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Circadian clocks are endogenous oscillators that control many physiological processes and confer functional and adaptive advantages in various organisms. These molecular oscillators comprise several interlocked feedback loops at the gene expression level. In plants, the circadian clock was recently shown to be organ specific. The root clock seemed to involve only a morning loop whereas the shoot clock also includes an evening loop in a more complex structure. My work aimed at refining the differences and similarities between the shoot and root clocks, using a combination of experimental and theoretical approaches. I developed an imaging method to obtain more data from the shoot and root clocks over time in various conditions. Some previous results were confirmed: the free running periods (FRPs) are longer in roots compared to shoots under constant light (LL). In addition, the amplitude of clock gene expression rhythms is lower in roots compared to shoots. However, the expression of several evening genes is circadian in roots, contrary to previous conclusions. This was confirmed with qPCR, and was observed in both light- and dark-grown roots. Yet light affects clock gene expression in roots, so an automatic covering system was designed to keep the roots in darkness and obtain data in more physiological conditions. Clock genes behaved differently in shoots and light-grown roots that were in the same environmental conditions, and may be differentially affected by blue and red light. However shoot and root clocks were more similar under constant darkness (DD). My imaging and RT-qPCR data, together with new microarray results and preliminary studies on clock mutants suggest that shoot and root circadian systems may have a similar structure but different input pathways. Entrainment is a fundamental property of circadian systems, which can be reset by cues such as light/dark (LD) cycles. I demonstrated that light can directly entrain the root clock in decapitated plants. The root clock could be entrained by a broad range of T cycles using low light intensity. In addition, rhythms were preferably entrained by low light than by any putative signal from shoots in experiments using conflicting LD cycles of different strengths. My results indicate that direct entrainment by LD cycles could be the main mechanism that synchronise the shoot and root clocks at constant temperature. This is physiologically relevant because dark-grown roots can perceive light channelled by the exposed tissues, in a fibre optic way. I also showed for the first time that clock and output genes could be rapidly entrained by temperature cycles in roots. Several mathematical models of the shoot circadian clock were used to try and fit the root clock data by optimising some parameters. The best set of parameters gave a good qualitative fit to root data under LD, LL and DD. It reproduced the long FRP observed in roots under LL and captured the entrainment under LD with lower amplitude in roots. The parameters that were changed for these simulations were all related to light input, which supports the idea of similar clock structures in shoots and roots but with different input pathways. Together my results confirmed that the plant circadian clock is organ specific and suggest that it is organ autonomous.

Musculoskeletal conditions and exercise-induced pulmonary haemorrhage are commonly diagnosed in Thoroughbred racehorses worldwide, and have serious consequences for racehorse welfare and the racing economy. Despite increasing interest in the study of genetic susceptibility to disease from the veterinary research community as a whole over past decades, the Thoroughbred has been largely ignored as a study group. The availability of software capable of complex genetic analyses using large, unbalanced pedigrees has made the study of genetic susceptibility to disease a realistic prospect for veterinary researchers. This study aimed to complete preliminary analyses of the genetics of a number of important musculoskeletal conditions, and of exercise-induced pulmonary haemorrhage, in two different Thoroughbred populations. Multivariable regression analyses were performed to identify important environmental risk factors for each condition in each population, and heritability analyses were conducted. Genetic correlations between disease conditions were also investigated. Fracture, tendon injury, suspensory ligament injury, osteoarthritis and EIPH/epistaxis were found to be heritable traits in the Hong Kong population. Distal limb fracture, SDFT injury and epistaxis were also found to be heritable in the UK Thoroughbred population. Most heritability estimates were small or moderate in magnitude. Selective breeding strategies that identify those animals with low genetic risk could play a part in future efforts to reduce the incidence of these conditions, in conjunction with favourable environmental manipulations based on research evidence. Due to low heritability, most of the conditions studied here would reduce in incidence slowly if selective breeding were implemented, thus strategic environmental manipulations would be warranted alongside such longer-term efforts to provide effective incidence reductions. A number of conditions were found to be positively genetically correlated, suggesting that risk reduction through breeding could reduce the risk of multiple diseases concurrently. For example, fracture and osteoarthritis were found to be positively genetically correlated (0.85 – 0.89) in the Hong Kong racehorse population. However, using the Hong Kong Thoroughbred population dataset, EIPH/epistaxis and tendon injury were negatively genetically correlated, which suggests that reduction in genetic risk of one of these may lead to increased genetic risk of the other. iii Measures of the durability and performance of racehorses were investigated to assess whether they were heritable traits in the UK and Hong Kong racehorse populations, and to assess their relationship to the disease conditions studied. Selection based on more holistic measures of horse health and longevity such as ‘career length’ could be a more attractive prospect for stakeholders, as this could forego the need to select for many different traits individually. Career length, number of starts over the career, and the level of earnings were all heritable traits in both populations. These holistic traits were found to have variable relationships with the disease conditions studied in each population. These analyses are the first to assess the genetic contribution to risk for many important diseases in the Thoroughbred. They provide a starting point from which further investigations into the applicability of genetic manipulations could yield realistic and achievable tools for racing stakeholders to use to ‘improve’ the breed in future.

This study set out to sequence the genome of Corynebacterium pseudotuberculosis (Cp) 3/99-5, an ovine strain isolated from a naturally-occurring case of caseous lymphadenitis (CLA) in Scotland. The isolate was sequenced and assembled by 454 Life Sciences, and then gap closure performed by ‘PCR bridging’. The resulting sequence consisted of three contigs with a length of 2,319,079 bp and a G+C content of 52.18%. The genome was then annotated and predicted to contain 2,153 coding sequences. Analysis of the coding sequences revealed the presence of several putative virulence factors, including four sortases with multiple sortase target proteins containing LPXTG motifs. A further two Cp strains, an Australian ovine and a North American equine isolate, as well as C. ulcerans NCTC 12077 were sequenced for comparison. Comparative genomics, both intra- and inter-species showed all the genomes to be highly homologous. However, the C. ulcerans genome is larger than the Cp genomes and is more distinct; it was found to be more similar to the equine Cp 1/06-A isolate which is the most diverged of the Cp isolates. Phylogenetic analyses of the Corynebacterium genus were performed using house-keeping loci but also secreted protein loci from Cp 3/99-5. Bayesian analysis of house-keeping loci distinguished the bacteria to a species level. Inclusion of secreted protein loci did not distinguish the isolates any further. The main objective of this work was to utilise the Cp genome sequence to identify potential diagnostic targets which could be used to augment the available ELITEST CLA or replace it. The ELITEST CLA is the only diagnostic test for CLA that exists on the commercial market in the UK. However, due to low specificity and sensitivity, it is only operated on a flock/group basis. Analyses of the Cp 3/99-5 genome identified several potential diagnostic candidates and seven protein targets were investigated further. Attempts were made to express these candidates as recombinant proteins, however, only two recombinants were successfully expressed and purified, Cp3995_0570 and CP40. The seroreactivity of these were then assessed by IgG ELISA using a panel of ten positive and ten negative CLA ovine sera. The sera were previously defined as positive or negative by PLD and whole cell ELISAs; both of which showed a significant difference between sera types. However, neither Cp3995_0570 nor CP40 distinguished between sera originating from Cp-infected and Cp-naïve animals.

Malaria, a parasitic disease caused by five species of the protozoan parasite Plasmodium, still kills an estimated 0.6 million people each year, almost all in the third world African countries. With renewed emphasis on global eradication of malaria, genome-based discovery of novel anti-transmission candidates has been identified as one of the priority research areas for the immediate future. The aim of this study was to exploit the benefits of a combination of classical forward/reverse genetics approaches, flow cytometry and high throughput whole genome sequencing to examine the molecular basis of gametocytogenesis in the rodent malaria parasite, P. berghei. Plasmodium is known to spontaneously generate gametocyte non-producing (GNP) mutants if asexually maintained for a long time. Using a sex-specific fluorescently labelled P. berghei parental line, ten parallel isogenic lineages were asexually maintained in mice by repeated mechanical passage for a year. Three, out of the potential ten lines, developed the GNP phenotype at the end of the study. The three GNP and their isogenic parental lines were sequenced using the Illumina platform and the sequence variations analysed. It was discovered that one single gene, a transcription factor with an AP2 domain (PBANKA_143750), was uniquely mutated in all the three de novo GNP mutants and also in the two pre-existing GNP mutants. The gene, called AP2-G, was thus implicated in regulating a switch associated with commitment to gametocytogenesis. Further conclusive evidence was generated using targeted AP2-G knockout studies (producing the GNP phenotype) and complementation studies in the AP2-G mutants (restoring the WT phenotype). AP2-G was also shown to recognize and bind to a conserved DNA motif in the selected gene promoters in a sequence-specific manner. Inhibition of this interaction by a synthetic customized polyamide compound, ISS-15, was also demonstrated in vitro. Collectively, the work done in the thesis (together with simultaneous independent evidence of involvement of the P. falciparum orthologue of AP2-G in gametocytogenesis) established AP2-G as the critical regulator of the commitment to gametocytogenesis in the form of a molecular switch.

Background: Host-associated microbial communities play a significant role in a species’ environmental interactions, often performing functions unachievable by the eukaryotic host, and is essential in developing a comprehensive understanding of the species and its impact on the local and global ecosystem. Earthworms (Lumbricina) habituate almost every type of soil environment globally, including sites of severe environmental stress and is an essential ecosystem engineer, central to healthy natural and agricultural soils. To date, only a singular symbiotic species (Verminephrobacter sp.) has been identified, but the earthworm impact on transient microbial communities and the surrounding soil microbiome is profound. Methods: Previous culture and molecular based studies found earthworm-associated microbiota unlikely however, this has not been explored using High Throughput Sequencing. Utilisation of Illumina, 454 and Ion Torrent sequencing has enabled production of the highest resolution microbial analysis of host-associated bacteria of any single eukaryotic species to date, including spatial bacterial localisation of the entire Lumbricus rubellus organism and impact analysis of a wide range of anthropogenic contaminants and environmental stressors on the basal microbiomic community. Results: A core bacterial community has been described which is distinct from the surrounding soil. A number of novel species have been associated with the earthworm crop, body wall and hindgut, contravening claims that the earthworm has limited or no impact on ingested soil bacteria. This demonstrate that the host properties impart significant effects on the transient population, demanding further analysis to determine potential symbiotic functionality. However, while a biologically important community has been described, the significant impact of anthropogenic contamination on the host microbiome must be considered given the observed eradication of the Verminephrobacter symbiont during the host’s exposure to arsenic and the potential subsequent implications on host health.

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.

Several human genetic diseases, including myotonic dystrophy type 1 (DM1) and Huntington disease (HD), are associated with inheriting an abnormally large unstable DNA simple sequence tandem repeat. These sequences mutate, by changing the number of repeats, many times during the lifetime of those affected, with a bias towards expansion. High repeat numbers are associated with early onset and disease severity. The presence of somatic instability compromises attempts to measure intergenerational repeat dynamics and infer genotype-phenotype relationships. Modelling the progression of repeat length throughout the lifetime of individuals has potential for improving prognostic information as well as providing a deeper understanding of the underlying biological process. Dr Fernando Morales, Dr Anneli Cooper and others from the Monckton lab have characterised more than 25,000 de novo somatic mutations from a large cohort of DM1 patients using single-molecule polymerase chain reaction (SM-PCR). This rich dataset enables us to fully quantify levels of somatic instability across a representative DM1 population for the first time. We establish the relationship between inherited or progenitor allele length, age at sampling and levels of somatic instability using linear regression analysis. We show that the estimated progenitor allele length genotype is significantly better than modal repeat length (the current clinical standard) at predicting age of onset and this novel genotype is the major modifier of the age of onset phenotype. Further we show that somatic variation (adjusted for estimated progenitor allele length and age at sampling) is also a modifier of the age of onset phenotype. Several families form the large cohort, and we find that the level of somatic instability is highly heritable, implying a role for individual-specific trans-acting genetic modifiers. We develop new mathematical models, the main focus of this thesis, by modifying a previously proposed stochastic birth process to incorporate possible contraction. A Bayesian likelihood approach is used as the basis for inference and parameter estimation. We use model comparison analysis to reveal, for the first time, that the expansion bias observed in the distributions of repeat lengths is likely to be the cumulative effect of many expansion and contraction events. We predict that mutation events can occur as frequently as every other day, which matches the timing of regular cell activities such as DNA repair and transcription, but not DNA replication. Mutation rates estimated under the models described above are lower than expected among individuals with inherited repeat lengths less than 100 CTGs, suggesting that these rates may be suppressed at the lower end of the disease causing range. We propose that a length-specific effect may be operating within this range and test this hypothesis by introducing such an effect into the model. To calibrate this extended model, we use blood DNA data from DM1 individuals with small alleles (inherited repeat lengths less than 100 CTGs) and buccal DNA from HD individuals who almost always have inherited repeat lengths less than 100 CAGs. These datasets comprise single DNA molecules sized using SM-PCR. We find statistical support for a general length-specific effect which suppresses mutational rates among the smaller alleles and gives rise to a distinctive pattern in the repeat length distributions. In a novel application of this new model, fitted to a large cohort of DM1 individuals, we also show that this distinctive pattern may help identify individuals whose effective repeat length, with regards to somatic instability, is less than their actual repeat length. A plausible explanation for this distinction is that the expanded repeat tract is compromised by interruptions or other unusual features. For these individuals, we estimate the effective repeat length of their expanded repeat tracts and contribute to the on-going discussion about the effect of interruptions on phenotype. The interpretation of the levels of somatic instability in many of the affected tissues in the triplet repeat diseases is hindered by complex cell compositions. We extend our model to two cell populations whose repeat lengths have different rates of mutation (fast and slow). Swami et al. have recently characterised repeat length distributions in end stage HD brain. Applying our model, we infer for each frontal cortex HD dataset the likely relative weight of these cell populations and their corresponding contribution towards somatic variation. By comparison with data from laser captured single cells we conclude that the neuronal repeat lengths most likely mutate at a higher rate than glial repeat lengths, explaining the characteristic skewed distributions observed in mixed cell tissue from the brain. We confirm that individual-specific mutation rates in neurons are, in addition to the inherited repeat length, a modifier of age of onset. Our results support a model of disease progression where individuals with the same inherited repeat length may reach age of onset, as much as 30 years earlier, because of greater somatic expansions underpinned by higher mutational rates. Therapies aimed at reducing somatic expansions would therefore have considerable benefits with regard to extending the age of onset. Currently clinical diagnosis of DM1 is based on a measure of repeat length from blood cells, but variance in modal length only accounts for between 20 - 40% of the variance in age of onset and, therefore, is not a an accurate predictive tool. We show that in principle progenitor allele length improves the inverse correlation with age of onset over the traditional model length measure. We make use of second blood samples that are now available from 40 DM1 individuals. We show that inherited repeat length and the mutation rates underlying repeat length instability in blood, inferred from samples at two time points rather than one, are better predictors of age of onset than the traditional modal length measure. Our results are a step towards providing better prognostic information for DM1 individuals and their families. They should also lead to better predictions for drug/therapy response, which is emerging as key to successful clinical trials. Microsatellites are another type of tandem repeat found in the genome with high levels of intergenerational and somatic mutation. Differences between individuals make microsatellites very useful biomarkers and they have many applications in forensics and medicine. As well as a general application to other expanded repeat diseases, the mathematical models developed here could be used to better understand instability at other mutational hotspots such as microsatellites.