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.

Hand hygiene is considered a key infection prevention strategy against the challenge of healthcare associated infections, as it prevents cross-transmission of microorganisms which may cause harm. Despite this, compliance amongst healthcare professionals is often poor. Considerable attention has been placed on developing interventions to increase hand hygiene, however known problems with measurement make determining improvement from established baselines difficult. This thesis addresses measurement through three research themes: The importance of meaningful data (Study 1), the potential for technology (Study 2), and the influence of human behaviour (Study 3). These are considered in relation to guidelines developed by the World Health Organisation (WHO) (My 5 Moments for Hand Hygiene). The thesis output provides recommendations for the healthcare setting, technology industry and research community by forming a new conceptual and integrated way of considering the measurement of hand hygiene compliance. A mixed methods approach was applied using a single case study methodology comprising three studies (two qualitative, one quantitative), based at a UK acute National Health Service Trust. Healthcare professionals involved in the current hand hygiene measurement process participated in all three studies (N=47). Methods included structured literature reviews, participant observation, one-to-one and group interviews, nonparticipant observation and analysis of existing case study site data. In Study 1 healthcare professionals identified a lack of clarity regarding feedback, and a lack of synergy between hand hygiene training and measurement. Combined with data accuracy flaws, their view was that the current hand hygiene measurement process produced meaningless data. Study 2 investigated healthcare professional views regarding the potential of technology to measure hand hygiene. It found that whilst current innovations are unable to detect all the WHO 5 Moments, healthcare professionals are interested in their potential to aid measurement and compliance. However they raised concerns about Fit for Purpose, anonymity and resistance, and over-reliance on technology and habituation. Interestingly participants suggested that hand hygiene across all WHO 5 Moments is not equal, expecting higher levels of adherence to Moments 2 and 3 than Moments 1, 4 and 5. Study 3 explored this, investigating the theory of Inherent and Elective hand hygiene behaviour. Inherent can be linked to Moments 2 and 3, through activities likely to stimulate an automatic “disgust” reaction within humans. Hand hygiene was significantly lower when healthcare professionals performed Elective rather than Inherent activities. The research developed Inherent and Elective theory further by proposing it as a lens with which to view the WHO 5 Moments and develop strategies for improved compliance. Understanding that hand hygiene is less likely at Elective activities, linked to Moments 1, 4 and 5 suggests these as key areas of focus for technology development. Acknowledging that hand hygiene may be more instinctive at Moment 2 and 3 may be useful when planning education, leading to reduced healthcare professional apathy towards hand hygiene. Involvement of healthcare professionals in exploring measurement processes and developing technologies for hand hygiene is proposed as key to ensure data produced by future methods of measurement is meaningful, vital to ensure desired behaviour change.

Waiting environments often represent the first interaction point of the healthcare journey and as such contribute to end-user overall experience. The design of these spaces should therefore make the experience as positive as possible. The body of evidence about the relationship between healthcare built-environments and end-user outcomes has grown rapidly in recent years. However, as opposed to inpatient and longterm care, few studies have focused on the design of outpatient settings and their waiting environments. In order to improve the waiting experience, it is necessary to understand how end-users perceive the design of the healthcare waiting environments. This research therefore aims to understand how end-users in the United Kingdom perceive the design of outpatient healthcare waiting environments (OHCWEs). A mixed methods research consisting of quantitative and qualitative techniques was developed to address key research objectives in four studies. In Study 1, end-user perceptions were explored through 24 photo-elicitation interviews. Content analysis of the data revealed that end-users described their perception of the design of OHCWEs using design descriptors and/or emotional, cognitive and associative terms. This contributed to the understanding of the content and language that participants used to describe the design. In Study 2a, 66 participants rated images on semantic differential scales. Using Principal Component Analysis, the level of pleasantness and typical healthcare appearance were extracted as two end-user main perceptions. As these two perceptions were found uncorrelated, a direct causal effect relationship between them could not be assumed. This challenged existing knowledge suggesting a positive effect on end-users related to untypical healthcare appearance. In Study 2b, learning from the review of the literature and from the studies 1 and 2a were consolidated to form a theoretical foundation for the research design of Study 3 to assess design attributes. In Study 3, participants (N=116) evaluated seven design attributes and their sub-attributes on perceived level of pleasantness using photo-realistic renderings which were specifically created. Conjoint Analysis revealed that wooden flooring, an open reception area, upholstered, single chairs that are arranged in rows, clear signage and additional features e.g. indoor plants or refreshment facilities were perceived most pleasant. The quantifiable measures about the contribution of each design attribute to perceived level of pleasantness extend existing knowledge in evidence-based design and hence represent conceptual contributions. In addition to the conceptual contributions, this research also contributes to the practical and methodological development of evidence-based design. The methodological framework provides a novel way of measuring end-user perceptions of the design in OHCWEs. The developed method allowed a more complete view on end-user insights which would not have been possible using traditional, pure methods. Additional learning about the design enabled the formulation of practical design recommendations to improve end-user perceptions of OHCWEs. Being able to assess the pleasantness of healthcare environments has the potential to improve the well-being of end-users.