Aims: Aspirin is recommended in secondary prevention (SP) in diabetes and macrovascular disease. Recommendation in primary prevention (PP) remains controversial as does the dose of aspirin prescribed. To ascertain whether these controversies are reflected in clinical practice, we conducted a survey of healthcare professionals' views on aspirin prescribing in diabetes. Methods: An anonymous online survey consisting of 26 questions (Likert scale) covering demographic characteristics and aspirin prescribing habits in primary prevention and secondary prevention was circulated via email. Results: 152 people responded with variable response rates: Primary care (96/152, 63%) - mixture of doctors/Diabetes Specialist Nurses; Secondary care were predominantly diabetes specialists (56/152, 37%). Primary prevention (PP): 39/103(37%) did not routinely prescribe aspirin whilst 16/121(13.2%) would consider using aspirin in all diabetes patients as primary prevention. Using Chi-square contingency tables showed that there were differences when prescribing aspirin with regards to hypertension as a risk factor in primary prevention between primary care (20/68[29.4%] opting for aspirin) and secondary care (24/49 [48.98%], p-value-0.03) and doctors and nurses (38/60 vs 16/58, p=0.0009) and also with microalbuminuria - primary care vs secondary care (15/67vs 21/48, p=0.015), and doctors versus nurses (26/60 vs 11/59, p=0.004). Nurses were less likely to prescribe aspirin as primary prevention in smokers (11/57[19.2%] vs 22/60 [36%]; OR-0.41 [0.16-1.03], p=0.042). Secondary prevention (SP): Despite no contraindications 8/125(6.4%) would not give aspirin. 75mg/day or 300mg/day preferred doses in various settings. There were no statistically significant differences between primary and secondary care (62/73 vs 47/52 or 84.9% vs 90.4%, p=0.36) but doctors prescribed aspirin more often compared to nurses (59/67 vs 51/85 or 60% vs 88.1%, p=0.006).In patients with history of peptic ulceration respondents recommended a) use of PPI cover in PP-37/103(35.9%) and SP-60/103(58.3%), b) enteric coated aspirin PP- 13/103(12.6%) and SP-11/103(10.7%), c) not use any anti-platelet agents in PP-53/103(51.5%) and SP-8/103(7.8%). Enteric coated aspirin recommended by respondents as follows: Always-8/109(7.3%), sometimes-16.5%, occasionally-37.6%, and never-35.8%. 89/103(86.5%) had stated their patients had raised issues with them regards aspirin use. 27/103(26.2%) would definitely take aspirin themselves if they had diabetes. The differences were not significant either in a primary prevention setting or a secondary prevention setting when primary care was compared to secondary care but doctors were more likely to prescribe aspirin with PPI cover or in the enteric coated form compared to nurses (48/57[84.2%] vs 23/46 [50%]; OR=0.188 (0.067-0.511), p<0.001). Conclusions: This survey confirmed that the controversy with regards to aspirin use particularly in primary prevention was reflected in a heterogeneous prescribing of aspirin in patients with diabetes. Further clarification and guidance on the optimum dose of aspirin in diabetes is required. b) Summary of Interventional Results Aims: To study the effects of aspirin at different doses on markers of oxidative stress, insulin resistance, dysglycaemia, endothelial function, and vascular inflammation in the primary prevention setting in a population with type 2 diabetes and high risk of cardiovascular disease. Methodology: Following baseline assessment subjects had aspirin (75mg, 300mg, 3.6 gm) or placebo (with minimum 2 week washout) and pre-intervention and post-intervention assessment of markers for metabolic indices (Blood pressure, weight, BMI, Fructosamine, Lipids, Creatinine),oxidative stress (TAOS, FRAP, & Glutathione assays), insulin resistance (HOMA), vascular inflammation (HsCRP, sVCAM-1), and endothelial function (photoplethysmography). Results: (reported in Mean±1SD or Median and Interquartile ranges) (See Table 28, P114) 17 Caucasians, 12 males, 5 females with age range between 40 and 75 years, completed the study. Mean age of the cohort was 57.4±9.1yrs (mean±1SD), with baseline characteristics summarized in Table-6 & Appendix B. Briefly HbA1c was 7.9±1.2%, blood pressure systolic- 130.8±11.5 mmHg & diastolic-73.95±6.97 mmHg, total cholesterol-4.57±1.01 mmol/l, and HDL-C-1.13±0.46 mmol/l. At baseline TAOS concentration was 59.3±9.7 (ascorbate equivalent antioxidant concentration micromolar or AEAC (μM)), total glutathione-302.2±183.3μM, FRAP- 0.86±0.23 (mM Fe II), HOMA-IR-1.41±1.04 Units, HOMA-S–76.27±45.20 %, Fructosamine- 282.9±50.6 μM/l, RI-GTN- 7.17% (3.17-12.83), RI-Salbutamol- 18.5% (13-21.5), Hs-CRP (15 subjects)=0.66 mg/L (0.41 to 2.06 mg/L, Median & IQR), and sVCAM-1 (15 subjects)=487.04 ng/ml (IQR = 450.4 to 572.3). There was a trend towards significance for the TAOS assay with an increase in antioxidant capacity but it did not reach significance. Reduced glutathione (GSH): p=0.12, oxidised glutathione (GSSG): p=0.38, or Glutathione ratio (GSH:GSSG): p=0.367 were not significantly different following any of the interventions. Differences in FRAP were nonsignificant following any of the interventions. Measurements of insulin resistance (HOMA-IR), and insulin sensitivity (HOMA-S) seemed to improve with aspirin 75mgs/day & 300mgs/day but did not reach significance (see figure 18, 19). Neither the different doses of aspirin nor placebo had a significant impact upon glycaemic control (Fructosamine, P=0.39), endothelial function (photoplethysmography, RI-GTN-p=0.36, RI-Salb-p=0.46), Vascular inflammation (Hs-CRPp> 0.05, sVCAM-1>0.05), fasting glucose, BMI, blood pressure, or lipid parameters. Multiple regression analyses showed a good correspondence between the metabolic factors at baseline but were not repeated with different doses as there were no significant differences demonstrated with any of the parameters. Conclusions: Aspirin at the doses studied and over the 2 week duration caused no significant changes in any of the markers studied. Good metabolic control (blood pressure, glycaemia, lipids), and widespread use of statins may be responsible for the lack of effect demonstrated.

The Tensins are a family of intracellular cytoskeleton interacting proteins that are involved in the regulation of cell motility and migration. Downregulation of Tensins has been observed in several cancers, indicating that it may be advantageous for tumour progression. In this study, an epigenetic basis for the observed downregulation of Tensin3 in human renal cell carcinoma (RCC) has been investigated, specifically the methylation state of the TNS3 gene promoter. The aims of this study were to: 1. Identify and validate a functional promoter for the human TNS3 gene that contains a CpG island within it; 2. quantify the methylation level of this region in RCC; 3. determine whether expression of Tensin3 could be altered through DNA demethylation treatment. Bioinformatic analysis revealed there to be a putative promoter for the human TNS3 gene, housing an 826-bp CpG island. A luciferase reporter assay demonstrated a functional minimal promoter activity for a 2000 bp sequence containing this island. For quantification of methylation in the TNS3 promoter, genomic DNA from RCC patients (tumour and adjacent non tumour) and a normal control group were analysed by bisulphite conversion followed by pyrosequencing analysis. This enabled quantitative determination of DNA methylation of individual CpG dinucleotides within the TNS3 gene promoter, out of a total of 43 analysed. Across the entire CpG stretch, RCC DNA showed significantly higher methylation level than non-tumour kidney DNA and normal control DNA (tumour n=12, non-tumour n=3; normal n=12; P<0.01, tumour vs. non-tumour; P<0.0001, tumour vs. normal). Out of the CpGs analysed, two CpG dinucleotides, CpGs 2 and 8, showed the most pronounced increases in methylation in tumour samples (P<0.0001). Furthermore, CpG 2 methylation negatively correlated with Tensin3 gene expression levels in RCC samples (P<0.005). In addition, pharmacological demethylation of cultured HK2 kidney cells with 5-aza-2’deoxycytidine caused a threefold upregulation of Tensin3 expression as measured by qRT-PCR. In conclusion, these results reveal a differential methylation pattern in the TNS3 promoter region occurring in human RCC, suggesting at least in part an epigenetic basis for the observed aberrant downregulation of Tensin3 in this disease.

Introduction: Non-specific low back pain is a common condition that continues to place a considerable burden on society. Various treatment approaches have emerged which are aimed at targeting non-specific low back pain and one of the most commonly recommended of these is spinal manipulative therapy, which is a central component of the chiropractic approach. However, despite observations in clinical practice in which some individuals respond well, results from clinical trials of treatment interventions for low back pain, such as chiropractic, are repeatedly seen to have small effect sizes. A plausible explanation for this is that low back pain may be considered a heterogeneous condition consisting of a number of subgroups of patients. Previously highlighted as a research priority, these subgroups and their predictive factors for outcome are beginning to be identified among low back pain patients receiving chiropractic treatment; however they are largely unstudied in the UK chiropractic patient population. The overall aim of this prospective cohort study was to attempt to identify predictors of outcomes in the short, medium and long term in low back pain patients undergoing chiropractic treatment in primary care settings throughout the UK. Methods: All practising members of the British Chiropractic Association were invited to participate in the study. Each chiropractor was required to recruit 10 consecutive low back pain patients. Patients were eligible for inclusion if they were between 18 and 60 years of age; presenting with a new episode of low back pain with or without leg pain; no treatment for low back pain within the previous 3 months; not pregnant; no contraindications to chiropractic care; a mobile phone user. All participating patients completed an informed consent form. Data were recorded utilising self report paper questionnaires by patients and chiropractors at baseline; and by patients only at the 4th visit, 3 months and 6 months follow-up. In addition, outcomes in the immediate short term were recorded from patients via text message on a daily basis for 7 days following the 1st visit. Baseline potential predictor variables encompassed demographics, clinical characteristic, clinical examination findings, work-related factors and psychosocial factors. The primary outcome was patient self-report global improvement. Patients were subgrouped according to the duration of the current episode of low back pain into acute (less than 2 weeks) and subacute/chronic (2 weeks or greater). Multivariate logistic regression analysis was used to construct prognostic models for baseline and change score variables at each follow-up. Results: Sixty-five chiropractors and 452 low back pain patients (222 acute; 230 subacute/chronic) participated in the study. The loss to follow-up at 6 months was approximately 65%. Almost 60% of patients participated in the text message study and the response rate was high (96%). The acute patients reported higher pain and disability at baseline; however a greater proportion of these patients were categorised as improved at each follow-up. The greatest drop in pain scores occurred in the 1st week in both subgroups. Several baseline predictor variables were independently associated with improvement at follow-up; however these differed between the subgroups with the exception of the patient-practitioner relationship. Early changes in pain were independently associated with improvement for the acute and subacute/chronic patients in the short and medium term. The discriminative ability of the baseline and change score prognostic models varied from weak to acceptable. Conclusion: The investigation presented here contributes to the body of research concerning prognostic factors, specifically those in the immediate short term, in the UK chiropractic LBP patient population and for being the largest study of its kind to date in the UK. Furthermore, this study highlights the potential impact of the patientpractitioner relationship on outcome in low back pain patients receiving chiropractic care. Although several baseline variables predicted improvement at follow-up, the importance of early change as a prognostic indicator is emphasised.

Background: Giardiasis is an intestinal diarrhoeal illness caused by the flagellate protozoan parasite Giardia intestinalis (synonymous with Giardia lamblia and Giardia duodenalis). Traditionally, giardiasis has been diagnosed in patients using faecal concentration and microscopy techniques. Non-microscopy based tests available for the laboratory diagnoses of giardiasis include recent innovations in polymerase chain reaction (PCR) and immunoassays with increased sensitivity. The laboratory diagnosis of giardiasis is complicated by the intermittent excretion of the parasite and asymptomatic presentation that sometimes occurs with this infection. Clinicians may on occasion treat patients for giardiasis on clinical suspicion alone when diagnostic tests have failed to identify Giardia intestinalis and some of the patients do get better putting into question the performance of the diagnostic test used. At the Hospital for Tropical Diseases (HTD) in London the ova, cysts and parasite microscopy (OCP-M) is the front line test for diagnosing giardiasis. Aim: The aim of this study was to critically analyse the performance of a commercial and a published real-time PCR diagnostic tests for their potential use as front line tests for the diagnosis of giardiasis in the clinical parasitology diagnostic laboratory at the HTD. Storage conditions that will allow the best yield of Giardia intestinalis DNA from stored faecal samples were also investigated in this study. Methods: In the absence of a gold standard, a composite reference standard (CRS) of enzyme immunoassay (EIA) and rapid membrane test (RMT) was used to evaluate the performance of Primerdesign Ltd. real-time PCR kit for Giardia intestinalis (which detects only assemblages A and B subtypes) and a real-time PCR assay using Verweij et al published primers (Verweij realtime PCR) which targeted the (SSU) rRNA gene. The two tests were compared with the OCP-M test in a diagnostic accuracy study using a nonprobability sampling technique with consecutive samples. Results: The Verweij real-time PCR which targeted the (SSU) rRNA gene showed a diagnostic sensitivity of 93.4 % (95 % CI: 86.2 to 97.5%) and a specificity of 74.7% (95% CI: 63.6 to 83.8%) with a limit of detection (LOD) of < 5 cysts/ml. The Primerdesign Ltd. real-time PCR which also targeted the gdh gene showed a diagnostic sensitivity of 61.5% (95% CI: 50.8 to 71.6%) and specificity of 98.7% (95% CI: 93.2 to 99.8%) with a limit of detection (LOD) of ≤ 114 cysts/ml. Also, with a serially diluted 1 in 10 dilutions of a known concentration Giardia intestinalis DNA solution, the Verweij real-time PCR produced efficiency (E) of 96% (the slope was - 3.414) with a correlation coefficient (R2) of 0.99 and a copy number variance predominantly less than 10% (< 10%). The Primerdesign Ltd. had E = 100% (the slope was -3.342), R2 = 0.95, and a copy number variance predominantly greater than 10% (> 10%). In this study, the OCP-M missed 16.5% Giardia positive stool samples contrasted with 6.6% missed by the Verweij real-time PCR. The Verweij real-time PCR therefore showed approximately 10% increase (i.e. 16.5% - 6.6%) in detection rate over the OCP-M and with an estimated detection limit of < 5 cysts/ml of stool, it also correctly identified 70% (14/20) of the discrepant cases as true positives. OCP-M identified 10% (2/20). When sensitivities were adjusted for the Verweij real-time PCR as a result of enhancement in the detection rate of the CRS, 19.3% (94.3% - 75% = 19.3%) more positive cases were noted. The Verweij real-time PCR proved to be more robust than the OCP-M and the Primerdesign Ltd. PCR and has therefore been shown to be more suited for deployment as a first line diagnostic test than the other two index tests. Even in combination with the OCP-M, the sensitivity remained unchanged at 93.4%. With its high specificity, the Primerdesign Ltd. Giardia PCR kit may be useful for partitioning clinical history for epidemiological studies but with LOD of ≤ 114 cysts/ml of stool and R2 < 0.99 when faecal samples are involved, it will require further optimisation for use on clinical samples. Up to the end of April 2013, a literature search showed no independent evaluation of this Giardia real-time PCR kit. Storage affects molecular analyses and from the findings of this study stool samples are best stored in industrial methylated spirit and kept at 4-6°C if they are to be used for real-time PCR for Giardia intestinalis detection. Alternatively they can be stored in the freezer at -20°C without industrial methylated spirit. Samples should however be tested within three months of storage. Conclusion: The reason why some patients get better when they are treated empirically following microscopy negative results for Giardia intestinalis may be found in the fact that, in this study, the OCP-M failed to detect 16.5% of positive cases. The Verweij real-time PCR performed better than the OCP-M and showed an improvement of 10% in Giardia intestinalis detection rate. The Primerdesign Ltd. Giardia PCR kit requires further optimisation for use on clinical samples. The Verweij real-time PCR was more robust than the OCP-M and the Primerdesign Ltd. PCR and therefore is more suited for use as a first line diagnostic test with best results obtained when stool samples are first treated with industrial methylated spirit, stored in the fridge at 4-6°C and tested within three months of storage. The Verweij real-time PCR assay may be used as a standalone test for in combination with the OCP-M, there was no improvement in the 93.4% sensitivity when it was used alone. The OCP-M, however, has the advantage of identifying the presence of other parasites.

The broad intention of this thesis was to understand which factors regulate the release of serotonin and noradrenaline throughout the brain. Multiple factors, such as ion channels, transporters and neurotransmitter receptors shape the release of serotonin and noradrenaline within strict spatial and temporal windows. I was interested in how fast inhibition mediated by GABA_ARs may influence LC and DRN neuronal excitability. Therefore, within this thesis I localised distinct GABA_AR subunits to the cellular and sub-cellular compartments of neurochemically diverse cell types which comprise the networks of two major monoaminergic brain centres, the noradrenergic Locus Coeruleus (LC) and the serotonergic Dorsal Raphe Nucleus (DRN). The GABA_AR alpha1 subunit was predominantly localised to the non-principal, putative interneurons of the LC and DRN, whereas the GABA_AR alpha2 and alpha3 subunits were mainly localised to the principal monoaminergic cells. This apparent segregation suggests that the precise targeting of certain GABA_AR subunits to different cellular and sub-cellular compartments is important for shaping LC and DRN neuronal excitability, and thus the release of noradrenaline and serotonin. As these monoaminergic systems are engaged by stressor exposure (Swinny et al., 2010, Kirby et al., 2000, Kirby et al., 2007), and as they have been shown to have an important role in shaping mood (Stockmeier et al., 1998, Baumann et al., 2002), I was also interested to understand whether stressors engaged the GABAergic system to influence the release of monoamines. Moreover, I have demonstrated that a mild repeated stressor influences GABA_AR expression at the transcriptomic level, in a brain region and subunit specific manner and thus provide evidence for an important role of the GABA_AR alpha3 subunit in the processing of stressor related information via the DRN. The finding that the stress neuropeptide CRH, contacts putative inhibitory synapses of serotonergic and non-serotonergic neurons of the DRN provides further evidence for the potential role of GABAergic neurotransmission in shaping DRN neuronal excitability in response to stressors. Finally, through behavioural phenotyping I have been able to demonstrate that a stressor induced increase in GABA_AR alpha3 subunit expression in the DRN, parallels adaptive-like behavioural changes in response to a novel environment.

The effects of microbiologically influenced corrosion (MIC) can be very expensive to correct, dangerous to workers and its mechanisms are poorly understood. Understanding these processes is important so that they can be monitored and mitigated (Koch et al., 2001). It is now accepted that for the assessment of biocorrosion risks, the most powerful approach is to detect functional genes encoding the enzymes that play an important part in material deterioration (Schadt et al., 2004). The main aim of this study was to identify the microbial community present in corroded and non-corroded systems, and to detect genes that might be implicated in corrosion processes, particularly iron corrosion, so that a biochip could be designed for risk assessment of oil environments. In this thesis the microbial populations and their actives were assessed using sequencing and hybridisation techniques for three oil field sites, generating information that can help identify MIC risk. The final section of the thesis describes the development and design of functional gene probes, identified from hybridisation studies that might be included in a biochip for risk assessment in oil field environments. Microbial groups known to be involved in MIC, such as sulphate-reducing procaryota, iron-reducing bacteria, nitrate-reducing bacteria, hydrocarbon-degrading bacteria were detected, according to their 16S rRNA gene sequence, in the water injection system and production pipelines. In addition to these expected groups, sequences for Firmicutes, acetogens and methanogens were detected. Firmicutes, primarily Clostridium species, and Synergistetes sequences pre-dominated the corroded systems. Functional genes involved in biocorrosion, many of which belonged to the groups named above, were detected using the GeoChip, and a list of marker genes that can be utilised for biocorrosion monitoring has been proposed. Oligonucleotide probes for biochip development were either designed or selected from published sources. A quick and inexpensive method for probe evaluation during microarray development is described. A total of 16 probes, representing 15 genes were tested; all the probes exhibited similar hybridisation behaviour under standard conditions. The results presented in this thesis were part of an extensive EU project, BIOCOR, involving academic and industrial partners, on fundamental and applied aspects of microbial corrosion in oil field environments, which was funded to generate the knowledge needed to develop monitoring techniques for corrosion. The results presented in this thesis are the final report to the European Commission.

Background and Aims Type 2 diabetes is increasing in prevalence and is associated with a threefold increased risk of cardiovascular mortality despite management of the traditional risk factors. Novel risk factors have been hypothesised that contribute to the pathogenesis of both type 2 diabetes and atherosclerosis and include oxidative stress, inflammation and endothelial dysfunction. Thiamine has been shown to be an important cofactor in the attenuation of these novel risk factors and people with type 2 diabetes have been shown to be thiamine deficient. This study tested the hypothesis that thiamine supplementation may improve endothelial function, oxidative stress, vascular inflammation and insulin resistance in subjects with type 2 diabetes who have a high cardiovascular risk profile. Methods Subjects with type 2 diabetes underwent a randomised, double blind, placebo-­‐controlled crossover pilot study receiving 300mg daily of oral thiamine hydrochloride or placebo for eight weeks with a two week washout period. Measurements were taken for endothelial function (change in the reflective index post salbutamol using digital photoplethysmography, plasma cyclic GMP, plasma sVCAM-­‐1, urinary albumin: creatinine ratio), insulin resistance (HOMA-­‐IR), oxidative stress (glutathione ratio, CuPRAC-­BCS) and inflammation (hsCRP) at the beginning and end of treatment with both thiamine and placebo. Results 34 patients (20 male) completed the study. Mean age 61 ± 9.4 years, HbA1c 7.46 ± 0.88%, blood pressure 137/77 ± 18/9 mmHg, total cholesterol 4.01 ± 1.11 mmol/l, HDL cholesterol 1.00 ± 0.30 mmol/l, triglycerides 1.87 ± 1.39 3 mmol/l. The majority of the patients were on two or more glucose lowering therapies with 88% on metformin. Most of the patients were on other cardiovascular disease modifying medications (statins or antihypertensive agents). Treatment with thiamine demonstrated a significant increase in thiamine diphosphate levels (310 ± 82 nmol/l post thiamine vs. 178 ± 32 nmol/l post placebo, p=<0.001) but no significant difference in markers of endothelial function, insulin resistance, oxidative stress or inflammation or other metabolic markers. Conclusion In this cohort of patients treatment with 300mg per day of oral thiamine for eight weeks in well-­‐controlled type 2 diabetes at high cardiovascular risk, demonstrated no significant improvement in endothelial function, insulin resistance, oxidative stress or inflammation.

Aims The method for the confirmation of drugs of abuse for addiction testing within King’s College Hospital prior to 2008 was a labour intensive thin layer chromatography method. To replace this with a faster method more suited to future requirements, the laboratory bought a liquid chromatography system with ion trap mass spectrometric detection. The development of the routine analytical method and the implementation of this method within the laboratory using on-line solid phase extraction and Turboflow® sample preparation will allow the laboratory to operate successfully in the field of clinical drugs of abuse testing in the future. Method Analyses are performed on an ion trap mass spectrometer with an electrospray ion source following reversed phase liquid chromatography, initially using on-line solid phase extraction with a Jasco XLC® series autosampler and pump and later a Thermo Turboflow® on-line extraction method with a CTC Combi-Pal® autosampler and Agilent 1100 series liquid chromatography system. Elution of drugs and metabolites is performed with a multi-step gradient of ammonium formate buffer and acetonitrile, followed by regeneration of the extraction and analytical columns to starting conditions. Detection is achieved with a Thermo LCQ Fleet ion trap mass spectrometer with a combination of full spectrum survey scans, dedicated product ion scans, neutral loss scans and data dependent product ion scans in two analysis segments. Total run time is only 20 minutes, allowing a throughput of around 65 samples per day. Results The methods include the novel combination of the elimination of any hydrolysis step, on-line SPE or Turboflow extraction, detection of multiple drug groups, full spectrum analysis and library matching, the use of data dependent scans and ion trap mass spectrometry using MS3 and neutral loss scans. The methods developed were validated using a departmental method validation protocol and accepted for routine use. Simultaneous detection of over fifty analytes has been found possible in a range of clinically relevant drug groups, including opiates, amphetamines, methadone, propoxyphene, cocaine, ketamine and their metabolites. The use of neutral loss scans and product ion scans of phase 2 drug metabolites permits the addition of previously unidentified drugs and metabolites to the method, allowing the laboratory’s services to develop in line with requirements of the service. Quality is maintained through the use of standard operating procedures, staff training, quality control samples and external quality assessment. Conclusion Drugs of abuse testing is key for treatment and monitoring of drug addiction. The introduction of modern mass spectrometry techniques has reduced the turnaround time of routine analysis for a range of drugs and metabolites and increased the range of drugs that can be analysed. The methods introduced have revolutionised testing at King’s College Hospital and produced a method which is capable of evolving with the needs of the service to keep abreast of future requirements of the service.

Duchenne muscular dystrophy (DMD) is a hereditary, X-linked, muscle wasting disease with no known cure. It is caused by altered mechanical stability of muscle cell membranes combined with altered cell signalling and inflammatory infiltrations due the absence of the cytoskeletal protein dystrophin, the product of the DMD gene. Progressive muscle fibre degeneration combined with chronic inflammation leads to a severe functional impairment, progressive disability, and ultimately to premature death. DMD presents predominantly in skeletal muscles but brain and bone are also affected. Analyses in the mdx mouse, the most commonly used model of DMD, has led to the identification of an increased expression and function of the P2X7 purinoceptor in dystrophic muscle cells and muscles in situ. This ATP-gated receptor has been implicated in a number of human diseases that, combined with its well-known role in inflammatory cells, suggested that P2X7 upregulation might also be important for the pathogenesis of DMD. To test the role of the P2X7 purinoceptor in DMD pathogenesis and its potential as a target for treatment, two mdx/P2X7-/- double mutant mouse strains were generated and compared to the mdx mouse with respect to several critical disease parameters during an acute degenerative stage of disease. Histological, molecular, biochemical, and functional analyses revealed reductions in both muscle and non-muscle pathology in mdx/P2X7-/- mice, with significant improvements in key molecular and structural parameters. These included lower serum creatine kinase levels and decreased sarcolemma permeability to blood-born molecules, indicative of less sarcolemma damage. P2X7 ablation also resulted in increased minimum Feret’s diameter, a morphological indicator of muscle regeneration. While the fraction of muscle fibres with centralised nuclei was not significantly different, levels of myogenin, a protein indicator of muscle differentiation, were also higher in mdx/P2X7-/- mice. These changes were concomitant with an overall decreased inflammatory signature in mdx/P2X7-/- mice compared to mdx and an increase in muscle strength, in vitro. Examination of diaphragms (undergoing continuous degeneration/regeneration in the mdx mouse) from 20 month old mice also showed the increase in minimum Feret’s diameter and continued reduced inflammation in the mdx/P2X7-/- group. Aged heart tissue from these mdx/P2X7-/- also presented less inflammatory infiltrate and reduced fibrosis compared to mdx. Moreover, micro-CT analyses showed greatly reduced osteopenia in mdx/P2X7-/- bones when compared to 6 month mdx mice. Amelioration of all symptoms was proportional to the extent of receptor depletion, where single P2X7 isoform loss was less effective than the complete knockout. These observations support involvement of the P2X7 purinoceptor in the dystrophic pathology in this model of DMD and are consistent with the well-known role of this receptor in other disorders. P2X7 purinoceptors are likely to act via several different molecular pathways, for example increased Ca2+ influx that affects dystrophic cells directly as well as through reduced inflammation. Indeed, this study represents the first known analysis of an effect of P2X7 ablation on a chronic inflammatory phenotype localised to skeletal muscle. These data from the most widely used model of DMD suggest that the P2X7 receptor can be also involved in human pathology and specific receptor antagonists could be considered for targeted pharmacological intervention to delay progression of this lethal disease.

Glia express a variety of ion channels, but the precise subtypes expressed by astrocytes and oligodendrocytes has not been fully elucidated. The Kir7.1 subtype of inwardly rectifying potassium channels (K_ir) is highly expressed in retinal pigment epithelium and has been demonstrated in Purkinje neurons of the adult rat cerebellum and pyramidal neurons of the hippocampus, but it has not previously been identified in glia. Using quantitative real time PCR, an ion channel profile for the developing mouse optic nerve was constructed and K_ir7.1 was identified as one of the major ion channels present. Immunostaining revealed widespread expression of K_ir7.1 in glia and neurons in the mouse brain with the highest expression found in optic nerve oligodendrocytes. A major function of K_ir is to maintain the membrane potential of glia in the face of large ionic shifts associated with normal neuronal function and pathology. Oligodendrocytes are particularly susceptible to ischemia so the role of Kir7.1 in maintaining oligodendrocyte integrity during oxygen and glucose deprivation (ODG) in the isolated intact mouse optic nerve was examined, using the K_ir7.1 channel blocker VU590. Blockade of K_ir7.1 resulted in increased cell death of optic nerve oligodendrocytes in normoxic conditions by activating caspase -dependent apoptotic pathways and significantly augmented cell death induced by OGD. Moreover, intracellular calcium fluctuations dependent on store operated calcium entry in optic nerve glia were identified as a potential mechanism for the cellular stress induced by K_ir7.1 inhibition. The results presented within this thesis demonstrate functional expression of K_ir7.1 in glial cells, and indicate they are important in maintaining oligodendrocytic integrity in both physiological and pathological conditions.