Eukaryotic cells respond to extracellular stimuli by transmitting intracellular instructions via signalling pathways to coordinate appropriate responses. Mitogen-activated protein kinase (MAPK) pathways are often used to transmit these instructions to regulate gene expression, where Ternary Complex Factors (TCFs) are among their nuclear targets. Elk-1 is the founding member of the TCF family of transcription activators. The mechanism of function and regulation of Elk-1 has been extensively studied, therefore providing a paradigm for signal-induced transcription. The activity of Elk-1 is influenced by Post-Translational Modifications (PTMs), such as phosphorylation and sumoylation. Elk-1 ubiquitylation has also been reported in vitro, however little work has been done on this modification of Elk-1. This thesis sought to reveal the mechanism of regulation and function of Elk-1 ubiquitylation. Elk-1 was demonstrated to be both monoubiquitylated and polyubiquitylated in vitro and in cells. Using size exclusion chromatography and the dominant negative nedd8 conjugating E2 enzyme Ubc12, several features of the Elk-1 specific E3 ligases have been revealed in vitro. In cells, ternary complex formation was shown to be important for monoubiquitylation. Furthermore monoubiquitylated Elk-1 is diminished following ERK-mediated phosphorylation, hence activation, in response to mitogen stimulation. It was also demonstrated that an Elk-1 derivative that exhibits strong monoubiquitylation level also exhibits a reduced capability to transactivate gene expression at the Serum Responsive Element (SRE), indicating a negative role of monoubiquitylation on Elk-1 transcriptional ability.

To clarify the role of one of the general chromatin remodelling factors-the histone acetylase Gcn5p in NER, I undertook experiments with <italic>&Delta;gcn5 </italic> and <italic>&Delta;tup1gcn5</italic> strains.

Colorectal cancer is one of the most clinically significant types of cancer due to both high incidence and mortality. Despite the well-established role of aberrant Wnt signalling in initiation and progression of colorectal cancer, therapies that specifically target the Wnt pathway are exceptionally limited. The development of such therapies is, in part, restricted by the toxicity of Wnt signalling inhibition for adult tissue homeostasis. Given this limitation, therapies that target downstream components and target genes of the Wnt pathway are required to minimise potential toxicity. The chromatin remodelling ATPase subunit Brg1 has been found to interact with β-catenin and facilitate transcriptional program driven by the Wnt pathway. In this thesis I aimed to explore Brg1’s potential as a therapeutic target in Wnt driven intestinal tumourigenesis. To achieve this, I conditionally deleted Brg1 in the murine small intestinal epithelium under normal physiological conditions and in the context of aberrant Wnt signalling using a range of transgenic mouse models. Additionally, I investigated the potential toxicity of Brg1 inhibition by analysing the effects of Brg1 loss in a range of epithelial tissues from the gastrointestinal tract and bladder. The results presented in this thesis demonstrate that Brg1 deficiency impedes Wnt-driven tumourigenesis in the murine small intestinal epithelium via attenuation of Wnt signalling and the elimination of stem cell derived tumours, which have a higher tumourigenic potential. Additionally, Brg1 was found to play a major role in the maintenance of normal small intestinal stem cell homeostasis, as loss of Brg1 in the small intestinal epithelium resulted in ablation of the stem cell population. The impact of Brg1 deficiency on homeostasis of other tissues of the gastrointestinal tract and bladder exhibited a remarkable diversity, from induction of epithelial hyperplasia to very subtle alterations in cell differentiation. Overall, the results presented in this thesis portray Brg1 as a promising potential therapeutic target in Wnt-driven neoplasia and suggest that specific targeting of the Brg1/β-catenin interaction rather than the catalytic activity of Brg1 may help to avoid any Wnt-independent toxicity of Brg1 inhibition.

Normal and pathological passaged BMdSCs were successfully grown to confluence over 3 passages and induced to differentiate down the osteogenic and adipogenic lineage with the formation of mineral deposit and multidroplet cell clusters respectively. JMML BMdSCs had a significantly increased growth rate and MDS BMdSCs had a significantly increased number of small colonies in comparison to normal. Lymphoma BMdSCs had an increased CD105, CD49b and CD45 antigenic expression; myeloma BMdSCs had an increased CD105 antigenic expression, and the lymphoproliferative BMdSCs had a significantly reduced CD90, CD105 and CD49b expression in comparison to normal. With osteogenic induction, AML and lymphoma (mineralised) cultures had a reduced osteocalcin gene expression in comparison to normal. With adipogenic induction, AML and lymphoma cultures showed reduced multidroplet cell cluster formation with comparable gene expression to normal.

ATP dependent chromatin re-modeling factors have previously been shown to play a pivotal role in the regulation of gene expression in several model organisms, including yeast, fruit fly and human. When encountered with a nutrient depleted environment Dictyostelium discoideum enter a process of multicellular development which requires the correct temporal and spatial expression of a large subset of genes. Here it is shown that two of these ATP dependent chromatin re-modelling factors, 1NO80 and CHDC, are required for the correct expression of developmental genes of Dictyostelium discoideum and subsequent multicellular morphogenesis. These factors are identified as having a key role in the earlier stage of aggregation and cellular chemotaxis towards the developmental chemoattractant cAMP. Genetic disruption of genes encoding major subunits of these complexes, arp8 and chdC, both result in a decreased ability to form correct fruiting bodies, also showing a marked decrease in chemotactic ability. In each case, these defects are seen to occur through different mechanisms, indicating the role of multiple pathways in the regulation of Dictyostelium chemotaxis. Interestingly, both mutant cell lines are also responsive to the neuropsychiatric treatment drug lithium and are shown to affect elements of the inositol signaling pathway.

The aim of this study was to investigate the genetic basis of selectivity in invasion of the red blood cells by the human malaria parasite Plasmodium falciparum. Multiple invasions of a single host red blood cell by more than one merozoite, which can be described or assessed in terms of the selectivity index (SI), has been reported to be related to the severity of malaria disease. In this study, selectivity index, defined as the ratio of the number of multiply-infected red cells observed to that expected from random invasion, as modelled by a Poisson distribution was determined for certain clones of P.falciparum. SI was measured under static and shaking culturing conditions for P. falciparum clones 3D7 and HB3 and 18 progeny clones derived from a genetic cross between these two parasite clones. P. falciparum clone 3D7 was found to have a significantly lower SI than HB3 under both static and shaking culture conditions. There was no relationship between SI and days in continuous culture for clone 3D7 under shaking and static conditions; the phenotype therefore appears to be stable over time. The genetic basis of the difference in selectivity index between P. falciparum clones 3D7 and HB3 was investigated in progeny clones from a cross between these two clones, to ascertain the inheritance pattern of the phenotype. Under static conditions, ten progeny clones had a selectivity index lower than either parent, one progeny clone had higher selectivity index than both parent, and six progeny clones had selectivity index intermediate between the parents . Under shaking conditions, fifteen progeny clones were observed to have a selectivity index lower than either parent. These observations suggest the involvement of more than one parasite gene in selectivity index. A Quantitative Trait Locus (QTL) analysis was performed in order to identify genomic regions influencing SI in the progeny clones. The highest LOD score of 5.06 was obtained for a QTL on chromosome 13 for SI measured in parasites cultured under shaking conditions. This QTL denoted, PF_SI_1, extends for approximately 100kb on chromosome 13 and contains 19 open reading frames. This finding indicates the presence of a gene or genes on chromosome 13 that influence the parasite’s selection of erythrocytes for invasion.

Malaria continues to be a major global health problem due to high mortality and morbidity rate in endemic regions. An. gambiae s.s is the major vector in endemic African countries. About 198 million cases of malaria were recorded globally in 2013 and this have led to over 584 000 deaths. Different measures have been implemented in order to reduce and control the transmission rate. However, the drug resistant parasites and insecticide resistant mosquitoes have created problems towards achieving this goal. The use of the sterile insect technique and genetically modified mosquitoes as a control measure seems very promising but requires massive releases of males that are vigorous and competitive for the strategy to be realistic. Thus, there is need to understand the genetics and ecology of male mosquitoes with reference to their reproductive investments particularly for the laboratory reared An. gambiae due to inbreeding effects in colonized strains. Here we developed a qPCR technique based on TaqMan assay to quantify male sperm investment and used the assay to examine the effects of hydric stress on sperm investment by males and sperm maintenance in mated females. From two inbred strains of An. gambiae s.s, we generated heterotic supermales and tested for inbreeding and heterosis effects on sex peptide and sperm investments by males in large and individual male mating cages to determine male reproductive success. No evidence of heterosis was found in the large group mating cages except in sperm activity. However, in the individual male mating cages, the heterotic supermales achieved higher reproductive success than the inbred strains. They produced more eggs and fathered numerous larvae. Furthermore, inbreeding affects the size of the sex peptide deposited and survival in inbred males. Conclusively, heterosis could be the quickest method to produce vigorous and competitive laboratory reared males for vector control projects where male reproductive success is required.

Premenstrual dysphoric disorder (PMDD) is a distressing and disabling syndrome causing a significant degree of impairment on daily functioning and interpersonal relationships in 3-8% of the women. With the convincing evidence that PMS is inheritable and that serotonin is important in the pathogenesis of PMS, and failure of initial studies to demonstrate significant associations between key genes controlling the synthesis, reuptake and catabolism of serotonin and PMDD, the main aim of this thesis was to target the functional polymorphisms of serotonin receptors.

The Malpighian tubule of Drosophila melanogaster is a valuable epithelial model for developmental, physiological and genetic studies. A set of 700 P{GAL4} enhancer-trap lines of D. melanogaster was screened for patterned -galactosidase reporter gene expression in the Malpighian tubules. Of these, around 20% show some internal patterning within tubules, and 1% appeared to be specific to tubule cell subpopulations. Staining patterns were compared and used to chart the patterns of gene expression in the tubule. By counterstaining nuclei with ethidium bromide, it proved possible to assemble a numerical map of the cell types in each tubule subdomain. It was found that tubules could be subdivided into at least five sub-domains and multiple cell types defined by gene expression. Remarkably, the patterns of gene expression and the numbers of both principal and secondary ("stellate") cell types within each domain, are reproducible to single-cell precision between individual animals. The numbers of cells, both in the whole tubule and in individual compartments, are practically invariant both between individuals of a particular line and between lines. Comparison with the regions previously identified by morphological or physiological techniques, revealed that a genetic boundary can always be found which corresponds with the known division. In addition, several new subdomains are proposed from enhancer trap analysis alone; additional experiments confirm the physiological significance of the proposed subdivisions. This is the first epithelium to be subjected to such a genetic analysis, and these results confirm the utility of enhancer trapping as a means of identifying genetic boundaries which can subsequently be shown to correspond with functional tissue domains

Campylobacter jejuni is a leading cause of human bacterial enteritis worldwide. Consumption of contaminated poultry meat is considered a major source of infection. The use of virulent bacteriophages as a form of biocontrol to specifically reduce this pathogen in poultry (phage therapy) is a promising intervention that does not rely on antimicrobials and therefore circumvents the emergence of antibiotic-resistant Campylobacter strains. In order to achieve this, a better understanding of the mechanisms involved in phage-host interactions at the molecular level would assist in the development of the strategy and the selection of bacteriophages. The main objective of this study was to therefore examine such interactions between Campylobacter and its virulent phages. To achieve this, the transcriptional response of C. jejuni to phage infection was investigated, along with the role of a Type II restriction-modification system during phage infection of Campylobacter. These studies were conducted using the highly phage-sensitive Campylobacter strain, C. jejuni PT14, in conjunction with a number of group II and III bacteriophages (Eucampyvirinae). Transcriptome studies (RNA-Seq) revealed a phage-induced host response that included a demand for iron and oxygen. This was highlighted by the up-regulation of several siderophore-based iron acquisition genes and down-regulation of genes associated with a number of anaerobic electron transport pathways that utilise alternative electron acceptors to oxygen. In addition, the pattern of gene regulation also suggested apo-Fur regulation of the iron-responsive and flagellar biogenesis genes. This host response has been proposed to occur as a consequence of the reduction of ribonucleotides to form deoxyribonucleotides during phage DNA replication. This process is catalysed by the enzyme ribonucleotide reductase and requires iron and oxygen during the formation of a reactive di-iron centre within the β-subunit of the enzyme. Unusually knock-out mutants of a Type II restriction-modification system had a negative impact on phage replication. The A911_00150 mutant displayed pleiotropic changes in motility, cell based invasion and the ability to colonise chickens. Transcriptome analysis highlighted down-regulation of the genes required for the synthesis of the bacterial flagellum.