Immunological disorders, including those of an autoimmune nature, cause chronic morbidity and disability. These conditions are not well controlled with current therapies and issues, including the lack of specificity of action, lead to a number of adverse drug effects. Thus the search for alternative immune modulating agents is a well sought after objective. Recently, the quorum sensing signal molecules, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and 2-n-heptyl-3-hydroxy-4(1H)-quinolone (PQS), isolated from Pseudomonas aeruginosa, were found to possess the ability to modulate the immune response. The immune modulatory activity of PQS suggests that an evaluation of its synthetic analogues may provide better understanding of the structural components that influence its activity and may lead to the development of novel therapeutic agents. Additionally, to allow an effective SAR study to be attempted with PQS a more efficient synthetic route has to be developed. To address these issues, the research in this thesis firstly investigates a number of synthetic routes towards the preparation of PQS and its synthetic analogues. The successful synthetic routes yielded analogues with alterations to the main structural components of PQS, namely the alkyl side chain, N-substitution, 3-hydroxyl group and substitution on, and in, the carbocyclic ring. All analogues were characterised for identity and purity, then their immune modulatory activity assessed in a concanavalin A mitogen stimulated murine cell proliferation assay and their cytotoxicity using a dye exclusion assay. Bioactivity was shown to be dependent on the length of the 2-akyl side chain, with extensions to the chain more tolerated than a reduction. The nature of the substitution at the 3-position has an influence on activity, whereas a proton on the nitrogen is not essential. The less polar and more lipophilic molecules displayed increased bioactivity. The core quinolone structure can tolerate insertion of extra heteroatoms the heterocyclic ring and substitution of chlorine on the carbocyclic ring, while retaining an acceptable level of activity. A similar trend was observed in the quinazolinone derivatives.

Cardiovascular disease is the leading cause of mortality. As blood pressure is one of the most important risk factors for cardiovascular disease, effective management of hypertension is critical in reducing this risk. In addition to high blood pressure, however, several factors have been identified as predictors of future cardiovascular events. These include high cholesterol, cigarette smoking, obesity and diabetes. Taken together, these traditional risk factors do not entirely explain the risk. Thus, many novel risk factors have been proposed for risk prediction of cardiovascular disease. Microalbuminuria is one such factor. Microalbuminuria is defined as excretion of albumin in the urine above the normal level but less than gross proteinuria. As excretion of albumin exhibits high variability due to many confounders (such as urinary tract infection and strenuous exercise), diagnosis of microalbuminuria should be ideally based on screening of multiple samples using either 24-hour urine collection or first-morning voids. Much evidence suggests that microalbuminuria is a reflection of generalised endothelial dysfunction. This is supported by the observation that microalbuminuria is strongly associated with cardiovascular disease. My main aim was to study microalbuminuria in people with hypertension attending specialist clinics. Microalbuminuria has been investigated extensively in diabetes and in patients with renal disease. However, the available information on the association of microalbuminuria with hypertension has many limitations since many studies had small sample size, restricted population or were confounded by potential misdiagnosis of microalbuminuria by the use of single samples. This has led to uncertainty about the prevalence of microalbuminuria in hypertension, where reported prevalence ranges from 4.7% to 58%, and probable underestimation of its clinical significance. I addressed these issues by conducting a series of studies in 1059 hypertensive subjects attending the Glasgow Blood Pressure Clinic or the Aberdeen Hypertension Clinic. Each patient was invited to provide an early morning urine specimen for the assessment of albuminuria. Urinary tract infection was tested using urine strips and, where positive, samples were discarded. If the first sample showed increased albumin excretion, two further samples were requested. Albuminuria (microalbuminuria or gross proteinuria) was diagnosed when two out of the three samples showed increased albuminuria. Two definitions of microalbuminuria were used in the analysis, a conventional definition with the threshold used by most therapeutic guidelines and a new definition that accounts for low excretion of albumin. All patient information was obtained from case-records. In the first study, I showed that microalbuminuria by the conventional definition was present in 9.5% of non-diabetic hypertensive subjects without renal impairment. Another 10% of this cohort had microalbuminuria by the new definition. Compared with people with normal urinary albumin, individuals with microalbuminuria by both definitions (n= 786, after excluding those with diabetes or severe renal impairment) had significantly higher blood pressure, higher pulse pressure, increased levels of inflammatory markers, poorer renal function, higher triglycerides levels and used more cardiovascular drugs. In a second study, the association of microalbuminuria with clinical characteristics was investigated. Subjects with microalbuminuria had increased prevalence of risk factors / co-morbidities such as left ventricular hypertrophy (19.2% in normoalbuminuria versus 29.7% and 34.8% for microalbuminuria by the new and the conventional definitions, respectively), ECG abnormalities and cardiovascular disease. In addition, people with microalbuminuria had higher risk scores for subsequent cardiovascular events using two risk calculators, the Framingham and the Joint British Societies equations. In a subcohort with controlled blood pressure and without co-morbidities or risk, microalbuminuria (by combining the two definitions) was found in 14%. Compared with those with normoalbuminuria, subjects with microalbuminuria had higher blood pressure, poorer renal function, higher blood glucose and higher levels of inflammatory markers although the limited sample size precluded statistical significance. In a further study, the independent association of microalbuminuria with different risk factors was evaluated using multivariate testing. Systolic blood pressure, serum creatinine, left ventricular hypertrophy and fasting triglycerides were among factors linked with microalbuminuria. The risk of microalbuminuria increased in people with poorly controlled blood pressure. I also found that microalbuminuria was associated strongly with left ventricular hypertrophy [odds ratio 1.87 (95% CI, 1.12 - 3.12) for a composite of both definitions- the combined definition] and cardiovascular abnormalities [odds ratio 1.72 (95% CI, 1.05 - 2.80) for the combined definition]. In a fourth study, the reproducibility of microalbuminuria screening was investigated. I discovered that a large proportion of people who had increased urinary albumin excretion on first sample was categorised as normoalbuminuria based on the result of multiple samples (48% at the Glasgow Clinic and 41% at the Aberdeen Clinic). This indicates that even after controlling microalbuminuria confounders, multiple testing can be recommended for more accurate diagnosis. In the final study, I demonstrated that subjects with microalbuminuria by both definitions attending the Glasgow Blood Pressure Clinic had relatively high blood pressure and pulse pressure at first visit and subsequently. This finding indicates that subjects with microalbuminuria require particularly rigorous blood pressure management to achieve blood target blood pressure. Furthermore, individuals with microalbuminuria may be at risk for cardiovascular disease greater than that in those with normoalbuminuria since the eventual blood pressure remained higher in these subjects. Together with the observations that microalbuminuria is associated with clustering of cardiovascular risk factors, my finding support the importance of even small increase in urine albumin excretion as an indicator of eventual cardiovascular disease. In conclusion, microalbuminuria is found in one-fifth of subjects with essential hypertension. Although my investigation was observational, the large sample size, the use of multiple samples and allowance for the effects of potential confounders enhances the precision of the results. Moreover, subjects involved in this study represent a real hypertension population with few restrictions. My findings support the value of microalbuminuria as a tool to identify subjects at high risk for cardiovascular disease. Before routine screening can be recommended, these observations require confirmation in clinical trials and prospective studies with long-term follow up. Linkage of the records of the patients who participated in this series of studies with national morbidity and mortality statistics offers one approach with the potential to test the clinical relevance of my findings.

Introduction The work described in this thesis looks particularly but not exclusively at two recently developed molecules which have dual enzyme inhibitor activity. Omapatrilat, a molecule which inhibits both angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP), and SLV 306 (active metabolite KC12615) a compound with both NEP and endothelin converting enzyme (ECE) inhibiting properties. Neurohumoral activation characterises the complex chronic heart failure syndrome. Clearly there is value in antagonizing neurohumoral systems likely to have detrimental effects in patients with heart failure, while simultaneously augmenting potentially desirable neurohumoral mediators. However enzyme inhibitors which act on multiple vasoactive mediators with complex interactions have unpredictable effects. Omapatrilat has received much attention following demonstration of a powerful hypotensive effect but a higher than expected incidence of angioedema in patients with hypertension or heart failure. GW660511X is another dual ACE/NEP inhibitor at an earlier stage in development. The pharmacodynamic profile of neither the ACE inhibitor activity nor the NEP inhibitor activity of GW 660511X has been fully described in humans. SLV 306 is the first orally available molecule of its kind and its NEP and ECE inhibitory properties have not previously been demonstrated in humans either in vitro or in vivo. The intention of this thesis is further characterisation of these molecules and their therapeutic potential while utilising their inhibitory properties in further investigation of the human neurohumoral system. I specifically consider the possible mediators of the impressive hypotensive effects of these molecules and of the unexpected and potentially life threatening side effects associated with them. Having demonstrated the complex neurohumoral substrate of these molecules I go on to report, for the first time in heart failure patients, the benefits of a more specific approach to neurohumoral manipulation using a recently developed renin inhibitor, aliskiren. Aliskiren has been shown to offer haemodynamic benefit in patients with hypertension but has not previously been given to patients with chronic heart failure. Methods 1) Small resistance arteries from patients (n=89) with coronary artery disease but normal left ventricular function were studied using wire myography. The vasopressor response to various neurohormones in the presence of omapatrilat, KC12615 (the active metabolite of SLV 306) and other vasoactive enzyme inhibitors is reported. 2) Following pilot studies of the pressor response to intravenous infusion of big ET-1 (n=6) and pharmacokinetic modeling of orally dosed SLV 306 in healthy volunteers (n=29), the effect of 3 doses of SLV 306 and placebo given at four separate visits one week apart, on the pressor and neurohumoral response to intravenous infusion of big endothelin-1 in healthy volunteers (n=15) is compared. 3) The effect of 2 oral doses of GW660511X and a single dose of the ACE inhibitor ramipril, given on three separate visits one week apart, on the pressor and neurohumoral response to an intravenous infusion of angiotensin I in healthy volunteers (n=16) is compared. 4) Finally, the neurohumoral and blood pressure response to aliskiren an orally active, long acting renin inhibitor is compared with placebo for one week and the ACE inhibitor ramipril for 6 weeks, in patients with left ventricular systolic dysfunction (n=27). Results 1) In patients with coronary artery disease but normal left ventricular systolic dysfunction; the vasodilator response to bradykinin was augmented by omapatrilat, KC 12615, phosphoramidon (NEP/ECE inhibitor), captopril (ACE inhibitor), and thiorphan (NEP inhibitor). Of note the augmentation is no greater with omapatrilat than captopril and in arteries taken from patients prescribed ACE inhibitor, KC 12615 does not augment the response. The vasodilator response to adrenomedullin was augmented by omapatrilat, KC 12615 and phosphoramidon. The vasoconstrictor response to angiotensin I was inhibited by omapatrilat and captopril and the vasoconstrictor response to endothelin-1 was inhibited by KC 12615 and phosphoramidon. 2) In healthy volunteers, SLV 306 caused a dose dependent attenuation of the hypertensive and reflex bradycardia response to big ET-1. There was also a dose dependent increase in ANP, big ET-1 and the ratio big ET-1: ET-1 but no increase in ET-1 following big ET-1 infusion. 3) In healthy volunteers, there was no notable change in blood pressure (pre angiotensin I infusion) and no significant inhibition of pressor response to angiotensin I following administration of GW660511X. Ramipril 10mg was associated with a reduction in blood pressure (pre angiotensin I infusion) and inhibition of the response to angiotensin I. There was significantly greater reduction in ACE activity with ramipril than GW660511X. GW660511X but not ramipril led to a dose dependent increase in plasma ANP concentration. 4) In patients with chronic heart failure, aliskiren suppressed plasma renin activity and reduced plasma angiotensin II. Ramipril in comparison caused an increase in renin activity and no change in angiotensin II. There were no significant changes in blood pressure with either treatment. Conclusion I have demonstrated the ACE and NEP inhibitory properties of omapatrilat and for the first time in humans, the ECE and NEP inhibitory properties of SLV 306, in vitro in patients with coronary artery disease but normal left ventricular dysfunction. I found no additional augmentation of bradykinin by omapatrilat or SLV 306 over and above that offered by ACE inhibition but significant augmentation by both dual inhibitors of adrenomedullin. This contradicts the suggestion that bradykinin has a role in the incidence of angioedema offers adrenomedullin as an alternative mediator. Adrenomedullin augmentation may also contribute significantly to the hypotensive effects of these molecules. I have demonstrated for the first time in humans the ECE and NEP inhibitory properties of SLV 306 in vivo. GW660511X is shown to inhibit NEP but to a much lesser extent ACE. Of note the comparison made is with full dose of a powerful pure ACE inhibitor. Any inhibition of ACE activity in contrast to the study of pure NEP inhibitors is consistent with the belief that dual inhibition offers additional benefit. Finally I have demonstrated for the first time in patients with chronic heart failure the renin inhibitor activity of aliskiren, confirming attenuation of the renin angiotensin aldosterone pathway consistently from its origin and in contrast the rise in renin activity seen with ACE inhibitors.

This study aimed to construct a database of 1500 newly diagnosed patients with epilepsy referred to the epilepsy unit at the Western Infirmary in Glasgow between 1982 and 2005. These patients commenced their first ever epilepsy treatment at the unit. The database included demographic, clinical and investigational information together with a detailed account of every drug regimen applied starting from the first AED prescribed until the last follow up appointment. Using this database, I was able to identify the efficacy and tolerability of different AEDs in relation to various demographic, clinical and pharmacological characteristics. This analysis provides a better understanding of the natural history of treated epilepsy, an informational aid for the future prescription choice of drug and/or drug combination according to different patient characteristics and facilitates the study of patients with intractable seizures from a pharmacological point of view.

It has been proposed that the use of cross-over or dose escalation designs for dose ranging studies in combination with more informative analysis could lead to a better characterisation of the dose response relationship. In example presented, the dose response relationship for the 3-hydroxy-3-methylglutaryl Coenzyme A inhibitors (HMG COA) inhibitor, simvastatin, was estimated from a cross-over study which covered the current recommended dose range (10 to 40 mg). Analysis using nonlinear mixed effects modelling approach demonstrated that the selected doses only covered 20% (70% to 90%) of the upper part of the estimated dose response relationship. It was concluded that a lower dose strength would be required to allow adjustment within the log-linear portion of the dose response relationship. The clinical implications of potential relationships between the pre-treatment cholesterol level and the model parameters were explored through prediction and simulation. On simulating the relationship between dose and the percentage of patients who would achieve reductions to below a recognised target concentration, it was found that a different set of dosages may better optimise clinical response. Where strict experimental design is invalidated by study design or restricted recruitment, the resulting data can be unbalanced and not easily analysed by standard statistical methods. In the example presented, the number and size of doses of dofetilide used to test for PK/PD differences between patients with ischaemic heart disease (ISH) and healthy volunteers were different. A population PK/PD modelling approach was implemented, and no difference between the two groups could be detected. The Cmax and peak QTc ranged were predicted to be narrower following a fixed dose regimen in comparison to a dose per kilogram regimen. However, after incorporating the PK/PD variability, this was not predicted to manifest into an overall increase in the risk of Torsades de Pointes.

Background: The transition away from secondary school is an important time for adolescents, when identity is shaped and autonomy is increased. It is likely that this period can be particularly worrisome for people with intellectual disabilities (IDs) and it is possible that these worries will have an impact on mental well-being. This study sought to shed light on the content and implications of worries during the approach to transition. Methods: Twenty-five participants with mild to moderate IDs and 27 participants without IDs, all aged 15 to 18, were recruited from schools in the West of Scotland. Participants were interviewed using a Worry Interview that had been adapted from previous research carried out with young adults with IDs. They also completed a measure of rumination and distress that related to their most salient worries. Anxiety was measured using the Glasgow Anxiety Scale for People with an Intellectual Disability (GAS-ID). Results: Content analysis of the interviews identified differences between the worries of the two groups of participants that may represent differences in life experiences. The distress that was linked to the worries was positively correlated with anxiety in both groups. The ID group were significantly more anxious than the non-ID group. Conclusions: Consideration should be given to the specific worries of adolescents in the approach to transition. Doing so may allow solutions for their concerns to be identified, thus easing distress and leading to an overall impact on mental well-being. Limitations of the study and ideas for future research are discussed.

The lipid-modifying qualities of nicotinic acid, a B3 vitamin, have been exploited for many years to prevent cardiovascular disease and its associated mortality. Despite its widespread availability and clinical use, the clinical target and precise molecular mechanism by which nicotinic acid acts remains elusive. In order to maximise nicotinic acid’s full potential as a lipid-modulating drug, overcome its related side effects and further develop novel and more potent drugs it is vital to understand its molecular mechanism of action. Fifty years on from its arrival on the market, receptors which this drug acts upon have recently been identified as the G protein-coupled ‘nicotinic acid receptors’. A family of three highly homologous receptors, HM74, HM74A and GPR81 are characterised by their differing affinities for nicotinic acid. Comparison of these homologues revealed a high degree of similarity between them, with the greatest similarity between HM74 and HM74A. The main structural difference between these receptors is the longer C-terminal tail of HM74. As the C-terminal tail of GPCRs is often implicated in receptor regulation it was hypothesised that the differences in this region may result in differential regulation of HM74 and HM74A. This hypothesis was tested by examining the regulation and desensitisation characteristics of each receptor in a heterologous expression system. Both HM74 and HM74A failed to interact with β-arrestin 2 or internalise in response to nicotinic acid. However, both receptors were phosphorylated in an agonist-dependent manner. HM74 but not HM74A was demonstrated to desensitise as result of prolonged nicotinic acid exposure. The importance of the C-terminal region was further analysed by the use of chimeric nicotinic acid receptors, in which the C-terminal tail of HM74 and HM74A were exchanged. It was shown that the C-terminal tail of HM74 may be implicated in the desensitisation characteristics of this receptor. Furthermore, it was shown that HM74 and HM74A display differential characteristics with respect to ERK1/2 phosphorylation.

Background Statins are inhibitors of the rate-limiting enzyme, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, in cholesterol biosynthesis. As such, they have been widely used in clinical practice as cholesterol lowering agents to reduce morbidity and mortality from coronary artery disease. There is evidence from clinical studies and in vitro experiments that statins have additional anti-inflammatory properties in atherosclerotic disease, which are unrelated to their lipid lowering activity. Clinical studies have previously suggested that statins might show a beneficial clinical effect in inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis. Furthermore, preliminary data obtained in models of pulmonary inflammation suggest that the effects manifest in rheumatoid patients can be achieved also in asthma. A proof of concept study was designed to test the hypothesis that atorvastatin improves asthma control and airway inflammation in adults with asthma. Methods Fifty four adults with allergic asthma were recruited to a 22-week crossover randomised controlled trial comparing the effect on asthma control and airway inflammation of oral atorvastatin 40 mg daily with that of a matched placebo. Each treatment was administered for 8 weeks separated by a 6-week washout period. The primary outcome was morning peak expiratory flow. Secondary outcomes included spirometry, asthma control questionnaire (ACQ) score, asthma quality of life questionnaire (AQLQ), provocation concentration to methacholine (PC20) and inflammatory markers: exhaled nitric oxide, sputum differential cell count, sputum supernatant and serum inflammatory markers such as interleukin-6 (IL-6), IL-5, IL-8, sICAM-1, TNF-α, leukotriene B4 (LTB4) and high sensitivity C-reactive protein (hsCRP), and blood lymphocyte proliferation. Results At 8 weeks, the change in mean morning PEF, as compared with baseline, did not differ between the atorvastatin and placebo treatment periods [mean difference -0.5 L/min, 95% CI -10.6 to 9.6, p=0.921]. No statistically significant effect of atorvastatin was seen in evening PEF, or methacholine responsiveness (PC20). Out of all spirometry results, only post-salbutamol FVC showed a statistically significant result, which was slightly lower in the atorvastatin group [treatment difference -0.1L, 95% CI -0.2 to 0.0, p=0.037]. There was also no change in ACQ or AQLQ. No change was seen in exhaled nitric oxide. The total cell counts recovered from sputum were similar after atorvastatin compared to after placebo treatment. After 8 weeks, the mean absolute and relative sputum macrophage count was significantly reduced after atorvastatin compared to placebo [mean absolute difference -44.9x104 cells, 95% CI -80.1 to -9.7, p=0.029]. There was a reciprocal increase in the relative proportion of sputum neutrophils [mean proportion difference 13.1%, 95% CI 1.8 to 24.4, p=0.025], but there were no significant changes in the absolute count of these cells or the counts and proportions of the other sputum cell phenotypes under atorvastatin treatment. The sputum concentrations of inflammatory cytokines and mediators were similar after atorvastatin compared to after placebo treatment other than LTB4 which was significantly reduced [mean difference -88.1 pg/mL, 95% CI -156.4 to -19.9, p=0.014]. No significant difference was seen in the concentration of any serum marker of inflammation between atorvastatin and placebo treatment periods. The change in hsCRP was of borderline significance [mean difference -0.65 mg/L, 95% CI -1.38 to 0.09, p=0.082], but there were no changes in sICAM-1, TNF-α, IL-5, IL-6 and IL-8. There was no significant difference in lymphocyte proliferation. The biochemical effects of atorvastatin therapy were reflected in significant reduction in concentration of serum lipids; cholesterol (mean difference -1.71 mmol/l, 95% CI -1.94 to -1.48 p<0.0001), and HDL-cholesterol (mean difference -0.14 mmol/l, 95% CI -0.26 to -0.02 p=0.026), but not triglycerides. There were significant, albeit modest, increases in mean bilirubin, AST and ALT. There was no difference in compliance, assessed by number of tablets returned and by biochemical results. There was no correlation between changes in LTB4 or IL-8 and sputum macrophage count, sputum neutrophil count, or PEF. The only correlation observed between the variables that were compared was between sputum macrophages and neutrophils. Adverse event rates were similar in patients taking atorvastatin compared with placebo. Equal numbers of patients were lost to follow-up in both arms of the study. One patient died of unrelated causes while taking the placebo medication. Conclusions There were no clinically important improvements in a range of clinical indices of asthma control after eight weeks of treatment with atorvastatin despite expected changes in serum lipids. There were however changes in airway inflammation and in particular, a reduction in the absolute sputum macrophage count after atorvastatin compared to placebo and an associated reduction in sputum LTB4 and a trend towards lower CRP. The lack of any evidence of clinical benefit of atorvastatin in allergic asthma confirms and extends the findings of a smaller randomised placebo controlled crossover trial of simvastatin in 16 subjects with asthma, which showed no change in clinical outcomes or inflammatory markers. It is unlikely that altering duration of treatment, washout period or type of statin used would have changed the outcome of the study. However, as all patients were receiving inhaled corticosteroid as part of their asthma therapy, it is possible that this may have masked any modest anti-inflammatory effects of the statin. Baseline asthma inflammation may also have been too low to show any significant improvement. Despite the postulated anti-inflammatory actions of statins, it seems that they may not be appropriate for the inflammatory phenotype associated with atopic asthma. The reduction in alveolar macrophage count found in patients with allergic asthma may however have relevance to the treatment of chronic lung diseases such as COPD in which alveolar macrophage function has been implicated in the pathogenesis.

In the last few decades, evidence has been accumulating for a role for xanthine oxidoreductase (XOR)-generated toxic reactive oxygen species (ROS) in a variety of pathological conditions that affect different organ systems. This enzyme in mammals exists in two inter-convertible forms: xanthine dehydrogenase (XDH) (the predominant intracellular form under physiological conditions) and xanthine oxidase (XO). A combination of XO and its oxidizable substrate xanthine (X) (or hypoxanthine (HX)) is widely used as a model to produce ROS and to study their effects in a variety of cell culture studies. However, the effect of the combination of XOR and the reduced nicotinamide adenine dinucleotide (NADH) in cell cultures is much less studied. NADH is another oxidizable substrate for XOR that binds to a different site on the enzyme from that of X binding. The aim of this project was to investigate some aspects of the in vitro toxicity of XOR, which might provide more insights into its in vivo toxicity. The main investigation was a comparison between the well studied X / XO and the much less studied NADH / XO toxicity models. Also, secondary studies were undertaken to investigate those aspects of X / XO toxicity where there are uncertainties about them. These studies were performed using primary cell cultures. Cell cultures are now widely used to study different diseases, and although they have their drawbacks, they have their advantages over the in vivo studies. For this project, primary cultures of cerebellar granule neurons (CGNs) were used. In the beginning, some problems were encountered with CGNs. The main problem was the immediate damage induced to the neurons (including those in the control groups) at the intervention/experiments day (i.e. day 8 or 9 after plating) by manipulating the cultures (i.e. aspirating the culture medium, adding treatment and control vehicles, and adding the restoration medium). After several months of investigation, it was serendipitously discovered that the immediate damage seen in the neurons (including those in the control groups) when they are manipulated at the experiments/intervention day was due to glutamate excitotoxicity (through activating its N-methyl-D-aspartate (NMDA) receptors). The source of glutamate was the fresh serum which is present at 10% V/V in the fresh culture medium that is added to the cultures at that day. After solving this problem, it was possible to conduct reliable experiments to investigate XO toxicity models. Regarding investigating XO toxicity, it was found that both of the X / XO and NADH / XO combinations were toxic to cultures of CGNs. However, the concentration of NADH needed to cause the toxicity was much higher than that of the other substrate, X, which is in agreement with previous cell-free experiments that showed that NADH is a much weaker substrate than X for the bovine milk XO used here. Blocking the site of X binding on XO prevented X / XO toxicity, but did not prevent NADH / XO toxicity. On the other hand, blocking the site of NADH binding prevented both X / XO and NADH /XO toxicities. Another difference between the two systems was that deactivating either superoxide or hydrogen peroxide (both are ROS) generated by XO prevented NADH / XO toxicity, whereas although deactivating hydrogen peroxide prevented X / XO toxicity, deactivating superoxide generated from this combination did not. In the NADH / XO system, an extracellular metal contaminant (likely contaminating XO powder/preparation) seemed to be involved in the toxicity. The two toxicity models were similar in the mediation of toxicity by intracellular iron ion. In X / XO toxicity, although superoxide generated extracellularly from the combination has no role in the toxicity, intracellularly produced superoxide seemed to play a role. Conclusions: 1. Culturing/experimental conditions have been optimised for viability studies in CGNs cultures. 2. The combination of NADH and XO induces damage to CGNs, where although blocking the NADH binding site prevents this damage, blocking the X binding site does not. It is feasible that the oxidation of NADH by some forms of XOR (other than the one used here) that are known to be very efficient in oxidizing NADH might produce in vivo toxicity. 3. A possibility raised by this study is that a metal (like the metal contaminant proposed to play a role in NADH / XO toxicity in this study) might contribute to XOR toxicity in vivo. 4. Intracellular superoxide often mediates XOR toxicity. 5. The results add support to many previous studies which suggested that intracellular hydroxyl radical (or a similar species) is involved in XOR toxicity.

The prolyl oligopeptidase family enzymes are a group of medically significant proteins distributed in all kingdoms of life that have several unifying structural and functional features. Members of the family hydrolyse short peptides, normally no more than 30 amino acids long. This strict substrate specificity is regulated by β-propeller domains that restrict access to the active site. Conformational changes allow access to the active site by small peptides but block access to larger, structured peptides. Mechanistic details such as these were revealed when the structure of prolyl oligopeptidase was solved by X-ray crystallography in 1998. Since then, numerous crystal structures of family members have been solved, increasing understanding of the structure-function relationships of these important enzymes. Many members of the family are either proven or potential drug targets. As such, structural characterization of these enzymes and their interactions facilitates the design of pharmaceutical compounds. With this in mind, this research was undertaken, using X-ray crystallography to further understand the interactions between particular members of the prolyl oligopeptidase family and inhibitor compounds. Four specific, potent inhibitors were visualized in complex with the active site of prolyl oligopeptidase. All four inhibitors follow a similar template which was shown to be successful when one of them advanced to phase 3 clinical trials as a treatment for Alzheimer‟s disease. The structure of oligopeptidase B from Trypanosoma brucei, which is involved in cell invasion leading to Trypanosomiasis, was solved to 2.5Å, revealing a number of interesting features and enabling various avenues of study. Finally, a plant derived glutamyl endopeptidase known to be regulated by endogenous inhibitors was overexpressed, purified to a high degree and shown to be correctly folded and active. Establishing this procedure allows for the commencement of crystal trials, as well as study by other biophysical methods.