Doctor of Philosophy / Division of Biology / Mark C. Ungerer / Nuclear genome size varies immensely across flowering plants, spanning nearly 2400-fold. The causes and consequences of this vast amount of variation have intrigued biologists since it became clear that nuclear DNA amount did not reflect organismal complexity (the so-called C-value paradox). In my dissertation I utilize wild sunflower species in the genus Helianthus to examine the role of transposable elements (TEs), and in particular, long terminal repeat (LTR) retrotransposons, in generating genome size variation and whether variation in genome size influences aspects of plant growth and development across multiple organizational levels. The genus Helianthus provides an excellent system for studying these questions given four-fold variation in nuclear DNA content among diploid species and well-resolved phylogenetic relationships. Utilizing short-read Illumina data and sequence information from a diverse panel of Helianthus annuus (common sunflower) full-length LTR retrotransposons, I found that nuclear genome size in Helianthus species is positively correlated with repetitive DNA, and LTR retrotransposon subtypes generally show similar patterns in genomic abundance across taxa. Helianthus species with the largest genomes, however, exhibit large-scale amplification of a small number of LTR retrotransposon subtypes. Measuring aspects of plant growth and development at cell-, organ- and whole plant-levels in a panel of diploid Helianthus species that vary 4-fold in nuclear genome size, I found that genome size is negatively correlated with cell production rate, but that this negative correlation does not persist at higher organizational levels. Taken together, these results provide insights into the mechanisms contributing to genome size evolution in plants and the organizational level at which genome size may impact growth patterns and developmental rates. Genome expansion in wild sunflowers is influenced most significantly by amplification of a small number of TEs and not necessarily by a greater diversity of TEs. Genome size is strongly negatively correlated with cell production rate but this relationship weakens at higher organizational levels, such as that of organ and whole-plant development.

<p>The complete and faithful duplication of the genome is essential to ensure normal cell division and organismal development. Eukaryotic DNA replication is initiated at multiple sites termed origins of replication that are activated at different time through S phase. The replication timing program is regulated by the S-phase checkpoint, which signals and repairs replicative stress. Eukaryotic DNA is packaged with histones into chromatin, thus DNA-templated processes including replication are modulated by the local chromatin environment such as post-translational modifications (PTMs) of histones.</p><p>One such epigenetic mark, methylation of lysine 20 on histone H4 (H4K20), has been linked to chromatin compaction, transcription, DNA repair and DNA replication. H4K20 can be mono-, di- and tri-methylated. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7 and subsequent di-/tri- methylation is catalyzed by Suv4-20. Prior studies have shown that PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which may be partially attributed to defects in origin selection and activation. Meanwhile, overexpression of mammalian PR-Set7 recruits components of pre-Replication Complex (pre-RC) onto chromatin and licenses replication origins for re-replication. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 impacts the replication program on a genomic scale. Finally, the methylation substrates of PR-Set7 include both histone (H4K20) and non-histone targets, therefore it is necessary to directly test the role of H4K20 methylation in PR-Set7 regulated phenotypes. </p><p>I employed genetic, cytological, and genomic approaches to better understand the role of H4K20 methylation in regulating DNA replication and genome stability in Drosophila melanogaster cells. Depletion of Drosophila PR-Set7 by RNAi in cultured Kc167 cells led to an ATR-dependent cell cycle arrest with near 4N DNA content and the accumulation of DNA damage, indicating a defect in completing S phase. The cells were arrested at the second S phase following PR-Set7 downregulation, suggesting that it was an epigenetic effect that coupled to the dilution of histone modification over multiple cell cycles. To directly test the role of H4K20 methylation in regulating genome integrity, I collaborated with the Duronio Lab and observed spontaneous DNA damage on the imaginal wing discs of third instar mutant larvae that had an alanine substitution on H4K20 (H4K20A) thus unable to be methylated, confirming that H4K20 is a bona fide target of PR-Set7 in maintaining genome integrity. </p><p>One possible source of DNA damage due to loss of PR-Set7 is reduced origin activity. I used BrdU-seq to profile the genome-wide origin activation pattern. However, I found that deregulation of H4K20 methylation states by manipulating the H4K20 methyltransferases PR-Set7 and Suv4-20 had no impact on origin activation throughout the genome. I then mapped the genomic distribution of DNA damage upon PR-Set7 depletion. Surprisingly, ChIP-seq of the DNA damage marker γ-H2A.v located the DNA damage to late replicating euchromatic regions of the Drosophila genome, and the strength of γ-H2A.v signal was uniformly distributed and spanned the entire late replication domain, implying stochastic replication fork collapse within late replicating regions. Together these data suggest that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains, presumably via stabilization of late replicating forks.</p><p>In addition to investigating the function of H4K20me, I also used immunofluorescence to characterize the cell cycle regulated chromatin loading of Mcm2-7 complex, the DNA helicase that licenses replication origins, using H4K20me1 level as a proxy for cell cycle stages. In parallel with chromatin spindown data by Powell et al. (Powell et al. 2015), we showed a continuous loading of Mcm2-7 during G1 and a progressive removal from chromatin through S phase.</p> / Dissertation

Nucleosome positioning is involved in a variety of cellular processes, and it provides a likely substrate for species evolution and may play roles in human disease. However, many fundamental aspects of nucleosome positioning remain controversial, such as the relative importance of underlying sequence features, genomic neighbourhood and trans-acting factors. In this thesis, I have focused on analyses of the divergence and conservation of nucleosome positioning, associated substitution spectra, and the interplay between them. I have investigated the extent to which nucleosome positioning patterns change following the duplication of a DNA sequence and its insertion into a new genomic region within the same species, by assessing the relative nucleosome positioning between paralogous regions in both the human (using in vitro and in vivo datasets) and yeast (in vivo) genomes. I observed that the positioning of paralogous nucleosomes is generally well conserved and detected a strong rotational preference where nucleosome positioning has diverged. I have also found, in all datasets, that DNA sequence features appear to be more important than local chromosomal environments in nucleosome positioning evolution, while controlling for trans-acting factors that can potentially confound inter-species comparisons. I have also examined the relationships between chromatin structure and DNA sequence variation, with a particular focus on the spectra of (germline and somatic) substitutions seen in human diseases. Both somatic and germline substitutions are found to be enriched at sequences coinciding with nucleosome cores. In addition, transitions appear to be enriched in germline relative to somatic substitutions at nucleosome core regions. This difference in transition to transversion ratio is also seen at transcription start sites (TSSs) genome wide. However, the contrasts seen between somatic and germline mutational spectra do not appear to be attributable to alterations in nucleosome positioning between cell types. Examination of multiple human nucleosome positioning datasets shows conserved positioning across TSSs and strongly conserved global phasing between 4 cancer cell lines and 7 non-cancer cell lines. This suggests that the particular mutational profiles seen for somatic and germline cells occur upon a common landscape of conserved chromatin structure. I extended my studies of mutational spectra by analysing genome sequencing data from various tissues in a cohort of individuals to identify human somatic mutations. This allowed an assessment of the relationship between age and mutation accumulation and a search for inherited genetic variants linked to high somatic mutation rates. A list of candidate germline variants that potentially predispose to increased somatic mutation rates was the outcome. Together these analyses contribute to an integrated view of genome evolution, encompassing the divergence of DNA sequence and chromatin structure, and explorations of how they may interact in human disease.

Muscadinia rotundifolia est une espèce de la famille des Vitaceae. C’est un sous-genre du genre Vitis, le deuxième sous-genre étant celui des Euvitis qui comprend l’espèce cultivée Vitis vinifera (2n=38). M. rotundifolia (2n=40) est une source de résistance aux maladies très importante pour l’amélioration de la vigne. Son génome commence seulement à être décrit avec deux cartes génétiques récemment publiées. Ma thèse a consisté à utiliser des ressources génomiques chez M. rotundifolia cv Regale (banque BAC, collection de séquence d’extrémités de BAC ou BES et séquences de BACs) pour caractériser le génome de cette espèce en comparaison avec celui de V. vinifera. Les résultats obtenus ne montrent pas de différence importante entre les génomes des deux espèces en termes de composition du génome en bases (GC%), en séquences codantes ou en éléments répétés. De même, à une échelle globale, la famille de gènes NBS-LRR semble être similaire en termes de nombre et de balance entre les sous-familles. A une échelle plus fine cependant (carte physique et séquences de BAC), des remaniements relativement importants sont observés dans des régions portant cette famille de gènes, aboutissant parfois à des contenus différents en gènes, de région normalement homologues : duplication différentielles de gènes, présence/absence de gènes. / Muscadinia Rotundifolia is a species of the Vitaceae family. It is a sub-genus of the Vitis genus along with the Euvitis sub-genus, which the cultivated species Vitis vinifera belongs to. M. rotundifolia (2n=40) is a very important source of resistance to diseases in grapevine breeding programs. Its genome is only starting to be described with the recent publication of two genetic maps. The present study aimed at using M. rotundifolia cv Regale genomic resources (BAC library, BAC end sequences or BES, BAC sequences) in order to characterize the genome of this species in comparison with the genome of V. vinifera. The results showed that there is no striking difference between the two species in term of base composition (GC %), repeats frequency and gene space. The NBS LRR gene family also seems to be globally quite similar between the two species in terms of numbers and balance between subfamilies. At a finer scale (physical map and BAC sequence), frequent rearrangements are observed in genomic regions carrying the NBS-LRR gene family sometimes clearly associated with a different gene content between the two species in homologous regions: differential gene duplication, presence/absence of genes.

La génomique comparative est l'étude des relations structurales et fonctionnelles entre des génomes appartenant à différentes souches ou espèces. Cette discipline offre ainsi la possibilité d'étudier et de comprendre les processus qui façonnent les génomes au cours de l'évolution. Dans le cadre de cette thèse, nous nous sommes intéressés à la génomique comparative des bactéries et plus particulièrement aux méthodes relatives à la comparaison des séquences complètes d'ADN des génomes bactériens. Ces dix dernières années, le développement d'outils informatiques permettant de comparer des génomes entiers à l'échelle de l'ADN est devenu une thématique de recherche à part entière. Actuellement, il existe de nombreux outils dédiés à cette tâche. Cependant, jusqu'à présent, la plupart des efforts ont été dirigés vers la réduction du temps de calcul et l'optimisation de la mémoire au détriment de l'évaluation de la qualité des résultats obtenus. Pour combler ce vide, nous avons travaillé sur différents problèmes statistiques soulevés par la comparaison de génomes complets bactériens. Notre travail se divise en deux axes de recherche. Dans un premier temps, nous nous sommes employés à évaluer la robustesse des alignements de génomes complets bactériens. Nous avons proposé une méthode originale fondée sur l'application de perturbations aléatoires sur les génomes comparés. Trois scores différents sont alors calculés pour estimer la robustesse des alignements de génomes à différentes échelles, allant des nucléotides aux séquences entières des génomes. Notre méthode a été expérimentée sur des données génomiques bactériennes réelles. Nos scores permettent d'identifier à la fois les alignements robustes et non robustes. Ils peuvent être employés pour corriger un alignement ou encore pour comparer plusieurs alignements obtenus à partir de différents outils. Dans un second temps, nous avons étudié le problème de la paramétrisation des outils de comparaisons de génomes entiers. En effet, la plupart des outils existants manquent à la fois de documentation et de valeurs par défaut fiables pour initialiser leurs paramètres. Conséquemment, il y a un besoin crucial de méthodes spécifiques pour aider les utilisateurs à définir des valeurs appropriées pour les paramètres de ces outils. Une grande partie des outils de comparaisons de génomes complets est fondée sur la détection des matches (mots communs exacts). Le paramètre essentiel pour ces méthodes est la longueur des matches à considérer. Au cours de cette thèse, nous avons développé deux méthodes statistiques pour estimer une valeur optimale pour la taille des matches. Notre première approche utilise un modèle de mélange de lois géométriques pour caractériser la distribution de la taille des matches obtenus lorsque l'on compare deux séquences génomiques. La deuxième approche est fondée sur une approximation de Poisson de la loi du comptage des matches entre deux chaînes de Markov. Ces méthodes statistiques nous permettent d'identifier facilement une taille optimale de matches à la fois pour des séquences simulées et pour des données génomiques réelles. Nous avons également montré que cette taille optimale dépend des caractéristiques des génomes comparés telles que leur taille, leur composition en base ou leur divergence relative. Cette thèse représente une des toutes premières études dont l'objectif est d'évaluer et d'améliorer la qualité des comparaisons des génomes complets. L'intérêt et les limites de nos différentes approches sont discutés et plusieurs perspectives d'évolution sont proposées. / Comparative genomics is the study of the structural and functional relationships between genomes belonging to different strains or species. This discipline offers great opportunities to investigate and to understand the processes that shape genomes across the evolution. In this thesis, we focused on the comparative genomics of bacteria and more precisely, on methods dedicated to the comparison of the complete DNA sequences of bacterial genomes. This last decade, the design of specific computerized methods to compare complete genomes at the DNA scale has become a subject of first concern. Now, there exist many tools and methods dedicated to this task. However, until now, most of the efforts were directed to reduce execution time and memory usage at the expense of the evaluation of the quality of the results. To fill this gap, we worked on different statistical issues related to the comparison of complete bacterial genomes. Our work was conducted into two directions. In the first one, we investigated the assessment of the robustness of complete bacterial genome alignments. We proposed an original method based on random perturbations of the compared genomes. Three different scores were derived to estimate the robustness of genome alignments at different scales, from nucleotides to the complete genome sequences. Our method was trained on bacterial genomic data. Our scores allow us to identify robust and non robust genome alignments. They can be used to correct an alignment or to compare alignments performed with different tools. Secondly, we studied the problem of the parametrization of comparison tools. Briefly, most of the existing tools suffer from a lack of information and of reliable default values to set their parameters. Consequently, there is a crucial need of methods to help users to define reliable parameter values for these tools. Most of the comparison tools are rooted on the detection of word matches. The key parameter for all these tools is the length of the matches to be considered. During this thesis, we developed two statistical methods to estimate an optimal length for these matches. Our first approach consisted in using a mixture model of geometric distributions to characterize the distribution of the length of matches retrieved from the comparison of two genomic sequences. The second approach is rooted on a Poisson approximation of the number of matches between two Markov chains. These statistical methods allow us to easily identify an optimal length for the matches from both simulated and real genomic data. We also showed that this optimal length depends on the characteristics of the compared genomes such as their length, their nucleotide composition, and their relative divergence. This thesis represents one of the earliest attempts to statistically evaluate and to improve the quality of complete genome comparisons. The interest and limitations of our different methods are discussed and some perspectives are proposed.

Wang, Xingyan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 126-131). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.I / ABBREVIATIONS --- p.II / ABSTRACT --- p.V / 摘要 --- p.VIII / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- CLINICAL DISEASE CAUSED BY M.TB --- p.1 / Chapter 1.1.1 --- Tuberculosis (TB) --- p.1 / Chapter 1.1.2 --- Pathogen: Mycobacteria tuberculosis (M. TB) --- p.2 / Chapter 1.2 --- HOST DEFENSE AGAINST M.TB --- p.4 / Chapter 1.2.1 --- Overview --- p.4 / Chapter 1.2.2 --- Specific pathways --- p.6 / Chapter 1.3 --- GENETIC PREDISPOSITION OF HOST TO INFECTION --- p.12 / Chapter CHAPTER 2 --- OVERVIEW AND AIM OF THIS PROJECT --- p.14 / Chapter 2.1 --- GWAS REPLICATION --- p.14 / Chapter 2.2 --- CANDIDATE GENES REVEALED IN GWAS OF OTHER GRANULOMATOUS INFLAMMATORY DISEASES (GLD) --- p.14 / Chapter 2.3 --- CHROMOSOME 17 CHEMOKINE CLUSTER REGION --- p.15 / Chapter CHAPTER 3 --- REPLICATION STUDY OF TB GWAS --- p.16 / Chapter 3.1 --- INTRODUCTION --- p.16 / Chapter 3.1.1 --- TB GWAS study --- p.16 / Chapter 3.1.2 --- Aims of this part --- p.16 / Chapter 3.2 --- MATERIAL AND METHODS --- p.17 / Chapter 3.2.1 --- Case and control samples --- p.17 / Chapter 3.2.2 --- DNA extraction --- p.18 / Chapter 3.2.3 --- Genotyping of the SNPs --- p.19 / Chapter 3.2.4 --- Statistical analysis --- p.21 / Chapter 3.3 --- RESULTS --- p.23 / Chapter 3.3.1 --- Description of studied samples --- p.23 / Chapter 3.3.2 --- Results of case-control study for replication studies of TB GWAS --- p.23 / Chapter 3.4 --- DISCUSSION --- p.28 / Chapter CHAPTER 4 --- GENETIC VARIANTS IN GRANULOMATOUS INFLAMMATORY DISEASES --- p.32 / Chapter 4.1 --- INTRODUCTION --- p.32 / Chapter 4.1.1 --- Granulomatous inflammation --- p.32 / Chapter 4.1.2 --- Diseases characterized by granulomatous inflammatory --- p.34 / Chapter 4.1.3 --- Shared immune mechanisms in GiDs --- p.38 / Chapter 4.1.4 --- Genome-wide Association Studies (GWAS) in GiD --- p.38 / Chapter 4.1.5 --- Hypothesis of this part --- p.41 / Chapter 4.2 --- MATERIAL AND METHODS --- p.43 / Chapter 4.2.1 --- Case and control samples --- p.43 / Chapter 4.2.2 --- DNA extraction --- p.44 / Chapter 4.2.3 --- Tag SNP selection --- p.44 / Chapter 4.2.4 --- Genotyping of tagging SNPs --- p.45 / Chapter 4.2.5 --- Statisitical analysis --- p.45 / Chapter 4.3 --- RESULTS --- p.55 / Chapter 4.3.1 --- Description of TB case samples --- p.55 / Chapter 4.3.2 --- Primary endpoint case-control results --- p.56 / Chapter 4.3.3 --- Secondary endpoint case-only studies results --- p.67 / Chapter 4.3.4 --- Haplotype analysis --- p.78 / Chapter 4.4 --- DISCUSSION --- p.83 / Chapter 4.4.1 --- ATG16L1 gene with TB susceptibility --- p.83 / Chapter 4.4.2 --- Associations in case-only studies (Interaction effects) --- p.83 / Chapter 4.4.2.1 --- Age and pathogenesis of TB --- p.83 / Chapter CHAPTER 5 --- STUDIES IN THE CHEMOKINE-GENE CLUSTER AND A MIRNA SNP STUDY --- p.89 / Chapter 5.1 --- INTRODUCTION --- p.89 / Chapter 5.1.1 --- Genetic susceptibility to TB in familial cases --- p.89 / Chapter 5.1.2 --- Familial studies suggested linkage at 17qll.2 --- p.89 / Chapter 5.1.3 --- Chemokines --- p.90 / Chapter 5.1.4 --- Studies of SNP rs2910164 of microRNA-146a (miRNA-146a) --- p.91 / Chapter 5.2 --- MATERIAL AND METHODS --- p.92 / Chapter 5.2.1 --- Case and control samples --- p.92 / Chapter 5.2.2 --- DNA extraction --- p.92 / Chapter 5.2.3 --- TagSNP selection --- p.92 / Chapter 5.2.4 --- Genotyping of tagging SNPs --- p.93 / Chapter 5.2.5 --- PCR-RFLP --- p.93 / Chapter 5.2.6 --- Statistical analysis --- p.94 / Chapter 5.3 --- RESULTS --- p.100 / Chapter 5.3.1 --- PCR-RFLP results of the three SNPs --- p.100 / Chapter 5.3.2 --- Description of TB case samples --- p.102 / Chapter 5.3.3 --- Primary endpoint case-control results --- p.103 / Chapter 5.3.4 --- Secondary endpoint case-only studies results of CCL genes --- p.109 / Chapter 5.4 --- DISCUSSION --- p.120 / Chapter 5.4.1 --- Genetic association of SNPs with severity of TB --- p.120 / Chapter 5.4.2 --- Smoking and immunity --- p.121 / Chapter CHAPTER 6 --- FINAL CONCLUSION AND PROSPECT FOR FUTURE WORK --- p.122 / Chapter 6.1 --- CONCLUSION --- p.122 / Chapter 6.2 --- LIMITATION OF THE STUDIES --- p.124 / Chapter 6.3 --- FUTURE WORKS AND PROSPECT --- p.125 / REFERENCES --- p.126

Genome sequencing is emerging as a powerful tool to identify the molecular mechanism of cancer progression. However, the software tools to define genomic and post-genomic mutations are just in its infancy. We have used a novel software algorithm to analyse the cancer genome by DNAseq and expressed cancer genome arising from transcription by RNAseq to define dominant sources of potentially expressed tumour-specific mutations and oncogenic targets. We focus primarily on the rare human pleomorphic sarcoma as a disease of high unmet clinical need but use a range of cancer models to accelerate the development of the pipeline. First, we applied next generation sequencing of whole exomes of tumour tissues and two matched normal tissues (blood and “normal” tumour adjacent tissue) from a small set of patients to define parameters for use of the new software. The approaches identified significant mutations in tumour relative to germline DNA, but also in normal adjacent tissue, relative to normal germline, consistent with known field cancerization. Thus, in setting up the larger sequencing screen in the subsequent set of twenty cancer pleomorphic sarcoma cancer patients, whole exome sequencing was performed on tumour tissue and their matched normal adjacent tissues, rather than germline blood derived DNA, to define truly tumour-specific mutations. This approach provided sets of recurrent non-synonymous mutations in tumour tissue such as a transmembrane protease and suggests potential therapeutic targets for future focus that are highly tumour specific in pleomorphic sarcoma. A major problem with using DNA genomics only to define drugable landscapes in cancer is that the tumour genome is static and the mutations do not reflect the expressed cancer landscape at the time of surgery. Thus, in a smaller subset of patients we also applied shotgun RNAseq to determine the number of expressed mutated genes. We defined within the parameters chosen, from 8-17% of the mutated genome is expressed as defined at the RNA level. However, to our surprise, there were an order of magnitude more RNA mutations that were not DNA encoded suggestive of RNA editing events. Each patient showed elevated RNA edits that were independent of each other suggesting a highly-patient, cancer-specific perturbation in the specificity of the RNA editing machinery. We thus developed a cancer cell model to validate the RNA-editing software and we found we could recapitulate some of the RNA edits observed in clinical tumour tissue, in particular the signalling kinase in the MAP kinase-kinase-kinase-kinase super-family. It was interesting that RNA edits can often cluster in exon-intron boundaries suggesting a link to splicing and allows us to begin to produce “rules” for RNA editing. These data provide future direction to understand the role of RNA editing, as well as DNA encoded mutations, as mutagenic events and possible drugable targets in cancer signalling. Lastly, novel or orphan mutant proteins observed in human cancers, whether from DNA encoded mutant proteins or from RNA-edited driven mutant protein synthesis require new tools and technologies to discover new oncogenic signalling mechanisms. We developed an SBP-tagged affinity purification method in combination with label-free SWATH mass spectrometry to identify a novel binding protein for the gain-of-function mutant protein in a key metastatic gene, ELMO1. This identified an elevated interaction with another oncogenic protein encoded by AGR2 gene and validates this proteomics discovery platform to further advance function of new mutated proteins. In conclusion, we have applied and validated newly emerging software to begin to interrogate cancer tissue from patients of unmet clinical need in order to define new mechanisms of cancer progression and to define possibly new or better drug targets for new therapies. The data identified highly recurrent genome encoded mutations in human pleomorphic sarcoma and a potentially novel, targetable landscape represented by RNA editing driven mutant protein production. This will provide a foundation for future work on making better choices to advance our ability to improve patient management in human pleomorphic sarcoma.

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.