Introdução: Ao consideramos que o crescimento craniofacial no indivíduo com fissura labiopalatina (FLP) é influenciado pela própria fissura e pelas cirurgias para sua correção, surgem questionamentos importantes quanto ao uso de medidas normativas obtidas com indivíduos que não apresentaram FLP para interpretar achados na fissura. Em 1957, Subtelny publicou medidas da extensão (ETV) e espessura (EPV) do palato mole e da profundidade da velofaringe (PN) e seus achados são usados como normas de referência para interpretar medidas na FLP. As normas foram calculadas a partir de telerradiografias obtidas ao longo de 18 anos em 30 norte-americanos sem fissura. No gerenciamento da FLP medidas das estruturas e espaços velofaríngeos podem contribuir para o diagnóstico da disfunção velofaríngea e documentação sistemática de resultados do tratamento. Objetivo: O objetivo principal deste estudo foi estabelecer ETV, EPV, e PNF na FLP operada e fala normal. Metodologia: As medidas foram determinadas a partir de telerradiografia em norma lateral para sujeitos com fissura transforame incisivo unilateral operada (FTIUO) que obtiveram fala normal após palatoplastia. A razão entre a medida de profundidade da nasofaringe e extensão velar (RPE) também foi estabelecida e os achados foram comparados com os dados de Subtelny. Os prontuários dos pacientes estudados foram consultados para confirmar fala normal no dia da radiográfica. As medidas foram realizadas com o programa Dolphin Imaging, versão 11.0, com análise de Arnett-Gunson FAB. Medidas da média e desvio padrão foram usadas para descrição dos achados com apresentação em tabelas e gráficos. O teste t não pareado foi usado para testar a hipótese de existência de diferença estatisticamente significante entre as medidas deste estudo e as normas de Subtelny, considerando-se estatisticamente significantes os resultados onde p < 0,05. Resultados: Foi analisado um total de 260 telerradiografias, sendo que 120 do gênero feminino e 140 masculino, com idades entre 5 e 14 anos. Comparando-se as medidas com os dados de Subtelny, observou-se: a) diferenças significantes para a medida ETV dos 05 aos 11 anos e 13 anos, sugerindo que o palato de pacientes com FTIUO com fala normal é mais curto do que de Subtelny na maioria das idades estudadas; b) diferenças significantes para a medida EPV nas idades de 5, 11, 12 e 14 anos, sugerindo que o palato de pacientes com FTIUO com fala normal é mais espesso do que de Subtelny, em algumas das idades estudadas; c) diferenças significantes para a medida PNF dos 05 aos 13 anos, sugerindo que espaço nasofaríngeo em pacientes com FTIUO com fala normal é mais estreito do que de Subtelny na maioria das idades estudadas; e d) diferença estatisticamente significante na RPE para as idades de 05 a 09 anos e 12 e 13 anos, sugerindo potencial para fechamento velofaríngeo e, portanto, corroborando o critério de fala normal. Discussão: Este estudo revelou medidas de EPV, ETV, e PNF para indivíduos com FTIUO significantemente diferentes daquelas propostas por Subtelny. Mesmo na presença de palato mais curto os pacientes estudados apresentavam fala normal sugerindo fechamento velofaríngeo adequado. Este achado pode ser decorrente de aumento de tecido linfático na população com FTIUO, o que foi confirmado com a medida de PNF que indica nasofaringe significantemente mais estreita na população estudada. O palato mole neste estudo foi muito mais espesso nos pacientes com fissura o que pode ser resultado da cicatrização velar ou uso de procedimento cirúrgico envolvendo a sobreposição de músculos o que explica aumento de tecido mole na área estudada. Conclusão: Como muitos dos achados deste estudo foram significantemente diferente das normas estabelecidas por Subtelny, sugere-se que as medidas propostas em 1957 não são representativas da população com FTIUO e fala normal. Propõe-se a importância de estabelecerem-se medidas normativas das estruturas e espaços velofaríngeos para populações específicas, de forma a contribuir para documentação de resultados do tratamento e complementar o processo diagnóstico e a definição da conduta para tratar a DVF. Telerradiografias são obtidas sistematicamente para os pacientes com FLP ao redor dos 8 anos na maioria dos centros craniofaciais ao redor do mundo, as medidas estudadas podem (e deveriam) ser obtidas para todos os pacientes (com e sem fala normal) nesta idade. / Introduction: Considering that craniofacial growth in individuals with cleft lip and palate (CLP) is affected by the cleft and the surgeries to correct the anomaly, important questions can be made regarding interpreting clinical findings for individuals with CLP using normative data obtained with a population without CLP. In 1957, Subtelny published data about the length (LSP) and width (WSP) of the soft palate and depth of the nasopharynx (DNP) and his findings are used until today as reference for interpretation of measures of velopharyngeal structure and space in the population with CLP. Subtelnys norms were calculated using cephalometric X-rays (CEPH) obtained during the first 18 years of life of 30 North-American speakers without CLP. While managing CLP measures of velopharyngeal structures and spaces can contribute in the diagnosis of velopharyngeal dysfunction (VPD) and systematic documentation of treatment outcome. Objective: The primary objective of this study was to establish LSP, WSP, and DNP in individuals with CLP and normal speech. Methodology: The measures of LSP, WSP and DNP were determined using CEPHs obtained from a group of individuals with operated unilateral cleft lip and palate (OUCLP) and normal speech after primary palatoplasty. The ratio between the measures of DNP and LSP (RDL) was also calculated and all findings were compared to Subtelnys. All CEPHs in a UCLPs data bank were considered for this study. Patients charts were reviewed to confirm normal speech. The measures were obtained using Arnett and Gunson FAB cephalometric analysis in Dolphin Imaging program (version 11.0). Mean and standard deviation measures were used to describe the findings which are presented in tables and graphs. Students t-test (unpaired) was used to test the hypothesis that this studys findings were significantly different than Subtelnys, with significance established at p<0.05. Results: A total of 260 CEPHs were studied, 120 from females and 140 from males between 5 and 13 years of age. Comparing this studys measures to Subtelnys we observed: a) significant difference in LSP between 5 and 11 years and 13 years, suggesting that the soft palate in individuals with OUCLP with normal speech is significantly shorten than Subtelnys for most ages studied; b) significant difference in WSP at 5, 11, 12 and 14 years of age, suggesting that the soft palate in individuals with OUCLP with normal speech is significantly thicker than the palates in Subtelnys for some age groups; c) significant difference in DNP between 5 and 13 years of age, suggesting that nasopharyngeal space in individuals with OUCLP with normal speech is significantly narrower than Subtelnys, for most ages studied; d) significant difference in RPE was found between 5 and 9 years and at 12 and 13 years of age suggesting potential for velopharyngeal closure and, therefore, corroborating criteria of normal speech. Discussion: This study revealed measures of LSP, WSP and DNP significantly different for individuals with OUCLP and normal speech when compared to Subtelnys norms for several age groups. Even though a much shorten soft palate was found (LSP), all patients studied presented with normal speech indicative of adequate velopharyngeal closure. This finding may be related to enlarged lymphatic tissue (adenoids) in the population with OUCLP and is supported in this study by reduced measures of DNP which revealed nasopharynx significantly narrower for the individuals with OUCLP when compared to the norms. The soft palate in this study was thicker which may be the result of velar scarring or the use of a surgical procedure involving muscle transpositioning during palatal repair which can explain more soft tissue in the area studied. Conclusion: Most findings for this study were significantly different than the reference norms established by Subtelny, suggesting that the norms, proposed in 1957 are not representative of population with OUCLP with normal speech. Establishing population specific normative data about size of velopharyngeal structures and spaces is important and can contribute adding an information about the outcome of cleft repair and also complement the diagnostic process supporting the identification of the best treatment for VPD. Since CEPH are systematically obtained for patients with CLP around 8 years of age in most craniofacial centers around the world, measures of LSP, WSP, DNP and RDL can (and should) be obtained for all patients(with and without normal speech) at this age.

This thesis is the construct of a computational system for studying the nasopharyngeal carcinoma (NPC) using high-throughput sequencing data. The system involves several components, including discovery of gene fusion in NPC cell line, construction of Esptein-Barr virus (EBV) genome, and evaluation on contaminated sequencing data alignment approaches. We successfully discovered a gene fusion (UBR5-ZNF423) in a NPC cell line (C666-1) which was verified by lab experiments and found in 8.3% of primary tumors. It was discovered the regulation of this gene affect the growth of cancer cell. We constructed the EBV genome in C666-1. It serves as an important reference for studying this important NPC cell line, which was the only NPC cell line in the world for a long time. We also evaluated three mapping approaches. Two of them are designed to filter out potential mouse contamination reads on human sequencing data, which can originate from NPC human-in-mouse xenografts. We found that special care should always be applied to contaminated data. Although direct mapping can give acceptable results if in most cases, the combined-based approached is suggested. It can effectively reduce false positive variants and maintain good enough numbers of true positive variants. Filtering approach is an alternative to the combined-based approach that can also effectively reduce contamination when memory is not sufficient. / 本論文利用電腦有系統地研究鼻咽癌，當中的數據利用了高通量測序技術來定序。其中章節包括在鼻咽癌胞系中尋找融合基因、組建潛藏於人體可引致鼻咽癌的EB病毒基因組、還有評價幾種可處理受污染序列的序列排列方法。我們成功地在鼻咽癌胞系（C666-1）中發現出一個融合基因（UBR5-ZNF423），並在實驗中確定此成果，其中發現在原發腫瘤中有8.3%的樣本中找出此融合基因。此外，也發現這融合基因調控會影響到癌細胞的生長。C666-1鼻咽癌胞系在過往有一段很長的時間裡，都是全世界唯一的鼻咽癌胞系，因此它有非常重要的參考價值，在此研究，我們組建了在C666-1裡的EB病毒基因組，使它作為研究C666-1的參考樣本。另外，我們評價了三種處理排列的方法，其中兩種的設計能過濾部分人類序列數據當中老鼠基因組的污染，老鼠基因組的污染可以來自於異種移植，即把人類癌細腫瘤移植於老鼠身上種植，我們建議在情況許可下都使用特殊的處理方法而不是直接作序列排列。直接作序列排列數據雖然已有合理的表現，但相比之下組合基因組式序列排列方法能有效減少錯誤肯定的遺傳變異，並同時保留足夠多正確肯定的遺傳變異，所以組合基因組式序列排列方法應在情況許可下都使用它。過濾式序列排列方法也是一種特殊的處理方法，它也能有效減少錯誤肯定的遺傳變異，它對記憶體的需求比組合基因組式序列排列方法少，可在電腦的記憶體不足時使用它。 / Tso, Kai Yuen. / Thesis M.Phil. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 112-120). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Detailed summary in vernacular field only.

Man, Cheuk Him. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 219-263). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xvi / List of Publications --- p.xvii / Chapter Ch.l --- Introduction --- p.1 / Chapter 1.1 --- Aim of study --- p.1 / Chapter 1.2 --- Literature review --- p.3 / Chapter 1.2.1 --- Nasopharyngeal carcinoma (NPC) --- p.3 / Chapter 1.2.1.1 --- Structure and function of nasopharynx --- p.3 / Chapter 1.2.1.2 --- Histopathology of NPC --- p.3 / Chapter 1.2.1.3 --- Epidemiology of NPC --- p.4 / Chapter 1.2.2 --- Etiology of NPC --- p.6 / Chapter 1.2.2.1 --- Genetic factors --- p.6 / Chapter 1.2.2.2 --- Environment factors --- p.13 / Chapter 1.2.2.3 --- Epstein-Barr virus (EBV) infection --- p.14 / Chapter 1.2.3 --- Therapeutic treatment of NPC --- p.24 / Chapter 1.2.3.1 --- Radiotherapy (RT) --- p.24 / Chapter 1.2.3.2 --- Chemotherapy --- p.25 / Chapter 1.2.4 --- Notch signaling pathway --- p.26 / Chapter 1.2.4.1 --- Notch receptors and their ligands --- p.26 / Chapter 1.2.4.2 --- Activation of Notch signaling pathway --- p.29 / Chapter 1.2.4.3 --- Regulators of Notch signaling pathway --- p.32 / Chapter 1.2.4.4 --- Effectors of Notch signaling pathway --- p.32 / Chapter 1.2.5 --- Role of Notch signaling pathway in tumorigenesis --- p.33 / Chapter 1.2.5.1 --- Cell proliferation --- p.34 / Chapter 1.2.5.2 --- Cell survival --- p.35 / Chapter 1.2.5.3 --- Angiogenesis --- p.36 / Chapter 1.2.5.4 --- Cell invasion and metastasis --- p.36 / Chapter 1.2.6 --- Notch and oncogenic virus --- p.37 / Chapter 1.2.7 --- Crosstalk between Notch and other signaling pathways --- p.38 / Chapter 1.2.7.1 --- NFkB signaling pathway --- p.38 / Chapter 1.2.7.2 --- Ras signaling pathway --- p.39 / Chapter 1.2.7.3 --- Wnt signaling pathway --- p.40 / Chapter 1.2.7.4 --- Akt signaling pathway --- p.40 / Chapter 1.2.7.5 --- ErbB2 signaling pathway --- p.41 / Chapter 1.2.8 --- Notch as therapeutic target for cancer --- p.41 / Chapter Ch.2 --- Materials and Methods --- p.45 / Chapter 2.1 --- "Cell lines, xenografts and primary tumors" --- p.45 / Chapter 2.1.1 --- Cell lines --- p.45 / Chapter 2.1.2 --- Xenografts --- p.46 / Chapter 2.1.3 --- Primary tumors --- p.48 / Chapter 2.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.50 / Chapter 2.2.1 --- Sample preparation for RT-PCR --- p.50 / Chapter 2.2.1.1 --- RNA extraction --- p.50 / Chapter 2.2.1.2 --- Quantitation of total RNA --- p.50 / Chapter 2.2.2 --- Conventional RT-PCR --- p.51 / Chapter 2.2.3 --- Quantitative RT-PCR --- p.51 / Chapter 2.3 --- Western immunoblot --- p.55 / Chapter 2.3.1 --- Protein extraction --- p.55 / Chapter 2.3.2 --- SDS-PAGE and immunoblotting --- p.55 / Chapter 2.4 --- Immunohistochemistry --- p.59 / Chapter 2.5 --- Cloning and plasmid DNA preparation --- p.62 / Chapter 2.5.1 --- Polymerase chain reaction (PCR) and purification of PCR products --- p.62 / Chapter 2.5.2 --- Restriction enzyme double digestion --- p.65 / Chapter 2.5.3 --- Ligation of plasmid and insert sequence --- p.65 / Chapter 2.5.4 --- Bacterial transformation --- p.66 / Chapter 2.5.5 --- Plasmid DNA extraction --- p.66 / Chapter 2.5.6 --- DNA sequencing --- p.67 / Chapter 2.6 --- Transient transfection of NPC cell lines --- p.67 / Chapter 2.7 --- Drug treatment on NPC cell lines --- p.69 / Chapter 2.8 --- Cell proliferation assays --- p.71 / Chapter 2.8.1 --- WST-1 assay --- p.71 / Chapter 2.8.2 --- BrdU assay --- p.71 / Chapter 2.9 --- Flow cytometry analysis --- p.72 / Chapter 2.9.1 --- Sample preparation --- p.72 / Chapter 2.9.2 --- Cell cycle analysis by propidium iodide staining --- p.73 / Chapter 2.9.3 --- Apoptosis analysis by AnnexinV-PI staining --- p.73 / Chapter 2.10 --- Apoptosis analysis by Caspase-3 activity assay --- p.74 / Chapter 2.11 --- RBP-Jk reporter assay --- p.75 / Chapter 2.12 --- NFKB1 reporter assay --- p.77 / Chapter 2.13 --- Dual luciferase reporter assay --- p.77 / Chapter 2.14 --- Expression array --- p.78 / Chapter 2.15 --- Statistical analysis --- p.79 / Chapter Ch.3 --- Characterization of Notch Signaling Molecules in NPC --- p.80 / Chapter 3.1 --- Introduction --- p.80 / Chapter 3.2 --- Results --- p.81 / Chapter 3.2.1 --- "Expression of Notch ligands, receptors, effectors and regulators in NPC cell lines and xenografts" --- p.81 / Chapter 3.2.2 --- "Expression of Notch ligands, receptors, regulators and effectors in NPC primary tumors" --- p.104 / Chapter 3.3 --- Discussion --- p.111 / Chapter 3.3.1 --- Overexpression of Jagl and D114 in NPC --- p.112 / Chapter 3.3.2 --- Overexpression of Notch receptors in NPC --- p.114 / Chapter 3.3.3 --- "Downregulation of Negative regulator, Numb, in NPC" --- p.116 / Chapter 3.3.4 --- Overexpression of Notch effectors in NPC --- p.117 / Chapter 3.4 --- Summary --- p.119 / Chapter Ch.4 --- Mechanisms of Activation of Notch Signaling Pathway in NPC --- p.120 / Chapter 4.1 --- Introduction --- p.120 / Chapter 4.2 --- Results --- p.122 / Chapter 4.2.1 --- EBV mediated Notch activation --- p.122 / Chapter 4.2.1.1 --- No effect of EBERs and EBNA1 on the expression of Notch Components --- p.122 / Chapter 4.2.1.2 --- LMP1 induces expression of Notch components --- p.129 / Chapter 4.2.1.3 --- LMP2A induces expression of Notch components --- p.133 / Chapter 4.2.2 --- Effect of CXCR4 on Notch signaling pathway in C666-1 --- p.137 / Chapter 4.3 --- Discussion --- p.139 / Chapter 4.3.1 --- EBV-mediated induction of Notch components --- p.139 / Chapter 4.3.2 --- Regulation of Notch expression by CXCR4 signaling pathway --- p.142 / Chapter 4.4 --- Summary --- p.145 / Chapter Ch.5 --- Investigation of the Oncogenic Role of Notch3 --- p.146 / Chapter 5.1 --- Introduction --- p.146 / Chapter 5.2 --- Results --- p.148 / Chapter 5.2.1 --- Effect of knockdown Notch 1 by siRNA on the growth of C666-1 --- p.148 / Chapter 5.2.2 --- Effect of knockdown Notch3 by siRNA on the growth of C666-1 --- p.151 / Chapter 5.2.2.1 --- Effect of knockdown Notch3 by siRNA on the RBP-Jk promoter activity of C666-1 --- p.153 / Chapter 5.2.2.2 --- Effect of knockdown Notch3 by siRNA on the proliferation of C666-1 --- p.155 / Chapter 5.2.2.3 --- Effect of knockdown Notch3 by siRNA on cell cycle progression of C666-1 --- p.158 / Chapter 5.2.2.4 --- Effect of knockdown Notch3 by siRNA on resistant to apoptosis in C666-1 --- p.160 / Chapter 5.2.3 --- Investigation of the anti-proliferation effect of therapeutic agents targeting Notch signaling pathway in NPC cells --- p.168 / Chapter 5.2.3.1 --- "Effect of DAPT on the proliferation of HEK293T, C666-1 and HK-1" --- p.168 / Chapter 5.2.3.2 --- Effect of AMD3100 on Notch signaling pathway and proliferation of NPC cells --- p.172 / Chapter 5.2.4 --- Study of downstream targets of Notch3 in NPC cells --- p.178 / Chapter 5.3 --- Discussion --- p.200 / Chapter 5.3.1 --- Oncogenic role of Notch3 in C666-1 --- p.200 / Chapter 5.3.2 --- Potential therapeutic approach in treating NPC via Notch inhibition --- p.206 / Chapter 5.3.2.1 --- "Gamma secretase inhibitor, DAPT" --- p.206 / Chapter 5.3.2.2 --- "CXCR4 antagonist, AMD3100" --- p.207 / Chapter 5.4 --- Summary --- p.209 / Chapter Ch.6 --- General Discussion --- p.210 / Chapter Ch.7 --- Conclusion --- p.217 / Reference --- p.219 / Appendices --- p.263 / Appendix 1 Summary of immunohistochemical staining results on 23 primary NPC samples --- p.264 / Appendix 2 Summary of 581 selected genes from the expression array --- p.265

Choi, Pui Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 101-122). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Xu, Meng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 112-129). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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

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

Tong Hung Man Joanna. / "June 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 137-149). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Based on matching analysis between different EBV strains, we found two nucleotide variations in miR-BART21 and four nucleotide changes in miR-BART22. Interestingly, two nucleotide variations upstream of mature miR-BART22 likely favor its biogenesis by Drosha/DGCR8 processing and we experimentally confirmed this augmentation by in-vitro Drosha digestion, and thus may underline the high and consistent expression of miR-BART22 in NPC tumors. / Infection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Many viral gene products including EBNA1, LMP1 and LMP2 have been implicated in NPC tumorigenesis, although the de novo control of these viral oncoproteins remain largely unclear. / MicroRNAs (miRNAs) are a class of small, non-coding RNAs with a size around 18--24 nucleotides with significant roles in regulating gene expression by either transcriptional silencing or translational suppression. As gene regulators, recent miRNA studies have emphasized the contribution of aberrant miRNA expression in cancer development. The recent discovery of EBV encoded viral miRNAs (ebv-miRNAs) in lymphoid malignancies has prompted us to examine the NPC-associated EBV miRNAs. In this study, we have systematically examined the NPC associated EBV genome for viral-encoded miRNA expression. By constructing small cDNA libraries from a native EBV positive NPC cell line (C666-1) and a xenograft (X2117), we screened about 3000 clones and detected several small EBV fragments, within which two novel ebv-miRNAs in the BARTs region were identified. These two newly identified miRNAs, now named miR-BART21 and miR-BART22, were proven to be abundantly expressed in most NPC samples by both Northern blot and QRT-PCR analysis. / Taken together, this thesis shows that two newly identified EBV-encoded miRNAs are highly expressed in latent EBV infection in NPC. Frequent expression of miR-BART22 can be explained partially by a specific EBV strain that is associated with NPC in our locality. Our findings emphasize the role of miR-BART22 in modulating LMP-2A expression. Because LMP-2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells (CTLs), down-modulation of LMP-2A expression by mir-BART22 may permit escape of EBV-infected cells from host immune surveillance. / We attempted to predict the potential viral and cellular targets of miR-BART21 and miR-BART22 by public available computer programs, miRanda and RNAhybrid. A number of potential cellular mRNA targets were suggested, although many failed to be validated by luciferase reporter assay. However, we found a putative miR-BART22 binding site in the LMP2A-3'UTR. Although the LMP-2A transcript is consistently detected in NPC, only 6 out of 26 (23%) primary NPC tumors show weak LMP-2A expression by immunohistochemistry (IHC). The expression levels of miR-BART22 and LMP-2A mRNA have also been determined in eleven of these tumors. Interestingly, the LMP-2A mRNA expression level did not directly correlate with protein expression, and relatively low expression levels of miR-BART22 miRNA were observed in all 3 LMP-2A positive-primary tumors. The suppressive effect of miR-BART22 on LMP-2A was also experimentally validated by a series of dual luciferase reporter assays using reporter constructs containing the putative or mutated recognition site at the LMP-2A 3'UTR. By co-transfection of different amounts of miR-BART22 with the LMP-2A-3'UTR expression vector in reporter assay, we confirmed that miR-BART22 suppressed the LMP-2A protein level in a dose-dependent manner. Furthermore, transfection of miR-BART22 into HEK293 cells that had been stably transfected with pcDNA3.1-LMP-2A, which contains a complete LMP-2A ORF and 3'UTR, readily suppressed levels of the LMP-2A protein. / Lung, Wai Ming Raymond. / Adviser: To Ka Fai. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 197-226). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

In brief, mRNA expression profiling of candidate genes in each locus was performed using semi-quantitative RT-PCR in a panel of ESCC and NPC cell lines, normal tissues and immortalized epithelial cell lines. Genes downregulated in cancer cells but with high expression in normal tissues and immortalized epithelial cells were subjected to promoter methylation analysis using methylation-specific PCR (MSP), bisulfite genomic sequencing (BGS) and pharmacological demethylation treatment. Genes with tumor-specific downregulation and methylation were further selected as candidates and their tumor suppressive roles were verified via functional studies. / In conclusion, RAB39 and WDRX, epigenetically silenced in multiple cancer cell lines, were identified as novel TSG candidates in this study. Meanwhile, the tumor suppressive functions of ADAMTS8 were further validated, proving the efficiency of this integrative approach. Further study on these novel TSG candidates may help to elucidate the detailed molecular mechanisms for ESCC and NPC, and provide novel therapeutic targets and biomarkers. / In this study, RAB39 and WDRX were identified as candidate TSGs in 11q22.3 and 15q21.3, respectively. Both genes were broadly expressed in normal tissues and immortalized epithelial cell lines, but significantly downregulated and methylated in multiple cancer cell lines. Demethylation treatment with 5-Aza-2'-deoxycytidine restored their mRNA expression, indicating that CpG methylation directly contributed to their transcriptional inactivation. Methylation of RAB39 and WDRX was detected in primary ESCC and NPC, but rarely observed in normal tissues, implicating that their tumor-specific methylation might be used as biomarkers. Ectopic expression of both genes significantly inhibited the clonogenicity of multiple cancer cell lines, supporting their potential roles as functional TSGs. Moreover, WDRX repressed WNT/beta-catenin signaling, underscoring a possible anti-tumorigenic mechanism for it. In addition, ADAMTS8 was revealed to inhibit clonogenicity of NPC and ESCC cell lines, acting as a negative modulator for ERK pathway and a potential pro-apoptotic metalloprotease. / Inactivation of tumor suppressor genes (TSGs) contributes to the genesis of cancers including esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC), two prevalent causes of death in Hong Kong. Apart from genetic abnormalities, epigenetic disruptions including CpG methylation represent another major mechanism for TSG inactivation. Promoter methylation of multiple TSGs was detected in different cancer types, suggesting that it could be utilized as therapeutic target or biomarker for disease diagnosis and prognosis. / TSGs are often located at frequently deleted chromosomal regions and subjected to tumor-specific methylation, making it possible to use an integrative epigenetic and genomic approach combining array comparative genomic hybridization (aCGH) with epigenetic profiling to screen for novel TSGs. Previous aCGH revealed that several loci in 11822.3, 15q14, 15q21.1 and 15q21.3 underwent frequent copy number loss in ESCC cell lines. Loss of heterozygosity (LOH) of these regions was also reported in other cancers, indicating that TSGs might reside within them. The aim of this study was thus to identify the candidate TSGs in these loci and study their anti-tumorigenic roles. In addition, the tumor suppressive function of ADAMTS8, a silenced 11q25 candidate TSG previously identified in our lab via this approach, was also studied. / Li, Jisheng. / Adviser: Qian Tao. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 136-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.