This work outlines the use of Lewis acidic boranes in a variety of different reactions, mainly in the activation of unsaturated substrates such as allenes, alkynes, ketones, aldehydes and imines. The activation of allenes toward frustrated Lewis pairs showed that a formal 1,4-addition product prevails, trapping the product as the zwitterionic s-cis diene species. Conversely, reacting these same substrates with B(C6F5)3 alone allows two distinct reactive pathways; either σ-activation to yield the scarcely observed 1,2- carboboration product or π-activation followed by dealkylation to generate the γ-lactone. Following this second activation mode, the reaction between propargyl esters, amides, carbamates and carbonates with a variety of homo- and hetero-leptic boranes as well as borocations promotes the 5-exo-dig cyclisation which, depending on the substrate, was either isolated as the oxazolium or dioxaborinine heterocycle. In addition, utilising the Lewis acid PhBCl2 in this reaction interestingly produces the hitherto unreported 1,3-haloboration of alkynes. B(C6F5)3 was also shown to be hugely successful in a number of other ring closing reactions, specifically the 6-endo-dig cyclisation of alkynyl ester derivatives to form various pyrones, isocoumarins and pyryliums. Of particular interest is the development of a new methodology for catalytic carbon-carbon bond formation through alkyl group transfer. In this case, benzyl, α-methylbenzyl and benzhydryl functionalised alkynyl esters underwent the expected cyclisation using catalytic B(C6F5)3 to form the lactone however, 1,5-migration occurs from oxygen to carbon to generate the corresponding γ-functionalised pyrones and isocoumarins. Finally, a diverse array of reduction chemistry was performed by attenuating the Lewis acidity at boron to specialise its function in catalysis. Other work focused on the exploitation of novel processing technologies to improve conventional batch-type main group reactivity via continuous-flow chemistry. Additionally, biologically inspired dihydropyridines were used in the formation of thermally stable borohydrides as well as elemental hydrogen surrogates in boron mediated transfer hydrogenation pathways.

Reverse micelles are self-organized aggregates formed by a surfactant in a non-polar solvent or oil. The presence of a water pool in the polar core of reverse micelles is of considerable advantage in protein extraction. A lot of researches have been done with ionic reverse micelles applied in protein extraction. However, this ability of non-ionic reverse micelles has not been fully understood and therefore requires more research. In this project, different surfactants (anionic AOT, cationic CTAB, non-ionic triblock L61 copolymer) were investigated for their ability to form RM and for their application in protein extraction. It was found that lysozyme could be extracted using an AOT RMS, but not with a CTAB RMS. For the first time, an aromatic solvent, p-xylene, was used for the extraction of lysozyme and it was found that the AOT in p-xylene RM system resulted in the higher lysozyme activity (73.81 %) compared to an AOT/isooctane RM system (43.2 %). The effect of different salts (KCl, KF, KBr) on the FE and BE of BSA was investigated using the CTAB in mixture of 1-bromooctane, 1-hexanol and petroleum ether. The results indicated that KCl gave the highest extraction efficiency of 64 % as compared to around 40 % with both cases of KF and KBr. The secondary structure of extracted BSA was maintained with KCl only. L61 pluronics polymers was investigated for its reverse micelles forming ability and it was established that small reverse micelles with a maximum W0 of 4 was formed. Because of the small size of L61 reverse micelles, lysozyme could not be extracted but was precipitated out when combined with the co-surfactant AOT. The activity of the recovered lysozyme from the precipitate was maintained (66% as compared with native lysozyme). Moreover, if L61 was used as a co-surfactant with AOT reverse micelles, extraction efficiency was improved (88 %) and the activity of the extracted ly-sozyme was increased (56 %) as compare to extraction with an AOT system alone (46 %). These studies thus gives useful insights in the role of individual and mixed surfactant systems in the extraction and precipitation of proteins.

Fragmentation of molecules by collision-induced dissociation (CID) is not well-understood, making interpretation of MS/MS spectra difficult and limiting the effectiveness of software tools intended to aid mass spectral interpretation. An approach is required which is tailored to each individual molecule and improves the ‘chemical sense’ of the software. It was hypothesised that the bonds which break during CID of protonated molecules are the bonds which are elongated, and hence weakened, as a result of conformational changes induced by protonation. Bond length changes for a test set of molecules were calculated using quantum chemistry software. Density Functional Theory (DFT) or Austin Model 1 (AM1) or both were used to energy minimise the structures of the neutral molecules and their corresponding protonated molecules (protonated at all possible sites). Bonds which elongated to the greatest extent after protonation were compared to the bonds which were found to cleave to give the product ions in the CID spectra of these compounds. Quantum chemistry modelling was also applied to the deprotonated molecules. AM1 calculated bond lengths were found to be similar to those generated by DFT and have the advantage of being rapidly obtained. All the polarised bonds which cleaved were calculated to elongate significantly, thus achieving a 100% success rate in the prediction of bond cleavage as a result of protonation on a heteroatom. The proton is mobile across the molecule, leading to fragmentation when the proton reaches a site where it causes significant bond elongation, provided the molecule has sufficient internal energy. Cleavage of carbon-carbon bonds was not predicted. The success rate for predicting bond cleavage in deprotonated molecules was 48%, suggesting this approach cannot be applied reliably for these anions. AM1 calculated bond length change acts as a descriptor for predicting polarised bond cleavage in protonated pseudo-molecular ions having the potential to be incorporated in mass spectral interpretive software to increase the accuracy of prediction of CID spectra.

Four new N-isopropylacrylamide (NIPAM) based particles have been synthesized and characterized. The first experimental chapter reports that fluorescent poly(Nisopropylacrylamide-co-5% vinyl cinnamate) microgels were deposited on different solid surfaces to produce new environmental responsive surfaces. The mass of microgel particles deposited on glass pre-treated with acid, glass pre-treated with base, quartz, stainless steel, gold and Teflon at 25°C and 60°C was determined using fluorescence spectroscopy. The factors affecting the adsorption/desorption of the microgel particles were also investigated. The study shows that the solid surface charge is the most significant factor, followed by that of surface roughness and temperature; meanwhile, the hydrophobicity/hydrophilicity of the surface was the least significant. Fluorescent temperature/pH responsive p(NIPAM-co-5% acrylic acid)-rhodamine B particles were studied. The results confirm the attachment of rhodamine B to the microgel particles. The long chains attached to the particles caused the particles to be poly-dispersed which suggests non-uniformity of particle size and behavior. Fluorescent temperature/pH responsive poly(N-isopropylacrylamide-co-5% lucifer yellow) (p(NIPAM-co-5% LY)) microgel particles were tested for toxicity. The particles were negatively charged, mono-dispersed and were approximately 250 nm in diameter at 15°C. The toxicity of different concentrations of p(NIPAM-co-5% LY), p(NIPAM) and NIPAM monomer was tested on two cell lines (HeLa and Vero). The results show that the two particles maintain cell viability over 80% (for both cell lines HeLa and Vero) up to a concentration of 3 mg/mL while NIPAM monomer showed a cell viability of over 80% at a concentration equal to or less than 0.3 mg/mL. The fourth experimental chapter explores the opportunity of using microgel particles as an emulsifier. p(NIPAM-co-5% acrylic acid)-hexenol particles with both hydrophilic and hydrophobic groups in their molecular structure were used to stabilize two emulsions (tricaprylin-in-water and hexadecane-in-water). The stability of the emulsions increased with increasing the concentration of the microgel. For the tricaprylin-in-water emulsion, a concentration of 0.3% w/v of the novel microgel particles managed to stabilize the emulsion for a week. In case of the hexadecane emulsion in water, 0.45% w/v microgel particles could show an increase in the emulsion stability and a decrease in the creaming.

The main objective of this study was to investigate different manufacturing processes claimed to promote inclusion complexation between different drugs and cyclodextrins (econazole and α-cyclodextrin; indomethacin and methyl-β-cyclodextrin; olanzapine and methyl-β-cyclodextrin; flurbiprofen and methyl-β-cyclodextrin) in order to enhance the apparent solubility and dissolution properties of drugs. Specifically, the effectiveness of supercritical carbon dioxide processing for the preparation of solid drug-cyclodextrin inclusion complexes was investigated and compared to other preparation methods. Nitrate, besylate, sulfosalicylate dihydrate and maleate salts of econazole were synthesised. The solid drug-cyclodextrin inclusion complexes were prepared by physical mixing, freeze drying from aqueous solution and processing with supercritical carbon dioxide. The complexes were evaluated by scanning electron microscopy, differential scanning calorimetry, X-ray powder diffraction, 1H-nuclear magnetic resonance (nuclear Overhauser effect correlation spectroscopy and inversion recovery T1 measurement experiments), and dissolution rate studies. Inclusion yield (%) studies of econazole base into α- and methyl-β-cyclodextrin were conducted in supercritical carbon dioxide to investigate the influence of pressure, temperature and contact time on the inclusion. All the working parameters (pressure, temperature and contact time) played a significant role in the inclusion of econazole base into cyclodextrins. Isothermal titration calorimetric studies of econazole besylate and sulfosalicylate dihydrate salts and α-cyclodextrin confirmed the formation of complexes between the salts and α-cyclodextrin in a 1:1 stoichiometry. Different degrees of crystallinity were observed in the analyses of products prepared by various methods, suggesting the possibility of drug-cyclodextrin interactions of different efficiencies, which may give rise to different degrees of inclusion formation and/or crystallinity of the sample. Nevertheless, products obtained by the freeze-drying and supercritical carbon dioxide-inclusion methods were among the ones showing the highest interaction between the drug and the cyclodextrin. All systems based on α-cyclodextrin and methyl-β-cyclodextrin exhibited greater drug release profiles than the drug alone. Solid state complexation using supercritical carbon dioxide processing proved to be useful complexation method for econazole and its salts into α-cyclodextrin; indomethacin, olanzapine and flurbiprofen into methyl-β-cyclodextrin. The freeze drying method produced highly amorphous and rapid dissolving complexes; however, it was characterised by long, energy-intensive processing steps. Supercritical carbon dioxide inclusion method was shown to be an efficient approach for the preparation of solid-state inclusion complexes. It is an efficient and economic process that allows the formation of solid complexes based in strong intermolecular forces in high yield in a single step avoiding the use of organic solvents and the problems associated with their residues.

The aim of the research reported herein was to identify and develop a dried blood spot (DBS) direct analysis technique that could support high sample throughput quantitative bioanalysis in a regulated drug development environment. An initial literature review, coupled with proof of concept testing of the most prominent direct analysis techniques coupled to mass spectrometers (MS), resulted in direct elution (direct extraction of DBS via a confined solvent, producing a liquid extract) being selected as the most suitable technique to develop for this application. Direct elution technology was then developed into fully automated techniques with sufficient functionality to enable compatibility with high sample throughput quantitative bioanalysis. Proof of concept robustness data demonstrated that direct elution, despite the lack of sample clean up, was a reliable technique which had no detrimental effects on detector or chromatographic performance compared to conventional wet plasma extraction and analysis. A proof of concept investigation also demonstrated that a method of improving internal standard (IS) performance by spraying IS solution onto DBS samples prior to extraction, allowed the analyte of interest and IS to be co-extracted, while retaining adequate analytical performance. The foregoing proof of concept data was then combined to produce a fully automated DBS direct elution instrument designed to introduce sample extracts into a LC-MS/MS system. This instrument incorporated a 500 DBS card capacity, an intelligent visual recognition system, a dynamic IS applicator module, and a highly effective wash system that virtually eliminates carryover. Ultimately, this work led to the production of a fully automated DBS direct elution system that is now commercially available. Subsequent research focused on optimising the system, and using this technology to address some of the issues that are currently inhibiting the development of DBS usage in drug development applications, namely haematocrit (HCT) based assay bias, and the decreased sensitivity on offer from DBS sampling. It was demonstrated that using the IS sprayer enabled the IS to integrate sufficiently with the DBS sample prior to extraction to nullify HCT based recovery bias. The direct elution mechanism was also optimised with a view to maximising assay sensitivity while retaining acceptable analytical and chromatographic (LC-MS/MS) performance. Generic optimised direct elution conditions were developed which demonstrated that increases in assay sensitivity of up to 30 fold (compared to conventional manual extraction methods) were possible using a set of representative small molecule compounds.

Bitter pit is an important physiological disorder of many apple cultivars where the low uptake and poor distribution of calcium within the cortex of apples pervades. Controlled atmosphere storage and application of 1-MCP (SmartFreshSM) can delay the onset of bitter pit symptoms by delaying maturity and senescence; however, significant losses may occur in long-term stored apples. It is hard to detect internal bitter pit using external examination alone. Previous studies have focused on improving pre-harvest prediction and curative treatments before harvest. Present prediction models are based on history of orchards, mineral analysis 2-3 weeks before harvest and quality assessments and monitoring over storage time. This study aimed to identify a greater understanding of the storage potential of fruit based on destructive standard quality assessments, biochemical and molecular analysis, also a non-destructive monitoring method by chlorophyll fluorescence at the point of harvest and monitoring during storage for developing more reliable prediction models to improve storage management. The role of free and conjugated calcium in maintaining cellular integrity and the relationship between biochemical and fluorescence changes and development of bitter pit were investigated. A diagnostic model based on comparison of changes of ascorbic acid during storage was developed. Another diagnostic model based on changes in the proportion of calcium oxalate content during storage in comparison with harvest was developed to identify samples with higher propensity to bitter pit. Also chlorophyll fluorescence was investigated as a non-destructive method for monitoring fruit during storage and prediction models for detecting changes in the maturity of fruit and developing bitter pit and reduction of fluorescence during storage as an alert to identify incidence of bitter pit were developed. Furthermore, changes in gene expression profiles of a limited number of genes like calmodulin showed the differences in patterns of transcripts between apples suffering from bitter pit and healthy apples. All the suggested methods have potential of being commercialised and applied practically to improve apple fruit store management. It would be possible to build a multi variate model for predicting the onset of bitter pit development in apple by combination of two or more suggested diagnostic tools.

Leishmania parasites are the causative agent of a wide range of human diseases known as leishmaniasis. The progression of disease pathogenesis is dependent on the ability of intracellular parasites to disseminate between human macrophages. It is assumed that parasites could exit macrophages following cell lysis. However, there is still a major gap in knowledge in understanding the exact mechanism pertaining to this critical step. This has been partially caused by the lack of reliable experimental models to study this stage of host-parasite interaction. This research aims to elucidate the mechanism behind cell-tocell spreading of Leishmania in vitro. A link between host cell apoptosis and Leishmania propagation was initially established by determining the effect of apoptotic induction on infection over a period of 96 hours. The result showed that 60-90% of cells were inoculated with L. aethiopica and L. mexicana within 72 and 96 hours infection. Although, the concentration of viable cells was greatly reduced as compared to non-induced samples, a heavily infected cell population was obtained with this approach. Hence, this population of L. aethiopica and L. mexicana infected cells at 72 hours was used to infect differentiated THP-1 macrophages and validated as a model for infection spreading. Amastigotes spreading was detected within 12 hours of coculture as confirmed by flow cytometry and live cell imaging techniques. Interestingly, the live imaging revealed inter-cellular extrusion of L. aethiopica and L. mexicana from infected to a recipient cells without cell lysis. To address whether parasite extrusion is linked to apoptotic induction, this model was used to detect expression of several apoptotic markers. The results showed that L. aethiopica (but not L. mexicana) dissemination was correlated with host apoptotic induction, as there was a significant increase (p < 0.05) in phosphatidyl serine (PS) externalization and active caspase-3 expression. In addition, western blot analysis demonstrated that L. aethiopica spread was associated with downregulation of three major apoptotic signaling pathways: AKT, NF-κB and PKC-δ. Further comparative proteomic analysis of this model supported these findings and showed that during L. aethiopica spreading, a number of host physiological processes were subverted. Specifically caspase-3 and caspase-9 activation strengthens the involvement of mitochondria in initiation of apoptosis. Interestingly, chitinase-3-like protein (inducer of IL-13 production) and butyrophilin (inhibitor of IFN-γ and IL-12) were markedly increased confirming the downregulation of inflammatory response. Finally, elevated increase in glutathione reductase and metallothionein-2 proteins, which are involved in scavenging free radicals, suggests that anti-microbial activity is also downregulated during L. aethiopica spreading. In conclusion, by developing and validating for the first time a credible in vitro spreading model in human macrophages, this research has clarified the mechanism of Leishmania spreading. L. ethiopica promotes host cell apoptosis in order to disseminate from cell-to-cell without activating inflammatory and anti-microbial responses. In particular, apoptotic induction takes place via downregulation of AKT and NF-κB, release of cytochrome C and consequent activation of caspase-3 and PS exhibition, independently from PKC-δ. This model clearly fits with the silent spread of parasites that takes place in the first weeks (to months) of infection in mice model. Interestingly, the fact that L. mexicana utilizes a non-apoptotic strategy to spread suggests that alternative mechanisms of parasitic extrusion also exist underlying the necessity of species specific investigations. Understanding the molecular basis of Leishmania spreading contributes to filling a current gap in knowledge in the biology of Leishmania infection strategy of this very successful parasite. Moreover, it has the potential to open up a novel set of targets for drugs and vaccine development against this increasingly widespread neglected disease.

Ion mobility-mass spectrometry experiments have been conducted to measure the drift-time and calculate collision cross-sections (CCSs) using travelling wave ion mobility spectrometry, and determine the CCS using drift-tube ion mobility spectrometry systems of analytes. The aim of the study was to identify if predictive approaches could facilitate rapid and definitive assignment of charge location sites and chemical structure. Molecular modelling was conducted to determine the energy minimised/geometry optimised structures and charge distribution of the protonated molecules studied. The geometry and charge distribution data were utilised in subsequent ion mobility calculations using two main methods 1) projection approximation and 2) trajectory method. Fluoroquinolone antibiotics were investigated as previous literature had postulated the ion mobility separation of charge location isomers differing only by their protonation site with little expected difference in their geometry (see Chapter 2). Projection approximation prediction of theoretical CCSs (tCCSs) for the singly protonated molecules of norfloxacin (with the proton assigned to all possible oxygen or nitrogen-containing protonation sites to generate candidates) revealed < 2 Å2 difference in tCCSs based on molecular modelling. In stark contrast the experimental CCS (eCCS) demonstrated > 10 Å2 difference between different components. The product ion spectra are consistent with the hypothesis of charge location isomer mobility separated components. Investigations with other fluoroquinolones, with both drift-tube ion mobility and travelling wave ion mobility, and using the trajectory method, remain consistent with the hypothesis of charge location isomers (see Chapter 3). A larger scale study sought to probe the accuracy of tCCSs over a large number of small molecule drug structures. If tCCSs accurately predict eCCSs, then tCCSs could be used to identify compounds and isomers based on their CCSs (see Chapter 4). Finally, software was developed to considerably accelerate the calculation of trajectory method tCCSs from 8-100 times faster than existing published approaches depending on available computing infrastructure (see Chapter 5). In summary this research project has explored whether eCCSs and tCCSs may be useful as a key structural tool alongside other traditional measurements including chromatographic retention time and m/z.

The first section of this thesis describes the development of a one-pot Pd(II)- catalysed Overman rearrangement, Ru(II)-catalysed ring closing enyne metathesis reaction and a hydrogen bond-directed Diels-Alder reaction for the diastereoselective synthesis of C-5 substituted aminobicyclo[4.3.0]nonanes in good yields. To explore the late stage diversification of these compounds, further work investigated a one-pot synthesis of a benzyldimethylsilyl-derived analogue. The synthetic utility of this compound was demonstrated using C–C bond coupling reactions, for the late stage synthesis of a range of sp3-rich bicyclononane scaffolds with up to six stereogenic centres. The second section of this thesis describes the development of a one-pot two-step procedure for aryl C–H amination using iron and copper catalysis. Firstly, a mild and highly regioselective method for the bromination (and chlorination) of arenes via iron(III) triflimide activation of N-bromosuccinimide (or N-chlorosuccinimide) was developed. The scope of both processes was explored for the synthesis of a wide range of aromatic compounds and natural products. The one-pot iron(III)- catalysed bromination/Cu(I)-catalysed N-arylation was then studied. After optimisation, the one-pot two-step process allowed the synthesis of a large library of para-aminated aryl compounds in high yields as single regioisomers.