Compression therapy remains the only treatment option for venous ulcers with <100% success. A better understanding of the mechanisms regulating venous ulcer healing might give rise to better treatments. Urokinsae plasminogen activator receptor (uPAR), a GPI anchored 3‐domain protein exists either attached to cell wall or in soluble forms (suPARI‐III, suPARI & suPARII‐III fragments). Cleavage of uPAR results in a number of nonproteolytic functions that may be important in wound healing. Our aim was to determine whether suPAR and its fragments are present in the environment of venous ulcers and whether this receptor has a role in ulcer‐healing. Ulcer exudates and ulcer tissue biopsies were obtained from patients with confirmed venous leg ulcers. Venous ulcers that healed within 6months of compression therapy were classified as healers (H) while those that did not heal in this time were defined as non‐healers (NH). Time resolved fluorescence immunoassays (TR‐FIA) were validated for measurement of suPAR fragments. TR‐FIAs specifically detected their antigens in wound exudates, but not in tissue homogenates. All forms of suPAR fragments were detected within venous ulcer exudates. Levels of all 3 suPAR fragments were significantly higher in exudates obtained from H compared to NH. ELISA analysis of uPA and plasminogen acitvator inhibitor (PAI‐1) antigen revealed that levels were similar in ulcer exudates and tissue homogenates from H and NH. 9 Treatment of scratched keratinocyte cultures with exudates from H resulted in a greater coverage of the scratch compared with NH. Depletion of suPAR from exudates obtained from H resulted in cell death. Results from this study have shown the presence of suPAR fragments within venous ulcers with higher presence of suPAR fragments in H compared to NH. This data suggests a role of suPAR and its fragments in ulcer healing, although the precise mechanisms involved remain to be identified. Measurement of suPAR fragments may provide a prognostic indicator and a therapeutic target for the treatment of venous ulcers.

At least eighty percent of skin cancer cases could be avoided through the adoption of simple precautions such as using high factor sunscreen (Cancer Research UK, 2001), yet the incidence of the disease continues to rise rapidly (De Vries, Bray, Coebergh & Parkin, 2003). Skin cancer is now the most common cancer (Cancer Research UK, 2005) and it is striking that there are now more deaths from skin cancer in the UK than in Australia (Cancer Research UK, 2004). A reversal of the increase in skin cancer rates has been identified as a major target in a number of policy documents (Health of the nation Green paper, 1998; NHS Cancer plan, 2000; Health protection in the 21 St century, 2005). Most recently, the NHS Cancer reform strategy (2007) makes a commitment to increasing funding for skin cancer awareness programmes. However, education alone is unlikely to solve the problem. Awareness about the risk of skin cancer and sun-protection practices is generally high whilst behaviour remains inconsistent (e. g. Eadie & MacAskill, 2007)

Five studies are described, examining the validity of the Palmar Sweat Index (PSI) as an alternative to traditional measures of electrodermal activity (EDA). A review of research using the PSI identified tour topics which needed to be addressed, before the PSI could be accepted as an alternative to measures of EDA. The first of these topics concerns the reliability of the PSI. A preliminary analysis confirmed that the PSI could be scored reliably. The second topic to be examined, was the relationship between the PSI and measures of EDA. The PSI was found to correlate significantly with several parameters of EDA. These results provide some support for models of EDA involving a single effector. More importantly, the PSI response was observed to show rapid recovery, and in one study the PSI was observed to show adaptation over the course of the session, while skin conductance level did not. The difference in the temporal patterning of the responses shown by the two measures provides an explanation for previous reports of a dissociation between the PSI and EDA. The final topics to be examined concerned the effects of psychological stress and anxiety, respectively, on the PSI. Stressful cognitive tasks were observed to lead to increased palmar sweating. Previous claims that the PSI may decrease in response to stress were not supported. More ecologically-valid stressors were less consistently associated with elevated levels of sweat gland activity. There was some support for a relationship between the PSI and experienced anxiety. It is suggested that this may explain the raised sweat gland activity observed during stressful tasks. Data are also presented from three collaborative studies. This data was collected by other workers and demonstrates the utility of the PSI for applied clinical research.

Cutaneous scars can be itchy, painful, disfiguring, psychologically damaging and even pathological. The myofibroblast cell phenotype, that differentiates from fibroblasts during healing, is central to the formation of normal and pathological scars. However, there remains no reliably effective therapy that prevents scarring. This research explores the efficacy and mechanism of action of a potential new antiscarring agent - insulin. Research at the RAFT Institute of Plastic Surgery has demonstrated that insulin inhibits differentiation of skin fibroblasts into myofibroblasts in both human and mouse cell cultures an in a murine in vivo wound healing model. The aim of this thesis is twofold. Firstly, to determine insulin's mechanism of action, in order to exploit it in future therapies. Several potential avenues were explored contemporaneously and included relatively inconclusive assessment of insulin's effects on EDA-fibronectin, FAK kinase, stress fibres and Thy-1. The most promising potential mechanism of insulin's action was its interaction with the cytokine transforming growth factor (3 (TGF-P) in particular the production of autocrine TGF-p and its activation. Human fibroblasts were cultured under various conditions and insulin's action investigated using techniques including immunohistochemistry, proliferation assays, multiplex and real time RT-PCR mRNA analysis, ELISA and SDS PAGE protein electrophoresis and Western blot analysis. The second aim is to test insulin's antiscarring efficacy in humans by means of a prospective randomised controlled trial. The procedure of bilateral breast reduction was chosen as it meant patients were administered both insulin and placebo treatments therefore allowing intrapatient controls. Scars were assessed 3 months after surgery using digital imaging, clinical scar scales, panel assessments, and multiphoton microscopy analysis of moulds of the scars. These studies have enabled progress in the development of a novel antiscarring therapy based on the antifibrotic properties of insulin.

Systemic sclerosis is a multisystem connective tissue disease characterised by skin thickening and widespread, but variable, visceral fibrosis. The aetiopathogenesis is likely to involve immunological activation and microvascular dysfunction leading to excessive accumulation of extracellular matrix (ECM) with increased production of collagen type I in lesional tissues. This implies a dysregulated repair process probably as a consequence of aberrant crosstalk between fibroblasts and inflammatory cells. It has been proposed that a hierarchical cascade of soluble mediators in which initial induction of proinflammatory cytokines expressed by the inflammatory infiltrate may lead to expression of profibrotic mediators including TGFβ. A salient feature of the inflammatory response is directional migration of leucocytes into subendothelial tissues orchestrated by chemokines in a spatially and temporally-regulated multistep process. Work described in this thesis explores the expression of chemokine, monocyte chemoattractant protein-3 (MCP-3/CCL7) in SSc and in murine models for SSc: type 1 tight skin mouse (Tsk1) and a transgenic mouse strain (TβRIIΔk) in which there is fibroblast-directed disruption of TGFβ signalling. The hypothesis that crosstalk between MCP-3 and TGFβ may modulate the signalling response in the fibrotic microenvironment was also explored. Overexpression of MCP-3 was demonstrated on cDNA expression profiling and protein analysis of neonatal Tsk1 and TβRIIΔk fibroblasts. This was supported by immunohistochemical studies on dermal tissues. Similar upregulation dermal patterns of MCP-3 protein expression were observed in the early stage of diffuse cutaneous SSc. Activation of collagen reporter genes by MCP-3 in transgenic mouse fibroblasts and wildtype neonatal mouse fibroblasts harbouring proα2(Ι)collagen promoter reporter gene constructs is mediated via sequences within the proximal promoter and is partly dependent on TGFβ. This coinduction between the two factors in the fibrotic response is also demonstrated by activation of TGFβ signalling pathways by MCP-3 leading to type I collagen secretion. In addition, MCP-3 gene expression is stimulated by TGFβ. Comparison of downstream signalling pathways that regulate collagen gene activation by both cytokines confirms the central role of MAPK pathway activation in mediating the effects of both factors. An additive effect of these two agonists was demonstrated for key TGFβ-regulated genes on comparative microarray analysis. Overall, these results demonstrate that overexpression of MCP-3 is a key biochemical feature of early stage SSc and murine models of SSc, and suggest a novel role for this chemokine as a profibrotic mediator in addition to its role in regulating leucocyte recruitment. Furthermore, there is a potentially important interplay between MCP-3 and TGFβ in modulation of the signalling response in the fibrotic microenvironment.

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Ultraviolet (UV) B therapy can induce complete clearance of psoriasis and often leads to prolonged remission following a treatment period. UVB phototherapy is a commonly used, highly effective treatment modality, but despite this its mechanism of action remains poorly understood. This thesis investigates the importance of keratinocyte apoptosis in UVB-induced clearance of psoriasis using a novel approach, and employs a mathematical model to explore the effects of this on epidermal homeostasis. The in vivo effects of two wavelengths of UVB were compared, one of which is clinically effective in clearing psoriasis (311nm), and one which is ineffective (290nm) even at high doses. This allowed investigation of which effects of UVB are relevant to plaque clearance; eliminating „bystander‟ effects such as erythema. The study showed significant keratinocyte apoptosis in lesional psoriatic epidermis following 311nm UVB compared to 290nm UVB; peaking 16-24h post irradiation. To determine clinical significance, a computational model of psoriatic epidermis was created utilising histological and kinetic parameters. The model predicted that apoptosis should occur in both stem and TA cells to account for plaque remodelling. This was confirmed and quantified experimentally, with real-time assays determining the rate of keratinocyte apoptosis. These data were fed back into the model and demonstrated that the observed level of keratinocyte apoptosis was sufficient to account for complete remodelling of psoriatic plaques in response to therapeutic UVB. The data was supported by gene array studies, which showed differential regulation of apoptotic genes at early time-points following 311nm rather than 290nm UVB. Finally, the wavelength dependence of UVB-induced apoptosis was examined, and only wavelengths which can clear psoriasis (i.e. greater than 300nm & less than 320nm UVB) induced significant epidermal apoptosis. In summary, this thesis demonstrates that keratinocyte apoptosis is a key mechanism of psoriatic clearance in response to UVB.

No description available.