Src-family kinases (SFKs) control a wide variety of biological processes, from cell proliferation and differentiation to cytoskeletal rearrangements. Abnormal SFK activation has been implicated in a wide variety of cancers and is associated with metastatic behaviour. SFKs are maintained in an inactive state by inhibitory phosphorylation of their C-terminal region by C-terminal Src kinase (CSK). In this study, Drosophila ASPP (dASPP) has been characterized and identified as a regulator of dCsk activity. dASPP is the homolog of the mammalian ASPP proteins, which are known to bind to and specifically stimulate the pro-apoptotic function of p53. dASPP is a positive dCsk regulator. Firstly, dASPP mutants show similar phenotypes to dCsk mutants, which are partially rescued by dSrc64B loss of function. Secondly, dASPP loss of function enhances the specific phenotypes of dCsk mutants in wing epithelial cells. Thirdly, dASPP physically interacts with dCsk to potentiate the inhibitory phosphorylation of dSrc42A. Taken together, these results suggest that dASPP has a role in maintaining epithelial integrity through dCsk regulation (Langton et al., 2007). This work also provides the initial characterization of Boa (Binder of dASPP). Boa physically interacts with dASPP and is important for maintaining high levels of dASPP at the apical membrane. This may be achieved by regulating the stability or localization of dASPP. The data suggests that Boa is important for dASPP function as it also genetically interacts with dCsk. dASPP or Boa loss of function results in similar phenotypes, although there are important differences indicating that Boa has other roles besides dASPP regulation. In another project, I have examined the transcriptional control of DIAP1 (Drosophila Inhibitor of Apoptosis 1) by the Ras pathway. Preliminary results suggest that the Ras pathway up-regulates DIAP1 transcription and this may be mediated by the Ttk69 transcription factor.

The proteolytic degradation of articular cartilage in load-bearing joints is a key pathological step in the progression of arthritis, a process mediated by enzymes called collagenases (specifically MMP-1 and MMP-13). My research has focused on the transcriptional regulation of these enzymes in cartilage cells (chondrocytes) in response to a pro-catabolic stimulus which mimics the complex milieu of elevated cytokines found within the arthritic joint. Activating Protein (AP)-1 transcription factors, specifically the c-Fos/c-Jun heterodimer, have previously been shown to be crucial in collagenase gene regulation. c-Fos/c-Jun gene expression, protein production and collagenase promoter enrichment studies identified a temporal deficit between transient c-Fos/c-Jun peak following 1 hour stimulation and the initiation of collagenase gene transcription following 6 hours stimulation. Protein synthesis inhibitor studies indicated that although c-Fos/c-Jun are indeed important, they are not the sole regulators of collagenase gene expression. DNA microarray studies highlighted a number of genes that contributed to transcriptional regulation early within this temporal deficit. Collagenase gene expression was assessed following the siRNA-mediated silencing of these factors. This confirmed that new factors, not previously associated with collagenase gene regulation, were demonstrated to have a significant role in initiating their transcription. This included factors such as activating transcription factor (ATF)3 and early growth response (EGR)2 which demonstrated differential regulation of the collagenase genes, with their silencing affecting MMP13 expression alone. Having identified a number of contributing factors, I then assessed their temporal gene expression and protein production. Comparisons to c-Fos/c-Jun induction confirmed that a number of these factors were transient, similar to AP-1, yet they peaked following longer durations of stimulation. Subsequent siRNA gene silencing of c-Fos and c-Jun led to decreased expression of some of these factors. This demonstrated that these factors may be regulating collagenase expression indirectly by controlling the expression of other transcription factors that, themselves contribute to the regulation of collagenase gene. The present study improves our understanding of how collagenases are regulated in chondrocytes in response to pro-catabolic stimuli. With an improved knowledge of regulation it may be possible to specifically abrogate aberrant collagenases expression in disease. Moreover, by exploiting the differential regulation of collagenases exhibited by some of these factors, there is the potential to mitigate the side effects associated with broad-spectrum collagenase inhibition, thereby removing the barrier to successful treatment.

There are two main mechanisms of tolerance, one in the thymus and one in the periphery. Anergy, a peripheral mechanism, is a state of hypo-responsiveness where T-cells fail to respond to antigenic stimulus. A breakdown in immunological self-tolerance leads to autoimmunity and so provides an exciting research area for therapeutic intervention in autoimmune disease. Differential display studies comparing anergic and activated CD4+ T-cells identified claudin domain containing protein 1 (CLDND1) to be differentially expressed between these two states. In addition, preliminary experiments performed in our lab identified CLDND1 as a potential negative regulator of CD4+ T-cell activation. The aim of this study was to identify the role of CLDND1 in CD4+ T-cells. Antibodies against CLDND1 were raised and validated before use to determine CLDND1 expression in immune cell subsets and during T-cell activation. The function of CLDND1 in T-cells was investigated using gene silencing or over-expression techniques. CLDND1 expression was also sought in the autoimmune disease, rheumatoid arthritis (RA), to identify whether CLDND1 may be involved in disease pathogenesis. Antibodies were successfully raised against CLDND1 and CLDND1 was found to be transiently up-regulated during CD4+ T-cell activation. CLDND1 gene silencing attempts, while successful at the RNA level, did not translate to a reduction in CLDND1 protein, suggesting CLDND1 may be regulated independently of gene transcription. Over-expression studies were consistent with CLDND1 being a negative regulator of T-cell proliferation or an inducer of cell death, depending on the activating stimulus used. CLDND1 expression was found to correlate with rheumatoid factor (RF) status in early RA patients and may suggest a role for CLDND1 in the disease setting. Some findings identify similarities between CLDND1 and other proteins, providing links for functional pathways and a plethora of further avenues of research.

Two approaches to the provision of pan-European paging services are now becoming established. One of these relies on satellite-based services to be provided by INMARSAT, BT and others at L-Band. The second, the European Radio Messaging Service (ERMES), is a terrestrial system currently under development and scheduled to operate at 169 MHz. Both of these systems may however encounter a problem in delivering adequate signal strength within multi-storey buildings. In the case of the satellite systems due to the necessarily limited transmitter power, and in the terrestrial case due to increasing use of RF opaque glass in new building construction. One solution could be to transpond paging signals down through the building from the roof. Since ERMES is a multichannel service, it is technically feasible to transpond both satellite and ERMES originated paging signals on whatever ERMES channel can be identified as free of other users. The work described here is the result of an extensive radio wave propagation measurement campaign carried out for both approaches. Propagation effects on satellite-mobile systems in the lower microwave part of the spectrum have been the subject of considerable study in the last few years. Most of the interest has been centered at frequencies near 900 MHz and 1.5 GHz, where tree attenuation is considered one of the dominant effects for rural and suburban areas. Since several frequency bands have been considered for future mobile satellite systems, it was thought appropriate to perform measurements of the attenuation induced by trees on satellite-like paths over a wide range of frequencies (1-4 GHz). These measurements could then be used to evaluate the comparable performance of mobile satellite systems at different elevation angles and over a wide frequency range. They could also provide a further insight into the applicability of the empirical formula on vegetation effects quoted in the CCIR report AB/5.

Research on the health effects of temperature has expanded greatly in recent years, mainly due to the occurrence of extreme weather events and predicted climate change scenarios. The development of appropriate statistical methodology has been an important component of this research, and standard approaches, primarily based on multi-city time series regression analysis, are now well established. However, particular aspects of temperature-health associations, such as the non-linear and delayed relationship and the joint handling of multi-city data, still pose important niet hodological problems. During my PhD research, I have contributed to the development of statistical methods that, address two particular limitations of traditional approaches, focusing on the development of two modelling frameworks: distributed lag non-linear models and multivariate meta-analysis. The former is a class of models that specify simultaneously non-linear and delayed exposure-response relationships in time series data, while the latter is an extension of traditional metaanalysis for the combination of multiple correlated outcomes across studies, that is also applicable to multi-parameter associations. These methods are placed within the traditional two-stage approach that, is adopted in tcuiperat, ure-health studies. The first stage is city-specific, with analyses deriving the estimated relationship within each city. The second-stage is meta-analytical procedure for combining the results from the first stage. I have implemented these modelling frameworks in two packages within the statistical environment R. In this PhD thesis I present a series of publications which summarize my research work. Their content focuses on three key aspects: the development of the statistical methodology, the implementation of the software, and the application of the methods to real data. The papers are preceded by an epidemiological and statistical introduction to the topic, and followed by a final discussion where I illustrate potential future developments and provide some conclusions. These methodological advancements contribute several improvements over standard methods that are applied to investigate temperature-health associations in time series data, and may be easily extended to other research fields and study designs.

Systemic acidosis is associated with bone loss and impaired bone mineralisation. The aim of this PhD project was to further investigate the action of extracellular pH on the function of osteoclasts and osteoblasts. I showed that blood-derived human osteoclasts exhibited a highly reproducible acid-activation response, with maximal activation close to pH 7.0, and little activity at blood pH (7.4). These experiments also provided strong evidence that accessory cells, such as osteoblasts or stromal cells, are not required for acid-activation of resorption. The pH-activation profile of human osteoclasts was similar to that of the recently discovered KT-sensing human G- protein-coupled receptor OGR1. Expression of OGR1 and TDAG8 (another GPCR) was detected in human osteoclasts and was upregulated by low pH. I obtained evidence that the multifunctional receptor TRPV1, which senses protons, heat and capsaicin, was expressed by human osteoclasts and was also upregulated at pH 7.0. Moreover, I showed that the alkaloid capsaicin strongly stimulated osteoclasts in non- acidified conditions. To date, only pertussis toxin has been reported to activate osteoclasts without co-stimulation by H. Using mouse bone organ cultures I found that resorption-associated factors TRACP, cathepsin K and TRAF-6 were also upregulated by acidosis. The effect of PTH on human osteoclasts was also studied. I showed that PTH directly stimulates human osteoclasts in the absence of osteoblasts, but only when acid-activated. This finding suggests that the dogma that PTH stimulation of osteoclast is osteoblast-mediated may not be correct. Studies using primary rat osteoblast cultures showed that the formation of mineralised bone nodules is inhibited by acidosis. The same pH reduction, which increases Ca2+ and PO43" solubility of hydroxyapatite by 2- and 4-fold respectively, did not alter collagen production or osteoblast proliferation but decreased alkaline phosphatase activity and expression. Thus, the primary effect of acidosis on osteoblast function is to cause a selective inhibition of bone mineralisation. In conclusion, this study showed that the important "double negative" action of acidosis on bone cells is consistent with a pathophysiological role of bone as a reserve of base to buffer excess protons when the kidneys and lungs are unable to maintain acid-base balance within narrow physiological limits.

Angiopoietin-2 (Ang2) is a secreted ligand that promotes blood vessel destabilisation, remodelling, leakage and inflammation in response to pro-inflammatory activators. In binding to its primary receptor, Tie2, it functions as a competitive antagonist of the anti-inflammatory and pro-quiescent agonist angiopoietin-1 (Ang1). Ang2 is markedly elevated in conditions associated with vascular dysfunction and therefore development of an Ang2 inhibitor has multiple potential clinical applications. Ligand traps are developed from native receptor ectodomain fragments and offer improved pharmacokinetics over monoclonal antibodies. However production of an Ang2-trap requires manipulation of the endogenous Tie2 receptor to permit Ang2 but not Ang1 binding, and this objective formed the inspiration for this study. The aim was to test whether the somatic hypermutation (SHM) gene diversification activity of B cells could be combined with cell surface display to create a streamlined system for evolving binding proteins. The data presented demonstrates that a cell surface-expressed Tie2 mutant library can be generated via single transfection of a chimeric Tie2 ectodomain-cell surface display construct into the hypermutating DT40 cell line. Subsequently selection of Ang2-binding phenotypes via fluorescently labelled-angiopoietin binding assay and Fluorescence Activated Cell Sorting (FACS) was repeated iteratively prior to sequencing ofTie2 from recovered cells. This approach was successful in isolating mutant forms of Tie2 with altered binding characteristics. Directed evolution is one of the most powerful approaches for manipulating binding properties of proteins but traditionally has involved laborious techniques including iterative cycles of mutagenesis, expression and selection with inter-species translation issues. This combination of vertebrate 'in-cell' diversification and 'on-cell' binding and selection demonstrates a sustainable approach for evolution of any protein.

The PTR-MS is the well-established technique in the field of analysis of volatile compounds from air, food and breath. The principal advantage of the technique is real time analysis, high sensitivity and less fragmentation. However, this technique requires the samples in the gas phase and therefore, liquid samples cannot be analysed directly. The leading focus of this thesis was to develop a technique that will enable the use of the aqueous sample to extend the application of the PTR-MS. This thesis documents the design and calibration of the sample inlet systems and subsequent application development. The initial work dedicated to develop and calibrate the heated inlet system (HIS) to inject the aqueous sample, volatile organic solvent sample and headspace above the liquid sample. The subsequent development and calibration of the capillary inlet system (CIS) for the continuous injection of the aqueous sample is documented in this thesis. Applications for the environmental, food and diagnostic field were developed. In the environmental field, identification and calibration of the pesticide compounds from the water was performed. Acceptable sensitivity and limit of quantification (~ 1.0 μg/mL) was achieved for organophosphate pesticides used in the experiments. Further work is required to improve the sensitivity and lower the limit of quantification to ng/mL levels instead of μg/mL levels. Additional work is required for the detection and quantification for the chlorinated pesticides, as PTR-ToF-MS instrument used in this thesis is not able to detect these pesticides. For the food field, Scotch whiskies were successfully characterised based on their VOC profiles. Lastly, for the diagnostic field, urine headspace from five volunteers was analysed, to identify and to quantify different VOCs. Different VOCs (different ketones and aldehydes) were successfully identified and quantified. The precision of the measurement for acetone and acetaldehyde was 13.7% and 15.6%, respectively. Further work with bigger pool of volunteers, and a technique in which urine samples can be injected directly is required to establish the database for the VOCs and to improve the sensitivity of VOCs. Lastly, a two-stage technique was developed to identify isobaric compounds that are chemically different but have a similar mass. For example, butanal which is an aldehyde and butanone, which is a ketone, are chemically different but both compounds have a mass of 72 amu. In this thesis, as stated above, it is documented that liquid samples can be analysed by using PTR-MS, but further work is required to resolve the issues like contamination and handling of large sample volume.

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