Liver fibrosis has a range of aetiologies and is a global cause of mortality. A critical effect of liver fibrosis which also increases mortality is portal hypertension. The hepatic stellate cell is accepted as a major progenitor of liver myofibroblasts, which have been shown to be a major source of collagen and extracellular matrix proteins that disrupt liver architecture and function. Relaxin is a hormone involved in remodelling of extracellular matrix in the uterus and cervix and is known to increase renal blood flow in pregnancy. It has been implicated in the regulation of fibrosis in animal models and to modify the cell biology of hepatic stellate cells in vitro. I have demonstrated the profile of expression of relaxin receptors in primary human stellate cells (HSC), showing them to express RXFP-1, 3 and 4. Using a cAMP assay I confirm these receptors to be functional, with RXFP-1 positively and RXFP-3 and 4 negatively coupling to cAMP. The expression of RXFP-1 is coupled with the level of activation, demonstrating a possible role for H2-relaxin in the regulation of HSC. I have established a dynamic regulation of fibrotic mediators and HSC activation markers, including a reduction in α-SMA, TIMP-1 and TGF-β with increases in MMP-1 and MMP-2, consistent with H2-relaxin having potentially therapeutic antifibrotic effects by increasing the fibrolytic phenotype. In addition through the use of gel contraction assays I demonstrate that H2-relaxin reduces serum or endothelin-1 induced HSC contraction. Through the use of siRNA I have confirmed that H2-relaxin mediates its regulation of fibrotic mediators and HSC activation markers as well as the inhibition of gel contraction through the relaxin receptor RXFP-1. I have evidence to suggest that the inhibition of contraction may in part be via nitric oxide release in HSC. In conclusion I propose that RXFP-1 is a potential therapeutic target in end stage human liver disease, targeting fibrosis and portal blood hypertension via both resolution of the phenotypic collagen deposition and vascular constriction associated with the human hepatic stellate cell.

COPD (Chronic Obstructive Pulmonary Disease) worldwide has a prevalence of 10% in men and 8.5% in women. Exacerbations of COPD account for approximately 10% of all acute medical admissions. Projected prevalence figures suggest that by 2020 COPD will be the third leading cause of mortality worldwide thus imposing a significant burden on healthcare resources in the future. Acute exacerbations are not only responsible for a decline in the patient’s quality of life, but have a major socioeconomic impact. Following a pilot study that showed current smokers recover lung function much more slowly from their exacerbation than ex smokers, I initiated a properly powered prospective study to investigate the difference between the two groups. A total of 58 patients admitted with acute infectious exacerbations of COPD were recruited to the study to determine the effect of smoking status on their exacerbation. Throughout the admission lung function was measured. Sputum was cultured for bacteria, and PCR used to detect viral infection. Blood and sputum cells were analyzed by flow cytometry. Serum was collected for CRP levels. Ex-smokers recovered significantly more quickly than current smokers in all spirometric parameters (P<0.01), and were discharged sooner (mean 3.08 vs 5.59 days, P<0.001). Sputum culture was positive for more pathogenic bacteria in current smokers, especially H. influenzae, which was associated with a significantly higher CRP rise (p<0.05) than any other organism. CD8+ T cells predominated in the sputum of ex-smokers while CD4+ T cells were the dominant cell type in current smokers (p<0.01). Current smoking is a risk factor for more severe exacerbations, delayed recovery and prolonged hospitalization. This may result from a variety of factors including bacterial, immune mediated responses and systemic inflammation.

Smoking is the major risk factor for COPD. Smoking also has systemic effects and is considered as one of the risk factors for metabolic syndrome (MeS). It is unclear whether it is smoking per se or the systemic effects of COPD that cause metabolic syndrome in smokers. Smokers with and without COPD and non-smoking controls were studied by pulmonary function testing, skin prick tests, body composition, fasting glucose, CRP, and lipids analysis to diagnose MeS. This showed a gradual increase in prevalence of MeS, but with only difference between non-smokers on one hand and smokers with or without COPD on the other being significant. This suggested that smoking, rather than the systemic effect of COPD, was the cause of MeS. All smokers were then grouped and smokers and non-smokers compared in respect of lung function, inflammatory markers (CRP, a series of inflammatory cytokines), insulin resistance, and body composition. Smokers had increased central obesity and total body fat, which is in contrast to the common belief that smoking reduces weight. Male smokers demonstrated increased abdominal fat, while females showed an increase in total body fat. FEV1 was reduced when comparing all smokers with MeS and those without MeS, and there was a greater reduction in males who had a greater prevalence of MeS, but had better quality of life even though they smoked more. However, whilst smokers with MeS had higher levels of insulin resistance, as measured by Homeostasis Model Assessment (HOMA-R), none of the plasma inflammatory markers, except for IL-12, was raised, suggesting that these indices of inflammation were not the reason for MeS. Smoking is associated with a gradual decline in lung function in smokers with and without COPD. A previously recruited cohort of smokers with and without COPD and healthy non-smoking subjects were followed up over a period of 5 years. Amongst a whole series of measurements, including HRCT, measures of lung density/emphysema, only sputum neutrophilia (both absolute and percentage counts) predicted the annual decline in FEV1. In summary, this study suggests that there is an increased prevalence of MeS in smokers associated with insulin resistance caused by smoking but it fails to show an association between MeS and COPD. Smoking is also associated with central obesity and increased body fat, contributing to a reduction in FEV1. Sputum neutrophilia, but not smoking pack years or lung HRCT measurements, predicts the annual FEV1 decline in smokers.

The inflammatory response is critical to healing outcome after cutaneous injury. Our current understanding of the response to injury has been compiled from targeted studies on components expected to play a role. It was hypothesised that an unbiased approach to characterisation of soluble mediators within the injured tissue might identify additional components, thereby increasing our understanding of the cellular processes involved. The aim of this research was to develop and characterise a model of injury with the potential to identify novel mediators of the early response. Microdialysis was chosen as both the sampling method and the means by which the tissue was injured as probe insertion causes a single injury that can be sampled continuously without further damaging the tissue. Early injury responses were initially characterised in terms of changes in blood flow and known markers of the inflammatory response, using Laser Doppler Imaging and a bead-based cytokine flow cytometric assay, respectively. A shotgun proteomic analysis was then undertaken to characterise of the protein content of the fluid obtained using microdialysis, dialysate. The phosphorylation status of proteins was also characterised following the implementation and optimisation of a recently reported method that uses dendrimer conjugation chemistry to capture phosphopeptides. Blood flow and cytokine measurements in the dialysate confirmed the occurrence of a reproducible inflammatory response to the microdialysis injury. Proteomic analyses of dialysate suggests that it has a relatively simple protein composition and is dominated by highly abundant components. The identified proteins originate from both intra- and extracellular locations and play a range of roles, including regulation of coagulation, cellular ii communication, and the immune response. Several are likely to undergo post-translational phosphorylation and hence their phosphorylation status was also investigated. Using the phosphopeptide capture method, potentially novel phosphorylation sites were identified in two abundant proteins, albumin and apolipoprotein L1 at positions S603 and S314, respectively. Collectively, the data obtained in this investigation increase our knowledge of the proteins and processes involved in responses to injury, and suggest that microdialysis may be of some use for studies in this area. Further, analysis of protein phosphorylation in dialysate suggests that this is an informative approach that could shed light on extracellular signalling events that occur during the progression of the response to injury.

The aim of this study was to investigate the potential use of ELISA and LC-MS-MS in combination and as individual techniques, for the detection of drugs of abuse in biological matrices. Overall the LC-MS-MS method showed good correlation results for opiates compared to the GC-MS method. 6-MAM was however detected in more root segments and segments excluding roots by LC-MS-MS. Morphine was detected in a greater number of root segments by LC-MS-MS compared to GC-MS. However, morphine was detected in a greater number of segments excluding roots by GC-MS. Codeine and dihydrocodeine were also detected in a greater number of root segments and segments excluding roots by GC-MS. The cocaine results showed excellent qualitative correlation between the LC-MS-MS and GC-MS methods for cocaine and benzoylecgonine. The GC-MS method did not however extract greater concentrations of cocaine and its metabolites compared to LC-MS-MS due to the higher recovery of the drug group specific GC-MS method. Cocaethylene and EME were detected in some samples by LC-MS-MS method for opiates and cocaine and its metabolites compared to the GC-MS method; there may be some cases where the GC-MS method would detect the analytes where the LC-MS-MS method would not. This has been demonstrated in 3 samples for morphine and in 6 samples for codeine. The LC-MS-MS method analysed for and detected amphetamines in samples that were not tested for amphetamines by GC-MS. In one sample that was tested by both methods, amphetamine was detected in the root sample by LC-MS-MS where GC-MS failed to detect it. Also a greater concentration of amphetamine was extracted using the LC-MS-MS method in the segment without roots. The LC-MS-MS method was useful for the analysis of 17 drugs of abuse in post-mortem hair samples in forensic toxicology cases. Using this method, it is possible to obtain maximum information from one hair sample which is extremely useful when the sample weight is limited. The ability of the LC-MS-MS method to extract and analyse a greater number of drug groups from one hair sample highlights the advantages of using this method over GC-MS which targets individual drug groups and requires splitting of the sample. This method is particularly applicable for implementation in the forensic toxicology laboratory at the University of Glasgow where currently GC-MS methods that target individual drug groups are used for routine hair screening and confirmation.