近端股骨面積骨密度（包括股骨頸，Ward’s 三角及大轉子）。而採用pQCT 測量受試者非優勢側橈骨遠端容積骨密度，包括皮質骨密度及松質骨密度。結果顯示AIS 患者面積骨密度，皮質骨密度及松質骨密度在不同年齡段和月經時間分組中均低於正常對照。並且AIS 與正常對照皮質骨密度的差異隨著年齡增長越來越大，而松質骨密度差異則隨著年齡增長越來越小。 / 第二部分通過顯微CT 及組織形態測定研究AIS 及正常骨組織的骨礦化及骨微結構。採用顯微CT 檢測骨組織的三維結構參數，包括材料骨密度及骨微結構。未脫鈣骨組織的切片通過Goldner’s 染色進行組織形態學測量。結果顯示AIS患者的骨體積分數，骨小梁數目，骨小梁厚度及結構模型指數與正常對照之間均無顯著差異，而材料骨密度顯著低於正常對照。組織形態學分析結果顯示AIS中低礦化骨顯著多於正常對照。 / 第三部分旨在研究AIS 及正常對照血漿中骨橋蛋白水準及其與骨密度的關係。採用酶聯吸附免疫法測量AIS 患者及年齡匹配的正常對照血漿中的骨橋蛋白水準。血漿骨橋蛋白水準與骨密度的關係採用多元回歸分析。研究結果顯示AIS 患者及正常對照血漿骨橋蛋白水平均與年齡及月經時間呈負相關。AIS 患者的血漿骨橋蛋白水準顯著高於正常對照，並且與松質骨密度呈顯著負相關。 / 本研究第四部分旨在探討骨組織中的骨橋蛋白表達與骨形態學及骨礦化指標在AIS 及正常對照中的關係。骨組織中骨橋蛋白的表達採用半定量免疫組織化學法評估。研究結果顯示在AIS 中血漿骨橋蛋白水準與骨組織中骨橋蛋白的表達呈正相關。且AIS 骨組織中骨橋蛋白的表達也顯著高於正常對照。進一步的研究發現骨組織中骨橋蛋白的表達與材料骨密度呈負相關，而與低礦化骨量呈正相關。 / 本研究明確了AIS 中骨礦化水準低於正常對照，進一步證明AIS 患者中的皮質骨及松質骨密度下降可能與骨礦化的調控異常有關。本研究發現的骨橋蛋白與低骨密度及低骨礦化水準的關係，可以推測AIS 患者中異常升高的骨橋蛋白水準可能在骨礦獲取的調解中起重要作用。本系列研究提供證據支援AIS 患者中骨橋蛋白的異常表達可能影響了骨基質的礦化，從而導致低骨密度。本研究為AIS 中低骨密度可能的機制提供了全新的見解，並可能進一步解釋AIS 的發病機理及其發生，發展。 / Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional deformity of the spine occurring most commonly in girls between ages 10-16 during the pubertal growth spurt. Despite its high prevalence and clinical impact, etiology of AIS remains largely unknown. Among the number of proposed hypothesis and observations on the etiopathogenesis of AIS, low bone mineral density (BMD) is one of the most reported factor (Cheng et al. 1999; Hung et al. 2005; Cheung et al. 2006; Hui et al. 2011). However, the underlying mechanism of low BMD in AIS has not been sufficiently studied scientifically and its link to the etiopathogenesis is still not clear. From a previous pilot study, our group has reported the histological features of reduced osteoblastic activity in bone biopsy specimens obtained from AIS subjects intraoperatively, thus providing the early evidence of abnormal bone mineral acquisition and mineralization (Cheng et al. 2001). / Osteopontin (OPN) has been recognized as one the major non-collagen extracellular matrix proteins in bone and plays an important role in bone mineralization. Recent report suggested that AIS patients have higher OPN level than normal controls (Moreau et al. 2009). It was hypothesized that the low BMD in AIS is associated with abnormal bone matrix mineralization which may be related to abnormal expression of OPN in the plasma and at tissue level. / In this series of studies, the first part aimed to investigate the differential cortical and trabecular bone mineral density of AIS Vs normal controls. The non-dominant proximal femur areal BMD (aBMD) (femoral neck, Ward’s triangle and greater trochanter) of the subjects were measured with dual-energy x-ray absorptiometry (DXA). The volumetric bone mineral density (vBMD) in non-dominant distal radius was measured with peripheral quantitative computed tomography (pQCT) that allows accurate three dimensional assessment of the cortical and trabecular bone mineral density and other parameters of bone quality. AIS was found to have lower aBMDs, trabecular BMD (TBMD) and cortical BMD (CBMD) in different age groups and year since menarche (YSM) groups. Furthermore, the percentage difference of CBMD between AIS and controls was increased with age while a decreasing trend was observed in the TBMD. / The second part of the study investigated the bone mineralization and bone micro-architecture with micro-computed tomography (micro-CT) and histomorphometry study of bone biopsies obtained from AIS and normal controls. Three-dimensional structural parameters including material bone mineral density (mBMD) and bone architecture were evaluated by micro-CT. Bone histomorphometry was assessed by undecalcified sectioning with Goldner’s trichrome staining. mBMD of trabecular bone in AIS was found to be significantly lower than the normal control while no difference could be demonstrated in BV/TV, Tb.N, Tb.Th and SMI measurement between the two groups. It was also shown that the percentage of low-mineralized bone in AIS was significantly higher than that in normal controls. / The third part aimed to study the plasma OPN level and its association with the BMD in AIS Vs normal controls. Plasma OPN level in AIS and age-matched controls was measured by ELISA. With multivariate regression analysis, the plasma OPN level was found to be negatively correlated with Age and YSM in both AIS and normal controls. In addition, the plasma OPN level in AIS was significantly higher and correlated with the low trabecular BMD. / The fourth part of the study investigated the OPN expression in bone tissues level and its association with histomorphometric bone mineralization and bone micro-architectural parameters in AIS Vs normal controls. OPN expression in bone biopsy was semi-quantified by immunohistochemistry. It was found that the bone tissue OPN level was significantly higher in AIS and also positively correlated with plasma OPN level. In addition, in this pilot study, we found the trend that OPN expression in trabecular bone was negatively associated with mBMD, and positively with the percentage of low-mineralized bone. / The present study showed that AIS had lower bone mineralization than normal controls. The low cortical and trabecular BMD found in AIS is likely to be resulting from abnormal regulation of bone mineralization. The association of OPN with abnormal BMD and bone mineralization further suggested that abnormal OPN level might play an important role in affecting the bone mineral acquisition in AIS. All of these findings strongly supported the hypothesis that the low BMD in AIS is associated with abnormal bone matrix mineralization which could be related to abnormal expression of OPN. This study provided important additional insight into the possible mechanism of lower bone mineral density that might be linked to theetiopathogenesis, development and progression of the spinal deformity in AIS. / 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. / Sun, Guangquan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 143-160). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract and appendix A also in Chinese. / THE CHINESE UNIVERSITY OF HONG KONG --- p.I / ACKNOWLEDGEMENTS --- p.II / ABSTRACT --- p.IV / ABBREVIATION --- p.XI / TABLE OF CONTENTS --- p.XIII / LIST OF TABLES --- p.XVII / LIST OF FIGURES --- p.XIX / LIST OF PUBLICATIONS --- p.XXI / Chapter CHAPTER 1 --- STUDY BACKGROUND --- p.1 / Chapter 1.1 --- GENERAL OVERVIEW OF ADOLESCENT IDIOPATHIC SCOLIOSIS (AIS) --- p.2 / Chapter 1.1.1 --- NATURAL HISTORY --- p.4 / Chapter 1.1.2 --- CURRENT TREATMENTS --- p.6 / Chapter 1.1.2.1 --- Observation --- p.7 / Chapter 1.1.2.2 --- Bracing --- p.7 / Chapter 1.1.2.3 --- Surgical treatments --- p.9 / Chapter 1.1.3 --- CURRENT HYPOTHESIS ON THE ETIOLOGY OF AIS --- p.11 / Chapter 1.1.3.1 --- Genetic factors --- p.12 / Chapter 1.1.3.2 --- Neuromuscular impairment --- p.14 / Chapter 1.1.3.3 --- Abnormalities in skeletal development --- p.16 / Chapter 1.1.3.4 --- Low bone mineral density in AIS --- p.16 / Chapter 1.2 --- BONE MINERALIZATION --- p.18 / Chapter 1.2.1 --- Overview of bone mineralization --- p.18 / Chapter 1.2.2 --- Bone modeling --- p.18 / Chapter 1.2.3 --- Bone remodeling --- p.19 / Chapter 1.2.4 --- Factors affecting bone mineralization --- p.21 / Chapter 1.3 --- OSTEOPONTIN --- p.23 / Chapter 1.3.1 --- Structure of osteopontin --- p.23 / Chapter 1.3.2 --- Osteopontin - cellular and tissue distribution --- p.24 / Chapter 1.3.3 --- Osteopontin functions --- p.25 / Chapter 1.3.4 --- Osteopontin functions in bone --- p.25 / Chapter 1.3.5 --- Osteopontin and bone mineral density in human --- p.29 / Chapter CHAPTER 2 --- STUDY HYPOTHESIS AND PLAN --- p.31 / Chapter 2.1 --- INTRODUCTION --- p.32 / Chapter 2.2 --- HYPOTHESIS --- p.33 / Chapter 2.3 --- OBJECTIVES --- p.34 / Chapter 2.4 --- STUDY PLAN --- p.34 / Chapter CHAPTER 3 --- LOW BONE MINERAL DENSITY IN ADOLESCENT IDIOPATHIC SCOLIOSIS - AREAL VS VOLUMETRIC, CORTICAL VS TRABECULAR BONE MINERAL DENSITY --- p.36 / Chapter 3.1 --- INTRODUCTION --- p.37 / Chapter 3.2 --- SUBJECTS AND METHODS --- p.39 / Chapter 3.2.1 --- Subjects --- p.39 / Chapter 3.2.2 --- BMD Measurement --- p.40 / Chapter 3.2.3 --- Statistical Analysis --- p.41 / Chapter 3.3 --- RESULTS --- p.42 / Chapter 3.3.1 --- aBMD of AIS and normal controls by age groups --- p.42 / Chapter 3.3.2 --- TBMD and CBMD in AIS and normal controls by age groups --- p.42 / Chapter 3.3.3 --- aBMD in AIS and normal controls by year since menarche --- p.43 / Chapter 3.3.4 --- TBMD and CBMD in AIS and normal controls by year since menarche --- p.43 / Chapter 3.3.5 --- Correlation between CBMD & TBMD and chronological age or year since menarche --- p.44 / Chapter 3.3.6 --- Comparisons adjusted for chronological age or year since menarche --- p.44 / Chapter 3.4 --- DISCUSSION --- p.45 / Chapter 3.5 --- TABLES AND FIGURES --- p.50 / Chapter CHAPTER 4 --- ABNORMAL BONE MATRIX MINERALIZATION AND BONE MICROARCHITECTURE IN ADOLESCENT IDIOPATHIC SCOLIOSIS - A HISTOMORPHOMETRIC AND MICRO-CT STUDY --- p.60 / Chapter 4.1 --- INTRODUCTION --- p.61 / Chapter 4.2 --- SUBJECTS AND METHODS --- p.62 / Chapter 4.2.1 --- Subjects --- p.62 / Chapter 4.2.2 --- Micro-computed tomography --- p.63 / Chapter 4.2.3 --- Bone histomorphometry --- p.64 / Chapter 4.2.4 --- Statistical analysis --- p.68 / Chapter 4.3 --- RESULTS --- p.68 / Chapter 4.3.1 --- Results of micro-CT analysis --- p.68 / Chapter 4.3.2 --- Results of histomorphometric analysis --- p.69 / Chapter 4.3.3 --- Relationship of mBMD and percentage of low-mineralized bone --- p.69 / Chapter 4.4 --- DISCUSSION --- p.70 / Chapter 4.5 --- TABLES AND FIGURES --- p.74 / Chapter CHAPTER 5 --- PLASMA OSTEOPONTIN LEVEL AND ITS ASSOCIATION WITH BONE MINERAL DENSITY IN ADOLESCENT IDIOPATHIC SCOLIOSIS --- p.82 / Chapter 5.1 --- INTRODUCTION --- p.83 / Chapter 5.2 --- SUBJECTS AND METHODS --- p.84 / Chapter 5.2.1 --- Subjects --- p.84 / Chapter 5.2.2 --- Anthropometric assessment --- p.84 / Chapter 5.2.3 --- Plasma osteopontin measurement --- p.85 / Chapter 5.2.4 --- BMD Measurement --- p.86 / Chapter 5.2.5 --- Statistical Analysis --- p.86 / Chapter 5.3 --- RESULTS --- p.86 / Chapter 5.3.1 --- Comparison of anthropometric parameters between AIS and controls --- p.86 / Chapter 5.3.2 --- Correlation between OPN plasma level with age or YSM in AIS and controls --- p.87 / Chapter 5.3.3 --- Comparison of OPN plasma level between AIS and controls --- p.87 / Chapter 5.3.4 --- Correlation between OPN plasma level and curve severity in AIS --- p.87 / Chapter 5.3.5 --- Relationship between OPN plasma level and vBMD --- p.88 / Chapter 5.4 --- DISCUSSION --- p.88 / Chapter 5.5 --- TABLES AND FIGURES --- p.94 / Chapter CHAPTER 6 --- OSTEOPONTIN EXPRESSION IN BONE TISSUE AND ITS ASSOCIATION WITH BONE MATRIX MINERALIZATION IN ADOLESCENT IDIOPATHIC SCOLIOSIS - A PILOT STUDY --- p.102 / Chapter 6.1 --- INTRODUCTION --- p.103 / Chapter 6.2 --- SUBJECTS AND METHODS --- p.104 / Chapter 6.2.1 --- Subjects --- p.104 / Chapter 6.2.2 --- Micro-computed tomography --- p.104 / Chapter 6.2.3 --- Bone histomorphometry --- p.104 / Chapter 6.2.4 --- Semi-quantification of OPN expression in bone biopsy by immunohistochemistry --- p.105 / Chapter 6.2.5 --- Plasma osteopontin measurement --- p.107 / Chapter 6.2.6 --- Statistical Analysis --- p.108 / Chapter 6.3 --- RESULTS --- p.108 / Chapter 6.3.1 --- Comparison of anthropometric parameters between AIS and control subjects --- p.108 / Chapter 6.3.2 --- Comparison of OPN expression detected by immunohistochemistry in bone biopsy between AIS and control groups --- p.108 / Chapter 6.3.3 --- Comparison of histomorphometric and micro-CT results between AIS and control groups --- p.109 / Chapter 6.3.4 --- Relationship between plasma OPN level and OPN expression in bone biopsy --- p.109 / Chapter 6.3.5 --- Relationship between percentage of low-mineralized bone and OPN expression in bone biopsy --- p.109 / Chapter 6.3.6 --- Relationship between material bone mineral density and OPN expression in bone biopsy --- p.110 / Chapter 6.4 --- DISCUSSION --- p.110 / Chapter 6.5 --- TABLES AND FIGURES --- p.114 / Chapter CHAPTER 7 --- SUMMARY STUDY FLOWCHART, OVERALL DISCUSSION, CONCLUSIONS, LIMITATIONS AND FURTHER STUDIES --- p.119 / Chapter 7.1 --- SUMMARY OF THE STUDY FLOW CHART WITH KEY FINDINGS --- p.120 / Chapter 7.2 --- OVERALL DISCUSSION --- p.125 / Chapter 7.2.1 --- The novel findings on bone mineralization abnormality in AIS in this study --- p.125 / Chapter 7.2.2 --- OPN is a key modulator in AIS --- p.128 / Chapter 7.3 --- OVERALL CONCLUSIONS --- p.130 / Chapter 7.4 --- LIMITATION OF THIS STUDY AND FUTURE RESEARCH --- p.131 / Chapter APPENDIX A. --- CONSENT FORM OF AIS RESEARCH --- p.135 / Chapter APPENDIX B. --- CONSENT FORM OF BONE BIOPSY COLLECTION --- p.137 / Chapter APPENDIX C. --- MATERIALS AND REAGENTS INFORMATION AND PROTOCOL FOR SOLUTIONS PREPARATION --- p.138 / BIBLIOGRAPHY --- p.143

Lam Wun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 140-153). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ACKNOWLDGEMENT --- p.iii / TABLE OF CONTENT --- p.iv / ABBREVIATIONS --- p.x / LISTS OF TABLES --- p.xi / LISTS OF FIGURES --- p.xii / Chapter 1. --- INTRODUCTION / Chapter 1.1 --- Neurodegeneration and triplet repeat diseases --- p.1 / Chapter 1.2 --- Polyglutamine diseases --- p.2 / Chapter 1.3 --- Polyglutamine nuclear inclusions --- p.4 / Chapter 1.3.1 --- Kinetics of polyglutamine nuclear inclusion formation --- p.4 / Chapter 1.3.2 --- Roles of protein inclusions in neurodegeneration --- p.7 / Chapter 1.4 --- Polyglutamine pathogenic pathways --- p.8 / Chapter 1.4.1 --- Protein depletion theory --- p.9 / Chapter 1.4.2 --- Induction of apoptotic pathways --- p.13 / Chapter 1.5 --- Previous study on NI proteins --- p.14 / Chapter 1.6 --- Drosophila model for studying polyglutamine diseases --- p.15 / Chapter 1.6.1 --- Drosophila model for studying human diseases --- p.15 / Chapter 1.6.2 --- GAL4/UAS gene expression system --- p.15 / Chapter 1.6.3 --- Drosophila polyglutamine models --- p.17 / Chapter 1.7 --- Objectives of the study --- p.21 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Drosophila genetics --- p.22 / Chapter 2.1.1 --- Drosophila culture --- p.22 / Chapter 2.1.2 --- GAL4/UAS gene expression system --- p.22 / Chapter 2.1.3 --- Eye phenotypic analysis --- p.25 / Chapter 2.1.4 --- Polyglutamine fly models --- p.25 / Chapter 2.1.5 --- Generation and characterization of GFP-polyglutamine transgenic fly models --- p.25 / Chapter 2.2 --- Proteomic identification of nuclear inclusion proteins --- p.26 / Chapter 2.2.1 --- Proteomic identification of NI proteins by SDS-insolubility of NIs --- p.26 / Chapter 2.2.2 --- Proteomic identification of NI proteins by FA-solubility of NIs --- p.27 / Chapter 2.2.2.1 --- Approach overview --- p.27 / Chapter 2.2.2.2 --- Sample preparation for two-dimensional gel electrophoresis --- p.27 / Chapter 2.2.2.3 --- Two-dimensional gel electrophoresis --- p.29 / Chapter 2.2.2.4 --- Polyacrylamide gel staining --- p.31 / Chapter 2.2.2.5 --- Computer analysis of 2D patterns --- p.31 / Chapter 2.2.2.6 --- In-gel trypsin digestion --- p.32 / Chapter 2.2.2.7 --- Mass spectrometric analysis --- p.33 / Chapter 2.2.3 --- Detection of NIs by flow cytometry --- p.34 / Chapter 2.3 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.34 / Chapter 2.3.1 --- Sample preparation for SDS-PAGE --- p.34 / Chapter 2.3.2 --- SDS-PAGE --- p.35 / Chapter 2.4 --- Immunodetection --- p.36 / Chapter 2.4.1 --- Electroblotting --- p.36 / Chapter 2.4.2 --- Western blotting --- p.36 / Chapter 2.4.3 --- Filter trap assay --- p.37 / Chapter 2.5 --- Sav antibody production --- p.38 / Chapter 2.5.1 --- Sav peptide synthesis --- p.38 / Chapter 2.5.2 --- Rabbit immunization --- p.38 / Chapter 2.6 --- Cryosectioning and immunostaining of adult fly heads --- p.39 / Chapter 2.7 --- Alcohol dehydrogenase assay --- p.40 / Chapter 2.8 --- Semi-quantitative reverse transcription- Polymerase Chain Reaction --- p.41 / Chapter 2.8.1 --- Total RNA preparation from fly heads --- p.41 / Chapter 2.8.2 --- Reverse transcription- Polymerase Chain Reaction (RT-PCR) --- p.41 / Chapter 2.9 --- Reagents and buffers --- p.42 / Chapter 3. --- RESULTS / Chapter 3.1 --- Transgenic polyglutamine fly models --- p.48 / Chapter 3.1.1 --- Characteristics of MJD polyglutamine fly model --- p.48 / Chapter 3.1.1.1 --- Overexpression of expanded truncated human MJD proteins in Drosophila causes eye degeneration --- p.49 / Chapter 3.1.1.2 --- Overexpression of expanded truncated human MJD proteins in Drosophila results in nuclear inclusion formation --- p.49 / Chapter 3.1.1.3 --- Formic acid dissolves fly polyglutamine nuclear inclusions --- p.51 / Chapter 3.1.1.3.1 --- Formic acid dissolves fly polyglutamine NIs as shown by Western blot analysis --- p.51 / Chapter 3.1.1.3.2 --- Formic acid dissolves fly polyglutamine NIs as shown by filter trap assay --- p.53 / Chapter 3.1.2 --- Summary --- p.55 / Chapter 3.2 --- Proteomic identification of nuclear inclusion (NI) proteins --- p.56 / Chapter 3.2.1 --- Proteomic identification of NI proteins by SDS-insolubility of NIs --- p.56 / Chapter 3.2.2 --- Proteomic identification of NI proteins by FA-solubility of NIs --- p.63 / Chapter 3.2.2.1 --- Two-dimensional gels showing differential protein spots as potential NI proteins --- p.63 / Chapter 3.2.2.2 --- NI protein candidates identified by the 2D approach --- p.75 / Chapter 3.2.3 --- Study of polyglutamine NI proteins by flow cytometry analysis --- p.90 / Chapter 3.2.3.1 --- Detection of fly polyglutamine NIs by flow cytometry --- p.90 / Chapter 3.2.3.2 --- Characterization of a new GFP-polyglutamine fly model --- p.92 / Chapter 3.3 --- Characterization of the nuclear inclusion protein candidates --- p.96 / Chapter 3.3.1 --- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) --- p.96 / Chapter 3.3.1.1 --- Confirmation of GAPDH as a NI protein --- p.97 / Chapter 3.3.1.2 --- Discussion --- p.97 / Chapter 3.3.2 --- Receptor of activated protein kinase C (RACK1) --- p.99 / Chapter 3.3.2.1 --- Confirmation of RACK1 as a NI protein --- p.99 / Chapter 3.3.2.1.1 --- Colocalization of RACK1 with NIs --- p.99 / Chapter 3.3.2.1.2 --- Formic Acid extracts RACK1 from NIs --- p.101 / Chapter 3.3.2.2 --- Reduction of soluble RACK1 protein level in polyglutamine fly --- p.101 / Chapter 3.3.2.2.1 --- Soluble RACK1 protein level reduced in polyglutamine fly --- p.101 / Chapter 3.3.2.2.2 --- RACK1 transcript level remains unchanged in polyglutamine fly --- p.103 / Chapter 3.3.2.3 --- Overexpression of RACK 1 partially suppresses polyglutamine degeneration --- p.105 / Chapter 3.3.2.4 --- Discussion --- p.107 / Chapter 3.3.3 --- Warts (Wts) --- p.111 / Chapter 3.3.3.1 --- Overexpression of Wts partially suppresses polyglutamine degeneration --- p.111 / Chapter 3.3.3.2 --- Wts mutant slightly enhances polyglutamine degeneration --- p.113 / Chapter 3.3.3.3 --- Genetic analysis of Warts pathway in polyglutamine pathogenesis --- p.113 / Chapter 3.3.3.3.1 --- Overexpression of Salvador partially suppresses polyglutamine degeneration --- p.116 / Chapter 3.3.3.3.2 --- Hpo mutant slightly enhances polyglutamine degeneration --- p.119 / Chapter 3.3.3.3.3 --- Overexpression of DIAP1 partially suppresses polyglutamine degeneration --- p.119 / Chapter 3.3.3.4 --- Discussion --- p.121 / Chapter 3.3.4 --- Alcohol dehydrogenase (Adh) --- p.122 / Chapter 3.3.4.1 --- Adh activity is reduced in polyglutamine flies --- p.122 / Chapter 3.3.4.2 --- Overexpression of Hsp70 partially restores the reduced Adh activity in polyglutamine flies --- p.122 / Chapter 3.3.4.3 --- Discussion --- p.125 / Chapter 3.3.5 --- Genetic analysis of other NI protein candidates --- p.127 / Chapter 3.3.5.1 --- Overexpression of CG7920 protein partially suppresses polyglutamine degeneration --- p.127 / Chapter 3.3.5.2 --- Pten dsRNA slightly enhances polyglutamine degeneration --- p.129 / Chapter 3.3.6 --- Summary --- p.131 / Chapter 4. --- DISSCUSSION / Chapter 4.1 --- Protein depletion theory --- p.133 / Chapter 4.2 --- Comparison of different approaches for identification of NI proteins --- p.134 / Chapter 4.3 --- Long-term significance --- p.136 / Chapter 4.4 --- Future studies --- p.137 / Chapter 4.4.1 --- Characterization of other NI protein candidates --- p.137 / Chapter 4.4.2 --- Study of NI proteins by an alternative approach --- p.137 / Chapter 4.4.3 --- Study of NI proteins using other polyglutamine fly models --- p.137 / Chapter 5. --- CONCLUSION --- p.139 / Chapter 6. --- REFERENCES --- p.140

Wang Ying. / "July 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 250-297). / 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.

Li, Sai Kam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 162-179). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.ii / Acknowledgements --- p.iii / Table of Content --- p.iv / Abbreviations --- p.xi / List of Figures --- p.xiv / List of Tables --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Hepatitis B Virus / Chapter 1.1.1 --- General information --- p.1 / Chapter 1.1.2 --- Classification --- p.2 / Chapter 1.1.3 --- Virus life cycle and genome --- p.3 / Chapter 1.1.4 --- Hepatitis B virus X protein (HBx) --- p.7 / Chapter 1.2 --- Enigmatic functions of HB --- p.x / Chapter 1.2.1 --- HBx as a transactivator --- p.10 / Chapter 1.2.2 --- HBx as a cell cycle regulator --- p.12 / Chapter 1.2.3 --- HBx as an apoptosis modulator --- p.13 / Chapter 1.3 --- Etiology of HBV-mediated hepatocarcinogenesis --- p.14 / Chapter 1.4 --- Clinical mutants of HBV --- p.16 / Chapter 1.5 --- Hypothesis and aims of the research --- p.16 / Chapter 1.6 --- Basis of Tet-On system --- p.18 / Chapter CHPATER 2 --- EXPERIMENT MATERIALS / Chapter 2.1 --- Cell culture / Chapter 2.1.1 --- Cell-lines --- p.21 / Chapter 2.1.2 --- Culture medium --- p.22 / Chapter 2.1.3 --- Culture medium supplements --- p.23 / Chapter 2.2 --- Reagents for subcloning / Chapter 2.2.1 --- Reagents for polymerase chain reaction (PCR) --- p.24 / Chapter 2.2.2 --- Reagents for restriction enzyme digestion --- p.24 / Chapter 2.2.3 --- Reagents for ligation --- p.25 / Chapter 2.2.4 --- Reagents for electrophoresis --- p.25 / Chapter 2.2.5 --- Reagents for E. coli DH5a preparation --- p.25 / Chapter 2.2.6 --- Materials for bacterial culture work --- p.27 / Chapter 2.3 --- Reagents for subcellular localization study / Chapter 2.3.1 --- Reagents for cell staining --- p.28 / Chapter 2.3.2 --- Reagents for mounting slides --- p.29 / Chapter 2.3.3 --- Materials for site-directed mutagenesis --- p.29 / Chapter 2.4 --- Reagents for cell cycle analysis and cellular proliferation / Chapter 2.4.1 --- Reagents for cell cycle analysis --- p.29 / Chapter 2.4.2 --- Reagents for cellular proliferation study --- p.30 / Chapter 2.5 --- Reagents for protein expression study / Chapter 2.5.1 --- Cell lysis buffer --- p.30 / Chapter 2.5.2 --- Reagents for SDS-PAGE --- p.30 / Chapter 2.5.3 --- Reagents for Western blot --- p.33 / Chapter 2.5.4 --- Antibodies --- p.34 / Chapter 2.6 --- Reagents for gene expression study / Chapter 2.6.1 --- Reagents for RNA extraction --- p.36 / Chapter 2.6.2 --- Reagents for first strand cDNA synthesis --- p.37 / Chapter 2.6.3 --- Reagents for real-time PCR --- p.37 / Chapter 2.7 --- Reagents for establishment of Tet-On inducible stable cell-lines / Chapter 2.7.1 --- Reagents for MTT assay --- p.38 / Chapter 2.7.2 --- Reagents for selection of stable clones --- p.38 / Chapter 2.8 --- Vectors used in the project / Chapter 2.8.1 --- Vectors for subcellular localization study --- p.39 / Chapter 2.8.2 --- Vectors for establishment of Tet-on inducible cell-lines --- p.39 / Chapter 2.9 --- Primers used in the project / Chapter 2.9.1 --- Primers used for subcloning --- p.42 / Chapter 2.9.2 --- Primers used for site-directed mutagenesis --- p.43 / Chapter 2.9.3 --- Primers used in real-time chain polymerase reaction --- p.43 / Chapter CHAPTER 3 --- RESEARCH METHODS / Chapter 3.1 --- Subcloning of HBx and mutant genes into a green fluorescence protein (GFP) expression vector / Chapter 3.1.1 --- Amplification of HBxWt，HBxΔC44 and HBxAN60 genes --- p.45 / Chapter 3.1.2 --- Purification of PCR products --- p.46 / Chapter 3.1.3 --- Restriction enzyme digestion --- p.47 / Chapter 3.1.4 --- Ligation of gene products with pEGFP-C 1 vector --- p.47 / Chapter 3.1.5 --- Preparation of chemically competent bacterial cells E. coli strain DH5α --- p.47 / Chapter 3.1.6 --- Transformation of the ligation product into competent cells --- p.48 / Chapter 3.1.7 --- PCR confirmation of successful ligation --- p.48 / Chapter 3.1.8 --- Small scale preparation of bacterial plasmid DNA --- p.49 / Chapter 3.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.50 / Chapter 3.1.10 --- Large scale preparation of target recombinant plasmid DNA --- p.50 / Chapter 3.2 --- Subcellular localization pattern study / Chapter 3.2.1 --- Cell transfection --- p.51 / Chapter 3.2.2 --- Mitochondria and nucleus staining --- p.52 / Chapter 3.2.3 --- Epi-fluorescence microscopy --- p.53 / Chapter 3.2.4 --- Analysis of fluorescence images --- p.53 / Chapter 3.2.5 --- In vitro site-directed mutagenesis --- p.53 / Chapter 3.3 --- Cell cycle phase analysis with flow cytometry / Chapter 3.3.1 --- Cell transfection --- p.55 / Chapter 3.3.2 --- Cell staining --- p.55 / Chapter 3.3.3 --- Flow cytometry --- p.55 / Chapter 3.4 --- Cellular proliferation quantification by BrdU proliferation assay / Chapter 3.4.1 --- Cell transfection --- p.57 / Chapter 3.4.2 --- BrdU ELISA measurement --- p.57 / Chapter 3.5 --- Protein expression / Chapter 3.5.1 --- Cell lysate collection --- p.58 / Chapter 3.5.2 --- Quantification of protein samples --- p.59 / Chapter 3.5.3 --- SDS-PAGE --- p.59 / Chapter 3.5.4 --- Western blot --- p.60 / Chapter 3.5.5 --- Western blot luminal detection --- p.60 / Chapter 3.6 --- Gene expression / Chapter 3.6.1 --- Primer design --- p.61 / Chapter 3.6.2 --- Cell transfection --- p.61 / Chapter 3.6.3 --- RNA extraction --- p.61 / Chapter 3.6.4 --- Reverse transcription for first strand complementary DNA (cDNA) --- p.63 / Chapter 3.6.5 --- Quantitative real-time PCR --- p.63 / Chapter 3.7 --- Establishment of Tet-On inducible stable cell-lines / Chapter 3.7.1 --- Subcloning of HBx gene into pTRE2 vector --- p.64 / Chapter 3.7.2 --- Construction of WRL68/Tet-On stable cell-lines --- p.64 / Chapter 3.7.3 --- Construction of WRL68/Tet-On HBx and mutants expression cell-lines --- p.68 / Chapter 3.7.4 --- Characterization of Tet-On gene expression monoclones --- p.69 / Chapter 3.8 --- Statistical analyses --- p.70 / Chapter CHPATER 4 --- STUDY ON MITOCHONDRIA TARGETING / Chapter 4.1 --- Establishment of pEGFP-Cl-HBx and mutants constructs --- p.71 / Chapter 4.2 --- Transactivation C-terminus domain is essential for granular localization --- p.73 / Chapter 4.3 --- Wild-type HBx localizes in mitochondria --- p.76 / Chapter 4.4 --- C-terminal transactivation domain is sufficient for mitochondria targeting --- p.79 / Chapter 4.5 --- Mapping of the HBx region crucial for mitochondria targeting --- p.81 / Chapter 4.6 --- The 111-117 amino acids in HBx do not work as a signal peptide --- p.83 / Chapter 4.7 --- Site-directed mutagenesis identifies the key amino acid at 115 in HBx for mitochondrial targeting --- p.85 / Chapter CHAPTER 5 --- CELL PROLIFERATION AND REGULATION / Chapter 5.1 --- Alteration of S-phase distribution in cell cycle --- p.88 / Chapter 5.2 --- Analysis of DNA synthesis using BrdU proliferation ELISA --- p.92 / Chapter 5.3 --- Differential molecular regulation of cell cycle --- p.94 / Chapter 5.4 --- Regulation of the mRNA expression levels of cyclin-dependent kinases inhibitors p2raf/cipl and p27kipl --- p.98 / Chapter CHAPTER 6 --- TRANSACTIVATION AND RAS/RAF/MAPK PHOSPHORYLATION / Chapter 6.1 --- Determination of p53-dependency of p21、vaf/cipl expression --- p.101 / Chapter 6.2 --- Ras/Raf/MAPK pathway activation by HBx variants / Chapter 6.2.1 --- ERK1/2 phophorylation by HBx variants --- p.104 / Chapter 6.2.2 --- ERK inhibition blocks the regulation effect on p53Wt and p21waf/cipl --- p.107 / Chapter 6.3 --- Transactivation activity on oncogenes/ proto-oncogenes / Chapter 6.3.1 --- Effect on c-myc (NM´ؤ002467) mRNA expression --- p.109 / Chapter 6.3.2 --- Effect on RhoC (NM_017744) and Rabl4 (NM´ؤ016322) mRNA expression --- p.112 / Chapter CHAPTER 7 --- CONSTRUCTION OF TET-ON INDUCIBLE CELL-LINES / Chapter 7.1 --- Establishment of WRL/Tet-On monoclonal cell-lines Page / Chapter 7.1.1 --- Determination of geneticin selection dosage --- p.116 / Chapter 7.1.2 --- Selection of the best WRL/TOn clone using luciferase assay --- p.118 / Chapter 7.2 --- Establishment of inducible WRL/TOn/Gene monoclonal cell-lines / Chapter 7.2.1 --- Determination of hygromycin selection dosage --- p.120 / Chapter 7.2.2 --- Selection of positive WRL/TOn/Gene clones with viral genes --- p.122 / Chapter 7.3 --- Characterization of TOXDC1 cell-line / Chapter 7.3.1 --- Cell morphology --- p.125 / Chapter 7.3.2 --- Growth pattern of TOXDC1 --- p.126 / Chapter 7.3.3 --- HBxAC44 induced p21waf/cipl mRNA expression --- p.127 / Chapter 7.3.4 --- Doxycycline concentration dependent HBxAC44 expression in TOXDC1 --- p.129 / Chapter CHAPTER 8 --- DISCUSSION / Chapter 8.1 --- Selection of cell model / Chapter 8.1.1 --- Selection of cell models --- p.130 / Chapter 8.1.2 --- Selection of truncation mutant --- p.131 / Chapter 8.2 --- Differential sub-cellular localization of HBx and its variants / Chapter 8.2.1 --- Mechanisms of mitochondria targeting --- p.132 / Chapter 8.2.2 --- Mitochondria as site of HBx-induced apoptosis --- p.134 / Chapter 8.2.3 --- Stimulation of calcium release from mitochondria by wild-type HBx --- p.135 / Chapter 8.3 --- Cell cycle distribution profiling and its regulations / Chapter 8.3.1 --- Cell cycle pattern and cell proliferation --- p.136 / Chapter 8.3.2 --- Differential cell cycle molecular pathway activation --- p.138 / Chapter 8.4 --- Ras/Raf/MAPK mediated transactivation by HBxWt and its mutants / Chapter 8.4.1 --- p53-mediated p21waf/cipl expression --- p.142 / Chapter 8.4.2 --- ERK-mediated p21waf/cipl and wild-type p53 mRNA expression --- p.143 / Chapter 8.4.3 --- Regulation of oncogenes/ proto-oncogenes expression --- p.147 / Chapter 8.5 --- General discussions on differential effects of HBxWt and HBxAC44 --- p.149 / Chapter 8.6 --- Establishment of Tet-On/HBxAC44 cell-line TOXDC1 --- p.153 / Chapter 8.7 --- Conclusions --- p.154 / Chapter 8.8 --- Future Prospects / Chapter 8.8.1 --- From mitochondria targeting to calcium signaling --- p.157 / Chapter 8.8.2 --- Construction of a complete cell cycle regulation pathway --- p.158 / Chapter 8.8.3 --- Elucidation of the transcriptional transactivation regulation --- p.159 / Chapter 8.8.4 --- To make the best use of the Tet-on stable cell-line TOXDC1 --- p.159 / Chapter 8.8.5 --- Study with other carboxy-terminal truncation mutants --- p.160 / Chapter 8.8.6 --- In vivo study --- p.160 / REFERENCES --- p.162

子宮頸癌是女性的主要癌症殺手，而人類乳頭瘤病毒 (HPV) 則是子宮頸癌形成的必要條件之一。HPV16型及HPV18型是全球最普遍的高危型HPV；而另一方面，HPV52及HPV58兩型在東亞地區的流行程度比世界其他地區為高。 / 過往有科學研究顯示HPV病毒載量的高低是引致高度癌前病變的重要決定因素，也有研究指出病毒載量與病變的嚴重程度成正比例，但同時亦有研究指兩者並無關係。HPV基因組可以兩種物理形態存在：游離型及整合型。HPV的E2基因可對E6及E7致癌基因產生重要的調節作用，而當HPV病毒與宿主染色體整合後，可使E2基因斷裂，因而令控制E6及E7致癌基因表達的負反饋基制失效。 / 本研究假設高病毒載量及由HPV基因組整合所造成的E2基因斷裂，並非引致子宮頸癌的僅一途徑。本研究分析了在不同程度的子宮頸細胞病變下，HPV16型、18型、52型及58型的病毒載量及基因整合情況。其中，有關HPV16型的研究部份更深入地探討了E6/7 mRNA的轉錄水平、E2和LCR的序列變異及E2結合位點的甲基化情況，最終希望能找出除了病毒基因整合之外的另一種致癌機理。 / 本研究的結果顯示，在不同HPV型所引致的子宮頸細胞病變中，病毒載體及病變程度之間的關係也存有差異；而根據管家基因的數量來為細胞DNA標準化，對準確分析不同程度子宮頸細胞病變的實驗結果至關重要。本研究的一項重要發現是部份侵襲性癌細胞只含有游離型HPV基因組；而在只含游離HPV基因組的侵襲性子宮頸癌樣本中，有三種E6/E7 mRNA的抄錄本水平與只含整合型基因組的樣本相若，反映在只含游離型HPV基因組的侵襲性子宮頸癌樣本中，E6/ E7 mRNA的表達量亦有上調。最重要的是，此表達量的上調並非由基因整合或E2基因斷裂所引致。 / 在只含有游離型病毒基因組的侵襲性子宮頸癌樣本中，E6及E7致癌基因表達上調的另一種機理，很可能是HPV16啟動區內E2結合位點上的CpG位點出現甲基化。這項觀察解釋及支持了當E2蛋白因結合位點甲基化而失去對E6及E7基因轉錄的抑制功能時，E6及E7致癌蛋白仍能保持高水平，而兩種蛋白產生協同作用，令細胞轉型及出現癌變。總結之言，本實驗也肯定了HPV整合並非導致子宮頸癌形成的唯一機理。 / Cervical cancer is a major cause of cancer-related death in women worldwide. Human papillomavirus (HPV) is essential, though not sufficient, to cause cervical cancer. HPV16 and HPV18 are the most prevalent high-risk types worldwide, whereas, HPV52 and HPV58 also show a notable higher prevalence in East Asia than in other parts of the world. / Studies have suggested that HPV viral load is an important determinant for the development of high-grade lesions. While some studies observed a positive correlation between viral load and disease severity, others have reported no association. The HPV genome can exist in two physical forms, episomal or integrated. The E2 gene, encoded by HPV has an important role in the regulation of E6 and E7 viral oncogenes. When HPV integrates into the host chromosome, it may result in disruption of the E2 gene thereby its control on the expression of the E6 and E7. / The hypothesis for this study was that high viral load and disruption of E2 gene associated with integration of HPV into the host genome was not the only pathway leading to cervical cancer development. In this study, the viral load and integration profile for HPV types 16, 18, 52 and 58 among different severity of cervical lesions were analyzed. Further detailed studies were performed on HPV16 with emphases on E6/E7 mRNA transcript levels, E2 and LCR sequence variation and the methylation status of two E2 binding sites. The ultimate aim was to determine what other alternative mechanisms exist apart from viral integration to drive the oncogenicity of HPV that lead to the development of cervical cancer. / The results showed that the relationship between viral load and disease varied between different HPV types and that normalization of cellular DNA input using a housekeeping gene was crucial for accurate interpretation among different cervical lesion grades. A key finding from this study was that a substantial proportion of invasive cervical carcinomas were found to contain the purely episomal form of the HPV genome. The levels of the three E6/E7 mRNA transcripts species in invasive cervical carcinomas containing the pure episomal form of the viral genome were found to be similar to those with pure integrated forms. This observation suggested that invasive cervical carcinoma samples containing the episomal form of the HPV genome were also mediated by the up-regulated E6/E7 mRNA expression. More importantly, this up-regulation in E6/E7 mRNA expression did not depend on integration and disruption of the E2 gene. / The alternative mechanism that up-regulated of the expression of E6 and E7 oncogene found in invasive cervical carcinoma samples harbouring the episomal form of the viral genome was likely to be a consequence of methylation of CpG sites in the two E2 binding sites at the promoter region of HPV16. This observation explained and supported that the repressive role of E2 on E6 and E7 transcriptional regulation was abolished due to methylation of the E2 binding sites, and that a sustained level of the E6 and E7 oncoproteins was maintained, working in synergy in cell transformation and in carcinogenesis. These observations confirmed the hypothesis that HPV integration was not the only mechanism leading to the development of cervical cancer. / 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. / Cheung, Lai Ken Jo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 233-248). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.I / Abstract of thesis --- p.IV / 論文摘要 --- p.VII / Publications --- p.IX / Contents --- p.X / Figures --- p.XV / Tables --- p.XVIII / Abbreviations --- p.XIX / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cervical Cancer --- p.2 / Chapter 1.1.1 --- Cervical Cytology Screening --- p.3 / Chapter 1.1.2 --- Classification System for Cervical Squamous Cell Dysplasia --- p.4 / Chapter 1.1.3 --- Histological Grading of Cervical Lesions --- p.6 / Chapter 1.1.4 --- Development of Cervical Cancer --- p.6 / Chapter 1.2 --- Structure of HPV --- p.7 / Chapter 1.1.1 --- HPV Genome Organization --- p.8 / Chapter 1.1.2 --- The E1 Protein --- p.10 / Chapter 1.1.3 --- The E2 Protein --- p.10 / Chapter 1.1.4 --- The E4 Protein --- p.13 / Chapter 1.1.5 --- The E5 Protein --- p.13 / Chapter 1.1.6 --- The E6 Protein --- p.14 / Chapter 1.1.7 --- The E7 Protein --- p.14 / Chapter 1.1.8 --- The L1 Protein --- p.15 / Chapter 1.1.9 --- The L2 Protein --- p.16 / Chapter 1.1.10 --- The Long Control Region --- p.17 / Chapter 1.3 --- HPV and Cervical Cancer --- p.19 / Chapter 1.3.1 --- HPV is an Etiological Cause of Cervical Cancer --- p.19 / Chapter 1.3.2 --- Establishment of HPV Infection --- p.20 / Chapter 1.3.3 --- Regulation and Control of HPV Viral Gene Transcription --- p.23 / Chapter 1.3.4 --- Viral Oncogene Expression by Alternative RNA Splicing --- p.23 / Chapter 1.3.5 --- DNA Methylation in Viral Oncogene Expression --- p.24 / Chapter 1.3.6 --- The Roles of E6 and E7 Protein in Cervical Carcinogenesis --- p.26 / Chapter Chapter 2 --- Controversies and Hypothesis --- p.33 / Chapter 2.1 --- Controversies in Mechanism of Cervical Carcinogenesis --- p.34 / Chapter 2.1.1 --- Viral Integration and Risk of Cervical Cancer Development --- p.34 / Chapter 2.1.2 --- Viral Load and Risk of Cervical Cancer Development --- p.35 / Chapter 2.2 --- Hypothesis of Study --- p.37 / Chapter 2.2.1 --- Study Design --- p.38 / Chapter Chapter 3 --- Materials and Methods --- p.41 / Chapter 3.1 --- Patient Recruitment and Sample Preparation --- p.42 / Chapter 3.1.1 --- Study subject recruitment --- p.42 / Chapter 3.1.2 --- Collection of cytology samples --- p.43 / Chapter 3.1.3 --- Collection of cervical biopsy samples --- p.44 / Chapter 3.2 --- Nucleic Acid Extraction and Preparation --- p.44 / Chapter 3.2.1 --- Extraction of DNA from cervical cytology samples --- p.44 / Chapter 3.2.2 --- Extraction of DNA from cervical biopsy samples --- p.45 / Chapter 3.2.3 --- Extraction of RNA from cervical cytology samples --- p.45 / Chapter 3.2.4 --- Extraction of RNA from cervical biopsy samples --- p.46 / Chapter 3.3 --- Detection and Genotyping of Human Papillomavirus --- p.46 / Chapter 3.4 --- Determination of Viral Load using Real-Time Polymerase Chain Reaction --- p.47 / Chapter 3.4.1 --- Optimization of HPV16, 18, 52 and 58 E7 real-time PCR --- p.48 / Chapter 3.4.2 --- Optimization of housekeeping gene real-time PCR --- p.50 / Chapter 3.4.3 --- Determination of HPV16, 18, 52 and 58 viral load --- p.50 / Chapter 3.5 --- Determination of HPV Genome Physical Status --- p.53 / Chapter 3.5.1 --- HPV E2 gene primer design --- p.53 / Chapter 3.5.2 --- Optimization of HPV16, 18, 52 and 58 E2 Real-time PCR --- p.56 / Chapter 3.5.3 --- Determination of the HPV genome physical status --- p.59 / Chapter 3.6 --- Evaluation of Housekeeping Genes for Normalization of Viral Gene Expression --- p.62 / Chapter 3.6.1 --- Optimization of housekeeping gene real-time PCR --- p.62 / Chapter 3.6.2 --- Quantitation of RNA and DNase treatment --- p.66 / Chapter 3.6.3 --- cDNA synthesis from the extracted RNA --- p.67 / Chapter 3.6.4 --- Detection of five housekeeping gene levels from cervical cytology samples by real-time PCR --- p.67 / Chapter 3.6.5 --- Data analyses --- p.68 / Chapter 3.7 --- Quantitation of HPV16 mRNA Transcripts --- p.69 / Chapter 3.7.1 --- Preparation of RNA from CaSki cells --- p.69 / Chapter 3.7.2 --- Amplification of mRNA transcripts from CaSki cells --- p.69 / Chapter 3.7.3 --- Amplification of artificial mRNA transcript E6*II --- p.73 / Chapter 3.7.4 --- Gel purification of mRNA transcript amplicons --- p.73 / Chapter 3.7.5 --- Cloning of E6 mRNA transcripts --- p.74 / Chapter 3.7.6 --- Confirmation of the mRNA transcript inserts --- p.74 / Chapter 3.8 --- Quantitation HPV16 E6 mRNA Transcript Levels Using Real-Time PCR --- p.79 / Chapter 3.8.1 --- mRNA transcript primer and probe design --- p.79 / Chapter 3.8.2 --- Optimization of real-time PCR for the detection of mRNA transcripts --- p.82 / Chapter 3.8.3 --- Determination of mRNA transcript levels from invasive carcinomas --- p.83 / Chapter 3.8.4 --- Normalization of mRNA transcript expression with a housekeeping gene --- p.84 / Chapter 3.9 --- Sequence Variation of the HPV16 E2 and Long Control Region --- p.84 / Chapter 3.9.1 --- Identification of sequence variation of the E2 gene --- p.84 / Chapter 3.9.2 --- Identification of sequence variation of the long control region --- p.87 / Chapter 3.1 --- Detection of Methylation Status of E2BS1 and E2BS2 on the LCR using Pyrosequencing --- p.87 / Chapter 3.10.1 --- Bisulfite DNA conversion --- p.87 / Chapter 3.10.2 --- Amplification of E2 binding site regions on the LCR --- p.88 / Chapter 3.10.3 --- Purification of PCR product prior to pyrosequencing --- p.92 / Chapter 3.10.4 --- Quantitation of methylation using pyrosequencing --- p.92 / Chapter Chapter 4 --- Results --- p.93 / Chapter Hypothesis 1 --- p.94 / Chapter Results of Study Part: 1 --- p.95 / Chapter 4.1 --- Human Papillomavirus Type 16 Viral Load and Genome Physical Status --- p.96 / Chapter 4.1.1 --- E7 viral load --- p.96 / Chapter 4.1.2 --- Viral genome physical status --- p.100 / Chapter 4.1.3 --- E2 disruption site --- p.105 / Chapter 4.2 --- Human Papillomavirus Type 18 Viral Load and Genome Physical Status --- p.107 / Chapter 4.2.1 --- E7 viral load --- p.107 / Chapter 4.2.2 --- Viral genome physical status --- p.110 / Chapter 4.2.3 --- E2 disruption site --- p.113 / Chapter 4.2.4 --- Infection status --- p.116 / Chapter 4.2.5 --- Adeno/adenosquamous carcinoma versus squamous cell carcinoma --- p.119 / Chapter 4.3 --- Human Papillomvirus Type 52 Viral Load and Genome Physical Status --- p.120 / Chapter 4.3.1 --- E7 viral load --- p.120 / Chapter 4.3.2 --- Viral genome physical status --- p.123 / Chapter 4.3.3 --- E2 disruption site --- p.126 / Chapter 4.3.4 --- Infection status --- p.129 / Chapter 4.4 --- Human Papillomavirus Type 58 Viral Load and Genome Physical Status --- p.131 / Chapter 4.4.1 --- E7 viral load --- p.131 / Chapter 4.4.2 --- Viral genome physical status --- p.133 / Chapter 4.4.3 --- E2 disruption site --- p.134 / Chapter 4.4.4 --- Infection status --- p.137 / Chapter 4.5 --- Summary of Study Part 1: --- p.140 / Chapter Hypothesis 2 --- p.141 / Chapter Results of Study Part 2: --- p.142 / Chapter 4.6 --- Housekeeping Gene mRNA Expression Level --- p.143 / Chapter 4.6.1 --- Expression levels across different grades of cervical lesion --- p.143 / Chapter 4.6.2 --- Expression stability of housekeeping genes --- p.145 / Chapter 4.7 --- Summary of Study Part 2: --- p.149 / Chapter Results of Study Part: 3 --- p.150 / Chapter 4.8 --- HPV16 mRNA Transcript Expression Level --- p.151 / Chapter 4.8.1 --- HPV16 viral genome physical status --- p.151 / Chapter 4.8.2 --- HPV16 E2 disruption site --- p.151 / Chapter 4.8.3 --- Expression level of E6/E7 mRNA transcripts --- p.155 / Chapter 4.8.4 --- Expression level of E6/E7 mRNA transcripts and viral genome physical status --- p.157 / Chapter 4.8.5 --- Expression level of E6/E7 mRNA transcripts and E2 gene disruption status --- p.161 / Chapter 4.9 --- Summary of Study Part 3: --- p.163 / Chapter Hypothesis 3 --- p.165 / Chapter Results of Study Part 4: --- p.166 / Chapter 4.1 --- HPV 16 E2 Gene Sequence Variation --- p.167 / Chapter 4.10.1 --- Sequence variation of E2 gene --- p.167 / Chapter 4.10.2 --- Sequence variation and viral genome physical status --- p.168 / Chapter 4.10.3 --- Sequence variation in the E2 binding sites --- p.169 / Chapter 4.10.4 --- Sequence variations of E2 in HPV16 cancer derived cell lines --- p.170 / Chapter 4.11 --- HPV16 Long Control Region Sequence Variation --- p.174 / Chapter 4.11.1 --- Sequence variation of LCR --- p.174 / Chapter 4.11.2 --- Sequence variation and viral genome physical status --- p.175 / Chapter 4.11.3 --- Sequence variation in E2 binding sites --- p.176 / Chapter 4.11.4 --- Sequence variation of LCR in HPV16 cancer derived cell lines --- p.176 / Chapter 4.12 --- Summary of Study Part 4: --- p.183 / Chapter Hypothesis 4 --- p.185 / Chapter 4.13 --- Methylation Status of E2 Binding Sites --- p.187 / Chapter 4.13.1 --- Proportion methylation in E2 binding sites --- p.187 / Chapter 4.13.2 --- Methylation in invasive carcinomas according to the viral genome physical status --- p.191 / Chapter 4.14 --- Summary of Study Part 5: --- p.195 / Chapter Chapter 5 --- Discussion --- p.196 / Chapter 5.1 --- Viral Load --- p.197 / Chapter 5.2 --- Viral Integration --- p.200 / Chapter 5.2.1 --- HPV16 Viral Load and Physical Status --- p.201 / Chapter 5.2.2 --- HPV18 Viral Load and Physical Status --- p.204 / Chapter 5.2.3 --- HPV52 Viral Load and Physical Status --- p.207 / Chapter 5.2.4 --- HPV58 Viral Load and Physical Status --- p.210 / Chapter 5.2.5 --- Viral Load and Physical Status Summary --- p.214 / Chapter 5.3 --- HPV16 E6/E7 mRNA Transcript and Genome Physical Status --- p.215 / Chapter 5.4 --- HPV16 E2 Sequence Variation and Genome Physical Status --- p.218 / Chapter 5.5 --- HPV16 LCR Sequence Variation and Genome Physical Status --- p.222 / Chapter 5.6 --- Methylation of HPV16 E2 Binding Sites and Genome Physical Status --- p.225 / Chapter 5.7 --- Conclusions --- p.230 / Chapter 5.8 --- Implication of Current Findings and Future Work --- p.231 / References --- p.233

No description available.

Strongyloidiasis stercoralis hyperinfection presenting as vasculitic-like skin lesions is rare. An autoinfection cycle allows intestinal strongyloidiasis, usually a benign infection, to persist for many decades. We report a woman with disseminated S stercoralis infection presenting as nonpalpable purpuric skin rash and syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Upon admission, she was treated with corticosteroids for her vasculitic skin lesions, which then worsened her status. When the diagnosis was recognized, steroids were stopped, thiabendazole treatment was instituted, and she gradually recovered. Serious or fatal infection can occur in patients with strongyloidiasis who were treated with immunosuppressive drugs. Stool specimen screening and/or serological tests for S stercoralis infection in patients who require immunosuppressive therapy helps to prevent complications before embarking on such treatment. Unexplained hyponatremia, severe hypoalbuminemia without proteinuria, and unusual skin rashes, especially over the lower aspect of the abdomen and upper aspects of the thighs, in persons living in areas endemic to S stercoralis should raise suspicion of S stercoralis infection.

Indiana University-Purdue University Indianapolis (IUPUI) / The impact of tobacco smoking on human health is well documented. The influence of smoking on tooth loss and cardiovascular diseases was investigated in the current study via both epidemiology and in vitro studies. From analyzing the 2006 Behavioral Risk Factor Surveillance System (2006 BRFSS) database, we confirmed that smoking was significantly associated with the number of teeth lost in a dose-dependent manner and smoking cessation reduced the risk when compared to those subjects continuing to smoke. In addition, the virulence factors related to caries were compared between Streptococcus mutans and Streptococcus gordonii in response to cigarette smoking condensate (CSC) treatment. We observed that S. gordonii was more susceptible to CSC treatment than S. mutans. CSC significantly enhanced S. mutans sucrose-dependent and independent adherence. Western blot assays revealed that several bacterial surface proteins including glucosyltransferase (GTF), glucan-binding proteins and antigen I/II, were significantly upregulated for the treated S. mutans. These findings suggested that the oral environment with CSC may favor a cariogenic dominant composition, which may increase the risk for smokers to develop caries. We also found that smoking and oral health status modified each other and synergistically increased the risk of CVD and this joint effect was more pronounced among the youngest age group using the 2006 BRFSS database. To further understand the joint effect, we conducted an in vitro study to investigate bacterial attachment to fibronectin and endothelial cells in response to smoking condensate treatment. Our study clearly demonstrated CSC significantly enhanced S. mutans attachment to both soluble and immobilized fibronectin as well as endothelial cells. Furthermore, our data suggested that bacteria possessed several adhesins that bound to host tissues and endothelial cells also had multiple receptors for bacterial attachment. Among these adhesins, antigen I/II seemed essential for bacterial attachment to endothelial cells without CSC. The knowledge of bacterial attachment to host tissues in the presence of CSC may help in developing different preventive or therapeutic strategies against attachment and colonization of the host by S. mutans.