No description available.

Abdominal Aortic Aneurysms (AAAs) are multi-genic, slow growing, degenerative vascular lesions resulting in focal aortic dilations. Following diagnosis, growth is monitored and the aneurysm surgically repaired when dilatation reaches 5.0 - 5.5 cm. Pharmacological treatments regressing or reducing growth rate remain a clinical ideal. Epidemiological evidence shows an inverse correlation between plasma High Density Lipoproteins (HDLs) and incidence of AAA, suggesting a role in the pathoaetiology This thesis investigates the role of HDLs in experimental AAA. Reconstituted HDL (rHDL - CSL-111) prevented AAA formation and reduced established AAA in mouse models. I used a hepatospecific adenoviral vector AdA-l; to induce production of human ApoA-l, increasing plasma HDL particles. Whilst infection with AdA-l significantly increased circulating human ApoA-l, it did not reduce established Ang Il-induced AAA or atheroma at the aortic root. Due to the complexity of HDL particles I elected to concentrate on mimicking its anti-inflammatory effects, rather than investigate alternate methods of increasing HDLs. One of HDL’s key anti-inflammatory effects is the prevention of Toll Like Receptor 4 (TLR4)-mediated inflammation, implicated in AAA development. I attempted to prevent AAA formation with a novel small molecule TLR4 antagonist, IAXO-102. IAXO-102 reduced TLR4-mediated aortic inflammation after three days of Ang II infusion; prevented AAA formation, and early rupture. Cytokines produced by TLR4-mediated inflammation stimulate other pro- inflammatory signalling pathways, such as the CD40/TRAF6 pathway. I investigated the effect of a CD40/TRAF6 inhibitor (687702), on experimental AAA. Following 3 days of Ang II infusion, treatment with Compound 6877002 made no significant difference to inflammatory markers associated with AAA; and no difference to vascular macrophage infiltration, a key cell mediator in AAA development. x HDLs are known to inhibit TLR4-mediated inflammation through up-regulation of Activating Transcription Factor 3 (ATF3), I investigated rHDLs manipulation of this negative feedback loop in HUVEC, and found stimulating HUVEC with rHDL, as well as with the TLR4 ligand LPS, induces ATF3 expression. Whilst surprisingly IAXO-102 did not prevent LPS stimulated ATF3 expression, 6877002 did, suggesting CD40/TRAF6 is essential for ATF3 expression. Through investigating the mechanisms in which HDL prevents AAA, I have highlighted the importance of TLR4 mediated inflammation in AAA and identified a potential pharmaceutical target.

Pain catastrophising is an exaggerated negative mental set brought to bear during actual or anticipated pain experience (Sullivan et al., 2001b). People with high pain catastrophising were reported to perceive stronger pain intensity, attribute more pain to others, and solicit higher levels of social support from others when exposed to pain, relative to low pain catastrophisers (Sullivan et al., 2001b, Quartana et al., 2009). Three important models of pain catastrophising, the appraisal model, the attentional model, and the communal coping model, have been proposed to investigate the influence of pain catastrophising on pain-related outcomes. However, the neural basis of pain catastrophising in the social-emotional context among healthy people is poorly understood. This thesis utilised neuroimaging methods and novel experimental paradigms to explore effects of pain catastrophising on behavioural and cortical responses to pain-related stimuli in healthy people. It also investigates the associations between pain catastrophising and structural brain features. A comprehensive review of previous experimental findings was performed to identify novel research questions. Behavioural, eye movement, EEG and MRI data for 6 unique studies were collected. Chapter One features a review of relevant theories, studies, and findings pertaining to pain catastrophising. The specific research problems and hypotheses investigated in the thesis are explicitly described. Chapter Two describes the theory of the EEG, MRI and eye tracking methods used in the experimental chapters of the thesis. Chapter Three outlines the methods and materials used for each individual study. Chapter Four describes the experimental findings of the thesis. In the first study, a paradigm using a varying level of background noise was applied to evaluate the sensitivity to pain cues in high and low pain catastrophisers. No significant differences were found. In the second and third study, the eye tracking method and a dot-probe paradigm were used to measure the attentional processing to pain-related stimuli. High pain catastrophisers responded to probes after pain scenes slower compared to low pain catastrophisers. In the fourth study, ERP data revealed that high pain catastrophisers exhibited differences in ERP components and source activation patterns during the observation of pain pictures. The first four studies of this thesis reported that high pain catastrophisers attributed stronger pain to pain in others. In the fifth study, LEP data showed that high pain catastrophisers reduced perceived pain during viewing of comforting hand postures, and displayed enhanced ipsilateral operculo-insular activation to pictures not showing comforting gestures. In the final study of the thesis, a morphological analysis of cortical and subcortical structures was performed using high-resolution T1-weighted MR images. It demonstrated that alterations to the morphology of selected cortical regions and the dorsal striatum were associated with pain catastrophising. Chapter Five discusses the findings of each individual study in light of previous research and the implications and inferences that can be drawn from the data. Chapter Six represents a general discussion of the main findings of the thesis. This chapter examines how the findings of each individual study relate to the theories of pain catastrophising. The limitations of the thesis and the implications of the findings for future research are also discussed.

Avian metapneumovirus (AMPV) is an enveloped negative sense single stranded RNA virus which is a major endemic respiratory pathogen of global domestic poultry. Since reverse genetic (RG) techniques have been applied to this pathogen several reports have investigated the effects of single and multiple genomic mutations and gene deletions or insertions on viral biology. The aim of this study was to gain a better understanding of the viral capacity to accept and in some cases express homologous and heterologous extra sequences. Initially an AMPV subtype A was modified to introduce a homologous 200 bp sequence within the G gene and this recombinant was suggested to be used as a positive control for validating all stages of a previously established RT-nested PCR. Different Green Fluorescent Protein (GFP) AMPV recombinants were then prepared each one containing the reporter gene in a different intergenic position and then were assessed for expression, stability and viability. In particular, quantification of the expression was calculated using a quantitative ELISA. All recombinants showed high stability, while good viability was observed in all the positions, except when GFP was inserted between nucleocapsid (N) and phosphoprotein (P). The highest expression was detected in the virus with the insertion between N and P as expected, according to the transcriptional model for non-segmented negative stranded viruses. However GFP was produced at high levels even when inserted at the trailer end intergenic positions. Poor expression was seen for all the other positions. The vectoring abilities of subtype A strains were further investigated to accept and express foreign genes, specifically GFP gene and both spike (S1) and nucleocapsid (N) genes of infectious bronchitis virus (IBV). After viruses had been recovered by RG, all recombinants were proven to express the inserted genes efficiently and were all found to be highly stable during passage in vitro. Subsequently IBV recombinants were tested as candidate vaccines by eye-drop inoculation of one-day-old chickens. When chicks were challenged with IBV, partial protection results were observed, as assessed by greater motility of tracheal cilia from animals receiving the recombinants. Finally the development of a new RG system was attempted in order to extend this type of studies to the B subtype. This subtype is distributed worldwide and growing field evidence suggests it to be more able to infect commercial chickens compared to subtype A. For this reason it would be convenient to have an RG available also for B viruses. The construction of a DNA copy of the viral genome was attempted using site-directed mutagenesis and ligation techniques, resulting in more than 85% of the genome cloned. Unfortunately full genome cloning proved to be not possible, as severe problems were encountered, including construct instability and cloning bacteria intolerance to viral sequences.

Multi-drug resistant (MDR) Streptococcus pneumoniae are a major public health concern worldwide. In Malawi a resource poor country, even the simplest antimicrobials remain a precious commodity. Given the limited number of antimicrobials available for the management of MDR invasive pneumococcal disease (IPD) measures need to be implemented to limit the spread of resistance. In order to design such measures it is essential that we gain a better understanding of the evolution of antimicrobial resistance in this setting. The purpose of this thesis was to assess the molecular basis and mode of dissemination of antimicrobial resistance in S. pneumoniae with the aim of identifying a biomarker of antimicrobial resistance that could be used to design a diagnostic PCR to assist epidemiological surveillance of antimicrobial resistance and inform treatment policy. Malawi introduced the 13-valent pneumococcal conjugate vaccine (PCV13) in 2011. To provide baseline data to assess the impact of PCV13 all invasive pneumococci isolated from children admitted to Queen Elizabeth Central Hospital, Malawi 2004-2006 were serotyped and subjected to antimicrobial susceptibility testing. The data suggested PCV13 will not provide protection against 61% of penicillin resistant pneumococci and if serotype replacement occurs following the introduction of PCV13, the incidence of penicillin resistant IPD could therefore increase Over 130 resistant and susceptible pneumococcal isolates from carriage and invasive disease were subjected to whole genome sequencing. The employment of an in vitro and in silco analytical approach established that S. pneumoniae employs a diverse array of antimicrobial resistance mechanisms, the dissemination of which is likely to be driven by high antimicrobial consumption. A relatively high incidence of antimicrobial resistant was observed in serotype 1 pneumococci, the most common cause of IPD in Malawi. This serotype is not usually associated with resistance in other geographic locations, the short duration of serotype 1 carriage is assumed to limit the chance it has to acquire resistance mechanisms via recombination. Interestingly the resistance mechanisms employed by serotype 1 had been acquired through multiple recombination events. Recombination was evidenced to contribute to >90% of the variation in the serotype 1 genomes. To allow the identification of resistance biomarkers free from any preconceptions about which genes are involved in resistance, multiple antimicrobial resistant lineages were generated in vitro. Isolates were sequenced at several time points as resistance developed. Comparison of the resistant isolates to the wild type isolates identified single nucleotide polymorphisms in 46 genes, 40 of these genes have not previously been implicated in antimicrobial resistance. The role of these genes in resistance warrants further investigation. The analysis suggests that rather than a single biomarker future research needs to identify multiple biomarkers; the dynamic nature of this organism means that it can adopt one of many routes to antimicrobial resistance.

Neospora caninum is an economically significant parasitic protozoan causing the disease neosporosis in cattle and dogs. Although a close relative of the zoonotic apicomplexan Toxoplasma gondii, the two organisms exhibit differing host ranges and infection dynamics. T. gondii is a model organism that has been much studied, and a great deal is known about the genes and proteins involved when it invades a host cell. This thesis explores protein expression in the proliferative and invasive tachyzoite stage of N. caninum, in particular the expression of proteins pertaining to the apical complex of organelles; those responsible for entry and establishment within a host cell. Almost 20 % of the predicted proteome has been identified by this analysis to be expressed in the tachyzoite stage, with approximately 50 % of the predicted repertoire of apical proteins being detected. The discovery of differences between these two parasites’ highly syntenic genomes could lead to a better understanding of the process by which T. gondii is able to cause disease in humans, while N. caninum has not been observed to do so. One finding of the recent genome sequencing and annotation project in N. caninum was that a key T. gondii virulence determinant, rhoptry gene 18 (ROP18) was pseudogenised in N. caninum. This finding was investigated further in this thesis to demonstrate that the pseudogenisation of ROP18 was conserved across a range of N. caninum isolates and that in vitro, N. caninum was not able to subvert the murine interferon-gamma (IFN-γ) immune response using ROP18 in the way that virulent T. gondii tachyzoites do. The tissue-dwelling Coccidia have a multi-stage life cycle which includes a latent tissue cyst-encapsulated stage called the bradyzoite. Tachyzoites convert to this more quiescent form when induced by cellular stress, and are able to remain as such for long periods, even years. At times of weakened host immunity, bradyzoites can recrudesce to produce an active infection, which can cross the placenta in a pregnant animal to infect the foetus. This a major route by which N. caninum infection is maintained within cattle herds, therefore the biology of stage conversion from tachyzoite to bradyzoite and vice-versa is of interest to researchers. An RNA-Seq analysis of cultured tachyzoites and bradyzoites identified a marked reduction in rhoptry gene expression, and differing expression profiles of other invasion-related genes from the micronemes and dense granules. Overall, these data identify proteins released from the apical organelles in N. caninum and give an insight into the different repertoires expressed by the tachyzoite and bradyzoite life stages. Furthermore, a comparison between N. caninum and T. gondii predicted apical proteomes indicates that although most genes are shared in a one-to-one orthologous relationship between the two organisms, there are a small number of differences which may turn out to be important to the biology of the parasite, as in the case of ROP18.

Obesity in the UK leisure population of horses and ponies is a growing problem with major welfare implications. To date, research into the associations between obesity and metabolic disease such as insulin dysregulation and laminitis remain ongoing. To improve our understanding of obesity in this species, the current thesis was designed to address several related objectives ranging from psychological aspects of obesity to the role of key determinants of energy balance in the setting of obesity. Implementing dietary restriction to reverse obesity requires an owner to correctly recognise obesity in their animal, knowledge of which is lacking for the horse. A two-tier internet-based questionnaire was created and distributed through UK equine-based forums. Tier 1 utilised lateral photographic images of horses and ponies and demonstrated that only 11% of respondents (n = 546 total) correctly identified all overweight animals from a panel of 12 images. When assessing the suitability of horses and ponies for taking part in a range of activities, respondents considered it more appropriate for each animal to carry more weight/condition for competing in affiliated showing classes. Tier 2 (n = 177 responses) provided information regarding current management practices of horse-owners in the UK. The ability to quantify internal adiposity in live animals requires imaging technology which is not yet available for the horse. A semi-quantitative regional adipose-depot specific scoring system (EQUIFAT) was developed and tested. Associations between ante-mortem body condition score (BCS) and post-mortem EQUIFAT scores (n = 207 animals) revealed that retroperitoneal EQUIFAT score had strong positive associations with BCS, whilst omental had weaker associations and mesenteric and epicardial scores had no associations with BCS, indicating clear functional differences between regional adipose depots in the horse. Performing in-depth molecular biology studies using abattoir-derived samples requires knowledge of the time-frame of RNA degradation. RNA was found to remain intact up to 30 minutes and 2 hours post-mortem for adipose tissue and skeletal muscle ( n = 3 horses), respectively. The expression of myostatin, a key regulator of skeletal muscle mass and energy balance was evaluated in lean and obese horses and ponies (n = 6/group). Myostatin gene expression was increased in skeletal muscle of obese animals, with no difference at the protein level. Circulating myostatin concentrations were increased in obese animals. Adipocyte area was increased in adipose depots (retroperitoneal, omental, crest and tailhead) in obese animals, except for epicardial WAT. The expression of lipolytic proteins PLIN1 and HSL was reduced in retroperitoneal WAT of obese animals, with fewer differences noted between groups for other depots. Together, findings from this thesis indicate a misperception of obesity exists among horse-owners and enthusiasts. Functional differences between regional adipose depots and altered expression of key regulators of energy balance have been identified in obese horses and ponies.

No description available.

Genome-wide association studies have been successful in identifying novel susceptibility loci across a range of complex diseases. However, the underlying mechanisms by which associated variants influence disease or quantitative phenotypes is often undefined and the variance that they explain is almost invariably small. Next Generation Sequencing (NGS) has allowed rarer genetic variants, which may potentially contribute substantially to phenotypic variance, to be genotyped and imputed in larger samples than before. The analysis of these variants may prove vital in understanding the missing heritability of complex disease. This has inspired the development of methodology to detect effects from multiple rare variants (termed collapsing methods), which single variant approaches may have limited power to detect. This thesis consists of several novel approaches to analysing regions of-rare genetic variation. Chapter 2 demonstrates an innovative approach to filtering variants prior to applying collapsing methods. In chapters 3 and 4 this approach is implemented in conjunction with collapsing methods to networks and pathways of genes respectively, identifying association signals which would not be identified using conventional filtering methods or by analysing genes individually. A similar method is applied to regions which map to protein domains in chapter 5, although these analyses provided limited evidence of association which was not driven by single variant effects. Analyses in chapter 6 u:vestigate the application of collapsing methods to DNA methylation data. Analysing the combined effect of variants provided evidence of association with methylation levels which were not identified using single variant approaches. Conventional applications of collapsing methods using individual gene regions and filtering by variant consequence have typically resulted in underpowered analyses. Results in this thesis demonstrate that collapsing variants using alternative definitions of a functional unit, filtering by predicted deleterious impact and analysing their effect on epigenetic phenotypes can identify novel association signals. Whilst collapsing methods remain a relatively new paradigm in statistical genetics, future endeavours which analyse increasingly larger samples of low coverage sequence data should benefit from using the approaches presented in this thesis to uncover the genetic architecture of complex disease.

Obesity is a worldwide health issue that has reached epidemic proportions, and is defined as increased white adipose tissue mass. This increase in adiposity is caused by either hypertrophy of existing mature adipocytes, or hyperplasia of pre-adipocytes, leading to increased adipocyte numbers. The current study used the murine 3T3-L1 pre-adipocyte cell line to explore in vitro the differentiation process from pre-adipocytes to mature adipocytes, termed adipogenesis. Lipidomic analyses demonstrated a shift in the predominant lipid species present; from phospholipids in the pre-adipocytes, to triglycerides in the mature adipocytes. This was expected from the morphological changes known to occur in this cell line, from fibroblastic pre-adipocytes, to spherical lipid-loaded mature adipocytes. The production of various eicosanoids was also investigated, and their concentration was greatest during the pre-adipocyte stage. This profile was also seen with arachidonic acid, a precursor in eicosanoid synthesis. These changes in lipid metabolism and eicosanoid production appeared to be linked, allowing the differentiation process and lipid accumulation to continue. The obese state is also associated with a chronic low-grade inflammation, and so the effects of TNF-α and IL-6 intervention on adipocyte metabolism were investigated. Differences in lipid mobilisation caused by these pro-inflammatory agents were suggested due to increases or decreases observed in the concentrations of various triglyceride and fatty acid species. Increases were observed in the concentration of various detected eicosanoid species from the arachidonic acid cascade, mainly prostanoid species. Effects of the anti-inflammatory agent dexamethasone were also investigated in mature 3T3-L1 adipocytes. It was associated with increases in the concentration of both triglyceride and fatty acid species, suggesting possible increase in lipogenesis and/or decrease in lipolysis. Increases in the production of various eicosanoid species from the arachidonic acid pathway were also observed. The majority of these species are pro-inflammatory; however, PGE2 is known to have both pro-and anti-inflammatory effects, and this may help to explain these findings. In conclusion, the work presented in this thesis has revealed how adipocyte metabolism changes in the naturally occurring stages of adipogenesis, as well as in response to pro- and anti-inflammatory intervention. Associations were observed between adipokine gene expression, lipid metabolism and eicosanoid production; however, further work is required to confirm these links by identifying the underlying mechanisms involved.