Type 2 diabetes is associated with increased hepatic lipogenesis and glucose production. Enzymes of lipogenesis are co-ordinately induced by insulin and glucose. However, the enzyme glucose 6-phosphatase (G6Pc), which catalyses the final reaction in hepatic glucose production is repressed by insulin but induced by glucose and is markedly elevated in type 2 diabetes. Another gene that is repressed by insulin and induced by glucose in muscle is thioredoxin interacting protein (TXNIP), which is abnormally elevated in muscle in type 2 diabetes. TXNIP gene regulation in liver has not been reported. The induction of hepatic lipogenic enzymes by glucose is attributed to the transcription factor ChREBP-Mlx, whereas the glucose-induction of G6Pc is attributed to covalent modification of FOXO transcription factors by O- GlcNAc formed by the hexosamine biosynthesis pathway (HBP). The aim of this thesis was to investigate the role of the HBP in regulation by glucose of G6Pc and TXNIP gene expression in hepatocytes. This thesis investigated three commonly used methods to modulate HBP flux and covalent modification by O-GlcNAc. (1) An inhibitor of glutamine:fructose 6- phosphate amidotransferase (6-diazo-5-oxo-l-norleucine, DON), the rate limiting enzyme of the HBP, was established to be a valid tool to study glucose-regulated gene expression. (2) Substrates that enter the HBP after GFAT, such as glucosamine which is widely used to demonstrate links between HBP or O-GlcNAc modification and insulin resistance were shown to be invalid tools. (3) Inhibitors of O-GlcNAc modification or expression of O-GlcNAc transferase were of limited use to alter protein modification by O-GlcNAc. Glucose caused a larger induction of G6Pc and TXNIP mRNA in the absence of insulin than in its presence, and this induction could be largely accounted for by Mlx- dependent mechanisms (supporting involvement of ChREBP or MondoA) and by FOXO transcription factors. G6Pc and TXNIP expression were confirmed to be regulated by distinct mechanisms based on the induction of TXNIP but not G6Pc by the glucose analogue, 2-deoxyglucose, through an Mlx-independent mechanism. Insulin caused rapid translocation of both FOXO1 and FOXO3A from the nucleus to the cytoplasm. Both glucose and 2-deoxyglucose opposed the translocation of FOXO1 and FOXO3A by insulin, and they stimulated translocation of FOXO3A to the nucleus in the absence of insulin. Inhibition of HBP flux with the GFAT inhibitor had the following effects: (i) counteraction of the glucose-induction of both G6Pc and TXNIP mRNA; (ii) counteraction of glucose-induced translocation of ChREBP to the nucleus without affecting the signalling metabolite, fructose 2,6-bisphosphate; (iii) counteraction of glucose-induced translocation of FOXO1 and FOXO3A to the nucleus. A role for O- GlcNAc modification of both ChREBP and FOXO3A was supported by wheat-germ agglutinin precipitation. The results of this thesis support involvement of both HBP flux and O-GlcNAc modification of ChREBP and FOXO3A in glucose-regulated expression of G6Pc and FOXO3A but they do not support a role for glucosamine as an experimental tool to study glucose-induced insulin resistance.

The incidence of type 2 diabetes mellitus (T2DM) is rising throughout the world. Explicit evidence has demonstrated sedentary behaviour is a powerful but modifiable risk factor affecting glycaemic control and the incidence of diabetes complications. Despite this, current population estimates indicate the importance of physical activity is not effectively translated to the T2DM population. The aim of this thesis was therefore to investigate barriers and facilitators to physical activity behaviour among individuals with T2DM. An extensive literature review investigating evidence linking physical activity to the management of T2DM was performed, then followed on by three studies which 1) investigated the effects of a 12-week supported exercise programme among newly diagnosed patients, 2) explored factors that may be associated with sedentary or physical activity behaviour, and 3) compared and contrasted the perception and use of physical activity among patients who participated in an exercise programme and patients who received standard care. The findings of this thesis demonstrated that a supported exercise programme can help newly diagnosed T2DM patients achieve moderate-high intensity physical activity 3-5 days·week-1, improving glycaemic control through enhanced β-cell function associated with decreased insulin resistance and improved lipid profile. Exploratory investigation suggested self-efficacy to exercise, internal locus of control and physical activity advice may be important antecedents for physical activity behaviour. Furthermore, it also appeared to suggest that peer support and more comprehensive physical activity information and reinforcement, are key to satisfying psychological needs - autonomy, competence and relatedness - and internalising motivation for physical activity and exercise behaviour. Given the projected incidence of T2DM and prevalence of sedentary behaviour among this population, the findings from this thesis highlight the important role of physical activity and also the need for further research investigating supported exercise programmes and the development of more comprehensive physical activity guidelines for individuals with T2DM.

Haemopoietic recovery after high dose chemotherapy (HOC) in the treatment of haematological disease may be slow and/or incomplete. This is generally attributed to progressive haemopoietic stem cell failure, however we hypothesize that HOC induced defective haemopoiesis may be in part due to poor stromal function. Although chemotherapy is known to damage mature bone marrow stromal cells in-vitro, the extent to which marrow mesenchymal stem cells (MSC) are damaged by HOC in-vivo and in-vitro is unknown. To firstly address this question the physical characteristics and functional properties of marrow MSC derived from patients who have received chemotherapeutic treatment for various haematological diseases were investigated. Subsequently a suitable in-vitro treatment culture model was developed and the effects of chemotherapy exposure in-vitro using cell culture and proteomic techniques were shown. Results of this study demonstrate proliferative and phenotypic changes to patient MSC caused by HOC regimens. In contrast, the differentiation capacity, and ability to form functional marrow stroma after exposure to HOC in-vivo was equal to that of patient MSC studied prior to HOC. Chemotherapeutic exposure to MSC cultures in-vitro have confirmed changes seen in-vivo, with abnormalities evident in MSC proliferation, differentiation and also in their ability to support haemopoietic stem cell migration and repopulation after treatment. A reduced C044 expression on MSC, after exposure to cyclophosphamide, was also observed and the importance of this molecule shown through con-focal microscopy demonstrating its interaction with C034+ haemopoietic stem cells. Using proteomic techniques differences in protein expression by MSC, relating to changes in their function after chemotherapy exposure, were also observed after treatment with cyclophosphamide or melphalan. Finally the role of keratinocyte growth factor as a potential cyto-protective agent to MSC against damage caused by chemotherapy exposure was tested. Results indicated there was significant evidence to indicate that KGF was able to preserve reductions in C044 expression levels after MSC exposure. It was concluded that marrow MSC sustain prolonged injury due to recurrent courses of HOC in-vivo and in-vitro. However, the clinical importance of the chemotherapy induced defects we have observed must be determined through the initiation of prospective randomized trials of the effects of MSC co-transplantation on haemopoietic recovery in the setting of HOC with and without haemopoietic stem cell rescue.

Circadian regulation synchronises the sleep-wake cycle to the light-dark cycle. Light cues reach the hypothalamus, the site of the circadian clock, via the retino-hypothalamic tract. In turn, the hypothalamus projects to the pineal, regulating melatonin synthesis, which is high at night. The rhythms of plasma melatonin, its urinary metabolite 6-sulphatoxymelatonin (aMT6s) and plasma cortisol are reliable markers of ihe phase of the clock. Sleep-wake abnormalities are common in patients with cirrhosis and have traditionally been attributed to hepatic encephalopathy. Melatonin rhythm abnormalities have also been observed in these patients, and generally ascribed to impaired hepatic melatonin metabolism; their impact on sleep-wake behaviour remains unknown. The purpose of this thesis was to clarify the relationship between sleep-wake and neuropsychiatric disturbance in patients with cirrhosis and to determine the contribution of the circadian regulatory mechanisms. • Almost 70% of patients with cirrhosis showed significant sleep-wake disturbance. However, no relationship was observed between sleep-wake indices and the presence/degree of hepatic encephalopathy. • Patients with cirrhosis showed. evidence_ of impaired hepatic melatonin metabolism, namely reduced nocturnal melatonin clearance, delayed aMT6s peaks and significant correlations between circadian markers and indices of hepatic failure. • Patients with cirrhosis also showed evidence of central circadian disruption, with parallel delays in the onset of plasma melatonin/plasma cortisol rhythms and attenuated melatonin sensitivity to lignt. • Circadian rhythm delays were associated with delayed sleep habits, but not with impaired sleep quality. A subgroup of patients also exhibited a degree of desynchronisation between sleep and circadian timing. The association between circadian and sleep timing abnormalities observed in patients with cirrhosis is reminiscent of ‘delayed sleep phase syndrome', and probably susceptible to treatment. However, circadian deregulation does not offer a comprehensive explanation for the sleep quality impairment exhibited by these patients and alternative pathophysiological pathways should be explored.

Research into the association between migraine and menstruation has been hampered by lack of an agreed definition for 'menstrual' migraine. This thesis presents evidence for increased risk of migraine without aura on or between the two days before menstruation and the first three days of bleeding. Within individual women menstrual attacks differ from attacks at other times of the cycle, being longer and more severe. These findings led to the development of definitions for 'pure menstrual migraine' and 'menstrually-related migraine', which have subsequently been adopted by the International Headache Society. Further research identified an inverse relationship between oestrogen and migraine incidence. The follicular phase oestrogen rise was associated with reduced risk of migraine late luteal oestrogen 'withdrawal' at menstruation was associated with increased risk of migraine. In order to counteract the luteal phase oestrogen drop and prevent menstrual attacks, oestrogen supplements were used from the luteal phase oestrogen peak (day-6) through to the early follicular rise of endogenous oestrogen (day +2). Identification of ovulation using a fertility monitor enabled prediction of menstruation and accurate timing of oestrogen supplements, despite a wide inter- and intra-individual range in cycle length. The results showed that use of oestrogen supplements was associated with a significant reduction in migraine days compared to placebo. However, the benefits were offset by delayed oestrogen 'withdrawal' migraine. In women with migraine in the pill-free interval of combined hormonal contraceptives, there was a trend for oestrogen supplements to prevent 'menstrual' attacks, although the dose used was suboptimal. These findings support the hypotheses that menstrual migraine is a discrete clinical entity and is associated with oestrogen 'withdrawal'. Further, oestrogen 'withdrawal' migraine can be independent of menstruation and independent of ovulation. Oestrogen 'withdrawal' migraine can be prevented with oestrogen supplements, although the optimal regime has yet to be established.

The aim of the present PhD thesis was to investigate the impact of uncontrolled and controlled experimental diabetes on the neo-osteogenesis and bone regeneration potential following Guided Bone Regeneration (GBR). Wistar strain rats (n=128) were allocated in three experimental groups: a) streptozotocin-induced, uncontrolled diabetes; b) systemic insulin controlled diabetes; c) systemic health. The impact of the diabetic status on the neo-osteogenesis and on the bone regeneration potential were evaluated histometrically in GBR treated models of de novo mandibular bone formation and regeneration of critical size calvarial defects respectively. Genome-wide microarray analysis was conducted in tissue samples obtained from GBR treated calvarial defects during the early healing stages. Following application of the GBR therapeutic principle, significant neo-osteogenesis and regeneration of critical size osseous defects were observed histologically and morphometrically, even in the presence of uncontrolled diabetes. Nonetheless, the diabetic status was associated with lower outcome predictability, which was improved via systemic insulin mediated glycaemic control. Uncontrolled diabetes compromised the initial stages of intramembranous bone regeneration following GBR, as evidenced by aberrations in fibrin mesh organisation, inflammatory and mesenchymal cells influx and woven bone formation. In parallel, the uncontrolled diabetic condition featured downregulation of genes encoding chemoattractants and inflammatory mediators during the inflammatory phase of the GBR healing process. Further, pathways related to cell division, energy production and osteogenesis were underexpressed during the proliferative phase, while the NF-kB and Wnt signalling pathways were misregulated. The insulin controlled diabetic state enhanced granulation tissue formation and osteogenesis and upregulated genes encoded growth factors and for extracellular matrix proteins during the early healing phases. It is suggested that, although experimental diabetes may compromise the initial stages of osteogenesis, GBR treatment can provide an environment permissive for significant, even though delayed, bone formation. Insulin mediated glycaemic control may enhance the bone regeneration potential in the diabetic status. Genome-wide expression profiling revealed perturbed pathways in GBR healing depending on the metabolic status, which may be applied in the design of novel therapeutic strategies for the reconstruction of osseous defects in diabetic patients.

No description available.

No description available.

No description available.

No description available.