DNA is regularly subjected to endogenous and exogenous reagents that cause mutations that can be detrimental to a cell if they are not repaired. One class of enzymes responsible for DNA repair is the family of DNA glycosylases and their role is to remove damaged bases. Uracil DNA Glycosylase (UDG) is a member of this family and is highly specific, removing only uracil, an RNA base, from DNA. Uracil arises in DNA through misincorporation of deoxyuridine monophosphate (dUMP) creating an A.U base pair, or through deamination of cytosine resulting in a G.U base pair. Though UDG acts on A.U pairs, this is not it’s primarily role as A.U pairings are not mutagenic. However the G.U mispair is highly mutagenic and leads to a G.C to A.T transition on subsequent rounds of replication. UDG only reacts with uracil and has no activity at thymine since the 5-methyl group on the base is excluded from the active site. This thesis examines mutants of UDG that can cleave cytosine but not 5-methylcytosine. Methylation of cytosine at CpG sites leads to gene silencing and is an important epigenetic signal. Knowing the methylation state of cytosines will therefore be important for understanding gene control and may be beneficial for treating many diseases. The most common method for detecting cytosine methylation uses a bisulphite reaction followed by normal DNA sequencing methods. This process has several drawbacks and the aim of this work is to create an enzyme that is capable of distinguishing between5-methylcytosine and cytosine. It has been reported that mutation of a critical asparagine in UDG to an aspartate allows the enzyme to accommodate cytosine into its active site; generating a cytosine DNA glycosylase (CDG). Using the natural ability of UDG to distinguish between uracil and thymine due to the presence of the 5-methyl group, we hypothesised that the mutant enzyme should be able to discriminate between5-methylcytosine and cytosine, which differ by the presence or absence of a methyl group in the same position. E. coli and human CDGs were prepared and their ability to remove cytosine or 5-methylcytosine examined when placed in different sequence contexts. hCDG was generated through complete gene synthesis of hUDG followed by the N204D mutation. The corresponding mutation in E.coli (N123D) generates a highly cytotoxic enzyme that cannot even be cloned in pUC19. As L191 aids base flipping, mutation to alanine (L191A) renders the enzyme inactive; activity can then be rescued using a bulky synthetic nucleoside that occupies the base pair and forces the target base into an extrahelical conformation. The L191A mutation was followed by N123D to generate an expressible and functional eCDG, denoted eCYDG. We demonstrate that these mutants have cytosine glycosylase activity when the cytosine is mispaired or unpaired, but not when paired with guanine, and show no activity against5-methylcytosine in any context. The activity of these CDGs varies with the stability of the base pair, with the fastest cleavage rates being obtained with the least stable base pairs, and also varies with the local sequence context. As CDGs are able to discriminate between cytosine and 5-methylcytosine we began development of a real-time PCR assay for detection of 5-methylcytosine. This employed a hexaethylene glycol (HEG) linker opposite the target cytosine, as this produces one of the fastest cleavage rates and cannot be read by a DNA polymerase.

Chromatin is the complex of DNA, histones and non-histone proteins that make up chromosomes and the region of dispersed chromosomes during the interphases and S phase of the cell cycle. It is found inside cell nuclei in eukaryotic cells. There are many important proteins in the nuclei which are involved in DNA replication, gene expression, DNA repair, etc. Core histones, linker histones and HMGs are the most important proteins in this group. In this thesis, a new, quick and efficient method is developed, to extract and purify proteins from cell nuclei, which is named "forward technology". By using the "forward technology", ultra-pure native core histone octamers, dimers and tetramers were extracted from chick erythrocytes. By using the pure histone dimers and tetramers, the complexes of NAP1-dimer and NAP1-tetramer were obtained in collaboration with others. Crystals of pure histone octamers with a higher resolution than before were produced (Chapter 2). A low KCI/phosphate wash of chick erythrocyte nuclei removed up to 1 g of proteins without nuclei lysis (cePNE1 proteins). The high KCI/phosphate soluble fraction of the cePNE1 is rich in HMG proteins, peptidyl-prolyl isomerases (FKBP3) and heatshock protein 70 which were fractionated by cation-exchange chromatography and anion-exchange chromatography (Chapter 3). Valuable (in terms of function) high-molecular-weight proteins were enriched, and another group of nucleoproteins called cePNE5 is fractionated by the "forward technology" from chick erythrocyte nuclei. It was confirmed that HMGB proteins prefer to bind to core histone H2A-H2B dimers (Chapter 4). It has proved possible to fractionate HMG-rich PNE1 proteins separate from a linker- histone rich nucleoprotein extract, separate from pure core histones (not as oetamers) from human tissue culture cells. The method has been applied to human leukocytes obtained from the National Blood Service. This enrichment of particular groups of proteins will be useful for proteomic studies (Chapter 5).

Freshwater environments are threatened worldwide by external stressors and biodiversity decline, with major implications for ecosystem resilience. The genetic consequences so far have been neglected, especially for freshwater invertebrates, though their abundance, diversity, ease of sampling and functional importance renders them ideal candidates for genetic appraisal. For three freshwater invertebrates (Amphinemura sulcicollis, Isoperla grammatica and Baetis rhodani) novel microsatellite markers were developed so that genetic structure, and genetic diversity could be assessed throughout upland Wales. The aim was to investigate dispersal and the genetic response to environmental stressors. Genetic diversity in these species was compared to species diversity across whole macroinvertebrate assemblages to investigate what factors might cause a correlation between these fundamental levels of biodiversity. The demographic history of each species was also investigated with the aim of assessing whether reduced genetic diversity was due to bottlenecks and more broadly, what this indicates in terms of the populations’ resilience. Species differed in their genetic structure and genetic diversity. All three species showed effective dispersal and geneflow, with each species displaying panmixa across catchments in southern and mid-Wales. However, A. sulcicollis and I. grammatica revealed genetic isolation and reduced genetic diversity at specific northern sites. Genetic and species diversity were correlated positively only in A. sulcicollis, where isolation combined with a common driver were the likely cause. There was evidence of recent bottlenecks in all three species. All these results could be explained by an underlying genetic response to post-industrial acidification: reduced genetic diversity correlated significantly with acidity for A. sulcicollis, while reduced species diversity and genetic bottleneck signatures was consistent with chronic and episodic acidification across the Welsh region. Overall, these results show how a positive correlation between species and genetic diversity can never be assumed, and illustrate how assessments of genetic health expand insights available from traditional biodiversity assessment.

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.

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.

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.

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.

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.

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.

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.