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.

Regenerative medicine and tissue engineering are being revolutionised by developments in the field of stem cell science. Mesenchymal Stem Cells (MSCs) are emerging as a desirable tool in regenerative medicine and cell therapy due to their wide ranging differentiation potential, large expansion capacity, and their lack of immune rejection following transplantation. Early in vivo studies have demonstrated therapeutic effects of hMSCs; however to clinically exploit the potential of hMSCs, the adherent cell type must be expanded to therapeutically relevant lot sizes (109 to 1012 cells). Hence now there is a need to develop protocols for stable, controlled in vitro expansion, isolation and preservation of a homogenous population of functionally viable cells. Specifically a practical, clinically safe and scalable system which adheres to current GMP guidelines is required to develop reproducible and cost effective therapeutic products. Here we describe the design, manufacture and characterisation of biofunctionalised hydrogel microcarriers containing ECM derived adhesion peptides and a range of compressive moduli for adipose derived stem cell expansion. Microfluidic devices were employed to produce monodisperse spherical particles which were polymerised in situ. In addition, these microcarriers have tunable characteristics which make them a particularly useful tool for the systematic investigation of cellular responses. Microcarriers modified to contain fibronectin and laminin derived peptides supported ADSC attachment and growth in a concentration dependent manner. ADSCs cultured on peptide modified microcarriers were capable of differentiating into osteocytes, chondrocytes and adipocytes, indicating cells cultured on microcarriers maintained multipotency. Substrate compressibility was found to effect ADSC differentiation, corroborating previous literature reports. Bioreactor culture demonstrated successful ADSC expansion with fold increases in cell number far higher than have previously been reported in the literature. High cell seeding densities produced large quantities of viable cells. However, decreasing initial cell seeding density, increased the total fold expansion and reduced cell doubling rates.

Understanding how the germ layers are formed is one of the key questions of developmental biology. Abundant studies in the anuran amphibian Xenopus laevis have described that maternal and vegetally localised mRNAs for VegT and Vg1 contribute greatly to the formation of mesoderm and endoderm in the developing embryo. Within Xenopus mesendoderm gene-regulatory network (GRN), Wnt/β-catenin as well as Nodal and Mix family members have been shown to play important roles. The involvement of several members of the Nodal and Mix gene families with redundant functions makes the mesendoderm GRN surprisingly complex and difficult to study in Xenopus laevis. By contrast, mouse and humans have only single copies of Nodal and Mix. Since urodeles have an embryology that is basal to amphibians and that has most likely also been conserved during the evolution of amniotes, including mammals, we have investigated the Mix and Nodal genes in the urodele Axolotl in the hope that their gene families contained fewer members. We cloned one Mix and two Nodal orthologs from the axolotl and showed by Southern blot analysis that there are likely no further copies in the axolotl genome. Morpholino and rescue experiments furthermore showed that AxNodal-1, Mix and Brachyury play essential roles in mesoderm specification in axolotl embryos, suggesting that the urodele Axolotl has a more simplified mesendoderm GRN. In this context, we demonstrate that Mix acts to induce Brachyury expression during mesoderm induction. Mixl1 shRNA knowdown in mouse embryonic stem cells (mESCs) shows that Mixl1 is involved in the production of mesoderm in mESCs too. Analysis of the localisation of the VegT and Vg1 mRNAs in oocytes revealed that they are neither vegetally localised in the Axolotl, nor in the basal fish species lungfish and sturgeon. Furthermore, gain and loss of function assays examining the roles of maternal VegT and β-catenin demonstrated that VegT is not required for mesoderm induction, whereas β-catenin is necessary and sufficient for mesoderm induction by activating AxNodal-1 expression in the axolotl. As these results reveal additional similarities to the GRN in mammals they further support our hypothesis that the regulatory network in the axolotl is more closely related to that in amniotes rather than anuran amphibians.

Nucleotide excision repair (NER) is a versatile pathway of DNA repair that deals with a variety of DNA lesions, such as UV-induced DNA damage and interstrand crosslinks. In bacteria, the UvrABC system carries out NER. In human cells, XPF and XPG are two structure-specific endonucleases that act in NER. XPF is responsible for a 5' incision at the DNA lesion and XPG carries out the 3' incision. In Archaea, the third domain of life, most species have homologues of some eukaryal NER proteins. Interestingly, Haloferax volcanii encodes homologues of both the eukaryotic NER genes (XPF, XPG, XPB and XPD) and bacterial NER genes (uvrA, uvrB, uvrC and uvrD). In this study, the function of XPG, XPF and UvrA in H. volcanii is investigated. XPG is related to FEN1, a structure-specific 5' flap endonuclease that acts in Okazaki fragment maturation. H. volcanii has a single gene homologous to both XPG and FEN1. The helicase/nuclease hef gene in H. volcanii is the archaeal homologue of human XPF, but also shows homology to Mus81 and FANCM. Mus81 has been found to resolve joint molecules in yeast, while FANCM is required for the repair of interstrand crosslinks in vertebrates. The uvrA gene in H. volcanii is the archaeal homologue of bacterial uvrA, which encodes a protein that plays a vital role in NER at the DNA damage recognition step. This study demonstrates that in H. volcanii, UvrA is involved in the major pathway for repair of UV induced DNA damage. By contrast, Hef and UvrA are involved in two different pathways for the repair of mitomycin C induced DNA crosslinks. Fen1 and Hef have overlapping functions for the repair of DNA cross-links, but not oxidative damage. We also obtain a spontaneous suppressor sfnA, which can suppress the slow growth and MMC sensitivity, but not the UV sensitivity of fen1 deletion mutants. Using plasmid assays, it has been shown that the hef deletion mutant is deficient in accurate end-joining and homologous recombination, including both crossover and non-crossover recombination. In contrast, Fen1 has no significant role in accurate end-joining, but Fen1 may regulate the ratio of non-crossover recombination to crossover recombination.

IRF-1 (Interferon Regulatory Factor 1) is a transcription factor first identified as a regulator of Interferon expression. Two decades after its discovery, IRF-1 has been shown to be involved in numerous other pathways including apoptosis, cell cycle regulation, DNA damage/repair, immune cell development and inflammation. Transcriptional regulation of IRF-1 by a number of external agents has been extensively studied, however almost nothing is known about the posttranslational regulation of IRF-1 activity. In this study IRF-1 is shown to be phosphorylated at Thr180 by GSK3β (Glycogen Synthase Kinase 3β). Phosphorylated Thr180 promotes interaction with the ubiquitin E3 ligase SCFFbxw7u, (Skp1-Cu11-Fbxw7α) which increases turnover of IRF-1 protein. Phosphorylation dependent ubiquitination of IRF-1 was confirmed, as substitution of Thr180 to alanine reduced IRF-1 ubiquitination and increased stability. Enhanced phosphorylation of IRF-1 (by increasing GSK3β expression) promotes increased ubiquitination/degradation. Transactivation of the TRAIL (TNFα Related Apoptosis Inducing Ligand) promoter by IRF-1 was found to be dependent on GSK3β phosphorylation of Thr180 by use of reporter assays and inducible expression of IRF-1 in breast cancer cell lines. Importantly IRF-1 activity on the TRAIL promoter is dependent on proper turnover by the UPS (Ubiquitin Proteasome System), as chemical inhibition of the proteasome, or reduction in IRF-1 ubiquitination reduced activity in reporter assays. This suggests that phosphorylation of IRF-1 by GSK3β acts as a destruction signal through association with SCFFbxw7a. This signal dependent turnover of IRF-1 is required for proper transcriptional activation of the TRAIL promoter.

Sporophytic self-incompatibility is a genetically controlled inbreeding prevention mechanism, which is prevalent in the Brassicaceae, and has been reported in a variety of high profile species. Despite the benefits of preventing self-fertilization in terms of maintaining genetic diversity, variation in the strength of self-incompatibility (SI) has also been well documented, as has a shift from SI to inbreeding at the species and population levels. An important underlying driving force behind a switch to inbreeding could be the reproductive assurance provided by not requiring an unrelated mating partner for sexual reproduction. This could be beneficial for a species undergoing rapid colonization, because only a single individual is required to begin a sexually reproducing colony after a long-distance dispersal event (Baker’s law), which is characteristic of the plight of many species after the last glacial maxima. The purpose of my thesis was to evaluate the effects of variation in mating system on post-glacial colonization, using two model species that show intraspecific variation in outcrossing rates. The first, Arabidopsis lyrata, represents an excellent model system to assess post-glacial colonization history because it exhibits broad geographical and ecological ranges, and has a recently completed genome sequence. In North America, A. lyrata has further benefits as a model system, namely it exhibits variation in the strength of SI and shift to SC at the population level, which is not observed in Europe. The second species is Arabis alpina, which also appears to show population level variation in mating system strength in Europe based on variation in FIS. This has been putatively linked to colonization history after the last glacial maxima. Unlike in A. lyrata however, its mating system has not been characterized. Mating system delimitation in A. alpina has the potential to aid the interpretation of patterns of ecological genetic diversity, which may in part be influenced by local or regional stochastic changes to mating system variation. My first objective was to identify if A. alpina had a functioning SI system based on both self-fertilization experiments, and allozyme based outcrossing rate estimations. I found strong evidence to suggest the presence of a functional barrier preventing self-fertilization in A. alpina. I identified multiple putative SRK alleles (the female determinant of self-incompatibility), suggesting that the same type of sporophytic system seen in other Brassicaceae species governs SI in this species. I also demonstrated linkage of SI phenotype to some SRK genotypes by diallel crosses, strengthening the case for a functional SI system in this species. Further to this I demonstrate variation in mating system strength between populations, and autonomous inbreeding was seen in a single population. I note that the potential changes in SI status coincide with areas suspected to differ in post-glacial history based on allozyme diversity reported in previous work. While the number of populations sampled was insufficient to link mating system variation to colonization history in A. alpina, mating system variation has been more extensively characterized in North American A. lyrata, allowing more fine-scale resolution of population structure and post-glacial colonization history; an underlying objective of my thesis. I used three molecular marker systems (cpDNA, nuclear micro-satellites and allozymes) to assess these phylogeographic questions, and present evidence of three putative colonization routes for the Great Lakes region. These putative routes are congruent with those described in other species, particularly amphibians and reptiles. Further to this I considered the possible location of glacial refugia, and likelihood that plant taxa may have survived during Pleistocene glaciation in close proximity to the Laurentide Ice Sheet, particularly in Illinois, Indiana, Wisconsin and Minnesota, which may also be true for some animal taxa. I examined patterns of population structure, and scenarios that may have influenced this, and present support for the previously documented theory of multiple breakdowns in SI in this geographic region. My final objective was to assess the suitability of the three marker systems for phylogeographic reconstruction in A. lyrata, by comparing and contrasting the patterns of population structure, and colonization history suggested by each system. Levels of variation observed between the marker systems used varied, and I explored how these patterns complemented and contradicted each other. As expected, the nuclear micro-satellite loci represent the system with the greatest genetic diversity, but do not allow meaningful conclusions to be drawn regarding colonization history because of low levels of shared variation between populations. Conversely, the allozyme loci presented much lower levels of genetic diversity, but support population structuring conclusions based on both cpDNA data and previous studies of A. lyrata and other taxa in this area. The cpDNA marker (trnF) represents a somewhat contentious system to use for phylogeography in A. lyrata, as it contains a tandem array of highly variable, but complexly evolving duplications (pseudogenes). I concluded that these structural changes could be phylogenetically informative when pseudogene evolutionary relationships can be resolved This was based on variation in patterns of diversity, and the subsequent population structure change that occurred when using different methods of assessing trnF variation.

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.

Trypanosoma brucei is the causative agent of both sleeping sickness in humans and the related veterinary disease, Nagana. Both diseases have a wide distribution across sub-Saharan Africa and affect some of the poorest areas of the world. T. brucei can be segregated into three morphologically identical sub-species based on host, geography and pathology. T. b. brucei is limited to domestic and wild animals throughout sub-Saharan Africa and is non-infective to humans due to trypanosome lytic factors found in human serum. T. b. gambiense and T. b. rhodesiense are human infective sub-species, named due to their relative geographic locations. T. b. gambiense is the dominant form of the disease, causing over 90% of reported cases. Study of T. b. gambiense is complicated in that there are two distinct groups. Group 1 is invariably resistant to lysis and by far the more prevalent group. Group 2 T. b. gambiense exhibit a variable resistance phenotype and are only found at a small number of Côte d’Ivoire disease foci. There are two trypanosome lytic factors in human serum (TLF-1 & 2), both containing the proteins Apolipoprotein L1 (ApoL1) and Haptogoblin-related protein (Hpr). It has been conclusively demonstrated that the lytic component of TLF is ApoL1, although Hpr is required for maximal lysis by facilitating uptake of TLF particles via the HpHbR cell surface receptor. This thesis has exposed several features of the human infectivity phenotype in both groups of T. b. gambiense, an area of research for which data has been lacking due to the difficulty of working with the organism. Fluorescence microscopy indicated that group 1 T. b. gambiense exhibit avoidance of TLF-1 particles by down-regulating HpHbR receptor expression and function. However, they are also able to resist the effects of recombinant ApoL1, suggesting an additional neutralisation or compensatory mechanism. Due to group 1 T. b. gambiense avoidance of TLF-1, TLF-2 is the more important lytic particle for this sub-species group and future research must take this into consideration. Unlike group 1, group 2 T. b. gambiense displays a variable human serum resistance phenotype that involves a neutralisation or compensatory mechanism for ApoL1, with no significant avoidance of lytic particles. Despite the high variability of the phenotype of group 2 T. b. gambiense, Quantitative Trait Analysis (QTL) using twenty-five F1 progeny from a T. b. brucei / group 2 T. b. gambiense cross indicated a strong heritable component to human serum resistance largely determined by a 30 gene locus on chromosome 8. Finally, a six multi-locus genotype population analysis of a Côte d’Ivoire T. b. gambiense focus was conducted, revealing little relationship between the two groups of T. b. gambiense in the field. The differences in the human serum resistance phenotypes and population genetics of both groups of T. b. gambiense revealed both prior and during this study make it appear likely that the two groups have evolved distinct human serum resistance strategies.

In the model plant Arabidopsis thaliana, the circadian clock is believed to be composed of a number of coupled transcriptional negative feedback loops. The LATE ELONGATED HYPOCOTYL (LHY) gene is thought to form part of at least two of these transcriptional feedback loops, as well as playing a role in the perception of light signals by the clock. To better understand how multiple transcriptional feedback loops might be integrated in the transcriptional regulation of LHY, we have performed an analysis of the cis-regulation of this gene. Through deletion analysis of reporter gene constructs, we have identified a 957 basepair region of the LHY promoter which contains sufficient sequence to direct the characteristic expression profile of LHY. Furthermore, we provide evidence that at least two circadian signals converge on this region. Electrophoretic mobility shift assays identified four classes of candidate cis-elements within the LHY promoter including a poly-CTT tract, an AAAAA motif, a candidate MYB-binding site and a G-box motif. Through mutational analysis of these elements, we have been able to determine aspects of their in vivo regulatory function. We report that a G-box motif and the previously uncharacterized AAAAA element are implicated in the regulation of LHY transcription by light signals. In etiolated seedlings, the region of the LHY promoter containing the MYB-binding site motif and multiple copies of the poly-CTT motif mediates regulation of LHY by both light-responsive and circadian signals.

The aim of the work presented in this thesis was to assess the genetic variation present within the wild tomato species Solanum pennellii that can be adapted to improve the texture of the domesticated tomato species. Using a population of S. pennellii introgression lines, 23 significant QTL supporting intervals were identified. Nine of the QTL were significant in two growing seasons. Three QTL were identified for pericarp firmness. Lines containing the firmness QTL F-Sp 2.1 were used to create BC1 populations. Sensory analyses were used to correlate the instrumental texture measurements to those perceived during mastication. Repeated texture measurements were conducted on lines representing similar chromosomal regions from another wild tomato species Solanum habrochaites introgression line population. To better understand the genetic basis of one of the firmness QTL identified, the Syngenta tomato Affymetrix GeneChip was used to quantify the differential expression of S. pennellii genes within the QTL-introgression line through development in comparison to the recurrent parent S. lycopersicum L. cv M82. The microarray analyses were extended to the ripening mutants Cnr, nor and rin. Differential gene expression between the ripening mutants and the wild type Ailsa Craig were compared through development. Candidate genes for the firmness QTL and fruit development were nominated.