The identification of the causative mutation(s) in individuals with familial cancer syndromes informs their clinical management and allows cascade testing of family members, which informs their clinical management in turn. The advent of next generation sequencing (NGS) has revolutionised diagnostic genetic analysis, demonstrated by this thesis. Three novel NGS assays have been developed. The first two assays allowed more comprehensive analysis of two genetically heterogeneous tumours, phaeochromocytoma/parganglioma and renal cell carcinoma, by creation of NGS-based gene panel tests. These assays allowed increased detection of germline mutations at a lower cost per gene and reduced processing time compared to previous methods of analysis. The third assay also uses NGS but, instead, to more thoroughly analyse a single gene. The full gene region for VHL was examined at mosaic detection level, with a clinically actionable mutation identified in 18% of patients with von Hippel-Lindau disease in whom a mutation could not be identified by conventional analysis. The difficulty of providing more comprehensive genetic analysis is the concurrent increase in identification of variants of uncertain significance (VUSs). In depth variant analysis was conducted for all VUSs identified during this research. The reassignment of 17% of these VUSs as pathogenic or benign was enabled.

Inherited thrombocytopenias are a heterogeneous group of disorders characterised by abnormally low platelet counts, often with secondary qualitative defects in platelet function, which can be associated with abnormal bleeding. Next generation sequencing has only previously been employed in small-scale studies and for the confirmation of suspected variants. This study presents the first large-scale approach to fully categorise inherited thrombocytopenia. Ninety-five patients were recruited to the UK-Genotyping and Phenotyping (GAPP) study with inherited thrombocytopenia of unknown genetic aetiology. An average platelet count of 88x109/L was observed across all individuals. Platelet function testing revealed a secondary phenotypic defect in addition to the reduction in platelet count in 71% (61/86). Of the 95 patients, 69 patients, encompassing 47 index cases, were analysed by whole exome sequencing. A variant with a positive prediction of pathogenicity was identified in 40% (19/47) of patients and overall plausible candidate variants of disease were identified in 69% of index cases. Subsequently an inherited thrombocytopenia specific gene panel was developed to serve as a pre-screen for patients prior to whole exome sequencing. In total, candidate variants in genes previously known to be implicated with disease were identified in 77% (20/26) of patients analysed. In conclusion, the application of a combined phenotyping and genotyping approach to patients with inherited thrombocytopenia is an effective and efficient means of complete diagnosis. It also has the added benefit of identifying novel candidate variants in genes not previously known to cause disease that may further our understanding of the processes surrounding haemostasis through subsequent functional studies.

All eukaryotic cells possess mitochondrial DNA (mtDNA), which is maternally inherited through the oocyte, its replication being regulated by nuclear-encoded replication factors. It was hypothesised that mtDNA replication is highly regulated in oocytes, pre-implantation embryos and embryonic stem cells (ESCs) and that this may be disrupted following nuclear transfer (NT). MtDNA copy number decreased between 2-cell and 8-cell staged porcine embryos and increased between the morula and expanded blastocyst stages, coinciding with increased expression of mtDNA replication factors. Competent porcine oocytes replicated their mtDNA prior to and during in vitro maturation to produce and maintain the 100000 mtDNA copies required for fertilisation. Those oocytes in which mtDNA replication was delayed had reduced developmental ability. Expression of pluripotency-associated genes decreased as murine ESCs differentiated into embryoid bodies, although expression of mtDNA replication factors did not increase until the stage equivalent to organogenesis. Cross-species NT embryos in which the donor cell-derived mtDNA was replicated produced decreased developmental outcomes compared to those in which no mtDNA replication took place. Disruption of the strict regulation of mtDNA replication that occurs during early embryogenesis, as is likely following NT, may therefore contribute to the reduced developmental ability of embryos produced using such techniques.

ChlR1 is a DNA helicase implicated in diverse cellular processes including sister chromatid cohesion and DNA replication and repair. However, the mechanism by which ChlR1 participates in these processes is unknown. Data presented in this thesis show that siRNA-mediated depletion of ChlR1 causes increased sensitivity to chemically-induced replication stress. Treatment of ChlR1-depleted cells with hydroxyurea results in increased mono-ubiquitination of PCNA and increased chromatin-associated RPA, indicating stalled DNA replication. Furthermore, ChlR1 is recruited to chromatin following hydroxyurea treatment, supporting a role in the stabilisation of forks during replication stress. Fibroblasts derived from a Warsaw Breakage Syndrome (WABS) patient caused by mutation of ChlR1 (G57R) have both defective sister chromatid cohesion and G2 checkpoint following radiation-induced damage. Complementation with wild-type ChlR1 rescued this mutant phenotype while a known helicase dead mutant of ChlR1 (K50R) or the WABS-associated mutants G57R or ΔK897 did not. However, increased and prolonged Chk1 activation was observed in both K50R and ΔK897 complemented cells after treatment with hydroxyurea while the G57R was comparable to wild-type. These data suggest that the novel WABS mutation (G57R) may retain some wild-type ChlR1 activity and offer important insight into the molecular basis of the WABS phenotype.

The aim of this work is to provide geneticists with appropriate statistical methods and computer programmes for the analysis of human pedigree data in view of mapping genes on the human chromosomes, and discovering the origin of chromosomal abnormalities such as the autosomal trisomies, the 47,XXY Klinefelter's syndrome and the 46,XX men syndrome. J.H. Edwards' marker algebra is presented in detail as used in his computer programme (MARK III) that analyses linkage with Morton's lod method for normal diploids. The programme is also described with all its specifications. The cytological mechanisms leading to autosomal trisomy are described to show that the proportion of trisomics carrying three alleles from three of their grandparents is bound to be greater than zero for any locus anywhere on a trisomic chromosome. The use of A.W.F. Edwards' method of support is then demonstrated on various sets of data to definitely exclude the ABO, MN, P, Jk, Gc and Lp loci from chromosome no. 21, and the theory is extended to show that about 401 of 47,XXY men receive an extra X from their fathers and 60% from their mothers, and that in general 46,XX men are more likely to arise from 47,XXY zygotes that lose their Y chromosomes than by an interchange between the X and Y chromosomes of their fathers.

Biocatalysis processes use biologically-derived enzymes to perform fine-chemical synthesis. Whole-cell biocatalysis, using live microorganisms, offers protection against buffer conditions and denaturation, and allows turnover of effective enzymes. However, cells may still be damaged by reaction conditions. In nature, cell populations protect themselves by attaching to surfaces and producing a multi-component protective extracellular matrix. This multicellular mode of growth is termed a biofilm. Biofilms offer many advantages over individual free-floating cells which may be beneficial in whole-cell biocatalysis. The primary aim of this work was to develop a biofilm platform using non-pathogenic Escherichia coli strains as a generic host for various biocatalysis enzymes. To this end, a simple, inexpensive and reliable biofilm generation method was developed and optimised using quantitative assays and confocal laser scanning microscopy. Reporter gene technology was used to provide insight into the expression of the matrix component curli. Flow cytometry was employed to reveal curli expression heterogeneity in biofilm-forming populations. Biofilm-modulating plasmids were used to determine whether improvements could be made to the biofilm-forming strains and their relevant effects were observed. Lastly, three biocatalysis processes were tested in the biofilm biocatalyst with observation of effects on biofilm formation, curli expression and biocatalytic potential.

Agrobiodiversity are threatened due to habitat loss, land reclamation and fragmentation, spread of diseases and pests, genetic uniformity, genetic erosion, and other human activities. Crop wild relatives (CWR) are wild species that are more or less genetically related to crops that can be used to introgress useful genes for improvement of productivity, resistance to biotic and abiotic stresses and quality of cultivated crop. These valuable resources are threatened and untapped for crop improvements. Therefore, their conservation would be valuable and will contribute to maintaining and promoting the sustainability of crop diversity, facilitating agricultural production and supporting the increasing demand for food, feed and natural resources. This thesis tackle for the first time the diversity and conservation status of CWR in North Africa region. In order to achieve this goal, different methods, approaches and techniques were used. These are identifying CWR in the region (CWR checklist), prioritize the checklist, ex situ and in situ gap analyses, species distribution modelling, threat assessment using IUCN Red List categories, climate change assessment and molecular genetic analysis of wild barley (Hordeum vulgare subsp. spontaneum (C. Koch) Thell). The outcomes will assist in lay the foundations for future ex situ and in situ conservation, and subsequent use.

Alternative splicing occurs in more than 90% of human genes and is particularly abundant in the nervous system. It has been recognized that toxicity can be caused at the level of pre-mRNA processing and potentially lead to age-dependent neurodegeneration upon low-dose chronic exposure. ELAV (Embryonic Lethal Abnormal Visual system)/Hu family proteins are prototype RNA binding protein and gene specific regulators of alternative mRNA splicing in the nervous system. Analysis of mutants in ELAV family proteins shows overlapping and distinct functions during development and age-dependent neurodegeneration. Overexpression of ELAV family proteins further revealed that cytoplasmic localization of ELAV family proteins in associated with enhanced neurotoxicity. Intriguingly all Drosophila ELAV family proteins and mammalian Hu proteins can regulate neuron-specific alternative splicing of Drosophila neuroglian gene- a known ELAV target. The blood brain barrier (BBB) and efficient excretion are protective mechanisms making delivery of many drugs to the brain difficult in vivo. Therefore, I analyzed the roles of a number of key Organic Anion Transporter Protein (OATP) and Multi- Drug Resistance (MDR) proteins and established a sensitized genetic background for CNS drug delivery. To assess if xenobiotics can interfere with ELAV function leading to neurodevelopmental/neurodegenerative defects, I assessed ELAV regulation of its major target erect wing (ewg) using an ewg fluorescent reporter, which recapitulates endogenous ELAV-mediated splicing and allows rapid visualization of potential modulators. From a compound screen in a sensitized genetic background, I identified a number of xenobiotics that cause changes in ewg splicing, indicating interference with ELAV function. Importantly, these compounds also phenocopy specific characteristics of ELAV mutants. My approach demonstrates the potential for using Drosophila in drug screening and neurotoxicity assessments.

Tuberculosis is a global problem, with little change in antibiotic therapy over the last fifty years, but with the emergence of both multi-drug resistant disease and extensive drug resistant disease further treatments are needed to ensure successful management of the disease. Severe vitamin D deficiency is prevalent in patients with tuberculosis and the immunomodulatory mechanisms of elements of the vitamin D axis, including vitamin D binding protein (DBP) and vitamin D receptor (VDR) have been explored in part. Aims This thesis will ascertain the role of polymorphisms in vitamin D axis genes in determining response to vitamin D in tuberculosis patients. Additionally it will aim to determine whether the interaction between underlying genotype, baseline vitamin D level and other elements of the vitamin D axis has the potential to influence clinical outcome and further investigate in vitro effects of vitamin D and elements of the vitamin D axis in influencing the frequency and functionality of monocytes and T cells, both of which are key elements in the immune response to tuberculosis infection. Results Associations between vitamin D baseline and response to supplementation appear to have a genetic association with DBP, VDR and DHCR7 genotypes and varying DBP haplotypes appear to determine the level of DBP at baseline measurement. The effect of vitamin D has an immunomodulatory role in both monocyte response and T regulatory activity, with a clear effect of vitamin D on cytokine response. Conclusion There appears to be a role for vitamin D in the treatment of tuberculosis but further questions are raised regarding the benefits and risks of immune response modulation in an inflammatory/ cytopathogenic condition such as tuberculosis.

Lesions in DNA can lead to accumulation of mutations that may cause cancer progression and disease. DNA repair is essential to overcome these lesions maintaining genomic integrity and preserving the genetic information, following DNA damage a wide variety of post-translational modifications (PTM) take place and phosphorylation is an essential one. A key DNA repair factor is BRCA1, a tumour suppressor gene that encodes a protein that can form 3 different complexes implicated in several mechanisms of DNA repair, interestingly BRCA1 mutations can lead to an increased risk of having breast and ovarian cancer by the age of 70. An important binding partner of BRCA1 is PALB2 a cancer predisposition gene that is essential for accurate HR repair, however it is yet not well understood whether the BRCA1-PALB2 interaction is involved in other DNA repair mechanisms. An important role of BRCA1 is to promote replication fork stability by protecting nascent DNA at stalled replication forks from degradation, and this function is essential to preserve genomic integrity but it remains unclear how BRCA1 regulates replication fork stability and fork protection. In this thesis we suggest that the ability of BRCA1 to protect nascent DNA is regulated in an unexpected fashion through CDK phosphorylation at Serine 114 and PIN1-mediated conformational change. Our data also reveals dual roles of PALB2 and BRCA1 at stalled replication forks, as restart and protection are separate, and interestingly unclassified patient variants within the CDK-PIN1 regulated region of BRCA1 exhibit deleterious fork protection. Altogether findings in this thesis suggest a previously unrecognised regulation pathway where CDK1, 2-BRCA1-PIN1-PALB2 cooperate together to govern fork stability.