Nucleotide excision repair (NER) is critical for maintaining genome integrity. How chromatin dynamics are regulated to facilitate this process in chromatin is still under exploration. Htz1 (H2A.Z), a highly conserved histone H2A variant, is incorporated into the nucleosomes around the promoters of many genes in S. cerevisiae. Htz1 is involved in the maintenance of genome stability, DNA transcription activation, DNA replication and DNA repair. In this study, I employed Saccharomyces cerevisiae, as a model organism. In this thesis, I examined the role of histone Htz1 in the response to UV light at specific regions using a high resolution approach and throughout the entire yeast genome using chromatin-immunoprecipitation coupled to microarrays. htz1Δ and its deposition related mutants are more sensitive to UV and CPD removal was impaired in total DNA from both htz1Δ and swr1Δ strains. Acetylation of Htz1 at K3, 8, 10, 14 does not contribute to UV sensitivity or CPD removal from total DNA. CPD removal experiments at the MFA2 promoter and the HMRa1 coding region have showed that Htz1 has a role in NER and it likely only affects repair at local nucleosomes containing Htz1. My ChIP experiments at MFA2 and HMRa1 indicate that Htz1 enhances the binding of the HAT Gcn5 to Htz1 containing chromatin and this promotes histone H3 hyperacetylation in Htz1 nucleosomes in the MFA2 promoter after UV irradiation. As a result of this optimal level of histone H3 acetylation occur and the binding of Rad14 to damaged DNA is also enhanced at this region. My genome-wide study shows that UV does not influence the localization of Htz1 as it still resides at the pre-UV sites. Htz1 and H3 K9K14 acetylation is found to be associated with each other genome-wide. This is believed to be via the interaction between Htz1 and Gcn5. These results show that there is a positive role of Htz1 in promoting efficient GG-NER.

Activation of complement and its terminal pathway leads to the formation and insertion of the membrane attack complex (MAC) in the membranes of target cells. Complement is activated in tumours as is clear from the presence of complement activation products in cancer tissues. Over-expression of membrane bound complement regulators on tumour cells together with endogenous recovery mechanisms restricts complement activity and results in escape from lytic killing; nevertheless, sublytic MAC deposition is not without consequence. Sublytic MAC assembly on nucleated cells causes cell activation, secretion of extracellular matrix and pro-inflammatory cytokines and may cause protection from apoptosis. Signalling of these events is unclear. The global effects of sublytic MAC were addressed in the murine colon carcinoma cell line (CT26) through the use of microarray technology. Cells were exposed to sublytic complement attack using pooled normal human serum (pNHS) and compared to MAC-inhibited controls generated using pNHS containing the C5 inhibitor OmCI. Total RNA was extracted at 0, 1 and 12 hours post-exposure and subjected to microarray analysis using the Illumina platform. Top statistically significant changes were then identified and a list of genes upregulated at both time points was uploaded to MetaCore and a gene network generated. From this a number of co-regulated genes which converged on the EGFR were highlighted. These were cxcl1, amphiregulin and matrix metalloproteinases (Mmp) 3 and 13. Both the top statistically significant and network derived genes were validated using qPCR. Changes in protein levels were then tested using western blot analyses for Mmp3 and Areg. Inhibition of the MEK/ERK, and to a lesser extent PI3K/Akt, signalling suppressed the gene upregulation that occurred in response to MAC but inhibition of p38 and JNK had no effect, implicating a MEK/ERK- PI3K co-activation. MAC deposition and not C5a/C5aR axis signalling was shown to be responsible for the mmp3 gene upregulation response. Identification of MAC-mediated events and the signalling pathways involved may provide insight into the mechanisms by which complement activation influences tumour growth. In particular the data suggest that sublytic MAC deposition might promote a gene expression response which pushes the cells to a more aggressive phenotype by the upregulation of proliferative, survival, invasion and migratory signals. This in turn will inform strategies that seek to harness complement or complement regulation in tumour immunotherapy.

No description available.

genes show monoallelic parent-of-origin specific expression and have an important role in mediating adult behaviour. Previous research has indicated that maternally expressed Nesp and paternally expressed Grb10, which are expressed in overlapping brain regions, may have a role in mediating risk-taking and/or impulsive behaviours. Impulsivity and risk taking are natural parts of human behaviour; however pathological levels of impulsivity and risk-taking are recognised as clinical traits of many psychiatric disorders. The aim of the current research is to explicitly test whether these two oppositely imprinted genes influence impulsivity and/or risk-taking behaviour in mice by examining mouse models that lack functional copies of paternal Grb10 (Grb10+/p) and maternal Nesp (Nespm/+) in a number of tests of impulsivity and risk-taking. Unlike previous findings in Nespm/+ mice, Grb10+/p mice had the same propensity to explore a novel environment as wild type (WT) controls. However, in a measure of delay-discounting behaviour it was discovered that Grb10+/p mice were less likely to discount delayed rewards. This is in contrast to previous work with Nespm/+ mice, which discounted delayed rewards more steeply. This is the first demonstration that oppositely expressed imprinted genes antagonistically affect behaviour. To further explore these behaviours, a novel test of risk-taking was developed. Using predator odours a perceived ‘risky’ environment was created in order to measure the decision between fear and reward seeking. Using the Predator Odour Risk-Taking (PORT) task it was demonstrated that Nespm/+ and Grb10+/p mice showed comparable levels of risk-taking behaviour to WT littermates. Finally, immunofluorescence was used to discover that Nesp55 and Grb10 are not only expressed in the same brain regions, but are co-expressed in some cells, particularly in serotonergic neurons. This suggests that these imprinted genes may be influencing delay discounting behaviour via the same integral neurotransmitter systems.

Huntington’s disease is an autosomal dominant neurological disease caused by an elongated CAG repeat in exon 1 of the huntingtin gene. There is currently no cure and treatments are limited. The genetic mutation causes selective cell death of the medium spiny neurons which reside in the striatum of the basal ganglia. Current disease models don’t necessarily recapitulate all aspects of the human disease and so alternatives are needed. The advent of induced pluripotent stem cells (iPSC), has allowed for HD patient specific pluripotent stem cells to be derived, hence differentiation of these cells in vitro could provide a disease model for drug testing and investigation of disease pathology. Current protocols for differentiation of pluripotent stem cells into medium spiny neurons (MSNs) are often inconsistent and lead to low yields of MSNs. Directing differentiation through forced expression of transcription factors has been used to differentiate neurons from fibroblasts and pluripotent stem cells, often with increased efficiency. Utilising transcription factors vital in post-mitotic MSN development, this study has aimed to produce MSNs in vitro, by transfection of transcription factors or combinations thereof in a multicistronic plasmid into ventral forebrain neural progenitors. This study has involved the cloning and expression of 5 different transcription factors important in MSN development in iPSC-derived neural progenitors. Two of these transcription factors; NOLZ1 (ZNF503) and ISL1 were further investigated for their ability to differentiate neural progentiroes into MSNs. This study showed that transfection of ISL1 enabled differentiation of neurons to produce a higher proportion of cells resembling MSNs, characterised by co-expression of the MSN markers DARPP32 and CTIP2 and expressing FOXP1. The combination of NOLZ1 and ISL1 in transfection improved functional maturation of neurons, becoming increasingly spontaneously active and increased excitability, as well as responding to GABA and NMDA, with dopamine D1 agonist enhancement of NMDA currents.

Human T-cells play a major role in controlling and clearing Mycobacterial infections. The adaptive immune system deploys a complex network of specialised T-cell subsets in order to tailor an optimum immune response. Two categories of T-cells have been described that are characterised by the ligands they recognise: “conventional” T-cells (polymorphic, HLA-restricted, peptide-specific) and “unconventional” T-cells (non-polymorphic, restricted by HLA-like molecules, non-peptide-specific). Both T-cell categories were shown to be important for the elimination of cells infected with Mycobacterium tuberculosis (M. tuberculosis) and their role, specificities and functionalities are under active investigation in order to develop optimum vaccination strategies. A large interest in unconventional T-cells, such as MR1-restricted MAITs or CD1-specific T-cells, and their role in mycobacterial infections has recently arisen. I initiated my studies by dissecting T-cell responses generated during direct ex vivo boosting of PBMCs with antigen presenting cells that had phagocytosed Mycobacterium smegmatis (M. smegmatis). M. smegmatis is a non-pathogenic bacterium and is mainly eliminated by the innate immune system. However, T-cells might respond to M. smegmatis antigens and therefore play a role in clearing the pathogen. Using polychromatic flow cytometry, I successfully identified major CD3+ conventional and unconventional M. smegmatis-specific T-cell populations and evaluated their respective frequencies and distribution. The identification of a significant frequency of M. smegmatis-specific unconventional MAITs pushed me to further analyse the specificity of this interesting T-cell subset. At the time of my studies, the ligand(s) presented by MR1 to MAITs were still undiscovered. However, structural models of MR1 groove moiety provided evidences that MR1 could potentially present peptides to MAITs. Therefore, I attempted to identify the molecular and cellular mechanisms by which an M. tuberculosis-specific MAIT clone recognises peptide loaded on MR1 and to refold this MHC-like protein. Vaccination strategies have been mainly focusing on targeting CD8 T-cells, known to be essential for the host defence against mycobacterial infections. Therefore a huge effort is made to discover new immunodominant mycobacterial epitopes. Collaborators isolated the HLA-A*0201-restricted D454 T-cell clone specific to the LLDAHIPQL epitope derived from the highly immunogenic Esx-G protein. The LLDAHIPQL sequence is conserved across mycobacterial species thus offering potential for pan-mycobacterial vaccination. I aimed at proving that D454 TCR binds to HLA-A*0201-LLDAHIPQL. I successfully obtained an HLA-A*0201-LLDAHIPQL crystal structure, the first bacterially-derived HLA-peptide complex, and identified the key mechanisms involved in the molecular recognition of HLA-A*0201-LLDAHIPQL by a conventional TCR.

Tobacco use is the leading preventable cause of death worldwide. In order to address this epidemic, it is important that we have a thorough understanding of the aetiology of tobacco use and dependence. Twin and adoption studies have consistently demonstrated the importance of genetic factors in smoking behaviours. The advent of genome-wide technologies has greatly facilitated the search to determine which specific genetic factors contribute to tobacco use phenotypes. A locus within the nicotinic acetylcholine receptor gene cluster CHRNA5-A3-B4 has generated particular interest – that marked by variants rs16969968 in CHRNA5 and rs1051730 in CHRNA3. The primary aim of this thesis was to determine the role played by this locus in smoking-related behaviours, with an emphasis on phenotype refinement. A number of different approaches were utilised to address this objective, namely systematic review and meta-analysis, genetic epidemiology (including detailed phenotyping of smoking behaviour in adolescence), laboratory-based techniques, and genome-wide meta-analysis. Compelling evidence for a small, robust association was observed between the rs1051730/rs16966968 variants and daily cigarette consumption, equivalent to a per allele effect of approximately one cigarette per day. This effect was consistent across population sub-groups. Compelling evidence for an association between this locus and level of tobacco exposure was further illustrated through genome-wide meta-analysis of cotinine levels in current smokers. No association was observed between this locus and smoking initiation however, as examined in a prospectively assessed cohort using precisely defined phenotypes. An association between rs1051730/rs16969968 and smoking topography has yet to be explored. However, a full protocol was developed and piloted to investigate this. In addition, this research has also illustrated the importance of precise, objective, phenotype definition, an observation which has important implications for the fields of molecular genetics and epidemiology.

Hypertension is a common, highly heritable trait of complex aetiology. Multiple environmental and lifestyle factors contribute to blood pressure variation. Hence the study of hypertension causality is not straightforward. Genetic linkage studies have implicated a number of loci involved in blood pressure regulation and the development of hypertension. Candidate gene association studies, however, have not reported any reproducible associations. Early genome-wide association studies (GWAS) showed remarkable success in identifying validated common variants associated with common diseases such as coronary artery disease and type 1 diabetes. However, the first GWAS of hypertension showed little success. This was largely because of a lack of statistical power and insufficient genomic coverage. Furthermore, it is widely believed that the failure of one GWAS of hypertension was partly due to misclassification of controls that were not phenotyped for blood pressure. Subsequently, two large international consortia-run GWAS of blood pressure as a quantitative trait produced tangible results. The current study is a GWAS of hypertension using an extreme case-control design. It employed intensive phenotyping and extreme case-control definitions to select a sample of individuals from a restricted geographical area of relative homogeneity. The aim was to reduce misclassification bias and increase the likelihood of detecting any genetic effects. Cases were sampled from the Nordic Diltiazem study, and defined as individuals younger than 60 years with at least two consecutive measurements of systolic blood pressure (SBP) ≥ 160 mmHg or diastolic blood pressure (DBP) ≥ 100 mmHg. Controls were sampled from the prospective Malmö Diet and Cancer Study, and defined as individuals aged at least 50 years with SBP ≤ 120 mmHg and DBP ≤ 80 mmHg with no evidence of cardiovascular disease during ten years of follow-up. The groups represent, respectively, the upper 1.7% and lower 9.2% of the Swedish blood pressure distribution. Comparison of groups from the extreme tails of distribution increased statistical power by inflating observed effect sizes. With genome-wide SNP coverage we were able to adjust for population stratification using principal components analysis. Following quality control exclusions, a final set of 521,220 single nucleotide polymorphisms was available for analysis in 1,621 cases and 1,699 controls. Seventeen SNPs were associated with hypertension at a P < 1 × 10-5 threshold of significance, of which three attained genome-wide significance, defined as P < 5 × 10-7. The top hit, rs13333226, underwent a two stage validation process in a total of 14 independent cohorts. The combined odds ratio for the discovery cohort and all replication cohorts meta-analysed was 0.87 (95% CI 0.84 – 0.91, P = 3.67 × 10-11) with the minor G allele associated with a lower risk of hypertension. In total 21,466 cases and 18,240 controls were included. After adjustment for age, age2, sex, and BMI, and when the discovery cohort was excluded from analysis, the association remained significant. Estimated glomerular filtration rate (eGFR), a measure of kidney function, was available in seven of the cohorts. When the analysis was repeated with adjustment for eGFR the effect was marginally strengthened. rs13333226 is located in close proximity, at -1617 base pairs, to the uromodulin (UMOD) transcription start site. UMOD encodes uromodulin, also known as the Tamm-Horsfall protein. Uromodulin is produced predominantly in the thick ascending limb of the loop of Henle and is the most abundant protein in urine. Its function is unclear; however, variants in UMOD have been associated with chronic kidney disease. Clinical functional studies were conducted in three separate populations. The minor G allele of rs13333226 (associated with a lower risk of hypertension) was associated with lower urinary uromodulin excretion. Furthermore, in one sample following a low salt diet urinary uromodulin excretion was significantly lower in the presence of the G allele, whereas after a high salt diet genotype was no longer associated with urinary uromodulin. If this were verified, this would entail a gene-environment interaction. Our combined results suggest that UMOD may have a role in regulating blood pressure, possibly through an effect on sodium homeostasis. There is ample evidence of a strong, graded relationship between blood pressure and subsequent renal disease. Hence the current finding is biologically plausible. Information on kidney disease was not available for the discovery samples so this could not be explored. However, the association between rs13333226 and hypertension was not substantively altered by adjustment for eGFR in the seven validation cohorts in which it was recorded, suggesting that it is independent of renal function. In conclusion, we have performed a GWAS of hypertension using an extreme case-control design. The most significant hit was validated in a meta-analysis of the discovery sample and 14 additional cohorts. Moreover, functional studies showed a relationship between genotype and urinary protein excretion. Overall, we demonstrate that with careful methodological planning and phenotyping it is possible to generate replicable hypertension GWAS results in a relatively small sample size.

Chemical control of mosquito vectors, via indoor residual spraying or insecticide treated bed nets, is an integral component of malaria control strategies. Limited availability of insecticides licensed for public health and the rapid development of resistance in mosquito populations to these insecticides, in particular to some pyrethroids, may compromise vector control efforts. With the exception of mutations in the insecticide target sites, relatively little is known about the genetics of pyrethroid resistance in malaria vectors. In some populations candidate effector genes, e.g. cyp6p3 or cyp6m2 in An. gambiae s.s. from Akron, Benin, have been identified as being over expressed in resistant strains but the underlying mechanisms responsible for the increased expression remain unknown. In this study, a combination of quantitative PCR, genetic mapping and microarray tools were used to investigate the mechanisms responsible for pyrethroid resistance in two African major malaria vectors, Anopheles gambiae and An. arabiensis. The current work was unable to confirm an association of these known candidates in either a laboratory colony established from Akron or in recent field caught material. Therefore a genetic mapping approach was adopted using field collected mated females to generate F2 isofemale lines. A major QTL on chromosome 3R was identified which coincides with a genomic region previously implicated in pyrethroid resistance in East African populations. This is the first genetic mapping of insecticide resistance using natural out-bred populations of Anopheles and the advantages and limitations of this approach are discussed. In a second experiment, genetic loci involved in permethrin resistance in An. arabiensis were mapped by establishing genetic crosses between a permethrin resistant strain from Chad and a susceptible strain from Mozambique. A single QTL on chromosome 2R was identified in the F2 progeny that accounts for ~24% of the phenotypic variance. This QTL coincides with a large cluster of detoxification genes. Pyrethroid resistance is not associated with target-site mutations in this population. Finally, microarrays were used to identify genes differentially expressed between a backcross population, generated by crossing the F1 population from the resistant Chad strain and the susceptible Mozambique strain of An. arabiensis back to the parental resistant strain, with the susceptible strain. A number of candidate genes were identified, including the P450 genes cyp4h24 and cyp9j5, but neither of these were located within the boundaries of the QTL on 2R. These findings support the presence of metabolic resistance in this population and fine mapping of the identified QTL as well as further investigation of the microarray hits is warranted.

Neuronal dysfunction and cell death occurs during neurodegeneration. Animal models that express human disease genes and show neurodegenerative-like pathologies are widely used to study particular molecular systems in early neurodegenerative changes. Axonal transport (AT) is perturbed in several prevalent neurodegenerative diseases. The development of a Huntington’s Disease (HD) model in Drosophila melanogaster larvae is described, in which disease gene expression is directed to motor neurons (Chapter 2). This results in stalling and accumulation of AT vesicles in live animals and a locomotion defect after additional environmental stress. The cause of AT disruption and neuronal dysfunction in most cases of neurodegeneration is unknown, but it is associated with protein misfolding and aggregation that overrides cellular defences such as the heat shock protein (HSP) molecular chaperone system. In addition to HD, this applies to human tauopathies such as Alzheimer’s Disease (AD), which involve axonal misfolding and aggregation of tau. Increased throughput assays to test larval locomotion are developed (Chapter 3) in a Drosophila larval model of tauopathy, in which locomotion defects are detectable under normal environmental conditions. Candidate chemical modulation of this locomotion phenotype is described that targets HSP induction (Chapter 4). The chemicals used result in no detectable change in hsp70 level, lower total tau levels, and worsening of the locomotion defect phenotype. Tissue-specific elevation of hsp70 after hypoxic stress (Chapter 5) protects from acute behavioural disability and reduced survival in aged adult Drosophila expressing human tau in the nervous system. These studies indicate some therapeutic potential for HSP elevation in tau mediated pathology. Nevertheless, further work is required if chemical chaperone induction, and the roles of HSPs in axonal transport and homeostasis during chronic neurodegenerative and acute environmental stress, are to be further explored in these models