Chronic neuropathic pain (CNP) occurs as a consequence of injury to the nervous system. Despite recent advances, CNP lacks objective diagnostic criteria, is often unrelenting and refractory to treatment. The primary aims of this thesis are twofold; the identification of CNP biomarkers using both human cohorts and an animal model (spinal nerve ligation; SNL) of neuropathic pain, and to provide clarity on the role of GTP cylcohydrolase I (GCH1) in CNP. Analysis of GCH1 and related genes and metabolites was conducted. As biomarkers, nitrite/nitrate and neopterin did not differentiate controls from CNP patients. However, significant differences were observed with biopterins, whilst correlations were observed between GCH1, nitrite/nitrate and neopterin, which were notably stronger in patients than controls. Analysis in human cohorts and in the SNL model also inferred that downregulation of GCHFR may contribute to BH4 synthesis. In order to provide clarity on the role of the GCH1 pain protective haplotype, reporter gene assays were used. This demonstrated a potential regulatory role for the GCH1 5’ SNP (rs8007267). In silico prediction of transcription factor binding sites suggested that this may be mediated by the aryl hydrocarbon nuclear translocator. The use of electrophoretic mobility shift assays showed strong specific binding with probe pertaining to the major allele. Further analysis is required to elucidate transcription factor binding, potentially facilitated by 2D-PAGE and mass spectrometry. In order to further elucidate potential CNP biomarkers, microarray analysis and qRT-PCR were performed using blood obtained from CNP patients. Data refinement led to the isolation of 27 potential CNP biomarkers, of which several cross-validated between cohorts. Microarray data, literature evidence, and correlations with previous microarrays provided evidence suggestive of a role for TIMP1. Multiple other genes, including CASP5, TLR4, TLR5, MC1R and CX3CR1, were differentially regulated in CNP. Genes surviving microarray data refinement were subsequently analysed in the dorsal horn of Sprague Dawley and Wistar Kyoto rats after SNL. Several genes, including Dpp3, Mc1r and Timp1, were similarly differentially expressed in the rodent SNL model, which suggests that these genes may be involved in the pathophysiological mechanisms of CNP, and may also function as potential translational biomarkers of CNP. This work provides multiple avenues for expansion and further investigation. Clearly, the challenges associated with biomarker discovery in CNP states are considerable, though it is hoped that this thesis provides valuable insight and the necessary foundation for future work.

The purpose of the research was to evaluate the coefficient of performance (COP) of both 150 L split and integrated type air source heat pump (ASHP) water heaters via experimental analysis, statistical tests and mathematical modelling. The ASHP water heaters are used as a potential replacement of inefficient geyser for the production of sanitary hot water due to the excellent efficiency of COP ranging between 2 and 4 and also the capability of reducing the electrical energy consumption by 50-70%. Both types of ASHP water heaters together with a 150 L geyser that served as the control experiment were set up such that distinctive real-time simulated volumes of hot water (100, 50 and 150 L) were drawn off from each of the storage tanks per day over a full year. A data acquisition system (DAS) was designed and built comprising of power meters, flow meter, temperature sensors, ambient temperature and relative humidity sensors in order to monitor the electrical, thermo-physical and environmental contributions of the various hot water heating devices. The hot water set point temperature on each of the technologies was 55oC and the volume drawn off corresponded to the demand during the morning, afternoon and evening, respectively. This mimic the profile of a typical middle or highincome family (3-4 adults) in South Africa. The results depicted that the average annual COP, load factor, and energy saving of the split and integrated type systems was 2.95 and 2.45; 10.2 and 16.7% and 2.770 and 2.499 MWh while the simple payback period was 3.9 and 5.2 years, respectively. The reliefF test revealed that the predictors (ambient temperature and relative humidity) were secondary factors while the electrical energy consumed, the difference in the temperature of the refrigerant at the inlet and outlet of the compressor and condenser were the primary factors to the COP. The derived multiple linear regression models exhibited an excellent determination coefficient of over 90% between the calculated and modelled COP of both types of ASHP water heaters. Finally, the 2D multi-contour plots simulation was accurately used to show the variation of each predictors to the COP. Also, a simulation application to simultaneously compare the COP of both types of ASHP water heaters was developed in the Simulink environment utilising the derived mathematical models. Heat pump manufacturers and energy service companies can employ both the 2D multi-contour plots simulation and the simulation application to show the variation of the specific predictors with the COP and to predict the COP of both types of ASHP water heaters. Conclusively, the research provides substantial evidence for both policy makers and home owners to justify the techno-economic and social benefits of retrofitting a geyser with an ASHP water heater.

No description available.

In this thesis, iodoarene and chiral iodoarene reagents have been developed and used catalytically in the enantioselective cyclisation reaction of unsaturated esters, amides and β-amidoketones. The results in this thesis are divided into three parts. In the first part, the development of the catalytic enantioselective cyclisation of 4-methoxy but-3-enyl benzoate to the 3-hydroxytetrahydrofuran ester using enantiomerically pure chiral hypervalent iodine reagents generated in situ is described. A good enantioselectivity of 77% ee was obtained with moderate yield. In the second part, the development of catalytic conditions for the cyclisation of N-alkenylarylamides induced by iodoarenes leading to the formation of different ring sizes is demonstrated. In addition, the catalytic enantioselective cyclisation of N-alkenylarylamides employing chiral iodoarenes giving dihydrooxazines in good yields and moderate enantioselectivities of up to 64% is achieved. In the third part, the catalytic cyclisation of both propargyl amides and β-amidoketones using 2-iodoanisole to afford 2-oxazolines in good yields is described. Moreover, the catalytic enantioselective cyclisation of β-amidoketones using several chiral iodoarenes is detailed but, unfortunately almost no enantioselectivities was observed.

This thesis describes several approaches to the synthesis of novel iodonium salts. Firstly, attempts to synthesise a variety of novel alkynyl(aryl)iodonium salts (i-iii) is discussed. Most of these attempts were unsuccessful, however, useful information for future work was obtaine. The synthesis of iodonium salt (iii) was successful and this compound displayed some unusual reactivity. Several alkynyl(phenyl)iodonium trifluoroacetates were synthesised directly from commercially available iodobenzene diacetate and terminal acetylenes precursors with a range of substitution patterns in one step in excellent yields. These species were converted into alkenyl(arylsulfonyl)iodonium tetrafluoroborates in excellent yields by stereoselective Michael-type addition of arylsulfinic acids under protic conditions. Secondly, new reactivity of alkenyl(arylsulfonyl)iodonium tetrafluoroborates was investigated. Specially, the conversion of these selectively into aldehydes and vinyl chlorides using aqueous DMSO was achieved. Thirdly, two examples of stable enynyl(phenyl)iodonium trifluoroacetates were synthesised in excellent yields. These were converted into a new range of dienyl(aryl)iodonium salts. Finally, a computational study of alkynyl(aryl)iodonium salts was undertaken in an attempt to rationalise our experimental data on reactivity and stability.

The objective of this study was to investigate the acylative-cyclisation-decarboxylation reactions of enamino acids derived from 1,3-difunctional compounds. Remarkably little is known regarding the generality of these variants of the Zav’yalov pyrrole synthesis, despite their considerable scope for the synthesis of functionalised pyrroles. The cyclisation of diethyl 2-(1-carboxyalkylaminomethylene)malonates provided access to a range of 5-(un)substituted-4-acetoxypyrrole-3-carboxylates. However, in some instances the corresponding 4-ethoxypyrrole-3-carboxylates also accounted for up to 20% of the reaction product. 1-Acetyl-4-ethoxy-5-ethylpyrrole-3-carboxylate was characterised by X-ray crystallography. Some of the (aminomethylene)malonates from bifunctional α-amino acids provided anomalous products. For example, the glutamine-derived enamino malonate gave a 5-acetylpyrrolidin-2-one via a Dakin-West-type reaction. The asparagine-enamino malonate cyclised to 4-acetoxy-1-acetyl-5-cyanomethylpyrrole-3-carboxylate probably via an isosuccinimide intermediate. Several mechanisms for the formation of the pyrrole products have been discussed. A 13C-labelling experiment confirmed that the carboxyl function in the starting material is not incorporated in the product. Evidence for the involvement of a 1,3-oxazolium-5-olate (münchnone) accrued from cyclisation of diethyl 2-(1-carboxymethylaminomethylene)malonate with Ac2O in the presence of dimethyl acetylenedicarboxylate which provided a novel 1-alkenylpyrrole, characterised by X-ray crystallography. An alternative pathway supervenes in the Zav’yalov reaction when α,α-disubstitution of the amino acid prevents münchnone and thus pyrrole formation to afford an N-alkenyloxazolidin-5-one. Novel ethyl 4-(di)acetamido-5-(un)substituted-pyrrole-3-carboxylates and the corresponding 3-carbonitriles have been obtained in good yields via the cyclisation of ethyl 2-(1-carboxyalkylaminomethylene)cyanoacetates and (1-carboxyalkylaminomethylene)malononitriles respectively. Evidence for a different cyclisation pathway, in the former, involving intramolecular acylation of the enaminonitrile moiety was observed. Thus, ethyl (2R*,3S*)-1-acetyl-3-cyano-2,4-diacetoxy-5-methyl-2,3-dihydropyrrole-3-carboxylate was characterised by X-ray crystallography. A wide range of novel 2-alkanoyl- and 2-aroyl-3-(1-carboxyalkylamino)acrylonitriles has been obtained via aminomethylenation of β-ketonitriles. The products from their cyclisations (Ac2O-NEt3) were largely independent of the nature of the acyl group but determined by the substituent in the α-amino acid moiety. The 3-(1-carboxy-1-phenylmethylamino)acrylonitriles provided mixtures of 3-acyl-4-(di)acetamido-5-phenylpyrroles in which the 4-acetamido- derivatives predominated. Contrasting behaviour was displayed by the 3-(1-carboxyalkylamino)acrylonitriles derived from alanine, 2-aminobutyric acid and valine in which the cyclisation followed an unexpected course, via enaminone acylation, to the novel 4-acetoxy-1-acetyl-5-alkylpyrrole-3-carbonitriles in high yields. The acylative cyclisation of the 2-acyl-3-(1-carboxymethylamino)acrylonitriles furnished mixtures of pyrroles. In two cases, 3-acetamido-6-aryl-5-cyanopyran-2-ones, generated by a unique cyclisation pathway were isolated. The structure of the 6-phenyl- derivative was confirmed by unambiguous synthesis. The synthesis and acylative cyclisation of (Z)-2-benzoyl-3-(1-carboxyalkylamino)crotononitriles was investigated. Whereas the 3-(1-carboxyethylamino)- derivative provided 4-acetoxy-1-acetyl-2,5-dimethylpyrrole-3-carbonitrile exclusively, the 3-(1-carboxy-1-phenylmethylamino)crotononitrile afforded a mixture of pyrroles. A remarkable minor component was characterised as 4-acetoxy-1-benzoyl-2-methyl-5-phenylpyrrole-3-carbonitrile, the result of sequential [1,5]-benzoyl migrations of a 3H-pyrrole intermediate. The acylative cyclisation of 3-(1-carboxyalkylamino)-2-tosylacrylonitriles provides access to hitherto unknown 3-diacetamido-4-tosyl- and 3-acetamido-4-tosylpyrroles. Cyclisation of 2-(1-carboxyalkylaminomethylene)dibenzoylmethanes offers an excellent, complementary approach to access 5-(un)substituted-3-benzoyl-4-phenylpyrroles, to the existing tosylmethyl isocyanide-based protocols.

Reported herein is an investigation into palladium-catalysed -allylation employing sulfonamide nucleophiles. Anions of benzylsulfonamides have been shown to react with a series of allyl acetates in the presence of Pd0 catalysts, phosphine ligands and base at room temperature, enabling the synthesis of sp3-functionalised sulfonamides. The developed methodology has allowed access to a library of novel allylated sulfonamides, varying both amine substituent and allylic functionality. In addition, we have applied our methodology to a series of known sulfonamide drug targets, to demonstrate our reaction as a useful late-stage functionalisation tool, whilst populating chemical space. The performed mechanistic study using a stereospecific electrophile confirms benzylsulfonamides behave as soft carbon nucleophiles in the Tsuji-Trost reaction, as a ‘net retention’ of stereochemistry is observed (confirmed by X-ray crystallography). Moreover, the asymmetric synthesis of allylated sulfonamides is probed, although obtaining enantioselectivity a- to SO bonds is naturally difficult, due to the conformational preferences of sulfonamide carbanions. Traditional methods for direct -alkylation of sulfonamides require strong bases, reactive electrophiles, low temperatures and use of stoichiometric amounts of additives. Therefore, in addition to a catalytic method, we report an alternative method reacting benzylsulfonamides with allyl bromide electrophiles via a nucleophilic substitution reaction, using mild conditions (LDA, THF at –20 °C).

DNA profiling has become a universal technique for identifying individuals for evidential use in courts of law. In more complex cases such as sexual assaults, identifying the origin of a stain or sample offers valuable information as to the events that occurred. Currently, many ‘in service’ body fluid identification (BFID) techniques are presumptive, require significant sample volumes and generate false positives. As such, the development of a highly specific and reliable BFID technique would be highly beneficial to forensic scientists in provide more informative and reliable evidence. MicroRNAs (miRNA) are short, stable, non-coding RNA’s which modulate gene expression. Expression of some of these miRNA are body fluid specific, making them a potentially robust tool for BFID. The possibility for the integration of a robust, miRNA based BFID technology for forensic casework employing stem-loop reverse transcription and qPCR analysis was the theme of the research presented here. To be incorporated into the workflow of current forensic laboratories, the protocol must be able to be carried out alongside current techniques with limited addition of cost, equipment, analysts and time. A range of custom designed miRNA markers were analysed on vaginal material, menstrual blood, saliva, venous blood, semen, seminal fluid and skin. Screening indicated specificity of hsa mir-124 to vaginal material, hsa-mir-10a, 135a and 888 to semen, hsa-mir-412 and 507 to menstrual blood, hsa-mir-144-3, 144-5, 142 and 451 to blood and although highly expressed in saliva, hsa-mir-205 was also observed in vaginal material. Universal expression was observed in hsa-mir-93, 508, 1260b and SNORD 47 providing a means of normalisation through the designation of these markers as endogenous controls. A combined panel of markers are presented which were capable of identifying all body fluids, excluding skin from single stains. The panel was successful at identifying certain mixtures such as semen within vaginal material but was unable to confirm saliva presence within vaginal material. Screening of hsa-mir 205 within vaginal material uncovered the observation that hsa-mir-205 was impacted by the use of female contraception. Once a full BFID panel was generated the robustness of the markers was further analysed across the menstrual cycle. No significant difference (p > 0.001) was seen in markers highly expressed in vaginal material during screening (hsa-mir-124, 203a, 205). Expression of non specific markers highlighted the importance of the optimisation of input miRNA. Differential extraction of genetic material was found to be detrimental to miRNA sample integrity. As such, total DNA extraction was employed for vaginal swabs obtained from volunteers following unprotected sexual intercourse, markers hsa mir-10a, 135a and 888 were able to successfully detect semen presence for up to 96 hours. The data generated to date has highlighted a number of miRNA markers that provide a platform for BFID. The developed protocol is reliable and robust; requiring minimal optimisation and is capable of integration with current laboratory workflow with minimum implications to time and cost. The markers identified for BFID have been implemented within studies that are representative of real case scenarios, and have demonstrated their ability to be stable, specific and successful in the identification of certain body fluids. Overall, this research showcases a reliable and body fluid specific protocol capable of being performed alongside DNA profiling.

The current plan for the permanent disposal of Low Level and Intermediate Level nuclear wastes is the cement-based geological disposal where the wastes will be disposed off in geological disposal facility (GDF). The chemical evolution of the GDF is expected to cause cellulosic materials in the waste to degrade into cellulose degradation products (CDP) of which isosaccharinic acids (ISA) are the major components. ISAs are reported to form complexation reactions with radionuclides, potentially enhancing their migration out of the GDF. Recent studies have shown that microbial consortia indigenous to anthropogenic sites can potentially degrade these ISAs but have focused on the use of different nitrogen sources Ca(ISA)2 as analogue for CDP. This study therefore aimed at characterizing novel ISA degrading bacteria and investigate the metabolic potentials of microorganism within soil sediments from Harpur Hill site to biodegrade ISAs and assess the impact of using Ca(ISA)2 or CDP with NH4+ against NH4+ free systems on the chemical and microbial community evolution under conditions representative of the GDF. In a batch/fed microcosms, microorganisms within the soil sediment were able to biodegrade ISAs in pH ranging from 9-11.5 irrespective of the source. The microbial community evolution in these systems were however different suggesting that the type of carbon source and the presence of a nitrogen source impacted on the selection of these communities. The evolution of the microcosms gave rise to a complex methanogenic, polymicrobial communities where the degradation of ISAs led to the formation of acetic acid and gases including CH4, CO2 and H2. The formation of these gaseous products are likely to contribute to the pressurization of the GDF as a result of which the porosity and permeability factors should be taken into account in the formulation of the cement backfill materials. Molecular characterization of the ISA degrading communities and pure isolates (Exiguobacterium sp. strain Hud and Oceanobacillu sp. strain Hud) will allow for studies into genes associated with ISA degradation which is currently lacking in the literature. The Harpur Hill site presents a diverse pool of microorganisms with the metabolic potentials to degrade ISA hence it could be a good candidate site for the GDF.

Background: Recent developments in our understanding of the biology of cancer has provided the opportunity to develop targeted agents with more specific pharmacological activity against cancer cells. Despite this shift toward targeted drug discovery, the much hoped-for paradigm shift in cancer treatment has not been realised. Tumour heterogeneity, plasticity and genomic instability are issues that contribute to this problem. One approach to circumvent these issues is to adopt a phenotypic based approach to drug evaluation where compounds with multiple mechanisms of action leading to a desirable phenotypic effect can be identified. The challenge with such an approach is to retain selectivity toward cancer cells as opposed to non-cancer cells. Aims: The aim of this study is to apply a phenotype based drug evaluation program that incorporates a measure of selectivity to the preclinical evaluation of a series of novel organometallic complexes. Methods: In this study, a series of novel inorganic complexes were evaluated against cancer and non-cancer cell lines. The primary evaluation procedures involved chemosensitivity testing with compounds being selected for further studies based upon (i) potency (ii) an in vitro selectivity index (SI) defined as the IC50 for non-cancer cells divided by the IC50 for cancer cells and (iii) comparable or improved properties than cisplatin, oxaliplatin and carboplatin with respect to potency and selectivity. Those compounds that met the selection criteria were evaluated further with the initial aim of characterising key pharmacological events such as cell cycle effects and induction of apoptosis. Results and Discussion: Initial studies focused on the clinically approved platinum based with cisplatin and oxaliplatin being significantly more potent than carboplatin. Selectivity for cancer over non-cancer cells was observed with selectivity index (SI) values typically in the range of 0.85-9.71, 0.36-3.35 and 2.18-7.44 for cisplatin, oxaliplatin and carboplatin respectively. A total of 210 test compounds were evaluated in this thesis and of these, a total of 91 compounds exhibited potency values equal to or better than the platinates. In contrast however, only 64 compounds had superior SI values compared to platinates. Of these, the most promising compounds were a series of large molecular weight metallohelicates that exhibited potency (in the nM range) and SI values up to a maximum of 93 (nearly 28 times higher than the best performing platinum drug). Analysis of these compounds demonstrated that they do not induce apoptosis, but preliminary data suggests that they induce an autophagic death response. Conclusions: The results of this study have demonstrated that a phenotypic based drug evaluation process based upon potency and selectivity in vitro is capable of identifying novel chemical entities with promising properties. This screen has more discriminatory power than potency alone and the concept of an 'in vitro selectivity index' has proved valuable in identifying a series of novel metallohelicate compounds as potential anti-cancer drugs. Significant further work is required to identify their mechanism(s) of action and pharmacological properties but their potential ability to induce autophagic cell death over apoptosis is of interest.