Long-term depression is a long-lasting decrease in signalling between neurons in the central nervous system. It can be elicited via a number of mechanisms, including NMDA receptors and mGLuR receptors. NMDA-dependent LTD is caused by the endocytosis of AMPA receptors in response to stimuli, whereas mGLuR-dependent LTD acts via the action of kinases. Recent studies have demonstrated a novel form of LTD, which is dependent on serine protease action. Molecular changes in protein expression also accompany the generation of this new type of LTD, although the exact mechanism has not been determined. The aims of this current study are to determine the mechanism by which subtilisin could modulate LTD, and whether this effect was solely dependent on proteolytic action, or other factors. Subtilisin-mediated LTD was elicited in hippocampal slices of mice as an experimental model, and each slice was retained at the end of every experiment to enable protein levels to be analysed via immunoblotting. Initial experiments were carried out to evaluate the relationship between subtilisin activity and metalloprotease action, based on previous studies that demonstrated subtilisin proteolysis of the synaptic protein VAMP-1, a known target of the metalloprotease tetanus toxin. The putative link between these two proteases was tested by addition of zinc to the perfusing solution, which promotes the activity of zinc-based metalloproteases. Subtilisin however was completely unaffected by this change to extracellular levels of zinc. Experiments were also conducted in the presence of the metal ion chelators EDTA and captopril to determine the contribution of metal ions to subtilisin action. Addition of the general metal chelator EDTA failed to decrease subtilisin action, and the zinc-specific chelator captopril likewise did not display inhibitory action against subtilisin. Attempts were also made to produce a direct comparison between the action of subtilisin and tetanus toxin, however, preliminary experiments were unsuccessful as the tetanus toxin used in the experiment was unable to cause VAMP-1 proteolysis. The possibility that subtilisin action was solely dependent on their inherent serine protease function was also explored using the general serine protease inhibitor phenylmethanesulfonylfluoride (PMSF). The use of PMSF could provide a better indication of the link between LTD and subtilisin proteolysis, and also shed light on the order of protein degradation. Perfusion of PMSF-inactivated subtilisin prevented the proteolysis of proteins, and also prevented the generation of LTD and abolished the proteolytic activity of subtilisin. These results indicate a close link between the LTD- 2 inducing and proteolytic effects of subtilisin, and in conjunction with the metalloprotease experiments, suggest a serine protease basis for subtilisin-mediated LTD. These results suggested that subtilisin action was not based on metalloprotease degradation of VAMP-1 alone, but on a more general proteolytic effect involving other protein targets such as the netrin receptor Unc5H3 and the cytoskeletal protein actin. The specific role of the Unc5H3 and VAMP-1 proteins in the subtilisin process was studied using protein- specific antibodies to prevent proteolysis. Preincubation of hippocampal slices with VAMP-1 proteins did not prevent the proteolytic effects of subtilisin, nor could it prevent the generation of LTD. Preincubation of hippocampal slices with VAMP-1 antibody in the presence of either the cell permeabiliser triton, or the pore-forming chemical streptolysin also had no inhibitory effect on subtilisin action. Hippocampal slices were also preincubated with Unc5H3 antibodies to determine the importance of this protein to the activity of subtilisin; however, this did not change the proteolytic or electrophysiological effects of subtilisin. Addition of either triton or streptolysin to the preincubation solution containing Unc5H3 antibodies failed to prevent the effects of subtilisin on the hippocampal slice, a result identical to those obtained with VAMP-1 antibody preincubations. The importance of actin proteolysis in mediating subtilisin action was also assessed by addition of the actin stabiliser jasplakinolide to the aCSF used in perfusions to prevent actin degradation. Jasplakinolide was unable to prevent the onset of subtilisin-mediated LTD or its associated degradation of actin. In order to verify whether the effects of subtilisin represented a novel form of LTD, the LTD elicited by subtilisin was compared to that of more established methods of eliciting LTD, such as the mGluR agonist DHPG. Slices exposed to DHPG failed to elicit LTD, however, the GABA mimetic ethylenediamine (EDA) appeared to be a potent inducer of LTD. EDA produced a significantly smaller LTD effect in comparison to subtilisin, which was not accompanied by degradation of any proteins associated with subtilisin perfusion. It would be of interest to know whether other serine proteases could cause this LTD effect, and based on previous studies, it is known that members of the S8A subfamily and the S1A subfamily of serine proteases are capable of causing the same effects as subtilisin. To date much work has been performed on subtilisin, and other groups S8A serine proteases such as Proteinase-K. In comparison, very little is known about of the mechanism utilised by group S1A serine proteases in causing LTD. In order to gain a greater understanding of The possibility that other serine proteases could generate LTD was also investigated as part of this study, with particular emphasis on members of the S8A (to which subtilisin belongs) and S1A subfamily of serine proteases, which are biochemically similar to 3 members of the S8A subfamily. Previous studies have shown that proteinase-K and cadeprin, both members of the S8A, as well as α-chymotrypsin, a member of the S1A subfamily, can replicate this LTD effect. By studying the impact of α-chymotrypsin on fEPSP and protein degradation, it was hoped that this would provide a much more balanced view on the mechanisms behind the LTD-inducing effects of serine proteases. Previous experiments with subtilisin had ruled out the action of mGluRs or the role of electrically-stimulated LTD as the basis for subtilisin-mediated LTD. For this study these two factors were investigated using α-chymotrypsin in order to discover the different mechanisms utilised by these two serine proteases. Electrically-elicited LTD is known to be sensitive to the action of protein phosphatases, and in the light of this knowledge, experiments were conducted using the phosphatase inhibitors phenylarsine oxide (PAO) and sodium orthovanadate. Sodium orthovanadate did not affect the progression of α- chymotrypsin LTD; however, PAO did significantly decrease the magnitude of this type of LTD. This suggests a possible role for tyrosine kinase in this process, although more experiments would be necessary to provide a definite link as sodium orthovanadate had no effect on the action of chymotrypsin. The involvement of mGLuR in α-chymotrypsin LTD was tested using the p38 MAPK inhibitor SB203580. Perfusion of this chemical did not result in a noticeable decrease in α-chymotrypsin activities, and taken together with previous experiments in subtilisin, suggests that serine protease-mediated LTD is not solely dependent on these two pathways for their neuronal depressive actions. The results presented here strongly suggest that LTD caused by serine proteases such as subtilisin and α-chymotrypsin do not involve metalloprotease functions. Instead, LTD is closely linked with the proteolysis of a select number of proteins that are cleaved by the endogenous proteolytic functions of these serine proteases. Furthermore, serine protease- mediated LTD is not simply restricted to subtilisin alone, but can involve other members of the S8A subfamily of serine proteases such as α-chymotrypsin.

Viral respiratory infection exacerbates asthma symptoms in almost all patients with allergic asthma. Asthma symptoms in viral associated asthma exacerbation are often severe and require urgent care as well as hospitalisation. Corticosteroids are the mainstay treatment for asthma. However, they are less effective in treating virus associated asthma exacerbation. The main aim of the thesis is to determine the role of virus infection in airway allergic inflammation and then define the effects of corticosteroids in virus associated exacerbations of airway allergic inflammation. Mice sensitised and challenged with ovalbumin demonstrated most of the main features of asthma including lung cellular inflammation with eosinophilia, early phase asthmatic responses (EAR), late phase asthmatic responses (LAR), and airway hyperresponsiveness (AHR) to methacholine provocations. Treatment with either systemic (dexamethasone: DEX) or inhaled (fluticasone propionate: FP) corticosteroids in the murine ovalbumin allergic airways inflammation model attenuated inflammatory cells influx and eosinophilia, LAR, and the AHR. Influenza A (H1N1/PR8) is the most infective to mice compared to human parainfluenza virus type 3 (HPIV3), and a synthetic dsRNA, poly (I:C). Influenza infection in mice caused a significant increase of inflammatory cell influx in the airways with marked neutrophilia, and AHR. Ovalbumin challenge in the acute course of influenza infection on a murine model of allergic airways inflammation exacerbated the inflammatory cells influx, LAR, and AHR. Treatment with either DEX or FP attenuated the airway cellular inflammation, LAR, but not the AHR. Mice only infected with influenza were resistant to the corticosteroids (DEX and FP) treatment. DEX but not FP showed antiviral activity against HPIV3 and influenza A in vitro. These data suggest that influenza infection in a murine model of allergic airways inflammation exacerbates the inflammation and alters the sensitivity toward corticosteroids. It is also suggested that some elements in the influenza associated exacerbation of murine model of allergic airways inflammation are refractory or not regulated by corticosteroid treatment.

Commercially available multi-ingredient formulas are ingested by the recreationally trained population to optimise training outcomes; however, there remains no convincing evidence in regards to their effectiveness. Thus, the aim of this project was to analyse the effects of three different multi-ingredient supplements on the expected and marketed outcomes. It was hypothesised that the supplements would potentiate the desired body composition, performance and recovery outcomes as claimed. Study 1 was conducted to analyse the effects of combining a 12 weeks resistance training programme with the ingestion of a commercially available carbohydrate-protein-creatine based multi-ingredient supplement on strength performance and body omposition in recreationally trained males. It was hypothesised that the ingestion the multi-ingredient supplement would potentiate strength performance adaptations to a greater extent than a maltodextrin placebo. As a secondary hypothesis it was expected that ingesting the multi-ingredient supplement would benefit body composition outcomes in comparison to the placebo. Thirteen healthy male subjects were assigned to either a multi-ingredient formula (n=7) or a carbohydrate placebo (n=6). Both groups ingested the multi-ingredient supplement or placebo in the morning and immediately after training. Before and after the 12 weeks progressive resistance training; percentage body fat and fat free mass were determined. Maximum strength and repetitions to failure with 60% one repetition maximum on bench press and parallel squat were also assessed before and after the resistance training period. No significant increases in any of the performance or body composition variables were observed in either group. However, larger standardised effects sizes and magnitude-based inferences demonstrated that the addition of the multi-ingredient supplement to a 12 week progressive resistance training protocol could be effective to potentiate upper body maximum strength or muscular endurance performance, but not body composition outcomes. Study 2 was undertaken in order to analyse the acute effects of a commercially available carbohydrate and caffeine gel on intermittent sprint performance in twelve recreationally trained males. It was hypothesised that the combination of carbohydrate and caffeine in gel form would attenuate fatigue and decrease perception of effort when compared to the ingestion of carbohydrate gels alone and placebo gels. A secondary hypothesis postulated that the carbohydrate and caffeine gel would maintain blood glucose levels throughout the intermittent sprint test in regards to both the carbohydrate and placebo gels. Using a cross-over design, one 70 mL dose of gel containing either, 25 g of carbohydrate with or without 100 mg of caffeine or a non-caloric placebo was ingested on three occasions: one hour before, immediately prior and during the intermittent repeated sprint test. Blood glucose, rating of perceived exertion and fatigue index were analysed. The main finding was that ingesting the carbohydrate and caffeine gel one hour before, prior to and during an IST is effective at transiently reducing fatigue and RPE whilst maintaining higher glucose levels at the final stages of the exercise. Study 3 was conducted in order to analyse the acute effects of a commercially available carbohydrate and protein based multi-ingredient recovery formula on the recovery process and muscle damage, in 10 recreationally trained males, after performing a bout of intermittent sprint exercise. It was hypothesised that the ingestion of a carbohydrate and protein based multi-ingredient supplement, before, during and after an acute bout of intermittent repeated sprint exercise would promote recovery estimated through the attenuation of neuromuscular fatigue and markers of muscle damage respect to the ingestion of carbohydrate only or a low caloric placebo. As a secondary hypothesis the ingestion of the multi nutrient formula would attenuate a decline in sprint performance during the intermittent sprint test when compared to the carbohydrate and placebo conditions. Using a cross-over design, one 500 mL dose of a multi-ingredient recovery beverage, a carbohydrate beverage or a placebo beverage was divided in to 4 equal servings of 125 mL and ingested before each of the 4 blocks of the intermittent sprint test. A second full serving was ingested with 20 minutes of completing the intermittent sprint test. 15m sprint times, creatine kinase, myoglobin, and interleukin-6 were assessed before (pre), immediately post (post), 1 hour and 24 hour after exercise. Total sprint time measured during the intermittent protocol was not different between conditions. 15m sprint time was slower at post, 1 hour and 24 hour compared to pre without differences between conditions. Creatine kinase at 24 hour was lower in the multi-ingredient compared to both carbohydrate and placebo. Myoglobin increased in all three conditions at post, and 1 hour compared to pre, showing lower values at 1 hour for the carbohydrate and approached significance (p=0.060) for multi-ingredient compared to placebo condition. Interleukin-6 increased at both post and 1 hour compared to pre with no differences between conditions. Thus demonstrating, the ingestion of a multi-ingredient supplement before, during and immediately after a 90 min intermittent sprint test resulted in no effects on performance and fatigue. However, the accumulation of some biomarkers of muscle damage could be attenuated. It would appear that manufacturers make use of the research available to formulate multi-ingredient supplements as the supplements that showed efficacy in studies 1 and 2 possess the most established research. Whereas investigations and knowledge in to the acute effects of a carbohydrate-protein based multi-ingredient supplement on recovery from an intermittent sprint test (study 3) still need to be determined.

This thesis reports an ethnomethodological, conversation analytic study of communication between stroke patients and physiotherapists. The study sought to describe and explicate patterns of conduct by which therapists and patients communicate about treatment activities during therapy sessions. Analysis included a comparison between practices observed in the data and current published recommendations for good practice. The data consist of 74 treatment sessions that were video-recorded in four English hospitals. The 21 patient participants were undergoing inpatient rehabilitation for stroke. Most were recorded on four occasions over a two-week period. Their disabilities varied, but all could speak and understand at least short sentences in English. Each of the ten therapist participants was employed at senior level and used treatment approaches that are prevalent in the UK. Analysis involved repeated viewing of data and transcription of talk and body movement. It focused on three areas that emerged as central to physiotherapy interactions: The nature of treatment activities and of participation in them Achievement (success and failure) in these activities Reasons, goals and purposes underlying them Consistent with conversation analytic studies in other settings, we found that each communication practice in physiotherapy has a range of interactional effects, and that these are locally constructed and accomplished. Therefore, rather than generating „blanket prescriptions‟ about „good‟ and „bad‟ interactional practices, our study contributes to enhancing practitioners‟ understanding of the contingencies and underlying orientations that shape communication conduct, and raising their awareness of the effects of different means of achieving various interactional tasks in physiotherapy. We argue that these understandings can contribute to improvements in the practice and training of physiotherapy communication. Our study contributes to ethnomethodological and conversation analytic knowledge regarding methodological strategies for researching lay professional interactions, and to sociological understandings about the organisation of conduct in clinical interactions, particularly the role of orientations to managing physical incompetence and its implications.

Cytotoxic T lymphocytes are able to identify malignant cells by scanning for aberrant peptides presented on cell surface human leukocyte antigen (HLA) Class I molecules by virtue of an antigen binding receptor called the T-cell receptor (TCR). Peptides presented by HLA Class I complexes represent the largest array of tumour associated antigens (TAAs) and are therefore ideal targets for immunotherapeutic reagents. Cancer patients frequently mount T-cell responses to tumour-specific antigens, but these are in most cases ineffective at clearing the tumour. This is in part due to the low affinity of TCRs for self-antigens coupled with low-level expression of target peptides on the surface of cancer cells. To harness the exquisite antigen recognition property of TCRs for use as potential therapeutic proteins, the principal goal of this thesis was to generate ultra-high affinity TCRs against three clinically relevant HLA Class I melanoma-specific epitopes, including peptides derived from Melan-A/MART-1(26-35), gp100(280-288) and MAGE-A3(168-176). TCRs are membrane-bound disulphide (ds)-linked heterodimers consisting of an alpha and a beta chain. Each chain comprises three hypervariable or complementarity-determining region (CDR) loops, which assemble to form the antigen binding domains. As a general rule the CDR3 loops, and to a lesser extent the CDR1 loops, contact the peptide bound in the HLA groove and as such specificity is largely attributable to the CDR3 loops. The remaining CDR loops interact with the HLA surface and not the bound peptide. Each CDR loop was mutagenised using degenerative NNK oligonucleotides and expressed on the surface of bacteriophage as fusions to the phage coat protein pIII. Through a Darwinian process of in vitro evolution using pHLA ligand as the target molecule, mutated TCRs with improved affinity for pHLA were identified. TCRs engineered by phage display were produced as soluble ds-linked proteins and the contribution to affinity of each mutated CDR was measured by surface plasmon resonance (SPR). Using a combinatorial strategy, individual mutated CDRs were spliced into the same TCR molecule in a stepwise manner to further increase binding affinity. The final combination of mutated CDRs was shown to bind their cognate pHLA antigen with substantially improved KD values of 18 pM (Melan-A/MART-1(26-35)), 11 pM (gp100(280-288)) and 58 pM (MAGE-A3(168-176)), representing an increase over the wild-type TCR of approximately 1.8 million-fold, 1.7 million-fold and 3.7 million-fold respectively. In addition, having discovered an off-target binding profile for the high affinity MAGE-A3 TCR, the phage display methodologies were explored to 5 reestablish the specificity of this molecule. These results are significant because this has provided a platform on which, for the first time, to make TCR-based therapeutics. For example, the affinity enhanced gp100 TCR is currently undergoing clinical evaluation in a Phase I/II trial.

Interparticulate interactions play a significant role in determining the downstream behaviours of all pharmaceutical formulations and are therefore essential considerations when approaching formulation design. Inhalation product formulation in particular is inherently bound to an understanding of these forces. Delivery of drugs to the lower airways to treat conditions like asthma and COPD requires a particle size of below 5 micron. This implicitly demands micronization of the active pharmaceutical ingredients (APls) and this process renders many particles of large surface area with high surface energies and an auto-adhesive tendency. There is therefore a concurrent reduction in the flowability and dispersion properties of these systems. The interactive character predisposes agglomeration, flocculation or device retention and will compromise manufacture, stability, device function, and the aerosolization behavior of a formulation. Ultimately the ability of any aerosolized API to reach the deep airways is dependent upon adhesion force dynamics. As such, an appreciation of the forces of attraction and scale of particulate interactions within inhaler technology is critical if a successful drug delivery device is to be realized. The advancement of the atomic force microscope (AFM) as a force probing apparatus, has meant that it is now possible to measure the force of adhesion between two particles of interest. However these measurements could not easily be compared, because there is no simple means to account for differences in the contact regime (geometrics) between measurements. However, the development of the cohesive adhesive balance (CAB) approach by Begat, Morton, Stainforth and Price in 2004 has offered a means to negate this limitation. Using a colloidal probe microscopy (CPM) derived technique a particle of a selected material of interest (API, carrier molecule etc.) is attached to an AFM cantilever and ramped onto and off the surface of another material of interest (adhesion measurement), and to a surface of the same material as the tip (cohesion measurement). By graphically plotting the adhesive force values of a series of tips, as a function of the cohesive force values of the same tips, a representation of the relative particle interaction can be obtained. Quantitative information regarding the adhesive/cohesive nature of the interaction can then be extracted from the graph and a description of the interaction formulated that can be compared to other material combinations. The CAB work carried out to date has used recrystallized model substrates. These molecularly flat surfaces ensured there would be no difference between the contact geometry of a functionalised AFM probe and the adhesive and cohesive surfaces of the study respectively. In this fashion the only variable between the two measurements would be the chemical interactivity, and not the interactive surface area. However while using such methodology guarantees the validity of the approach, it is not necessarily a true representation of the materials 'in-situ' and requires more complex sample preparation and complex experimental design. For a variety of reasons this can be misleading in its own right. This thesis details the .investigation into the application of an adapted CAB approach in characterizing the force balance between APls for inhalation in their real state. In so doing, the aim was to see whether such a CAB would offer a quicker and simpler, yet relevant and informative assessment of a drug system force balance. It was hoped that said force balance could in turn be associated with a measurable impact upon the formulation performance of the characterised ingredients as measured 'in-vitro'. This interest was particularly directed at the lesser characterized pressurized metered dose inhaler (pMOI) systems. While these formulations are solvent based, it was of interest to identify whether a simple API to API challenge could infer a descriptive balance that could link to 'in-vitro' performance. Furthermore there was interest in evaluating the use of a range of surface specific imaging techniques to analyse the deposition dynamics of the combination formulations. It was hoped that by doing so, the localisation of the individual components within the binary deposits could again be associated back to the force balance of that system, and that an appreciation of the capability of the techniques involved would be gained. The work that follows therefore commences with the evaluation and description of the capacity for the CAB approach to be adapted to measure force relationships between real beclomethasone dipropionate (BOP) particles and pMDI component surfaces. From this assessment it was found that even with relatively smooth substrates, the combination of bulky functional particles and the inherent substrate roughness caused a critical failure in the CAB model. The parity between cohesive and adhesive geometries of contact was excessively stretched, leading to a loss of force normalisation which was reflected in uncorrelated CAB plots. As a consequence little could be confidently gleaned from the force data acquired, although there was the suggestion that the use of a fluorinated ethylene proplylene (FEP) coating reduced the adhesive interaction between the APls and the pMDI canister wall. This was then followed by an attempt to find a compromise between the model crystal substrates of a pure CAB process and the real particle morphologies that had caused the CAB model to fail. Using a compression process to form API powder compacts, in conjunction with small and discreet functional particles, a confident CAB was achieved for two combinations of APls selected on the basis of surface energy and physical stability analysis. Salbutamol sulphate was characterised with beclomethasone dipropionate, and salmeterol xinafoate with fluticasone propionate. Both combinations showed CAB plots with a sufficiently strong linear regression analysis to suggest a broad accuracy of force balance assessment. Both beta2-agonists showed cohesively dominated relationships with respect to the paired glucocortiocoids, while in reverse both glucocorticoids showed adhesively dominated relationships with the beta2-agonists. There was concern raised over the compression process of the powder discs, and its impact on the physicochemical state of the APls, with some thermodynamic evidence of polymorphic changes that required further work. The next chapter looks at the 'in-vitro' deposition performance of the two API combinations from a HFA134a pMDI system by analysis in an Andersen Cascade Impactor (ACI). The coformulation of salmeterol with fluticasone induced an improvement in the fine particle performance of fluticasone, with a concurrent decrease in the fine particle performance of salmeterol. This impact was hypothesised to be related to alterations in the structure and strength of particle-particle agglomerates. The impact on deposition performance of coformulating beclomethasone and salbutamol was unclear, as a critical unexplained loss of beclomethasone by total recovered mass was seen from all beclomethasone containing formulations. This instability of beclomethasone within the HFA134a system, precluded an accurate assessment of a direct impact on salbutamol deposition. The final chapter, compared a range of surface specific imaging techniques, including scanning electron microscopy (SEM), desorption electrospray ionization mass spectrometry (DESI), Raman spectrometry and time-of-flight secondary ion mass spectrometry (ToF-SIMS) in assessing the extent and nature of 'in-vitro' co-deposition from the salmeterol and fluticasone pMDI formulations. It was apparent that the deposition of the two APls on ACI plates was not likely to be directly comparable assessment of the incidence of particle co-deposition 'in-vivo' due to the forced nature of nozzle directed impaction. However the combination of techniques employed produced a wealth of physical and chemical data that did suggest that the two APls showed extensive co-ordination 'in-vitro'. Raman spectroscopy was able to identify individual particle character and showed frequent salmeterol and fluticasone particle combinations, but suffered from exceptionally long run times and anomalies from photoreactive surface elements. The use of a multivariate approach to ToF-SIMs analysis defined the strong co-association of the two APls, although could not differentiate particle to particle deposition. Multivariate curve resolution (MeR) was used and produced distinct components that segregated ions from both APIS from the background plate but not from each other. SEM imaging was able to define the morphologies of the deposited particles, but was unable to differentiate the two. DES I imaging showed the presence of the two APls together within several drug spots, but could not be used to investigate individual drug spots, and the distribution within, due to inadequate spatial resolution and differences in desorption efficacy. While the co-association of the two APls was observed, the lack of a comparator in another combination of APls made the link between deposition performance and force balance purely descriptive. It was unclear as to whether the force balance of the system lends itself to a particular increase in co-deposition behaviour. However it was apparent that the analytical techniques employed all had respective strengths and weaknesses as mapping tools, and with further work with other formulations could be used to provide a tailored formulation screening process, if subsequent links to force balances could be made.

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.