Protein kinase A (PKA) is an important enzyme inside the body; it is responsible for phosphorylation of gene regulatory elements and thus regulation of gene expression inside the nucleus. Malfunction of PKA affects transcriptional and translational levels of cell signaling ligands, leading to abnormal activity of various signaling pathways. PKA holoenzyme is composed of two regulatory and two catalytic subunits; four main regulatory subunit isoforms (R1α, R1β, R2α and R2β) and four main catalytic subunit isoforms (Cα, Cβ, Cγ and Prkx) of PKA have been identified. Mutations in these subunits lead to altered total PKA activities and PKAT-I to PKAT-II ratios, leading to diseases both in human and mice. These diseases include Carney Complex (CNC), fibrous dysplasia (FD) and Cushing syndrome. We studied the effect of PKA subunit mutations on intracellular PKA activities, PKAT-I to PKAT-II ratios, and bone and adrenal gland phenotypes in transgenic mouse models. Firstly, we generated whole-body transgenic mice single or double heterozygous for PKA regulatory subunits. Tail vertebral bone lesions including osteosarcomas, osteochondromas and osteochondrosarcomas were found in these mice and we found that mutations in different PKA subunits affect bone lesion formation, new bone generation, and bone organization and mineralization in mouse tail vertebrae. Elevated Cβ subunit expression in Parkar1a+/-Prkar2a+/- and Prkar1a+/-Prkar2b+/-double heterozygous mice leads to a less severe vertebral bone lesion phenotype, an increased osteogenic activity and a better bone regeneration activity. We then studied mice with tissue specific knock out of Prkar1a, the gene coding for type I regulatory subunit, specifically in adrenal cortex (AdKO). AdKO mice developed pituitary-independent Cushing syndrome with increased PKA activity. They also demonstrated increased plasma corticosterone levels resistant to dexamethasone suppression. Dietary treatment of both mice with bone lesions and mice with adrenal lesions with COX2 inhibitor Celecoxib led to partial rescue of phenotypes; this is due to inhibition of the positive feedback loop between PKA signaling and inflamasome pathway at COX2 induction level by Celecoxib. / 蛋白激酶A（PKA）是人體中重要的蛋白酶， 它通過燐酸化基因調控元件來實現對細胞核內基因表達的調節。PKA異常影響細胞內信號傳遞因子的基因轉錄和蛋白翻譯水平，從而導致各細胞信號通路的異常活動。PKA全酶由兩個調節亞基和兩個催化亞基組成，目前已經發現的有四個調節亞基 (R1α, R1β, R2α 和R2β) 以及四個催化亞基(Cα, Cβ, Cγ和Prkx)。發生在這些亞基中的基因突變會改變總的PKA活動水平，PKA-I 和PKA-II的比例，在人類和實驗鼠中引起疾病。這些疾病包括卡尼綜合症 （CNC），骨纖維性發育不良（FD）和庫欣綜合症。我們在轉基因鼠模型中研究PKA亞基突變對細胞中PKA總活性， PKA-I和PKA-II比例的影響，以及由此帶來的骨和腎上腺表型的改變和病變。我們首先製造了有一個或兩個PKA亞基雜合性缺失的全身轉基因鼠。在這些轉基因鼠中，我們發現了包括骨肉瘤，骨軟骨瘤和骨軟骨肉瘤在內的尾椎骨病變。研究發現在不同PKA亞基中的基因變異對實驗鼠尾椎骨病變的發生，新骨的形成和骨的結構和纖維化均有影響。在Prkar1a+/-Prkar2a+/-和Prkar1a+/-Prkar2b+/-實驗鼠中我們發現了較高的Cβ催化亞基表達，這兩個基因型因此具有更輕度的骨病變和更強的骨再生能力。我們繼續研究了在腎上腺中敲除了標記PKA 第一調節亞基的Prkar1a基因的實驗鼠 （AdKO）。AdKO實驗鼠中產生了與垂體無關的庫欣綜合症，並伴隨PKA活性的增加。它們還表現出耐地塞米松抑制的血漿皮質酮水平增加。對骨病變或腎上腺病變的實驗鼠通過飲食進行COX2抑制劑塞來昔布的治療可以部分緩解病變表型。這是由對PKA和炎性體的正反饋機制在COX2誘導步驟的抑制造成的。 / Liu, Sisi. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 115-130). / Abstracts also in Chinese. / Title from PDF title page (viewed on 09, September, 2016). / Detailed summary in vernacular field only.

Cheung Hoi Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 151-166). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.I / ABSTRACT --- p.II / ABBREVIATIONS --- p.IV / LIST OF FIGURES --- p.VII / LIST OF TABLES --- p.IX / CONTENTS --- p.X / Chapter CHAPTER ONE: --- GENERAL INTRODUCTION / Chapter 1.1 --- Non-coding RNAs in transcriptional output --- p.2 / Chapter 1.2 --- Diverse functions of non-coding RNAs --- p.5 / Chapter 1.3 --- HI9: imprinted non-coding RNA --- p.6 / Chapter 1.4 --- Objective --- p.7 / Chapter CHAPTER TWO: --- The ROLE OF H19 RNA IN MDR1 EXPRESSION OF HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- H19-Igf2 locus as a model for genomic imprinting --- p.10 / Chapter 2.1.2 --- HI9 as a non-protein coding regulatory RNA --- p.12 / Chapter 2.1.3 --- Controversial roles of H19 RNA --- p.13 / Chapter 2.1.4 --- Novel role of H19 RNA in drug resistance --- p.15 / Chapter 2.2 --- Materials and methods / Chapter 2.2.1 --- Materials --- p.17 / Chapter 2.2.2 --- Methods / Chapter 2.2.2.1 --- Cell culture --- p.19 / Chapter 2.2.2.2 --- Plasmid construction and stable cell transfection --- p.19 / Chapter 2.2.2.3 --- Transient gene transfection --- p.20 / Chapter 2.2.2.4 --- RNA isolation and RT-PCR --- p.21 / Chapter 2.2.2.5 --- MTT drug sensitivity assay --- p.22 / Chapter 2.2.2.6 --- Western blot analysis --- p.22 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Differential expression of H19 RNA in different human cancer cell lines --- p.24 / Chapter 2.3.2 --- R-HepG2 cells over-expressed P-glycoprotein and H19 RNA --- p.24 / Chapter 2.3.3 --- Development of H19-silenced cell lines in HepG2 cells by RNA interference --- p.26 / Chapter 2.3.4 --- Altered drug sensitivity in H19-silenced cells --- p.28 / Chapter 2.3.5 --- Expression of P-glycoprotein in H19-silenced cells --- p.31 / Chapter 2.3.6 --- Overexpression of H19 RNA in HepG2 cells --- p.34 / Chapter 2.3.7 --- Induction of H19 RNA and MDR1 in HepG2 cells --- p.34 / Chapter 2.4 --- Discussion / Chapter 2.4.1 --- H19 regulation of MDR1 associated drug resistance --- p.38 / Chapter 2.4.2 --- The puzzle of riboregulation in drug resistance --- p.40 / Chapter CHAPTER THREE: --- The ROLES OF PTB AND IMP1 IN H19-RELATED MDR1 EXPRESSION OF HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- H19 RNA binding proteins --- p.43 / Chapter 3.2 --- Materials and methods / Chapter 3.2.1 --- Materials --- p.46 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Cell culture --- p.48 / Chapter 3.2.2.2 --- Plasmid construction and stable cell transfection --- p.48 / Chapter 3.2.2.3 --- RNA extraction and RT-PCR --- p.48 / Chapter 3.2.2.4 --- MTT drug sensitivity assay --- p.48 / Chapter 3.2.2.5 --- Western blot analysis --- p.48 / Chapter 3.2.2.6 --- Real-time PCR analysis of gene expression --- p.49 / Chapter 3.2.2.7 --- DOX efflux assay --- p.49 / Chapter 3.3 --- Results / Chapter 3.3.1 --- PTB knockdown increased P-glycoprotein expression --- p.51 / Chapter 3.3.2 --- IMP1 knockdown decreased MDR1 /P-glycoprotein expression --- p.54 / Chapter 3.3.3 --- Altered drug sensitivity in IMP 1 -knockdown cells --- p.60 / Chapter 3.4 --- Discussion / Chapter 3.4.1 --- Antagonistic effect of PTB and IMP1 on H19/MDR1 expressions --- p.64 / Chapter 3.4.2 --- Complexity of riboregulation --- p.65 / Chapter CHAPTER FOUR: --- IDENTIFICATION OF H19 RNA BINDING PROTEINS FROM HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- Overview of RNA-protein interactions --- p.69 / Chapter 4.1.2 --- Methodology in the study of RNA-protein interactions --- p.71 / Chapter 4.1.3 --- Identification of RNA-binding proteins --- p.72 / Chapter 4.2 --- Materials and methods / Chapter 4.2.1 --- Materials --- p.75 / Chapter 4.2.2 --- Methods / Chapter 4.2.2.1 --- Screening of H19 cDNA from human placenta cDNA library --- p.78 / Chapter 4.2.2.2 --- Preparation of nuclear and cytoplasmic extracts from HepG2 cells / Chapter 4.2.2.3 --- In vitro RNA transcription and RNA labeling --- p.80 / Chapter 4.2.2.4 --- RNA electrophoretic mobility shift assay --- p.81 / Chapter 4.2.2.5 --- In vitro UV-crosslinking assay --- p.82 / Chapter 4.2.2.6 --- Preparation of RNA-affinity column and isolation of RNA binding proteins --- p.83 / Chapter 4.2.2.7 --- In-gel digestion and MALDI-TOF mass spectrometry --- p.84 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Screening of H19 cDNA and preparation ofH19 RNA --- p.86 / Chapter 4.3.2 --- Electrophoretic mobility shift analysis of H19 RNA with HepG2 cytoplasmic extract --- p.87 / Chapter 4.3.3 --- UV-crosslinking of H19 RNA with HepG2 nuclear and cytoplasmic extract --- p.90 / Chapter 4.3.4 --- Isolation of H19 RNA binding proteins by RNA-affmity chromatography --- p.94 / Chapter 4.3.5 --- Confirmation of PTB and IMP1 as H19 RNA binding protein --- p.96 / Chapter 4.3.6 --- MALDI-TOF mass spectrometric analysis of isolated H19 RNA binding proteins --- p.96 / Chapter 4.4 --- Discussion / Chapter 4.4.1 --- RNA-protein interactions: an initial step for mechanistic study --- p.99 / Chapter 4.4.2 --- In vitro and in vivo methods for isolation of RNA binding proteins --- p.101 / Chapter 4.4.3 --- Novel role of hnRNP M protein in H19 RNA binding --- p.103 / Chapter CHAPTER FIVE: --- THE ROLE OF PTB IN APOPTOSIS / Chapter 5.1 --- Introduction / Chapter 5.1.1 --- Overview of polypyrimidine tract-binding protein in RNA processing and post-transcriptional gene regulation --- p.106 / Chapter 5.1.2 --- Evidences of polyrimidine-tract binding protein in the regulation of apoptosis --- p.108 / Chapter 5.2 --- Materials and methods / Chapter 5.2.1 --- Materials --- p.111 / Chapter 5.2.2 --- Methods / Chapter 5.2.2.1 --- Cell culture --- p.114 / Chapter 5.2.2.2 --- Stable cell transfection in A431 cells --- p.114 / Chapter 5.2.2.3 --- Western Blot analysis --- p.114 / Chapter 5.2.2.4 --- MTT drug sensitivity assay --- p.114 / Chapter 5.2.2.5 --- DNA fragmentation assay --- p.115 / Chapter 5.2.2.6 --- Flow cytometry analysis of apoptosis --- p.115 / Chapter 5.2.2.7 --- Caspase activity assay --- p.116 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Taxol as an apoptosis inducer in HepG2 cells --- p.117 / Chapter 5.3.2 --- PTB was cleaved during Taxol-induced apoptosis --- p.118 / Chapter 5.3.3 --- PTB knockdown increased Taxol cytotoxicity and apoptosis --- p.118 / Chapter 5.3.4 --- Effect of PTB knockdown on drug sensitivity of cells --- p.121 / Chapter 5.3.5 --- Effect of PTB knockdown on other drug-induced apoptosis --- p.121 / Chapter 5.3.6 --- Effect of PTB knockdown on the basal expressions of genes in apoptosis pathway --- p.126 / Chapter 5.3.7 --- The role of caspase-9 activation in PTB-regulated apoptosis --- p.129 / Chapter 5.3.8 --- The effect of PTB knockdown on pro-caspase-9 expression and Taxol-induced apoptosis in A431 cells --- p.133 / Chapter 5.3.9 --- The role of PTB in the regulation of intrinsic apoptosis pathway --- p.136 / Chapter 5.4 --- Discussion / Chapter 5.4.1 --- The role of PTB in intrinsic apoptosis pathway --- p.138 / Chapter 5.4.2 --- PTB in regulation of pro-caspase-9 expression --- p.139 / Chapter CHAPTER SIX: --- GENERAL DISCUSSION AND CONCLUSION / Chapter 6.1 --- H19 as a potential target in anti-cancer gene therapy --- p.143 / Chapter 6.2 --- Conclusion --- p.144 / Chapter 6.3 --- Unanswered questions and future work --- p.145 / Chapter 6.4 --- A proposed model for H19 pathway --- p.148 / REFERENCES --- p.151

Gho Wai-Man. / Thesis submitted in: August 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 114-123). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.IV / ACKNOWLEDGEMENT --- p.VI / TABLE OF CONTENT --- p.VII / LIST OF TABLES --- p.XII / LIST OF FIGURES --- p.XIII / ABBREVIATIONS --- p.XVI / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Epidemiology --- p.2 / Chapter 1.3 --- Etiological factors --- p.6 / Chapter 1.3.1 --- Viral Hepatitis Infection --- p.6 / Chapter 1.3.1.1 --- Hepatitis B Virus (HBV) --- p.7 / Chapter 1.3.1.2 --- Hepatitis C Virus (HCV) --- p.9 / Chapter 1.3.2 --- Aflatoxin Exposure --- p.10 / Chapter 1.3.3 --- Alcohol Abuse --- p.11 / Chapter 1.3.4 --- Liver Cirrhosis --- p.12 / Chapter 1.4 --- Genetic alterations in hcc --- p.16 / Chapter 1.4.1 --- Chromosomal Gain --- p.16 / Chapter 1.4.2 --- Chromosomal Loss --- p.17 / Chapter 1.5 --- Discovery of common activating transcription factor 5 (atf5) down-regulations in hcc --- p.19 / Chapter 1.5.1 --- Chromosome 19 Aberration in HCC --- p.19 / Chapter 1.5.2 --- Discovery of High Frequency of　ATF5 Down-regulations --- p.19 / Chapter 1.5.3 --- Activating Transcription Factor Family --- p.20 / Chapter 1.6 --- Aim of thesis --- p.28 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.29 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- Chemicals --- p.30 / Chapter 2.1.2 --- Buffers --- p.31 / Chapter 2.1.3 --- Cell culture --- p.31 / Chapter 2.1.4 --- Nucleic acids --- p.32 / Chapter 2.1.5 --- Enzymes --- p.32 / Chapter 2.1.6 --- Equipment --- p.32 / Chapter 2.1.7 --- Kits --- p.33 / Chapter 2.1.8 --- Software and Web Resource --- p.33 / Chapter 2.2 --- Dna extraction --- p.34 / Chapter 2.2.1 --- Cell Lines --- p.34 / Chapter 2.2.2 --- Primary HCC --- p.34 / Chapter 2.2.3 --- Lymphocytic DNA --- p.35 / Chapter 2.3 --- Rna extraction --- p.36 / Chapter 2.4 --- Dna sequencing --- p.38 / Chapter 2.4.1 --- Polymerase Chain Reaction (PCR) --- p.38 / Chapter 2.4.2 --- Cycle Sequencing --- p.39 / Chapter 2.5 --- Dual-labeled fluirescence in situ hybridization (fish) --- p.41 / Chapter 2.5.1 --- FISH Probe Preparation --- p.41 / Chapter 2.5.1.1 --- Preparation of Human Bacterial Artificial Chromosome (BAC) --- p.41 / Chapter 2.5.1.2 --- Nick Translation --- p.41 / Chapter 2.5.2 --- FISH --- p.42 / Chapter 2.6 --- 5-aza-2'-deoxycytidine & trichostatin a treatment on cell lines --- p.43 / Chapter 2.7 --- Bisulfite modificaiton of dna --- p.43 / Chapter 2.8 --- Methylation-specific pcr (msp) --- p.44 / Chapter 2.9 --- Bisulfite dna sequencing --- p.44 / Chapter 2.10 --- Quantitative reverse transcription pcr (qrt-pcr) --- p.46 / Chapter 2.11 --- In-vitro and in-vivo functinal examination --- p.49 / Chapter 2.11.1 --- ATF5 Transfection --- p.49 / Chapter 2.11.2 --- Cell Growth Assay --- p.50 / Chapter 2.11.3 --- Xenograft Development --- p.51 / Chapter 2.12 --- codelink expression microarray --- p.51 / Chapter 2.13 --- Statistical analysis --- p.53 / Chapter CHAPTER 3 --- INACTIVATION OF MECHANISMS UNDERLYING ATF5 DOWN-REGULATION --- p.54 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and methods --- p.58 / Chapter 3.2.1 --- Cell Lines --- p.58 / Chapter 3.2.2 --- Mutational Analysis --- p.58 / Chapter 3.2.3 --- Copy Number Loss --- p.59 / Chapter 3.2.4 --- Epigenetic Control --- p.59 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Sequencing Analysis of A TF5 Gene --- p.67 / Chapter 3.3.2 --- FISH Analysis of ATF5 Copy Number --- p.73 / Chapter 3.3.3 --- Epigenetic Control of A TF5 Expression --- p.73 / Chapter 3.4 --- Discussion --- p.82 / Chapter CHAPTER 4 --- FUNCTIONAL EXAMINATION AND INVESTIGATION OF DOWNSTREAM TARGETS MODULATED BY ATF5 --- p.85 / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Materials and methods --- p.88 / Chapter 4.2.1 --- Cell Lines --- p.88 / Chapter 4.2.2 --- Plasmids and Transfection --- p.88 / Chapter 4.2.3 --- Cell Growth Assay --- p.88 / Chapter 4.2.4 --- Xenograft Development --- p.88 / Chapter 4.2.5 --- CodeLink Expression Microarray --- p.89 / Chapter 4.2.6 --- Quantitative RT-PCR --- p.90 / Chapter 4.2.7 --- Statistical analysis --- p.90 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Cell Proliferation --- p.91 / Chapter 4.3.1.1 --- In-Vitro Examination --- p.91 / Chapter 4.3.1.2 --- In-Vivo Examination --- p.91 / Chapter 4.3.2 --- Microarray A nalysis --- p.91 / Chapter 4.3.3 --- Correlation of A TF5 with Id-1 Expression --- p.103 / Chapter 4.4 --- Discussion --- p.106 / Chapter CHAPTER 5 --- PROPOSED FUTURE INVESTIGATIONS --- p.110 / Chapter 5.1 --- inactivation mechanisms of atf5 gene --- p.111 / Chapter 5.2 --- Molecular pathways modulated by atf5 --- p.112 / Chapter CHAPTER 6 --- REFERENCES --- p.114

Leung Wan-chi. / Thesis submitted in: November 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 276-284). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese Version) --- p.v / Acknowledgements --- p.viii / Tables of Contents --- p.ix / List of Abbreviations --- p.xxx / List of Figures --- p.xxxiii / List of Tables --- p.xlii / Chapter Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Peroxisome proliferator activator receptors --- p.1 / Chapter 1.2 --- Peroxisome proliferators --- p.6 / Chapter 1.2.1 --- Hepatomegaly --- p.9 / Chapter 1.2.2 --- Peroxisome proliferation --- p.11 / Chapter 1.2.3 --- Target genes regulation --- p.12 / Chapter 1.2.4 --- Hypolipidemic effect --- p.16 / Chapter 1.2.5 --- Hepatocarcinogenesis --- p.18 / Chapter 1.3 --- Mode of actions --- p.20 / Chapter 1.3.1 --- Oxidative stress --- p.21 / Chapter 1.3.2 --- Inhibition of apoptosis --- p.22 / Chapter 1.3.2 --- Increase in cell replication --- p.22 / Chapter 1.3.4 --- Alterations in cell cycle control --- p.23 / Chapter 1.4 --- Objectives --- p.23 / Chapter Chapter 2 --- Materials and Methods --- p.25 / Chapter 2.1 --- Animal tail-genotyping --- p.25 / Chapter 2.1.1 --- Materials --- p.25 / Chapter 2.1.2 --- Methods --- p.28 / Chapter 2.2 --- Animal treatment --- p.29 / Chapter 2.2.1 --- Materials --- p.29 / Chapter 2.2.2 --- Methods --- p.29 / Chapter 2.3 --- Serum cholesterol and tryiglyceride analysis --- p.30 / Chapter 2.3.1 --- Materials --- p.31 / Chapter 2.3.2 --- Methods --- p.31 / Chapter 2.3.2.1 --- Serum preparation --- p.31 / Chapter 2.3.2.2 --- Serum cholesterol analysis --- p.31 / Chapter 2.3.2.3 --- Serum triglyceride analysis --- p.32 / Chapter 2.4 --- Histological analysis --- p.32 / Chapter 2.4.1 --- Materials --- p.32 / Chapter 2.4.2 --- Methods --- p.33 / Chapter 2.5 --- Total RNA isolation --- p.34 / Chapter 2.5.1 --- Materials --- p.34 / Chapter 2.5.2 --- Methods --- p.34 / Chapter 2.6 --- DNase I treatment of total liver RNA --- p.37 / Chapter 2.6.1 --- Materials --- p.37 / Chapter 2.6.2 --- Methods --- p.37 / Chapter 2.7 --- Reverse transcription (RT) of mRNA and non- fluorescent PCR (non-fluoroDD PCR) --- p.38 / Chapter 2.7.1 --- Materials --- p.43 / Chapter 2.7.2 --- Methods --- p.43 / Chapter 2.8 --- Reverse transcription (RT) of mRNA and fluorescent PCR (fluoroDD PCR) --- p.44 / Chapter 2.8.1 --- Materials --- p.44 / Chapter 2.8.2 --- Method --- p.44 / Chapter 2.9 --- Fluorescent differential display (fluoroDD) --- p.45 / Chapter 2.9.1 --- Materials --- p.45 / Chapter 2.9.2 --- Methods --- p.45 / Chapter 2.9.2.1 --- FluoroDD gel preparation --- p.45 / Chapter 2.9.2.2 --- Sample preparation and electrophoresis --- p.45 / Chapter 2.10 --- Excision of differentially expressed cDNA fragments --- p.46 / Chapter 2.10.1 --- Materials --- p.46 / Chapter 2.10.2 --- Methods --- p.46 / Chapter 2.11 --- Reamplification of differentally expressed cDNA fragments --- p.48 / Chapter 2.11.1 --- Materials --- p.48 / Chapter 2.11.2 --- Methods --- p.50 / Chapter 2.12 --- Subcloning of reamplified cDNA fragmens --- p.50 / Chapter 2.12.1 --- Materials --- p.53 / Chapter 2.12.2 --- Methods --- p.53 / Chapter 2.12.2.1 --- Ligation --- p.53 / Chapter 2.12.2.2 --- Transformation --- p.53 / Chapter 2.12.2.3 --- Phenol-choloroform extraction --- p.54 / Chapter 2.12.2.4 --- Confirmation of insert size by EcoRI digestion --- p.54 / Chapter 2.12.2.5 --- Mini-preparation of plasmid DNA from recombinant clones --- p.55 / Chapter 2.13 --- Sequencing of subcloned cDNA fragments --- p.55 / Chapter 2.13.1 --- Materials --- p.56 / Chapter 2.13.2 --- Methods --- p.56 / Chapter 2.13.2.1 --- Sequencing of fluoroDD cDNA fragments --- p.56 / Chapter 2.13.2.2 --- Blast search against computer database --- p.57 / Chapter 2.14 --- Northern blot analysis of sequenced cDNA fragments --- p.57 / Chapter 2.14.1 --- Materials --- p.58 / Chapter 2.14.2 --- Methods --- p.58 / Chapter 2.14.2.1 --- Formaldehyde agarose gel electrophoresis of total RNA --- p.58 / Chapter 2.14.2.2 --- Preparation of DIG-labeled RNA probes for hybridization --- p.59 / Chapter 2.14.2.3 --- Preparation of PCR DIG-labeled cDNA probes for hybridization --- p.60 / Chapter 2.14.2.4 --- Hybridization and colour development --- p.60 / Chapter Chapter 3 --- Results --- p.62 / Chapter 3.1 --- Confirmation of genotypes by PCR --- p.62 / Chapter 3.2 --- Body weight changes --- p.62 / Chapter 3.3 --- Organ weight changes --- p.67 / Chapter 3.4 --- Serum cholesterol and triglyceride levels --- p.70 / Chapter 3.5 --- Liver histology --- p.78 / Chapter 3.6 --- Reverse transcription (RT) of mRNA and non-fluorescent PCR (non-flurroDD PCR) --- p.114 / Chapter 3.7 --- Reverse transcription (RT) of mRNA and fluorescent PCR (fluoroDD PCR) --- p.125 / Chapter 3.8 --- Reamplification of fluorescent differential display (FDD) fragments --- p.138 / Chapter 3.9 --- Subcloning of reamplifled FDD fragments --- p.162 / Chapter 3.10 --- Sequencing of subcloned cDNA fragments --- p.176 / Chapter 3.11 --- Northern blot analysis of sequenced cDNA fragments --- p.195 / Chapter Chapter 4 --- Discussion --- p.250 / Chapter 4.1 --- Body weight changes --- p.250 / Chapter 4.2 --- Organ weight changes --- p.251 / Chapter 4.3 --- Serum cholesterol and triglyceride levels --- p.253 / Chapter 4.4 --- Liver histology --- p.254 / Chapter 4.5 --- "Functions and roles of identified PPARa-dependent and Wy-14,643- responsive genes" --- p.255 / Chapter 4.6 --- Mechanism of PP-induced hepatocarcinogeneis --- p.270 / Chapter Chapter 5 --- Conclusions --- p.274 / References --- p.276 / Appendix A Tables of preparation of reaction mix --- p.285 / Table A1. Preparation of animal tail genotyping PCR reaction --- p.285 / Table A2. Preparation of DNase I treatment --- p.285 / Table A3. Preparation of reverse transcription of non-fluoroDD and fluoroDD --- p.285 / Table A4. Preparation of non-fluoroDD and fluoroDD RT-PCR --- p.286 / Table A5. Preparation of reamplification of differentially expressed cDNA fragments --- p.286 / Table A6. Preparation of PCR reaction for DNA sequencing --- p.286 / Table A7. Preparation of PCR reaction for RNA probe --- p.287 / Table A8. Preparation of PCR reaction for cDNA probe --- p.287 / Appendix B DNA sequences and sequencing alignments of FluoroDD Fragments --- p.288 / Chapter B 1.1: --- DNA sequence of cDNA subclone AA1#2 (AP1 & ARP2) using M13 forward (-20) primer --- p.288 / Chapter B 1.2: --- "Sequencing alignment of cDNA subclone AA1#2 with mouse peroxisomal delta 3, delta 2-enoyl-Coenzyme A isomerase (Peci) by BLAST searching against the National Center for Biotechnology Information database" --- p.288 / Chapter B 1.3: --- Summary of sequence alignment of cDNA subclone AA1#2 with mouse Peci --- p.288 / Chapter B 2.1: --- DNA sequence of cDNA subclone AA1#3 (AP1 & ARP2) using M13 forward (-20) primer --- p.289 / Chapter B 2.2: --- "Sequencing alignment of cDNA subclone AA1#3 with mouse peroxisomal delta 3, delta 2-enoyl-Coenzyme A isomerase (Peci) by BLAST searching against the National Center for Biotechnology Information database" --- p.289 / Chapter B 2.3: --- Summary of sequence alignment of cDNA subclone AA1#3 with mouse Peci --- p.289 / Chapter B 3.1: --- DNA sequence of cDNA subclone AA1#4 (AP 1 & ARP2) using Ml3 reverse primer --- p.290 / Chapter B 3.2: --- "Sequencing alignment of cDNA subclone AA1#4 with mouse peroxisomal delta 3, delta 2-enoyl-Coenzyme A isomerase (Peci) by BLAST searching against the National Center for Biotechnology Information database" --- p.290 / Chapter B 3.3: --- Summary of sequence alignment of cDNA subclone AA1#4 with mouse Peci --- p.290 / Chapter B 4.1: --- DNA sequence of cDNA subclone AA1#20 (AP 1 & ARP2) using Ml3 forward (-20) primer --- p.291 / Chapter B 4.2: --- "Sequencing alignment of cDNA subclone AA1#20 with mouse peroxisomal delta 3, delta 2- enoyl-Coenzyme A isomerase (Peci) by BLAST searching against the National Center for Biotechnology Information database" --- p.291 / Chapter B 4.3: --- Summary of sequence alignment of cDNA subclone AA1#20 with mouse Peci --- p.291 / Chapter B 5.1: --- DNA sequence of cDNA subclone AA4#1 (AP 1 & ARP2) using Ml3 forward (-20) primer --- p.292 / Chapter B 5.2: --- Sequencing alignment of cDNA subclone AA4#1 with mouse apolipoprotein A-V (Apoa5) by BLAST searching against the National Center for Biotechnology Information database --- p.292 / Chapter B 5.3: --- Summary of sequence alignment of cDNA subclone AA4#1 with mouse Apoa5 --- p.292 / Chapter B 6.1: --- DNA sequence of cDNA subclone AA4#9 (AP 1 & ARP2) using Ml3 reverse primer --- p.293 / Chapter B 6.2: --- Sequencing alignment of cDNA subclone AA4#9 with mouse apolipoprotein A-V (Apoa5) by BLAST searching against the National Center for Biotechnology Information database --- p.293 / Chapter B 6.3: --- Summary of sequence alignment of cDNA subclone AA4#9 with mouse Apoa5 --- p.293 / Chapter B 7.1: --- DNA sequence of cDNA subclone AA5#5 (AP 1 & ARP2) using Ml3 forward (-20) primer --- p.294 / Chapter B 7.2: --- Sequencing alignment of cDNA subclone AA5#5 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.294 / Chapter B 7.3: --- Summary of sequence alignment of cDNA subclone AA5#5 with mouse mitochondrion --- p.294 / Chapter B 8.1: --- DNA sequence of cDNA subclone AA6#1 (AP1 & ARP2) using Ml3 forward (-20) primer --- p.295 / Chapter B 8.2: --- Sequencing alignment of cDNA subclone AA6#1 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.295 / Chapter B 8.3: --- Summary of sequence alignment of cDNA subclone AA6#1 with mouse mitochondion --- p.295 / Chapter B 9.1: --- DNA sequence of cDNA subclone AA6#9 (AP 1 & ARP2) using Ml3 reverse primer --- p.296 / Chapter B 9.2: --- Sequencing alignment of cDNA subclone AA6#9 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.296 / Chapter B 9.3: --- Summary of sequence alignment of cDNA subclone AA6#9 with mouse mitochondrion --- p.296 / Chapter B 10.1: --- DNA sequence of cDNA subclone AA7#3 (AP 1 & ARP2) using Ml3 forward (-20) primer --- p.297 / Chapter B 10.2: --- Sequencing alignment of cDNA subclone AA7#3 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.297 / Chapter B 10.3: --- Summary of sequence alignment of cDNA subclone AA7#3 with mouse mitochondrion --- p.297 / Chapter B 11.1: --- DNA sequence of cDNA subclone AA7#5 (AP 1 & ARP2) using Ml3 reverse primer --- p.298 / Chapter B 11.2: --- Sequencing alignment of cDNA subclone AA7#5 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.298 / Chapter B 11.3: --- Summary of sequence alignment of cDNA subclone AA7#5 with mouse mitochondrion --- p.298 / Chapter B 12.1: --- DNA sequence of cDNA subclone AA10#1 (AP1 & ARP2) using M l3 forward (-20) primer --- p.299 / Chapter B 12.2: --- Sequencing alignment of cDNA subclone AA10#1 with mouse cysteine sulfinic acid decarboxylase (Csad) by BLAST searching against the National Center for Biotechnology Information database --- p.299 / Chapter B 12.3: --- Summary of sequence alignment of cDNA subclone AA10#1 with mouse Csad --- p.299 / Chapter B 13.1: --- DNA sequence of cDNA subclone AA10#1 (AP 1 & ARP2) using M13 reverse primer --- p.300 / Chapter B 13.2: --- Sequencing alignment of cDNA subclone AA10#1 with mouse cysteine sulfinic acid decarboxylase (Csad) by BLAST searching against the National Center for Biotechnology Information database --- p.300 / Chapter B 13.3: --- Summary of sequence alignment of cDNA subclone AA10#1 with mouse Csad --- p.300 / Chapter B 14.1: --- DNA sequence of cDNA subclone AA12#4 (AP1 & ARP2) using Ml3 forward (-20) primer --- p.301 / Chapter B 14.2: --- "Sequencing alignment of cDNA subclone AA12#4 with mouse acetyl-coenzyme A dehydrogenase, medium chain (MCAD) by BLAST searching against the National Center for Biotechnology Information database" --- p.301 / Chapter B 14.3: --- Summary of sequence alignment of cDNA subclone AA12#4 with mouse MCAD --- p.301 / Chapter B 15.1: --- DNA sequence of cDNA subclone AA12#4 (AP 1 & ARP2) using Ml3 reverse primer --- p.302 / Chapter B 15.2: --- "Sequencing alignment of cDNA subclone AA12#4 with mouse acetyl-coenzyme A dehydrogenase, medium chain (MCAD) by BLAST searching against the National Center for Biotechnology Information database" --- p.302 / Chapter B 15.3: --- Summary of sequence alignment of cDNA subclone AA12#4 with mouse MCAD --- p.302 / Chapter B 16.1: --- DNA sequence of cDNA subclone AB7#2 (AP3 & ARP3) using Ml3 forward (-20) primer --- p.303 / Chapter B 16.2: --- "Sequencing alignment of cDNA subclone AB7#2 with mouse UDP-glucuronosyltransferase 2 family, member 5 (UGT2b5) by BLAST searching against the National Center for Biotechnology Information database" --- p.303 / Chapter B 16.3: --- Summary of sequence alignment of cDNA subclone AB7#2 with mouse UGT2b5 --- p.303 / Chapter B 17.1: --- DNA sequence of cDNA subclone AB7#8 (AP3 & ARP3) using M13 reverse primer --- p.304 / Chapter B 17.2: --- "Sequencing alignment of cDNA subclone AB7#8 with mouse UDP-glucuronosyltransferase 2 family, member 5 (UGT2b5) by BLAST searching against the National Center for Biotechnology Information database" --- p.304 / Chapter B 17.3: --- Summary of sequence alignment of cDNA subclone AB7#8 with mouse UGT2b5 --- p.304 / Chapter B 18.1: --- DNA sequence of cDNA subclone AB17#16 (AP3 & ARP3) using M13 reverse primer --- p.305 / Chapter B 18.2: --- Sequencing alignment of cDNA subclone AB17#16 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.305 / Chapter B 18.3: --- Summary of sequence alignment of cDNA subclone AB17#16 with mouse mitochondrion --- p.305 / Chapter B 19.1: --- DNA sequence of cDNA subclone AB18#4 (AP3 & ARP3) using M13 forward (-20) primer --- p.306 / Chapter B 19.2: --- Sequencing alignment of cDNA subclone AB18#4 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.306 / Chapter B 20.1: --- DNA sequence of cDNA subclone AB18#4 (AP3 & ARP3) using M13 reverse primer --- p.307 / Chapter B 20.2: --- Sequencing alignment of cDNA subclone AB18#4 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.307 / Chapter B 20.3: --- Summary of sequence alignment of cDNA subclone AB 18#4 with mouse mitochondrion --- p.307 / Chapter B 21.1: --- DNA sequence of cDNA subclone AB19#2 (AP3 & ARP3) using M13 forward (-20) primer --- p.308 / Chapter B 21.2: --- Sequencing alignment of cDNA subclone AB 19#2 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.308 / Chapter B 21.3: --- Summary of sequence alignment of cDNA subclone AB19#2 with mouse mitochondrion --- p.308 / Chapter B 22.1: --- DNA sequence of cDNA subclone AB19#10 (AP3 & ARP3) using Ml3 reverse primer --- p.309 / Chapter B 22.2: --- Sequencing alignment of cDNA subclone AB 19#10 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.309 / Chapter B 22.3: --- Summary of sequence alignment of cDNA subclone AB19#10 with mouse mitochondrion --- p.309 / Chapter B 23.1: --- DNA sequence ofcDNA subclone AB22#9 (AP3 & ARP3) using M13 forward (-20) primer --- p.310 / Chapter B 23.2: --- Sequencing alignment of cDNA subclone AB22#9 with mouse peroxisome biogenesis factor 16 (Pexl6) by BLAST searching against the National Center for Biotechnology Information database --- p.310 / Chapter B 23.3: --- Summary of sequence alignment of cDNA subclone AB22#9 with mouse Pexl6 --- p.310 / Chapter B 24.1: --- DNA sequence of cDNA subclone AB22#9 (AP3 & ARP3) using Ml3 reverse primer --- p.311 / Chapter B 24.2: --- Sequencing alignment of cDNA subclone AB22#9 with mouse peroxisome biogenesis factor 16 (Pexl6) by BLAST searching against the National Center for Biotechnology Information database --- p.311 / Chapter B 24.3: --- Summary of sequence alignment of cDNA subclone AB22#9 with mouse Pexl6 --- p.311 / Chapter B 25.1: --- DNA sequence ofcDNA subclone AB24#9 (AP3 & ARP3) using Ml3 forward (-20) primer --- p.312 / Chapter B 25.2: --- Sequencing alignment of cDNA subclone AB24#9 with mouse Cyp4al4 by BLAST searching against the National Center for Biotechnology Information database --- p.312 / Chapter B 25.3: --- Summary of sequence alignment of cDNA subclone AB24#9 with mouse Cyp4al4 --- p.312 / Chapter B 26.1: --- DNA sequence of cDNA subclone AB24#9 (AP3 & ARP3) using M13 reverse primer --- p.313 / Chapter B 26.2: --- Sequencing alignment of cDNA subclone AB24#9 with mouse Cyp4al4 by BLAST searching against the National Center for Biotechnology Information database --- p.313 / Chapter B 26.3: --- Summary of sequence alignment of cDNA subclone AB24#9 with mouse Cyp4al4 --- p.313 / Chapter B 27.1: --- DNA sequence of cDNA subclone AB25#6 (AP3 & ARP3) using Ml3 forward (-20) primer --- p.314 / Chapter B 27.2: --- Sequencing alignment of cDNA subclone AB25#6 with mouse Cyp4a l4 by BLAST searching against the National Center for Biotechnology Information database --- p.314 / Chapter B 27.3: --- Summary of sequence alignment of cDNA subclone AB25#6 with mouse Cyp4al4 --- p.314 / Chapter B 28.1: --- DNA sequence of cDNA subclone AB26#17 (AP3 & ARP3) using Ml3 forward (-20) primer --- p.315 / Chapter B 28.2: --- Sequencing alignment of cDNA subclone AB26#17 with mouse Cyp4al4 by BLAST searching against the National Center for Biotechnology Information database --- p.315 / Chapter B 28.3: --- Summary of sequence alignment of cDNA subclone AB26#17 with mouse Cyp4al4 --- p.315 / Chapter B 29.1: --- DNA sequence of cDNA subclone AB26#3Q (AP3 & ARP3) using M13 reverse primer --- p.316 / Chapter B 29.2: --- Sequencing alignment of cDNA subclone AB26#30 with mouse Cyp4al4 by BLAST searching against the National Center for Biotechnology Information database --- p.316 / Chapter B 29.3: --- Summary of sequence alignment of cDNA subclone AB26#30 with mouse Cyp4al4 --- p.316 / Chapter B 30.1: --- DNA sequence of cDNA subclone AB29#7 (AP3 & ARP3) using Ml3 forward (-20) primer --- p.317 / Chapter B 30.2: --- Sequencing alignment of cDNA subclone AB29#7 with mouse catalase by BLAST searching against the National Center for Biotechnology Information database --- p.317 / Chapter B 30.3: --- Summary of sequence alignment of cDNA subclone AB29#7 with mouse catalase --- p.317 / Chapter B 31.1: --- DNA sequence of cDNA subclone AC1#1 (AP2 & ARP19) using Ml3 forward (-20) primer --- p.318 / Chapter B 31.2: --- Sequencing alignment of cDNA subclone AC1#1 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.318 / Chapter B 31.3: --- Summary of sequence alignment of cDNA subclone AC1#1 with mouse SPI --- p.318 / Chapter B 32.1: --- DNA sequence of cDNA subclone AC1#1 (AP2 & ARP 19) using Ml3 reverse primer --- p.319 / Chapter B 32.2: --- Sequencing alignment of cDNA subclone AC 1# 1 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.319 / Chapter B 32.3: --- Summary of sequence alignment of cDNA subclone AC1#1 with mouse SPI --- p.319 / Chapter B 33.1: --- DNA sequence of cDNA subclone AC1#2 (AP2& ARP 19) using M13 forward (-20) primer --- p.320 / Chapter B 33.2: --- Sequencing alignment of cDNA subclone AC 1#2 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.320 / Chapter B 33.3: --- Summary of sequence alignment of cDNA subclone AC1#2 with mouse SPI --- p.320 / Chapter B 34.1: --- DNA sequence of cDNA subclone AC1#2 (AP2& ARP 19) using M13 reverse primer --- p.321 / Chapter B 34.2: --- Sequencing alignment of cDNA subclone AC1#2 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.321 / Chapter B 34.3: --- Summary of sequence alignment of cDNA subclone AC1#2 with mouse SPI --- p.321 / Chapter B 35.1: --- DNA sequence ofcDNA subclone AC2#2 (AP2 & ARP19) using Ml3 reverse primer --- p.322 / Chapter B 35.2: --- Sequencing alignment of cDNA subclone AC2#2 with mouse bifunctional enzyme (PBFE) by BLAST searching against the National Center for Biotechnology Information database --- p.322 / Chapter B 35.3: --- Summary of sequence alignment of cDNA subclone AC2#2 with mouse PBFE --- p.322 / Chapter B 36.1: --- DNA sequence of cDNA subclone AC2#5 (AP2 & ARP19) using Ml3 reverse primer --- p.323 / Chapter B 36.2: --- Sequencing alignment of cDNA subclone AC2#5 with mouse catalase by BLAST searching against the National Center for Biotechnology Information database --- p.323 / Chapter B 36.3: --- Summary of sequence alignment of cDNA subclone AC2#5 with mouse catalase --- p.323 / Chapter B 37.1: --- DNA sequence of cDNA subclone AC2#6 (AP2 & ARP19) using Ml3 forward (-20) primer --- p.324 / Chapter B 37.2: --- Sequencing alignment of cDNA subclone AC2#6 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.324 / Chapter B 37.3: --- Summary of sequence alignment of cDNA subclone AC2#6 with mouse SPI --- p.324 / Chapter B 38.1: --- DNA sequence ofcDNA subclone AC4#3 (AP2 & ARP19) using Ml3 forward (-20) primer --- p.325 / Chapter B 38.2: --- Sequencing alignment of cDNA subclone AC4#3 with mouse Cyp2a5 by BLAST searching against the National Center for Biotechnology Information database --- p.325 / Chapter B 38.3: --- Summary of sequence alignment of cDNA subclone AC4#3 with mouse Cyp2a5 --- p.325 / Chapter B 39.1: --- DNA sequence ofcDNA subclone AC4#3 (AP2 & ARP 19) using M13 reverse primer --- p.326 / Chapter B 39.2: --- Sequencing alignment of cDNA subclone AC4#3 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.326 / Chapter B 39.3: --- Summary of sequence alignment of cDNA subclone AC4#3 with mouse SPI --- p.326 / Chapter B 40.1: --- DNA sequence of cDNA subclone AC7#5 (AP2& ARP 19) using M13 forward (-20) primer --- p.327 / Chapter B 40.2: --- Sequencing alignment of cDNA subclone AC7#5 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.327 / Chapter B 40.3: --- Summary of sequence alignment of cDNA subclone AC7#5 with mouse SPI --- p.327 / Chapter B 41.1: --- DNA sequence of cDNA subclone AD6#4 (AP2 & ARP 18) using Ml3 reverse primer --- p.328 / Chapter B 41.2: --- Sequencing alignment of cDNA subclone AD6#4 with mouse N-terminal Asn amidase (Ntanl) by BLAST searching against the National Center for Biotechnology Information database --- p.328 / Chapter B 41.3: --- Summary of sequence alignment of cDNA subclone AD6#4 with mouse Ntanl --- p.328 / Chapter B 42.1: --- DNA sequence of cDNA subclone AD6#10 (AP2 & ARP 18) using Ml3 forward (-20) primer --- p.329 / Chapter B 42.2: --- Sequencing alignment of cDNA subclone AD6#10 with mouse Cyp4al0 by BLAST searching against the National Center for Biotechnology Information database --- p.329 / Chapter B 42.3: --- Summary of sequence alignment of cDNA subclone AD6#10 with mouse Cvp4al0 --- p.329 / Chapter B 43.1: --- DNA sequence of cDNA subclone AD6#10 (AP2 & ARP18) using M13 reverse primer --- p.330 / Chapter B 43.2: --- Sequencing alignment of cDNA subclone AD6#10 with mouse Cyp4al0 by BLAST searching against the National Center for Biotechnology Information database --- p.330 / Chapter B 43.3: --- Summary of sequence alignment of cDNA subclone AD6#10 with mouse Cyp4al0 --- p.330 / Chapter B 44.1: --- DNA sequence of cDNA subclone AD8#2 (AP2 & ARP 18) using M13 forward (-20) primer --- p.331 / Chapter B 44.2: --- Sequencing alignment of cDNA subclone AD8#2with mouse Cyp4a l0 by BLAST searching against the National Center for Biotechnology Information database --- p.331 / Chapter B 44.3: --- Summary of sequence alignment of cDNA subclone AD8#2 with mouse Cvp4a10 --- p.331 / Chapter B 45.1: --- DNA sequence ofcDNA subclone AD8#7 (AP2 & ARP18) using Ml3 reverse primer --- p.332 / Chapter B 45.2: --- Sequencing alignment of cDNA subclone AD8#7 with mouse Cyp4al0 by BLAST searching against the National Center for Biotechnology Information database --- p.332 / Chapter B 45.3: --- Summary of sequence alignment of cDNA subclone AD8#7 with mouse Cyp4a10 --- p.332 / Chapter B 46.1: --- DNA sequence of cDNA subclone AD9#2 (AP2 & ARP 18) using Ml3 forward (-20) primer --- p.333 / Chapter B 46.2: --- Sequencing alignment of cDNA subclone AD9#2 with mouse Cyp4al0 by BLAST searching against the National Center for Biotechnology Information database --- p.333 / Chapter B 46.3: --- Summary of sequence alignment of cDNA subclone AD9#2 with mouse Cyp4al0 --- p.333 / Chapter B 47.1: --- DNA sequence of cDNA subclone AD9#3 (AP2 & ARP 18) using M13 reverse primer --- p.334 / Chapter B 47.2: --- Sequencing alignment of cDNA subclone AD9#3 with mouse Cyp4al0 by BLAST searching against the National Center for Biotechnology Information database --- p.334 / Chapter B 47.3: --- Summary of sequence alignment of cDNA subclone AD9#3 with mouse Cvp4a10 --- p.334 / Chapter B 48.1: --- DNA sequence ofcDNA subclone AF1#8 (AP10 & ARP13) using M13 forward (-20) primer --- p.335 / Chapter B 48.2: --- Sequencing alignment of cDNA subclone AF1#8 with mouse very-long-chain acyl-coA synthetase (VLACS) by BLAST searching against the National Center for Biotechnology Information database --- p.335 / Chapter B 48.3: --- Summary of sequence alignment of cDNA subclone AF1#8 with mouse VLACS --- p.335 / Chapter B 49.1: --- DNA sequence of cDNA subclone AF1#8 (AP 10 & ARP 13) using Ml3 reverse primer --- p.336 / Chapter B 49.2: --- Sequencing alignment of cDNA subclone AF1#8 with mouse very-long-chain acyl-coA synthetase (VLACS) by BLAST searching against the National Center for Biotechnology Information database --- p.336 / Chapter B 49.3: --- Summary of sequence alignment of cDNA subclone AF1#8 with mouse VLACS --- p.336 / Chapter B 50.1: --- DNA sequence of cDNA subclone AF21#5 (AP 10 & ARP 13) using M13 reverse primer --- p.337 / Chapter B 50.2: --- "Sequencing alignment ofcDNA subclone AF21#5 with mouse cell death-inducing DNA fragmentation factor, alpha subunit-like effector B (Cideb) by BLAST searching against the National Center for Biotechnology Information database" --- p.337 / Chapter B 50.3: --- Summary of sequence alignment of cDNA subclone AF21#5 with mouse Cideb --- p.337 / Chapter B 51.1: --- DNA sequence ofcDNA subclone AF25#6 (AP10 & ARP13) using M13 forward (-20) primer --- p.338 / Chapter B 51.2: --- Sequencing alignment of cDNA subclone AF25#6 with mouse major urinary protein 2 (MUPII) by BLAST searching against the National Center for Biotechnology Information database --- p.338 / Chapter B 51.3: --- Summary of sequence alignment of cDNA subclone AF25#6 with mouse MUP II --- p.338 / Chapter B 52.1: --- DNA sequence of cDNA subclone AF25#7 (AP 10 & ARP 13) using Ml3 reverse primer --- p.339 / Chapter B 52.2: --- Sequencing alignment of cDNA subclone AF25#7 with mouse major urinary protein 2 (MUP II) by BLAST searching against the National Center for Biotechnology Information database --- p.339 / Chapter B 52.3: --- Summary of sequence alignment of cDNA subclone AF25#7 with mouse MUPII --- p.339 / Chapter B 53.1: --- DNA sequence ofcDNA subclone AF30#4 (AP10 & ARP13) using M13 forward (-20) primer --- p.340 / Chapter B 53.2: --- Sequencing alignment of cDNA subclone AF30#4 with mouse mRNA for suppressor of actin mutations (SAC1 gene) by BLAST searching against the National Center for Biotechnology Information database --- p.340 / Chapter B 53.3: --- Summary of sequence alignment of cDNA subclone AF3Q#4 with mouse SAC1 --- p.340 / Chapter B 54.1: --- DNA sequence of cDNA subclone AF30#5 (AP 10 & ARP 13) using Ml3 reverse primer --- p.341 / Chapter B 54.2: --- Sequencing alignment of cDNA subclone AF30#5 with mouse mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.341 / Chapter B 54.3: --- Summary of sequence alignment of cDNA subclone AF30#5 with mouse mitochondrion --- p.341 / Chapter B 55.1: --- DNA sequence ofcDNA subclone AH1#6 (AP11 & ARP19) using M13 forward (-20) primer --- p.342 / Chapter B 55.2: --- Sequencing alignment of cDNA subclone AH1#6 with mouse EST by BLAST searching against the National Center for Biotechnology Information database --- p.342 / Chapter B 55.3: --- Summary of sequence alignment of cDNA subclone AH1#6 with mouse EST --- p.342 / Chapter B 56.1: --- DNA sequence of cDNA subclone AIl#5 (AP6 & ARP4) using Ml3 forward (-20) primer --- p.343 / Chapter B 56.2: --- Sequencing alignment of cDNA subclone AIl#5 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST searching against the National Center for Biotechnology Information database --- p.343 / Chapter B 56.3: --- Summary of sequence alignment of cDNA subclone All#5 with mouse SPI --- p.343 / Chapter B 57.1: --- DNA sequence of cDNA subclone AI1#5 (AP6 & ARP4) using Ml3 reverse primer --- p.344 / Chapter B 57.2: --- Sequencing alignment of cDNA subclone AIl#5 with mouse serine (or cysteine) proteinase inhibitor (SPI) by BLAST --- p.344 / Chapter B 57.3: --- Summary of sequence alignment of cDNA subclone AIl #5 with mouse SPI --- p.344 / Chapter B 58.1: --- DNA sequence of cDNA subclone AI18#6 (AP6 & ARP4) using Ml3 forward (-20) primer --- p.345 / Chapter B 58.2: --- Sequencing alignment of cDNA subclone AI18#6 with mouse argininosuccinate lyase (Asl) by BLAST searching against the National Center for Biotechnology Information database --- p.345 / Chapter B 58.3: --- Summary of sequence alignment of cDNA subclone AI18#6 with mouse Asl --- p.345 / Chapter B 59.1: --- DNA sequence of cDNA subclone AI18#6 (AP6 & ARP4) using M13 reverse primer --- p.346 / Chapter B 59.2: --- Sequencing alignment of cDNA subclone AI18#6 with mouse argininosuccinate lyase (Asl) by BLAST searching against the National Center for Biotechnology Information database --- p.346 / Chapter B 59.3: --- Summary of sequence alignment of cDNA subclone AI18#6 with mouse Asl --- p.346 / Chapter B 60.1: --- DNA sequence ofcDNA subclone AJ1#4 (AP6 & ARP14) using Ml3 forward (-20) primer --- p.347 / Chapter B 60.2: --- Sequencing alignment of cDNA subclone AJ1#4 with mouse carboxylesterase by BLAST searching against the National Center for Biotechnology Information database --- p.347 / Chapter B 60.3: --- Summary of sequence alignment of cDNA subclone AJ1#4 with mouse carboxylesterase --- p.347 / Chapter B 61.1: --- DNA sequence ofcDNA subclone AJ1#5 (AP6 & ARP14) using Ml3 reverse primer --- p.348 / Chapter B 61.2: --- Sequencing alignment of cDNA subclone AJ1#5 with mouse carboxylesterase by BLAST searching against the National Center for Biotechnology Information database --- p.348 / Chapter B 61.3: --- Summary of sequence alignment of cDNA subclone AJ1#5 with mouse carboxylesterase --- p.348 / Chapter B 62.1: --- DNA sequence ofcDNA subclone AJ2#10 (AP6 & ARP14) using M13 forward (-20) primer --- p.349 / Chapter B 62.2: --- Sequencing alignment of cDNA subclone AJ2#10 with peroxisomal acyl-coA oxidase (AOX) by BLAST searching against the National Center for Biotechnology Information database --- p.349 / Chapter B 62.3: --- Summary of sequence alignment of cDNA subclone AJ2#10 with mouse AOX --- p.349 / Chapter B 63.1: --- DNA sequence ofcDNA subclone AJ2#10 (AP6 & ARP14) using Ml3 reverse primer --- p.350 / Chapter B 63.2: --- Sequencing alignment of cDNA subclone AJ2#10 with peroxisomal acyl-coA oxidase (AOX) by BLAST searching against the National Center for Biotechnology Information database --- p.350 / Chapter B 63.3: --- Summary of sequence alignment of cDNA subclone AJ2#10 with mouse AOX --- p.350 / Chapter B 64.1: --- DNA sequence ofcDNA subclone AJ9#1 (AP6 & ARP 14) using Ml3 forward (-20) primer --- p.351 / Chapter B 64.2: --- Sequencing alignment of cDNA subclone AJ9#1 with mouse catalase by BLAST searching against the National Center for Biotechnology Information database --- p.351 / Chapter B 64.3: --- Summary of sequence alignment of cDNA subclone AJ9#1 with mouse catalase --- p.351 / Chapter B 65.1: --- DNA sequence ofcDNA subclone AJ9#1 (AP6 & ARP14) using Ml3 reverse primer --- p.352 / Chapter B 65.2: --- Sequencing alignment of cDNA subclone AJ9#1 with mouse suppressor of actin mutations (SAC1 gene) by BLAST searching against the National Center for Biotechnology Information database --- p.352 / Chapter B 65.3: --- Summary of sequence alignment of cDNA subclone AJ9#1 with mouse SAC1 --- p.352 / Chapter B 66.1: --- DNA sequence ofcDNA subclone AL2#8 (AP7 & ARP15) using M13 forward (-20) primer --- p.353 / Chapter B 66.2: --- Sequencing alignment of cDNA subclone AL2#8 with mouse hydroxy steroid (17-beta) dehydrogenase 11 (Hsdl7pil) by BLAST searching against the National Center for Biotechnology Information database --- p.353 / Chapter B 66.3: --- Summary of sequence alignment of cDNA subclone AL2#8 with mouse HSD17β11 --- p.353 / Chapter B 67.1: --- DNA sequence of cDNA subclone AL3#3 (AP7& ARP 15) using Ml3 forward (-20) primer --- p.354 / Chapter B 67.2: --- Sequencing alignment of cDNA subclone AL3#3 with mouse hydroxy steroid (17-beta) dehydrogenase 11 (Hsdl7pll) by BLAST searching against the National Center for Biotechnology Information database --- p.354 / Chapter B 67.3: --- Summary of sequence alignment of cDNA subclone AL3#3 with mouse HSD17β11 --- p.354 / Chapter B 68.1: --- DNA sequence of cDNA subclone AL3#3 (AP7& ARP 15) using M13 reverse primer --- p.355 / Chapter B 68.2: --- Sequencing alignment of cDNA subclone AL3#3 with mouse hydroxysteroid (17-beta) dehydrogenase 11 (Hsdl7β1l) by BLAST searching against the National Center for Biotechnology Information database --- p.355 / Chapter B 68.3: --- Summary of sequence alignment of cDNA subclone AL3#3 with mouse HSD17β11 --- p.355 / Chapter B 69.1: --- DNA sequence of cDNA subclone AO1#2 (AP5 & ARP 10) 356 using Ml3 forward (-20) primer --- p.356 / Chapter B 69.2: --- Sequencing alignment of cDNA subclone AO1#2 with mouse 356 adipose differentiation related protein (ADFP) by BLAST searching against the National Center for Biotechnology Information database --- p.356 / Chapter B 69.3: --- Summary of sequence alignment of cDNA subclone AO1 #2 with 356 mouse ADFP --- p.356 / Chapter B 70.1: --- DNA sequence ofcDNA subclone AO1#5 (AP5 & ARP10) 357 using M13 reverse primer --- p.357 / Chapter B 70.2: --- Sequencing alignment of cDNA subclone AO1#5 with mouse 357 carnitine O-octanoyltransferase (Crot) by BLAST searching against the National Center for Biotechnology Information database --- p.357 / Chapter B 70.3: --- Summary of sequence alignment of cDNA subclone AO1 #5 with 357 mouse Crot --- p.357 / Chapter B 71.1: --- DNA sequence ofcDNA subclone AO2#6 (AP5 & ARP10) 358 using Ml3 forward (-20) primer --- p.358 / Chapter B 71.2: --- Sequencing alignment of cDNA subclone A02#6 with mouse 358 RNase A family 4 (Rnase4) by BLAST searching against the National Center for Biotechnology Information database --- p.358 / Chapter B 71.3: --- Summary of sequence alignment of cDNA subclone AO2#6 358 with mouse Rnase4 --- p.358 / Chapter B 72.1: --- DNA sequence of cDNA subclone AO2#6 (AP5 & ARP 10) 359 using Ml3 reverse primer --- p.359 / Chapter B 72.2: --- Sequencing alignment of cDNA subclone A02#6 with mouse 359 RNase A family 4 (Rnase4) by BLAST searching against the National Center for Biotechnology Information database --- p.359 / Chapter B 72.3: --- Summary of sequence alignment of cDNA subclone A02#6 359 with mouse Rnase4 --- p.359 / Chapter B 73.1: --- DNA sequence ofcDNA subclone AO2#8 (AP5 & ARP10) 360 using Ml3 reverse primer --- p.360 / Chapter B 73.2: --- Sequencing alignment of cDNA subclone A02#8 with mouse 360 carnitine O-octanoyltransferase (Crot) by BLAST searching against the National Center for Biotechnology Information database --- p.360 / Chapter B 73.3: --- Summary of sequence alignment of cDNA subclone AO2#8 with 360 mouse Crot --- p.360 / Chapter B 74.1: --- DNA sequence ofcDNA subclone AO8#2 (AP5 & ARP10) 361 using M13 forward (-20) primer --- p.361 / Chapter B 74.2: --- Sequencing alignment of cDNA subclone A08#2 with mouse 361 RNase A family 4 (Rnase4) by BLAST searching against the National Center for Biotechnology Information database --- p.361 / Chapter B 74.3: --- Summary of sequence alignment of cDNA subclone AO8#2 with 361 mouse Rnase4 --- p.361 / Chapter B 75.1: --- DNA sequence of cDNA subclone AP4#4 (AP12 & ARP2) 362 using Ml3 forward (-20) primer --- p.362 / Chapter B 75.2: --- Sequencing alignment of cDNA subclone AP4#4 with mouse 362 mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.362 / Chapter B 75.3: --- Summary of sequence alignment of cDNA subclone AP4#4 with 362 mouse mitochondrion --- p.362 / Chapter B 76.1: --- DNA sequence ofcDNA subclone AP4#4 (AP12 & ARP2) 363 using Ml3 reverse primer --- p.363 / Chapter B 76.2: --- Sequencing alignment of cDNA subclone AP4#4 with mouse 363 mitochondrion by BLAST searching against the National Center for Biotechnology Information database --- p.363 / Chapter B 76.3: --- Summary of sequence alignment of cDNA subclone AP4#4 with 363 mouse mitochondrion --- p.363

Medulloblastoma (MB) and supratentorial primitive neuroectodermal tumor (stPNET) are pediatric embryonic brain tumors, which arise in a brain that is in the process of growth and development. They differ significantly from adult lesions and may involve unique genetic and epigenetic factors. However, the pathogenesis of these tumors is still elusive. My project consisted of four parts, investigating major genetic and epigenetic alterations of these tumors. / Multiple genetic studies have shown high frequency of loss (30--60%) on chromosome 8p in MBs. Microcell-mediated transfer of chromosome 8 suppressed tumorigenesis or the proliferation of colon and breast cancer cell, indicating that chromosome 8p is likely to include several TSGs in human cancers. In previous studies from our laboratory, results showed the frequency of loss on chromosome 8p is also rather high (66.7%). An overlapping HD region was identified in a 1.8cM interval on 8p22-23.1, between markers D8S520 and D8S1130, in two MBs (Yin et al., 2002), indicating that several candidate TSGs are located within or near this region. PinX1 on 8p23.1, a potential inhibitor of telomerase, is most likely the candidate TSG in MBs due to its location and function. To evaluate the genetic alterations of PinX1 and to investigate its role in MBs, the first part of my study is to perform mutation analysis in a series of 52 primary MBs, 3 MB cell lines and 4 primary stPNETs. Transcript expression of PinX1 was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) in microdissected tumors and normal cerebellum. Using the telomeric repeat amplification protocol (TRAP) assay, 19 MBs, 2 stPNETs and all 3 MB cell lines were analyzed for telomerase activity. No somatic point mutations and loss of expression of PinX1 were detected in our series, suggesting that PinX1 is not the target gene on 8p23.1 in MBs. Although we did not find a significant association between PinX1 expression and telomerase activity, the presence of telomerase activity in 16 of 22 MBs and 1 of 2 stPNETs indicate that telomerase activation is associated with the development of this malignant disease. Our study represents the largest series of MB examined by telomerase repeat amplification protocol (TRAP) assay. (Abstract shortened by UMI.) / Chang Qing. / "April 2005." / Adviser: Ho-Keung Ng. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0191. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 201-228). / 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.

背景和目的：胰腺導管腺癌（簡稱胰腺癌）是世界範圍內惡性程度最高的癌癥之一，目前它的5 年生存率不到5%。大部分的病人在診斷初期就已經發展到了局部浸潤或遠處轉移的階段，因此失去了根治性手術切除的机会。輔助性化療對於胰腺癌病人來說是一個首選的治療方案，但是目前只有一小部分病人對化療藥物有良好的反應，而臨床化療失敗常與腫瘤細胞對化療藥物產生耐藥有關。吉西他濱是目前臨床上常用的一線抗癌藥物，但是它的耐藥現象在胰腺癌病人中廣泛存在，也是阻礙其臨床應用的主要原因之一。盡管已經有很多研究致力於揭示吉西他濱在胰腺癌細胞中的耐藥機理，目前臨床上仍然沒有有效的方法應對吉西他濱耐藥。我們的研究主要是為了探討一些以前沒有报道過的參與吉西他濱耐藥機理的基因，借此揭示胰腺癌細胞的吉西他濱耐藥的深層機制，為臨床上的治療提供理論依據。 / 實驗方法：我們實驗室之前在胰腺癌細胞株Capan2 中用全基因組RNAi篩選的方法確定LLGL1 作為抑癌基因能增強吉西他濱在胰腺癌細胞中的細胞毒性。我們隨後用體外細胞毒性分析實驗和皮下腫瘤動物模型來驗證LLGL1 是否能增強吉西他濱的細胞毒性，用蘇木素-伊紅染色和原味末端轉移酶標記技術分析抑制LLGL1 的表達是否會影響吉西他濱誘導的細胞雕亡反應。我們還應用微陣列分析技術進一步探尋LLGL1 的下遊靶蛋白，用實時定量PCR(qRT-PCR) 、蛋白印跡法（western blotting）、熒光素酶檢測等技術來進一步證實LLGL1 與下遊靶蛋白的關系，用免疫組織化學方法探究LLGL1 下遊靶蛋白在胰腺癌組織中的表達情況，以及該蛋白與LLGL1 的表達相關性，還應用染色體免疫共沈澱的方法探討轉錄因子Sp1(pThr453) 和RNA 聚合酶 II 在LLGL1 下遊靶蛋白的啟動子上的富集情況。 / 實驗結果：LLGL1 能增強吉西他濱在胰腺癌中的細胞毒性，抑制該基因的表達能誘導胰腺癌細胞對吉西他濱的耐藥，而上調該基因的表達則會增強胰腺癌細胞對吉西他濱的細胞毒性反應。OSMR 是LLGL1 的下遊靶蛋白, 其在胰腺癌組織中的表達與LLGL1 呈負性相關，抑制OSMR 的表達可以逆轉由LLGL1表達下調引起的吉西他濱耐藥現象。OSMR 表達上調可以增強腫瘤幹細胞標記物CD44 和CD24 的表達。另外，在胰腺癌細胞中，抑制LLGL1 的表達能激活ERK2/Sp1 信號通路，導致磷酸化Sp1(pThr453)的表達升高。OSMR 啟動子既沒有TATA 元件也沒有INR 元件，但是有Sp1 结合元件可供Sp1 結合。磷酸化Sp1(pThr453)可以結合到OSMR 啟動子的Sp1 结合元件上，從而促使RNA 轉錄酶II 結合到該啟動子上，啟動OSMR 基因的轉錄。 / 結論：我們的研究發現：1，LLGL1 能增強吉西他濱在胰腺癌中的細胞毒性，抑制該基因在胰腺癌細胞中的表達能上調OSMR 的表達，並誘導吉西他濱耐藥；2，OSMR 的表達在胰腺癌組織中與LLGL1 呈負性相關；3，下調LLGL1的表達能激活ERK2/Sp1 信號通路，進一步導致磷酸化Sp1(pThr453)和RNA 轉錄酶II 在OSMR 啟動子上的聚集，最終促使OSMR 的高表達，而下調LLGL1的表達能抑制該調節通路，從而抑制OSMR 的轉錄。 / Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant cancers worldwide. Its 5-year survival rate is less than 5%, because most patients have already developed to the advanced stage of local invasion or distant metastasis once diagnosed, and missed the chances of curable surgical resection. Adjuvant chemotherapy is an alternative therapeutic strategy against PDAC. Yet, only very small proportion of patients could benefit from chemotherapy due to the innate and easily-acquired chemo-resistance in PDAC cells, especially to the first-line chemotherapeutic drug, gemcitabine. Many studies have been conducted to exploring the mechanisms underlying gemcitabine resistance in PDAC cells, but gemcitabine resistance is still the major obstacle impeding PDAC patients benefits from chemotherapy. Our studies aimed to investigate novel genes involved in gemcitabine response and to explore the undefined mechanisms generating gemcitabine resistance in PDAC cells. / Methods: Our colleagues previously performed genome-wide RNAi screening in gemcitabine-sensitive Capan2 cells. Lethal giant larvae homolog 1 (LLGL1) was identified as a potential gemcitabine-sensitizing gene which was then validated by our subsequent in-vitro drug cytotoxicity assay in LLGL1-inhibited Capan2 and SW1990 cells and in vivo subcutaneous xenograft mouse model. Hematoxylin & Eosin staining and terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling were applied for the assessment of apoptotic effects induced by gemcitabine in subcutaneous xenografts. We did gene expression microarray analysis to explore the potential downstream targets of LLGL1. Western blotting, qRT-PCR, and luciferase assay were applied to validate the downstream target of LLGL1 that were figured out by microarray analysis. We also did immunohistochemical staining to investigate the expression levels and correlationship of LLGL1 and its downstream target in PDAC specimens. Chromatin immunoprecipitation was performed to explore the enrichment of the transcriptional factor Sp1(pThr453) and RNA polymerase II (Pol II) at the promoter of the downstream targets of LLGL1. / Results: LLGL1 was identified as a gemcitabine-sensitizing gene, whose inhibition remarkably reduced gemcitabine response in gemcitabine-sensitive Capan2 and SW1990 cells, and ectopic expression induced gemcitabine response in gemcitabine-resistant PANC1 cells. Oncostatin M receptor (OSMR) was identified as a downstream target of LLGL1, whose expression was negatively correlated with LLGL1, and knockdown of OSMR significantly reversed gemcitabine resistance induced by LLGL1 inhibition in Capan2 and SW1990 cells. Additionally, activation of OSMR signaling was associated with the elevated expression of cancer stem cell markers, CD44 and CD24, both of which had already been identified to contribute to gemcitabine resistance in PDAC cells. Moreover, OSMR up-regulation induced by LLGL1 inhibition in SW1990 cells depended on the activation of ERK2/Sp1 signaling and subsequent accumulation of Sp1(pThr453) and Pol II at the TATA-less, INR-less but Sp1-binding-site-rich promoter of OSMR, while ectopic expression of LLGL1 in PANC1 cells inactivated ERK2/Sp1 signaling and subsequently reduced the enrichment of Sp1(pThr453) and Pol II at OSMR promoter. / CONCLUSIONS: Our studies revealed the novel tumor suppressive role of LLGL1 as a gemcitabine-sensitizing gene in PDAC cells. Loss of LLGL1 resulted in the activation of ERK2/Sp1 signaling and up-regulation of OSMR expression, and ultimately desensitized gemcitabine response in PDAC cells. More importantly, ectopic expression of LLGL1 disrupted such regulatory axis and improved gemcitabine response. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhu, Yinxin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 154-183). / Abstracts also in Chinese.

by Cheung Kwok Kuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 111-130). / Chapter Chapter 1 --- General introduction of human ovarian cancer / Chapter 1.1 --- Epidemiology --- p.1 / Chapter 1.2 --- Symptoms and diagnosis --- p.4 / Chapter 1.3 --- Etiology --- p.5 / Chapter 1.3.1 --- Factors associated with decreased risks --- p.6 / Chapter 1.3.2 --- Factors associated with increased risks --- p.8 / Chapter 1.4 --- Classification of ovarian cancer --- p.12 / Chapter 1.5 --- Molecular basis of ovarian cancer --- p.18 / Chapter 1.6 --- Project aim --- p.29 / Chapter Chaper 2 --- "DOC-2, a differentially expressed gene in human ovarian cancer" / Chapter 2.1 --- Introduction --- p.32 / Chapter 2.2 --- Materials and Methods --- p.35 / Chapter 2.2.1 --- Expression of DOC-2 in human ovarian tissues --- p.35 / Chapter 2.2.1.1 --- Preparation of specimen --- p.35 / Chapter 2.2.1.2 --- Immunohistochemical studies of the expression of DOC-2 protein in human ovarian tissues --- p.35 / Chapter 2.2.1.3 --- Quantitation of immunoreactivity --- p.38 / Chapter 2.2.2 --- Effect of DOC-2 transfection on growth rate of the ovarian cancer cell lineSKOV3 --- p.39 / Chapter 2.2.2.1 --- Cell line --- p.39 / Chapter 2.2.2.2 --- Transfection of DOC-2 to SKOV3 ovarian carcinoma cell line --- p.39 / Chapter 2.2.2.3 --- Growth curve of the transfected ovarian carcinoma cell lines --- p.40 / Chapter 2.2.3 --- In vivo tumorigenicity study --- p.42 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- Expression of DOC-2 in human ovarian tissues --- p.44 / Chapter 2.3.2 --- Effects of DOC-2 transfected gene on the growth rate of the human ovarian cancer cell line SKOV3 --- p.46 / Chapter 2.3.2.1 --- Standard curves for calculating cell density from absorbance --- p.46 / Chapter 2.3.2.2 --- The effect of DOC-2 transfection on the growth rate of the human ovarian cancer cell line SKOV3 --- p.47 / Chapter 2.3.3 --- In vivo tumorigenicity --- p.48 / Chapter 2.4 --- Discussion --- p.50 / Chapter Chapter 3 --- DOC-2 expression in mouse embryonic development / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.60 / Chapter 3.2.1 --- Expression of murine homolog of DOC-2 (p96) during mouse embryonic development --- p.60 / Chapter 3.2.1.1 --- Preparation of paraffin-embedded mouse embryo sections --- p.60 / Chapter 3.2.1.2 --- Preparation of OCT-embedded mouse embryo sections --- p.61 / Chapter 3.2.1.3 --- Immunohistochemistry of murine homolog of DOC-2 (p96) on mouse embryos --- p.61 / Chapter 3.2.2 --- Effect of antibody blocking for DOC-2 protein on the growth of embryonic kidney in vitro --- p.62 / Chapter 3.3 --- Results --- p.64 / Chapter 3.4 --- Discussion --- p.69 / Chapter Chapter 4 --- Apoptosis / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.1.1 --- Current methods for the detection of apoptosis --- p.74 / Chapter 4.1.1.1 --- Agarose gel electrophoresis --- p.75 / Chapter 4.1.1.2 --- Flow cytometric analysis --- p.76 / Chapter 4.1.1.3 --- 3-OH end labelling --- p.77 / Chapter 4.1.1.4 --- Nuclease assay --- p.78 / Chapter 4.1.2 --- Apoptosis in normal physiology and oncogenesis --- p.78 / Chapter 4.1.3 --- p53 and apoptosis --- p.80 / Chapter 4.1.4 --- bcl-2 and apoptosis --- p.83 / Chapter 4.2 --- Materials and Methods --- p.92 / Chapter 4.2.1 --- Expression of p53 and bcl-2 in human ovarian tissues --- p.92 / Chapter 4.2.1.1 --- Preparation of specimens --- p.92 / Chapter 4.2.1.2 --- Immunohistochemical studies of the expression of p53 and bcl-2 proteins in ovarian tissue --- p.92 / Chapter 4.2.2 --- In stiu terminal transferase-mediated dUTP nick and labelling (TUNEL) --- p.94 / Chapter 4.3 --- Results --- p.96 / Chapter 4.3.1 --- Expression of p53 and bcl-2 in human ovarian tissues --- p.96 / Chapter 4.3.2 --- Apoptosis in human ovarian tissues --- p.99 / Chapter 4.4 --- Discussion --- p.101 / Chapter Chapter 5 --- Concluding Remarks --- p.108 / References --- p.111 / Appendix --- p.131 / Figures and legend --- p.138

Background & aims. DNA methylation induced tumor suppressor gene silencing plays an important role in carcinogenesis. By using methylation-sensitive representational difference analysis, we identified paired box gene 5 (PAX5) being methylated in human cancer. PAX5 locates at human chromosome 9p13.2 and encodes a 391 amino acids transcription factor. However, the role of PAX5 in gastric cancer is still unclear. Hence, we analyzed its epigenetic inactivation, biological functions, and clinical implications in gastric cancer. / Conclusions. Our results demonstrated that PAX5 promoter methylation directly mediates its transcriptional silence and commonly occurs in gastric cancer. PAX5 gene can act as a functional tumor suppressor in gastric carcinogenesis by playing an important role in suppression of cell proliferation, migration, invasion, and induction of cell apoptosis. Detection of methylated PAX5 may be utilized as a biomarker for the prognosis of gastric cancer patients. / Methods. Methylation status of PAX5 promoter in gastric cancer cell lines and clinical samples was evaluated by methylation specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS). The effects of PAX5 re-expression in cancer cell lines were determined in proliferation, cell cycle, apoptosis, migration and invasion assays. Its in vivo tumorigenicity was investigated by injecting cancer cells with PAX5 expression vector subcutaneously into the dorsal flank of nude mice. Chromosome Immunoprecipitation (ChIP) and cDNA expression array were performed to reveal the molecular mechanism of the biological function of PAX5. / Results. PAX5 was silenced or down-regulated in seven out of eight of gastric cancer cell lines examined. A significant down-regulation was also detected in paired gastric tumors compared with their adjacent non-cancer tissues (n = 18, P = 0.0196). In contrast, PAX5 is broadly expressed in all kinds of normal adult and fetal tissues. The gene expression of PAX5 in the gastric cancer cell line is closely linked to the promoter hypermethylation status. In addition, the expression levels could be restored by exposure to demethylating agents 5-aza-21-deoxycytidine. Re-expression of PAX5 in AGS, BGC823 and HCT116 cancer cells reduced colony formation (P < 0.01) and cell viability (P < 0.05), arrested cell cycle in G0/G1 phase (P = 0.0055), induced cell apoptosis (P < 0.05), repressed cell migration and invasion (P = 0.0218) in vitro. It also inhibited tumor growth in nude mice (P < 0.05). The molecular basis of its function were investigated by cDNA expression array and demonstrated that ectopic expression of PAX5 up-regulated tumor suppressor gene P53, anti-proliferation gene P21, pro-apoptosis gene BAX, anti-invasion gene MTSS1 and TIMP1; and down-regulated anti-apoptosis gene BCL2, cell cycle regulator cyclinD1, migration related gene MET and MMP1. ChIP assay indicated that P53 and MET are direct transcriptional target of PAX5. Moreover, PAX5 hypermethylation was detected in 90% (145 of 161) of primary gastric cancers compared with 16% (3 of 19) of non-cancer tissues (P < 0.0001). After a median follow-up period of 15.4 months, multivariate analysis revealed that gastric cancer patients with PAX5 methylation had a significant poor overall survival compared with the unmethylated cases (P = 0.0201). / Li, Xiaoxing. / Advisers: Hsiang Fu Kung; Jun Yu. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 134-159). / 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.

[Truncated abstract] Ovarian cancer is currently the leading cause of death from gynaecological malignancies in women from developed countries. Serous ovarian cancer is the most prevalent type of all ovarian cancers, with the majority diagnosed in an advanced stage where treatment efficacy is reduced and patient survival is poor. Because of this fact, the development of improved detection and treatment strategies are necessary, with much research focussing on the complex molecular pathways involved in ovarian tumour growth as one potential avenue for intervention. Apoptosis, or programmed cell death, is one such area of investigation because currently successful cancer treatments induce apoptosis in tumour cells. Molecular analysis of apoptosis in both normal tissue and tumours has established a positive relationship between increased expression of secreted frizzled-related protein 4 (SFRP4) and apoptosis, however to date, very little research has focussed on the role of this gene in the ovary . . . An examination of SFRP4 and β-catenin expression in 163 primary serous ovarian carcinomas revealed high SFRP4 expression was associated with low β-catenin expression and conversely, low SFRP4 was associated with high β-catenin expression in the majority of the ovarian tumours analysed, reinforcing the inverse relationship observed in the ovarian cell lines. A positive trend was observed between cancer stage and the expression level of these proteins, with increased SFRP4 expression and reduced β-catenin expression as cancer stage increased. Additionally, patient survival revealed a trend towards increased survival among ovarian cancer patients who had tumours expressing low levels of SFRP4. Taken together, the novel findings of this study indicate that the increased expression of SFRP4 observed in a large proportion of serous ovarian cancers is a cellular response to down-regulate the level of β-catenin, and thus an attempt to maintain cellular homeostasis by counteracting the excessive proliferating signals present in these tumour cells.

Mdm2 and Mdm4 are critical negative regulators of the p53 tumor suppressor. Mdm4-null mutants are severely anemic and exhibit impaired proliferation of the fetal liver erythroid lineage cells. This phenotype may indicate a cell-intrinsic function of Mdm4 in erythropoiesis. In contrast, red blood cell count was nearly normal in mice engineered to express low levels of Mdm2, suggesting that Mdm2 might be dispensable for red cell production. In the first part of the thesis, we further explore the tissue-specific functions of Mdm2 and Mdm4 in the erythroid lineage by crossing the conditional Mdm4 and Mdm2 alleles to an erythroid-specific-cre (EpoRGFP-Cre ) knock-in allele. Our data show that Mdm2 is required for rescuing erythroid progenitors from p53-mediated apoptosis during primitive erythropoiesis. In contrast, Mdm4 is only required for the high erythropoietic rate during embryonic definitive erythropoiesis. Thus, in this particular cellular context, interestingly, Mdm4 only contributes to p53 regulation at a specific phase of the differientation program.<p><p>Moreover, a large body of evidence indicates that aberrant expression of either MDM2 or MDM4 impairs p53 tumor suppression function and consequently favors tumor formation. Overexpression of MDM2 was observed in 10% of 8000 human cancers from various sites, including lung or stomach, and MDM4 was found amplified and/or overexpressed in 10-20% of over 800 diverse tumors including lung, colon, stomach and breast cancers. Remarkably, selective MDM4 amplification occurs in about 65% of human retinoblastomas. In contrast, MDM2 amplifications are relatively rare (about 5%) in retinoblastomas, indicating that depending on the tumor context (cell type, initiating oncogene, …), MDM4, rather than MDM2, overexpression might be selected for as a more efficient mean of suppression of p53 function. As part of a large effort to better understand why different cell types require distinct combinations of mutations to form tumours, we will examine the molecular basis for selective up-regulation of Mdm4 in retinoblastomas. In this context, we have successfully generated 2 conditional transgenic mouse lines expressing either mycMdm2 or mycMdm4 driven by the PCAGGs promoters in the ROSA26 locus. Since a cassette containing a floxed transcriptional stop element is inserted upstream of the transgenes, we can achieve tissue-specific expression and spatio-temporal regulation of the transgenes by using different Cre and CreER. By the use of N-terminal myc-tag fused with the transgenes, we are able to compare the expression levels of the transgenes. Finally, due to C-terminal IRES-GFP element, we can easily identify transgene expressing cells. One of our aims is to use this Mdm4 conditional transgenic mouse line as the first, non-chimeric, mouse model of retinoblastoma that can be used as an appropriate preclinical model to improve treatment of this disease.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished