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Topical photodynamic therapy (PDT) elicits a therapeutic response in both non melanoma skin cancer and immune-mediated skin disorders. Production of reactive oxygen species may result in direct cell death; however PDT-induced inflammatory and immune responses may also contribute to the therapeutic effects. The aims of my thesis were to examine for evidence of topical PDT-induced apoptosis and leucocyte trafficking, and the mechanisms underlying their mediation, in human skin.

Basal Cell Carcinoma (BCC) is the most common type of skin cancer; its development is associated with mutational inactivation of the PTCH1 tumour suppressor gene leading to deregulation of the Hedgehog pathway through increased expression of the downstream GLI (GLI1 and GLI2) transcription factors. The high expression of GLI1 and GLI2 has also been shown to be maintained through a positive feedback mechanism in human keratinocytes, but this has partly relied upon mRNA expression and has not been fully explored at the protein level. Moreover, expression analysis in human BCCs has largely relied upon in situ hybridization or RT-PCR and no studies have investigated co-expression of the GLI1 and GLI2 proteins. Whereas an active mutant of GLI2 (GLI2DN) has been shown to increase endogenous GLI1 protein expression in normal human keratinocytes, preliminary evidence suggests that ectopic GLI1 suppresses endogenous GLI2 protein expression, which refutes the presence of a positive GLI feedback loop. In human BCC samples, there was a relatively high degree of heterogeneity in the expression pattern of GLI1 and GLI2 with the various antibodies employed and this was also affected by how the tissue was processed i.e. frozen versus formalin-fixed. Surprisingly, many BCCs displayed strong epidermal GLI1 expression that was decreased in the basal layer and tumour islands. In most tumours GLI1 and GLI2 showed areas of co-expression, but this was not uniform and in many areas one protein was more abundant than the other. Moreover, there was no clear difference in the expression level of GLI1 or GLI2 between BCC subtypes (aggressive versus non-aggressive). Finally, one antibody (GLI1 H-300) detected strong GLI1 expression in mesenchymal cells surrounding tumour islands (in frozen samples) but this did not correlate with epithelial Sonic Hedgehog ligand expression, suggesting a lack of paracrine Hedgehog signalling in BCC biology. In summary, this project has shown that GLI regulation and expression are more complex than previous studies have suggested and that research in this field is hampered by a lack of consistency and reliability of commercial GLI antibodies.

The master-regulators of an epithelial-mesenchymal transition (MR-EMT) have a pivotal role in the regulation of carcinoma development, promoting transformation and generating a migratory and invasive phenotype. Within epithelial cells, the ZEB proteins are co-regulated, jointly repressed by the miR-200 family of microRNAs. However, here it is demonstrated that the expression and regulation of the MR-EMT in malignant melanoma cell lines appears to be fundamentally different, with a hierarchical organisation identified. ZEB2 and SNAIL2 were found to be expressed in melanocytes, whilst ZEB1 and TWIST1 expression was acquired by a sub-set of malignant melanoma cell lines. Melanoma-initiating mutations within B-RAF and NRAS were shown to reversibly promote expression of ZEB1 and TWIST1 at the expense of ZEB2 and SNAIL2. Additionally, ZEB2 and SNAIL2 were identified up-stream of ZEB1 and TWIST1 within the MAPK signalling cascade, with ZEB2 functioning as a repressor of ZEB1. Furthermore, ZEB2 and SNAIL2 were found to positively regulate expression of MITF, a marker of melanocyte differentiation. In contrast, ZEB1 repressed expression of MITF and was the primary transcriptional repressor of E-cadherin, an adhesion molecule vital for the interaction between differentiated melanocytes and keratinocytes. Previously, within epithelial cell lines, all the MR-EMT have been identified as transcriptional repressors of E-cadherin. However, ZEB2 and SNAIL2 were co-expressed with E-cadherin within melanocytes and melanoma cell lines and, along with TWIST1, were not able to independently induce E-cadherin re-activation following repression. Surprisingly, ZEB2 became a repressor of E-cadherin in conjunction with ZEB1. Finally, E-cadherin expression was also shown to be controlled in a ZEB1-dependent manner by the transcriptional co-repressor BRG1, the ATPase subunit of the SWI/SNF chromatin remodelling complex, and by the presence of DNA methylation at the E-cadherin promoter. Indeed, DNA methylation was identified as a possible factor controlling the success rate of metastatic colonisation in melanoma cells, allowing for the dynamic re-expression of E-cadherin at the secondary site. These data demonstrate that in malignant melanoma the expression and regulation of the MREMT is fundamentally different to that of epithelial tumours, with the MR-EMT structured hierarchically, with opposing regulatory functions.

Tiam1 (for T-lymphoma invasion and metastasis-inducing protein) belongs to the Rho GEF family of proteins. In response to growth factors and cell-substrate interactions, Tiam1 selectively activates Rac. Using a two stage chemical carcinogenesis protocol (DMBA/TPA) it was previously shown that mice lacking Tiam1 are resistant to the development of Ras-induced skin tumours, suggestmg an important role for Tiam1/Rac signalling in tumourigenesis in vivo.

The aim of the work presented in this thesis was to develop vectors for gene transfer, that are based on herpes simplex virus type 1, for possible application in the field of surgery.

Ultraviolet radiation (UVR) is a complete carcinogen, inducing skin cancer via DNA photodamage that can lead to mutagenesis, and promoting its growth via photoimmunosuppression (PI). The omega-3 polyunsaturated fatty acid (n-3 PUFA) eicosapentaenoic acid was shown in murine studies to protect against PI and UV-induced skin cancer although the mechanism is uncertain. The principal objectives of this thesis were to (i) examine whether n-3 PUFA can protect against a clinical model of PI in healthy humans and (ii) explore whether the underlying mechanism could be abrogation of UV-induced depletion of antigen-presenting Langerhans cells (LC) from the epidermis, and/or impact on immunomodulatory cytokines. Nickel (Ni) allergic females (n=79) were randomized to 3 months of daily supplementation with 5g n-3 PUFA (70% eicosapentaenoic acid, EPA; 10% docosahexaenoic acid, DHA) or the placebo medium chain triglyceride, GTCC. Local PI was clinically assessed post supplementation using the nickel contact hypersensitivity (Ni CHS) model. In each volunteer, Ni patches were applied to 3 skin sites that were irradiated for 3 consecutive days with UV-doses of 1.89, 3.82 & 7.59J/cm2 respectively. CHS responses were measured and compared to responses of control patches applied on unirradiated skin using a reflectance erythema meter. In the same subjects, assessments of cellular and biochemical mediators of PI were made pre and post supplementation. At 24hr post irradiation with an erythemal UV-dose (4 minimal erythemal doses) to upper buttock skin, half the subjects (n=39) had skin punch biopsies taken and the other half (n=40) had suction blisters raised on this irradiated skin and on unirradiated skin of the contralateral buttock. Epidermal sheets were prepared from the punch biopsies and immunohistochemically stained to assess UV-induced LC numbers. Levels of immunomodulatory cytokines were analysed in the suction blister fluid using Luminex multiplex assay kits. To evaluate compliance and bioavailability, blood samples were taken from all volunteers, pre and post supplementation and EPA% weight in red blood cell membranes was examined using gas chromatography. Post supplementation, EPA %wt was significantly higher in the active group compared to control: mean 3.61% ± 0.22% (SEM) vs. 0.93% ± 0.06% (p<0.001). 3 volunteers showed evidence of non-compliance and were excluded from further analysis. Compared to placebo, evidence for protection against local PI of Ni CHS was apparent post n-3 PUFA at all UV doses, reaching statistical significance at the UV-dose of 3.8J/cm2 (p<0.05). No significant difference in post-UV epidermal LC numbers after supplementation was seen between active and placebo groups, with a % fall following UV of 76.61 ± 3.39% (SEM) in the active group and 73.52 ± 5.24% (SEM) in the control group. When intragroup comparisons were made pre vs. post supplementation, a similar increase in UV-induced LC depletion from the epidermis was seen in both groups, reaching statistical significance following n-3 PUFA (p=0.018). Levels of interleukins IL-10 and IL-8, and of TNF-α, increased post-UVR in both active and control groups pre-supplementation, with no changes occurring following supplementation. In conclusion, supplemental EPA was bioavailable and evidence of protection against clinical PI of Ni CHS was seen in the actively treated group. However, no evidence was found that this abrogation of PI was mediated through a reduced effect of UV on migration of epidermal LC or the immunomodulatory cytokines examined. This original study gives the first evidence that dietary n-3 PUFA may protect against clinical PI, and potentially skin cancer promotion, in humans. Further research is needed to confirm this finding, and to examine the underlying mechanisms, which could involve other immunoregulatory cells of the skin, such as dermal dendritic cells and T regulatory cells and other mediators of UV-immunosuppression including the prostanoids, which may be modified by n-3 PUFA.