VEZF1 is a highly conserved vertebrate transcription factor that is essential for mammalian development. The gene regulatory functions of VEZF1 are largely undetermined. The generation of human cells depleted or absent of VEZF1 would greatly assist the study of VEZF1 functions and mechanism of action. This study makes use of synthetic biology technologies to either repress or knock out VEZF1 gene transcription to enable further studies of VEZF1 function. This study explores various strategies to use engineered DNA-binding proteins to direct the repression or mutation of a gene of interest. Zinc Finger (ZF) and Transcription Activator Like Effector (TALE) proteins that specifically recognise DNA sequences at the VEZF1 gene promoter were constructed using modular or Golden Gate assembly methods. The ability of TALE fusion proteins to function in human cells was studied. An expression vector system was created to assemble TALE Repressor (TALER) fusion proteins. The use of TALERs allowed for the rapid assessment of TALE protein binding at their chromosomal targets in human cells. Transient expression of most of the assembled TALE repressor proteins resulted in reduced VEZF1 transcription. A subset resulted in very substantial VEZF1 repression, making them useful tools for the study of VEZF1 function. Functional TALE domains were assembled into TALE nuclease (TALEN) fusion proteins. TALEN expression vectors were developed to assemble TALEN proteins with optimised expression, cleavage activity and target specificity. Transient expression of TALEN proteins in human cells was used to direct the cleavage and error-prone DNA repair of the VEZF1 promoter. Following development of the assays used to detect TALEN-directed mutations, several functional TALEN pairs were identified. Some TALENs resulted in over 65% mutation rates, with some mutations removing the VEZF1 promoter. These TALENs will be useful for the development of VEZF1 knock out cell lines. Interestingly, our study reveals a correlation between TALE length and the activity of TALERs and TALENs that should be considered in the future application of TALE proteins.

Hippocampal neurogenesis occurs within the subgranular zone of the dentate gyrus and is important for learning and memory. Neurogenesis is impaired in many pathological conditions; an observation that may account for learning and memory deficits in patients suffering from these conditions. Studies on immune-deficient mice show reduced hippocampal neurogenesis and associated learning and memory impairments in mice devoid of CD4+ T lymphocytes. Neuropeptides are potential candidates for mediating neuro-immune interactions. Vasoactive Intestinal Peptide (VIP) is a neuropeptide, released by firing interneurons from the stem cell niche, that modulates hippocampal neurogenesis via VPAC1/2 receptors. VIP receptors are also present on T lymphocytes. Microglia are innate immune cells that regulate hippocampal neurogenesis. They are ideally placed to communicate with T lymphocytes that normally reside outwith the brain parenchyma. Given the nescience underlying T lymphocyte regulation of hippocampal neurogenesis, we sought to investigate the hypothesis that VIP modulates T lymphocytes to release cytokines to regulate hippocampal neurogenesis via interaction with microglia. We have shown that T lymphocytes supernatant increases the proliferation of hippocampal nestin-expressing cells. This effect is further enhanced under VIP treatment via VPAC1 receptor subtype. Examining possible cytokine involvement, we found that IL-4 mediates proliferation. Using Mac-1-SAP to deplete resident microglia, we demonstrated that supernatant acts primarily via microglia to increase supernatant effects. T lymphocytes induce microglia to upregulate cytokines and mediators such as IL-10 and BDNF. Phenotyping showed an additional neurogenic effect under VIP treated supernatant. Our results show VPAC1 receptor subtype expressed by CD4+ T lymphocytes mediates VIP proliferative effects on hippocampal precursor cells via IL-4 cytokine release. Microglia are key for mediating this effect via release of mediators. The findings of this study implicate a novel mechanism for VPAC1 CD4+ T lymphocyte receptor as a neuro-immune mediator of hippocampal neurogenesis, and from a therapeutic perspective, shows that the effect can be pharmacologically manipulated.

Throughout the world, control of parasitic nematodes in livestock has been compromised by the emergence and spread of anthelmintic resistance. Teladorsagia circumcincta is the most important gastrointestinal nematode parasite of small ruminants in temperate regions and the major resistant species in the United Kingdom (UK). In most cases the genetic factors which underpin resistance to broad-spectrum anthelmintics are still poorly understood. Recent work conducted independently in New Zealand (NZ) and Scotland has implicated the involvement of a particular P-glycoprotein (Pgp) gene, Tci-pgp-9, in multiple-anthelmintic resistance in T. circumcincta. The focus of this study is to further characterise Tci-pgp-9 and its possible role in ivermectin (and multi-drug) resistance using two UK field isolates of T. circumcincta, one which is anthelmintic susceptible (MTci2) and another that is multiple-anthelmintic resistant (MTci5). The generation of full-length cDNA sequence data from these isolates allowed genetic comparisons which identified the presence of nine non-synonymous SNPs in the Tci-pgp-9 coding sequence of the MTci5 isolate. The 3.8 kb, Tci-pgp-9 transcript from the MTci2 and MTci5 isolates shared 95.5 % identity at the nucleotide level and 99.5 % identity at the protein level. Twelve sequence variants were identified in the first internucleotide binding domain, designated Tci-pgp-9-IBDA, some of which shared a high level of identity with sequence variants identified in near-isogenic NZ strains. Multiple allelic variants were present in the majority of individuals, but a reduction in the number of allelic variants present in individuals of MTci5 relative to the MTci2 isolate was evident. A further reduction in the number of alleles present in individuals was also observed in individuals derived from an IVM treated population of MTci5, suggesting that IVM treatment applied purifying selection pressure. Quantitative real time PCR analysis showed a 3.7-fold increase in Tci-pgp-9 gene copy number in the MTci5 isolate relative to the MTci2 isolate, which was consistent with a 3.4-fold increase observed in the NZ study. None of the common haplotypes identified were unique to any given isolate, and the relationship between haplotype and copy number was not straightforward. This study provides evidence that Tci-pgp-9 is under anthelmintic selection, but the precise role of this specific P-glycoprotein gene, and its alleles, in the phenotypic expression of anthelmintic resistance in T. circumcincta remains to be determined.

Hodgkin lymphoma (HL) is one of the most common haematological malignancies in the Western world. Although HL responds favourably to cytotoxic therapy in the majority of cases, late side-effects such as secondary malignancies and cardiovascular disease are becoming of great concern, particularly in younger patients. The current challenges are to maintain treatment efficacy whilst reducing side-effects and to develop biomarkers to predict response to treatment(s). Protein degradation pathways in HL cells have remained largely unstudied in this malignancy and represent an opportunity for more targeted therapy. This thesis describes the activities of protein degradation pathways in HL-derived cell lines and their sensitivities to inhibition. The results suggest that the use of proteasome and HDAC6 inhibitors, alone or in combination, may be of clinical benefit in the treatment of HL in the future. The presence of p62 was used to monitor protein handling stress; however, its diverse expression patterns in HL-derived cell lines and in paraffin-embedded HL biopsy material preclude its use as an informative biomarker in HL. p62 was found to traffic between the cytoplasm and nucleus in HL-derived cell lines and its association with a DNA damage marker in both cellular compartments implicate it as a chaperone for the cytoplasmic degradation of nuclear proteins. A previously unreported nuclear expression of the lysosomal enzyme cathepsin B in HL-derived cell lines was identified, and this may have implications for the aberrant transcriptional profile of these cells given the regulatory activities of other members of this family of cysteine proteases. The non-lymphoid origin of the HD-MyZ cell line, a putative HL-derived cell line, was corroborated by phenotypic differences in protein handling pathways in comparison to accepted HL-derived cell lines. The activities and insensitivity to inhibition of protein handling pathways in this cell line suggest that it will be an interesting model for studying alternative protein degradation pathways. The presence of a number of small cell populations with cancer stem cell (CSC)-like characteristics was confirmed in HL-derived cell lines and, although these populations require further characterisation, this has implications for HL patients who suffer relapse.

Survival of an organism depends on its perception and response to external stressors such as infection, osmotic stress (ionic or desiccation) or xenobiotic stress. At the cellular level, stress is perceived and relayed via signal transduction pathways that alter transcription or establish new transcriptional programmes that modulate physiology at the whole-organism level to regain homeostasis and promote survival. The vital function of epithelial tissues (e.g., kidneys in vertebrates and Malpighian tubules in insects) is systemic balance of nutrient, solute and water levels. Additionally, epithelial tissues act in sensing stress and relaying signals for adaptation and tolerance to stress. The PhD work presented here is to delineate the roles of Relish and cGMP-dependent kinases in mechanisms of epithelial stress handling using a genetically tractable epithelium, the Drosophila Malpighian tubule, as an in vivo model for stress sensing and response. Relish, a transcription factor, is the insect orthologue of the mammalian NF-kB. It regulates the insect’s innate immune pathway and is highly expressed in D. melanogaster tubules. We show that Relish expressed in Malpighian tubules modulates organismal tolerance to osmotic stress caused due to a high salt diet (salt stress). In order to determine the genes that are involved in salt stress tolerance, Affymetrix Drosophila GeneChips (microarrays) were run with RNA isolated from the wild-type and Relish mutant tubules from flies fed either on normal food, or on ‘salt food ‘. The transcriptomic data was analysed to find genes that were dependent and independent of Relish in response to stress. Additionally, the data also revealed that during salt stress, with the loss of Relish related signalling pathway, the other stress response pathways, in particular, the c-Jun kinase pathway is hyper-activated. This suggests (1) a potential cross-talk occurring between Relish and other stress response pathways, and (2) a redundancy in stress response pathways, for adapting to salt stress. These data demonstrate a novel role for Relish in salt tolerance in Drosophila melanogaster. Moreover, under unstressed conditions, expression of 448 genes was significantly altered and a reduced basal fluid secretion rates were observed in Relish mutant tubules. This suggests that basal Relish activity is required for optimal working of the tissue. In addition, a study to elucidate the immune and osmotic stress-associated roles of cyclic guanosine monophosphate (cGMP)-dependent kinases - Dg1 and Dg2 - in tubules was carried out. Salt stress and desiccation stress survival assays in flies with targeted knock down of each of Dg1 and Dg2 genes in tubule principal cells showed an opposing stress phenotype for these two stressors. No immune phenotype was observed on infection with non-lethal gram negative bacteria. This showed that the cGMP-associated osmotic stress response mechanisms were beneficial or detrimental to survival of organism, depending on the type of stressor and downstream effectors. The understanding gained from this in vivo approach of studying stress pathways in Drosophila Malpighian tubules can be further explored through a systems biology approach .This, together with combinatorial gene knockdown studies to reveal stress network “hubs”, may be applied to development of potential targets of insecticides and in biomedical sciences.

Cancer cells have been noted to have an altered metabolic phenotype for over ninety years. In the presence of oxygen, differentiated cells predominately utilise the tricarboxylic acid (TCA) cycle and oxidative phosphorylation to efficiently produce energy and the metabolites necessary for protein and lipid synthesis. However, in hypoxia, this process is altered and cells switch to a higher rate of glycolysis and lactate production to maintain their energy and metabolic needs. In cancer cells, glycolysis is maintained at a high rate, even in the presence of oxygen; a term described as “aerobic glycolysis”. Tumour cells are rapidly dividing and have a much greater need for anabolism compared to normal differentiated cells. Rapid glucose metabolism enables faster ATP production as well as a greater redistribution of carbons to nucleotide, protein, and fatty acid synthesis, thus maximising cell growth. Recently, other metabolic changes, driven by mutations in genes related to the TCA cycle, indicate an alternative role for metabolism in cancer, the “oncometabolite”. This is where a particular metabolite builds up within the cell and contributes to the tumorigenic process. One of these genes is isocitrate dehydrogenase (IDH) IDH is an enzyme that forms part of the tricarboxylic acid (TCA) cycle and converts isocitrate to α-ketoglutarate (α-KG). It exists in three isoforms; IDH1, IDH2 and IDH3 with the former present in the cytoplasm and the latter two in the mitochondria. Point mutations have been identified in the IDH1 and IDH2 genes in glioma which result in a gain of function by converting α-KG to 2-hydroxyglutarate (2HG), an oncometabolite. 2HG acts as a competitive inhibitor of the α-KG dependent dioxygenases, a superfamily of enzymes that are involved in numerous cellular processes such as DNA and histone demethylation. It was hypothesised that the IDH1 mutation would result in other metabolic changes in the cell other than 2HG production, and could potentially identify pathways which could be targeted for therapeutic treatment. In addition, 2HG can act as a potential competitive inhibitor of α-KG dependent dioxygenases, so it was hypothesised that there would be an effect on histone methylation. This may alter gene expression and provide a mechanism for tumourogenesis and potentially identify further therapeutic targets. Metabolic analysis of clinical tumour samples identified changes associated with the IDH1 mutation, which included a reduction in α-KG and an increase in GABA, in addition to the increase in 2HG. This was replicated in several cell models, where 13C labelled metabolomics was also used to identify a possible increase in metabolic flux from glutamate to GABA, as well as from α-KG to 2HG. This may provide a mechanism whereby the cell can bypass the IDH1 mutation as GABA can be metabolised to succinate in the mitochondria by GABA transaminase via the GABA shunt. JMJ histone demethylases are a subset of the α-KG dependent dioxygenases, and are involved in removing methyl groups from histone tails. Changes in histone methylation are associated with changes in gene expression depending on the site and extent of chemical modification. To identify whether the increase in 2HG and fall in α-KG was associated with inhibition of histone demethylases a histone methylation screen was used. The IDH1 mutation was associated with an increase in methylation of H3K4, which is associated with gene activation. ChiP and RNA sequencing identified an increase in H3K4me3 at the transcription start site of the GABRB3 subunit, resulting in an increase in gene expression. The GABRB3 subunit forms part of the GABA-A receptor, a chloride channel, which on activation can reduce cell proliferation. The IDH1 mutation was associated with an increase in GABA and GABRB3 subunit of the GABA-A receptor. This raises the possibility of GABA transaminase as a potential therapeutic target. Inhibition of this enzyme could reduce GABA metabolism, potentially reducing any beneficial effect of the GABA shunt in IDH1 mutant tumours, and increasing activation of the GABA-A receptor by increasing the concentration of GABA in the brain. This in turn may reduce cell proliferation, and could be achieved by using Vigabatrin, a GABA transaminase inhibitor licensed for use in epilepsy.

After being transported into Europe during the Neolithic, mouflon (Ovis aries musimon) became extinct from mainland Europe, but remnant populations persisted and became feral on the Mediterranean islands of Corsica and Sardinia. These populations have been used for reintroductions across continental Europe during the last 200 years. This thesis aimed to investigate the global and local ancestry of European mouflon and domestic sheep, to investigate signals of artificial and natural selection in their genomes, and to develop analytical frameworks and informatic tools to aid similar analyses using SNP array data. I describe the development of software that allows rapid investigation of genome-wide SNP data to infer effective population size trajectories using patterns of linkage disequilibrium. I inferred the absence of widespread sheep introgression in extant European mouflon populations although signals of recent introgression were recorded in one enclosed Sardinian mouflon population. By applying a novel approach to aid the investigation of local genomic ancestry data, signals of mouflon ancestry in sheep could be inferred and were found to be related to biological functions involved with innate immunity processes with bitter taste recognition being identified in two breeds known for their broad dietary choices. By investigating signals of positive selection and local adaptation in feral and domestic sheep using novel locus-specific empirical p-value inference, traits with selection signatures such as fertility, pigmentation and behaviour were identified in sheep, while traits involved with stature - probably related to mating success - were found in mouflon. Signals of local adaptation to environmental variables were not detected, which is likely to be due to the inadequate sample available, determined by post-hoc analysis.

Background Chronic pancreatitis (CP) is a disease of fibrosis of the pancreas for which alcohol is the main causative agent. However, only a small proportion of alcoholics develop chronic pancreatitis. Genetic polymorphism may affect pancreatitis risk. Aim To determine the factors required to classify a chronic pancreatic population and identify genetic variations that may explain why only some alcoholics develop chronic pancreatitis. Methods The most appropriate method of diagnosing CP was assessed using a systematic review. Genetics of different populations of alcohol-related chronic pancreatitics (ACP) were explored using four different techniques: genome-wide association study (GWAS); custom arrays; PCR of variable nucleotide tandem repeats (VNTR) and next generation sequencing (NGS) of selected genes. Results EUS and sMR were identified as giving the overall best sensitivity and specificity for diagnosing CP. GWAS revealed two associations with CP (identified and replicated) at PRSS1-PRSS2_rs10273639 (OR 0.73, 95% CI 0.68-0.79) and X-linked CLDN2_rs12688220 (OR 1.39, 1.28-1.49) and the association was more pronounced in the ACP group (OR 0.56, 0.48-0.64)and OR 2.11, 1.84-2.42). The previously identified VNTR in CEL was shown to have a lower frequency of the normal repeat in ACP than alcoholic liver disease (ALD; OR 0.61, 0.41-0.93). Homozygosity of the normal variant was more common in ALD than ACP (OR 0.53, 0.3-0.96) or Healthy Controls (OR 0.55, 0.3-1.00)). The NGS discovery phase lead on to validation of the 21 most significant SNPs with Sequenom array. This showed significance difference between ACP and ALD in allele frequency of the synonymous SNP, PRSS1_rs6666, (OR 1.99, 1.46-2.72) Conclusion A range of potential exonic and intronic sites have been identified that have association with a predisposition to developing chronic pancreatitis. These findings show that further work is justified to fully assess the interaction of the different polymorphisms and their phenotypic significance in development of the disease.

No description available.

The myeloproliferative disorders (MPDs) are a group of haematopoietic stem cell diseases, characterised by proliferation of one or more cells of the myeloid lineage. Several lines of evidence have highlighted the importance of aberrant tyrosine kinase signalling in the pathogenesis of these disorders. Cloning of rare chromosomal translocations and point mutation analysis in the MPDs has identified diverse deregulated tyrosine kinase genes, notably PDGFRA, PDGFRB, FGFR1 and JAK2. However the majority of atypical MPDs still remain to be characterised and identification of patients harbouring fusions, particularly those involving the PDGF receptors is of increasing importance, as they are likely to be responsive to targeted therapy with imatinib. I am investigating MPD patients for abnormalities affecting tyrosine kinase signalling, and have used three approaches, translocation cloning, expression analysis and SNP array analysis to detect regions of loss of heterozygosity (LOH). Thus far, by translocation cloning I have identified a previously undescribed partner gene fused to PDGFRB and two new PDGFRA fusion genes. I have also designed two reverse transcriptase PCR (RT-PCR) assays and a cDNA MLPA assay to detect over-expression of specific tyrosine kinases screening approximately 200 patients. Each assay identified all patients previously diagnosed with known fusions. Additionally, two patients identified with overexpression of PDGFRB have been found to have cryptic ETV6-PDGFRB fusions and overexpression of PDGFRA in one patient lead to the discovery of a previously undescribed fusion involving a novel partner gene (KIF5B). Recent evidence has indicated that acquired isodisomy is a novel mechanism by which mutations in cancer may be reduced to homozygosity. Typically, acquired isodisomy is associated with oncogenic changes rather than tumour suppressor genes, eg. the activating JAK2 V617F mutation and 9p aUPD. I have undertaken a screen using Affymetrix 50K SNP arrays for regions of acquired isodisomy as a means to identify genomic regions that may harbour novel oncogenes in different subgroups of MPD patients. Large tracts of homozygosity (defined as >20Mb running to a telomere), strongly suggesting acquired isodisomy, were seen in 40% aMPD patients. The homozygous tracts encompassed diverse genomic regions in aMPD, but two common regions (3 cases for each region) were identified at 7q and 11q. Mutations in the CBL ubiquitin ligase gene were discovered in all three aCML patients with 11q aUPD as well as in an additional 23 MPD patients following further screening.