Over the last few years stimulant substances, such as amphetamine-type stimulants (ATS) and synthetic cathinones (SC), have dramatically increased the frequency of lethal intoxications. The spread of these stimulant drugs has caused a complex challenge to the forensic toxicology community. Most analytical methods focus on detection and quantification of a specific class of drugs instead of an extensive variety of compounds. A method for the detection and quantification of 29 ATS and SC drugs in a single procedure was carried out using solid-phase extraction (SPE), pentafluoropropionic anhydride (PFPA) agent and gas chromatography—mass spectrometry (GC−MS). The method was validated in accordance with SWGTOX guidelines using human urine samples. The limits of detection (LOD) and lower limits of quantification (LLOQ) were between (0.5 and 10) ng mL−1, and (5 and 50) ng mL−1, respectively. The linearity range was between 50 and 2000 ng mL−1 with a R2 >0.990 for 20 compounds. The bias and RSD were ≤20%, and no interferences or carryover were observed. The recovery was 80 to 120% for the majority of analytes. Prior to testing the substances, the GC−MS was initially optimised in terms of the oven and injector port temperatures. The sensitivity and selectivity of the GC−MS were then improved using acidified methanol and derivatisation agents. Six acylation reagents were compared and investigated using PFPA, trifluoroacetic anhydride (TFA), chlorodifluoroacetic anhydride (CLF2AA), heptafluorobutyric anhydride (HFBA), acetic anhydride (AA) and propionic anhydride (PA). The derivatisation method was optimised by modifying incubation time and temperature during the reaction and evaporation stages. Several parameters were used to evaluate the performance of the reagents, including the number of ions, relative ion ratio, peak area values, number of unique ions with some validation parameters. The reagents were further inspected using recovery through SPE in whole blood. The results of the comparison study showed that PFPA was the favoured reagent. All the derivatisation reagents were suitable for use on cathinones. Long term stability was investigated for the 29 stimulant compounds in human urine specimens over a period of 381 days at room temperature (RT), refrigerator (4°C) and freezer (−20°C) conditions. ATS were stable under all conditions, and all tested substances were stable at freezer conditions. Most SC at RT had lost more than 20% of the compound after two days, and had completely disappeared after a month. Most SC’s at refrigerator temperatures were unstable after day 21, and gradually decreased until undetected between days 77 and 349. The substances were stable on the autosampler for three days. No concentration-dependent variations were observed. Half-lives of selected drugs were briefly discussed. A sample preparation method that meets green analytic chemistry (GAC) requirements is desirable. Therefore, a method using solid phase microextraction (SPME) tips were initially developed via 13 processing steps and then validated using GC−MS in urine for eight ATS and SC substances. LOD and LLOQ were (5−25) ng mL-1, and (25−100) ng mL-1, respectively. The bias and RSD were < 15% error with R2 ≥0.992 for all analytes. Applying green analytical chemistry (GAC) parameters, the procedure had minor effects on health, waste and safety proportionate to LLE and SPE by adding the only microscale amounts of methanol and salt. Attention to the prevalence of new psychoactive substances (NPS) such as SC, is significant to the justice system and the forensic toxicology community. The prevalence of SC was studied using 273 urine specimens collected from Riyadh City in Saudi Arabia. The cathinone compound estimation prevalence rate was 1.01%. No other cathinones were identified. Further prevalence studies should be conducted in the future using a larger sample size and incorporating more drug substances and metabolites.

Azetidines are important class of heterocyclic organic compounds in drug discovery and natural product synthesis. Several natural products and pharmaceuticals are containing azetidine precursors such as, penaresidin and Azelnidipine. Iodine mediated cyclisation of homoallylamines were found to furnish iodoazetidines at 20 °C and further reaction with amine nucelophiles afforded aminoazetidines in high yields. The cyclisation of various homoallylamines which different substitution patterns using molecular iodine to furnish new classes of compounds with interesting biological applications were studied. The iodocyclisation of 3-methyl substituted homoallylamine were found to deliver γ-lactam compound in moderate yield as a mixture of diastereoisomers, the reaction conditions were optimised, and the separation into single diastereoisomers and modification in to pyrrolidine ring were investigated. The tricyclic furan bispyrrolidines were obtained in relatively low yields when 3-phenyl substituted homoallylamines were cyclised using the same strategy. The cyclisation of homoallylhydrazines to deliver pyrazoline compounds in high yield was studied in detail. Finally, the biological activity of some of the synthesised compounds were investigated in zebrafish embryo developmental assays and showed intriguing biological responses. Some of the synthesised compounds were sent to Syngenta and Lilly Company for further investigation.

This project was undertaken to increase the understanding of platelet function, with paticular emphasis on abnormal platelets in diabetes mellitus, and improving the quality of platelet concentrates for transfusion. {1} Potential platelet antagonists, including PGE1, verapamil, insulin and hirudin, were added to platelet concentrates in an attempt to improve the recovery and shelf-life of the concentrate. Each "preservative" caused some improvement in platelet concentrate quality, as measured by functional tests, and radio-immunoassay for the platelet activation marker, B- thromboglobulin. The best results were obtained with the addition of PGE1, which facilitated recovery of all samples to which it was added, suggesting a cheap way of ensuring consistently good platelet concentrates. {2} Various investigations were carried out regarding abnormal platelet function in diabetes mellitus. No significant differences were found in the responses of platelets from diabetics and age-matched controls to the calcium-channel blocking drug, Verapamil, in vitro. Similarly, the capacity of diabetic platelets to produce malondialdehyde, a by-product of thromboxane A2 synthesis, was not significantly different from control platelets. The use of insulin in aggregometry studies showed, surprisingly, that insulin could have a pro-aggregatory effect on platelets from diabetics, but not those from healthy controls. In addition, evidence for the existence of a platelet aggregation-enhancing factor in the plasma of diabetics and older controls was obtained. {3} Extensive tests to investigate the nature of spontaneous platelet aggregation (SPA) in whole blood have established the existence of two types of SPA, (i) ADP-dependent, and (ii) ADP-independent. The results obtained suggest a major role for erythrocytes in the development of inappropriate platelet aggregation.

Plenoptic imaging is a technology by which a three dimensional representation of the world can be captured in a single image. Previous research has focused on the technology itself, with very little focusing on applications of the technology. This thesis presents an investigation into its potential application to the field of retinal imaging, with the aim of producing three dimensional images of the retina at a cheaper cost than the current gold standard of retinal imaging, optical coherence tomography. Both a theoretical and practical approach have been utilised through the means of computational simulations and plenoptic imaging through the use of a commercial camera. Significant steps have been taken towards the overall goal, forming a strong foundation from which future projects will benefit.

Cardiovascular disease (CVD) is the most common cause of death worldwide, accounting for 31% of the annual deaths per year. The term cardiovascular disease refers to many different disorders of the heart and blood vessels, the most prolific of which is coronary heart disease (CHD). The root cause of CHD is atherosclerosis, which is the build-up of plaque in the intima of the arteries, restricting the blood supply which reaches the heart. If left untreated, the plaques lining the vessel can rupture and cause thrombosis and myocardial infarction (MI). Taking into account an individual’s risk-benefit ratio, a suitable treatment is considered with the aim to prevent MI from occurring. The two main revascularisation strategies are coronary artery bypass graft (CABG) and percutaneous coronary intervention (PCI), the latter being the most frequently used; within the UK 80% of revascularisation is performed by PCI. A PCI procedure is non-invasive and involves the deployment of an intravascular stent into the diseased vessel, which acts as a permanent scaffold that mechanically re-widens the vessel. However, despite the prolific use of intravascular stents within the clinic, there are significant problems associated with this revascularisation technique, such as in-stent restenosis (ISR) and late stent thrombosis (LST). ISR is the re-narrowing of arteries after stent deployment, characterised by a neointima growth within the lumen of the vessel. ISR is a significant clinical problem, because it can lead to repeat revascularisation procedures and often patients present frequently with unstable symptoms, fulfilling the criteria for an MI diagnosis. ISR occurs as a complex wound healing response caused by the stent deployment. Denudation and tearing of the endothelium and the mechanical stress endured by the vascular smooth muscle cells (VSMC) is a stimulus for hyperplasia. VSMC de-differentiate from the contractile to synthetic states and proliferate and migrate into the lumen of the vessel, secreting ECM which forms the bulk of the neointima. The neointima growth will continue to form until the endothelium has re-established itself. As a treatment to prevent ISR, drug-eluting stents (DES) were introduced which were coated with anti-proliferative drugs, to prevent ISR from occurring. DES proved to be very successful at preventing ISR; however elevated rates of LST were associated with these stents. This elevated event rate for DES by LST is thought to be caused as a result of delayed re-endothelialisation and by inflammatory reactions to the polymer used in the coating of DES. The aim of this study was consequently to investigate methods for delivery and coating of novel therapeutics, adenoviruses and miRNA onto stent surfaces. This would allow investigation into the prevention of ISR using these therapeutics, taking advantage of the localised stent setting. It was hoped that local delivery would provide significant advantages over systemic delivery by decreasing the dosage required, avoiding systemic side-effects and increasing delivery to the targeted area, thereby enhancing the therapeutic effect. For the development of coating methods for adenovirus eluting stents, the majority of the work conducted was done using Ad5 as a model virus vector. Three different methods were investigated and evaluated in vitro; deposition onto polyelectrolyte multilayer (PEM) surfaces, by direct conjugation with a modified poly(lactic acid) (PLA) surface through covalent bond formation and collagen gel entrapment. The development of coating methods for miRNA eluting stents focussed initially on collagen gel entrapment; however it was discovered that direct application onto a PLA surface provided a system whereby excellent delivery of the miRNA could be achieved. This methodology was extensively investigated and evaluated in vitro from stent material surfaces, and in vivo in both the porcine and murine stenting models. The results presented here have extended current methodology for both miRNA and adenovirus eluting stents. To the best of our knowledge, this is the first time that miRNA eluting stents have been used in these animal models and therefore contributes significantly to the field of miRNA-based therapeutics.

Ultrasonic transducers have been widely used in the medical applications. High frequency array transducers have recently attracted many research interests, since it provides not only the electronic beamforming but also the very fine image resolution. The transducers are usually either annular or linear arrays. This study concentrates on the design of both types of array in high frequency applications. The annular array is firstly investigated, and an optimized design of array geometry is developed including the kerfs influence which is usually neglected in the design of high frequency arrays. The improved imaging performance is found by this optimized design. More interestingly, the conventional annular array with element having the same surface area has a relatively large sidelobe closest to the main lobe. Two methods are applied to suppress the sidelobe. The method of using high-impedance single matching to enlarge the element bandwidth only shows very limited lobe suppression, however, a novel matching approach is found. Another method of changing the array geometry from equal area elements to equal width ones successfully suppressed the sidelobe. The imaging results suggest that the novel equal width array shows a good improvement. The linear array is then analyzed. The kerfs influence again is found to be significant for the array especially for high frequency. It is essential to use a high attenuated kerfs material in the linear array design. More importantly, the limitation of current fabrication techniques forces the linear array to concede a high grating lobe in the imaging field if it operates in a relatively high frequency. A novel linear array is developed to suppress the grating lobe without using advanced fabrication techniques. The imaging results show that the novel linear array has a general reduction of -6dB for the lobe suppression. The novel design made the linear array operate in a very high frequency without strong grating lobe, and the array can be fabricated by current techniques.

There is a requirement to assess the safety of chemicals to both 'man' and the environment. Traditionally this was determined through the use of animal testing. However, there is an increased need to develop alternatives to animal testing for the determination of toxicity due to ethical and legislative reasons. One approach to replacing the use of animals is the application of computational methods. These include Quantitative Structure-Activity Relationships ((Q)SARs), which are the formalisation of the relationship of the effects (e.g. toxicity) for a series of chemicals and their physico-chemical and structural properties. Most QSARs for toxicity require knowledge of a chemicals hydrophobicity. Traditionally hydrophobicity has been characterised by the logarithm of the octanol/water partition coefficient (log P). Current experimental and predictive methods are limited in terms of applicability for compounds with extreme log P values, compounds ionised under the conditions of analysis and surface active agents. An alternative technique to assess hydrophobicity is Immobilised Artificial Membrane High Performance Liquid Chromatography (IAM-HPLC). The IAM stationary phase was developed initially to mimic biological membranes more realistically than octanol/water partitioning. This study has collated published literature values for the IAM retention index (kIAM), including details of the experimental procedure, into a database. The database includes 1910 values for 647 compounds. The effect of variability of experimental procedure on reported values was investigated. Key experimental parameters were identified that ensure comparable log kIAM values. An IAM-HPLC method was optimised; the HPLC method covers a range of hydrophobicities (log P of -1.35 to 6.03) and includes both unionised and ionised compounds under the conditions of analysis. Additionally the method has been demonstrated to be robust across system of analysis, column and stationary phase batch. The assessment of robustness increases confidence in the log kIAM (pH 7.4) values for 66 aliphatic and aromatic compounds determined as part of this work. Methods to predict log klAM (pH 7.4) were investigated. Both a fragment and correction factor method, based on theoretical structural features, and a 'classical' descriptor based QSAR approach, was applied to both the experimental log kIAM (pH 7.4) values determined in this work and comparable values collated from the literature. QSARs have been developed using log klAM as a descriptor to predict the ability of a chemical to cross the skin barrier and to predict various acute aquatic toxicity endpoints, using published skin absorption and ecotoxicity data respectively.

Introduction: Acute lymphoblastic leukaemia (ALL) is the commonest childhood cancer. In the early trials of ALL treatment, central nervous system (CNS) relapse was a common occurrence - the introduction of CNS-directed therapy in the 1970s was associated with the largest single improvement in outcome for childhood ALL. Today, despite universal intensive CNS-directed therapy - with significant associated toxicity - the CNS is involved in around 50% of ALL relapses, with approximately 50% of these being isolated CNS (iCNS) relapse. Whilst many factors increase risk of CNS relapse, few are specific for CNS relapse. Discovery of specific risk factors for CNS relapse would allow increased therapy for children at high risk, and potentially less CNS-directed therapy for those at low risk. Relatively little is known about the biological differences between systemic and CNS ALL. In the CNS, leukaemic cells form plaques adherent to the leptomeninges, bathed in low-nutrient, low-oxygen cerebrospinal fluid (CSF). It was hypothesised that leukaemic cells adapt metabolically to this nutritionally poor CNS microenvironment, and these metabolic adaptations may be targets for specific therapy and/or specific biomarkers for CNS relapse. Findings: Transcriptional analysis of ALL cell lines from CNS and spleen in a mouse xenograft model, and of ALL cells retrieved from the CSF at CNS relapse of ALL, have shown the upregulation of cholesterol biosynthesis as a key adaptation to the CNS niche. Analysis of transcriptomic data from the bone marrow or peripheral blood from children with ALL at diagnosis have shown the potential for upregulated cholesterol biosynthesis (and, independently, upregulated IL7R) as a significant risk factor for CNS relapse of ALL. To support this finding, metabolomic analysis found evidence of changes in CSF cholesterol in the presence of CNS leukaemia, and of increased mevalonate (a cholesterol precursor) and cholesterol in ALL cells retrieved from the CNS in a xenograft model. Therapeutic targeting of CNS ALL in vivo with statins resulted in a CNS-specific increase ALL disease burden. Untargeted metabolomic analysis of CSF shows differences between children with ALL (either at diagnosis or on maintenance therapy) and non-ALL controls, and between children with ALL at diagnosis and the same children on maintenance therapy. Creatine abundance was significantly different in children with ALL at diagnosis compared with both other groups (1/3 lower at diagnosis than either on maintenance or non-ALL controls). This change in creatine and persisted on analysis of CSF from mice with and without leukaemia. On analysis of CSF from children at CNS relapse with ALL there is evidence of increased reduced creatine at time of CNS relapse in 3 of 4 patients. Conclusions: There is evidence to confirm the hypothesis that ALL cell adapt metabolically to the CNS niche. Cholesterol biosynthesis was identified as a key pathway upregulated in CNS ALL, and upregulated cholesterol biosynthesis in ALL cells at diagnosis was found to be a key risk factor for CNS relapse of ALL. In addition, clear changes in the CSF metabolome related to both ALL and ALL therapy were shown, and a new potential marker for the presence of CNS ALL identified. Prospective analyses in independent cohorts are required to determine the clinical utility of these novel strategies for prediction of CNS relapse risk.