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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.

Melanoma is notoriously resistant to immuno- and even targeted chemotherapeutic strategies despite recent advances in drug development. The overall mortality of melanoma correlates with its ability to metastasise. Breslow thickness or the depth of invasion remains the most useful prognostic indicator, thereby linking the ability of the cells to invade with their propensity to metastasise. Invasion occurs early during tumour development, but the factors driving this process remain poorly understood. There is a growing appreciation that chemotaxis plays an important role in driving the migration and invasion of melanoma cells, but the key stimuli are not known. Through the generation and validation of an improved chamber for cancer cell chemotaxis, melanoma cells are shown to create chemotactic gradients that drive or disperse themselves outwards with remarkable efficiency. This process is driven by strikingly positive chemotaxis and depends on the melanoma reaching a critical density to generate the gradient. The principal attractant is the inflammatory signal lysophosphatidic acid (LPA). Unexpectedly, it is active across all stages of melanoma evolution and LPA is both necessary and sufficient for chemotaxis in 2D & 3D assays. Growth Factors were previously considered to play essential roles in driving directed migration, but instead facilitate LPA chemotaxis. Sampling across the margins of melanomas in vivo, gradients of LPA are reliably identified, which are capable of driving accurate chemotaxis. This not only confirms the physiological importance of the results, but also is the first time a chemoattractant gradient has been measured in vivo. The corollary of these findings is that, provided with an external homogenous source of LPA, a large enough melanoma will degrade the local LPA to generate an outward gradient. Therefore it is the ability to degrade the gradient that acts as the signal to drive chemotaxis and invasion rather than the presence of LPA per se. In the chambers, cells are observed dispersing in a wave and in addition to driving efficient melanoma invasion, this may be responsible for the patterning of melanocytes across the skin during embryogenesis. Ultimately, identifying key aspects of and targeting the LPA-axis may prove a novel prognostic indicator and therapeutic target for invasion and metastasis.

Human cancer stem cells are proposed to play a critical role in tumour initiation and maintenance by their exclusive ability to regenerate the tumour. Thus cancer stem cells share many of the properties of normal stem cell including self-renewal and ability to give rise to progeny which undergo tissue-specific differentiation. Thus we hypothesised that by determining the normal patterns of tissue differentiation within cancer we could identify tumour type specific factors that promote differentiation, for therapeutic development. Therefore the aim of this study is to define patterns of human hair follicle differentiation in human basal cell carcinoma (BCC) in order to elucidate potential drug-able targets that can promote tumour specific differentiation. To test this hypothesis we analysed 20 different hair follicle specific differentiation markers, which define distinct layers within the normal adult hair, in six different human BCC samples using RT-PCR with normal hair follicle tissue as control. For the 12 specific keratin genes expressed in the BCC, we analysed expression by immunofluorescence on 20 different BCC samples, using hair follicle samples as positive controls. Our findings suggest that human BCC demonstrates both inward and upward differentiation patterns similar to the hair follicle, with expression of: outer root sheath (K5,14,16,and k17), companion layer (K75), inner root sheath (K26,27,28,71,72,and k74), and cuticle (K32,35,82,and k85); but not hair shaft (K31) markers. Consistent with these findings we observed the mutually exclusive relationship between expression of the early differentiation marker K19 and cell proliferation in the hair follicle and BCC. Similarly, expression of the outer root sheath keratins coincided with nuclear translocation of both GLI1 and NFIL-6, suggesting that BCC also share normal hair follicle tissue regulatory pathways. To further test the hypothesis that normal tissue factors observed in the hair follicle regulate BCC differentiation we have developed an in vitro BCC assay. Using this tissue culture model we hypothesised that BCC’s are stuck in the telogen part of the hair follicle cycle, resulting from autocrine expression of bone morphogenic proteins 2 and 4. Inhibition of BMP signalling by addition of noggin as well as addition of TGF-β to BCC colonies in tissue culture led to further induction of inner root sheath, cuticle and medulla keratins. In summary we have shown that BCC exhibit hair follicle differentiation, which is similarly regulated, but is stuck in telogen arrest and can be rescued by addition of noggin and TGF- β2.

Introduction: Malignant Melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Breslow thickness remains the best predictor of metastasis and Sentinel Lymph Node Biopsy is the only method of detecting nodal spread in clinically node negative patients. Surgery is the only effective therapy. Angiogenesis – the growth of new vessels from pre-existing vasculature - is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. Anti- angiogenic isoforms of VEGF have been demonstrated previously to be protective with regard to metastasis. The aims of this thesis were to determine whether VEGF expression within the tumour may allow prediction of the nodal status. Furthermore another aim was to determine whether via the “Seed and Soil” theory, by examination of angiogenic and lymphangiogenic profiles of the tumour and node can we determine that the tumour may control the microenvironment around the Sentinel Node? Finally, as a cohort of false negative patients emerged with a higher mortality rate than their true negative and true positive patient cohort counterparts, could any further patterns be established by performing the same experiments on these patients? Methods: Archived human tumour and corresponding Sentinel Node samples were used and immunohistochemistry was used to investigate the role of pro and anti angiogenic isoforms of VEGF, VEGF-C, LYVE-1 and CD31 within these patients. Results: VEGF-C expression was significantly increased in the intranodal component of positive Sentinel Lymph Nodes (p < 0.01 Bonferroni). This increased expression appeared to be independent of tumoural influences and no strong evidence for the “Seed and Soil” theory was proved. A significantly higher number of lymphatic vessel counts were identified within node negative patients (p < 0.05 ANOVA). No further significant findings were defined on examination of the false negative cohort of patients. Conclusions: This study has shown that positive Sentinel Lymph Nodes exhibit high levels of intranodal VEGF-C. This expression does not appear to be related to tumoural influences. It would therefore appear that VEGF-C expression within Sentinel Nodes warrants further investigation and may aid diagnosis of spread or represent a target to slow or even prevent the onset of metastasis.

Leishmania parasites are digenetic protozoans which infect human hosts and are causative agents of a series of diseases known under the name of leishmaniasis. Macrophages represent the main host. Hence the interaction between Leishmania and macrophages is a fundamental step in the development of the disease. Many studies have been undertaken to understand early stages of the parasite interaction with macrophages; however, few have investigated the later stages of infection. This study was undertaken to develop an experimental model to examine the fate of the parasites when they leave the safe environment represented by their host macrophage. Primarily, the study investigated how Leishmania spread to neighbouring cells without being recognized and killed by the immune system defences. Three Old World species of Leishmania parasites: L. aethiopica, L. major and L. tropica, all responsible for the cutaneous form of the disease, were used. A model of infection was described using two cell lines well known for supporting infection: THP-1 and U937. Axenic amastogotes for L. aethiopica parasites were obtained and used to identify drugs active against the infection. On the basis of the information available in the literature, a model was suggested involving interaction of intracellular parasites with the host cells’ apoptotic machinery. Specifically it was suggested that Leishmania parasites were able to induce incomplete activation of apoptosis in the host cells. This hypothesis was confirmed by the findings that during infection an increased number of host cells showed two features associated with early apoptosis but not the one associated with the later stage. Results were validated in peripheral blood derived human macrophages. The information obtained from comparative proteomics analysis of the infection confirmed that Leishmania regulates apoptotic processes. On the basis of the results obtained a model was presented to explain how induction of apoptosis allows intracellular amastigotes to spread unrecognised to uninfected macrophages without inducing an inflammatory response or losing the host cell’s protection.

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.

Breast cancer is the single most prevalent form of cancer in the UK today, accounting for around 16% of all diagnoses, and around 31% of diagnoses in women. The survival rates are good, however the prognosis is heavily dependent on the stage to which the cancer has progressed at diagnosis. In order to help accurately determine this stage, the sentinel lymph node of patients undergoing tumour resection surgery is removed and examined cytologically for the presence of cancerous cells. This examination of the lymph node is currently the rate-limiting step in the operation as a whole. There is evidence in the literature to suggest that cancerous tissue has a measurably different infrared spectrum from healthy tissue owing to chemical and morphological differences in the tissue. There is further evidence to suggest that in the visible and near infrared region, the spectra of healthy lymph node tissue is different from that of cancerous tissue. This thesis details a project, performed in collaboration with a surgical team at St Mary's Hospital, Newport, Isle of Wight, to analyse spectral images taken in the visible and near infrared, of biopsied lymph node tissue. In the course of the project, an unsupervised classificaton technique, based on an extension to the well establised 'spectral angle', was developed to analyse the spectral images. Psoriasis affects 2-3% of the UK population causing itchy and/or painful plaques on the skin. One of the main treatments for psoriasis is UV phototherapy, exposure to which is a risk factor for burning and the development of cancers. This thesis details an investigation into the possibility of developing a targeted UV phototherapy system based on spectral imaging to delineate the plaques and a proposed new UV laser for treatment.

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.