Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 164-183). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 164-183). Also available on the Internet.

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

TAX1 Binding Protein 2 (TAX1BP2) has been found to be a centrosome duplication regulating protein. Previous findings have demonstrated that over-expression of TAX1BP2 suppresses centrosome over-duplication. Recently, our lab has revealed that TAX1BP2 is a novel tumor suppressor in hepatocellular carcinoma (HCC) regulated by cyclin-dependent protein kinase 2 (CDK2), nevertheless, the molecular mechanism of how TAX1BP2 regulates centrosome duplication and the link between its centrosome duplication regulatory ability and the tumor suppressing property remain elusive. With the aim to understand the roles of TAX1BP2 in HCC, the present study intended to investigate the link between centrosome duplication regulating ability and tumor suppressing property. Polo-like kinase 4 (PLK4) is a special member of the Polo-like kinase family as its structure is diverged from other family members. Instead of having two Polo-boxes, it carries one Polo-box and one cryptic Polo-box. It has been shown that PLK4 is involved in the formation of centrioles, an important component of centrosome, and is a key regulator of centrosome duplication. Based on the functional similarity, it was hypothesized that PLK4 may function as a regulator of TAX1BP2. To define if PLK4 regulate TAX1BP2, the interaction between PLK4 and TAX1BP2, both in vivo and in vitro, was first confirmed using affinity pulldown and co-immunoprecipitation assays. To understand the significance of the physical interaction, in vitro and in vivo kinase assay were used to study the phosphorylation activity between PLK4 and TAX1BP2. It was demonstrated that TAX1BP2 is a potential substrate of PLK4. Centrosome duplication assay was also performed to investigate if over-expression of PLK4 abolished the centrosome over-duplication suppressing ability of TAX1BP2. In order to delineate the signaling pathway of TAX1BP2, the interaction between TAX1BP2 and its cellular interacting partners was investigated in this study. Ten proteins were isolated as potential interacting partners of TAX1BP2 using Tandem affinity purification (TAP) coupled with Mass Spectrometry protein fingerprinting. Two of the ten proteins, the Ezrin and Mortalin, were confirmed to be binding partners of TAX1BP2 using affinity pull-down assay and TAP, respectively. The identification of the interacting partners suggested that TAX1BP2 may modulate centrosome duplication via alteration of the subcellular localization of Mortalin. These findings helped to delineate the signaling pathway of TAX1BP2 and enabled the better understanding of the roles of TAX1BP2 in tumor suppressor function of HCC. In summary, we demonstrated that TAX1BP2 contains a centrosome duplication regulatory domain (CDRD) and its centrosome duplication regulating ability is critical for its tumor suppressing property. Moreover, three novel interacting partners of TAX1BP2, including Ezrin, PLK4 and Mortalin, are identified. Our findings provide a new insight into the roles of TAX1BP2 in centrosome duplication, hepatocarcinogenesis and metastasis. / published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Thesis (Ph. D. in Cancer Biology)--Vanderbilt University, May 2005. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 143-162).

Dab2是一個細胞內接頭蛋白和腫瘤抑制因子。在小鼠胚胎中，應用免疫熒光染色技術，從E8.5-E11.0 Dab2發現表達於肌節的生皮肌節中。從E8.5 E9.5，Dab2表達於生皮肌節的中部。在E10.5，Dab2表達於生皮肌節的腹外側唇部，與肌肉發育的早期標誌基因Pax3和 Myf5共定位。從E11.5-E14.5，Dab2表達於四肢與軀體的肌肉中,Dab2在出生後小鼠肌肉中的表達逐漸減弱。此外，因為肌肉正常發育需要很多細胞信號的調節並且Dab2已經發現調節MAPK, TGF-β和 Wnt信號轉導通路。這些發現預示了Dab2在肌肉發育和分化中可能具有重要作用。 / 為了進一步研究它在肌肉發育中的作用，非洲爪蟾的胚胎和C2C12 肌原細胞在此研究中分別被用作體內和體外的研究模型。原位雜交結果揭示非洲爪蟾的Dab2基因表達於其胚胎的肌節中，並與肌肉發育的標誌基因XPax3, XMyoD, XMef2c和 XMyos共定位於此。用morpholino敲低XDab2 在非洲爪蟾胚胎中的表達，下調了許多肌肉發育標誌基因的表達，例如：XPax3, XMyf5, XMef2c, XMyoS 和XAC100。與此同時，免疫熒光技術也檢測到MHC(MF20)和12/101在肌節中的表達下調。 / 來源於小鼠肌肉衛星細胞的C2C12肌原細胞系被用作體外模型來檢測Dab2基因在骨骼肌發育和分化中的作用。在C2C12肌原細胞被誘導分化形成肌管的過程中，Dab2基因在RNA和蛋白水平的表達被誘導性的升高。Dab2基因超表達能夠加速肌原細胞的融合，從而增加肌小管的形成。利用miRNA敲低Dab2基因的表達能夠減緩肌原細胞的融合，從而減少肌小管的形成。利用慢病毒shRNA技術我們得到了2個Dab2穩定敲低細胞系，命名為克隆5-2和克隆5-7。這兩個克隆具有減少或抑制減少或抑制肌小管形成的特點。蛋白免疫印跡實驗表明，磷酸化p38 MAPK的表達在這兩個克隆中被抑制。在克隆5-2中超表達Dab2基因能夠恢復肌小管的形成。這個研究表明Dab2基因在肌小管的形成過程中具有至關重要的作用。 / 利用Affymetrix微陣列技術，我們檢測並分析了在克隆5-2和對照細胞中差異表達的基因。235個探針（155個基因）的顯示出超過2倍的差異表達。在這155個基因中，127個基因下調表達，28個基因上調表達。熒光定量PCR結果顯示出與微陣列結果相一致的結果。這些差異表達基因的功能發現與肌肉系統的發育和功能具有顯著地聯系。它影響了與肌肉收縮，橫紋肌的收縮，肌前體細胞的分化和肌肉發育相關功能的基因。基因網絡分析結果揭示，在克隆5-2中Mef2c基因的下調表達可能是一個導致肌細胞分化抑制的原因。 Mef2c基因在克隆5-2中超表達能夠拯救肌細胞的分化。 / 總括來說，體內和體外實驗共同表明Dab2基因是一個肌肉發育和分化的正調控基因。 / Dab2 is an intracellular adaptor protein and a tumor suppressor. In mouse embryos, Dab2 was found to be expressed in the dermomyotome of somites from E8.5 to E11.0 using immunofluorescence staining, with expression first detected in the medial aspect of the dermomyotome at E8.5 and then co-localized with the early muscle markers Pax3 and Myf5 at the ventrolateral lip of the dermomyotome at E10.5. From E11.5 to E14.5, Dab2 was expressed in muscle masses of limb buds and the trunk. Dab2 expression in skeletal muscles was gradually decreased after birth. These observations suggested potential roles of Dab2 in the skeletal muscle myogenesis. In addition, since the normal development of skeletal muscles requires proper signal transduction, and Dab2 has been known to be involved in the MAPK, TGF-β and Wnt signaling pathways, Dab2 may therefore be important for the muscle development. / To determine the role of Dab2 in the skeletal muscle development, Xenopus laevis embryos and C2C12 myoblasts were employed as in vivo and in vitro models, respectively. In situ hybridization results showed that XDab2 was expressed in somites of Xenopus embryos and co-localized with the muscle markers XPax3, XMyoD, XMef2c and XMyos. Knockdown of XDab2 expression with antisense morpholinos down regulated the expression of several muscle markers in somites including XPax3, XMyf5, XMef2c, XMyoS and XAC100. Down-regulation of MHC and 12/101 were also observed in whole mount preparations and transverse sections of XDab2 morpholino-injected embryos after immunohistochemical staining. / The C2C12 cell line derived from mouse muscle satellite cells was then employed as an in vitro model to determine the role of Dab2 during early muscle development. When C2C12 myoblasts were induced to differentiate into myotubes, Dab2 expression was simultaneously increased at RNA and protein levels. Dab2 over-expression after transfection with Dab2 plasmids resulted in enhanced myoblast fusion and increased numbers of myotubes. Conversely, suppression of Dab2 expression with miRNAs resulted in reduced myoblast fusion and decreased numbers of myotubes. Lentiviral shRNA-mediated Dab2 stable knockdown reduced myotube formation in 2 representative stable clones, clone 5-2 and clone 5-7. Western blot analysis showed that expression of phospho-p38 MAPK was down-regulated in clone 5-2 and 5-7. Dab2 re-expression through plasmid-mediated transient transfection in clone 5-2 could partially restore the myotube formation. These observations therefore suggested that Dab2 plays essential roles in the formation of myotubes. / Comprehensive profiling of differentially expressed genes was performed with the Affymetrix microarray analysis between the Dab2-knockdown clone 5-2 and the C2C12 parental cell line. As compared to the parental cells, the clone 5-2 showed significant changes in the expression of 235 probe sets representing 155 genes (p<0.05) with 2 folds or greater changes. Among the 155 genes, 127 were down-regulated, while 28 up-regulated. qRT-PCR results were found to be consistent with the microarray results. Functions of the differentially expressed genes were found to be significantly associated with the development and functions of the muscular system. Knockdown of Dab2 affected the genes involved in muscle contraction, the contraction of striated muscle, differentiation of muscle precursor cells, and the development of skeletal muscle fibers. A network analysis and a gene expression study revealed that Mef2c down-regulation was related to the inhibition of myogenic differentiation in the clone 5-2. Furthermore, forced expression of Mef2c in the clone 5-2 could rescue the myogenic differentiation. / In conclusion, these results indicated that Dab2 is positive regulator of the skeletal muscle development and differentiation both in vivo and in vitro. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Shang, Na. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 211-227). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgements --- p.vi / Table of contents --- p.vii / Abbreviation --- p.xiii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Characterizations of the Dab2 gene --- p.1 / Chapter 1.2 --- The role of Dab2 in Wnt/ beta-catenin signaling --- p.2 / Chapter 1.3 --- The role of Dab2 in TGF beta signaling --- p.3 / Chapter 1.4 --- The role of Dab2 in Ras-MAPK signaling --- p.4 / Chapter 1.5 --- The role of Dab2 in protein trafficking and endocytosis --- p.5 / Chapter 1.6 --- Dab2 expression and its functions. --- p.7 / Chapter 1.7 --- Somite and skeletal muscle development --- p.8 / Chapter 1.8 --- The formation of the somite and its structure --- p.9 / Chapter 1.9 --- The formation of dermomyotome and its function --- p.10 / Chapter 1.10 --- The formation of myotome and its function --- p.11 / Chapter 1.11 --- The formation of muscle fibers and musculatures --- p.12 / Chapter 1.12 --- The formation of satellite cells and its function in skeletal muscle differentiation --- p.12 / Chapter 1.13 --- The gene expression during skeletal muscle development and differentiation --- p.13 / Chapter 1.14 --- Dab2 genetically modified mice --- p.16 / Chapter 1.15 --- Objectives of this research --- p.17 / Chapter Figures and legends --- p.21 / Chapter Chapter 2 --- Expression of Dab2 in the mouse somites and skeletal muscles --- p.32 / Chapter 2.1 --- Introduction --- p.32 / Chapter 2.2 --- Materials and Methods --- p.34 / Chapter 2.2.1 --- Mouse embryos and tissue isolation --- p.34 / Chapter 2.2.2 --- Histological preparation of embryos and tissues --- p.34 / Chapter 2.2.3 --- Immunostaining using Tyramide signal amplification kits --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Dab2 expression in somites of the mouse embryos --- p.36 / Chapter 2.3.2 --- Dab2 expression in skeletal muscles of embryonic and postnatal mice --- p.36 / Chapter 2.3.3 --- Co-localization of Dab2 and Pax3 immunoreactivities with double immunofluorescence staining --- p.37 / Chapter 2.3.4 --- Co-localization of Dab2 and Myf5 immunoreactivities with double immunofluorescence staining --- p.38 / Chapter 2.3.5 --- Co-localization of Dab2 and Myogenin immunoreactivities with double immunofluorescence staining --- p.38 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.5 --- Summary --- p.42 / Chapter Table 2.1 --- p.44 / Chapter Figures and Legends --- p.45 / Chapter Chapter 3 --- Dab2 is a positive regulator of skeletal muscle development in Xenopus embryos --- p.58 / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and Methods --- p.61 / Chapter 3.2.1 --- RNA extraction --- p.61 / Chapter 3.2.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.61 / Chapter 3.2.3 --- Gene cloning and sequencing analysis --- p.61 / Chapter 3.2.4 --- Transformation --- p.62 / Chapter 3.2.5 --- Plasmid mini and midi-preparation --- p.62 / Chapter 3.2.6 --- Frogs and embryos handling --- p.63 / Chapter 3.2.7 --- Synthesis of mRNA for microinjection --- p.64 / Chapter 3.2.8 --- Microinjection --- p.64 / Chapter 3.2.9 --- Synthesis of DIG-labeled anti-sense RNA probe --- p.65 / Chapter 3.2.10 --- Whole mount in situ hybridization (WMISH) and whole mount immunohistochemical localization --- p.65 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Cloning of Xenopus Dab2 long isoform and the sequence analysis --- p.67 / Chapter 3.3.2 --- Phylogenetic analysis --- p.67 / Chapter 3.3.3 --- RT-PCR analysis of Xenopus Dab2 (XDab2) expression --- p.68 / Chapter 3.3.4 --- Xenopus Dab2 spatial and temporal expression examined by WMISH analysis --- p.68 / Chapter 3.3.5 --- Dab2 expression in somites and its colocalization with myogenic transcription factors --- p.69 / Chapter 3.3.6 --- XDab2 knockdown led to down-regulation of myogenic transcription factors and muscle markers at the RNA level --- p.70 / Chapter 3.3.7 --- XDab2 knockdown led to down-regulation of muscle markers at the protein level --- p.70 / Chapter 3.3.8 --- XDab2 overexpression led to up-regulation of XPax3, XMyf5 and XMyoS --- p.71 / Chapter 3.4 --- Discussion --- p.72 / Chapter 3.5 --- Summary --- p.77 / Chapter Table 3.1 --- p.78 / Chapter Figures and Legends --- p.79 / Chapter Chapter 4 --- Potential roles of Dab2 in C2C12 myoblast differentiation --- p.99 / Chapter 4.1 --- Introduction --- p.99 / Chapter 4.2 --- Materials and Methods --- p.101 / Chapter 4.2.1 --- Cell culture and differentiation in vitro --- p.101 / Chapter 4.2.2 --- Cell sample preparation --- p.102 / Chapter 4.2.3 --- Real-time PCR --- p.102 / Chapter 4.2.4 --- SDS-PAGE --- p.103 / Chapter 4.2.5 --- Western blotting and immunodetection --- p.104 / Chapter 4.2.6 --- Plasmids used for transient over-expression --- p.105 / Chapter 4.2.7 --- Generation of miRNAs targeting at Dab2 --- p.105 / Chapter 4.2.8 --- C2C12 differentiation after transfection --- p.106 / Chapter 4.2.9 --- Immunohistochemical staining for myotubes --- p.106 / Chapter 4.2.10 --- Lentiviral shRNA mediated Dab2 stable knockdown --- p.107 / Chapter 4.2.10.1 --- shRNA Lentiviral Transduction Particles and sequence information --- p.107 / Chapter 4.2.10.2 --- Optimization of puromycin treatment on C2C12 myoblasts --- p.107 / Chapter 4.2.10.3 --- Determination of the optimal MOI for C2C12 --- p.108 / Chapter 4.2.10.4 --- Lentivirus transduction method --- p.109 / Chapter 4.2.10.5 --- Stable cell line generation --- p.109 / Chapter 4.2.11 --- Rescue experiments --- p.109 / Chapter 4.2.12 --- Serum starvation and FGF treatment --- p.110 / Chapter 4.2.13 --- Microarray and data analysis --- p.110 / Chapter 4.3 --- Results --- p.113 / Chapter 4.3.1 --- Expression of Dab2 during myogenesis --- p.113 / Chapter 4.3.2 --- Generation of miRNAs targeting at Dab2 --- p.113 / Chapter 4.3.3 --- Improvement of the transfection efficiency --- p.114 / Chapter 4.3.4 --- Knockdown efficiencies of the 4 miRNAs --- p.114 / Chapter 4.3.5 --- Down-regulation of Dab2 expression by transient transfection inhibited C2C12 differentiation --- p.115 / Chapter 4.3.6 --- Up-regulation of Dab2 expression by transient transfection enhanced myogenic differentiation --- p.116 / Chapter 4.3.7 --- Lentivirus-mediated Dab2 stable knockdown inhibited myotube formation --- p.117 / Chapter 4.3.8 --- Re-expression of Dab2 partially restored myogenic differentiation in the clone 5-2 --- p.120 / Chapter 4.3.9 --- Dab2 knockdown affected the MAPK signaling pathway --- p.122 / Chapter 4.3.10 --- Transcriptome and network analysis revealed changes of gene expression patterns in the C2C12 cell line after Dab2 knockdown --- p.123 / Chapter 4.3.11 --- Mef2c down-regulation was related to the inhibition of the myotube formation in the clone 5-2 --- p.126 / Chapter 4.4 --- Discussion --- p.128 / Chapter 4.4.1 --- Dab2 expression was found to be induced upon differentiation and down-regulated after myotube formation --- p.128 / Chapter 4.4.2 --- Dab2 was found to be a positive regulator of C2C12 differentiation --- p.129 / Chapter 4.4.3 --- Dab2 knockdown affected the MAPK signaling pathway --- p.131 / Chapter 4.4.4 --- Potential roles of Dab2 in myogenic differentiation revealed by transcriptome and network analysis --- p.133 / Chapter 4.4.5 --- Mef2c down-regulation may be involved in the inhibition of myogenic differentiation after Dab2 knockdown --- p.135 / Chapter 4.5 --- Summary --- p.138 / Chapter Table 4.1 --- p.141 / Chapter Table 4.2 --- p.142 / Chapter Table 4.3 --- p.143 / Chapter Table 4.4 --- p.144 / Chapter Table 4.5 --- p.147 / Chapter Table 4.6 --- p.148 / Chapter Table 4.7 --- p.149 / Chapter Figures and Legends --- p.150 / Chapter Chapter 5 --- Conclusions and discussion --- p.192 / Chapter 5.1 --- Dab2 expression in somites and skeletal muscles of mouse embryos --- p.192 / Chapter 5.1 --- Dab2 as a positive regulator for skeletal muscle development in Xenopus embryos in vivo --- p.194 / Chapter 5.3 --- Dab2 as a positive regulator of skeletal muscle development in vitro --- p.196 / Chapter 5.3.1 --- Dab2 was found to be a positive regulator of C2C12 differentiation --- p.196 / Chapter 5.3.2 --- Dab2 knockdown affected the MAPK signaling pathway --- p.198 / Chapter 5.3.3 --- Potential functions of Dab2 revealed by transcriptomeand network analysis --- p.200 / Chapter 5.3.4 --- Mef2c down-regulation was closely related to the inhibition of myogenic differentiation upon Dab2 knockdown --- p.202 / Appendix I --- p.204 / Appendix II --- p.205 / References --- p.211

Yuen, Ka Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 160-185). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.IV / ACKNOWLEDGEMENTS --- p.VI / LIST OF ABBREVIATIONS --- p.VII / TABLE OF CONTENTS --- p.VIII / LIST OF TABLES --- p.XII / LIST OF FIGURES --- p.XIII / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- Pseudomalignant nature of the placenta --- p.2 / Chapter 1.1 --- Overview --- p.2 / Chapter 1.2 --- "Proliferation, migration and invasion behaviour" --- p.3 / Chapter 1.3 --- Gene expression --- p.4 / Chapter 1.3.1 --- Angiogenic factors --- p.5 / Chapter 1.3.2 --- Growth factors --- p.5 / Chapter 1.3.3 --- Proto-oncogenes --- p.6 / Chapter 1.3.4 --- Tumour suppressor genes --- p.8 / Chapter CHAPTER 2: --- Epigenetics --- p.10 / Chapter 2.1 --- Overview --- p.10 / Chapter 2.2 --- DNA methylation in mammals --- p.11 / Chapter 2.3 --- Regulation of DNA methylation machinery --- p.12 / Chapter 2.4 --- Role of DNA methylation --- p.13 / Chapter 2.5 --- Aberrant DNA methylation --- p.16 / Chapter 2.6 --- DNA methylation in normal cells --- p.17 / Chapter 2.6.1 --- X-chromosome inactivation --- p.17 / Chapter 2.6.2 --- Genomic imprinting --- p.18 / Chapter 2.6.3 --- Cell-type-specific methylation --- p.19 / Chapter 2.6.4 --- Placental-specific methylation --- p.20 / Chapter 2.7 --- Aim of Thesis --- p.21 / Chapter SECTION II: --- MATERIALS AND METHODOLOGY --- p.23 / Chapter CHAPTER 3: --- Materials and methods --- p.24 / Chapter 3.1 --- Preparation of samples --- p.24 / Chapter 3.1.1 --- Collection of placental tissues --- p.24 / Chapter 3.1.2 --- Preparation of blood cells --- p.25 / Chapter 3.1.3 --- Preparation of cell lines --- p.25 / Chapter 3.1.4 --- Treatment of JAR and JEG3 with 5-aza-2'-deoxycytidine (5-aza-CdR) and Trichostatin A (TSA) --- p.26 / Chapter 3.2 --- Nucleic acid extraction --- p.26 / Chapter 3.2.1 --- DNA extraction from tissue samples --- p.26 / Chapter 3.2.2 --- DNA extraction from blood cells --- p.29 / Chapter 3.2.3 --- RNA extraction from cell lines --- p.30 / Chapter 3.3 --- Methylation analysis --- p.31 / Chapter 3.3.1 --- Principles of bisulfite modification --- p.31 / Chapter 3.3.2 --- Bisulfite Conversion --- p.32 / Chapter 3.3.3 --- Primer design for methylation-specific polymerase chain reaction / Chapter 3.3.4 --- Methylation-specific polymerase chain reaction (MSP) --- p.33 / Chapter 3.3.5 --- Primer design for bisulfite sequencing --- p.34 / Chapter 3.3.6 --- Cloning and bisulfite genomic sequencing --- p.35 / Chapter 3.4 --- Quantitative measurements of nucleic acids --- p.39 / Chapter 3.4.1 --- Principles of real-time quantitative PCR --- p.39 / Chapter 3.4.2 --- Real-time quantitative MSP --- p.42 / Chapter 3.4.3 --- Real-time reverse transcriptase (RT)-PCR --- p.42 / Chapter 3.5 --- MALDI-TOF mass spectrometry (MS) --- p.43 / Chapter 3.5.1 --- Principle of homogeneous MassEXTEND assay and MALDI-TOF MS --- p.43 / Chapter 3.5.2 --- Methylation-sensitive restriction enzyme digestion and homogeneous MassEXTEND assay for APC and H19 --- p.46 / Chapter SECTION III: --- A SEARCH FOR HYPERMETHYLATED TUMOUR SUPPRESSOR GENES IN THE HUMAN PLACENTA --- p.48 / Chapter CHAPTER 4: --- Screening on TSGs and non TSGs --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.2 --- Materials and methods --- p.50 / Chapter 4.2.1 --- Sample collection --- p.50 / Chapter 4.2.2 --- Sample processing and DNA extraction --- p.50 / Chapter 4.2.3 --- Experimental Design --- p.51 / Chapter 4.3 --- Results --- p.63 / Chapter 4.3.1 --- Identification of hypermethylated TSGs by methylation-specific PCR screening --- p.63 / Chapter 4.3.2 --- Validation of hypermethylated TSGs by bisulfite sequencing --- p.69 / Chapter 4.4 --- Discussion --- p.77 / Chapter CHAPTER 5: --- Methylation status of TSGs in different tissues --- p.80 / Chapter 5.1 --- Introduction --- p.80 / Chapter 5.2 --- Materials and methods --- p.81 / Chapter 5.2.1 --- Sample collection --- p.81 / Chapter 5.2.2 --- Sample processing and DNA extraction --- p.81 / Chapter 5.2.3 --- Experimental design --- p.81 / Chapter 5.3 --- Results --- p.86 / Chapter 5.3.1 --- Methylation patterns of TSGs in non-placental fetal tissues --- p.86 / Chapter 5.4 --- Discussion --- p.90 / Chapter SECTION IV: --- FUNCTIONAL IMPLICATION OF HYPERMETHYLATED TUMOUR SUPPRESSOR GENES IN THE PLACENTA --- p.94 / Chapter CHAPTER 6: --- Imprinting checking --- p.95 / Chapter 6.1 --- Introduction --- p.95 / Chapter 6.2 --- Materials and methods --- p.96 / Chapter 6.2.1 --- Sample collection --- p.96 / Chapter 6.2.2 --- Sample processing and DNA extraction --- p.97 / Chapter 6.2.3 --- Experimental design --- p.97 / Chapter 6.3 --- Results --- p.100 / Chapter 6.3.1 --- Imprinting checking of H19 by enzyme digestion on placental tissues --- p.100 / Chapter 6.3.2 --- Imprinting checking of　APC by enzyme digestion on placental tissues --- p.101 / Chapter CHAPTER 7: --- CORRELATION OF HYPERMETHYLATION AND GENE EXPRESSION --- p.107 / Chapter 7.1 --- Introduction --- p.107 / Chapter 7.2 --- Materials and methods --- p.108 / Chapter 7.2.1 --- Sample preparation and processing --- p.108 / Chapter 7.2.2 --- DNA and RNA extraction from cell lines --- p.108 / Chapter 7.2.3 --- Experimental design --- p.108 / Chapter 7.3 --- Results --- p.111 / Chapter 7.3.1 --- Methylation status of APC in choriocarcinoma cell lines --- p.111 / Chapter 7.3.2 --- Demethylation of APC in choriocarcinoma cell lines --- p.114 / Chapter 7.4 --- Discussion --- p.115 / Chapter SECTION V: --- CONSERVATION OF METHYLATION IN PLACENTA ACROSS DIFFERENT SPECIES --- p.118 / Chapter CHAPTER 8: --- Methylation analysis of hypermethylated TSG homologues in the placentas of the mouse and rhesus monkey --- p.119 / Chapter 8.1 --- Introduction --- p.119 / Chapter 8.2 --- Materials and methods --- p.120 / Chapter 8.2.1 --- Sample collection --- p.120 / Chapter 8.2.2 --- Sample processing and DNA extraction --- p.120 / Chapter 8.2.3 --- Experimental design --- p.120 / Chapter 8.3 --- Results --- p.124 / Chapter 8.3.1 --- Methylation status of TSGs in rhesus monkey and murine placental tissues --- p.124 / Chapter 8.4 --- Discussion --- p.136 / Chapter SECTION VI: --- CONCLUDING REMARKS --- p.138 / Chapter CHAPTER 9: --- Conclusion and future perspectives --- p.139 / Chapter 9.1 --- Pseudomalignant nature of placenta at the epigenetic level --- p.139 / Chapter 9.2 --- Functional implication of TSG hypermethylation --- p.140 / Chapter 9.3 --- Significance of hypermethylated TSGs in the placental evolution --- p.142 / Chapter 9.4 --- Clinical implication of TSG hypermethylation --- p.143 / Chapter 9.5 --- Future perspectives --- p.145 / APPENDIX I COMPLETE BISULFITE SEQUENCING DATA FOR HYPERMETHYLATED TSGS --- p.147 / APPENDIX II BISULFITE SEQUENCING DATA FOR PTEN --- p.156 / APPENDIX III BISULFITE SEQUENCING DATA OF LOCI NOT SHOWING HYPERMETHYLATION --- p.158 / REFERENCES --- p.160

BRCA1 preserves genome integrity through both homology-directed repair (HDR) and stalled fork protection (SFP). In vivo, BRCA1 exists as a heterodimer with the BARD1 tumor suppressor, and both proteins harbor a C-terminal BRCT domain with a phospho-recognition surface. Most pathogenic lesions of BRCA1 and BARD1 disrupt their respective BRCT domains, and BRCA1 BRCT phospho-recognition is required for its tumor suppression activity. Here we evaluate mice with mutations (Bard1S563F and Bard1K607A) that ablate Bard1 BRCT phospho-recognition. Although not affecting HDR, these mutations impair BRCA1/BARD1 recruitment to stalled replication forks, resulting in stalled fork degradation, chromosomal instability, and sensitivity to PARP inhibitors. However, Bard1S563F/S563F and Bard1K607A/K607A mice are not tumor-prone, indicating that ablation of SFP activity alone is insufficient for spontaneous tumor susceptibility. Nevertheless, since SFP, unlike HDR, is also impaired in Brca1/Bard1 heterozygous-mutant cells, SFP and HDR may contribute to distinct stages of tumor development in BRCA1/BARD1 mutation carriers.