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.

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.

Transposable elements account for the almost half of the sequence encoded by mammalian genomes, which become silenced during early embryonic development. This thesis sought to explore the hypothesis of the involvement of the RNAi pathway in the silencing of transposable elements in mammals, predominantly through the identification of transposon-associated RNA of -20-25nt using a gel blotting technique. Initially cell lines of embryonic and tumour origin were analysed. This lead to the identification of several previously unreported transposon-associated RNA ranging from 70-90nt. However, it was not until a more detailed analysis of the embryonic cell lines, with the induction of differentiation in cell culture that several discrete RNA of -20nt were detected for the mouse transposons L1 and B2. The differentiation of embryonic cell lines in culture also serendipitously lead to the detection of several short 55 rRNA of -22-26nt, these were also later detected in several human cell lines of breast cancer origin and healthy breast tissue. Intriguingly the -20-26nt repeat-associated identified were predominantly observed after two-days of differentiation in cell culture in several cell lines and often coincided with an ethidium bromide stainable band of -19nt. The latter may indicate a large proportion of these RNAs. Further analysis of the B2 and 55 rRNA repeat-associated short RNA revealed both to have reduce accumulation in Dicer-null embryonic stem cells, implicating a possible association with a known component of the RNAi pathway. Dicer was also observed to process the longer -50-80nt 55rRNA to -20-26nt in vitro. BLAST was also used to identify possible mRNA targets for the short B2 and 55 rRNA. One of these, the mRNA encoding sialic acid acetylesterase (SIAE) was consistently observed to be reduced with the accumulation of the short RNA using end-point RT-PCR, consistent with targeting through the RNAi or a similar pathway. However, no further links with the RNAi pathway were established, with no targeting detected for the short B2 or 55 rRNA using dual luciferase sensor assays. The repeat-associated RNA identified in this thesis are among the first of their type and further work will be required to establish what relevance they have to the RNAi pathway and transposon regulation.

The G-protein coupling specificity of D2-like dopamine receptors was investigated using both receptor-G protein fusions and membranes of cells in which pertussis toxin-resistant mutants of individual Gi-family G proteins could be expressed in an inducible fashion. A range of ligands displayed agonism at the long isoform of the human dopamine D2 receptor. However, varying degrees of efficacy were observed for individual ligands as monitored by their capacity to load [35S] GTPγS onto each of Gi1, Gi2, Gi3 and Go1. By contrast, S-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine (S-(-)-3PPP) was a partial agonist when Go1 was the target G protein but an antagonist/inverse agonist at Gi1, Gi2, Gi3. In ligand binding assays dopamine identified both high and low affinity states at each of the dopamine D2 receptor-G protein fusion proteins and the high affinity state was eliminated by guanine nucleotide. S-(-)-3PPP bound to an apparent single state of the constructs where the D2 receptor was fused to Gi1, Gi2 or Gi3. However, it bound to distinct high and low affinity states of the D2 receptor-Go1 fusion with the high affinity state being eliminated by guanine nucleotide. Similarly, although dopamine identified guanine nucleotide-sensitive high affinity states of the D2 receptor when expression of pertussis toxin-resistant forms of either Gi2 or Go1 was induced, S-(-)-3PPP identified a high affinity site only in the presence of Go1. These results demonstrate S-(-)-3PPP to be a protean agonist at the D2l receptor and may explain in vivo actions of this ligand. Furthermore, in agreement with previous studies, the ability of the dopamine D2l receptor to couple promiscuously to Gαi1-3, and Gαo1 was demonstrated. However, despite high homology between dopamine D2l and D3 receptors, the G protein-coupling specificity of the D3 receptor has not been well characterised. Again using both receptor-G protein fusions and membranes of cells in which pertussis toxin-resistant mutants of individual Gi-family G proteins could be expressed in an inducible fashion, we confirmed the selective coupling of the D3 receptor to Gαo1. A range of ligands displayed agonism at the D2l receptor and the D3 receptor when coupled to Gαo1. As a general trend, agonists, including dopamine, displayed a higher potency at the D3 receptor. This perhaps reflects the role of D3 as an autoreceptor. Of particular interest was the demonstration that S-(-)-3PPP has both a higher efficacy and potency at the D3 receptor when coupled to Gαo1. The investigations into dopamine receptor-G protein coupling highlighted the utility of the [35S]GTPγS binding assay as a method of directly measuring receptor catalysed nucleotide exchange on the α subunit of G proteins. However, the expense associated with the use of radiolabels makes this assay less attractive, particularly for high-throughput screening programmes. In an attempt to develop a non-radioactive assay equivalent to the [35S] GTPγS assay an immunisation programme was initiated to generate antibodies selective against the active (GTP bound) conformation of G proteins. 4 way primary screening of 1632 hybridomas generated from mice immunized with GTPS-loaded Gi1 and isolated using an automated robotic colony picker, identified 3 antibodies that interacted with the constitutively active Q204L but neither the constitutively inactive G203A nor wild type form of Gi1. This profile extended to other closely related Gi-family G proteins but not to the less closely related Gs and Gq/G11 families. Each of these antibodies was, however, also able to identify wild type, GDP-bound Gi- family G proteins in the presence of AlF4- which mimics the presence of the terminal phosphate of GTP and hence generates an active conformation of the G protein. Stimulation of cells co-expressing a wild type Gisubunit and the dopamine D2 receptor with the agonist ligand nor-apomorphine also allowed these conformation selective antibodies to bind the G protein. Such reagents allow the development of label- free assays for G protein-coupled receptor-mediated activation of Gi- family G proteins.

Smokers with asthma display reduced responses to both inhaled and oral corticosteroids with associated increased symptoms, accelerated decline in lung function and increased use of health care services. Little work has been undertaken to address the possible causes of this reduced response and to find effective replacement therapies. Therefore this thesis was carried out with the aim of identifying potential mechanisms and new therapies for this group. The oral bronchodilator theophylline has been suggested as a treatment for corticosteroid insensitivity due to its ability to increase HDAC activity in-vitro. I undertook an exploratory proof of concept clinical trial based on the hypothesis that low dose theophylline would restore corticosteroid sensitivity in smokers with asthma through theophylline induced recovery of HDAC activity. Low dose oral theophylline added to inhaled corticosteroid increased pre-bronchodilator lung function and reduced symptoms of asthma whilst low dose theophylline given alone reduced symptoms but had no effect on pre-bronchodilator lung function. This research provides a foundation for future studies designed to examine the efficacy of theophylline in smokers with asthma. Agonists of the nuclear hormone receptor peroxisome proliferator activated receptor-γ (PPARγ) have been demonstrated to be effective at reducing inflammation in both in-vitro and animal models of asthma. Therefore to examine the hypothesis that PPARγ stimulation would reduce the inflammation present in smokers with asthma I undertook an exploratory, proof of concept clinical trial using the PPARγ agonist rosiglitazone. Treatment with rosiglitazone was associated with a trend to improvement in FEV1 and improvement in a marker of small airway lung function and as such may provide an alternative treatment for small airways obstruction in conditions such as asthma and chronic obstructive airways disease. This trial will enable powering of future confirmatory studies. Altered cytokine profiles, specifically the combination of increased interleukin (IL)-2 and 4, are observed in asthmatic subjects with corticosteroid insensitivity. Based on this work I examined the hypothesis that the altered response to corticosteroids in smokers with asthma was associated with an altered cytokine milieu including raised levels of IL-2 and 4. Smokers with asthma, characterised as corticosteroid resistant by oral corticosteroid trial, demonstrated significantly raised sputum supernatant IL-6 levels and raised levels of a number of other sputum cytokines compared to non smokers with asthma. This altered phenotype suggests cigarette smoking in asthma may be associated with a deviation to Th1 mediated inflammation and could provide an explanation for the reduced corticosteroid response of smokers with asthma. The cell type/s responsible for both this shift in immunological phenotype and production of increased levels of sputum cytokines is unclear and will require further study. Previous in-vitro and in-vivo research has identified altered histone acetylation patterns in subjects with relative corticosteroid resistance. Therefore I examined the hypothesis that smokers with asthma displayed reduced responses to corticosteroids as a result of a cigarette smoke induced reduction in histone de-acetylase (HDAC) activity. Smokers with asthma provided sputum macrophages and blood for peripheral blood borne monocytes to examine total HDAC activity. Sputum and blood macrophage total HDAC activity was equivalent in smokers and non-smokers with asthma. Therefore reduced blood total HDAC activity does not appear to explain the altered corticosteroid response in this group. However the number of sputum macrophages obtained may have been too low to allow conclusive examination of this endpoint. Another consideration is that contamination of the sample due to the technique used may be altering the signal obtained. Further work either through modification of sputum induction techniques to increase macrophage number or bronchoscopic sampling is required to conclusively address the role of alveolar macrophage HDAC activity in the reduced corticosteroid response displayed by smokers with asthma. Exhaled nitric oxide has been exploited as a useful exploratory and confirmatory endpoint in asthma. However exhaled nitric oxide, measured using standard flow rates and methodology, is unhelpful in smokers with asthma as cigarette smoking is associated with a marked reduction in exhaled nitric oxide levels in the majority of subjects. Recent research has demonstrated that measurement of exhaled nitric oxide at multiple flow rates followed by mathematical modelling reveals increased levels of alveolar nitric oxide that were unaltered by current smoking. Therefore to examine the hypothesis that smokers with asthma display altered levels of alveolar nitric oxide and flow independent parameters compared to non-smokers with asthma I carried out a cross-sectional study. Alveolar nitric oxide, determined by linear modelling, was significantly reduced in smokers with asthma compared to non smokers with asthma. The concentrations observed were within the range for normal subjects and therefore this method does not overcome the problems inherent in measuring exhaled nitric oxide at standard flows. The use of non-linear modelling did demonstrate parity between smokers and non-smokers with asthma for alveolar nitric oxide. Nitric oxide flux was lower in smokers with asthma when derived by both linear and non-linear modelling and displayed sensitivity to oral corticosteroids. Therefore nitric oxide flux is worthy of further investigation as an exploratory endpoint in smokers with asthma. In conclusion treatment of smokers with asthma with low dose theophylline alone, the combination of low dose theophylline and inhaled corticosteroid and the PPARγ agonist rosiglitazone was associated with clinical improvements and further clinical trials to assess the role for these treatments in the management of smokers with asthma are justified. Smokers with asthma display an altered sputum cytokine profile with raised levels of the proinflammatory cytokine IL-6, equivalent blood total HDAC activity and reduced alveolar nitric oxide compared to non-smokers with asthma. Sputum HDAC activity requires further development before it can be confidently employed as a method of assessing total pulmonary HDAC activity.

African trypanosomiasis is a disease caused by the Kinetoplastida parasites Trypanosoma brucei rhodesiense and T. b. gambiense. The distribution of the disease is split geographically with T. b. rhodesiense found in eastern sub-Saharan Africa and T. b. gambiense in the west of the continent. Current treatment for this fatal disease is wholly unsatisfactory with problems such as extreme toxicity, affordability and the emergence of resistance. The case for the generation of new potential chemotherapies is compelling and urgent. Phosphodiesterase (PDE) enzymes degrade the secondary signalling molecule cyclic adenosine monophosphate (cAMP) to AMP by hydrolysis, thereby modulating and regulating the signal transduction to the effector proteins. The phosphodiesterase enzymes in the PDEB family in T. brucei were shown to be essential to the host-infective bloodstream forms and validated as good drug targets using RNA-interference (Zoraghi, R. and Seebeck, T., 2002; Oberholzer, M., 2007). Prompted by these findings, two series of putative trypanosomal PDE inhibitors, from different sources, were thoroughly assessed in this project for their anti-trypanosomal activity and their intracellular effects on the trypanosome. The whole-cell in vitro efficacy for each compound, against T. brucei wildtype and the drug-resistant strain TbAT1 knockout, was established by the standard resazurin reduction assay. 25 compounds from Series 1 had EC50 values below 0.5 µM, with 7 under 100 nM and the most active having an EC50 value of 5.8 ± 3.4 nM. For the much smaller Series 2 (GJS Compounds), the most active compound was GJS-128 with an EC50 value of 79.4 ± 10.3 nM. This demonstrates that a number of compounds from both series have potent in vitro activity against trypanosomes that is better than or equal to the current chemotherapeutic compound diminazene, and some Series 1 compounds are on a par with pentamidine and melarsoprol. No major cross-resistance was displayed by the TbAT1 knockout strain to either Series 1 or the GJS series. Similarly, a panel of Series 1 compounds tested against the B48 strain (resistant to pentamidine and melaminophenyl arsenical drugs), and also against Trypanosoma equiperdum wildtype and diminazene resistant (PBR) strains, showed no major cross-resistance displayed by the other resistant strains. This suggests that there would also be little or no cross-resistance from refractory strains in the field, and also that the compounds are active against multiple Trypanosoma species. A small panel of Series 1 compounds were also tested for efficacy against trypanosomes in infected mice. 4 daily doses of 20 mg/kg bodyweight of Compound 48 significantly reduced parasitaemia by approximately 60% compared to untreated controls, however higher concentrations were not tolerated by the mice so a cure could not be demonstrated. A high-throughput method for monitoring the speed of action of test compounds on trypanosomes in real time was developed, based on the fluorescence of propidium iodide when bound with DNA. Optimisation of the protocol to 96-well plates and low cell densities provided higher resolution and accurate traces of the lysis of trypanosomes in a cell suspension compared to previously used methods, as well as a greatly increased capacity. The propidium iodide assay could also be converted to provide end-point EC50 values that were directly comparable to those established by the standard resazurin reduction assay. The majority of Series 1 compounds did not increase the intracellular concentration of cAMP on incubation with bloodstream form trypanosomes; those that did only induced a minor elevation of the intracellular concentration of the signalling molecule. Since genetic disruption to phosphodiesterase enzymes resulted in large increases in cAMP levels (Oberholzer, M. et al, 2007; Zoraghi, R. and Seebeck, T., 2002), the lack of increase in cAMP by the Series 1 compounds strongly suggest that they do not sufficiently inhibit the PDEs in live trypanosomes and kill the cells via an alternative pathway. In contrast, incubation with the GJS compounds did result in significant increases in intracellular cAMP concentration with the most active being GJS-128 recording an approximately 3-fold increase in cAMP over 3 hours at just 30 nM. The concentrations that begin to increase cAMP level are consistent with the EC50 values for trypanosomes cultured in vitro (this study), and is also in line with inhibition data of recombinant TbrPDEB enzymes (work conducted by Dr. Herrmann Tenor, ALTANA Pharma, and Prof. Thomas Seebeck, University of Bern). This gives a clear and consistent link between the cause of cAMP rise (inhibition of PDEB by GJS compounds) and the effect of that concentration increase on bloodstream form trypanosomes (cell death), demonstrating that the GJS series are inhibitors of trypanosomal PDEs and chemically validate PDEs as drug targets for potential new chemotherapies against African trypanosomiasis. The effect of PDE inhibition on the physiology of the bloodstream form trypanosomes was also investigated. Flow cytometry analysis and the assessment of DNA configuration by fluorescence microscopy after DAPI staining determined that PDE inhibition by GJS-128 resulted in a precise block of the cell cycle in cytokinesis. The replicating trypanosome synthesized and segregated its DNA into two nuclei and kinetoplasts as normal and proceeded to initiate the physical separation of mother and daughter cells. The cleavage furrow between the old and new flagella progressed normally until the point of abscission, at which point division was halted with only a small section of plasma membrane connecting the two almost separated cells. Both cells appeared viable and underwent subsequent rounds of DNA replication, segregation and attempted physical separation that was always blocked near completion. This indicates cAMP signalling plays an important role in the correct physical separation of the replicating bloodstream form trypanosomes. A trypanosome cell line resistant to GJS-128 was developed by chemical mutagenesis and continuous culture with gradually increasing, but sub-lethal concentrations of the PDE inhibitor. This cell line, termed R0.8, was >15-fold less sensitive to GJS-128 and displayed significant cross-resistance to the other GJS compounds, as well as to stable, membrane permeable cAMP analogues. The mode of resistance was investigated by comparing the cAMP profile of the R0.8 and parental wildtype strains on incubation with GJS-128. No major differences were observed suggesting that both the adenylyl cyclase and phosphodiesterase activities remained unchanged in the PDE inhibitor-resistant strain. In support of this, the sequencing of TbrPDEB1 and TbrPDEB2 in both strains, while uncovering the loss of heterozygosity in the R0.8 line, revealed no mutations that would impact on enzyme function or inhibitor binding in the resistant cell line. These data strongly suggest that the adaptation resulting in resistance to PDE inhibitors is located in the effector proteins downstream of the PDEs and adenylyl cyclases in the cAMP signalling pathway. Identifying a compound that inhibits phosphodiesterases in trypanosomes and elevates cAMP concentrations, along with the generation of a PDE inhibitor-resistant cell line will allow more detailed examination of all aspects of the cAMP signalling pathway in T. brucei and across the Kinetoplastida. Phosphodiesterases have also been demonstrated to be chemically inhibitable in trypanosomes and could prove to be the target of a new generation of chemotherapies against African trypanosomiasis.

Epilepsy affects 50 million people world-wide. Since 1982, the Western Infirmary Epilepsy Unit has provided a specialist service for over 6900 people with suspected and established seizure disorders. The twelve studies detailed in this thesis discuss the management of epilepsy in different patient populations, and explore beneficial and adverse effects of antiepileptic drugs (AEDs). AED development has allowed advances in pharmacological treatment of localisation-related epilepsies. Thus, outcomes were investigated in 550 such patients followed-up at the epilepsy clinic over 13 years (Paper i). Of these, 312 (57%) became seizure-free on medication. Those with hippocampal sclerosis had the poorest outcome (p<0.01), and a higher incidence of febrile convulsions (p<0.001). Although many patients benefited from AED therapy, results may be biased, given this cross-sectional study analysed data from both newly diagnosed patients, and those with drug-resistant seizures. Many people with epilepsy take more than one AED, although supportive evidence is sparse. Hence, polytherapy outcomes in 2881 patients registered with the Epilepsy Unit database were examined (Paper ii). Of these, 1617 (56%) were seizure-free, with 332 (21%) taking more than one AED (287 on two, 86%; 42 on three, 13%; 3 on four, 1%). There were 40 duotherapy and 28 triple therapy combinations resulting in seizure freedom. Therefore, combining two or three, but rarely four AEDs may be useful for patients not responding to monotherapy. Because this was a retrospective analysis of newly treated patients and those with refractory epilepsy, the analysis was subject to bias. Lack of a control group was also a weakness. Epilepsy Unit staff are therefore now examining similar outcomes in a large population of newly treated patients only. To establish the place of recently marketed AEDs in clinical practice, four studies examined prospectively the efficacy and tolerability of the novel agent, topiramate, in uncontrolled epilepsy. Adjunctive topiramate was administered in 170 patients with refractory seizures (Paper iii). Seizure frequency and adverse events were monitored. Patients were followed-up until seizure freedom for ≥ 6 months, ≥ 50% or <50% seizure reduction, intolerable side-effects, or lack of efficacy occurred. Seizure freedom was achieved in 39 (23%) patients. A ≥50% reduction in seizure frequency was reported in 80 (47%) others. Doses were often lower than those in regulatory studies. Efficacy as monotherapy was also demonstrated. Using the same end-points, topiramate was added to AED regimens of 64 patients with learning disabilities and epilepsy (Paper iv). Remission from seizures was established in 16 (25%). In similar fashion, levetiracetam was started in 156 patients with uncontrolled epilepsy (Paper v). Of these, 40 (26%) became seizure-free, many on low doses. When the drug was added to AED regimens in 64 patients with learning disabilities, 24 (38%) became seizure-free for at least 6 months (Paper vi). Caregiver quality-of-life scores improved significantly with levetiracetam (p<0.001). It is important to recognise that for all four audits results may be biased due to their observational nature, and the fact that they were undertaken at a single centre, with no control group. For patients with learning disabilities, small numbers, and retrospective baseline recordings for some could also have introduced bias. In Papers vii, viii and ix, findings from longitudinal studies in teenagers, people with learning disabilities and epilepsy, and newly diagnosed elderly patients attending the Epilepsy Unit, are reported. At the Teenager Clinic, 301 adolescents were reviewed over four years (Paper vii). Epilepsy was excluded in 135 (45%), five taking AEDs. A single seizure occurred in 22 others. In the 144 with epilepsy, seizure freedom for ≥ 12 months was attained in 76 (53%), but outcomes were poorer than expected. Neuroimaging was abnormal in 27 (43%). Newly diagnosed patients fared better than those taking treatment (p<0.05). More teenagers with primary generalised seizures (60%) attained remission, compared to those with focal-onset seizures (46%) (p<0.02). The retrospective natures of the analysis, and lack of control group may have biased results, thus making statistical conclusions inaccurate. Findings suggested the need for improved services. Over four years, 214 patients with learning disabilities and refractory epilepsy were followed-up (Paper viii). Although it is generally thought these individuals’ seizures are difficult to control, 59 (43%) became seizure-free for ≥ 12 months with AEDs. There was no change in quality-of-life scoring during this time, and no relationship between extent of learning disability and seizure control. The observational nature of the audit, and lack of control group may have biased results. Currently, there are few data on elderly people with epilepsy. Thus, outcomes over a 20-year period in 117 newly diagnosed senior citizens were examined (Paper ix). After starting AED treatment, 93 (79%) became seizure-free for ≥ 12 months, 73 (62%) with their first drug. Prognosis was better than in younger patients, and for those with fewer pre-treatment seizures (p=0.0078). Again bias may have been introduced because of the study’s observational nature and lack of control group. The final studies concentrate on AED-related adverse effects (Papers x, xi and xii). Bone changes have been reported with AED use. Hence, the relationship between bone mineral density, and long-term AED treatment in 78 older adults (47 post-menopausal women, 31 men), taking hepatic enzyme-inducing or non-inducing AEDs, was explored in a case-controlled study (Paper x). Men had significantly lower bone mineral density than controls at the lumbar spine (p<0.01), and neck of femur (p<0.005). Women had statistically reduced bone mineral density at the femoral neck (p<0.05). It was concluded that long-term AED treatment is an independent risk factor for reduced bone mineral density in people with epilepsy. As sodium valproate may be associated with metabolic changes and polycystic ovarian syndrome, hormone profiles were compared in 76 young men and women taking sodium valproate or lamotrigine monotherapy, to assess whether a pharmacological effect of valproate was responsible (Paper xi). Results revealed only four obese females exhibiting biochemical characteristics of polycystic ovarian syndrome (p=0.05), with obese patients of both sexes (p=0.01), and valproate-treated men (p=0.03) having higher insulin concentrations. Results are not significant when corrected for multiple comparisons. It can therefore be concluded that no differences in metabolic indices between patients taking sodium valproate or lamotrigine existed. To examine further effects on androgenic hormones, and the efficacy and tolerability of sodium valproate and lamotrigine monotherapy, a randomised, prospective study in 225 patients was performed (Paper xii). Patients were recruited if they presented with a minimum of two unprovoked seizures of any type, or a single seizure and underlying neuropathology. Of patients with partial-onset seizures, 81 received sodium valproate and 80 were randomised to receive lamotrigine. Of those with idiopathic generalised epilepsies, 30 received sodium valproate and 34 took lamotrigine. Seizure-free rates were identical in both arms at twelve months between the valproate and lamotrigine cohorts. There was a trend towards superiority for valproate (57% seizure-free) over lamotrigine (35% seizure-free) for patients with idiopathic generalised epilepsies (p=0.09), but a converse separation of outcomes for localisation-related epilepsies (43% seizure-free with valproate, 53% seizure-free with lamotrigine, p=0.24). More patients taking sodium valproate withdrew due to adverse events (p=0.046), eight because of weight gain.