Being one of the most rapidly advancing cancer imaging techniques in recent years, Positron Emission Tomography (PET) represents a standard of excellence with respect to sensitivity and resolution for the non-invasive in vivo molecular imaging of solid tumours via the detection of radiotracer molecules. Amongst these radiotracers, radiolabelled fluorinated nucleosides such as 3’-Deoxy-3’-[18F]-fluorothymidine (18F-FLT) has been widely recognized as a key, specific biomarker for tumour cellular proliferation. Current methods for the commercial production of [18F]FLT are characterized by low overall yields and time-consuming high-performance liquid chromatography (HPLC) purification. These disadvantages could be rectified by the development of a fast, efficient synthetic route to FLT that could enhance the productivity and reduce the reaction time of the fluoridation step necessary for the synthesis of short-lived radioisotope, 18F (t1/2=110 min) installed nucleoside. This project focussed mainly on the development of a new efficient chemical synthetic route to FLT. The synthesis and in vitro evaluation of a series of pro-nucleotide (ProTide) analogues of FLT as new potential therapeutic agents was also carried out. Various studies regarding FLT synthesis have been carried out on the cold (non-radioactive) 19F and radioactive 18F isomer by varying and optimizing conditions for the incorporation of different protecting groups and also different fluoridation reactions by nucleophilic displacement. Further optimization studies were made for the fluoridation step and the synthesis of [18F]FLTProTide analogues as new diagnostic PET imaging agents by variation of different chemical parameters on the phosphoramidate group was attempted. The synthesis of 19F- FLT based ProTides as new therapeutic agents were initiated by the introduction of the phosphoramidate group at the 5’-position of the furanosyl group of thymidine under basic conditions followed by fluoridation to generate the desired analogues. In addition to that, biological evaluation of the newly developed 19F-FLT ProTide analogues for anti-HIV 1 and anti-HIV 2 activities was undertaken on CEM cells. The results in those models indicated that the synthesized compounds were less potent than the parent nucleoside FLT. However in TK- (HIV-2) cells, the analogues retained biological activity in contrast to FLT. This suggested that the FLT ProTides bypassed the first phosphorylation step. However, the therapeutic in vitro evaluation for anti-tumour activity on L1210, CEM and HeLa cells showed no significant activity.

In this PhD project we show for the first time that novel dideoxy bicyclic pyrimidine nucleoside analogues (ddBCNAs) with L-chirality represent promising antiviral candidates for use against pox and measles viruses. We suggest a mechanism of action based on a cellular target. Our lead compound (Cf2642, with side chain C9H18–O–C5H11) is active against vaccinia virus (a surrogate poxvirus for smallpox) and measles virus, with IC50 concentrations of 0.19 and 7.5 µM, respectively. This is a 60-fold enhancement over cidofovir (viral DNA polymerase inhibitor; IC50 of 11.5 µM against VACV). A structure activity relationship was established, which was similar for both viruses, indicating a common and specific mechanism of action. Cf2642 does not inhibit HSV-1/2, influenza, adeno or yellow fever viruses. The mechanism of action for the ddBCNAs has been investigated and, though not defined, has been narrowed down. Based on our observations of drug activity in cell lines derived from various sources, we have suggested a cellular target for the ddBCNAs, most likely cellular membrane compartments or the proteins located therein. Though inhibition of vaccinia is observed within two hours of infection, we have shown that the ddBCNAs are unlikely to be entry inhibitors. Acidification of the extracellular medium was observed but, whilst it may be linked to the mechanism of action, this is not the cause of the antiviral effects. With a possible cellular target, toxicity was carefully evaluated. We have not observed significant cytotoxicity in any of our cell models. Antivirals active against cellular targets are less subject to viral resistance, which may develop rapidly with virus-targeting drugs. This could be critical since, there are currently no effective measles antiviral drugs available on the market, and resistance to measles RNA polymerase inhibitors and the potential antipoxviral drug cidofovir has already been described.

Consideration of broader outcomes of disease, especially those exclusively experienced and reported by the patient, such as HRQOL, is not only consistent with the ‘whole person’ view of health contained in the 1948 WHO definition, but is also a prerequisite to building health-care systems that are responsive to the needs of the patients. For chronic skin diseases, such as hyperhidrosis, these provide a useful indicator of how a patient feels and functions disease for both practical and methodological reasons. The aims of this study therefore were to investigate the impact of hyperhidrosis on patients’ HRQoL using a mix of qualitative and quantitative methods. In addition, a further aim was to develop and validate a disease-specific instrument for assessing HRQoL in hyperhidrosis. In pursuing the above aims, the feasibility of applying online social networking sites for outcomes research in dermatology was assessed. Patients were recruited through online social networking communities related to hyperhidrosis for all stages of the study. Interviews, focus groups and surveys were used for collecting qualitative data from patients (n = 71) to understand quality of life issues of patients, and to provide the content of the new instrument. Dermatologists (n= 5) and patients (n=7) took part in the content validation of the HidroQoL©. Item reduction and the development of the scale’s structure was carried out through several field-testing studies (n: USA, 559; UK, 115), using the item response theory (IRT) Rasch model and factor analyses. Further psychometric testing was performed in a separate study (n = 241). Distribution-based methods were applied in establishing minimum clinically important difference (MCID). A thematic analysis of the qualitative data collected produced 29 quality of life themes and 102 sub-themes, forming the content for the initial 49-item HidroQoL©. The two expert panels judged the instrument as content valid, with a few suggestions. The Rasch analysis modelling led to the collapsing of response categories (from five to three) and the reduction in number of items (from 49 to 18), to ensure a perfect model fit. Factor analyses supported both a single- and a two-factor structure. In subsequent construct validation study the HidroQoL correlated with the DLQI (rs = 0.572, p < 0.01) and the Skindex-17 (rs = 0.551, p < 0.01). Reliability was high (Cronbach alpha = 0.9; test-retest ICC = 0.93). The scores were sensitive to change in patients’ disease severity (standard response mean = 0.8, 95% C.I: 0.34-1.27). The scale banding proposed for the HidroQoL score is as follows: 0 – 1, no effect at all; 2 – 11, small effect; 12 – 22, moderate effect; 23 – 32, large effect; 33 – 36, very large effect. The MCID values were 1.94 – 3.07, for generalised v hyperhidrosis, 2.16 – 4.36, for axillary hyperhidrosis, 2.15 – 3.39, for palmo-plantar hyperhidrosis. An MCID of three is currently being proposed for all types of hyperhidrosis. This study has provided the initial evidence supporting the appropriateness of the content of the HidroQoL and validity of inferences from its scores for assessing HRQoL in hyperhidrosis. In addition, the availability of MCID estimates for the HidroQoL will facilitate its clinical interpretation in both research and routine clinical practice. This study has also demonstrated how CTT and IRT can be integrated in the development and validation of a new generation of HRQoL instruments, using social network for patient recruitment.

RNA viruses are a major cause of disease that in the last fifteen years counted for frequent outbreaks, infecting both humans and animals. Examples of emerging or ri-emerging viral pathogens are the Foot-and- Mouth disease virus (FMDV) for animals, Chikungunya virus (CHIKV), Coxsackie virus B3 (CVB3) and Respiratory Syncytial virus (RSV) for humans, all responsible for infections associated with mild to severe complications. Although both vaccines and small-molecule compounds are at different stages of development, no selective antiviral drugs have been approved so far, therefore for all four these viruses improved treatment strategies are required. Promising targets are the viral non-structural proteins, which are commonly evaluated for the identification of new antivirals. Starting from the study of different viral proteins, several computer-aided techniques were applied, aiming to identify hit molecules first, and secondly to synthesise new series of potential antiviral compounds. The available crystal structures of some of the proteins that play a role in viral replication were used for structure- and ligand-based virtual screenings of commercially available compounds against CVB3, FMDV and RSV. New families of potential anti-CHIKV compounds were rationally designed and synthesized, in order to establish a structureactivity relationship study on a lead structure previously found in our group. Finally, a de-novo drug design approach was performed to find a suitable scaffold for the synthesis of a series of zinc-ejecting compounds against RSV. Inhibition of virus replication was evaluated for all the new compounds, of which different showed antiviral potential.

Bacterial drug resistance is an increasingly serious problem that threatens public health and researchers need to develop new drugs. The biosynthetic pathway of the bacterial peptidoglycan is a known and good target for the development of novel antibacterial agents. This research project focused on the first lipid-linked step of peptidoglycan biosynthesis. The enzyme required for this step is Phospho-MurNAc-pentapeptide Translocase MraY. Our aim was to screen for novel inhibitors of MraY. A continuous fluorescence MraY assay was developed and optimised to test known and potential inhibitors such as nucleoside natural products, antimicrobial peptides and structurally new small molecule potential inhibitors. The fluorescence assay was adapted to a high-throughput fluorescence assay in microtitre plate format and around 2,000 compounds were screened from the diversity set of the National Cancer Institute (NCI) against MraY in order to identify novel inhibitors. However, around 22 % of the test compounds from the diversity set interfered with fluorescence. Therefore, a radiochemical MraY assay was developed as an independent method. We eventually identified one potential MraY inhibitor from the diversity set, the naphthylisoquinoline alkaloid, michellamine B, with IC50 values of 400 and 340 μg/ml against E. coli and B. subtilis MraY respectively. The compound showed antibacterial activity against B. subtilis with an MIC value of 16 μg/ml. It was established that MraY inhibition from the pacidamycin producer, S. coeruleorubidus, was detectable directly from culture supernatants by the fluorescence and by the radiochemical MraY assays. Therefore, we tested culture supernatants and cell extracts from various Streptomyces strains. MraY inhibition was observed using cell extracts from Streptomyces venezuelae, and higher levels of inhibition were observed from a gbnB/gbnR S. venezuelae mutant, though it was not possible to identify the active species present. Following an earlier report of halogenated fluoresceins identified from a combined MraY/MurG screen, we also tested several halogenated fluoresceins, and found that phloxine B, a tetra-bromo fluorescein analogue, was an inhibitor of E. coli MraY with an IC50 value of 32 μM, and also inhibited MraY from P. aeruginosa, B. subtilis, M. flavus and S. aureus with IC50 values ranging between 100 and 210 μg/ml.

The pharmaceutical industry has been criticised for a lack of innovation associated with the drug discovery and development process, for example when compared with the computer or music industries. In fact, bringing a new medicine to the market requires, on average, the screening of up to 10 000 molecules, an expense in the range of $500 million-$2 billion and roughly 10-15 years of research. Such a situation not only has a direct impact on the health and life expectancy of every single human being on the planet, but also indicates that alternative strategies for drug development should be investigated. In this thesis, studies of direct formulation-membrane interactions, both in a high throughput (HT) manner and at a nanometre scale, were initially identified as an important approach that could offer advantages for in vitro-in vivo correlations of in-man drug behaviours. Subsequently, supported lipid bilayers (SLBs) of physiologically-relevant lipid compositions were indicated as experimental models of preference for pre-clinical drug development. For that reason, the characterisation and assessment of physicochemical and behavioural properties of the model SLBs at a nanometre scale, as well as development of an SLB microarray for HT applications were the focus of this research. Here, the optimisation and characterisation of model lipid films was performed using atomic force microscopy (AFM), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). Additionally, the AFM-investigated assessment of the interactions between model SLBs and formulation components (e.g. Pluronics®, siRNA, DNA polyplexes) enabled both the correlation of in vitro observations with literature-reported in vivo performances of the components of interest and the development of hypotheses with regards a number of phenomena in biology. Furthermore, the development of a SLB microarray prototype suitable for HT applications is reported. Directly, this research improves: the understanding of SLB behaviours and experimental investigation at a nanometre scale of the mechanisms of interactions between membranes and: Pluronics®, nucleic acids and their complexes, as well as the technology of SLB microarray development. Indirectly, this research contributes towards the progress in a number of research areas within pharmaceutical sciences, potentially resulting in new scientific disciplines, such as immunolipidomics or nanopharmacology.

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.