Part 1. The ecology of Ixocles trianguliceps Birulav 1895- Ixodes trianguliceps and its small mammal hosts have previously been proposed as serving an occult reservoir role in the cycling of Loupine-ill virus. The tick was collected from three host species: C. glareolus, M. agrestis and A. sylvaticus. The main study area was at Balerno, Ididlothian where it was the only species of 'tick recovered. Regular collections were made from July 1974 until August 1976. C. glareolus had the greatest frequency of parasitised hosts and the heaviest burdens; the males were more heavily burdened than the. females. Such sex preference was not found for the other hosts. Larvae infest the hosts throughout the year, burdens being maximal in June and minimal in July, a second, much lower, peak occurs in October. Females are active from early spring and nymphs from late spring until autumn. The ear pinna is the area of the body most heavily infested by larvae and nymphs, particularly the inner surface with voles and the outer surface with the mouse. While larvae are overdispersed on all three species of host, nymphs and females are overdispersed on glareolus only, and this host alone of the three maintains the instars in appropriate numbers and proportions to qualify it as an independent maintenance host. While C. glareolus could maintain the tick population, M. agrestis and A. sylvaticus could not and may even be deleterious to the tick population. Larvae have a pre-feeding diapause while attached to the host from December until February. There is no evidence that larvae are active during winter as reported by other workers. Diapause is abandoned when infested hosts are brought into the laboratory and engbrý6memt to repletion is slower on all three hosts than it is during the rest of the year. At any time of the year larvae take longer to feed on A. s1vaticus than on the voles. only 9% of field captured larvae completed development and it is proposed that the majority of engorged larvae enter a pre-developmental diapause. Similar conclusions are reached for nymphs and females. For larvae and nymphs which develop at constant temperature without manifest dispause delays the relationship between incubation temperature and developmental velocity is linear and direct, Part 2. Prevalence of Louping-ill in northern Scottish cattle., In a survey of cattle in the northern counties and inner islands of the west coast of Scotland, 4529 sera were tested for immunoglobulin G to Louping-ill, using antigen derived from Louping-ill strain 31 The incidence of antisera in herds increases from the west of the country to the east. In a stable herd structure there is a significantt steady increase in antibody with age. It is suggested, that cattle are useful indicators of Louping-ill, but their role in maintenance of the disease remains an unknown factor.

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In the face of climate change and increasing anthelmintic resistance, sustainable control strategies for nematodes in livestock are needed. A prerequisite to the application of these strategies is an understanding of the ecology of the major gastrointestinal nematode (GIN) species, which forms the theme of this study. This thesis investigates the effect of microclimatic factors on the ecology and epidemiology of GIN in small ruminants. It focuses on the two most important climatic variables that determine the behaviour of the free-living stages of GIN : temperature and moisture. The rationale for the work and the methods used are summarised in Chapters 1 and 2. The effects of constant and fluctuating temperatures and moisture availability on the development and survival of infective larvae (L3) of Haemonchus contortus and Teladorsagia circumcincta were first investigated by conducting controlled experiments (Chapter 3). A significant part of the thesis then investigates the effect of moisture on the migration of L3 from faeces, which has barely been studied previously. This was first examined using controlled experiments on the effect of artificial rainfall on larval migration in laboratory environments (Chapter 4). These experiments revealed that a threshold faecal moisture content (FMC} was needed to release L3, and that adding more rainfall over this threshold had no effect on larval migration. The effect of current and future climate warming scenarios on opportunities for migration of L3 was examined using glasshouse experiments (Chapter5). Results indicated no significant difference in the rate of migration between baseline and warmed (+3°C} conditions, with migration being possible for 3-6 hours after a single rainfall event. Results of the controlled experiments were used to devise a simple model to predict the larval availability of H. contortus, which was tested in the field by comparing faecal egg counts (FEC} on sheep farms with meteorological data (Chapter 6). The field studies provided some qualitative validation of the predictions, but were limited by ability to differentiate H. contortus from other nematode species in faecal samples. Two rapid and simple methods for the diagnosis of H. contortu5 were consequently developed, based on egg morphology and differential development success in culture, to support future and more precise field studies (Chapter 7). There were three sequential steps to achieve the overall aims of the thesis, starting with laboratory experiments, then experiments on turf in controlled environments (experimental greenhouses) and finally validation with field data. The final goal of these efforts was to develop a mathematical model to predict the time course of larval availability under manipulated and natural climates, thus providing evidence to support the design of climate-driven GIN control strategies. While achieving robust and useful models for this purpose is an ongoing endeavour, the results presented in t his thesis provide new understanding of the effects of climate on larval availability, especially the role of rainfall in supporting migration of infective nematode larvae from faeces. In particular, it is clear that in drier regions of the world, water availability through rainfall will impose stronger effects on larval translation onto pasture than will higher temperatures. This thesis presents a renewed focus on micro-climatic conditions for parasite transmission, understanding of which will be essential to predicting effects of climate change on livestock disease from GIN.

The cell survival pathway autophagy, sequesters and degrades long lived and damaged proteins, recycling for reuse as biosynthetic building blocks. Autophagy is often dysregulated in disease pathologies, evidences shows both loss or gain of autophagy function can provide survival advantage. Constitutive activation of the KRas pathway occurs in cancer, with mutant Ras inducing autophagy for survival. Microarray datasets reveal that KRas mutant cancers increased MAP4K3 mRNA, which encodes a serine/threonine specific protein kinase that regulates mTORC1 signalling, in response to nutrients. Since nutrient signalling and autophagy are closely linked, the potential role for MAP4K3 in autophagy regulation, through mTORC1 in KRas mutant colorectal cancer cells, was investigated. MAP4K3 protein expression was elevated in KRas mutant (HCT116) cells compared to isogenic KRas wild type (HKH2) cells. Although silencing of MAP4K3 using shRNA decreased cell proliferation and metabolic activity in both cell lines, MAP4K3 silencing specifically diminished basal autophagic flux in KRas mutant cells, revealing a potential role for MAP4K3 in the regulation of autophagy. Loss of MAP4K3 expression after shRNA silencing led to decreased hVps34 activity and LC3 processing, both of which are essential for autophagy activation and progression. Furthermore, inhibition/silencing of MAP4K3, revealed a feedback mechanism that led to increased ERK1/2 phosphorylation in both cell lines. Combined Silencing of MAP4K3 with the MEK inhibitor PD184352 resulted in preferential sensitisation of KRas mutant cells to apoptotic cell death. This thesis identifies the novel function of MAP4K3 in autophagy control, through control of the hVps34 complex, furthermore the autophagy pathway is up-regu lated in KRas mutant cells and its inhibition sensitises KRas mutant cells towards MEK inhibitors. Thus, therapeutic targeting of MAP4K3 could be combined with Ras pathway inhibition to selectively target KRas mutant cancers.

This thesis considers the field of ecological immunology, specifically considering life-history trade-offs between reproductive investment and self-maintenance. Classical immunology considers the physiological mechanisms behind the function of the immune system; both in a state of disease and at times of health. Ecological immunology makes the transition from the study of biochemical pathways and the molecular mechanisms involved, to an integrated study of these components in an ecological context, having been shaped by evolution. Variation in immune investment is observed across taxa and within populations. Despite the clear fitness benefits to efficient immune function, immune investment is costly. Therefore, investment in immunity must be balanced with investing in other life-history traits. This research considers how immune function and reproductive investment are balanced in different scenarios. Using burying beetles (Nicrophorus vespilloides) as a model system, this study investigated immune and reproductive strategies in a species that exhibits costly biparental care and also exploits a microbe rich environment. This thesis includes experiments designed to contribute to areas that are currently understudied in the literature: social immunity, how specifically parental care is balanced with immune function, changes in immune function with age, and the fitness consequences of changes in the balance of trade-offs.

T helper 17 (Th17) cells potently produce interleukin (IL)-17, which is essential for Th17 cell-mediated pathogen clearance. Failure to regulate Th17 cells can increase Th17 cell numbers and IL-17 production, and is associated with autoimmune disease pathology. Therefore, understanding how Th17 cell responses are controlled may improve treatments in instances of Th17 cell dysregulation. Investigations in mice and humans have mainly studied the cytokine signals that determine Th17 cell responses. However, the strength of TCR signalling has previously been shown to be a further factor capable of determining effector T cell development. The central hypothesis of my thesis is, therefore, that the strength of TCR stimulation is also capable of regulating Th17 cell responses. I also investigated if T-cell density, a parameter often overlooked in investigations, can also affect Th17 cell responses. Cell density has been shown previously to be capable of modulating many parameters including the expression of certain Th17 cell-related transcription factors. To assess the effect of T-cell stimulation strength on Th17 cell responses, human CD4+ T-cells were activated with high or low strength stimuli administered by bead-bound antibodies which activate the TCR/CD3 complex and the costimulatory molecule CD28, or by monocyte-derived dendritic cells pulsed with decreasing superantigen concentrations. Experiments were performed in the presence of pro-Th17 cell cytokines IL-1β, TGFβ and IL-23. In both systems low strength TCR stimulation profoundly and significantly promoted Th17 cell responses, both proportionately and absolutely. The enhancement of Th17 cell responses by low TCR stimulation only occurred in the presence of co-stimulation through CD28. Furthermore, it was revealed by chromatin immunoprecipitation that low strength stimulation promoted Th17 cell responses by allowing binding of a Ca2+ regulated transcription factor NFATc1 to the IL-17 promoter in a Ca2+ dependent manner. To investigate how low strength T-cell stimulation might promote human Th17 cell responses in vivo, 20 healthy donors were genotyped for a single nucleotide polymorphism within the gene Protein Tyrosine Phosphatase-N22 (PTPN22), which is highly associated with the autoimmune diseases type I diabetes and rheumatoid arthritis. PTPN22 encodes a TCR signalling molecule, Lyp, which in minor allele carriers confers both reduced TCR and Ca2+ signalling. Culture of genotyped memory CD4+ T-cells with anti-CD3/anti-CD28 stimulation and pro-Th17 cell cytokines revealed a trend indicating that the presence of the minor T allele promoted both IL-17 and IFN-g production but decreased regulatory IL-10 secretion. Regulation of Th17 cells by T-cell density was explored by culturing memory CD4+ T-cells at decreasing T-cell densities in the presence of either pro-Th17 or pro-Th1 cell cytokines. Low T-cell densities profoundly promoted Th17 cell responses both proportionately and absolutely. No effect was observed on the IFNy response within cultures containing pro-Th1 cell cytokines, suggesting that T-cell density specifically affects Th17 cell responses. STAT3 activation, important for IL-17 expression, can be regulated by cell density. Analysis of STAT3 activation by western blot revealed higher STAT3 activation in low density cultured T-cells compared to high density cultured T-cells, which may provide an explanation for the increased Th17 cell responses observed. The data within this thesis provide interesting and novel mechanisms by which human Th17 cell responses are regulated. I have demonstrated that Th17 cell responses are favoured by both low strength TCR stimulation and low T-cell density. These data highlight the diversity of factors capable of affecting Th17 cell responses in vitro; factors of which in the majority of studies have been overlooked.

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Autophagy is a catabolic mechanism by which cytoplasmic components are sequestered and transported by a double-membrane vesicle called an autophagosome to the lysosome for degradation. This recycling of organelles and macromolecules provides the cell with amino acids in times of nutrient deprivation though we do not fully know how the process is triggered or controlled. It is a highly regulated process in mammalian cells and its deregulation has been shown to contribute to multiple diseases. In order to find new regulators of mammalian autophagy, I performed a genome-wide screen using the Dharmacon human siRNA library in a stable human cell line expressing GFP-LC3, a specific marker for autophagosomal membranes. First I incubated the cells with the siRNA pools then I starved the cells of amino acids. This initiated the formation of GFP-LC3-labelled autophagosomes that I quantified using the Cellomics VTiScan microscope and accompanying software. I measured the effect of specific siRNA-mediated knock-down on multiple parameters including spot count. Accounting for cell death and normalising the data, I generated a Z-score for each siRNA pool and retested the best 500 autophagy-increasing and 500 autophagydecreasing siRNAs as above. The 190 strongest siRNA pools were deconvoluted leaving 20 hits that reproduced the phenotype with three or four out of four duplexes. These 20 hits were then assayed for endogenous LC3 lipidation in a different cell line and the ability of their siRNA to reduce mRNA levels was determined. Four increasers of GFP-LC3 spots increased endogenous LC3 lipidation, suggesting that these proteins are either negative regulators of autophagy or inhibit the maturation or degradation of autophagosomes. Five decreasers of GFP-LC3 spots also inhibited endogenous LC3 lipidation and I have characterised two of these proteins required for autophagy. SCOC colocalises with early autophagy markers and may be providing a scaffold for autophagy machinery. WAC, through its reported binding partners, may be playing a role in both the autophagic and ubiquitin/proteasome pathways.

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