The growing problem of insecticide resistance is jeopardising current pest control strategies and current insecticide development pipelines are failing to provide new alternatives quickly enough. Metabolomics offers a potential solution to the bottleneck in insecticide target discovery. As a proof of concept, metabolomics data for permethrin exposed Drosophila melanogaster was analysed and interpreted. Changes in the metabolism of amino acids, glycogen, glycolysis, energy, nitrogen, NAD+, purine, pyrimidine, lipids and carnitine were observed along with markers for acidosis, ammonia stress, oxidative stress and detoxification responses. Many of the changed metabolites and pathways had never been linked to permethrin exposure before. A model for the interaction of the observed changes in metabolites was proposed. From the metabolic pathways with the largest changes, candidate genes from tryptophan catabolism were selected to determine if the perturbed pathways had an effect on survival when exposed to permethrin. Using QPCR it was found that all genes in the entire pathway were downregulated by permethrin exposure with the exception of vermilion suggesting an active response to try and limit flux through tryptophan catabolism during permethrin exposure. Knockdown of the tryptophan catabolising genes vermilion, cinnabar and CG6950 in Drosophila using whole fly RNAi resulted in changes in susceptibility to permethrin for both topical and oral routes of exposure. Knockdown of the candidate genes also caused changes in susceptibility when the insecticides fenvalerate, DDT, chlorpyriphos and hydramethylnon were orally administered. These results show that tryptophan catabolism knockdown has an effect on surviving insecticides with a broad range in mode of action. Symptoms that occur in Drosophila during exposure to the different insecticides were also noted. To gain further understanding into the mechanisms affecting survival, tissue specific knockdown was performed revealing tissue and gender specific changes in survival when vermilion, cinnabar and CG6950 are knocked down. Metabolomics was performed on the knockdown strains to determine the efficacy of the knockdowns on tryptophan catabolism and to identify any knock-on effects. The results indicate that tryptophan metabolite induced perturbations to energy metabolism and glycosylation also occur in Drosophila along with apparent changes in the absorption of ectometabolites. As the knockdown of vermilion, cinnabar and CG6950 tended to result in reduced susceptibility to insecticides, they would make poor targets for insecticidal compounds, however, they may be the first examples of genes that are not directly involved in insecticide metabolism or cuticle synthesis that increase insecticide tolerance in Drosophila. As the first metabolomics data set showed evidence for oxidative stress during permethrin exposure, preliminary work was begun for identifying the tissue specificity and timing of oxidative stress in both Dipterans and Lepidopterans using Drosophila and Bombyx mori as models. In Drosophila oxidative stress did not begin immediately suggesting that the insecticide itself is not a cause, however, a rapid increase in oxidative stress occured over a six hour period after a day of oral exposure implicating catabolites of permethrin. Bombyx were highly susceptible to permethrin showing oxidative stress in the Malpighian tubule and silk gland when exposed. This study has shown that metabolomics is highly effective at identifying pathways which modulate survival to insecticide exposure. It has also brought insight into how insecticide induced pathology may cause death. Data has also been generated which could help characterize the putative transaminase CG6950.

Research in label free methods for biological analysis has brought interesting developments. Cell impedance spectroscopy has been one of the promising outcomes. It allows the measurement of cell proliferation and motility whereby it is possible to study wound healing and cell behavior in vitro. This thesis presents the progress towards an 8-well impedance measurement setup that uses conducting polymers as electrode material in cell impedance spectroscopy. A step by step fabrication of devices with PEDOT:PSS electrodes is described along with the hardware and software, developed and integrated, to perform impedance measurements of cell cultures. Electrochemical analysis was performed for PEDOT:PSS and Au electrodes to compare the two materials for use in cell impedance spectroscopy. PEDOT:PSS electrodes showed lower interfacial impedance and reach electrochemical equilibrium faster than Au electrodes. It was observed through electrochemical impedance analysis that the lower interfacial impedance is due to the low charge transfer resistance of PEDOT:PSS. MDCK cell proliferation experiments were performed using both types of electrode materials to provide a comparative study. The impedance measurement results showed differences between the two materials that led to a different kind of electrical model for the changes measured due to cell proliferation. Curve fitting results to the electrical model provided an understanding of the cell-substrate interactions and the capabilities of cell impedance spectroscopy. The application of cell impedance spectroscopy to human embryonic stem cells was also explored. The impedance changes of pluripotent stem cells during differentiation to trophoblasts were measured and analyzed. Analysis of changes to the phase values in the frequency spectrum show that by measuring the frequency where the phase is minimum, it is possible to distinguish between the two cell types. It provides a new method of using cell impedance spectroscopy to study stem cells behavior in real time and help researchers in the maintaining of stem cell cultures in the lab. Another new application of cell impedance spectroscopy to determine cell types based on the flexibility of the cytoskeleton was also explored. Some preliminary data is presented in the last chapter.

The research presented aimed to investigate potential of subcritical fluid technology to effect Cl- release and transform compounds of copper alloy artefacts of cultural significance. The work intended to determine the most effective pH, temperature, and time combination subcritical treatment variables to: 1. transform or solvate insoluble or sparingly soluble copper compounds containing Cl - CuCl and Cu2(OH)3Cl (atacamite and clinoatacamite); 2. examine impact on typical patina compounds Cu2O and ‎Cu2CO3(OH)2 and to establish optimal conditions for their retention throughout treatment. Additionally, the research expected to offer guidance on the values of the operational parameters to use when applying subcritical treatment to archaeological copper alloy objects. The series of experiments yielded preliminary results on solvation, extraction, chemical transformation, and physical modification of the predominant corrosion products found on copper alloy artefacts. Experiments were completed using analogue pressed pellets of corrosion products, naturally corroded copper coupons and archaeological artefacts, with specific focus on corrosion profiles, metallography and microstructure. The results of extraction show significant amounts of Cl- are removed and thus the reactivity of objects is reduced. While this study conclusively demonstrated subcritical treatment is capable of both removal and transformation of Cl-bearing compounds commonly present in copper alloy objects, it cannot be recommended for treating archaeological objects based on these results alone. Aesthetic and physical changes are unpredictable and may be unacceptable. Accepting these changes cannot directly be balanced against the proven effectiveness of subcritical treatment for removing Cl, nor its rapid treatment time.

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.