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Subclinical thyroid dysfunction (SCTD), characterised by abnormal serum thyrotrophin concentrations (TSH) with normal free thyroxine (FT\(_4\)), is regularly encountered in primary care. The clinical manifestations of SCTD are not well established, particularly in older individuals in whom SCTD, co-morbid conditions and symptoms frequently occur. This thesis clarifies the clinical expression and pathophysiological consequences of SCTD in the elderly with reference to existing evidence and a cross-sectional study evaluating thyroid function (TF), health status and specific symptoms in community dwelling individuals aged 65 years and above. TF for 2870 participants was categorised, 2703 (94%) euthyroid, 138 (4.8%) subclinical hypothyroidism and 29 (1%) subclinical hyperthyroidism. No significant differences in the prevalence of individual symptoms, pairs of symptoms or multiple symptoms were observed between TF groups. In the presence of individual or multiple symptoms, health status scores were significantly lower. In conclusion, symptoms and impaired health status were not associated with SCTD in this study. Results suggest that assessment of symptoms and health status does not aid clinical decision making with respect to management of SCTD in the elderly. Coupled with weak evidence demonstrating pathophysiological consequences in SCTD, overall findings suggest this population is unlikely to benefit from treatment for SCTD.

COPD is a leading cause of morbidity and mortality worldwide and it is believed that the neutrophil is key to the pathology. Evidence to date suggests that neutrophils migrate less accurately in patients with COPD, although the precise mechanisms by which this occurs have not been defined. We have shown that COPD neutrophils migrate faster (chemokinesis) but less accurately (chemotaxis) in various chemokine gradients. It appears to be an intrinsic cell defect, as incubation of healthy neutrophils in COPD plasma did not alter migratory dynamics. This phenomenon does not occur in other respiratory diseases and is unrelated to age, disease severity, smoking status or the presence of emphysema. Furthermore, there were no differences in markers of neutrophil activation or maturity, although degranulation markers were higher in COPD. Expression of certain chemokine receptors was lower on quiescent COPD neutrophils, but these differences were abolished after stimulation. Receptors localised to the leading edge effectively in COPD neutrophils and there were no differences in receptor shedding. PI3K phosphorylation was increased in COPD and inhibition of γ and δ isoforms improved chemotaxis. Neither Akt phosphorylation nor intracellular calcium signalling was altered. Simvastatin also improved chemotaxis of COPD neutrophils but CXCR1/2 inhibitors did not.

Human adipose tissue acts as a multi-functional endocrine organ. It has the capacity to control energy homeostasis through changes in peripheral and central mechanisms. However, with increasing weight gain, adipose tissue can more than double its original mass, leading to pathogenic consequences including chronic low grade inflammation and insulin resistance mediated by adipocytokines. These changes within adipose tissue can induce secondary detrimental effects on other organs, such as muscle and liver; which also produce hormones that can affect energy homeostasis. The inflammatory response appears modulated by dietary factors and nutrients, including lipids and glucose, as well as gut derived bacteria, in the form of endotoxin. This thesis sought to investigate the role of nutrients, such as lipids (fatty acids) and glucose, on inflammatory responses, energy expenditure and insulin resistance, in vitro and in whole body human physiology. Specifically, studies examined the impact of duration and frequency of nutrient exposure as well as the impact of novel hormones on central and peripheral metabolism in healthy and insulin resistant states. Nutrients, such as fatty acids, were noted to have an immediate impact on the inflammatory response in both murine and human differentiated pre-adipocytes in vitro. Follow-on whole body physiology studies examined lean and overweight/obese (OW/OB) subjects, given isocaloric high fat meals as either 2 large meals or 5 smaller meals over 24 hours. These studies showed minimal effects on blood profiles in the lean subjects post-meal, whilst the OW/OB subjects took much longer to regain their normal lipid and glucose profiles. However, the frequency of the meals in both cohorts had no specific or significant impact on energy expenditure, inflammation, glucose/insulin levels or lipid profile. Analysis of factors that could affect adipose tissue metabolism and whole body physiology, such as irisin in muscle and FGF21 in liver, highlighted that insulin resistance status, BMI and lipid profiles could, at times, influence their circulating levels. This further highlighted the impact of adipose tissue mass, function and cross-talk on health. In summary, both in vitro and in vivo findings highlighted that ‘over-nutrition’ with lipids and glucose could trigger an inflammatory response, which was more pronounced with weight gain. Secondly, this thesis provided evidence that the frequency or duration of exposure to nutrients was less important than the total amount of nutrients consumed. Taken together, these studies emphasise the importance of focusing on interventions to examine nutrient composition, and to reduce overall nutrient intake, rather than the frequency of meals to improve metabolic health in obesity.

The purpose of this work was to study the effects of reactive oxygen species (ROS) on DNA and to investigate the relevance of ROS-induced DNA damage in systemic lupus erythematosus (SLE). Using model systems of ROS generation, it was found that DNA was damaged by ROS at all levels of its structure, causing strand breaks, base modifications and conformational changes. Hydrogen peroxide, a ROS generated during inflammation in vivo, produced a characteristic type of site-specific damage dependent on the DNA-bound metal ion catalysis of its degradation. 8-hydroxydeoxyguanosine (8OHDG), a modified DNA base, was used as a marker of oxidative damage to investigate the role of DNA damage in the aetiopathogenesis of SLE. Excretion of this adduct was detected in normal urine and is believed to arise from normal oxidative metabolic processes. In patients with active rheumatoid arthritis, this level of 8OHDG excretion was significantly elevated. In contrast, in SLE patients with inflammatory activity, 8OHDG was undetectable in the urine. Investigation of the mechanism responsible for this showed that SLE cells had aberrant removal of 8OHDG from DNA following oxidative stress in vitro compared to normal cells, and that ROS-denatured DNA accumulated in circulating immune complexes associated with the disease. SLE is also characterised by circulating anti-DNA antibodies. These antibodies were found to bind better to ROS-DNA than to native double-stranded DNA. Furthermore, ROS-DNA was able to stimulate lymphocytes to produce anti-DNA antibodies. The pattern of DNA damage seen in SLE patients was typical of that induced by hydrogen peroxide in vitro. This suggests that inflammation generates ROS which cause DNA damage. As a result of defective repair within cells, ROS-DNA is released into the circulation following cell death which can form complexes with anti-DNA antibodies. In addition, the ROS-DNA can stimulate further anti-DNA antibody production by acting directly on cells thus perpetuating the disease process and contributing to immune complex deposition, a deleterious manifestation of the disease process.

Osteoarthritis (OA) is a chronic and highly prevalent degenerative disease of the synovial joint leading to cartilage destruction and bone remodelling. The current management of end-stage OA is joint replacement, however, this procedure is not suitable for a subset of patients hence there is a growing need for alternative treatments and technologies to address this limitation. One such approach to this problem is the application of cell-based therapies that regenerate areas of damaged cartilage. Recently discovered articular cartilage progenitor cells (CPC) have been hallmarked as a potential cell source for repair and/or regeneration of damaged articular cartilage. Initial focus was on the characterisation of human CPC isolated from healthy donors and compared with OA derived CPC and patient matched OA Bone Marrow Mesenchymal Stem Cells (BM-MSCs). Comparison of all cell types showed similar morphology and proliferative capacity. In addition, all cell types isolated showed positive expression of the putative mesenchymal stem cell makers; CD-90, CD-105 and CD-166 while lacking expression of CD-34. All cell types investigated showed successful osteogenic, chondrogenic and adipogenic differentiation, hence providing evidence of the mesenchymal stem cell properties of isolated CPC. A gene profiler array was used to identify the expression of Wnt pathway genes from RNA isolated from CPC cell lines originating from healthy and OA cartilage. Interestingly, the expression of Dkk-1 was observed to have the highest up-regulation in OA-derived CPC. The role of Dkk-1 was further studied in a number of CPC and chondrocyte cell lines from healthy and OA cartilage. It was found that normal CPC cell lines showed homogenously low expression and secretion of Dkk-1, however, OA-derived CPC cell lines exhibited a heterogeneous expression and secretion of Dkk-1. In a pellet culture model of chondrogenic differentiation, CPC cell lines secreting high levels of Dkk-1 failed to undergo chondrogenic differentiation, measured by diminished expression of chondrogenic differentiation markers, Type II collagen, ACAN and Sox-9 at both molecular and protein levels. Immunolocalisation of Dkk-1 in OA osteochondral plugs showed peri-cellular expression in chondrocytes located in all zones and around migratory endothelial cells invading articular cartilage where there was a quantifiable increase of blood vessel invasion. This later observation was further studied through a series of experiments to investigate the role of Dkk-1 in relation to endothelial cell migration and angiogenesis using an in vitro model of angiogenesis and migration/invasion assays. A novel finding emerged from these studies, which provides evidence for a pro-angiogenic and pro-migratory role of Dkk-1 and to a lesser extent Dkk-2 in human endothelial cell lines. A novel in vitro Transwell co-culture model was developed to study the interaction between chondrocytes and endothelial cells mimicking the osteochondral interface. A novel finding from these studies included the observation that normal or OA-derived chondrocytes appeared to induce an endothelial to mesenchymal transformation (EndMT) of the co-culture endothelial cells. This was assessed by a loss of the endothelial cobble stone morphology and a down-regulation of key factors implicated in endothelial cell phenotype, including VE-cadherin, Tie-2, e-NOS, PDGF-AA and PECAM-1. As endothelial cells lost their phenotype they adopted a spindle morphology and expressed mesenchymal cell markers including: Lumican, Snail, α-SMA, Vimentin and MMPs. Interestingly, this was also associated with an increase in Dkk-1 expression. To confirm a role for Dkk-1 in this process endothelial cells were cultured in the presence of Dkk-1 and were found to undergo EndMT when compared to the control. In summary, this thesis has uncovered several interesting differences in CPC phenotype. In addition, my results suggest that Dkk-1 has potential as a biomarker of OA pathology. This thesis highlights further the complex role of the Wnt Pathway and in particular Dkk-1 may play a role in the pathogenesis of osteoarthritis.

A typical absence is a non-convulsive epileptic seizure that is a sole symptom of childhood absence epilepsy (CAE). It is characterised by a generalised hyper-synchronous activity (2.5-5 Hz) of neurons in the thalamocortical network that manifests as a spike and slow-wave discharge (SWD) in the electroencephalogram. Although CAE is not a benign form of epilepsy, its physiological basis is not well understood. In an attempt to make progress regarding the mechanism of SWDs, I built a large-scale computational model of the thalamocortical network that replicated key cellular and network electric oscillatory behaviours. Model simulation indicated that there are multiple pathological pathways leading to SWDs. They fell into three categories depending on their network-level effects. Moreover, all SWDs had the same physiological mechanism of generation irrespective of their underlying pathology. They were initiated by an increase in NRT cell bursting prior to the SWD onset. SWDs critically depended on the T-type Ca2+ current (IT) mediated firing in NRT and higher-order thalamocortical relay cells (TCHO), as well as GABAB synaptic receptor-mediated IPSPs in TCHO cells. On the other hand, first-order thalamocortical cells were inhibited during SWDs and did not actively participate in their generation. These cells, however, could promote or disrupt SWD generation if they were hyperpolarised or depolarised, respectively. Importantly, only a minority of active TC cells with a small proportion of them bursting were necessary to ensure the SWD generation. In terms of their relationship to other brain rhythms, simulated SWDs were a product of NRT sleep spindle (6.5-14 Hz) and cortical δ (1-4 Hz) pacemakers and had their oscillation frequency settle between the preferred oscillation frequencies of the two pacemakers with the actual value depending on the cortical bursting intensity. These modelling results are discussed in terms of their implications for understanding CAE and its future research and treatment.