BACKGROUND Arrhythmia is a cardiovascular disorder which can lead to several complications. Over the past decade the introduction of many new drugs has raised concerns about their questionable benefits and cost-effectiveness. Classification of antiarrhythmic drugs has not been fully resolved. Although numerous clinical trials have been conducted, the value of antiarrhythmic drugs in many indications remains controversial. Two meta-analyses of clinical trials addressing the indication of quinidine (Class I) for maintenance of sinus rhythm after cardioversion have suggested high efficacy rates but increased mortality relative to placebo. Several overviews which were conducted to evaluate the impact of antiarrhythmic therapy on improving survival post acute myocardial infarction, have defined a turning point in the management strategy from Class I to Class III drugs, particularly amiodarone and sotalol, due to the unfavourable mortality outcome with the former Class. MAJOR AIMS This thesis was conducted with three major aims: 1) To assess both qualitatively and quantitatively the benefits and risks associated with flecainide (Class Ic), amiodarone (Class III), and sotalol (Class III & II) in treatment of chronic atrial fibrillation, acute medical or surgical supraventricular arrhythmias, and life-threatening ventricular arrhythmias developing post acute myocardial infarction; 2) To produce an overall summary estimate of effectiveness and probabilities of incidence of adverse effects, which can be useful for subsequent incorporation in cost-effectiveness analysis; 3) To validate the usefulness of various therapeutic outcomes implemented by general treatment guidelines. OVERVIEW OF THESIS A meta-analysis was carried out to compare the efficacy and safety of three antiarrhythmic agents (flecainide, sotalol, and amiodarone) in maintaining sinus rhythm after cardioversion of chronic atrial fibrillation. 42 of 119 clinical trials retrieved satisfied the predefined inclusion criteria. Data from 17 amiodarone trials (5 randomised, and 12 uncontrolled), 8 sotalol trials (6 randomised, and 2 nonrandomised), and 19 flecainide trials (8 randomised, 4 nonrandomised controlled, and 6 uncontrolled) were pooled separately after testing for homogeneity of treatment effect across the trials. Although the pooled rate difference in proportion of patients remaining in sinus rhythm between amiodarone and placebo (2 trials) was statistically nonsignificant (RD3mon = 16.1 %, 95% CI = -29.7 to 61.7, P>0.05), the pooled effect compared to Class IA drugs (3 trials) demonstrated significant differences at all time intervals (RDs were 20.5%, 31 %, and 28.8% at 3, 6, and 12 months respectively). Aggregating sotalol efficacy data in randomised or nonrandomised controlled trials has yielded highly significant effect in favour of sotalol as compared to placebo and equal effect as compared to Class IA and Class IC at all time points. Furthermore, comparison of flecainide to placebo or Class IA has revealed a highly superior effect in favour of flecainide. The calculated summary statistics (ORpeto, ORMH, RD, and RR) for the incidence of mortality and pro arrhythmia in the full-exposure group in amiodarone and sotalol trials were not significant, affirming the safety of those two drugs. In flecainide placebo-controlled trials, the ORMH for mortality and proarrhythmia were 1.8 (95% CI, 1.2-2.7, P=0.002), and (95% CI, 4.23-10.6, P<0.00l) respectively, thus indicating low benefit-risk ratio for flecainide as compared to amiodarone. The validity of this meta-analysis was examined by assessment of publication bias using funnel-plots. A funnel-plot of the amiodarone clinical trials displayed the shape of an 'inverted funnel', thus suggesting an evidence of low retrieval bias. However, due to the small sample size identified (18 trials only), a firm conclusion with regard to absence of publication bias could not be drawn. Evolving strategies for management of newly occurring supraventricular arrhythmias were reviewed. A meta-analysis was undertaken to determine the most effective agent for prompt cardioversion to sinus rhythm. Flecainide efficacy relative to placebo was confirmed by pooling data from 5 placebo-controlled trials (OR3hrs, 7.2; 95% CI, 4.7 to 11.1; Z=8.9; and OR8hrs, 5.5; 95% CI, 3.6 to 8.4; Z=7.85). However, pooling the data from three amiodarone, placebo-controlled trials at 3 and 8 hour-intervals demonstrated a nonsignificant effect (OR3hrs, 1.3; 95% CI, 0.7-2.4; Z=0.85; and OR8hrs, 1.03; 95% CI, 0.6-1.8, Z=0.12). All individual odds ratios for intravenous sotalol compared to placebo were highly significant with pooled OR at 1 hour of 8.8 (95% CI, 4.7-16.5; Z=6.8). The effect sizes of the three agents on mean ventricular response rate was estimated for both converted and unconverted patients. Whilst the effect size of flecainide versus placebo was not statistically significant at any time point, those of sotalol and amiodarone were statistically and clinically meaningful for both converted and unconverted patients. It is suggested that for acute cardioversion, intravenous flecainide or sotalol should be initially implemented. Intravenous amiodarone can be subsequently introduced for controlling the ventricular rate in persistent unconverted patients. Recent meta-analyses of randomised controlled trials of secondary prevention of myocardial infarction by antiarrhythmic agents have questioned the validity of using arrhythmia suppression as a substitutive end point for mortality. A meta-analysis examining the effect of sotalol and amiodarone for prevention of death post acute myocardial infarction was undertaken. In addition to single point estimates of pooled odds ratios of total mortality and sudden death, a meta-analysis of survival data which included censored end points was employed. An attempt was made to reconstruct the life tables in individual trials of amiodarone. The Kaplan-Meier percentages were recalculated and pooled at specific time points to reproduce the final meta-analytic survival curves of total mortality and sudden death. The meta-analysis confirmed the clinical efficacy of amiodarone for prolonging the survival in patients with congestive heart failure or myocardial infarction. The nonparametric log-rank odds ratio method was applied to raw actuarial data deduced from published Kaplan-Meier graphs as well as data generated by curve fitting. Pooling each set of data separately has yielded highly significant log-rank ORs for total mortality in the first set of four trials with censoring (log-rank OR at 102 months, 0.598; 95% CI, 0.43 to 0.83; Z = -3). However, log-rank ORs from data generated by curve fitting of data from a further three trials, were nonsignificant up to 48 months (log-rank OR, 0.87; 95% CI, 0.72 to 1.06, Z = -1.4). Merging of the two data sets has suggested strong evidence of efficacy for improving survival in terms of both total mortality and sudden death.

Professional practice guidelines direct health care professionals to include patients in the decision-making process and to establish collaboration for therapeutic goal setting. Currently, little is known about the interaction between patients and professionals during this process. The aim of this study is to shed light on goal setting practices in physiotherapy. Twenty-eight consenting patients seeking physiotherapy for their musculoskeletal problems and their therapists were videotaped during three consecutive sessions. Sequences related to goal setting were selected, and Conversation Analysis was chosen to analyse patient-therapist interactions. The data comprise fifteen episodes in which therapists enquire explicitly about goals. Findings show that two assumptions underlie these enquiries: a) that patients have a goal in mind, and b) that they are able to articulate it. My data indicate that this is not straightforwardly the case in practice. Patients orient in their responses to epistemic dimensions related to issues of whether they have access to this knowledge, and whether they treat themselves as entitled to know about goals. When patients respond to therapists’ enquiries, they use a variety of interactional resources to convey their epistemic orientation. I further found that therapists use different strategies for following-up patients’ responses: these have different implications for patients’ continued talk. My analysis also shows that a goal can only be treated as acceptable by therapists when it is amenable to improvement by physiotherapy. My study indicates that the process of goal setting is not as straightforward as policy documents suggest. In actual practice it requires addressing and managing underlying assumptions and epistemic dimensions. A better comprehension of the interaction between physiotherapists and patients will contribute to better understand the limitations of current goal setting theory, and how and why current policies on goal setting may not have the desired effect.

The endocannabinoid system (ECS) comprises the endocannabinoids (ECs), including anandamide (AEA) and 2-arachidonoyl glycerol (2AG), which interact with the G protein-coupled type-1 and type-2 cannabinoid receptors(CB1 and CB2 respectively). The ECS is thought to have a role in a number of central processes including neuroinflammation, neurogenesis, neuroprotection, learning and memory. Due to its influence on a diverse number of processes, it has been suggested that modifying the ECS may be therapeutically beneficial in Alzheimer's disease (AD). AD is an age-related neurodegenerative disorder characterised by the presence of extracellular amyloid beta (Ab) plaques and intracellular neurofibrillary tangles (NFTs) resulting in impairments in learning in memory. The aim of this thesis was to determine the status of the brain ECS in the APPswe/PS18E9 mouse model of AD and wild type littermates at 4, 6 and 8 months of age and the performance of these animals in a behavioural test battery. The results of this study indicated that APPswe/PS18E9 animals were hyperactive compared to their wildtype counterparts at all ages and that they also displayed deficits in behavioural flexibility. EC levels increased with age in both wild type and APPswe/PS18E9 mice. Cannabinoid receptor coupling was increased in the frontal cortex and striatum of APPswe/PS18E9 mice relative to wildtype. This study concluded that the status of the brain ECS is altered in AD. Modifications to the performance of the ECS were made in the form of chronic administration of a CB1 receptor antagonist (SR141716A1rimonabant) and a CB2 receptor agonist (JWH133). Chronic administration of SR141716A was able to reverse some learning impairments in APPswe/PS18E9 animals. In contrast, chronic administration of JWH133 resulted in impaired memory extinction in both wildtype and APPswe/PS18E9 mice. The results support the potential benefit of modulating the endocannabinoid system in the treatment of memory impairment in AD.

The purine nucleoside adenosine is considered an important endogenous anticonvulsant, which exerts its actions via adenosine receptors. Adenosine can be released per se, or as ATP, which is then broken down to adenosine via the action of extracellularly located ectonucleotidases. ATP is a signalling molecule in its own right, which can activate ionotropic P2X and metabotropic P2Y receptors. While the role of adenosine and its receptors in epilepsy is well established, little data is known about the role of the P2 receptors. The aim of this work was threefold: (i) to what extent the P2 receptors modulate seizure-like activity in vitro, (ii) to detect the release of ATP and/or adenosine during seizure-like activity, and (iii) to establish the cellular source of the purines released. To do so, I used two NMDA receptor dependent models of electrically (high-frequency stimulation in Mg2+-free medium)- or chemically (6 mM K+ in Mg2+-free medium)- induced seizure-like events (SLE), and a model of bursting based on the activation of group I metabotropic glutamate receptors (GI mGluR). In my NMDA receptor-dependent models, I show that some P2 receptors partially aggravate SLEs, but their contribution is dwarfed compared to the powerful action of the adenosine A1 receptors. Accordingly, the minimal contribution of P2 receptors was reflected in my inability to detect ATP using microelectrode biosensors, despite my attempts to boost the amount of extracellular ATP using two ecto-ATPase inhibitors. GI mGluR-dependent bursting was partially blocked by the P2Y1 receptor antagonist MRS2179. Biosensor data revealed small, CA3 regionspecific ATP release. In contrast, larger quantities of adenosine were detected in all models used. Work with mice modified to express different levels of adenosine kinase (ADK) revealed that ADK plays an important role in regulating the amount of extracellular adenosine and seizure parameters. Furthermore, dn SNARE mice, in which astrocytic vesicular release of purines is selectively blocked, showed small amounts of SLE-related adenosine produced. My data suggest that P2 receptors partially contribute to seizure activity. Furthermore, I have confirmed the strong anticonvulsive action of adenosine, which is likely released from astrocytes, and tightly regulated by ADK. Thus, work contained in this thesis will hopefully contribute to the on-going attempts to establish adenosine-based epilepsy therapies.

Broad-Spectrum Chemokine Inhibitors (BSCIs) are a novel type of anti-inflammatory drug, discovered by Fox and colleagues, which act through the receptor SSTR2. Chapter 1 consists of the story of the development of current BSCIs and a literature review of existing SSTR2 ligands. In Chapter 2 a series of receptor probes were synthesised based on existing SSTR2 ligands, BSCIs and a hybrid of the two. Biological data were gained determining their SSTR2 binding ability and their extent of leukocyte migration inhibition. In Chapter 3 a series of small molecules were synthesised based on the structure of highly potent BSCIs. Once again biological data were gained determining their SSTR2 binding ability and their extent of leukocyte migration inhibition. In Chapter 4 a series of BSCIs were synthesised which contained substituted aromatic groups using an iron-cross coupling reaction. Biological data were gained to determine these compounds SSTR2 binding ability. Further iron cross-coupling reactions were carried out to determine the scope of these reactions and their applications in medicinal synthetic chemistry. This works has gained evidence to support a split binding site theory for SSTR2. Somatostatin and BSCIs bind in slightly different area of the binding site, and through functional selectivity somatostatin structural analogues can exert an anti-inflammatory effect while somatostatin does not.

Since the discovery of cisplatin, numerous attempts have been made to emulate its activity while reducing its collateral toxicity. Coordination complexes based on a wide number of transition metals have been developed in the search for improved bioavailability, selectivity and reduced adverse side-effects. Ruthenium(II) complexes have been widely developed in this field as a viable alternative to platinum chemotherapeutics. This thesis is concerned with the synthesis, characterization and biological evaluation of three series of novel half-sandwich complexes of the general formula [RuII(arene)(X)(YZ)]n+. These piano-stool RuII complexes have been designed as to allow the fine-tuning of their chemical and biological properties. In the first two series, the arene unit has been varied between p-cymene, biphenyl and terphenyl to investigate the correlation between hydrophobicity and antiproliferative activity, while the N,N-imino pyridine chelating ligand, YZ, has been modified to include either a higher number of aromatic units that could allow better DNA intercalation or substituent groups that could affect the overall charge distribution in the complex. Finally, the monodentate ligand, X, is either chloride or iodide. These compounds have been fully characterised by NMR, MS and elemental analysis. Their aqueous behaviour has been investigated together with the extent of 9-EtG binding, as an indication of the possible interaction with nucleobases. The antiproliferative activity of these novel RuII complexes was determined, several of them show promising IC50 values, in the low μM range, against ovarian, colon, lung and breast cancer cell lines, in many cases the activities observed are better than cisplatin. The pathways for cellular accumulation were investigated. Complexes with an I as the monodentate ligand, X, exhibit partial energy-independent uptake. Overall results indicate that the novel RuII complexes synthesised in this thesis are most likely to be multi-targeted and that their mechanism of action depends to a great extent on the nature of the monodentate ligand, X. Two particularly active complexes in these series include the impy-NMe2 ligand as YZ chelate. These have been compared to their isostructural azopyridine analogues and also to their OsII equivalents. In this case, experiments were designed to study the activation of landmark events that lead to apoptosis, allowing contrasting the effects of different metal centres (Ru vs Os), isoelectronic ligands (impy-NMe2 vs azpy-NMe2) and monodentate ligands (Cl vs I). Results indicate that the molecular pathway followed by the iodido complexes is p53-independent. In comparison, the chlorido analogues activate the intrinsic apoptotic pathway and their activity relies on the existence of this tumour suppressor. DNA intercalation was also evaluated as a possible mechanism of action. Finally, the third series includes inactive RuII complexes with tetrahydroquinoline derivatives, which were found to enhance the activity of platinum drugs in clinical use. These promising preliminary results in the use of RuII complexes in combination therapy open a world of possibilities for the dose-reduction of platinum-chemotherapeutics.

The interplay between protein structure, flexibility, mobility and function is a central concern in structural biology. Here, we have studied the interactions of two different proteins, HIV-1 protease and cyclophilin A (CypA), with a variety of ligands. HIV-1 protease is a key modern drug target due to its role in the lifecycle of the virus HIV-1. CypA is a multifunctional protein which acts principally as an enzyme during protein folding, but also as the primary binding partner for the immunosuppressant drug cyclosporin A (CsA). Using a computational approach we have studied the manner in which different drugs affect the flexibility of HIV-1 protease. We have used experimental and computational approaches to investigate the binding effect of CsA on the structure, flexibility and mobility of CypA. The wealth of structural data available, particularly from X ray crystallography studies, creates an opportunity for computational scientists to provide data on flexibility and mobility in order to augment the structural data and inform future experiments. We use rapid computational tools to model the flexibility (first) and mobility (froda) with a variety of structural data as input. We characterised, with first, the effect of ligand binding on the rigidity of more than 200 X-ray crystal structures of HIV-1 protease. A similar approach has been applied to the structural dataset available for CypA. In addition, we have used froda to simulate the mobility of the protein. A new procedure, of tracking surface exposure during large-scale simulations of protein motion and combining the information with rigidity analysis data, was used to try to predict results of hydrogen-deuterium exchange NMR experiments (HDX). Experimentally, we have characterised CypA and its binding with CsA and subsequently performed NMR spectroscopy including HDX on both the unliganded CypA and the CypA-CsA complex. Small changes in chemical shift and the HDX folding core were observed upon ligand binding. This is the first study of the CypA-CsA complex using HDX. Our method for predicting the results of HDX for CypA is an improvement on the first-only approach, but we find that it is not yet sufficiently sensitive to predict the effects of ligand binding on CypA using these experiments. It is hoped that this work will contribute to the general understanding of ligand interactions, the particular interactions involving HIV-1 protease and CypA, and the applications of computational simulation using rigidity analysis and large-scale coarse-grained motion.

Clostridium difficile is the most common cause of hospital acquired infections. While the current treatments of choice, antibiotics, are generally effective in promoting recovery ,the increased incidence of C. difficile infections and treatment failure associated with antibiotic resistance combined with the emergence of hypervirulent strains highlights the need to develop therapeutic approaches that specifically target the pathogen without causing collateral damage to the protective microbiota. Several non-antibiotic approaches are currently being investigated, such as bacteriophage therapy. For this reason, we attempted to isolate C. difficile specific lytic bacteriophages which could form the basis of a treatment for C. difficile. While we were unable to isolate lytic phages, we were able to isolate twelve temperate bacteriophages from twenty-three clinical isolates of C. difficile using mitomycin C. Unfortunately we failed to identify a susceptible host strain capable of supporting the replication of these phages. This failure may in part be due to repeated episodes of phage infection over time, which have resulted in the emergence of “phage resistant” species mediated by systems such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). Employing a PCR-based approach using primers specific for the lysin genes of five previously isolated C. difficile phages, we found evidence to suggest that repeated bacteriophage infection is a common event for clinical isolates of C. difficile. Our inability to isolate a lytic bacteriophage prompted us to adopt an alternative approach in which we used endolysin enzyme of five previously identified C. difficile phages as recombinant protein. These lysins showed broad spectrum activity against the vegetative forms of a large collection of C. difficile ribotypes with little or no activity against other species, supporting their potential as therapeutic agents. We also identified a genome associated lysin (CD630, YP_001088405), which lysed vegetative C. difficile in a similar manner to the phage derived lysins. We also cloned and expressed a spore cortex lytic enzyme (SleC) which targeted the cortex of C. difficile spores. Unfortunately this enzyme was inactive against intact spores, suggesting that the outer layers of the spore act as a permeability barrier. The results of this study showed in vitro the applicability of endolysins against the vegetative form of C. difficile and the activity of spore cortex lytic enzyme against coatless spores, offering interesting perspectives for evaluation of the antibacterial activity of a mixture of endolysin and spore cortex lytic enzyme.

No description available.

The rapid growth of ketamine and amphetamine misuse worldwide has led to the development of methods for the detection and analysis of ketamine and amphetamines in biological specimens. Most methods previously developed in forensic toxicology for the detection of ketamine and amphetamines used GC-MS. The present work developed alternative methods based on LC-MS/MS. Ketamine was chosen as the drug of interest because there are no data currently available on the extent of ketamine abuse in Malaysia even though a large amount of ketamine has been seized by the Malaysian Royal Police, while amphetamines are the most widely abused synthetic drugs in South East Asia including Malaysia. The study addressed some of the challenges facing the forensic toxicologist, such as the need to use new technology (LC-MS/MS) and improve sensitivity and selectivity in forensic toxicology analysis through efficient sample preparation techniques. The general requirements of method validation, including as the parameters linearity, limit of detection (LOD) and Lower Limit of Quantification (LLOQ), recovery, precision and matrix effects were observed. Three main techniques were used in the study: enzyme-linked immunosorbent assay (ELISA), liquid chromatography tandem mass spectrometry (LC-MS/MS) and molecularly imprinted polymer solid phase extraction (MISPE). MISPE is a new extraction technique in forensic toxicology applied to biological samples. Initially work was carried out on the optimization, development and validation of the Neogen® ELISA for screening ketamine and norketamine in urine. The Neogen® ketamine ELISA kit was found to be adequately sensitive and precise for ketamine screening at a cut-off concentration of 25 ng/mL. The ELISA test was shown to be highly specific to ketamine and demonstrated minimal (2.1%) cross-reactivity to its main metabolite norketamine compared to ketamine. Subsequently, an LC–MS/MS confirmation method for ketamine and norketamine in urine samples was developed and validated with application of the method to urine samples from chronic ketamine users in Malaysia. The method demonstrated good linearity, LOD, LOQ, accuracy and precision and had acceptable matrix effects. The efficiency of ELISA as a screening method at cut-off of 25 ng/ml and LC-MS/MS as a confirmation method at 2 ng/ml was evaluated. These methods complemented each other and both ELISA and LC-MS methods were 100% sensitive and specific with no false positive results for ketamine and norketamine in urine samples. The results demonstrated that a combination of these two methods can be reliably used for routine screening and confirmation of ketamine and norketamine in urine specimens. Preliminary data from this study provided information on the concentrations of ketamine and norketamine typically found in urine samples collected from individuals frequenting pubs in Malaysia. The main work in this thesis involved molecularly imprinted polymer materials which were used as sorbents in solid phase extraction (MISPE). Ketamine was used as a model substance for novel in-house synthesised MIPs as no anti-ketamine MIP have previously been reported and because the ketamine structure is suitable for the synthesis of molecularly imprinted polymers. The study was intended to improve the selectivity and sensitivity of the extraction method (MISPE) prior to LC-MS/MS analysis. Evaluation of polymer imprinting was carried out using HPLC-UV. MIP extraction and LC-MS/MS analysis were applied to the determination of ketamine and norketamine in hair samples and compared with a conventional SPE-based method. MISPE extraction was selective and sensitive with fewer matrix effects than the conventional SPE method and could also be applied to norketamine, the principal metabolite of ketamine, due to the group-selective binding nature of the MIP, but not to structurally dissimilar analytes such as PCP and tiletamine. MISPE was superior to conventional SPE for trace detection of ketamine and norketamine in hair, in terms of improved sensitivity, lower limits of detection and reduced matrix effects. In addition, the commercial product Amphetamine SupelMIPTM was evaluated for identification of amphetamines in post mortem blood coupled with LC-MS/MS analysis. This work assessed whether the MIP, sold by the manufacturer for the extraction of amphetamines in urine, could also be used for whole blood. The results demonstrated that the MIP can be used successfully for the determination of amphetamines in post mortem blood. The recoveries of five amphetamines were lower than with a comparable GC-MS method but the LODs and LLOQs of the LC-MS/MS method were better and suitable for detection of low levels amphetamines in post mortem blood. Further optimisation is needed to develop an improved protein precipitation method prior to MISPE. Liquid Chromatography Electro-Spray Ionization Mass Spectrometry (LC–ESI-MS) was used with the MISPE and SPE methods for detection and quantification of ketamine, norketamine and amphetamines in urine, whole blood and hair samples. LC-ESI-MS was found to be easy to use and could detect lower concentrations of drugs and gave reproducible results for all the methods developed in this thesis.