Kaposi's sarcoma-associated Herpesvirus (KSHV; HHV -8) is associated with multiple malignancies, including Kaposi's sarcoma. KSHV has two distinct life cycle phases, latent persistence and lytic replication. In contrast to other oncogenic Herpesviruses, lytic replication plays an important part in tumourigenicity and pathogenesis of KSHV. Therefore, it is essential to study the molecular mechanisms which regulate lytic replication to fully understand KSHV pathogenesis. This in turn may lead to novel therapies, which could become an important strategy for the treatment of KSHV -associated diseases. Post-transcriptional regulation of RNA biogenesis is fundamental to KSHV lytic gene expression. The KSHV ORF57 protein plays an essential role in viral RNA processing, transcription, splicing, mRNA stability, nuclear mRNA export and translation. To date, it is unknown how ORF57 co-ordinates these many roles. Functional diversity of a protein can be achieved by post-translational modifications. We demonstrate that ORF57 is post-translationally methylated and inhibition of methylation has a dramatic effect on the ability of ORF57 to export intronless viral RN A out of the nucleus. This work shows that hypomethylation of ORF57 enhances its ability to bind RNA. Attempts were made to find ORF57 residues which are methylated and results identified PRMT5, a cellular protein methyltransferase as well as the putative demethylase MINA, which interact with ORF57. Furthermore, a proteomic-based approach was used to identify ce~lular proteins which are changed in their intracellular distribution or abundance upon ORF57 expression. This SILAC-based approach highlighted proteins and pathways affected by ORF57. Data showed changes in RNA processing pathways previously unknown to be affected by ORF57, e.g. mRNA polyadenylation and nucleoskeleton rearrangements. In addition, changes of putative, novel components of the human TREX complex were found. Data presented will provide a valuable resource not only to enhance our understanding of this viral pathogen but also to show new routes for drug intervention.

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Malignant melanoma is a relatively rare disease, but it is the most fatal form of skin cancers. In UK alone, it represents 10% of all skin cancers and its incidence has increased four times during the last three decades. The survival rate of melanoma is inversely proportional to the tumour thickness, so early detection with surgical removal of thin lesions is vital for successful treatment. Many dermatologists have advocated the development of computer- assisted diagnosis (CAD) for early detection of melanoma, due to its objectivity and the potential it could provide for self-screening. Numerous computer-based methods have been developed for melanoma diagnosis. However, better accuracy and robustness are demanded for computer-based systems to be trustworthy enough for routine clinical applications. With the aim of improving the existing CAD systems, the present study develops several innovative techniques to achieve accurate and reliable computer-based diagnosis for melanoma. Based on the clinical evidence, a new sub-segmentation scheme for cutaneous lesions is firstly proposed to allow the isolation of normal skin, suspicious lesion areas, and interesting darker areas inside the lesion, simultaneously. This scheme is much different from traditional segmentation techniques, where only lesion areas and non-lesion areas are separated. It has been found that the isolated darker areas within the lesion are of great diagnostic importance, such that they are useful in characterising the malignancy of cutaneous lesions. Melanin index and erythema index, which respectively characterise the amount and distributions of melanin and haemoglobin components within human skin, are computed from conventional RGB images according to the optical theory of human skin. These biological indices are employed to generate new colour variegation descriptors for melanoma diagnosis. Experiments show that the derived chromophore indices can accurately describe pigmentation distributions, and tend to be less influenced by external imaging complexities (e.g. light conditions and optical sensor parameters) than the conventional image intensities such as RGB colour values. A novel asymmetry analysis algorithm based on the global point signatures (GPSs) is developed to quantify the shape and pigmentation asymmetry of cutaneous lesions, simultaneously. In contrast to existing methods for asymmetry analysis, the newly proposed method results in only one pair of reflective symmetry axes. This is consistent with the asymmetry of cutaneous lesions as perceived by the human eye, and circumvents the problem of yielding two different pairs of symmetry axes when shape and colour asymmetries are evaluated separately. In addition, the new asymmetry descriptor is approved to be invariant to rigid transformations, and robust to non-rigid deformations. This suggests that the GPS-based asymmetry descriptor not only benefits for the computer-assisted diagnosis of melanoma, but also has good potential for follow-up monitoring of suspicious cutaneous lesions for high-risk Caucasian population. Finally, an innovative CAD system for melanoma is developed using the extended Laplacian Eigenmap. This system incorporates, for the first time, clinically important metadata into a completely automatic classification process for melanoma diagnosis. Algorithm performance is evaluated on both 2D dermoscopy images and 3D data obtained from our Skin Analyser device. The proposed CAD system achieves 90.97% sensitivity and 86.42% specificity with the dermoscopy database, and 94.12% sensitivity and 88.99% specificity with the Skin Analyser database. The diagnostic accuracy obtained by our system is superior to most of the results from other existing CAD systems for melanoma.

UVA irradiation penetrates deep into the epidermis producing reactive oxygen species (ROS) that are responsible for damaging cellular components. Many phenolic compounds scavenge ROS and chelate transition metal ions that promote oxidation. In this thesis the effect of five polyphenols, namely, epicatechin gallate (ECG), luteolin, luteolin-4'-O-glucoside, luteolin-7-O-glucoside and trolox were investigated in terms of their ability to modulate UVA-mediated oxidative damage in human skin fibroblasts (HSF) at two different doses of irradiation (250 and 500 kJ/m2). The cytotoxicity of each compound was evaluated by both the MTT and LDH assays using a polyphenol concentration of 30 muM which was not cytotoxic. At both doses of UVA irradiation, an increase in apoptosis was observed. Each of the phenolic compounds tested reduced the extent of apoptosis except for luteolin-4'-O-glucoside and trolox. UVA irradiation of HSF increased lipid peroxidation (PV and MDA-TBARS) Overall, luteolin and its glucosides demonstrated the greatest protection in terms of reducing lipid peroxidation at both UVA doses. These flavones were more effective than ECG, which in turn was more protective than trolox. Trolox only reduced PV at the lower irradiation dose. UVA irradiation caused an increase in protein cross-linkage (dityrosine formation) shown by increased fluorescence, and in collagen I and IV degradation in HSF. All (poly)phenols reduced protein cross-linkage at the high UVA dose but did not prevent collagen I and IV degradation. Both luteolin and its 7-O-glucoside interacted effectively with Fe3+ and Cu2+ indicating the importance of both 3,4-dihydroxy group and the 4-oxo group on the B and C rings, respectively. Luteolin-4'-O-glucoside showed a lower interaction with the two transition metals highlighting the importance of the B ring catechol group. ECG also interacted effectively with Cu2+ but not Fe3+ whereas trolox showed the least interaction with these metal ions. These results show that UVA causes apoptosis and damage to lipids and proteins in HSF. This damage may be reduced by the inclusion of luteolin and its associated glucosides, as well as ECG, to HSF prior to irradiation. However, trolox was generally ineffective. Overall the efficacy of the (poly)phenols investigated in terms of their ability to prevent UVA induced damage was related to their structural features and their ability to interact with metal ions.

Malignant melanoma is an aggressive cancer that originates from melanocytes, the pigment producing cells of the skin. The incidence of melanoma is increasing and the outcome for patients with advanced disease remains poor. New therapies for melanoma are in urgently needed as no current systemic treatment is effective. Interactions between the nervous system and epidermal melanocytes have been suspected on the basis of their common embryological origin from the neural crest. Both melanocytes and melanomas are innervated by autonomic nerves with acetylcholine and noradrenaline acting as transmitter molecules. It is now well established that the purine nucleotide adenosine triphosphate (ATP) is a co-transmitter with noradrenaline in sympathetic nerves and with acetylcholine in parasympathetic nerves. ATP acts on extracellular receptors which have been characterised to consist of a number of subtypes. ATP acting on these receptors is involved with both rapid signalling in neurotransmission and also long term signalling in cell proliferation, differentiation and apoptosis.

The c-myc proto-oncogene plays a key role in cell proliferation, malignant transformation and apoptosis. Previous work at RAFT demonstrated that c-myc expression levels are an accurate prognostic marker in melanoma. Ribozymes are catalytic RNA molecules that cleave specific mRNA sequences to prevent gene expression. A ribozyme was constructed targeting the c-myc translation initiation site. A375M melanoma cells were transfected using liposomes and growth was assessed using a MTS growth assay, c-myc expression was measured with immuno-staining and flow-cytometry. Ribozyme treatment of A375M melanoma cells reduced c-myc expression when compared to non-specific controls (p < 0.001). Ribozyme treatment also significantly reduced growth of A375M cells in-vitro compared to untreated controls (p < 0.001). Ribozymes were investigated in combination with interferon and cis- platinum. A prospective investigation of c-myc expression in 117 melanomas was undertaken using immunostaining and flow cytometric analysis. Clinico-pathological details were also studied. Analysis revealed that high c-myc expression was associated with a worse prognosis (p=0.043). Multivariate analysis demonstrated c-myc as an independent prognostic marker when measured against other routine parameters including Breslow thickness. To facilitate rapid screening for molecular alterations in melanoma, a tissue micro-array was created using historical paraffin embedded specimens. 126 tumours were included in the array block representing all stages of disease progression. Analysis was performed on a variety of genes associated with c-myc examining prognostic significance. High expression of p27 and p53 were significantly associated with improved survival (p < 0.05). Cyclins A, E and D1 correlated with disease progression (p < 0.05). Conclusions. Ribozymes can effectively block c-myc gene expression in vitro and may have a role in the treatment of melanoma. Flow cytometric analysis of c-myc expression provides a new important independent prognostic marker for melanoma. Tissue array technology is a simple and powerful tool in the search for new prognostic markers in melanoma.

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