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As our understanding of the cancer genome has progressed, traditional chemotherapeutic agents are being replaced, in part, by targeted therapies. Development of these therapies is driven by our understanding of genetic vulnerabilities harboured by tumours. The aim of this project is to identify novel synthetic lethal interactions within melanoma. Synthetic lethality describes a relationship between two genes, where loss of either of the pair is compatible with cellular viability, but simultaneous loss of both genes induces cell death. This approach can therefore exploit somatically acquired mutations to specifically kill tumour cells and to define new therapeutic targets. Abstract In order to do this, we selected a panel of putative synthetic lethal interactions and built a bespoke multiplex CRISPR-Cas9 library to interrogate 1192 gene-pair interactions. We deployed this library on a panel of melanoma cell lines and a retinal pigment epithelial cell line as a normal comparator. We went on to develop a bioinformatic pipeline with which to analyse the paired screen output and using this pipeline we have identified a number of novel synthetic lethal relationships. Using low throughput assays we validated a number of the interactions found by our screen. Finally, using human tumour expression data, we identified a candidate pair with potential therapeutic relevance. Using external datasets and isogenic knockout models we have further validated this relationship and performed mechanistic experiments to explore the role of these genes within the cell and generate a hypothesis to explain why loss of these two genes results in cell death.

The construction industry has been accused of being, at its worst, wasteful, inefficient and ineffective, wasting over £1 billion in 1999 due to errors and rework (Nicholson, 1999). Competitive pressures from within industry, as well as external political, economic and other considerations are forcing the industry to re-examine and improve its modus operandi (Anumba et al, 2000). The message is that to survive and prosper, the industry must change its management and practice. Nevertheless it took the publication of both the Latham (1994) and Egan (1998) Reports, both advocating the use of performance measurement, for the industry to recognise that it needed to modernise in order to tackle the severe problems facing it. However, in order for it to improve, the Construction Industry is faced with a bewildering range of business improvement models and approaches, all with their unique features, to use on their quality journey, all proclaiming to improve organisational capability and results. Hence, the aim of this research is to provide guidance on how these business improvement models and approaches can be used to support and improve bottom-line results to achieve business excellence. The scope of this research is to focus on three industry sectors and companies: Construction (Morgan Est Pic), automotive (Lear, a first tier supplier) and aerospace (Rolls Royce), as proof of concepts. An analysis of the overlap between the business improvement models and approaches, conducted in the literature review, confirms the need for this research. The initial concept adopted in this research is to pursue and evaluate the most popular business improvement models; ISO 9001; the European Foundation for Quality Management (EFQM) Excellence Model and the American Baldrige Model and the business improvement approaches such as Total Quality Management (TQM), Business Improvement Review (BPIR), Six Sigma and other "tools" that help Managers understand the business, such as Balanced Scorecards and the Process Classification Framework (PCR). Accordingly, a theoretical evaluation model was developed to demonstrate where the approaches support the business improvement models. The model was subjected to the empirical feedback of expert interviews and case studies and to confirm its suitability for use within the construction industry. The now developed evaluation model demonstrated that these models and approaches are "TQM" with ISO 9001 providing the support and consistency between them all. Furthermore, the results of a questionnaire survey revealed that ISO 9001 can be the starting point for the journey towards world-class performance. It provides the platform for taking the organisation forward by achieving control over leadership, customer focus and continual improvement. However, it needs to be implemented with a view to excellence rather than compliance. The main finding from this research is that the construction sector still needs to provide major investment in improvement models and approaches to deliver bottom-line results and achieve business excellence.

Heptadentate ligands form lanthanide (III) complexes, which allow an increased hydration state about the metal centre, resulting in an improvement in the relaxivity of the contrast agents administered. A series of analogues based upon the compound (2) was pursued, incorporating aromatic functionalised groups, consequently increasing the hydrophobicity of the complexes. The aryl moiety significantly enhances the binding to human serum albumin (HSA), consequently improving the observed relaxivity. Relaxivity enhancements were seen for each of the carboxylate complexes in the presence of increasing serum protein concentrations. (DIAGRAM-Chemical structure of the ligand aD03A) (2)The phosphonate analogue of the aD03A ligand (2) was identified as a target and synthesised as a key ligand whose lanthanide (III) complex possesses a higher peripheral negative charge, repelling the attraction towards anions, potentially maintaining higher relaxivity values in serum solutions. A trifluoromethanesulphonamide moiety was introduced into the aD03A moiety that exhibited a high binding affinity to human serum albumin resulting in high values for the observed relaxivity. The solution structures of the diastereoisomers for the model albumin binding complex (S)-[Eu-(EOB)-(DTPA)](^2-) were analysed by (^1)Ή NMR, CD and CPL spectroscopy. The major species observed were found to possess similar (^1)Ή NMR paramagnetic shifts and emission spectra, consistent with a 9-coordinate structure involving one bound water molecule.

In recent years great strides have been made in our understanding of the secretory pathway. Much of the new information regarding regulated exocytosis has come from biochemical studies of neuronal cells and tissues. The aim of the work described in this thesis was to determine if cells of the haematopoietic lineage use the same granule docking and fusion machinery as that used for neurotransmitter release and to investigate the function of candidate genes involved in granulocyte degranulation. Western blotting across eosinophil subfractions failed to identify known components of the synaptic vesicle fusion machinery. cDNA library screening resulted in the isolation of VAMP2 and cellubrevin cDNA clones from eosinophil and RBL-2H3 cDNA libraries. Tagged VAMP2 or cellubrevin cDNA constructs transiently expressed in RBL-2H3 cells localised to the endocytic pathway. To determine if key gene products thought to play a role in regulated exocytosis affected RBL-2H3 cell degranulation, a functional assay utilising flow cytometry was developed. This enabled the effect of overexpression of GFP-tagged ARF and PLD 1b on RBL-2H3 degranulation to be determined. Using this approach the GDP-bound mutant of ARF6 inhibited ionomycin stimulated degranulation while overexpression of wild-type PLD 1b increased granule fusion and localised to RBL-2H3 granules. Therefore PLD1b and ARF6 are implicated in granulocyte degranulation.