Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, no drugs that target MYCN have yet been developed. Here, by combining a whole genome shRNA library screen and Master Regulator Inference Algorithm (MARINa) analysis, we identified Transcription Factor Activating Protein 4 (TFAP4) as a novel synthetic lethal interactor with MYCN amplification in neuroblastoma. Silencing TFAP4 selectively inhibits MYCN amplified neuroblastoma growth both in vitro and in xenograft mice models. TFAP4 expression is inversely correlated with patient survival in MYCN-high neuroblastoma. Mechanistically, silencing TFAP4 induces neuroblastoma differentiation, as seen by increased neurite outgrowth, and up-regulation of neuronal markers. TFAP4 regulates a downstream signature similar to the signature of the oncogene anaplastic lymphoma kinase (ALK). Taken together, our results validate TFAP4 as an important master regulator in MYCN amplified neuroblastoma and a novel synthetic interactor with MYCN amplification. Thus, TFAP4 may be a novel drug target for neuroblastoma treatment.

Background: The obesity epidemic is the greatest public health problem of our time, and exerts an enormous health and economic burden by acting as a risk factor for multiple disorders and all-cause mortality. While environmental and social factors certainly contribute to the complex etiology of obesity, there is strong evidence of a substantial genetic component. The majority of obesity genes are involved the leptin-melanocortin receptor pathway governing energy homeostasis, but mutations affecting this circuit are often untreatable and rare, and an improved understanding of other genetic risk factors could aid in the development of novel therapies. In this thesis I study two obesity candidate genes with unclear direct relevance to disease: 1) rare structural variation at the 16p11.2 BP4-BP5 locus and 2) common variation in the Fat Mass and Obesity-Associated (FTO) gene. Methods: 1) I analyzed disinhibited eating measurements from families with 16p11.2 copy number variation (CNV) carriers, to test whether eating in the absence of hunger (EAH) and loss of control (LOC) eating behaviors mediate the dosage-dependent CNV-BMI relationship. 2) Using association data from a study of over 20,000 African Americans and 1,145 functional annotations from the Encyclopedia of Non-coding Elements (ENCODE) and Roadmap Epigenomics projects, I statistically fine-mapped the FTO locus to identify the SNP(s) and cellular contexts underlying the association between FTO and obesity. Results: 1) EAH due to external triggers mediates over 30% of the 16p11.2 deletion’s effect on obesity, while other EAH and LOC behaviors were not significant mediators. This result was independent of IQ deficits and autism related to the CNV, as well as parents’ feeding behaviors and practices. 2) Given 51 FTO SNPs’ association statistics, correlation, and overlap with functional annotations, rs9927317 and rs62033405 had the highest posterior probability of association with obesity. Obesity-associated SNPs may regulate expression of FTO and/or nearby genes through the activity of enhancers and 5’ ends of transcribed genes in the substantia nigra of the brain, bone chondrocytes, and white adipose. Conclusions: These results may help pinpoint the specific genes, regulatory elements, and cellular contexts through which the 16p11.2 and FTO loci exert their effects on obesity.

目的：症狀性癲癇通常是由大腦損傷引起的，但大多數人患有這種腦損傷的人不會進而發展為癲癇，這表明，有些人擁有癲癇的易感基因。全基因組關聯研究（GWAS）是一種找到複雜疾病潛在基因的有效方法。最近關於對中國人口症狀性癲癇的GWAS報導了一個在1q32.1的CAMSAP1L1基因 (也叫做CAMSAP2) 上最顯著的單核苷酸多態性rs2292096 [G] （p=1.0×10⁻⁸，OR=0.63）。除了這個SNP，我們還選擇另一個在ERBB4基因上的潛在SNP rs13021324 [C]（p=5.8×10⁻⁵，OR=0.73）作為我們的研究。 CAMSAP1L1編碼一種細胞骨架蛋白，但是，它是否具有任何神經特定功能尚未知曉。 ERBB4是一個精神分裂症的易感基因，也有文章報導了ERBB4的突變引起了早期肌陣攣性腦病。我們推測，這兩個基因會影響人發展成為症狀性癲癇的易感性，以上的單核苷酸多態性會影響基因的表達或功能。這項研究的目標是，探索CAMSAP1L1的一些功能，和比較基因型之間以及癲癇患者和對照組之間這兩個基因的表達。 / 方法：在癲癇患者樣品中，我們對CAMSAP1L1 的SNP（rs2292096）和ERBB4的SNP（rs13021324）進行基因分型，首先將從癲癇患者的冰凍組織中提取RNA和蛋白質，他們的表達水平將分別通過實時PCR和蛋白印跡來測量，看家基因GAPDH用於測量對照，然後將不同的單核苷酸多態性與相應的表達水平關聯來觀察單核苷酸多態性是否會影響表達水平。同時，我們收集了匹配的癲癇患者和普通人的海馬和顳葉的石蠟包埋樣品，我們將通過免疫組化來測量樣品之間CAMSAP1L1這種蛋白表達水平是否不同。此外，對於基因CAMSAP1L1，我們將使用SH-SY5Y細胞進行雙螢光實驗，嘗試看到它在細胞中的位置，同時通過轉染來研究它對於神經突增長的一些功能。 / 結果：在顳葉有關SNP rs2292096（CAMSAP1L1）的RNA表達相關分析中，GG基因型患者呈現出高表達的趨勢（p=0.024）。而在相同的蛋白關聯分析中，則沒有看到這種趨勢（p=0.568）。在免疫組化試驗中，海馬部位的CASMAP1L1對照組的表達有高於癲癇組的趨勢（p=0.059）。而在顳葉，這兩組中觀察到的差異很小（p=0.12）。 在rs13021324（ERBB4）在RNA關聯表達分析中，攜帶等位基因C的患者的表達有高於T等位基因的一些趨勢, 但是不是很明顯。雙螢光試驗中，我們看到CAMSAP1L1在神經突的表達，它與β-微管蛋白有部分的表達重疊，在CAMSAP1L1轉染實驗中，近60的基因被抑制，在0μM和25μM RA 分化的SH-SY5Y細胞中，CAMSAP1L1的siRNA引起的基因沉默顯著增強了神經突以及分支的生長。 / 結論：在GWAS中發現的CAMSAP1L1基因上SNP的 顯著型p值表明，SNP和癲癇之間的遺傳性關聯可能是由於特定SNP標記的單體型的蛋白質表達水平和功能所決定的。我們的數據表明，在SNP rs2292096中，GG基因型的人倾向于有較高的表達，但需要更多的實驗來證實這一結果。CAMSAP1L1會抑制神經突生長。對於基因ERBB4，在C等位基因型中輕微高ErbB4的表達和我們的GWAS以及和以前的研究相符。 / Purpose: Symptomatic epilepsy is initiated by a brain insult, but most people suffering brain insults do not go on to develop epilepsy, indicating that some people are genetically predisposed to epilepsy after such insults. Genome wide association study (GWAS) is an effective way to find genes that contribute to diseases. The most significant SNP in a recent GWAS of symptomatic epilepsy in the Chinese population was rs2292096 [G] (p=1.0×10⁻⁸, OR=0.63), in the CAMSAP1L1 gene, also known as CAMSAP2. We chose to study this SNP as well as another SNP associated with epilepsy in the same GWAS, rs13021324 [C] (p=5.8×10⁻⁵, OR=0.73), which is in the epilepsy candidate gene ERBB4. CAMSAP1L1 encodes a cytoskeletal protein; however, it is not yet known to have any neurologically-specific function. ERBB4, a schizophrenia-susceptibility gene, has been reported to be mutated in a case of early myoclonic encephalopathy. We hypothesize that these two genes affect the predisposition of people to develop symptomatic epilepsy, and that the above SNPs influence gene expression or function. The objectives of this study are to elucidate some functions of CAMSAP1L1 and to explore whether the expression of the two genes will be affected by different genotypes. / Methods: One CAMSAP1L1 SNP (rs2292096) and one ERBB4 SNP (rs13021324) were genotyped in epilepsy patients. RNA and homogenates were prepared from frozen hippocampus and temporal lobe tissue from epilepsy patients. CAMSAP1L1 and ERBB4 RNA and protein levels were measured by real-time PCR and western blotting with reference to the housekeeping gene GAPDH, and expression levels were compared among genotypes of the above SNPs. We performed immunohistochemistry in paraffin-embedded sections to compare CAMSAP1L1 protein levels between epilepsy and control subjects in hippocampus and temporal lobe. In addition, we used human SH-SY5Y neuroblastoma cells to examine CAMSAP1L1 localization and function, performing double immunofluorescence for CAMSAP1L1 and tubulin and measuring the effect of CAMSAP1L1 knockdown on neurite outgrowth. / Results: In analysis of rs2292096 (CAMSAP1L1) in the temporal lobe, patients with the GG genotype showed higher expression of RNA (p=0.024), but not of protein (p=0.57). Immunohistochemistry showed a tendency toward lower expression of CAMSAP1L1 in epilepsy patients than in control subjects (p=0.059) in hippocampus but not in temporal lobe (p=0.12). Expression analysis of rs13021324 demonstrated non-significant tendencies toward higher expression of ErbB4 with C allele than the T allele for RNA and protein in hippocampus and temporal lobe. Double immunofluorescence showed CAMSAP1L1 expression on neurites, partially overlapping with β-tubulin. CAMSAP1L1 siRNA transfection of SH-SY5Y cells treated with or without retinoic acid reduced the CAMSAP1L1 protein level nearly 60% and stimulated neurite outgrowth, as measured by outgrowths, processes and branches compared with the control siRNA group. / Conclusion: The association of CAMSAP1L1 and ERBB4 SNPs with epilepsy is likely due to linkage disequilibrium with SNPs that affect functions or levels of CAMSAP1L1 or ErbB4. Our data suggest that the rs2292096 GG genotype, which reduces risk of symptomatic epilepsy, tends to increase expression of CAMSAP1L1, but more subjects are needed to confirm this result. CAMSAP1L1 represses neurite outgrowth. A tendency toward higher expression of ErbB4 with the C allele is consistent with the GWAS finding that the C allele decreases risk and with a report that ErbB4 levels are decreased in epilepsy. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Shuai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 77-94). / Abstracts also in Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgements --- p.V / List of figures --- p.VI / List of tables --- p.VIII / List of abbreviations --- p.IX / Table of contents --- p.XII / Chapter Chapter One --- Introduction --- p.1 / Chapter 1.1 --- Introduction to epilepsy --- p.1 / Chapter 1.1.1 --- Epilepsy types and epidemiology --- p.1 / Chapter 1.1.2 --- Drug-resistant epilepsy --- p.2 / Chapter 1.1.3 --- Treatment for epilepsy --- p.3 / Chapter 1.2 --- Genetics of epilepsy --- p.5 / Chapter 1.2.1 --- Genetic mechanism underlying epilepsy --- p.5 / Chapter 1.2.2 --- Gene expression in epilepsy --- p.8 / Chapter 1.2.3 --- Genomics in epilepsy --- p.10 / Chapter 1.2.4 --- Animal models --- p.11 / Chapter 1.3 --- Methods to identify variants affecting disease susceptibility --- p.12 / Chapter 1.3.1 --- Traditional methods to identify genetic loci for disease susceptibility --- p.12 / Chapter 1.3.2 --- Genome wide association study (GWAS) --- p.12 / Chapter 1.3.3 --- A GWAS of symptomatic epilepsy in the Chinese population --- p.14 / Chapter 1.4 --- Functions of the CAMSAP family --- p.18 / Chapter 1.5 --- Functions of ErbB4 --- p.21 / Chapter 1.6 --- Hypothesis and proposed plan for expression study of CAMSAP1L1 and ERBB4 --- p.22 / Chapter 1.6.1 --- Proposed hypothesis of CAMSAP1L1 and ERBB4 in symptomatic epilepsy --- p.22 / Chapter 1.6.2 --- Objectives of the study --- p.22 / Chapter 1.6.3 --- Significance of the study --- p.23 / Chapter 1.7 --- Study scheme --- p.23 / Chapter Chapter Two --- RNA expression analysis of SNPs rs2292096 and rs13021324 --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and methods --- p.24 / Chapter 2.2.1 --- Materials --- p.24 / Chapter 2.2.2 --- Genotyping --- p.25 / Chapter 2.2.3 --- RNA extraction --- p.26 / Chapter 2.2.4 --- Reverse transcription PCR --- p.27 / Chapter 2.2.5 --- Real-time PCR --- p.27 / Chapter 2.2.6 --- Data analysis --- p.29 / Chapter 2.3 --- Results and discussions --- p.29 / Chapter 2.3.1 --- Genotyping --- p.29 / Chapter 2.3.2 --- Real-time PCR --- p.31 / Chapter 2.4 --- Conclusion --- p.35 / Chapter Chapter Three --- Protein expression analysis of rs2292096 and rs13021324 --- p.37 / Chapter 3.1 --- Introduction --- p.37 / Chapter 3.2 --- Materials and methods --- p.37 / Chapter 3.2.1 --- Materials --- p.37 / Chapter 3.2.1 --- Protein extraction --- p.38 / Chapter 3.2.3 --- Western blot --- p.38 / Chapter 3.2.4 --- Western blot for quantification of protein samples --- p.39 / Chapter 3.3 --- Results and discussion --- p.40 / Chapter 3.3.1 --- CAMSAP1L1 band confirmation --- p.40 / Chapter 3.3.2 --- ErbB4 immunobloting --- p.43 / Chapter 3.4 --- Conclusion --- p.48 / Chapter Chapter Four --- Immunohistochemistry analysis of CAMSAP1L1 --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.2 --- Materials and methods --- p.49 / Chapter 4.2.1 --- Materials --- p.49 / Chapter 4.2.2 --- Immunohistochemistry --- p.50 / Chapter 4.2.3 --- Quantification of FFPE samples by Image Pro-Plus --- p.51 / Chapter 4.3 --- Results and discussion --- p.52 / Chapter 4.3.1 --- CAMSAP1L1 immunohistochemistry --- p.52 / Chapter 4.3.2 --- CAMSAP1L1 protein quantification between epilepsy and control groups --- p.54 / Chapter 4.4 --- Conclusions --- p.57 / Chapter Chapter Five --- Double immunofluorescence and CAMSAP1L1 siRNA transfection in SH-SY5Y cells --- p.58 / Chapter 5.1 --- Introduction --- p.58 / Chapter 5.2 --- Materials and methods --- p.59 / Chapter 5.2.1 --- Materials --- p.59 / Chapter 5.2.2 --- Double immunofluorescence --- p.60 / Chapter 5.2.3 --- CAMSAP1L1 siRNA transfection --- p.60 / Chapter 5.2.4 --- Gene knockdown assay --- p.61 / Chapter 5.2.4 --- MTT cell viability assay --- p.62 / Chapter 5.2.5 --- Neurite outgrowth assay --- p.62 / Chapter 5.3 --- Results and discussion --- p.63 / Chapter 5.3.1 --- Double immunofluorescence --- p.64 / Chapter 5.3.2 --- CAMSAP1L1 knockdown assay by western blot --- p.64 / Chapter 5.3.3 --- MTT cell viability assay --- p.66 / Chapter 5.3.4 --- Neurite outgrowth --- p.67 / Chapter 5.4 --- Conclusion --- p.72 / Chapter Chapter Six --- Overall conclusion and prospects --- p.73 / Chapter 6.1 --- Overall conclusion --- p.73 / Chapter 6.1.1 --- CAMSAP1L1 --- p.73 / Chapter 6.1.2 --- ErbB4 --- p.74 / Chapter 6.2 --- Future work --- p.74 / Chapter 6.3 --- Prospects --- p.75 / References --- p.77

讀寫障礙是最普遍的一種學習障礙（80%)，影響全球大約一成的學童。讀寫障礙患者於閱讀及書寫能力方面出現困難，而這並非因為患者本身的智力、學習動機或學習機會引致。對於引至讀寫障礙的理仍未清楚，但在西方人士的遺傳研究方面已發現多個與讀寫障礙相聯的基因位點及基因。本研究針對其中4個基因位點（DYX1 ’ DYX2 ’ DYX3 ’ DKX8)及其覆蓋的11基因測試了 131讀寫障礙的中國人家庭與讀寫障礙的關聯性。是項研究從國際人類基因組單體型圖(HapMap)中選擇標籤單核苷酸多型性(Tag-SNPs)及選擇以往報告與讀寫障礙有關的單核苷酸多型性進行測試。並在DYXZa基因（rs3743205 ’ padjusted =0.0072' OR = 0.08 ( 95% CI: 0.01 - 0.64 ))私MRPL19 (風險單体型rs2422229-rs7570229,風險單体型T-G, Padjusted=0.0020, OR = 2.345 (95% CI: 1.402 - 3.923))發現與讀寫障礙有正關聯性。單核苷酸多態性亦與閱讀的幾個特徵相關：DYX1C1 ( rs3743205 )與快速命名（Digit Rapid Naming ) ’語音記憶（Non-word repetition)，字型結構的左右逆轉（Left-Right Reversal )相關；KIAA0319 ( rs2760157 rs807507 )與語音意識（Onset Detection )相關；MRPL19 ( rs2422229-rs7570229 ) 與字型結構的部首位置(Radical Position)相關；SFPQand ZMYM4 ( rs3738697 - rs12093076 )與詞彙決策（Lexical Decision )相關。本研究是首個對於讀寫障礙的中國人族群進行的的基因研究（Lim et al. 2011) ’結果有助提供我們了解讀寫障礙在使用不同語言的族群的情況。 / 在過往歐洲的研究樣本中’位於KAA0319基因5'-上游的基因變異重複地被找出與讀寫障礙有關的閱讀特徵有關。過往研究亦指出位於KIAA0319的假定調控區上的單核苷酸多態性(SNP)顯示與KIAA0315的基因表達有關。唯本項研究於這區域並無發現陽性結果。為了找出這區域中與讀寫障礙有關聯但未被本研究策略選擇使用基因組單體型圖的標籤-單核苷酸多態性(Hapmap Tag-SNPs)的基因變異，本研究對KAM0319基因的5'-上游進行了基因组重测序。其中發現的3個短序變異（-121的rs6456625，-128到- 1 5 4 的r s 7 1 8 1 5 1 4 3 及- 1 5 7 的6 > A )出現了不同的榮光素酶報告基因活動，當中單体型A-DEL-A的活動訊號最高，而G-INS-G則最低。然而，它們的等位基因和單体型基因出現率於讀寫障礙樣本與對照組沒有顯著不同。 / 至今有關對KIAA0319基因抑制的行為研究仍然不足。本研究亦對位於果繩的CG7565基因，即KIAA0319的同源基因’進行特性分析。CG7565在果繩的發展階段出現了不同的表達水平及基因剪接形式。本研究使用了UAS-RNAi糸統和飛行模擬器對CG7565基因抑制的果繩的行為變化作出了分析，結果顯示於泛神經基因抑制晰/+; e/aV-Gal4/+; 3707/+及晰/+; e/av-Gal4/+; 8396/+的果蠅視覺模式記憶出現了缺陷。當CG7565在果繩大腦中央複合區的神經元F5(扇形體)和R2/R4m(摘球体)被基因抑制時，果繩視覺模式記憶亦出現了缺陷。是次有關M/PL119的遺傳關聯研究跟以往MRPL19中5'-上游與讀寫障礙的關聯報導的一致’顯示這可能是真正的致病序列變異的位置。在M/PL119上的假定調控區進行基因突變分析顯示，在其中一個讀寫障礙的樣本中發現一個新的序列變化(-647 T>G)，而在對照組則沒有發現此變化。計算機預測模型分析估計這個序列變化會取消了熱休克轉錄因子1的結合位點。攜帶了 G等位基因的調控區會增加榮光素酶的活動。這種變異的作用必須得到進一步的證實。我們亦觀察到在其中兩個讀寫障礙樣本中出現了非孟德爾遺傳，在一個個体身上帶有3或4種單倍型的基因。基因拷貝數目變異或基因轉換可能是一個引至這種現象的因素。 / Developmental dyslexia is a learning disability characterized by difficulties in acquisition of reading and writing skills not due to intelligence, motivation or schooling. Being the most common form of learning disability (80%), it affects 10% of schoolchildren worldwide. Research delineating genetic factors in developmental dyslexia identified loci and candidate genes in Caucasian populations, although disease mechanisms are still unknown. Four loci covering eleven genes (DYX1, DYX2, DYX3, DYXS) were tested for association in 131 Chinese families with dyslexic children in our study. Tagged-SNPs selected from International HapMap Consortium and reported SNPs were used as markers for this study. Positive associations with dyslexia were found in two genes, DYX1C1 (rs3743205, padjusted=0.0072, OR =0.08 (95% CI: 0.01 - 0.64)) and MRPL19(rs2422229-rs7570229, risk haplotype T-G,Padjusted=0.0020, OR = 2.345 (95% CI: 1.402 一 3.923)), in our study. SNPs associated with several reading-related traits were also identified: DYX1C1(rs3743205) associated with Rapid Naming (Digit Rapid Naming), Phonological Memory (Non-word repetition),Orthographic skill (Left-Right Reversal); KIAA0319 (rs2760157-rs807507) with honological awareness (Onset Detection); MRPL19 (rs2422229-rs7570229) with Orthographic knowledge (Radical Position); SFPQ and ZMYM4 (rs3738697 - rs12093076) with Orthographic knowledge (Lexical Decision). This is the first genetic study in Chinese dyslexia (Lim et al.2011), and results provide knowledge into dyslexia in populations using different languages. / Variants located 5' upstream of KIAA0319 were consistently reported for association with DD reading-related traits in European samples. A SNP in the putative promoter of KIAA0319 showed functional significance in KIAA0319 expression. However, no positive result of this region is found in this study. Resequencing of the 5' upstream of KIAA0319 was done to reveal potentially associated variants not selected using current strategies in genetic association (Hapmap Tagged-SNPs). A short sequence fragment of 3 variants (-121 rs6456625, -128 to -154 rs71815143 and -157 G>A) show differential luciferase activities, haplotype A-del-A have highest signal, G-Ins-G the lowest. However, allele and haplotype frequencies in dyslexia samples were not significantly different from controls. / Direct behavioral study of KIAA0319-knockdown is still inadequate. A homolog of KIAA0319, CG7565 in Drosophila, was characterized. Differential gene expression and splicing forms were observed during Drosophila development stages. Using UAS-RNAi system and flight simulator to study behavioral change in CG7565-knockdown Drosophila showed pan-neural knockdown lines w/+; elav-Gal4/+; 3707/+ and w/+; elav-Gal4/+; 8396/+ are defective in visual pattern memory. The study of neuronal specific knockdown showed this memory was impaired when CG7565 was selectively knocked down in F5 neuron (fan-shaped body) and R2/R4m (ellipsoid body) of the central complex in Drosophila brain. / Our genetic association study of MRPL19 agrees with reports of the association of 5' upstream of MRPL19 with DD, showing that true causative sequence variants may lie here. Mutational analyses of the putative promoter of MRPL19 revealed a novel sequence change T>G at -647 in a dyslexic sample not found in controls. In-silico analysis indicates a binding site of heat shock factor-1 that is predicted to be abolished by this variant. Luciferase activity increased in the promoter carrying the G allele. The role of this variant must be confirmed. Non-Mendelian Inheritance was observed in 2 individual dyslexic samples with 3 and 4 types of haplotypes. Copy number variation or gene conversion may be a factor. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lim, King Poo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 201-225). / Abstracts also in Chinese. / Dedication --- p.I / Abstract --- p.II / 摘要 --- p.IV / Acknowledgements --- p.V / Table of contents --- p.VI / List of Figures --- p..XI / List of Tables --- p.XIV / List of Abbreviations --- p.XVII / Chapter 1. --- Chapter 1 --- p.1 / Genetic Association of dyslexia-candidate genes in Chinese children with dyslexia --- p.1 / Chapter 1.1. --- Introduction --- p.2 / Chapter 1.2. --- Prevalence --- p.4 / Chapter 1.3. --- Definition --- p.7 / Chapter 1.4. --- Theories of developmental dyslexia --- p.9 / Chapter 1.4.1. --- Phonological deficit theory --- p.9 / Chapter 1.4.2. --- Double deficit hypothesis --- p.10 / Chapter 1.4.3. --- Cerebellar deficit theory --- p.11 / Chapter 1.4.4. --- Magnocellular deficit theory --- p.12 / Chapter 1.4.5. --- Deficits in Chinese people with dyslexia --- p.12 / Chapter 1.5. --- Neurobiological aspects of Dyslexia --- p.15 / Chapter 1.5.1. --- Postmortem studies --- p.15 / Chapter 1.5.2. --- Structural Neuroimaging studies of dyslexia --- p.16 / Chapter 1.5.3. --- Functional Neuroimaging studies --- p.17 / Chapter 1.5.4. --- fMRI results in Chinese --- p.18 / Chapter 1.6. --- Genetics of Dyslexia --- p.19 / Chapter 1.6.1. --- Familial studies --- p.19 / Chapter 1.6.2. --- Twin studies --- p.21 / Chapter 1.6.3. --- Mode of Inheritance --- p.24 / Chapter 1.6.4. --- Genetic mapping of disease gene --- p.26 / Linkage analysis --- p.26 / Association study --- p.29 / Molecular genetic findings in dyslexia --- p.33 / Chapter 1.6.5. --- Statement of Research Rationale --- p.47 / Chapter 1.6.6. --- Objectives --- p.48 / Chapter 1. --- Chapter 2 --- p.49 / Genetic association of dyslexia-candidate genes in Chinese children with dyslexia --- p.49 / Chapter 2.1. --- Introduction --- p.50 / Chapter 2.2. --- Materials and methods --- p.51 / Chapter 2.2.1. --- Subjects --- p.51 / Chapter 2.2.2. --- DNA extraction and genotyping --- p.54 / Chapter 2.2.3. --- SNP marker selection --- p.55 / Chapter 2.2.4. --- Statistical analyses --- p.57 / Chapter 2.3. --- Results --- p.59 / Chapter 2.3.1. --- DYX1C1 --- p.59 / Single marker analysis --- p.59 / Haplotype analyses --- p.62 / Chapter 2.3.2. --- KIAA0319 --- p.67 / Association of KIAA0319 with Chinese dyslexic children --- p.67 / Association of KIAA0319 with reading related traits --- p.67 / Chapter 2.3.3. --- DCDC2 --- p.74 / Association of DCDC2 with Chinese dyslexic children --- p.74 / Chapter 2.3.4. --- MRPL19 and C2orf3 --- p.78 / Haplotypes located within 5' upstream of MRPL19 are significantly associated with DD --- p.78 / Association of the 5' upstream variants with reading related traits --- p.79 / Chapter 2.3.5. --- KIAA0319L and its surrounding genes --- p.85 / Association of KIAA03190L and its surrounding genes with Chinese dyslexic children --- p.85 / Chapter 2.3.6. --- Gene-Gene interaction analyses --- p.88 / Chapter 2.3.7. --- Parent-of-origin analysis --- p.92 / Chapter 2.4. --- Discussion --- p.94 / Chapter 2.4.1. --- DYX1C1 variant associated with DD and reading skills --- p.94 / Chapter 2.4.2. --- KIAA0319 associated with phonological awareness in Chinese --- p.99 / Chapter 2.4.3. --- DCDC2 is not associated with DD in Chinese children --- p.108 / Chapter 2.4.4. --- Association of MRPL19 and C2ORF3 in a Chinese sample --- p.111 / Chapter 2.4.5. --- Association of KIAA03190L and its surrounding genes with Chinese children with dyslexia --- p.115 / Chapter 2.4.6. --- Gene-Gene interaction --- p.118 / Chapter 2.4.7. --- Parent-of-origin --- p.119 / Chapter 2.5. --- Summary --- p.122 / Chapter 3. --- Chapter 3 --- p.126 / Resequencing analyses and characterization of 5' upstream of KIAA0319 --- p.126 / Chapter 3.1. --- Introduction --- p.127 / Chapter 3.2. --- Materials and Methods --- p.129 / Chapter 3.2.1. --- DNA samples --- p.129 / Chapter 3.2.2. --- DNA re-sequencing --- p.129 / Chapter 3.2.3. --- KIAA0319 Promoter constructs --- p.131 / Chapter 3.2.4. --- Luciferase Reporter Assays --- p.133 / Chapter 3.2.5. --- In-silico sequence analyses --- p.133 / Chapter 3.3. --- Results --- p.134 / Chapter 3.4. --- Discussion --- p.143 / Chapter 4. --- Chapter 4 --- p.148 / Characterization of CG7565, a homolog of KIAA0319, in a Drosophila model --- p.148 / Chapter 4.1. --- Introduction --- p.149 / Chapter 4.2. --- Methods and Materials --- p.152 / Chapter 4.2.1. --- Drosophila stock --- p.152 / Chapter 4.2.2. --- Sequence analyses --- p.153 / Chapter 4.2.3. --- RNA extraction and quantitative reverse-transcription PCR (RT-PCR) --- p.153 / Chapter 4.2.4. --- Behavioral Assays --- p.156 / Visual pattern memory assays --- p.156 / Optomotor Response Assays --- p.158 / Visual Discrimination Analyses --- p.159 / Chapter 4.3. --- Results --- p.160 / Chapter 4.4. --- Discussion --- p.174 / Chapter 5. --- Chapter 5 --- p.181 / Mutational analyses of 5' region of MRPL19 in children with dyslexia --- p.181 / Chapter 5.1. --- Introduction --- p.182 / Chapter 5.2. --- Materials and Methods --- p.183 / Chapter 5.2.1. --- DNA samples --- p.183 / Chapter 5.2.2. --- High resolution melting analyses (HRM) --- p.183 / Chapter 5.2.3. --- MRPL19 promoter constructs --- p.185 / Chapter 5.2.4. --- In-silico sequence analyses --- p.186

by Zeng Naiyan. / "December 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 185-213) / 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.

Dexamethasone (DEX) and triamcinolone acetonide (TA) are widely used in clinical practice for ocular anti-inflammation. The most common side effect of these two corticosteroids is the rise of intraocular pressure that leads to death of the retinal ganglion cells, a feature of POAG. We investigated the differential gene expression profiles induced by DEX and TA treatment in human trabecular meshwork (hTM) cells using microarray technology. A number of genes differentially expressed in hTM cells were identified under DEX and TA treatment, mainly involving in proteolysis, cell adhesion and acute phase response. Five genes (MYOC, GAS1, SENP1, ZNF343 and SOX30) were commonly differentially expressed in both DEX and TA treatment. It indicates that DEX and TA may share similar effect on hTM cells, which may associate with the onset of ocular hypertension. / In one Chinese juvenile onset POAG (JOAG) family with autosomal dominant inheritance, a novel locus at 15822-q24 (GLC1N) was identified using genome-wide scan, supported by clinical, linkage, and haplotype transmission data. The critical region covered a genetic distance of 16.6 Mb. To search for disease genes within this new JOAG locus, we screened NR2E3, SMAD6 and CLN6 for mutations. However, no mutations was found in the family members. We attempted a new gene-based SNPs genotyping approach to search for susceptibility genes to JOAG in this novel locus by using 97 unrelated JOAG patients and 99 unrelated control subjects. Significant association was identified in a set of 6 adjacent SNPs out of 122 gene-based SNPs. Among them, one non-synonymous SNP rs3743171 in the SLC24A1 gene was incompletely segregated in the JOAG family. Our findings indicate the mutation in other regions of SLC24A1 may be responsible for JOAG in this family, or another gene in this region may be the actual cause of glaucoma. / Primary open angle glaucoma (POAG) is a leading cause of visual impairment and blindness worldwide. Genetic factors play a major role in the etiology of POAG. This thesis describes our investigations of the POAG causative genes using genome-wide DNA scanning by linkage/association analysis and RNA level scanning by microarray technology. / Wang Danyi. / "September 2006." / Adviser: Calvin Chi Pui Pang. / Source: Dissertation Abstracts International, Volume: 68-08, Section: B, page: 5155. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 139-181). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Tsang, Wan Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 156-165). / Abstracts in English and Chinese. / Abstract --- p.iii / Abstract in Chinese (摘要） --- p.V / Acknowledgement --- p.vi / Table of Content --- p.vii / List of Figures and Tables --- p.xiv / List of Abbreviations --- p.xvii / Chapter Chapter 1 --- Introduction --- p.Page / Chapter 1.1 --- Developmental Dyslexia --- p.1 / Chapter 1.1.1 --- Study of Developmental Dyslexia --- p.1 / Chapter 1.1.2 --- Genetic Basis of Developmental Dyslexia --- p.2 / Chapter 1.1.3 --- Susceptibility Gene for Developmental Dyslexia --- p.3 / Chapter 1.2 --- Dyslexia-Susceptibility Gene KIAA0319-Like --- p.5 / Chapter 1.2.1 --- Association Studies of KIAA0319-Like --- p.5 / Chapter 1.2.2 --- KIAA0319-Like Gene and Protein --- p.6 / Chapter 1.2.3 --- Functional prediction of KIAA0319-Like Protein --- p.10 / Chapter 1.3 --- Potential Interacting Partners of KIAA0319-Like Protein --- p.12 / Chapter 1.3.1 --- Nogo Receptor 1 (NgR) --- p.12 / Chapter 1.3.2 --- SH2B Adaptor Protein 1 (SH2) --- p.13 / Chapter 1.3.3 --- FEM-1-like death receptor binding protein (FEM) --- p.14 / Chapter 1.3.4 --- Adaptor-related protein complex 2，mu 1 subunit (AP2) --- p.14 / Chapter 1.4 --- Notch Signaling Pathway and KIAA0319-Like --- p.16 / Chapter 1.5 --- Previous Research on KIAA0319-Like --- p.18 / Chapter 1.6 --- Research Objectives --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.Page / Chapter 2.1 --- Gene Cloning --- p.21 / Chapter 2.1.1 --- Cloning of Human KIAA0319-Like into expression vectors --- p.21 / Chapter 2.1.2 --- Gel Extraction of PCR Product --- p.22 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.22 / Chapter 2.1.4 --- Ligation of gene products with vector --- p.23 / Chapter 2.1.5 --- "Cloning of Human SH2, FEM, AP2 and NgR gene" --- p.23 / Chapter 2.1.7 --- Preparation of chemically competent E.coli strain DH5a --- p.26 / Chapter 2.1.8 --- Transformation of the ligation product into competent cells --- p.26 / Chapter 2.1.9 --- Diagnostic PCR for confirmation of successful ligation --- p.27 / Chapter 2.1.10 --- Small scale preparation of bacterial plasmid DNA --- p.27 / Chapter 2.1.11 --- DNA sequencing of the cloned plasmid DNA --- p.28 / Chapter 2.1.12 --- Large scale preparation of target recombinant plasmid DNA --- p.28 / Chapter 2.2 --- Cell Culture --- p.30 / Chapter 2.2.1 --- Culture medium --- p.30 / Chapter 2.2.2 --- Cell lines --- p.30 / Chapter 2.2.3 --- Freezing and thawing cells --- p.31 / Chapter 2.3 --- DNATransfection --- p.32 / Chapter 2.3.1 --- Transfection of HEK293 Cells with LipofectaminéёØ Reagent --- p.32 / Chapter 2.3.2 --- Transfection of Neuronal Cells with Tranfas´tёØ Reagent --- p.32 / Chapter 2.4 --- Immunocytochemical Staining --- p.33 / Chapter 2.5 --- Immunohistochemical Staining --- p.34 / Chapter 2.6 --- Western Blotting --- p.36 / Chapter 2.6.1 --- Collection of Cell Lysate --- p.36 / Chapter 2.6.2 --- Collection of Mouse Tissue Lysate --- p.36 / Chapter 2.6.3 --- Protein concentration determination by Bradford Protein Assay --- p.36 / Chapter 2.6.4 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.37 / Chapter 2.6.5 --- Semidry Transfer to PVDF Membrane --- p.37 / Chapter 2.6.6 --- Blocking and Antibody Probing --- p.38 / Chapter 2.6.7 --- Enhanced chemiluminescence (ECL) --- p.38 / Chapter 2.6.8 --- Synthetic peptide based neutralization --- p.39 / Chapter 2.7 --- Quantitative Real-Time PCR --- p.40 / Chapter 2.7.1 --- RNA extraction from Cell culture --- p.40 / Chapter 2.7.2 --- Genomic DNA Elimination and Reverse Transcription --- p.40 / Chapter 2.7.3 --- Quantitative Real-Time PCR --- p.41 / Chapter 2.8 --- Coimmunoprecipitation --- p.42 / Chapter 2.9 --- Luciferase Reporter Assay --- p.43 / Chapter 2.10 --- Wound-Healing Assay --- p.44 / Chapter Chapter 3 --- Distribution of KIAA0319-Like Protein --- p.Page / Chapter 3.1 --- Study of the Distribution of KIAA0319-Like Protein --- p.45 / Chapter 3.1.1 --- Anti-KIAA0319-Like Antisera H635 --- p.45 / Chapter 3.1.2 --- Affinity Purification of Anti-KIAA0319-Like Antisera H635 --- p.45 / Chapter 3.2 --- C57/BL6 Mouse Tissue Expression Pattern of KIAA0319-Like --- p.48 / Chapter 3.3 --- C57/BL6 Mouse Brain Regional Expression Pattern of KIAA0319-Like --- p.50 / Chapter 3.4 --- Immunostaining of Sagittal Section of C57/BL6 Mouse Brain --- p.52 / Chapter 3.5 --- Mouse Brain Co-staining with KIAA0319-Like Protein and Markers --- p.54 / Chapter 3.6 --- Subcelluar Localization of KIAA0319-Like Protein in Human Neurons --- p.57 / Chapter 3.7 --- Discussion --- p.59 / Chapter 3.7.1 --- Affinity Purification of Anti-KIAA0319-Like Antisera --- p.59 / Chapter 3.7.2 --- Mouse Tissue Expression Pattern of KIAA0319-Like --- p.59 / Chapter 3.7.3 --- Mouse Brain Regional Expression Pattern of KIAA0319-Like --- p.59 / Chapter 3.7.4 --- Brain Cell Types expressing KIAA0319-Like Protein --- p.61 / Chapter 3.7.5 --- Subcellular Localization of KIAA0319-Like Protein --- p.62 / Chapter 3.7.6 --- Future Perspectives --- p.63 / Chapter Chapter 4 --- KIAA0319-Like Modulates Neuronal Migration --- p.Page / Chapter 4.1 --- Study of the effect of KIAA0319-Like on Neuronal Migration --- p.64 / Chapter 4.2 --- Establish Stable Cell Lines with Altered KIAA0319-Like Expression --- p.65 / Chapter 4.2.1 --- Cloning of siRNA into Lentiviral Vector pLVTHM --- p.65 / Chapter 4.2.2 --- Lentivirus packaging in HEK293 Cells --- p.67 / Chapter 4.2.3 --- Checking of Lentivirus by HEK293 Cells Transduction --- p.70 j / Chapter 4.2.4 --- Lentiviral Transductions to Neuronal Cells --- p.70 / Chapter 4.2.5 --- Fluorescent-Activated Cell Sorting of Transduced Cells --- p.70 / Chapter 4.2.6 --- KIAA0319-Like Expression Changes in Stable Cell Lines --- p.71 / Chapter 4.3 --- Effects of KIAA0319-Like Overexpression on Neuronal Migration --- p.75 / Chapter 4.4 --- Effects of KIAA0319-Like Knockdown on Neuronal Migration --- p.77 / Chapter 4.5 --- Effect of Anti-KIAA0319-Like Antibody on Neuronal Migration --- p.79 / Chapter 4.6 --- Discussion --- p.81 / Chapter 4.6.1 --- Stable Cell line with altered KIAA0319-Like Level --- p.81 / Chapter 4.6.2 --- Disruption of KIAA0319-Like expression affects Cell Migration --- p.81 / Chapter 4.6.3 --- Antibody Blockade of KIAA0319-Like Inhibits Cell Migration --- p.81 / Chapter 4.6.4 --- Possible existence of Multiple Regulatory Pathways --- p.82 / Chapter 4.6.5 --- Possible Limitations of Expression alternations by Transduction --- p.83 / Chapter 4.6.6 --- Possible Susceptibility Polymorphism of KIAA0319-Like --- p.83 / Chapter 4.6.7 --- Future Perspectives X --- p.84 / Chapter Chapter 5 --- Interacting Partners of KIAA0319-Like Protein --- p.Page / Chapter 5.1 --- Study of Interacting Partners of KIAA0319-Like Protein --- p.85 / Chapter 5.1.1 --- Identification of Potential Interacting Partners of KIAA0319-Like --- p.85 / Chapter 5.1.2 --- Identification of Domains involved in Interactions --- p.86 / Chapter 5.1.3 --- Subcloning of KIAA0319-Like Protein Domain Deletion Mutant --- p.86 / Chapter 5.2 --- Interaction between NgR and KIAA0319-Like Protein --- p.92 / Chapter 5.2.1 --- Reciprocal Coimmunoprecipitation of NgR for KIAA0319-Like --- p.92 / Chapter 5.2.2 --- Colocalization of NgR and KIAA0319-Like Protein --- p.94 / Chapter 5.2.3 --- Interaction between NgR and KIAA0319-Like Deletion Mutants --- p.97 / Chapter 5.3 --- Interaction between SH2 and KIAA0319-Like Protein --- p.99 / Chapter 5.3.1 --- Coimmunoprecipitation between SH2 and KIAA0319-Like --- p.» 99 / Chapter 5.3.2 --- Colocalization of SH2 and KIAA0319-Like Protein --- p.101 / Chapter 5.3.3 --- Interaction between SH2 and KIAA0319-Like Deletion Mutants --- p.103 / Chapter 5.4 --- Interaction between FEM and KIAA0319-Like Protein --- p.105 / Chapter 5.4.1 --- Coimmunoprecipitation between FEM and KIAA0319-Like --- p.105 / Chapter 5.4.2 --- Colocalization of FEM and KIAA0319-Like Protein --- p.107 / Chapter 5.4.3 --- Interaction between FEM and KIAA0319-Like Deletion Mutants --- p.109 / Chapter 5.5 --- Interaction between AP2 and KIAA0319-Like Protein --- p.111 / Chapter 5.5.1 --- Coimmunoprecipitation between AP2 and KIAA0319-Like --- p.111 / Chapter 5.5.2 --- Colocalization of AP2 and KIAA0319-Like Protein --- p.113 / Chapter 5.5.3 --- Interaction between AP2 and KIAA0319-Like Deletion Mutants --- p.115 / Chapter 5.6 --- Discussion --- p.117 / Chapter 5.6.1 --- The Interaction of KIAA0319-Like with NgR and SH2 Protein --- p.118 / Chapter 5.6.2 --- Interaction between FEM and KIAA0319-Like Protein --- p.121 / Chapter 5.6.3 --- Interaction between AP2 and KIAA0319-Like Protein --- p.121 / Chapter 5.6.4 --- Future Perspectives --- p.122 / Chapter Chapter 6 --- KIAA0319-Like Association with Notch Pathway --- p.Page / Chapter 6.1 --- Study of KIAA0319-Like association with Notch Pathway --- p.123 / Chapter 6.1.1 --- Luciferase Reporter System for quantifying y-Secretase Activity --- p.123 / Chapter 6.2 --- Effects of KIAA0319-Like Expression Alternations on y-Secretase Activity in HEK293 Cells --- p.126 / Chapter 6.3 --- Effects of Increasing KIAA0319-Like Protein Level in Culture Medium on y-Secretase Activity in HEK293 Cells --- p.128 / Chapter 6.4 --- Effects of KIAA0319-Like Expression Alternations on y-Secretase Activity in SH-SY5Y Cells --- p.130 i / Chapter 6.5 --- Effects of KIAA0319-Like Expression Alternations on y-Secretase Activity in Neuro-2a Cells --- p.132 / Chapter 6.6 --- Effect of Notch Blockade on SH-SY5Y Cells Migration Rate --- p.134 / Chapter 6.7 --- Effect of KIAA0319-Like Expression changes on Notch Inhibited SH-SY5Y Cells Migration Rate --- p.136 / Chapter 6.8 --- Discussion --- p.139 / Chapter 6.8.1 --- Luciferase Reporter System for quantifying y-Secretase Activity --- p.139 / Chapter 6.8.2 --- Cell-Type Specific association of KIAA0319-Like expression level and y-Secretase activity --- p.139 / Chapter 6.8.3 --- Entry Point of KIAA0319-Like to the Notch Signaling Pathway --- p.140 / Chapter 6.8.4 --- Functional relationship between KIAA0319-Like and Notch --- p.141 / Chapter 6.8.5 --- Future Perspectives --- p.142 / Chapter Chapter 7 --- Discussions and Conclusions --- p.Page / Chapter 7.1 --- Discussions --- p.143 / Chapter 7.1.1 --- Further Evidence for KIAA0319-Like Association with Dyslexia --- p.143 / Chapter 7.1.2 --- Possible Pathways connecting KIAA0319-Like with Cell Motility --- p.145 / Chapter 7.1.3 --- Nogo Receptor Pathway association with Cell Motility --- p.146 / Chapter 7.1.4 --- Notch Signaling Pathway association with Cell Motility --- p.146 / Chapter 7.1.5 --- Combined Effects of NgR and Notch Pathway on Cell Migration --- p.149 / Chapter 7.1.6 --- Cellular Compartment for Interactions of KIAA0319-Like --- p.151 / Chapter 7.2 --- Conclusions --- p.153 / Chapter Chapter 8 --- References --- p.156 / Chapter Chapter 9 --- Appendix --- p.166

抑制腫瘤的基因（又稱抑癌基因）之表達失活，是導致癌變的重要機制之一。除了基因突變之外，越來越多研究證明抑癌基因的關閉轉錄，是由於抑癌基因啟動子區的CpG島甲基化所致。本論文的研究確定了兩個候選抑癌基因ADAMTS8和TUSC14，在多種腫瘤細胞株中經常因動子區的CpG島甲基化而下調或停止表達，這有別於它們在正常組織中廣泛表達的情況。沉默細胞株在脫氧核糖核酸甲基轉移酶的抑製劑5-氮-2'-脫氧胞苷(5-aza-2′-deoxycytidine; Aza) 或與組蛋白去乙酰化酶抑製劑曲古抑菌素A (trichostatin A, TSA)的去甲基化作用下，能恢復這兩個抑癌基因的表達，因而證明了啟動子甲基化是直接導致其表達下調及沉默的機制。 / 論文的第一部分，主要調查ADAMTS8啟動子區在原發腫瘤樣本被甲基化的比率，並研究其腫瘤抑制功能。含血小板凝血酶敏感蛋白基序的解整聯蛋白金屬蛋白酶 (ADAMTSs) ，在各種癌症中的表達異常已有報導。然而，它們在腫瘤的職能作用仍然模糊不清。本研究發現，異位表達ADAMTS8誘導細胞凋亡，因而顯著抑制腫瘤細胞克隆形成的能力,。這些都突顯其抑制腫瘤的功能。此外，作為分泌蛋白酶的ADAMTS8，能夠透過減少表皮生長因子受體(EGFR) 蛋白的磷酸化，抑制EGFR / MEK / ERK信號通路，並進一步破壞肌動蛋白應力纖維的組織，抑制腫瘤細胞的遷移性。 / 論文的第二部分，集中於研究一個未知功能的基因TUSC14，這基因的蛋白質編碼具有氨基末端蛋白質相互作用域 (BTB/POZ domain)及C₂H₂乙炔鋅指結構。TUSC14的異位表達能抑制腫瘤細胞克隆的形成，但這種抑制作用會在删除蛋白中的BTB/POZ或C₂H₂乙炔鋅指結構功能域後消失。因此證實了TUSC14蛋白同時需要BTB / POZ和C₂H₂乙炔鋅指結構兩個功能域來抑制腫瘤生長。此外，TUSC14具有抑制NF-kB轉錄的功能，其功能不但依賴於组蛋白去乙酰基酶(HDAC)，並且與c-MYC和cIAP-2等NF-κB靶基因下調表達相關。TUSC14的抑癌功能，包括抑制腫瘤生長與增加細胞凋亡，與其減少c-MYC及抗凋亡基因cIAP-2的表達，效果一致。進一步的分析發現，TUSC14與HDAC1和P65於蛋白質複雜免疫共沉澱實驗中，有物理相互作用。此外，染色質免疫沉澱實驗顯示TUSC14透過與c-MYC和cIAP-2的相互作用，抑制其基因啟動子區的轉錄功能。結果表明，TUSC14是通過招募HDAC至NF-κB靶基因的啟動子區這機制，來抑制NF-kB靶基因的轉錄，以達至抑制癌細胞生長和誘導癌細胞凋亡的效果。因此，TUSC14的沉默是破壞癌細胞中NF-kB信號通路負調控(negative regulation)的重要因素。 / 綜上所述，本研究鑒定了兩個在多種腫瘤細胞因表觀遺傳沉默效應而表達下調或沉默的抑癌基因ADAMTS8和TUSC14，並證實它們具有抑癌功能。 / Inactivation of tumor suppressor genes (TSGs) is one of the critical mechanisms leading to carcinogenesis. Apart from genetic mutations, a growing number of TSG has been shown to be silenced through promoter CpG methylation. In this thesis, we identified two candidate TSGs: ADAMTS8 and TUSC14 that are frequently downregulated or silenced in multiple carcinoma cell lines by promoter methylation while broadly expressed in normal tissues. Expression of these two genes was restored after treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (Aza) or in combination with a histone deacetylase inhibitor trichostatin A (TSA), suggesting promoter-methylation directly contributes to their silencing. / In the first part of the thesis, prevalence silencing of ADAMTS8 was detected in primary tumor samples. Expression of many disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) was reported to be dysregulated in various cancers. However, their functional roles in tumorigenesis remain obscure. This study revealed that ectopic expression of ADAMTS8 markedly inhibits tumor cell clonogenicity by inducing apoptosis, underscoring its function as a tumor suppressor. Furthermore, ADAMTS8, as a secreted protease, inhibits EGFR/MEK/ERK signaling pathway by reducing their phosphorylation, further resulting in the disruption of actin stress fiber organization and suppression of tumor cell motility. / The second part of the thesis focused on a novel gene TUSC14 which encodes a protein with BTB/POZ domain and C₂H₂zinc-fingers. Ectopic expression of TUSC14 suppresses colony formation of cancer cells but this inhibitory effect is abolished with the deletion of BTB/POZ domain or C₂H₂ zinc-fingers. This suggested that both BTB/POZ domain and C₂H₂ zinc-fingers are required for inhibiting tumor cell clonogenecity. In addition, TUSC14 functions as a transcriptional repressor of NF-kB pathway that is dependent on HDAC. Suppression of NF-κB transcriptional activity by TUSC14 expression correlates with the downregulation of NF-κB target genes including c-MYC and cIAP-2. Reduction of c-MYC and anti-apoptotic cIAP-2 agrees well with the consequent growth suppression and enhanced apoptosis following the ectopic expression of TUSC14. Further analyses showed TUSC14 physically interacts with HDAC1 and p65 via co-immunoprecipitation assay. Preliminary ChIP assay showed that TUSC14 associates with gene promoters of c-MYC and cIAP-2 for their transcription repressions. These results revealed that TUSC14 represses NF-kB activity through recruiting HDAC to the NF-kB target genes; and transcription repression of NF-kB represents a mechanism for TUSC14 to mediate its growth inhibitory and apoptosis-inducing effects in cancer. Hence, silencing of TUSC14 contributes to the lost of negative regulation on NF-kB signaling in cancer. / In summary, this study demonstrated that ADAMTS8 and TUSC14 are functional tumor suppressors that are epigenetically silenced in multiple tumors. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Choi, Ching Gee. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 140-153). / Abstract also in Chinese. / Abstract --- p.i / Chinese abstract --- p.iv / AcknowledgEments --- p.vii / List of Figures --- p.ix / List of Tables --- p.xi / LIST OF ABBREVIATIONS --- p.xii / List of PUBLICATIONs --- p.xiv / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- Overview of cancer epigenetics --- p.1 / Chapter 1.2 --- Epigenetic events --- p.2 / Chapter 1.2.1 --- DNA methylation --- p.2 / Chapter 1.2.2 --- Histone modifications --- p.5 / Chapter 1.2.3 --- The interdependence of DNA methylation and histone modifications --- p.8 / Chapter 1.3 --- Epigenetic alterations in cancer --- p.9 / Chapter 1.3.1 --- Genome-wide DNA hypomethylation --- p.9 / Chapter 1.3.2 --- CpG island promoter hypermethylation silencing of tumour suppressor genes in tumorigenesis --- p.10 / Chapter 1.3.3 --- Aberrations of histone modifications --- p.11 / Chapter 1.4 --- Causes for epigenetic deregulation in cancer --- p.14 / Chapter 1.5 --- The interplay of genetic and epigenetic aberration in cancer progression --- p.21 / Chapter 1.6 --- Epigenetic inactivation of tumor suppressor genes in cancer --- p.23 / Chapter 1.7 --- Clinical implications of epigenetic research --- p.27 / Chapter 1.7.1 --- Epigenetic modifications as biomarker for cancer diagnosis --- p.27 / Chapter 1.7.2 --- Targeting epigenetic modifications as therapeutics towards cancers --- p.29 / Chapter 1.8 --- Roles of ADAMTS proteins in cancer --- p.32 / Chapter 1.8.1 --- Introduction on ADAMTS metalloproteases --- p.32 / Chapter 1.8.2 --- Deregulation of ADAMTS protein in cancer --- p.34 / Chapter 1.9 --- Roles of BTB/POZ-ZF family of transcription factors in cancer --- p.36 / Chapter 1.9.1 --- Introduction on BTB/POZ-ZF Family --- p.36 / Chapter 1.9.2 --- BTB/POZ-ZF functions as transcription repressors --- p.37 / Chapter 1.9.3 --- Many BTB/POZ-ZF proteins are important players in tumorigenesis --- p.39 / Chapter 1.9.4 --- The role of BTB/POZ-ZF in tumor initiation and progression --- p.40 / Chapter CHAPTER 2 --- Aims of this study --- p.44 / Chapter CHAPTER 3 --- General Methodology --- p.46 / Chapter 3.1 --- Cell Culture --- p.46 / Chapter 3.1.1 --- Growth and maintenance of cells --- p.46 / Chapter 3.1.2 --- Mammalian cell transfection --- p.46 / Chapter 3.1.3 --- Drug and stress treatments --- p.47 / Chapter 3.2 --- DNA and RNA extraction --- p.47 / Chapter 3.3 --- Semi-quantitative RT-PCR and Real-time PCR --- p.48 / Chapter 3.4 --- CpG island and Transcription factor binding sites analysis --- p.49 / Chapter 3.5 --- Methylation-specific PCR (MSP) and Bisulfite genomic sequencing (BGS) --- p.49 / Chapter 3.6 --- Bacterial transformation and Plasmid extraction --- p.50 / Chapter 3.6.1 --- Heat-shock transformation --- p.50 / Chapter 3.6.2 --- Mini-scale preparation of plasmid DNA --- p.51 / Chapter 3.6.3 --- Preparation of endotoxin-free plasmids --- p.52 / Chapter 3.7 --- DNA cycle sequencing --- p.52 / Chapter 3.8 --- Indirect immunofluorescence for subcellular localization study --- p.54 / Chapter 3.9 --- Colony formation assay --- p.54 / Chapter 3.10 --- Cell cycle analysis --- p.55 / Chapter 3.11 --- Apoptosis assay --- p.56 / Chapter 3.12 --- Co-immunoprecipitation and Western blot --- p.56 / Chapter 3.13 --- Chromatin immunoprecipitation (ChIP) --- p.58 / Chapter 3.14 --- Dual Firefly and Renilla luciferase reporter gene assay --- p.59 / Chapter 3.15 --- Statistical analysis --- p.60 / Chapter CHAPTER 4: --- Characterization of the Tumor Suppressive Functions of ADAMTS8 --- p.61 / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Materials and Methods --- p.64 / Chapter 4.2.1 --- Tumor samples --- p.64 / Chapter 4.2.2 --- Expression of ADAMTS8 --- p.64 / Chapter 4.2.3 --- Immunofluorescence staining of ADAMTS8 --- p.64 / Chapter 4.2.4 --- Detection of secreted ADAMTS8 in culture medium --- p.65 / Chapter 4.2.5 --- Collection of conditioned medium and Western Blotting --- p.66 / Chapter 4.2.6 --- Wound healing assay --- p.66 / Chapter 4.3 --- Result and Discussion --- p.69 / Chapter 4.3.1 --- Frequent ADAMTS8 methylation in primary carcinomas --- p.69 / Chapter 4.3.2 --- ADAMTS8 is a secreted protease --- p.70 / Chapter 4.3.3 --- ADAMTS8 inhibits phosphorylation of pEGFR --- p.73 / Chapter 4.3.4 --- ADAMTS8 suppresses cell migration --- p.77 / Chapter 4.4 --- Summary --- p.81 / Chapter CHAPTER 5: --- Epigenetic Alterations of TUSC14 Gene in multiple carcinomas --- p.83 / Chapter 5.1 --- Introduction --- p.83 / Chapter 5.2 --- Materials and Methods --- p.84 / Chapter 5.2.1 --- Cell lines --- p.84 / Chapter 5.2.2 --- Normal and primary tumor tissues --- p.85 / Chapter 5.3 --- Results and Discussion --- p.86 / Chapter 5.3.1 --- Expression profiling of TUSC14 in normal tissues and tumor cell lines --- p.86 / Chapter 5.3.2 --- Frequent inactivation of TUSC14 by promoter CpG methylation --- p.90 / Chapter 5.3.3 --- Pharmacologic and genetic demethylation restores TUSC14 expression --- p.94 / Chapter 5.3.4 --- Frequent TUSC14 methylation in primary tumors --- p.96 / Chapter 5.4 --- Summary --- p.98 / Chapter CHAPTER 6 --- Characterization of the Tumor Suppressive Functions of TUSC14 --- p.99 / Chapter 6.1 --- Introduction --- p.91 / Chapter 6.2 --- Materials and Methods --- p.100 / Chapter 6.2.1 --- Gene cloning and plasmids construction of TUSC14 --- p.100 / Chapter 6.2.2 --- Drug and stress treatments of cells --- p.100 / Chapter 6.3 --- Results and Discussion --- p.102 / Chapter 6.3.1 --- TUSC14 localizes to nuclear speckles --- p.102 / Chapter 6.3.2 --- TUSC14 inhibits clonogenicity --- p.107 / Chapter 6.3.3 --- Expression of TUSC14 induces apoptosis in tumor cells --- p.110 / Chapter 6.3.4 --- TUSC14 alters cell cycle progression --- p.112 / Chapter 6.3.5 --- TUSC14 acts as a transcriptional repressor of multiple genes --- p.114 / Chapter 6.3.6 --- TUSC14 represses NF-кB activity through an HDAC-dependent mechanism --- p.118 / Chapter 6.3.7 --- The effect of TUSC14 on the expression of downstream targets of NF-κB Signaling --- p.120 / Chapter 6.3.8 --- TUSC14 co-immunoprecipitates with HDAC1 and p65 --- p.124 / Chapter 6.3.9 --- ChIP analysis of promoters of TUSC14-regulated genes --- p.127 / Chapter 6.4 --- Summary --- p.130 / Chapter CHAPTER 7 --- General Discussion --- p.133 / Chapter CHAPTER 8 --- Conclusions --- p.138 / References --- p.140

Ng, Hin Yi Winnie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 137-140). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / List of Tables --- p.iii / List of Figures --- p.iv / Abstract in English --- p.vi / Abstract in Chinese --- p.ix / Table of Contents --- p.xi / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Medulloblastoma (MB) --- p.1 / Chapter 1.1.1 --- Definition of Medulloblastoma --- p.1 / Chapter 1.1.2 --- Pathological Classification --- p.2 / Chapter 1.1.3 --- Current Treatment --- p.3 / Chapter 1.1.4 --- Molecular Pathology --- p.4 / Chapter 1.1.5 --- Molecular Classification of MB --- p.7 / Chapter 1.2 --- MicroRNAs (miRNAs) --- p.9 / Chapter 1.2.1 --- Biogenesis --- p.9 / Chapter 1.2.2 --- Functions --- p.10 / Chapter 1.2.3 --- MicroRNAs & Cancers --- p.10 / Chapter 1.2.4 --- Aberrant Expressions of MicroRNAs in Medulloblastoma --- p.12 / Chapter 1.2.5 --- MiR-106b-25 Cluster in MB --- p.13 / Chapter 1.2.6 --- miR-106b-25 Cluster in Regulating Target Genes --- p.15 / Chapter 1.2.7 --- Application of Regulatory miRNAs --- p.16 / Chapter 1.3 --- Target Gene Identification --- p.18 / Chapter 1.3.1 --- Recent Molecular Advances in Target Gene Identification --- p.18 / Chapter 1.3.2 --- Importance of Target Gene Identification --- p.19 / Chapter CHAPTER 2: --- AIMS OF STUDY --- p.21 / Chapter CHAPTER 3: --- COMPUTATIONAL TARGET PREDICTION --- p.23 / Chapter 3.1 --- Introduction- Computational Approach --- p.23 / Chapter 3.2 --- Methods --- p.27 / Chapter 3.2.1 --- Prediction Algorithms --- p.27 / Chapter 3.2.1.1 --- EIMMo2 --- p.27 / Chapter 3.2.1.2 --- miRDB --- p.27 / Chapter 3.2.1.3 --- miR-Tar-miRanda --- p.28 / Chapter 3.2.1.4 --- miR-Tar-RNAhybrid --- p.28 / Chapter 3.2.1.5 --- Diana-microT --- p.29 / Chapter 3.2.1.6 --- Pic-Tar --- p.29 / Chapter 3.2.1.7 --- TargetScan 4.2 --- p.29 / Chapter 3.2.2 --- Cell Culture --- p.30 / Chapter 3.2.2.1 --- Cell Lines --- p.30 / Chapter 3.2.2.2 --- Cell Counts --- p.31 / Chapter 3.2.3 --- Transfections --- p.31 / Chapter 3.2.3.1 --- Transfection of MicroRNA Inhibitors --- p.31 / Chapter 3.2.3.1.1 --- Transfection Efficiency of Lipofectamine2000 --- p.32 / Chapter 3.2.3.1.2 --- Transfection of MicroRNA Inhibitors for Real-time PCR --- p.32 / Chapter 3.2.3.1.3 --- Transfection of MicroRNA Inhibitors for Western Blotting --- p.33 / Chapter 3.2.3.2 --- Co-transfection of Plasmid and MicroRNA Inhibitors --- p.33 / Chapter 3.2.3.2.1 --- Blocking Efficiency of MicroRNA Inhibitors --- p.33 / Chapter 3.2.3.2.2 --- Co-transfection of Target Gene Expression Vector and MicroRNA Inhibitors --- p.34 / Chapter 3.2.4 --- Real-time PCR Amplification --- p.35 / Chapter 3.2.4.1 --- Total RNA Extraction from Cell Lines --- p.35 / Chapter 3.2.4.2 --- Stemloop miRNA Taqman qRT-PCR Analysis --- p.36 / Chapter 3.2.4.3 --- Reverse Transcription --- p.37 / Chapter 3.2.4.4 --- Real-time PCR Target Gene Expression --- p.38 / Chapter 3.2.5 --- Cloning of Potential Target Genes into pMIR Luciferase Expression Vector --- p.39 / Chapter 3.2.5.1 --- High-Fidelity PCR Amplification of yUTRs --- p.41 / Chapter 3.2.5.2 --- PCR Purification of Amplified PCR Product --- p.42 / Chapter 3.2.5.3 --- Restriction Enzyme Digestions --- p.42 / Chapter 3.2.5.4 --- Ligation of 3'UTR to Expression Vector --- p.43 / Chapter 3.2.5.5 --- Transformation --- p.43 / Chapter 3.2.5.6 --- Preparation of the Cloned Plasmid --- p.43 / Chapter 3.2.5.7 --- Sequencing of the Cloned Plasmid --- p.44 / Chapter 3.2.6 --- Site-directed Mutagenesis --- p.45 / Chapter 3.2.7 --- Dual-Luciferase Assay --- p.47 / Chapter 3.2.8 --- Western Blot Analysis --- p.47 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- Expression Levels of miR-106b-25 Cluster in MB Cell Lines --- p.49 / Chapter 3.3.2 --- Evaluation of Transfection Efficiency Using Lipofetamine2000 --- p.51 / Chapter 3.3.3 --- Blocking Efficiency of MicroRNA Inhibitors --- p.52 / Chapter 3.3.4 --- Target Prediction List --- p.53 / Chapter 3.3.5 --- Recognition Sites of Potential Targets --- p.55 / Chapter 3.3.6 --- Expression Levels of ZNFX1 in MB Cell Lines --- p.56 / Chapter 3.3.7 --- Transcriptional Regulation of ZNFXl and DNAJB12 --- p.57 / Chapter 3.3.8 --- Verification of Potential Target Genes --- p.59 / Chapter 3.3.9 --- Identification of Critical Target Sites --- p.61 / Chapter 3.3.10 --- Effects of Anti-microRNA Inhibitors on ZNFX1 Protein Levels --- p.66 / Chapter 3.4 --- Discussion --- p.67 / Chapter CHAPTER 4: --- EXPERIMENTAL APPROACH IN INDENTIFYING POTENTIAL TARGETS --- p.77 / Chapter 4.1 --- Introduction- Experimental Approach --- p.74 / Chapter 4.2 --- Methods --- p.79 / Chapter 4.2.1 --- Isolation of cDNA Clone Library --- p.79 / Chapter 4.2.1.1 --- Preparation of Cytoplasmic Extracts --- p.79 / Chapter 4.2.1.2 --- Reverse Transcription Using Endogenous miRNA as Primers --- p.81 / Chapter 4.2.1.3 --- Collection of Polynucleotides --- p.82 / Chapter 4.2.1.4 --- Synthesis of Second-strand cDNAs --- p.82 / Chapter 4.2.1.5 --- PCR Purification of Double-stranded cDNAs --- p.83 / Chapter 4.2.1.6 --- Restriction Endonuclease Digestion --- p.84 / Chapter 4.2.1.7 --- Ligation to Adaptor --- p.85 / Chapter 4.2.1.8 --- PCR Amplification with Biotin-labelled miRNA PCR Primers --- p.86 / Chapter 4.2.1.9 --- Capture of Biotin-labelled PCR Fragments --- p.88 / Chapter 4.2.1.10 --- Introducing NotI Recognition Sequences --- p.88 / Chapter 4.2.1.11 --- Cloning into the pCR2.1 Vector --- p.89 / Chapter 4.2.1.12 --- Ligation of the cDNA Fragments and the pCR2.1 Vector --- p.90 / Chapter 4.2.1.13 --- Transformation --- p.90 / Chapter 4.2.1.14 --- Preparation of Purified Plasmids --- p.91 / Chapter 4.2.1.15 --- Sequencing Analysis of the cDNA Clone Library --- p.91 / Chapter 4.2.2 --- Real-time PCR Target Gene Expression in Cell Lines --- p.92 / Chapter 4.2.3 --- Real-time PCR Target Gene Expression Upon Inhibition of miR-106b --- p.92 / Chapter 4.2.4 --- Cloning of Potential Target Genes into pMIR Luciferase Expression Vector --- p.93 / Chapter 4.2.5 --- Site-directed Mutagenesis --- p.94 / Chapter 4.2.6 --- Luciferase Reporter Assay --- p.94 / Chapter 4.3 --- Results --- p.95 / Chapter 4.3.1 --- Sequencing Analysis of the cDNA Clone Library --- p.95 / Chapter 4.3.2 --- Expression Levels of Candidate Genes in MB Cell Lines --- p.100 / Chapter 4.3.3 --- Effects of Anti-miR-106b Inhibitors on 3'UTR of Target Genes --- p.101 / Chapter 4.3.4 --- Verification of Candidate Genes --- p.103 / Chapter 4.3.5 --- Verification of Target Sites with Site-directed Mutagenesis --- p.104 / Chapter 4.4 --- Discussion --- p.107 / Chapter CHAPTER 5: --- FUNCTIONAL ASSAYS --- p.111 / Chapter 5.1 --- Introduction- Functional Investigation of miR-106b-25 Cluster --- p.111 / Chapter 5.2 --- Methods --- p.113 / Chapter 5.2.1 --- Cell Culture --- p.113 / Chapter 5.2.2 --- Over-expression of miR-106b Mimic --- p.113 / Chapter 5.2.3 --- MTT Assay --- p.114 / Chapter 5.2.4 --- IC50 of Cisplatin --- p.115 / Chapter 5.2.5 --- MTT Assay with Cisplatin Treatment --- p.115 / Chapter 5.2.6 --- Cell Cycle --- p.116 / Chapter 5.2.7 --- BrdU Cell Proliferation Assay --- p.117 / Chapter 5.2.8 --- Wound Healing Assay --- p.117 / Chapter 5.3 --- Results --- p.119 / Chapter 5.3.1 --- Effects of Inhibition of miR-106b-25 Cluster on Cell Growth. --- p.119 / Chapter 5.3.2 --- Cell Cycle Distribution Analysis --- p.121 / Chapter 5.3.3 --- Sensitivity to Cisplatin --- p.123 / Chapter 5.3.4 --- Cell Proliferation Assay --- p.124 / Chapter 5.3.5 --- Cell Motility --- p.126 / Chapter 5.3.6 --- Efficiency of Over-expression Using miR-106b Mimic --- p.129 / Chapter 5.3.7 --- Effects of miR-106b on Cell Growth --- p.130 / Chapter 5.4 --- Discussion --- p.131 / Chapter CHAPTER 6: --- CONCLUSION --- p.135 / REFERENCE --- p.137

Suen, Wai Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 117-128). / Abstract also in Chinese. / ACKNOWLEDGEMENTS --- p.ii / LIST OF ABBREVIATIONS --- p.iii / ABSTRACT --- p.v / 摘要 --- p.vii / LIST OF PUBLICATIONS --- p.viii / TABLE OF CONTENTS --- p.ix / Chapter CHAPTER 1. --- INTRODUCTION --- p.1 / Chapter 1.1. --- Nature of Telomeres / Chapter 1.1.1. --- Telomere structure --- p.1 / Chapter 1.1.2. --- Importance of telomeres --- p.2 / Chapter 1.1.3. --- Telomere length attrition during replicative senescence --- p.3 / Chapter 1.1.4. --- Telomere length maintenance --- p.6 / Chapter 1.1.5. --- "Oxidative stress, inflammatory process and telomere length" --- p.7 / Chapter 1.1.6. --- Telomere attrition rate --- p.8 / Chapter 1.2. --- "Age, Gender and Telomere Length" / Chapter 1.2.1. --- Age and telomere length --- p.10 / Chapter 1.2.2. --- Gender difference of telomere length --- p.10 / Chapter 1.3. --- Health Status and Telomere Length --- p.13 / Chapter 1.3.1. --- Coronary heart diseases --- p.13 / Chapter 1.3.2. --- Cancers --- p.14 / Chapter 1.3.3. --- Infections and chronic inflammation --- p.15 / Chapter 1.3.4. --- Bone mineral density --- p.16 / Chapter 1.3.5. --- Neurodegenerative diseases --- p.17 / Chapter 1.3.6. --- Frailty and mortality --- p.19 / Chapter 1.4. --- "Lifestyles, Environment and Telomere Length" --- p.21 / Chapter 1.4.1. --- Obesity --- p.21 / Chapter 1.4.2. --- Smoking --- p.22 / Chapter 1.4.3. --- Physical activity --- p.23 / Chapter 1.4.4. --- Diet --- p.23 / Chapter 1.4.5. --- Psychological stress --- p.24 / Chapter 1.4.6. --- Socioeconomic status --- p.24 / Chapter 1.5. --- Methods of Measuring Telomere Length --- p.27 / Chapter 1.6. --- Aims and Hypotheses of the Study --- p.31 / Chapter 1.6.1. --- Aims --- p.31 / Chapter 1.6.2. --- Hypotheses --- p.31 / Chapter CHAPTER 2. --- SUBJECTS AND METHODS --- p.33 / Chapter 2.1. --- Subjects Recruitment --- p.34 / Chapter 2.2. --- Interview --- p.34 / Chapter 2.3. --- Anthropometry --- p.35 / Chapter 2.4. --- DNA Extraction and Storage --- p.35 / Chapter 2.5. --- Telomere Length Measurement --- p.37 / Chapter 2.5.1. --- Terminal restriction fragment analysis --- p.37 / Chapter 2.5.2. --- Quantitative real-time PCR --- p.39 / Chapter 2.6. --- Self-perceived Health --- p.46 / Chapter 2.7. --- Medical History --- p.46 / Chapter 2.8. --- Bone Mineral Density --- p.47 / Chapter 2.9. --- Frailty Index --- p.47 / Chapter 2.10. --- Mortality Rate --- p.50 / Chapter 2.11. --- Smoking --- p.50 / Chapter 2.12. --- Physical Activity --- p.51 / Chapter 2.13. --- Dietary Intakes --- p.51 / Chapter 2.15. --- Socioeconomic Status --- p.52 / Chapter 2.16. --- Statistical Analysis --- p.53 / Chapter CHAPTER 3. --- RESULTS AND DISCUSSIONS --- p.55 / Chapter 3.1. --- Demographics --- p.55 / Chapter 3.2. --- Telomere Length Distribution --- p.64 / Chapter 3.2.1. --- Age and telomere length --- p.64 / Chapter 3.2.2. --- Gender and telomere length --- p.68 / Chapter 3.3. --- Health Status and Telomere Length --- p.71 / Chapter 3.3.1. --- Self-perceived health --- p.71 / Chapter 3.3.2 --- History of diseases --- p.74 / Chapter 3.3.3. --- Bone mineral density --- p.80 / Chapter 3.3.4. --- Frailty index --- p.86 / Chapter 3.3.5. --- Mortality rate --- p.91 / Chapter 3.4. --- "Lifestyles, Environment and Telomere Length" --- p.94 / Chapter 3.4.1. --- Smoking --- p.94 / Chapter 3.4.2. --- Physical activity --- p.99 / Chapter 3.4.3. --- Diet --- p.104 / Chapter 3.4.4. --- Socioeconomic status --- p.109 / Chapter CHAPTER 4. --- CONCLUSIONS --- p.115 / Chapter 4.1. --- General conclusions --- p.115 / Chapter 4.2 --- The Significance of telomere length in the elderly --- p.115 / Chapter 4.3. --- Future Works and Prospect --- p.116 / References --- p.117