The aim of this Ph.D. was to develop advanced methods for quantitative proteomics and use these methods to investigate the presence of protein biomarkers of sperm performance, differential expression of sperm membrane proteins and differential expression of E.coli proteins. Quantitative analysis of E.coli generated analytical samples that were analysed with multiple mass spectrometers and with multiple software packages. Through these samples an optimal label free quantitative proteomic workflow was generated and software was thoroughly tested to determine the optimal software to be used for data analysis on varying biological questions. Identification of protein(s) that correlate with increased or decreased fertility would be economically beneficial. Currently semen samples are subject to quality control where general movement and morphological defects are studied, but this does not always correlate with the ejaculate passing a post cryopreservation quality control check or that specific bull generating offspring. Identification of a protein or set of proteins with abundance variation in bulls of known high or low fertility would allow lower fertility bulls to be removed from the breeding programme at an early age, reducing rearing costs, and would allow longitudinal health monitoring of individual bulls. Discovery of differentially expressed proteins in the membrane of sperm with the X or Y chromosome would allow the generation of a method to separate the two sperm populations. This will be beneficial as most livestock farmers would prefer offspring of a specific sex, either to sell or replenish animal stock. Quantitative analysis of proteins present in bovine seminal plasma led to the identification and quantitative comparison of the seminal plasma proteins present in two breeds of bull, Holstein and Belgian Blue and a quantitative comparison of the seminal plasma from two domestic farm animal species, bovine and porcine. Intra species comparisons determined no quantitative variation between the two breeds, while the inter species comparison determined variation between the proteins present in both species seminal plasma and the corresponding amounts of proteins present in both species. A quantitative comparison was performed to determine the expression of proteins from two strains of E.coli, a wild type strain (MG1655) and a genome depleted strain (MDS66), this led to the confirmation of gene deletions in the genome depleted strain due to their lack of protein products in mass spectrometric analysis, and the identification of proteins that were differentially expressed due to pleiotropic effects of these genome deletions. To investigate the proteins expressed in the sperm membrane a mass spectrometer compatible enrichment method was generated and membrane proteins were identified, quantified and compared between sperm expressing X and Y chromosomes. This study did not lead to the determination of any proteins with differential expression in the X or Y bearing sperm.

The Apicomplexan parasite Toxoplasma gondii is an obligate intracellular parasite. Infection by T .gondii causes the disease toxoplasmosis, which is one of the most prevalent parasitic diseases of animals and humans. It has been 100 years since the first discovery of the parasite in 1908; research on T. gondii has been carried out in many scientific disciplines consistently expanding the understanding of this parasite. In the last ten years, the developments of EST, microarray, genome sequencing and continuing efforts towards genome annotation has centralized the focus of T. gondii research on the understanding of gene expression and gene functions on the genome scale. Equipped with the technical advances in mass spectrometry and bioinformatics, proteomics has become established as an integral component in the post-genomics era by providing first-hand data on the functional products of gene expression. In this study, three complementary proteomic strategies, 1-DE, 2-DE and MudPIT, have been used to characterise the proteome of T. gondii tachyzoites. Protein identifications have been acquired for more than two thousand (2252) unique release 4 genes, representing almost one third (29%) of the predicted proteome of all life cycle stages. Functional predictions for each protein were carried out, which provided valuable insights into the composition of the expressed proteome and their potential biological roles. The T. gondii proteomic data has been integrated into the publically accessible ToxoDB, where 2477 intron-spanning peptides provided supporting evidence for correct splice site annotation of the release 4 genome annotation. The incompleteness of the release 4 genome annotation has been highlighted using peptide evidence, confirming 421 splice sites that are only predicted by alternative gene models. Analysis has also been carried out on the proteomic data in the light of other genome wide expression data. The comparison of the proteome and transcriptome of Toxoplasma and other Apicomplexa parasites has revealed important discrepancies between protein and mRNA expression where interesting candidates have been highlighted for further investigation. A preliminary DIGE study has been developed to characterize protein expression changes in T. gondii grown in the presence or absence of glucose. In conclusion, this study has demonstrated the importance of proteomic applications in understanding gene expression profiles and regulation in T. gondii and highlighted the importance and potential of proteogenomic approaches in genome annotation process. The importance of temporal and quantitative proteomics as well as the future of systems biology has been discussed.

Canine atopic dermatitis (cAD) is a common and severe pruritic, inflammatory skin disease that can be considered a naturally-occurring, spontaneous model of human Atopic Dermatitis (hAD). The genetics of cAD are poorly understood and therefore the aim of this project was to investigate the genetic factors involved in the pathogenesis of cAD and to identify specific gene associations with cAD within and between dog breeds. It was hoped that this study would further strengthen the evidence that dogs are a suitable model hAD. Using dogs as a model to study the genetic basis of AD is advantageous because dog breeds form genetically isolated populations exhibiting strong linkage disequilibrium (LD). In contrast to humans where LD across the genome is weak (10-100 kb), domestic dog breeds have strong LD which extends over long distances (0.8 -5 Mb). This is highly advantageous in genetic studies because fewer genetic markers and smaller sample sizes are needed to find disease associations in dogs. To study the genetic basis of cAD a dual approach of candidate gene association study and genome wide association study (GWAS) was used. This gave not only a novel unbiased approach but also used information from previous studies on which gene selection was based. Therefore increasing the likelihood that the causative genes involved in cAD pathogenesis could be identified. This thesis demonstrated altered mRNA expression in 54 genes out of 22,000 transcripts by mRNA microarray in cAD. Further to this qPCR was used to confirm the microarray results and quantify gene expression in potential cAD candidate genes. This approach identified 11 genes with altered expression in cAD. The qPCR results were further correlated 2 with the clinical outcomes: Canine Atopic Dermatitis and Severity Index (CADESI-03) and number of responses on intra-dermal allergen tests. Eleven novel SNPs and 1 novel microsatellite were identified by transgenomic WAVE analysis. The microsatellite was further typed in 659 dogs and but no association with cAD was found. A GWAS with 22,362 SNPs was performed. The significant results were validated by Sequenom along with the SNPs from the candidate gene study (literature selected genes) in 659 dogs across 8 breeds. In total, 232 SNPs across 54 genes and 41 intergenic regions were genotyped on Sequenom. From this 45 putative associations were found in various breeds. A large number of these associations had relevant functions to AD and/or previous association with hAD.

Campylobacter spp. are the most common causes of bacterial gastroenteritis in humans worldwide, and although poultry and cattle are considered major sources of Campylobacter spp., infection has also been associated with dogs. In order to investigate the potential zoonotic risk to humans, dog faeces were examined for the presence of Campylobacter spp. from several different dog populations including; vet-visiting, boarding, rescue and hunt dogs. The Campylobacter spp. prevalence, and species distribution was determined for all studies, and some studies were analysed for possible risk factors for Campylobacter spp. carriage in dogs. Longitudinal studies were carried out on kennelled dogs to investigate shedding patterns, and possible transmission. All C. jejuni, and 41 C. upsaliensis isolates from these studies underwent multilocus sequence typing (MLST), along with nine C. upsaliensis isolates originating from human clinical cases, in order to identify possible sources of infection, and assess the potential zoonotic risk to humans. Additionally a pilot study was performed to annotate a plasmid as part of a C. upsaliensis genome project. The findings of this thesis found that the overall prevalence of Campylobacter spp. ranged from 0-73%, although the majority of studies had a prevalence greater than 30%. The prevalence and species distribution differed depending upon the dog population. Kennelled dogs generally demonstrated the highest overall Campylobacter spp. prevalence, whilst the greatest species diversity was found in hunt dogs. C. upsaliensis dominated in most of the populations sampled, except for two hunt kennels where C. lari and C. jejuni dominated. The prevalence of C. jejuni was relatively high in some of the rescue and hunt kennels, reaching 20% and 26% respectively, whereas in vet-visiting and boarding dogs it was relatively low, 1.2-9%. Longitudinal studies indicated that the majority of dogs entered the kennels already carrying Campylobacter spp. but when possible transmission events occurred they often involved C. jejuni. Rescue dogs appeared to be exposed to sources of C. jejuni before and after entry to the kennel, but boarding dogs were only exposed after entry. The shedding of C. jejuni in dogs appeared to be over short durations, whereas dogs that carried C. upsaliensis shed the bacterium in nearly every sample. Data suggested that dogs carried the same C. upsaliensis strain throughout the study, providing further evidence that the species may act as a commensal in dogs. Further to this no associations could be made between Campylobacter spp. carriage, specifically C. upsaliensis, and disease in dogs in any of the studies. Younger dogs were significantly more likely to carry C. upsaliensis than older dogs in the vet-visiting study (OR for every additional month 0.99) and living with another dog carrying Campylobacter spp., was significantly associated with Campylobacter spp. carriage in dogs. A considerable amount of genetic diversity was observed within the C. jejuni and C. upsaliensis isolates originating from dogs, and MLST results suggested that strains of both species were the same, or highly similar to strains found in humans. This suggests that there may be common sources of infection for both humans and dogs and that dogs remain a potential zoonotic risk to humans. Although only a small number of household dogs carry C. jejuni, infected dogs should still be considered a potential zoonotic risk to humans, particularly if the dogs originate from kennelled or hunt kennel populations where the prevalence may be higher. Dogs remain a significant reservoir of C. upsaliensis, but the relationship between the presence of C. upsaliensis and gastroenteritis in both dogs and humans is still unclear.

The aim of this thesis was to investigate the mechanisms that mediate the effects of beta-adrenergic agonists (BA) and Growth Hormone (GH) in sheep, by examining the changes in skeletal muscle transcriptome and blood metabolome in order to identify the predominant metabolic mechanisms by which muscle hypertrophy was mediated. Male lambs (120 days old) were all fed a high protein/energy feed ad-libitum, with the GH group (n=10) receiving a single subcutaneous injection of bovine GH (3.75mg/kg body weight, POSILAC, Monsanto) on day 1; the BA group (n=10) receiving BA (cimaterol) at 10mg/kg feed, whereas the control group (CO, n=11) only had the ad-libitum feed. After 6 days sheep were slaughtered, plasma and samples of the Longissimus dorsi (LD), Supraspinatus (SS) muscles and liver were collected. The effect of treatments on the LD transcriptome was assessed on a subset of samples (n=3 from each treatment) via a cross-species approach using the Affymetrix Human U133+2 GeneChip array (47K human microarray). Verification of identified differentially expressed genes and proteins was by quantitative RT-PCR or western blotting, respectively, on all animals. Metabolomics analysis of plasma samples was carried out by Metabolon Inc. (USA) using GC/MS and LC/MS/MS platforms. BA, but not GH, significantly (P<0.05) increased muscle weights and this was associated transition to large fast-glycolytic muscle fibre types. In GH, but not BA treated animals, there was an increase in liver weights (P<0.001). This was associated with an increase in the whole liver content of glycogen (P<0.001), protein (P<0.01), and lipid (P<0.05) content. Analysis of the LD transcriptome of the treated sheep identified 477 and 316 transcripts were significantly altered (P<0.05 and 1.5 fold change) by BA and GH respectively, relative to controls. This muscle was selected as it is a commercial valuable muscle and is commonly used for muscle biochemical studies therefore this would allow us to make comparisons to other studies, including our own. In addition it is a fast glycolytic muscle fibre type there could be compared against SS muscle (oxidative muscle fibre type). BA decreased the expression of genes involved with oxidative phosphorylation and upregulated those serine biosynthetic pathways. Subsequent qRT-PCR analysis showed a BA induced increase in expression of phosphoglycerate dehydrogenase (PHGDH) (P<0.05) and phosphoserine-aminotransferase (PSAT) (P<0.05) mRNA in both LD and SS but not liver. In LD there was also an increase (P<0.001) in PHGDH protein in muscle from BA treated sheep relative to GH treated sheep. Up-regulation of this pathway has been previously reported in cancer cells which has a tendency to be associated with an increase in gene expression of a specific isoform of the glycolysis enzyme pyruvate kinase (PKM2) which has reduced activity. Total PKM and PKM1 and PKM2 isoforms were increased in the SS and LD of BA treated sheep (P<0.05). Previous studies in cancer cells have suggested that increases in serine synthesis are mediated by changes in PKM2 expression and associated enzyme activity. The lack of a differential increase in PKM2 suggested that the regulation of muscle PK in BA treated animals was not critical to the potential increase in serine synthesis capacity. No clear change in PKM gene expression suggested this was not the mechanism by which the serine synthesis pathway was stimulated. There was an increase (P<0.05) in the expression the mitochondrial form of phosphoenolpyruvate carboxykinase (PCK2) in the LD of BA treated sheep, which might be expected to increase gluconeogenic potential thereby increasing intermediates that could be used for serine synthesis. There was no effect of this gene on sheep treated with GH. An increase in the gene expression of asparagine synthetase (ASNS) was also seen in the muscles of BA but not GH treated sheep (P<0.001) and there was no effect on their livers, which further suggested that BA was influencing the production of nonessential amino acids. Metabolomics analysis showed that products of triacylglycerol breakdown, glycerol and free fatty acids, were all elevated in the plasma of both BA and GH treatments, indicating lipolytic effects but the increase in the free fatty acid profile were more pronounced with GH treatment (P<0.05). Likewise GH rather than BA had a greater impact on elevating plasma glucose and associated metabolites such as pyruvate (P<0.05). There was no effect of either treatment on plasma serine or asparagine concentrations. However there was a decrease in glycine (P<0.05) and glutamine (P<0.05) in GH relative to control, with BA decreasing histidine (P<0.05) and methionine (P<0.01) relative to control. Cell culture experiments were carried out in the myogenic C2C12 cell line to determine if the genes associated with the GH and BA response in sheep were affected during myogenesis and whether there was an effect of des (1-3) IGF-I and dibutyryl cyclic adenosine monophosphate (dbcAMP) that stimulates GH and BA signalling pathways respectively. During differentiation, without treatment, gene expression of PHGDH and PSAT enzymes declined (P<0.05), which might be expected as cells move from a proliferative to a terminally differentiate state. There was no clear effect of treatment on genes associated with the serine synthesis pathway suggesting that the effects of BA, in particular, are on muscle fibres rather than differentiating cells. Of the two growth promoters examined in this thesis BA appears to be the most potent in skeletal muscle. A clear effect of this agent was an increase in the gene expression of the serine biosynthetic pathway, which has been shown to be upregulated in various cancers and, in this pathology, is thought to be a novel mechanism for hyperplastic growth. The associated changes in the expression of genes such as ASNS and PCK2 indicate that their co-ordinated upregulation could be mediated via endoplasmic reticulum stress response mechanisms. Unlike GH, BA does not appear to have a major effect upon the systemic mobilisation of nutrients, but instead seems to targets muscle fibres, activating muscle biosynthetic pathways that potentially provide the substrates required for growth.

This thesis develops the scant existing literature which explores the ethical justifications for living animal donation in the veterinary setting and contributes to the considerable research on social and ethical aspects of human living donation. The work argues that whilst some justifications for human living donation are not-transferable, others may be adapted and applied to the veterinary setting. The unique social context of veterinary donation is analysed, using novel empirical analysis to refine and contextualize the ethical arguments made. The research methods entail an innovative comparative ethical analysis and a qualitative empirical study which are integrated using an empirical bioethics approach. The main justificatory arguments for human living donation are identified as informed consent and donor best interest. These arguments stem from a human’s acknowledged rights to bodily integrity and the human medical professional’s duty not to harm their patients. The reduced capacity of animal donors means that donor consent arguments are not directly transferable to the veterinary setting and an animal owner’s informed consent is shown to have reduced moral authority. Whilst animal donors may lack comparable bodily rights, veterinary living donation practices can conflict with a veterinary surgeon’s professional obligations. A comparable justification for living animal donation is argued to exist only when the procedure is in the donor animal’s best interest. An empirical analysis of canine blood donation to a UK animal blood bank develops understanding of the social context of living animal donation. The analysis indicates that animal owners may not always be motivated by the best interest of donor animals; furthermore, consistent themes such as trust, optimism and human-animal comparisons have implications for the quality of a donor owner’s consent. These ethical and empirical findings are used to construct an ethical framework for living animal donation with detailed provisions for owner consent, donor best interest, donor harm and benefit, recipient benefit, fairness and transparency. The ethical framework is used to argue for the development of regulatory approaches to companion animal blood banking and feline renal transplantation in the UK. The work also has wider implications for veterinary ethics, veterinary policy and the social and ethical understanding of human living donation.

The way we assess readiness for slaughter in beef cattle has not progressed in the past 200 years, with subjective visual and manual assessments of cattle still the primary mechanisms used to determine peak condition, resulting in less than half of all cattle carcases meeting a UK premium classification. Current losses to the UK Beef industry are estimated at approximately £12.5 million per year through the sending of over-fat and poorly conformed cattle to the abattoir. With global population rapidly increasing, there is a fundamental need to provide more food efficiently and effectively from the resources we have. Therefore, successfully reducing wastage and improving carcase quality across the UK beef industry through accurate assessment and selection of beef cattle for slaughter is an important step forwards towards a sustainable future for beef production. The EUROP system of bovine carcase classification dictates which carcases are most desired for the current market, with those failing to meet market specification subject to a penalty. The aim of this research project was therefore to provide farmers with an objective tool using a binary logistic regression model, to combine fat and morphometric measurements taken from live cattle, in order to help predict which cattle are most likely to achieve a desired carcase classification and therefore most suitable for slaughter. Through the use of a series of binary logistic regression models, it was discovered that out of 15 measurements taken from cattle, a combination of pelvis height, pelvis width, 10th and 12th rib fat point readings and the P8 fat point reading were best able to predict the likelihood of cattle meeting a UK premium carcase classification. In a later study, when breed was factored into the model on a larger, more commercial scale, this reduced the number of measurements required to just the pelvis width and 12th rib fat point reading, subsequently increasing the practicality to apply this research on-farm.

Broiler chicken has been bred for meat production and is characterised with a very fast skeletal muscle growth rate, whilst the layer type have been bred for the production of eggs. This study aimed to understand how intense breeding programmes have developed commercial meat type chickens that have resulted in a fast muscle growth phenotype by a comparative analysis of gene expression between fast-growing broiler and slow-growing layer type chicken. Two chicken trials were carried out. In Trial 1 from fast-growing broiler Ross 308 genotype (R) fast-growing breast Pectoralis major (RPM) and slow-growing leg Peroneus tertius (RPT) muscles were collected at day 14, 36 and 42 post-hatch. In Trial 2, from fast-growing broiler Ross 308 (R) or slow growing layer Hy-Line (H) Pectoralis major (either RPM or HPM) and Peroneus tertius (either RPT or HPM) were collected at day 4, 14, 23, 35 and 42 post-hatch. In Trial 1, there was a muscle type x age interaction in muscle weights with the RPM having the highest value at day 43 (P < 0.001). This effect was also reflected in the protein content with RPM having more protein than RPT. However, RPT had a higher DNA content per unit tissue weight (muscle type x age interaction, P=0.003) with highest value seen at day 43. This suggests that muscle cells of RPM are bigger than those in RPT and that as a result they contain more protein, potentially reflecting RPM hypertrophy compare to RPT. In Trial 1, genes associated with glycolysis, GAPDH and -enolase, were significantly higher in the faster growing RPM (P < 0.05). Genes involved in serine biosynthesis PHGDH, PSAT and PSPH, as well as P70S6K involved in protein translation, also had a significantly higher expression in the RPM when compared to the RPT (P < 0.05) indicating a greater rate of protein synthesis in the faster growing RPM. Expression of genes associated with smaller muscles (myostatin) or protein degradation (calpastatin, the specific endogenous inhibitor of calpain proteinases) were not different between muscles. LIM domain proteins, CSRP3 and FHL2, had a higher expression in the slower growing muscle, indicating the possibility of those two genes being negative regulators of muscle growth, or may be involved in the upregulation of muscle regenerative physiological processes, as a result of the strain on the leg muscles induced by physical activities during locomotion. In Trial 2 there was a three-way interaction between genotype, muscle type and age in muscle growth (P < 0.001) with the highest value seen at day 42 in the RPM. There was a 3-way interaction between genotype, muscle type and age in the expression of -enolase (P=0.036) with the highest value seen at day 42 in the HPM. For -Enolase mRNA expression, there was a separate genotype and a muscle type effect (both P < 0.001) with HPM and HPT been higher than RPM and RPT, and PM muscles having a higher expression than PT muscles in both genotypes. When the serine synthesis pathway was examined, there was a genotype x age interaction (P=0.046) for PSAT and PSPH gene expression, with the highest expression at day 35 in both muscles of the Ross 308 genotype for the former, whilst the latter had the highest expression in the RPM at day 35. For ASNS mRNA expression there were significant genotype (P=0.004) and muscle type effects (P=0.014), with the Ross 308 genotype having the higher expression and the PM being greater than PT. For P70S6K mRNA expression, PM was higher than PT (P=0.012). For P70S6K total protein there was a significant muscle type x age interaction (P=0.049), RPM and HPM being higher than RPT and HPT at days 14 and 35. For calpain activity only at day 35 was there significant genotype x isoform interaction (P < 0.001), with the Hy-Line genotype having a higher micro and milli calpain activity than the Ross 308 genotypes, irrespective of muscle. For trypsin, chymotrypsin and caspase – like activities of the proteasome, there was a significant genotype effects (P < 0.001). In all three assays Ross 308 muscles had a higher activity when compared muscles from Hy-Line at both time points. For the ubiquitin ligases there was a borderline muscle type x time interaction (P=0.05) in the expression of MAFbx mRNA, with the RPT and HPT having a higher expression than the RPM and HPM respectively at day 14. For MuRF1 mRNA there was a significant genotype x age interaction (P < 0.001) with the HPM and HPT having a higher expression than the RPM and RPT at day 14 and 35. For CSRP3 there was a genotype x age interaction (P < 0.001), with the highest expression seen in the HPT at day 42. There was also muscle type effect within genotypes with the RPT and HPT having a higher expression than the RPM and HPM (P < 0.001). Trial 2 indicated that there is an increase in protein synthesis which leads to clear increase in protein accretion in faster growing chicken muscles, thereby supporting the wealth of literature detailing protein turnover in chicken skeletal muscles. There also appears to be an interaction between protein synthesis and degradation, however in most cases protein synthesis seems to be more dynamic and these changes seem to appear around day 35. The novel findings of this study were the observed increase in the expression genes that could limit the synthetic capacity of non-essential amino acid (non-EEA) in fast growing muscles.

No description available.

This thesis presents a sociological analysis of the organisation and significance of thoroughbred horse-racing in Britain. It focuses on both the internal world of racing and the relationship between racing and the wider society. It argues that such an approach is necessary for an appreciation of the full meaning of horse-racing as a social institution. The study finds two major points of articulation between racing and wider social processes: first in terms of the role of racing in elite sociability and structuration; and second in terms of its location in working class culture, particularly as it is mediated through the working class betting tradition. The precise linkages, continuities and changes within these areas are explored in order both to amplify and qualify the conventional observation of a coalescence of interests in racing between otherwise sharply differentiated social strata. The analysis points to the conclusion that while the symbolic legacy of this observation may be strong, the evidence for this symmetry and its pervasiveness is now more tenuous and its implications for the general process of class identification heavily circumscribed. The analysis of the discrete world of horse-racing concentrates first upon the social production of the racehorse as reflected through the position of the stable worker. Evidence is presented which both casts doubt on received images of this process and indicates some erosion of the distinctive cultural output of racing which has customarily attracted a benign curiosity in outsiders. Secondly, attention is focused on developments in the control and administration of racing. In particular, the emergent role of the state in this process is shown to have reverberated through both the production and regulatory sectors of the industry, provoking a profound dislocation in the exercise of power. Such intervention is also demonstrated to have reacted upon the production and consumption of betting, precisely the activity which provided the original rationale for intervention in racing. While there are important elements of continuity in the organisation of racing, the thesis expresses the view that racing has passed over a watershed in the last two decades which in time may prove to have eroded its distinctive contribution to British society.