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Wong Pak Cheung Ronald. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 121-142). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract --- p.II / Abbreviation --- p.VI / List of figures --- p.IX / List of tables --- p.XI / Content --- p.XII / Content / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- The p53 tumor suppressor gene --- p.2 / Chapter 1.1.1 --- Structure and function --- p.2 / Chapter 1.1.2 --- Regulation of p53 stability and activity --- p.3 / Chapter 1.1.3 --- p53 and cell cycle arrest --- p.4 / Chapter 1.1.4 --- p53 and apoptosis --- p.4 / Chapter 1.2 --- Mutation in p53 gene --- p.9 / Chapter 1.2.1 --- Loss of function through dominant negative effect --- p.9 / Chapter 1.2.2 --- Gain-of-function through transactivation by mutant p53 --- p.10 / Chapter 1.2.3 --- Mutation in p53 and resistance to cancer therapy --- p.10 / Chapter 1.3 --- Objective of the study --- p.14 / Chapter Chapter 2: --- Mutant p53 induced interferon resistance and its regulation of the Jak/Stat pathway --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.1.1 --- IFN classification and biological activities --- p.16 / Chapter 2.1.2 --- IFN signaling --- p.17 / Chapter 2.1.3 --- IFN direct antitumor effect: cell cycle arrest and apoptosis --- p.18 / Chapter 2.1.4 --- IFN in immunotherapy --- p.20 / Chapter 2.1.5 --- Resistance to IFN therapy --- p.21 / Chapter 2.2 --- Materials and Methods --- p.24 / Chapter 2.2.1 --- Cell lines --- p.24 / Chapter 2.2.2 --- Drugs and antibodies --- p.24 / Chapter 2.2.3 --- Cell Proliferation assay- MTT assay --- p.24 / Chapter 2.2.4 --- Cell cycle analysis --- p.25 / Chapter 2.2.5 --- DNA fragmentation assay --- p.25 / Chapter 2.2.6 --- Western blot analysis --- p.26 / Chapter 2.2.7 --- "Combined treatment of IFNs and Jak inhibitors in MTT assay, DNA fragmentation assay and Western blot analysis" --- p.26 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Mutant p53-V143A and p53-R273H induced IFN resistance: the role of IFN induced apoptosis and cell cycle arrest --- p.27 / Chapter 2.3.2 --- IFN induction of apoptosis: a p53-independent and caspase-dependent pathway --- p.28 / Chapter 2.3.3 --- Mutant p53 regulation of Jak/Stat pathway --- p.36 / Chapter 2.3.4 --- Janus kinases (Jaks) and IFN-alpha sensitivity in Saos-2 cells --- p.41 / Chapter 2.3.5 --- Janus kinases (Jaks) and IFN-gamma sensitivity --- p.49 / Chapter 2.4 --- Discussion --- p.56 / Chapter 2.4.1 --- Mutant p53-V143 and p53-R273H induced IFN resistance in Saos-2 cells --- p.56 / Chapter 2.4.2 --- Role of Jaks in IFN sensitivity in Saos-2 cells --- p.57 / Chapter 2.4.3 --- IFN signaling in Saos-2 cells --- p.57 / Chapter 2.4.4 --- Jak2 and IFN induced apoptosis --- p.58 / Chapter Chapter 3: --- Mutant p53 induced drug resistance --- p.60 / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.1.1 --- The multidrug resistance (MDR) --- p.61 / Chapter 3.1.2 --- Anticancer drugs used in the study: action mechanisms and resistance --- p.67 / Chapter 3.1.3 --- Jak/Stat pathway and MDR --- p.68 / Chapter 3 .2 --- Materials and Methods --- p.72 / Chapter 3.2.1 --- Cell lines --- p.72 / Chapter 3.2.2 --- Drugs and antibodies --- p.72 / Chapter 3.2.3 --- Caspase 3 activity assay --- p.72 / Chapter 3.2.4 --- Cell Proliferation assay- MTT assay --- p.73 / Chapter 3.2.5 --- Cell cycle analysis --- p.73 / Chapter 3.2.6 --- DNA fragmentation assay --- p.73 / Chapter 3.2.7 --- Reverse transcription polymerase chain reaction --- p.73 / Chapter 3.2.8 --- Western blot analysis --- p.74 / Chapter 3.2.9 --- "Combined treatment of IFNs and Jak inhibitors in MTT assay, DNA fragmentation assay and Western blot analysis" --- p.74 / Chapter 3.3 --- Results --- p.75 / Chapter 3.3.1 --- Mutant p53 and drug sensitivity --- p.75 / Chapter 3.3.2 --- Mutant p53 and drug induced apoptosis and cell cycle arrest --- p.75 / Chapter 3.3.3 --- Classical drug resistance factors in mutant p53 induced drug resistance --- p.87 / Chapter 3.3.4 --- The role of Jaks in drug sensitivity of Saos-2 cells --- p.89 / Chapter 3.3.5 --- The role of Jaks in drug induced DNA fragmentationin Saos-2 cells --- p.89 / Chapter 3.3.6 --- Jak signaling and caspase activation in MTX induced apoptosis in Saos-2 cells --- p.100 / Chapter 3.3 --- Discussion --- p.108 / Chapter 3.3.1 --- Mutant p53-V143A and p53-R273H induced drug resistance in Saos-2 cells --- p.108 / Chapter 3.3.2 --- Role of Jaks in drug sensitivity in Saos-2 cells --- p.109 / Chapter 3.3.3 --- Jak/Stat signaling in Saos-2 cells --- p.109 / Chapter 3.3.4 --- Jak2 and MTX induced apoptosis --- p.110 / Chapter Chapter 4: --- General discussion --- p.112 / Chapter 4.1 --- Mutant p53 induced immunotherapy and chemotherapy resistance --- p.113 / Chapter 4.2 --- Gain of new function of mutant p53-V143A and p53-R273H in regulating Jak/Stat pathway leading to resistance to IFN and chemotherapeutic drugs --- p.114 / Chapter 4.3 --- The role of Jaks in MTX sensitivity --- p.114 / Chapter 4.4 --- Future work --- p.115 / Chapter 4.5 --- Perspective --- p.120 / References --- p.121

腫瘤發生過程中，遺傳和/或表觀遺傳異常都可導致抑癌基因(TSG)的失活。新TSG的鑒定和研究對理解癌症發展至關重要，并能提供潛在的治療靶點和腫瘤標誌物。本研究的目標是在人類腫瘤中鑒定新的被表觀遺傳異常沉默的TSG并進一步研究其抑癌的分子機理。 / 表觀遺傳修飾因子是重要的腫瘤相關基因。這裡，我研究了兩個作為功能性TSG的表觀遺傳修飾基因。首先，我發現一個在腫瘤中被甲基化和下調的候選TSG, PRDM5, 可通過抑制TCF/LEF依賴的轉錄和引起多個癌基因的表觀遺傳下調而抑制癌細胞增殖。其次，通過檢測24個表觀遺傳修飾基因在癌細胞系中的表達，我發現一個頻繁下調的新候選TSG，TUSC12。TUSC12在正常組織中普遍表達，但在腫瘤細胞系中被啟動子區甲基化下調，且原發腫瘤中也存在高頻TUSC12甲基化。TUSC12顯著抑制癌細胞克隆形成能力，但這種抑制效果因其PHD功能域的失活被部份削弱。TUSC12與轉錄抑制因子KAP1在細胞核中共定位，并可能通過招募HDAC相關複合體而抑制基因轉錄。 / 另外，我研究了一個通過以前鼻咽癌(NPC) aCGH鑒定得到的新3p14.2 TSG，ZNF312。啟動子區甲基化導致ZNF312在NPC細胞系和原發癌中的沉默。恢復ZNF312表達可抑制NPC細胞生長，并引起細胞週期阻滯和凋亡。作為一個轉錄抑制因子，ZNF312靶向EZH2和MDM2的啟動子區并下調它們的表達。 / 之前的基因數字表達分析已篩選出一些可能在腫瘤中低表達的基因。我研究了一個通過這種方法分離出的新TSG，TUSC45。相比正常組織，TUSC45表達在癌組織中顯著下降，而且TUSC45低表達與病人的低存活相關。TUSC45在多個腫瘤細胞系中也被下調，但它的基因組缺失卻不多見。啟動子區甲基化導致TUSC45在多數細胞系中的沉默，而且藥物或遺傳去甲基化能顯著激活它的表達。特別的，TUSC45在腫瘤組織而不在正常組織中被高頻率甲基化。誘導TUSC45表達可抑制細胞增殖，引起細胞凋亡、週期阻滯、和衰老，並上調一個關鍵抑癌基因p53。TUSC45以p53依賴的方式激活p53的靶基因，而TUSC45對p53缺失的H1299和HCT116/p53KO細胞沒有生長抑制作用。TUSC45與p53/MDM2複合物結合并正向調節p53蛋白穩定性。 TUSC45表達導致MDM2蛋白半衰期縮短，提示一種可能的TUSC45調節p53通路的機制。 / 本研究表明癌變過程中四個抑癌基因腫瘤特異的甲基化和沉默引起了多個細胞信號通路紊亂，並可作為潛在的腫瘤檢測標誌物。 / Tumor suppressor genes (TSGs) can be inactivated by genetic and/or epigenetic alterations during carcinogenesis. Identification and characterization of novel TSGs are critical for the understanding of cancer development, and provide potential targets for clinical treatment and biomarkers for tumor diagnosis. In this study, I aimed to identify novel TSGs epigenetically silenced in human cancers and further characterize the molecular basis of their anti-tumorigenic functions. / Emerging evidence highlights the importance of epigenetic modifiers as cancer genes. Here, I characterized two epigenetic modifier genes as functional TSGs. First, I found PRDM5, a candidate TSG methylated and downregulated in multiple cancers, suppressed tumor cell proliferation through inhibiting TCF/LEF-dependent transcription and inducing epigenetic repression of multiple oncogenes. Second, through expression profiling of 24 epigenetic modifiers, I identified a novel candidate TSG, TUSC12, showing frequent silencing in tumor cell lines. TUSC12 was broadly expressed in human normal tissues, but downregulated by promoter CpG methylation in tumor cell lines. Frequent TUSC12 methylation was detected in primary tumors as well. TUSC12 dramatically inhibited tumor cell clonogenicity, but this growth inhibitory effect was partially impaired by disrupting its PHD domain. TUSC12 colocalized with the transcription corepressor KAP1 in the nucleus and is likely to repress gene transcription through recruit HDAC-associated complex. / I also studied a novel 3p14.2 TSG, ZNF312, identified from previous aCGH profiling of nasopharyngeal carcinoma (NPC). Frequent promoter methylation silenced ZNF312 in NPC cell lines and tissues. Restored ZNF312 expression strongly suppressed NPC cell growth through inducing cell cycle arrest and apoptosis. Further, ZNF312 acted as a transcription repressor targeting the promoter regions of EZH2 and MDM2 and downregulating their expression. / Moreover, previous digital gene expression subtraction from cDNA libraries revealed a list of genes possibly downregulated in tumors compared to normal tissues. I characterized a novel TSG, TUSC45, initially isolated from this strategy. The expression of TUSC45 was significantly reduced in tumor tissues compared to normal tissues, with lower TUSC45 expression associated with poorer patient survival. Downregulation of TUSC45 in multiple tumor cell lines was also observed, while only infrequent genomic deletion was detected. In contrast, promoter methylation was responsible for TUSC45 silencing in most cell lines, in which pharmacologic or genetic demethylation can dramatically restore its expression. Remarkably, TUSC45 was frequently methylated in primary tumors but not in normal tissues. Further, TUSC45 suppressed anchorage-dependent and -independent tumor cell growth. Induced TUSC45 expression inhibited cell proliferation, induced apoptosis, cell cycle arrest and senescence, and lead to the upregulation of a key tumor suppressor p53. Moreover, TUSC45 activated p53 target genes in a p53-dependent manner. Forced TUSC45 expression in p53-null H1299 and HCT116/p53KO (knock out) cells showed no inhibitory effect on cell growth. Finally, TUSC45 interacted with p53/MDM2 complex and positively regulated p53 protein stability. The protein half-life of MDM2 was shortened by TUSC45, indicating a possible mechanism for TUSC45 modulation on p53 signaling. / In conclusion, this study showed the tumor-specific methylation and silencing of the four TSGs lead to the epigenetic disruption of multiple cell signalings during tumorigenesis and could potentially be used as biomarkers for cancer detection. / 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. / Shu, Xingsheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 121-140). / 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 / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / List of Publications --- p.xiv / Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction and Literature Reviews --- p.1 / Chapter 1.1 --- Tumor suppressor genes (TSGs) and the pathways they control --- p.2 / Chapter 1.1.1 --- Basic concepts about TSG --- p.2 / Chapter 1.1.1.1 --- The discovery of TSG --- p.2 / Chapter 1.1.1.2 --- Knudson’s “two-hit“ hypothesis --- p.3 / Chapter 1.1.1.3 --- New insights of the “two-hit“ model --- p.4 / Chapter 1.1.2 --- Key TSGs and their cellular pathways --- p.5 / Chapter 1.1.2.1 --- p53 pathway --- p.6 / Chapter 1.1.2.2 --- Rb pathway --- p.9 / Chapter 1.1.2.3 --- APC and Wnt/β-catenin pathway --- p.10 / Chapter 1.1.2.4 --- Chromatin regulators with tumor suppressive properties --- p.12 / Chapter 1.1.3 --- Methods for TSG identification --- p.17 / Chapter 1.2 --- The epigenetic abnormalities of TSGs in cancer --- p.19 / Chapter 1.2.1 --- Promoter CpG methylation --- p.20 / Chapter 1.2.1.1 --- Molecular basis of DNA methylation --- p.20 / Chapter 1.2.1.2 --- Silencing of TSGs by promoter methylation --- p.22 / Chapter 1.2.1.3 --- Mechanism of methylation-induced transcription repression --- p.23 / Chapter 1.2.1.4 --- Abnormal DNA methylation contributes to early stages of tumorigenesis --- p.25 / Chapter 1.2.2 --- Aberrant histone modification and chromatin remodeling --- p.26 / Chapter 1.2.3 --- Clinical applications of epigenetic biomarkers and therapeutic targets --- p.28 / Chapter 1.2.3.1 --- DNA methylation and histone modification as biomarkers for cancer diagnosis and prognosis --- p.28 / Chapter 1.2.3.2 --- Epigenetic targets for cancer treatment --- p.29 / Chapter Chapter 2 --- Aims and Design of This Study --- p.32 / Chapter Chapter 3 --- Materials and Methods --- p.34 / Chapter 3.1 --- Cell lines --- p.34 / Chapter 3.2 --- Human normal and cancer tissues --- p.35 / Chapter 3.3 --- DNA and RNA extraction --- p.35 / Chapter 3.4 --- Semi-quantitative and quantitative RT-PCR --- p.36 / Chapter 3.5 --- DNA methylation analysis --- p.37 / Chapter 3.5.1 --- Bisulfite modification of genomic DNA --- p.37 / Chapter 3.5.2 --- CpG island analysis --- p.37 / Chapter 3.5.3 --- Methylation-specific PCR (MSP) --- p.38 / Chapter 3.5.4 --- Bisulfite genomic sequencing (BGS) --- p.38 / Chapter 3.5.5 --- Pharmacologic demethylation treatment of cell lines --- p.38 / Chapter 3.6 --- Luciferase assay of gene promoter activity --- p.39 / Chapter 3.7 --- Multiplex genomic-DNA PCR --- p.39 / Chapter 3.8 --- Construction of expression plasmids and PCR-mediated mutagenesis --- p.40 / Chapter 3.9 --- Plasmid mini- and midi-preparation --- p.41 / Chapter 3.10 --- Creation of stable cell line for inducible gene expression --- p.42 / Chapter 3.11 --- Monolayer-culture and soft-agar colony formation assay --- p.42 / Chapter 3.12 --- Cell proliferation assay --- p.43 / Chapter 3.13 --- Flow cytometry analysis of cell cycle --- p.43 / Chapter 3.14 --- Apoptosis assay --- p.44 / Chapter 3.15 --- Senescence cell staining --- p.44 / Chapter 3.16 --- Western blotting --- p.45 / Chapter 3.17 --- Co-immunoprecipitation (Co-IP) --- p.46 / Chapter 3.18 --- Transcription factor activity assay --- p.46 / Chapter 3.19 --- Immunofluorescence microscopy --- p.47 / Chapter 3.20 --- Chromatin Immunoprecipitation (ChIP) --- p.47 / Chapter 3.21 --- Gene expression and copy number analysis using Oncomine database --- p.48 / Chapter 3.22 --- Protein half-life assay --- p.48 / Chapter 3.23 --- In vivo ubiquitination assay --- p.48 / Chapter 3.24 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Two Epigenetic Modifying Genes, PRDM5 and TUSC12, Suppress Tumor Cell Growth and are Silenced by Promoter Methylation in Human Cancers --- p.50 / Chapter 4.1 --- PRDM5 --- p.50 / Chapter 4.1.1 --- PRDM5 is a candidate TSG downregulated and methylated in multiple carcinomas --- p.50 / Chapter 4.1.2 --- PRDM5 is a stress responsive gene but its response is disrupted by promoter methylation --- p.53 / Chapter 4.1.3 --- PRDM5 suppresses cancer cell growth and proliferation --- p.54 / Chapter 4.1.4 --- PRDM5 inhibits TCF/LEF-dependent transcription and induced epigenetic repression of multiple oncogenes --- p.55 / Chapter 4.2 --- TUSC12 --- p.59 / Chapter 4.2.1 --- mRNA expression profiling of epigenetic modifying genes in tumor cell lines --- p.59 / Chapter 4.2.2 --- Promoter CpG methylation contributes to TUSC12 silencing in tumor cells --- p.61 / Chapter 4.2.3 --- Demethylation of TUSC12 promoter restores its expression --- p.63 / Chapter 4.2.4 --- TUSC12 methylation in primary tumor tissues --- p.65 / Chapter 4.2.5 --- TUSC12 expression inhibits anchorage-dependent and -independent tumor cell growth --- p.66 / Chapter 4.2.6 --- TUSC12 is an epigenetic modifier repressing transcription --- p.68 / Chapter 4.3 --- Discussion --- p.69 / Chapter Chapter 5 --- The Human Zinc Finger Protein 312 is a Novel Tumor Suppressor for Nasopharyngeal Carcinoma --- p.73 / Chapter 5.1 --- Identification of ZNF312 as a candidate 3p14.2 TSG --- p.74 / Chapter 5.2 --- Silencing of ZNF312 by promoter methylation in NPC cell lines --- p.75 / Chapter 5.3 --- ZNF312 is frequently downregulated and methylated in primary NPC tissues --- p.78 / Chapter 5.4 --- ZNF312 suppresses tumor cell clonogenicity --- p.79 / Chapter 5.5 --- ZNF312 is a transcription repressor --- p.80 / Chapter 5.6 --- ZNF312 regulates cell cycle progression, induces apoptosis, and inhibits cell stemness --- p.83 / Chapter 5.7 --- ZNF312 represses oncogene expression --- p.85 / Chapter 5.8 --- Discussion --- p.87 / Chapter Chapter 6 --- Identification of a Novel Tumor Suppressor Regulating p53 Signaling and Frequently Methylated in Multiple Tumors --- p.91 / Chapter 6.1 --- TUSC45 is broadly expressed in human normal tissues but frequently downregulated in tumor cell lines --- p.91 / Chapter 6.2 --- Reduced TUSC45 expression in primary tumors is associated with poor survival of patients --- p.94 / Chapter 6.3 --- TUSC45 is mainly silenced by promoter CpG methylation in tumor cell lines --- p.96 / Chapter 6.4 --- Pharmacologic or genetic demethylation reactivates TUSC45 in silenced cell lines --- p.98 / Chapter 6.5 --- TUSC45 is frequently methylated in primary tumors --- p.99 / Chapter 6.6 --- TUSC45 suppresses anchorage-dependent and -independent tumor cell growth --- p.101 / Chapter 6.7 --- Induction of TUSC45 expression inhibits tumor cell growth through inducing apoptosis, cell cycle arrest and senescence --- p.104 / Chapter 6.8 --- TUSC45 tumor-suppressive function is dependent on p53 signaling --- p.108 / Chapter 6.9 --- TUSC45 positively regulates p53 protein stability --- p.110 / Chapter 6.10 --- Discussion --- p.112 / Chapter Chapter 7 --- General Discussion --- p.116 / Chapter 7.1 --- General discussion --- p.116 / Chapter 7.2 --- Future perspectives --- p.118 / Reference List --- p.121

Chi Tian-yi. / "July 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 213-257). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Repair of damage to the DNA is essential for the maintenance of genomic stability, both during embryonic development and normal growth. The cell has therefore evolved a complex array of interconnected pathways to ensure the appropriate response to DNA damage is initiated, such as cell cycle checkpoint arrest, activation of DNA repair pathways or induction of apoptotic processes. These co-ordinated signal transduction pathways have been termed the DNA damage response (DDR). A previous study showed that ATR-dependent damage responses were frequently defective in cell lines from patients with Microcephalic Primordial Dwarfism (MPD) disorders. In this thesis I have further characterised ATR–dependent damage response signalling in several cell lines from patients with various MPD disorders. I have shown that novel mutations in PCNT, which encodes a structural centrosomal protein, result in an MPD disorder and have characterised the associated ATRdependent DNA damage responses. I also contributed to the identification of mutations in ORC1, encoding a component of the DNA replication Origin Recognition Complex, in further MPD patients and examined origin licensing and Sphase progression in the patient derived cell lines. As a novel finding, I observed defects in the ATR-dependent G2/M checkpoint response in these cells. Additionally, I have characterised novel mutations in ATRIP, a gene encoding the obligate partner of ATR, in Seckel Syndrome patients, denoting a novel genetic defect in this condition. Finally, I have explored the role of PLK1 and AurA kinase in ATRdependent G2/M checkpoint control and provided compelling evidence of misregulation of this pathway in various MPD-patient derived cell lines. Collectively these data provide important functional insights into the genetic defects that cause MPD disorders and further explore the link between defective ATR-dependent damage response signalling and microcephaly.

The INO80 complex is a large ATPase chromatin remodeller which contains 15 accessory subunits in S.cerevisiae. Its subunits include the highly conserved ATPases Ruvb1 and Ruvb2, the actin-related proteins Arp5, Arp8, Act1 and Arp4, Actin, and a number of IES (I̱noE̱ighty S̱pecific) subunits Ies1, Ies2, Ies3, Ies4, Ies5 and Ies6, in addition to subunits Nhp10 and Taf14. All 15 of the accessory subunits are assembled around a catalytic core component known as Ino80. The INO80 complex has roles in transcription, DNA repair, replication, and chromosome segregation. These roles are in addition to its traditional nucleosome remodelling activities and the dispacement of H2A.Z from chromatin. Recent studies in S. cerevisiae have identified the subunit Ies6 as a critical component of the INO80 complex. Deletion of IES6, which encodes the small accessory subunit, clearly mimics the deletion f the gene encoding the catalytic subunit, INO80. Surprisingly, only one domain within Ies6 has been formally identified based on sequence analysis. This domain belongs to the L1_C class of domains. Such domains are commonly associated with DNA binding activity and transcription factors. This stud has further characterised the Ies6 subunit both genetically and biochemically. Genetically, it has demonstrated that single point mutations at regions of proposed subunit-subunit interaction between the Arp5 or Rvb2 subunits, or within the YL1_C are not sufficient to disrupt Ies6 function. However, expression of a double point mutation, ies6(K114E/Y125A), in combination with rad50 deletion, caused a sensitivity to replication inhibition, but not chromosome segregation inhibition, indicating a potential separation of function in this utant due to the loss due of only one of the biological functions of Ies6. Biochemically, we have confirmed that DBA binding capacity of Ies6 resides within the YL_C domain. In addition, although it has been demonstrated that the removal of H2A.Z acetylation exacerbates the increase in cellular ploidy observed in ies6 null cells, we found that overall levels of H2A.Z acetylation were not influenced by the loss of Ies6. This indicates that the role of H2A.Z acetylation in chromosome segregation may only affect ploidy status upon the loss of Ies6. In addition, work on the R2TP complex (which contains the INO80 APases Ruvb1/Ruvb2, and subunits Tah1 and Phi1) has revealed the recruitment mechanism for the molecular chaperone, Hsp90, and the telomere length regulation protein, Tel2. Together, the R2TP complex, Hsp90 and Tel2 promote the stabilisation and maturation of multi-protein complexes. These include Phosphatidylinositol 3-kinase-related kinases (PIKKs, a family of kinases involved i Serine and Threonine phosphorylation), subunits of the INO80 complex and subunits of the SWR1 chromatin remodelling complex (a partner comlex to INO80 that incorporates H2A.Z into chromatin).

Over the last decade, major advances in the development and application of microarray-based comparative genomic hybridisation (aCGH) technology have significantly contributed to our understanding of Genomic Disorders. My aims here were to provide insight into the genotype to phenotype relationships of three Genomic Disorders; CUL4B-deleted X-Linked Mental Retardation (XLMR), Wolf-Hirschhorn Syndrome (WHS) and 16p11.2 Copy Number Variant Disorder. CUL4B encodes a structural component of the Cullin-RING-ligase 4-containing class of E3 ubiquitin ligases. CUL4B-deleted XLMR represents a syndromal form of mental retardation whereby patients exhibit other clinical features aside from the MR, such as seizures, growth retardation and disrupted sexual development. I used CUL4B-deleted patient-derived cell lines to investigate the impacts of CUL4B loss on mitochondrial function. I have shown that loss of CUL4B is associated with a distinct set of mitochondrial phenotypes, identifying CUL4B-deleted XLMR as a disorder associated with mitochondrial dysfunction. Furthermore, I have uncovered a reciprocal relationship between CUL4B and Cereblon, providing evidence of a potential role for the CUL4-CRBN E3 ligase complex in maintaining mitochondrial function. Deletion or duplication of the 16p11.2 region is associated with macro-/microcephaly respectively. Here, I have evaluated the cellular consequences of 16p11.2 CNV, specifically with regards KCTD13 expression, DNA replication and checkpoint activation. WHS is typically caused by a small hemizygous telomeric deletion of the 4p16.1 region. Haploinsufficiency of 4p16.1 is associated with microcephaly, growth retardation and complex developmental abnormalities. I investigated the impacts of LETM1 copy number change in WHS patient-derived cells. Here, I have shown that copy number change of LETM1 specifically segregates with mitochondrial dysfunction, likely underlying the seizure phenotype exhibited by the large subgroup of WHS patients whose deletions incorporate LETM1 as well as the rarer instances of the reciprocal duplication. In this thesis I use patient-derived cell lines from three Genomic Disorders as a fundamental tool providing new pathomechanistic insight into the clinical presentation of these conditions.

Mutations are the ultimate source of new genetic information and they can be neutral, harmful or beneficial. The ultimate fate of all mutations is either to be lost or to eventually become fixed in a population. In this thesis I investigate genome wide traces of natural selection in eukaryotes. I focus on the most common type of mutations, point mutations, in protein coding genes. I investigated whether there is adaptive evolution in 11 plant species comparisons by applying an extension of the McDonald Kreitman (MK) test and found little evidence of adaptive evolution. However, most of the investigated plant species have low effective population sizes (Ne) and the rate of adaptive evolution is thought to be correlated to Ne. I therefore extended my study using additional data from mammals, drosophilids and yeast to investigate the relationship between the rate of adaptive evolution and Ne. I found a highly significant correlation between the rate of adaptive evolution relative to the rate of neutral evolution (!a) and Ne. It has been proposed that evidence of adaptive evolution can be an artifact of fluctuating selection. I simulated a model of fluctuating selection, in which the average strength of selection acting upon mutations is zero. Under this model adaptive evolution is inferred using MK-type tests. However, the mutations which become fixed are on average positively selected. The signal of adaptive evolution is therefore genuine. Ne can not only vary between species but also across genomes. However, how much variation there is, and whether this affects the efficiency of natural selection, is unknown. I analysed 10 species and show that variation in Ne is widespread. However, this variation is limited, amounting to a few fold variation in Ne between most genomic regions. This is never-the-less sufficient to cause variation in the efficiency of selection.

Hox genes encode a family of evolutionarily conserved transcription factors involved in the activation of diverse cell differentiation programs along the antero-posterior axis of animals. Hox gene expression is controlled by a complex set of regulatory mechanisms which are still not fully understood. Despite this, misregulation of Hox gene expression can lead to severe developmental abnormalities and various forms of disease. This work addresses the way in which small non-coding RNAs (microRNAs, miRNAs) regulate Hox gene expression and function during development. To do this we use the Drosophila Hox gene Ultrabithorax (Ubx) as a paradigm for Hox gene function. Using a suite of genetic methods we first uncover a novel regulatory interaction between Drosophila Ubx and the miR-310C family of miRNAs during the development of the haltere, a small dorsal appendage involved in flight control. We also show that this miRNA cluster is required to fine tune Ubx expression. Furthermore, our data provides insight into the role played by Ubx during appendage development. Secondly, using a next generation RNA sequencing approach, we identify the full repertoire of miRNAs present in two serially homologous appendages of Drosophila – the wing and haltere. Our results show that these morphologically distinct appendages have divergent miRNA profiles, including miRNAs which display appendage-specific expression patterns. In addition, combining these profiles with available transcriptomic data enabled us to study how miRNAs are integrated into the Ubx gene regulatory networks that govern haltere development. This analysis suggests that haltere miRNAs reinforce the regulatory programmes installed by Ubx during haltere development. Our work therefore contributes to the understanding of the regulatory function of miRNAs during development and sheds light on the ways in which Hox gene expression can contribute to the formation of complex morphological structures.

Maintenance of the genome is essential for life to prosper. Regular insults to the genome are sustained by all cellular life and can foster genetic instability if left unrepaired. The most lethal genetic damage is a double strand break (DSB), the cleavage of the phosphate backbone on both strands of the DNA double helix. Two main pathways exist which provide mechanisms for coping with DSBs; precise repair utilising the identical sister chromatid as a template to recreate the broken segment (homologous recombination; HR), and direct fusion of the broken ends in the absence of an intact template (nonhomologous end joining; NHEJ). NHEJ was first characterised in eukaryotes, and an analogous system has been found to exist in bacteria during the past decade. The bacterial NHEJ pathway is composed of four key proteins; the DNA end binding Ku homodimer, a DNA Ligase, a DNA polymerase and a phosphoesterase (PE). The first results chapter of this thesis details the identification of an orthologous set of proteins in the archaeon Methanocella paludicola, and their subsequent isolation and characterisation. The second results chapter expands on the individual activities of the proteins by combining them, and asserting the ability of archaeal NHEJ to join discontinuous ends in vitro. The role of the PE has been unclear in the bacterial system, but in vitro assays described here suggest that the enzyme plays a role in processing NHEJ intermediates formed by the NHEJ polymerase. The PE is found to optimise repair intermediates for ligation, and to reverse potentially genotoxic DNA strand displacements. The final results chapter investigates the structural aspects of the archaeal NHEJ enzymes. Together these studies establish a functional NHEJ system in an archaeon for the first time, and expand our knowledge of the bacterial system by proposing a standard model of archaeo--‐prokaryotic NHEJ.