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.

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.

Background: Metastases are the major cause of death from melanoma, a skin cancer which has the fastest rising incidence of any malignancy in the Western world. Molecular pathways that drive melanoblast migration in development are believed to underpin the movement and ultimately the metastasis of melanoma. Aims: In this thesis we use genetically modified mice models to characterise two novel anticancer targets, P-Rex1 and focal adhesion kinase (FAK). Embryonic melanoblast migration is compared with cancer outcomes for each genetic modification. Results: Mice lacking P-Rex1, a Rac-specific Rho GTPase guanine nucleotide exchange factor (GEF), have a melanoblast migration defect during development evidenced by a white belly. These P-Rex1-/- mice are resistant to metastasis when crossed to a murine model of melanoma, an effect specifically channeled through loss of P-Rex1 GEF activity. FAK disruption compromises melanoblast cell numbers and migration in development, but has no long-term effect on melanocyte homeostasis. FAK-deleted mice have a divergent role in melanomagenesis, delaying primary melanoma onset whilst promoting metastasis following disease onset. Conclusions: We conclude that P-Rex1 and FAK play important roles in melanoblast embryology and melanoma development and progression. Both P-Rex1 and FAK represent interesting therapeutic targets for the treatment of cancer.

Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa are the main multi-drug resistant pathogens associated with deep wound infections which then may cause septicaemia. Treatment is problematic and re-infection is quite common. Free Living Amoebae (FLA), such as Acanthamoeba, are widely distributed in the environment and may also contaminate wounds. It is well known that Acanthamoeba feed on and protect bacteria. The role of Acanthamoeba in wound infections is not very well understood. It is possible that the presence of Acanthamoeba in wounds is one of the key factors for such re-infections. In this study, 140 wound swabs were collected to check for the presence of Acanthamoeba spp. Only one sample was positive for Acanthamoeba spp. Sequencing of the highly variable DF3 region of 18S rRNA gene for the sample showed that this isolate belongs to genotype T4. In addition, clinical isolates of MRSA and Pseudomonas from wound infections were used in this study. The results showed that MRSA and Pseudomonas were able to bind with, invade, survive and multiply inside Acanthamoeba species. One of the essential compounds for microorganisms to grow is iron. The role of iron chelators, including deferiprone and selected novel compounds based on hydroxyl pyridine moiety, was studied. Findings revealed that all novel iron chelators have an antimicrobial activity against both bacteria. In addition, all novel iron chelators were able to kill Acanthamoeba. Cytotoxic effects of MRSA, P. aeruginosa and Acanthamoeba were investigated using the KB epithelial cell line and mesenchymal stem cells (MSC) using a general caspase inhibitor. The results revealed that the ability of live bacteria to induce cell death was higher compared with heat-killed bacteria, bacteria conditioned medium (BCM) and Acanthamoeba conditioned media (CM). The exact trigger for the cell death in this study was not investigated but the relative contributions of apoptosis and necrosis were investigated using fluorescent technique, caspase inhibition and LDH assay. In conclusion, presence of Acanthamoeba in wounds could be the reason of prolong treatment and reinfection in wounds.

There is a requirement to assess the safety of chemicals to both 'man' and the environment. Traditionally this was determined through the use of animal testing. However, there is an increased need to develop alternatives to animal testing for the determination of toxicity due to ethical and legislative reasons. One approach to replacing the use of animals is the application of computational methods. These include Quantitative Structure-Activity Relationships ((Q)SARs), which are the formalisation of the relationship of the effects (e.g. toxicity) for a series of chemicals and their physico-chemical and structural properties. Most QSARs for toxicity require knowledge of a chemicals hydrophobicity. Traditionally hydrophobicity has been characterised by the logarithm of the octanol/water partition coefficient (log P). Current experimental and predictive methods are limited in terms of applicability for compounds with extreme log P values, compounds ionised under the conditions of analysis and surface active agents. An alternative technique to assess hydrophobicity is Immobilised Artificial Membrane High Performance Liquid Chromatography (IAM-HPLC). The IAM stationary phase was developed initially to mimic biological membranes more realistically than octanol/water partitioning. This study has collated published literature values for the IAM retention index (kIAM), including details of the experimental procedure, into a database. The database includes 1910 values for 647 compounds. The effect of variability of experimental procedure on reported values was investigated. Key experimental parameters were identified that ensure comparable log kIAM values. An IAM-HPLC method was optimised; the HPLC method covers a range of hydrophobicities (log P of -1.35 to 6.03) and includes both unionised and ionised compounds under the conditions of analysis. Additionally the method has been demonstrated to be robust across system of analysis, column and stationary phase batch. The assessment of robustness increases confidence in the log kIAM (pH 7.4) values for 66 aliphatic and aromatic compounds determined as part of this work. Methods to predict log klAM (pH 7.4) were investigated. Both a fragment and correction factor method, based on theoretical structural features, and a 'classical' descriptor based QSAR approach, was applied to both the experimental log kIAM (pH 7.4) values determined in this work and comparable values collated from the literature. QSARs have been developed using log klAM as a descriptor to predict the ability of a chemical to cross the skin barrier and to predict various acute aquatic toxicity endpoints, using published skin absorption and ecotoxicity data respectively.