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The study was designed to (i) investigate the behavioural characteristics of individuals with Rett syndrome and (ii) the impact that severity of behavioural and clinical symptoms has on family well-being. A cross-sectional study was conducted to assess differences in behavioural characteristics within subjects and across groups with rare genetic syndromes using standardised measurements. The sample was followed-up after 16 months to examine developmental changes. Three studies were conducted: a national survey of individuals with Rett syndrome and their families, direct behavioural observation of 11 individuals with Rett syndrome in the natural environment and a longitudinal follow-up of the national survey. Results confirmed that behaviours such as hand stereotypies, breathing abnormalities and sleep disturbances are typical of the syndrome. Other behaviours, such as autistic features, impulsivity and overactivity, self-injurious behaviour and depression were also investigated. Although the behaviours were reported in some of the participants, these were not typical of the syndrome. Although some trends were highlighted in the analysis of the longitudinal study, the behavioural features of the group were found to be stable over time. Family stress, anxiety and depression were found to be related to increased severity in areas such mood, fear/anxiety, body rocking and expressionless face and not related to the severity of the clinical phenotype. Results of the longitudinal family study were consistent with the cross-sectional study in that increased severity of behavioural problems is linked to worse maternal psychological well-being. Behaviours mostly seen in the group who were observed were: hand stereotypies, breathing abnormalities and self-injurious behaviours. Hand stereotypies were very frequent for the vast majority of the participants. However, analysis suggested that these stereotypies were less frequent when the girls/women were engaged in another activity using the hands. Breathing abnormalities were observed in the younger girls and the behaviour tended to attract adult attention.

Endothelium-derived hydrogen peroxide (H2O2) has been suggested to function as a freely diffusible endothelium derived hyperpolarizing factor (EDHF). However, in the rabbit vasculature, it has been shown that the electrotonic spread of endothelial hyperpolarization via myoendothelial and homocellular smooth muscle gap junctions is essential for nitric oxide (NO)-prostanoid-independent arterial relaxation. Therefore, a series of interlinked experiments, both mechanical and imaging, have been undertaken to investigate the role of H2O2 in vascular control, focusing on the mechanisms through which H2O2 may regulate intracellular endothelial calcium (Ca2+) homeostasis. These studies have shown that exogenous H2O2 does not directly mediate an EDHF-type response, but can potentiate electrotonically-mediated relaxations by facilitating the elevation of endothelial cell intracellular Ca2+ concentration ([Ca2+]i), thereby promoting the activation of hyperpolarizing endothelial Ca2+-activated potassium channels (KCa). Mechanistically, this potentiating effect of H2O2 involves enhanced depletion of the ryanodine-sensitive endoplasmic reticulum Ca2+ store, through inhibition of sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) activity, and therefore increased extracellular Ca2+ influx through store-operated Ca2+ entry. This effect of H2O2 is independent of the nature of the initiating stimulus, as it is observed with both the receptor-coupled agonist acetylcholine and the SERCA pump inhibitor cyclopiazonic acid. Paradoxically, however, H2O2 was also shown to exert inhibitory effects on NO-mediated endothelium-dependent relaxations. Additionally, arsenite was found to modulate vascular responses through the elevation of the endogenous endothelial-produced H2O2 that is secondary to the activation of NADPH oxidase. This thesis provides evidence that H2O2 is a physiological-important signalling molecule in endothelial Ca2+ homeostasis. The findings also give further insights into the mechanism underlying the compensatory role of the EDHF phenomenon to compromised NO-mediated response that are observed in diseased vessels.

At fertilisation in mammals a series of Ca2+ oscillations are initiated that activate development. These Ca2+ oscillations cause the reduction of mitochondrial NAD+ and flavoproteins, suggesting that they might also stimulate changes in cytosolic ATP levels. Many events at fertilisation are triggered that require ATP; however, the changes in ATP during fertilisation are poorly defined. In this thesis intracellular C a2+ and ATP levels in individual m ouse eggs were m easured by monitoring the fluorescence of a C a 2+ dye (Oregon green bapta dextran) and lum inescence of firefly luciferase. During fertilisation of m ouse eggs it w as found that there are two phases of increase in ATP in both the cytosol and the mitochondria, during the series of sperm-induced Ca2+ oscillations. The increase in ATP is Ca2+ dependent since it did not occur when Ca2+ oscillations were prevented by BAPTA injection and, were abrogated by extracellular Ca2+ chelation. Additionally, it w as not seen when eggs were activated by cycloheximide, which does not cause a Ca2+ increase. The ATP increase is likely to be caused by oxidative phosphorylation by the mitochondria since the ATP levels in substrate free media are recovered by the addition of pyruvate. This recovery is blocked by the pyruvate uptake inhibitor ar-Cyano-4-hydroxycinnamic acid. T hese data suggest that mammalian fertilisation is associated with a sudden but transient increase in cytosolic ATP via oxidative phosphorylation, and that Ca2+ oscillations are both necessary and sufficient to cause this increase in ATP. Work in this thesis has also investigated the functionality of the sperm factor PLC?. Using luciferase tagged PLC constructs, the Ca2+ oscillation inducing ability of a series of PLC? truncated constructs, PLC5 and PLCy have been established. Results show that PLC? activation of m ouse eggs cannot be reproduced by other PLCs and that the C2, EF1 and catalytic site on the X domain are all essential for causing C a2+ oscillations.

This simple, non-invasive test will reduce the patient numbers requiring the more time-consuming ultrasound examination, by screening out a high proportion of individuals who definitely do not have lower limb DVT. However, further signal processing methods should be investigated to improve the specificity of the test.

This study aimed to characterize the roles of the receptor tyrosine kinases (RTKs), epidermal growth factor receptor (EGFR) and ErbB2, and their downstream signalling pathways in diabetes-induced vascular dysfunction. Studies in the mesenteric vascular bed of streptozotocin (STZ)-induced diabetic rats showed that exaggerated vasoconstrictor response to noradrenaline and endothelin-1, and attenuated vasodilator response to carbachol in STZ-diabetic rats was normalized by treatment with either genistein (general RTK inhibitor) or with AG1478 (selective EGFR inhibitor). Higher levels of phosphorylated (p)EGFR and pErbB2 were seen in mesenteric tissues from diabetic rats, which was prevented by treatment with AG1478, AG825 (ErbB2 inhibitor) and with genistein, further suggesting a role for EGFR and ErbB2. Response to the endothelium-independent vasodilator sodium nitroprusside remained normal in tissue from STZ-diabetic animals, suggesting a dysfunction at the level of the endothelium. Studies in human endothelial-like ECV-304 cells grown in high glucose also showed alterations in EGFR and ErbB2 signalling. Glucose-induced upregulation of pEGFR and pErbB2 levels were accompanied by increased phosphorylation of the downstream molecule PKC and of eNOS at threonine 495, which inactivates the molecule and reduces NO production. Studies using isolated rat aortic tissue revealed that EGF has a vasodilator effect in endothelium-intact tissue but a vasoconstrictive effect in endothelium-denuded tissue. These studies along with cell culture studies also suggest that glucose changes EGFR signalling properties from it mediating a pro-relaxant effect in normal glucose to produce a pro-contractile effect in high glucose conditions. These results suggest an important role for EGFR, ErbB2 and PKC signalling in mediating diabetes-induced vascular dysfunction, and as such may represent novel therapeutic targets.

Epithelial mesenchymal transdifferentiation (EMT) has been shown to contribute to renal disease and tissue fibrosis and is known to be mediated by transforming growth factor-β (TGF-β). EMT involves loss of an epithelial phenotype and acquisition of a mesenchymal or myofibroblastic phenotype shown by up-regulation of α-smooth muscle actin (α-SMA). Assembly of hyaluronan (HA) has an important role in extracellular matrix formation and in maintaining the phenotype of different cells. HA has been shown to organize into cable structures or peri-cellular coats. Cable HA binds to inflammatory proteins and prevents their cell surface interaction and has anti-inflammatory properties, while peri-cellular coats make cells migratory. HA assembly is influenced by its interaction with hyaladherins and this study investigated the role of tumour necrosis factor-α stimulated gene (TSG)-6, one of the hyaladherins by assessing its interaction with HA, HABP and CD44 in proximal tubular cells (PTC) EMT. TSG-6 has an important role as an anti-inflammatory protein and is upregulated when stimulated with interleukin-1β (IL-1β) and TGF-β. In the presence of TGF-β, PTCs were demonstrated to be less migratory, with reduced E-cadherin and increased α-SMA expression suggesting TSG-6 may have important role in EMT. Both IL-1β and TGF-β induce increased expression of hyaluronan synthase (HAS) 2 and HA receptor, CD44. This also leads to loss of HA cables and increased assembly of an HA coat. Knockdown of TSG-6 gene in PTC leads to loss of HA cables and the peri-cellular assembly of HA coat was loose and scattered. These TSG-6 knockdown PTCs maintained its epithelial phenotype and TGF-β-mediated phenotypic transition was blocked. There was increased expression of CD44 and HAS2 in these TSG-6 knockdown cells and in subsequent experiments where CD44 was silenced with transfection, HAS2 expression was inhibited. This suggests that HAS2 expression was dependent on CD44 in the absence of TSG-6. These results collectively show that TSG-6 has an important role in EMT in PTCs.

Chronic wounds are a rapidly growing clinical problem across the world, expensive to healthcare systems and severely injurious to the quality of life of those affected. Such wounds have been shown to contain bacteria within biofilms which can be difficult to identify and treat, and are implicated in the lack of healing. The aim of this study was to improve our understanding of the characterisation of these bacterial communities within chronic wounds. This was achieved through the collection of 18 unique chronic wound samples of various phenotypes and 9 acute wound samples. The two bacterial species, Staphylococcus .aureus and Pseudomonas. aeruginosa, most commonly isolated from chronic wounds and known to be prolific biofilm formers and formidable human pathogens were the focus of this study. Both PCR and culture demonstrated the presence of one or both species in all chronic wound samples and the presence of S. aureus in some acute samples. Novel monoclonal antibodies were developed to TNase (of S.aureus) and LPS (of P. aeruginosa) and their ability to detect the bacteria in vitro in a simulated chronic wound environment was evaluated. The action of these monoclonal antibodies were calibrated and evaluated through the use of fluorescent microscopy, and direct and competition ELISA. Following this biofilms of S.aureus and P. aeruginosa both single species and mixed were evaluated utilising Peptide Nucleic Acid Flourescent In Situ Hybridisation, and the Confocal Laser Scanning Microscope to demonstrate the architecture of the biofilms produced and the locations of the bacteria within the biofilm. Microbiologically using both culture and PCR, the widespread presence of S. aureus and P. aeruginosa throughout human chronic wound samples was demonstrated. The ELISA and fluorescent microscopy illustrated the feasibility of MABs as a rapid and accurate detection system for identifying bacteria within chronic wounds. The PNA FISH accurately identified individual species in mixed biofilms through multiplex staining. It also visually demonstrated the tissue invasion of S. aureus, adhesive properties of P. aeruginosa and the synergy of these virulence factors in mixed biofilms. This study has met its aims in that it has provided further evidence of techniques which could be successfully applied to rapidly identify bacteria within a chronic wound environment, and to characterise the structure and composition of bacterial biofilms formed within these wounds. It provides a basis for the development of future clinically relevant rapid bacterial detection systems, and for the academic study of bacteria within the biofilm phenotype.

A separate but related investigated of a database of respiratory and cardiovascular admissions revealed a significant variation by category of road but the observed associations were not what were expected.

The interaction of recombinant TR with TRE-containing double-stranded DNA duplexes was monitored using the electrophoretic shift assay (EMSA). A protein titration allowed the calculation of the K& of TR for DNA. This quantitation of the affinity of TR for DNA was subsequently measured in the presence of known T3 analogues, thus providing the basis of a TR-DNA binding assay.