The human epidermis is a self-renewing stratified epithelial tissue that forms the outermost protective layer of the skin. The epidermis is comprised of a number of cell types, the most abundant of which are keratinocytes. Normal function of the epidermis requires that keratinocyte proliferation, differentiation, and apoptosis be precisely regulated and a failure to regulate these processes is a feature of many skin diseases. Although the precise mechanism by which epidermal homeostasis is regulated is still far from clear, much progress has been made in the characterisation of signaling pathways involved in normal epidermal function. A key group of signaling proteins that have been clearly implicated in epidermal function are the Rho family of small GTP-binding proteins. This thesis focuses on one member of the family, RhoE/Rnd3, and the analysis of the role it plays in the regulation of proliferation, differentiation, apoptosis and cell-cell adhesion in the epidermis. Use of RNA interference to specifically ‘knock-down’ expression of RhoE has led to the discovery of a novel role for RhoE in regulation of cell-cell adhesion and apoptosis. Loss of RhoE expression resulted in keratinocytes developing resistance to apoptosis mediated via either the intrinsic or extrinsic pathways. RhoE depletion was also associated with increased expression of desmosomal proteins and increased numbers of desmosomes. Resistance to apoptosis was shown to be a function of desmosome-mediated cell-cell adhesion and a component of demosomes – plakoglobin – was shown to play a key role in RhoE-mediated resistance to apoptosis.

Computational studies on light‐tissue interactions in medical treatment and diagnosis have offered deeper insights in the processes underlying laser treatments and fluorescence measurements. I apply this approach in the study of fluorescence detection and of laser therapy. First, I investigate three methods of fluorescence detection and the reported contrast between healthy skin and malignant tissue. I varied the concentration of haemoglobin in the target, the concentration of melanin in the epidermis, the scattering of light in the skin, the depth at which the target is located in the skin, the width of the target, the thickness of the target, the concentration of photosensitizer in the target, and the concentration of photosensitizer in the skin. My findings confirm previous clinical studies in that the auto‐fluorescence corrected fluorescence detection method generally shows a higher contrast than the other methods. The results support earlier clinical studies and are in accordance with expert experience. Second, I study laser therapy for psoriasis. In a series of simulations, I analyse three types of pulsed dye laser systems and one IPL system. The investigated biological effects are heat shock proteins, hyperthermic tissue damage and vasoconstriction of the microvasculature. The changes in the skin concern blood volume, blood oxygenation and scattering in the epidermis. The calculations show that there are some notable differences in the effect changes in the composition of psoriatic tissue has on the efficacy of laser and IPL therapy. Still, Inter‐device variance was more prominent than intra‐geometry variance. My study adds to the understanding of fluorescence detection of keratinocyte skin cancers, as well as that of laser therapy for psoriasis. Additionally, it offers potential avenues for increasing the efficacy and efficiency of these therapies.

In order to understand the role of the formulation components in topical drug delivery it is necessary to separate the various contributing factors, these include thermodynamic activity effects, changes in the ionisation state of the permeant and alteration of the membrane properties. Three model permeants, ibuprofen, salicylic acid and acetaminophen were selected to represent a range of physicochemical properties. Solubility and distribution behaviour of ibuprofen and salicylic acid was determined and demonstrated how the addition of cosolvent could impact upon the permeation of the two weak acids by altering the ionisation state of the permeants. The cosolvents, (typical formulation excipients) were water, propylene glycol ethanol, mineral oil, miglyol and Transcutol . To begin with binary combinations were tested, moving on to ternary combinations more representative of an actual formulation. Silicone membranes were used to investigate the diffusional properties of the model permeants. Similarities in the behaviour of the permeants in the selected solvents were observed. Ibuprofen was found to have a higher permeation rate than salicylic acid possibly because of the hydrophobic nature of the Silicone membrane. Analysis of diffusion profiles using a nonlinear curve fitting procedure revealed that the selected vehicles enhanced the permeation of ibuprofen and salicylic acid by increasing partitioning into the membrane. Acetaminophen was found to oxidise in the presence of hydrogen bonding solvents, and for this reason was eliminated from the study. Diffusion experiments were conducted using an established ATR-FTIR approach but the data interpreted using sophisticated chemometric approaches which allowed the deconvolution of the IR signals of all permeating species and the membrane. Using this approach it was possible to examine the individual profiles from multi-component formulations. Using data from traditional diffusion experiments alongside information obtained from ATR-FTIR diffusion experiments using a new method of analysis allowed a deeper insight into the role of the solvent in the permeation process. Data from ATR-FTIR experiments revealed that ethanol permeated silicone membrane at a faster rate than the other solvents studied. This finding was in line with evidence from Franz-type diffusion experiments in which flux was consistently higher from formulations containing ethanol. Where possible, the effect of the same vehicles on the permeability properties of human skin was examined. The vehicles selected were predominantly influencing the partition of the drug into the skin rather than the diffusion coefficient.

Consideration of broader outcomes of disease, especially those exclusively experienced and reported by the patient, such as HRQOL, is not only consistent with the ‘whole person’ view of health contained in the 1948 WHO definition, but is also a prerequisite to building health-care systems that are responsive to the needs of the patients. For chronic skin diseases, such as hyperhidrosis, these provide a useful indicator of how a patient feels and functions disease for both practical and methodological reasons. The aims of this study therefore were to investigate the impact of hyperhidrosis on patients’ HRQoL using a mix of qualitative and quantitative methods. In addition, a further aim was to develop and validate a disease-specific instrument for assessing HRQoL in hyperhidrosis. In pursuing the above aims, the feasibility of applying online social networking sites for outcomes research in dermatology was assessed. Patients were recruited through online social networking communities related to hyperhidrosis for all stages of the study. Interviews, focus groups and surveys were used for collecting qualitative data from patients (n = 71) to understand quality of life issues of patients, and to provide the content of the new instrument. Dermatologists (n= 5) and patients (n=7) took part in the content validation of the HidroQoL©. Item reduction and the development of the scale’s structure was carried out through several field-testing studies (n: USA, 559; UK, 115), using the item response theory (IRT) Rasch model and factor analyses. Further psychometric testing was performed in a separate study (n = 241). Distribution-based methods were applied in establishing minimum clinically important difference (MCID). A thematic analysis of the qualitative data collected produced 29 quality of life themes and 102 sub-themes, forming the content for the initial 49-item HidroQoL©. The two expert panels judged the instrument as content valid, with a few suggestions. The Rasch analysis modelling led to the collapsing of response categories (from five to three) and the reduction in number of items (from 49 to 18), to ensure a perfect model fit. Factor analyses supported both a single- and a two-factor structure. In subsequent construct validation study the HidroQoL correlated with the DLQI (rs = 0.572, p < 0.01) and the Skindex-17 (rs = 0.551, p < 0.01). Reliability was high (Cronbach alpha = 0.9; test-retest ICC = 0.93). The scores were sensitive to change in patients’ disease severity (standard response mean = 0.8, 95% C.I: 0.34-1.27). The scale banding proposed for the HidroQoL score is as follows: 0 – 1, no effect at all; 2 – 11, small effect; 12 – 22, moderate effect; 23 – 32, large effect; 33 – 36, very large effect. The MCID values were 1.94 – 3.07, for generalised v hyperhidrosis, 2.16 – 4.36, for axillary hyperhidrosis, 2.15 – 3.39, for palmo-plantar hyperhidrosis. An MCID of three is currently being proposed for all types of hyperhidrosis. This study has provided the initial evidence supporting the appropriateness of the content of the HidroQoL and validity of inferences from its scores for assessing HRQoL in hyperhidrosis. In addition, the availability of MCID estimates for the HidroQoL will facilitate its clinical interpretation in both research and routine clinical practice. This study has also demonstrated how CTT and IRT can be integrated in the development and validation of a new generation of HRQoL instruments, using social network for patient recruitment.

Examining 20 human wound bed biopsies (pilonidal sinus and venous leg ulcers), significant differences were observed between the subpopulations of wound macrophages in acute healing and chronic non-healing wounds. The results exhibit that within the acute wound there is an accumulation of early stage macrophages (mean 20.8, SD 8.6) differentiating from monocytes which become activated and contribute to the wound healing process. There being few early stage macrophages within the chronic wound (mean 10.4, SD 6.7) (p≤0.01). Chronic wounds in comparison demonstrated a significant accumulation of tissue macrophages (mean 34.0, SD 10.5) when compared to acute wounds (mean 10.9, SD 4.4) with limited wound healing (p≤0.01). The dermagraft (DG) study comprising of 53 patients, showed that applying a biologically active dressing (1-12 dressings over 12 weeks) and compression to the wound bed, exhibited complete closure (76%) or reduction in the size of the wounds at 12 weeks, compared to compression alone. Changes in the extracellular components and an array of inflammatory cells and cytokines in fifty three paired wound bed biopsies (106) with and without DG were examined at week 0 and week 6 of treatment. On histological investigation, DG exhibited an increase in the amount of collagen present and angiogenesis in the wound at week 6 of treatment. Although there were no significant changes in the lymphocyte counts in response to the application of DG, it was possible to demonstrate a significant increase in the number of stage macrophages at week 6 of treatment (p≤0.05) and a significant reduction in the tissue macrophage counts, at week 6 of treatment (p≤0.05) in patients treated with 4 pieces of DG. The levels of different cytokine expression within the wound bed at week 6 exhibited some changes but this was not significant, in response to DG treatment. This could be to the possible presence of proteinases within the chronic wound bed hydrolysing the cytokines produced by DG. From the results attained, it was able to conclude, for clinical use 4 pieces of DG at regular dosing intervals were sufficient to achieve wound contraction or closure. This dose regimen has not been taken forward for further pivotal studies. This thesis thus represents some of the first evidence in human tissue that macrophages may play role in wound healing, and in chronic wounds, a subpopulation of macrophages can be modified to stimulate these wounds towards healing.

Investigations into the function of mesenchymal-TG2 in epidermal formation and keratinocytes migration were established with varying success using coculture systems. Results indicate TG2 overexpression increases the rate of keratinocytes migration via an as yet unidentified soluble factor. Conversely, the presence of fibroblasts overexpressing this TG have demonstrated an inhibitory effect on keratinocyte migration.

Canine atopic dermatitis (cAD) is a common and severe pruritic, inflammatory skin disease that can be considered a naturally-occurring, spontaneous model of human Atopic Dermatitis (hAD). The genetics of cAD are poorly understood and therefore the aim of this project was to investigate the genetic factors involved in the pathogenesis of cAD and to identify specific gene associations with cAD within and between dog breeds. It was hoped that this study would further strengthen the evidence that dogs are a suitable model hAD. Using dogs as a model to study the genetic basis of AD is advantageous because dog breeds form genetically isolated populations exhibiting strong linkage disequilibrium (LD). In contrast to humans where LD across the genome is weak (10-100 kb), domestic dog breeds have strong LD which extends over long distances (0.8 -5 Mb). This is highly advantageous in genetic studies because fewer genetic markers and smaller sample sizes are needed to find disease associations in dogs. To study the genetic basis of cAD a dual approach of candidate gene association study and genome wide association study (GWAS) was used. This gave not only a novel unbiased approach but also used information from previous studies on which gene selection was based. Therefore increasing the likelihood that the causative genes involved in cAD pathogenesis could be identified. This thesis demonstrated altered mRNA expression in 54 genes out of 22,000 transcripts by mRNA microarray in cAD. Further to this qPCR was used to confirm the microarray results and quantify gene expression in potential cAD candidate genes. This approach identified 11 genes with altered expression in cAD. The qPCR results were further correlated 2 with the clinical outcomes: Canine Atopic Dermatitis and Severity Index (CADESI-03) and number of responses on intra-dermal allergen tests. Eleven novel SNPs and 1 novel microsatellite were identified by transgenomic WAVE analysis. The microsatellite was further typed in 659 dogs and but no association with cAD was found. A GWAS with 22,362 SNPs was performed. The significant results were validated by Sequenom along with the SNPs from the candidate gene study (literature selected genes) in 659 dogs across 8 breeds. In total, 232 SNPs across 54 genes and 41 intergenic regions were genotyped on Sequenom. From this 45 putative associations were found in various breeds. A large number of these associations had relevant functions to AD and/or previous association with hAD.

Microdialysis is a method of monitoring biochemistry of tissues in-vivo, and is established as a clinical and research tool for monitoring of other tissue such as liver and brain. The aim of this thesis was to investigate the use of the microdialysis method in monitoring the human dermis in healthy and burned skin, with a view to its use as a tool to detect progression of the burn wound. Ten healthy volunteers were used to establish a normal range for glucose, lactate, pyruvate and glycerol levels, and to assess the effect of temperature on results. Ten patients with burns <15%TBSA were recruited and microdialysis was undertaken in zones of coagulation and stasis and in unburned skin in each patient from recruitment until36 hours post-bum. A further 2 patients with burns >30%TBSA were also studied. Biochemical findings and the result of amino acid assays are reported. An attempt at correlating biochemical findings with Laser Doppler Images was made, to establish the role of the technique in predicting wound progression. However no wounds showed any evidence of progression in the areas of probe placement over the time of the study.

Introduction Squamous cell carcinoma of the auricle has an unusually high rate of lymph node metastases when compared to similar tumours at other sites. The lymph nodes affected are close to the base of the skull and in the neck. Development of metastasis carries a poor prognosis and most patients will subsequently die of failure of loco-regional control. Despite the likelihood of a poor outcome nothing can be done for patients prior to development of metastasis, as the risk of spread is not sufficiently high to warrant intervention in all patients. They are therefore treated with a ‘wait and see policy’ and only offered treatment once clinical evidence of metastatic spread is detected. This thesis sets out to examine what can be done, at the time of initial presentation with an auricular squamous cell carcinoma to identify patients who would benefit from treatment to the regional lymph node basins. Materials and Methods The thesis is divided into four separate studies. A systematic review examines the evidence available to date, an anatomical study examines the lymphatic drainage of the auricle in cadavers, a sentinel lymph node biopsy study examines the use of this technique to identify early tumour spread and a retrospective analysis of cases of auricular squamous cell carcinoma in our unit examines histopathological prognostic indictors of metastatic spread. Results The systematic review found that these tumours have a metastatic rate of about 11%. Patients developing metastasis usually die from failure of loco-regional control. Depth of tumour invasion, tumour size and mode of invasion seem to be potential indicators of metastatic risk. There is a strong argument for prophylactic intervention to the regional lymph nodes but there is no consensus of opinion as to when this should be carried out The anatomical study comprised 5 cadaveric dissections. They showed that the first echelon nodes draining the auricle lie in the superficial parotid gland, post-auricular/ mastoid nodal group and level II of the neck. There are anastamotic pathways around the mastoid and post-auricular nodes that could permit embolic tumour cells to bypass them. Five lymphatic pathways draining the auricle are described and some of these lie on the lateral and anterior surfaces of the mastoid bone and traverse the insertion of sternocleidomastoid. 28 cases of auricular squamous cell carcinoma were enrolled for sentinel lymph node biopsy. None of them were found to have any metastatic spread. One case showed non-viable tumour cells in a lymph node. There was a high incidence of complications (14%) directly related to the sentinel node biopsy procedure. The retrospective analysis identified 229 cases of auricular squamous cell carcinoma treated in our unit from 1992 - 2004. 212 of these cases had the primary pathology available for analysis. 24 (of 212) patients developed metastasis. 17 patients died as a result of their disease usually due to failure of control at the regional lymph node basin. Primary tumours with a depth of invasion greater than 8mm have metastatic rate of 56%. Tumours with a depth of invasion between 2-8mm and evidence of cartilage destruction, lymphovascular invasion or a non-cohesive invasive front have 24% metastatic rate. Tumours outwith these high-risk groups did not metastasise. Conclusions Elective lymph node dissections of the superficial parotid gland, post-auricular/mastoid and level II nodes should be considered in patients with primary auricular squamous cell carcinomas with a depth of invasion >8mm or a depth of invasion between 2 - 8 mm and evidence of cartilage destruction, lymphatic invasion or a non-cohesive invasive front. This should ideally be done as part of an observational study to evaluate the cost / benefit ratio for these patients. The neck dissection must clear the mastoid bone to a sub-periosteal level on its anterior and lateral surfaces. This will require the removal of the upper portion of sternocleidomastoid. Sentinel lymph node biopsy requires further study to evaluate it as a method for early detection of metastatic spread in auricular squamous cell carcinoma. This could be done as part of an observational study of elective neck dissections.

The decision whether or not to discharge an outpatient is vital in determining outpatient clinic attendance numbers, directly affecting overall patient care efficiency. A review of the factors influencing discharge decisions revealed that there was limited evidence of these factors and a lack of understanding how clinicians take discharge decisions. This project’s objectives were to describe the influential factors on discharge decisions from the clinicians’ and patients’ perspectives, to demonstrate the development and clinical evaluation of a novel "Traffic-light” design dermatology outpatient discharge information checklist to improve appropriateness and consistency in discharge decision-making. Semi-structured interviews were carried out with 40 consultant dermatologists across England. 148 influences were generated and thematically analysed manually and using NVivo10 software. A wide array of nonclinical factors, clinician-based, patient-based, practice-based and policy-based, influence discharge decision-making. Observations of 64 consultations and 56 semi-structured interviews with dermatology outpatients were carried out to understand their experience concerning the decision for their discharge. Twelve of 31 patients (39%) who were discharged considered their discharge inappropriate. A three-round Delphi exercise with 17 dermatology consultants (100% response) was carried out to reach agreement on what a high quality discharge information checklist should contain. There was strong inter-rater reliability (ICC=0.958) and fair inter-rater agreement (Fleiss Kappa=0.269). Thirteen items were identified that formed the "Trafficlight" design checklist. Twelve (67%) dermatology clinicians who evaluated the checklist found it useful. This study has demonstrated the importance of approaching discharge decision taking in an informed, structured manner. The checklist provide the basis for making discharge decisions more systematic, auditable and transparent, improving patient safety and optimising healthcare costs. These methods are potentially useful in other clinical disciplines.