Search

Broilers have high dietary requirements for protein as a major constituent of the biologically active compounds in the body and a vital nutrient for growth. In addition, protein and essential amino acids are essential for antibody formation and a well-functioning immune system, however protein is one of the most expensive nutrients in poultry diets. Therefore, considerable attention is being given to optimise (minimise) crude protein (CP) levels in poultry diets, including by applying the ideal protein concept (IP), which represents essential amino acid (AA) requirements as a ratio to lysine leading to an ideal dietary AA profile. However, since AA supplementation has been shown to improve resilience to sub-clinical pathogen exposure, the assumed generality of the IP concept may be challenged. The aim of this project was to investigate the effect of AA supplementation on resilience of broiler chickens to sub-clinical health challenges using IP-based rationing as the starting point. The first objective was to assess the sensitivity of broiler resilience to reused litter exposure and to supplementation of IP-based control diets with individual or a combination of threonine, arginine and glutamine (TAG) on performance, plasma biochemistry and organs weights. In the first study, TAG was included at the expense of corn starch to maintain iso-energetic conditions. Host responses to specific AA supplementation may be sensitive to microbiota exposure from reused litter, as immune and pathological responses could result in increased whole bird AA requirements that deviate from the ideal AA profile. Since the control diet was formulated on an IP-basis, it was hypothesised that AA supplementation improves performance in the presence but not in the absence of exposure to reused litter. Our results showed that reused litter exposure resulted in greater weight gain, better feed conversion ratio and heavier bursal weights compared to birds on clean litter. However, TAG birds reduced weight gain and feed intake relative to control birds. Since reused litter benefitted performance and the control diet was IP formulated, the absence of positive responses to AA supplementation would be consistent with AA tested being in excess and resulting in a cost rather than a benefit on growth performance and feed efficiency. To test this hypothesis, we assessed whether TAG supplementation leads to excess protein associated changes in caecal microbiome profiling and metabolites and litter characteristics of broilers exposed to reused litter. Our results confirmed that the impaired growth of birds fed TAG diets compared to control diets may have arisen from enhanced proteolytic fermentation, supported by elevated caecal iso-butyric acids levels and lowered abundance of bacteria known to dominate in well-performing birds. Overall, exposure to reused litter, particularly when it has no detectable levels of pathogenic bacteria, might be a source of recycled nutrients and exposure to direct fed microbials, which can benefit bird performance. The second objective was to investigate effects of TAG supplementation to IP-formulated low CP diets at the expense of glycine to maintain both iso energetic and iso-nitrogenous diets on resilience of broilers to a combined exposure to reused litter at placement and challenge with E. maxima at day 14. Growth performance at appropriate stages over the whole course of the trial was measured. In addition, organ weights and plasma biochemistry were measured at three time points, i.e. when birds had been exposed to reused litter only (10 days of age), at the acute phase following E. maxima challenge (20 days of age) and at the recovery phase (27 days of age). The low CP diets reduced bird performance compared to birds fed relatively high CP control diet (Chapter 4 and Chapter 5), which might have resulted from dietary electrolyte imbalance and limiting non-essential AA other than glycine. This further indicated a reduced ability to successfully apply the IP concept, particularly during the starter phase. In agreement with the first trial, placement on reused litter increased body weight. Though superimposing birds with sub-clinical coccidiosis reduced bird performance during the acute phase of the challenge, TAG supplementation to the low CP diets did not improve resilience. We further investigated the effect of TAG supplementation to low CP diets on apparent ileal nutrient digestibility, digestible nutrient intake and litter pH and moisture content. Our results showed that exposing birds to reused litter and sub-clinical coccidiosis did not impact digestibility measured at 10 and 27 days of age. On the other hand, TAG supplementation to low CP diets reduced apparent ileal CP digestibility, which might indicate that this specific AA supplementation caused AA imbalance, reduced AA availability and excess protein. Litter pH and moisture content for birds fed low CP diets were lower than those fed control diets, which confirms that lowering dietary CP could be a tool to reduce nitrogen excretion and ammonia emissions from poultry houses. The third objective was to test the effects of storage duration on microbial and physicochemical properties of reused litter to provide a possible basis to the perhaps unexpected beneficial growth response observed upon reused litter exposure, which deviated from previous studies. Litter samples, weekly collected over 28 days, showed a reduced litter pH and moisture content on day 28 compared to day 0. In addition, the major pathogen of concern in poultry, such as Salmonella spp, Clostridium perfringens, E. tenella and E. maxima, were not detected in our litter samples. The average copy number of 16S rRNA gene of total bacteria and FimH gene copy numbers of avian pathogenic E. coli reduced from day 0 to day 21 though then slightly increased at day 28. The average copy number of 18S rRNA of total fungi decreased from day 0 to day 28. These results confirmed that reused poultry litter showed variation in physicochemical properties and pathogen load over time, and may in part explain the variability in growth performance observed between studies when broilers are exposed to reused litter. In conclusion, TAG supplementation to IP-formulated standard or lowered protein diets did not improve resilience to sub-clinical enteric challenge but reduced bird performance. This reduced performance may have been due to reduced ileal digestibility of AA, resulting in increased excess protein and thus enhanced proteolytic fermentation. Lowering dietary CP while maintaining the IP concept and glycine requirements reduced bird performance, which could indicate the inability to apply the IP concept, particularly during the starter phase. Exposing birds at placement to reused litter in which Salmonella spp., C. perfringens and Eimeria spp. were below detection level, improved bird performance, indicating that reused litter might be a source of recycled nutrients and direct-fed microbiome rather than a source of pathogen challenge. Therefore, before employing reused litter exposure, detailed litter characterisation is required to optimise study design, including origin description, duration and storage condition between collection and usage.

The thesis has focused on the development of dry powder inhaler (DPI) formulations for pulmonary delivery of antidiabetic peptide and protein drugs such as insulin and glucagon-like peptide-1(7-36) amide (GLP-1). These drugs are generally administered via subcutaneous injection that interferes with the patients' lifestyle and affects patient compliance and adherence to treatment. The primary goal of the research was to assess the value of insulin and GLP-1 DPI formulations for inhalation use in people with diabetes as an alternative treatment option to injectable antidiabetic medications. The aerosolisation performance of insulin and GLP-1 dry powders from two different types of DPI formulations; carrier free DPI formulations (drug alone) and carrier-based DPI formulations (drug and carrier) were investigated using a next generation impactor. No studies have been reported on the development of carrier free GLP-1 DPI formulations and carrier-based DPI formulations containing excipient free insulin or GLP-1 for pulmonary delivery. Particle engineering such as spray drying and spray freeze drying were employed for the drug and carrier powder preparation, respectively as a formulation strategy to optimise the properties of both drug and carrier particles (e.g., particle size, morphology). In addition, drug particles were prepared in the absence of excipients to minimise the lung safety concern. Mannitol was selected as an alternative carrier to lactose which is the most used carrier in DPI formulations but associated with chemical incompatibility with proteins. Glycine or L-leucine selected as an excipient was added to mannitol carrier to optimise carrier properties (morphology). Spray drying demonstrated to reduce the particle size of insulin and GLP-1 powders to a suitable size range (aerodynamic diameter: ≤ 5 µm) for pulmonary delivery and modified the morphologies. This resulted in high fine particle fraction (insulin FPF: 77.36% ± 18.01%, GLP-1 FPF: 90.73% ± 1.76%) showing feasible for pulmonary delivery of spray dried insulin and GLP-1 dry powders. However, spray dried drug particles for inhalation (≤ 5 μm) were naturally cohesive (high degree of drug-drug agglomeration) due to the small particles associated with high inter-particulate forces between drug particles (drug-drug cohesive forces) therefore exhibited poor powder flow and low drug delivery efficiency (total drug deposition on throat and all impactor stages) from an inhaler device (Handihaler®) (insulin delivered dose: 38.64% ± 3.82%, GLP-1 delivered dose: 32.88% ± 7.00%). Spray freeze drying produced spherical and porous carrier powders with the particle size range between 50 µm and 130 µm suitable as DPI carriers regardless of the inclusion or absence of amino acids. However, surface properties (e.g., morphology and roughness) of spray freeze dried mannitol-based carriers were dependent on the type of amino acid and its concentrations. The novel amino acid-mannitol carriers prepared by spray freeze drying were employed to improve the aerosolisation performance of DPI formulations (e.g., powder flow). Carrier-based DPI formulations containing spray freeze dried glycine-mannitol carrier improved the powder flow of the cohesive drug particles and delivered higher drug dose from Handihaler® compared to drug particles alone in carrier free formulations (insulin delivered dose: from 38.64% ± 3.82% to over 57.0%, GLP-1 delivered dose: from 32.88% ± 7.00% to 45.92% ± 5.84%). This was attributed to the porous powders produced by spray freeze drying. Without the addition of the engineered spray freeze dried carrier to the formulation, spray dried drug particles showed poor flowability. Overall, both carrier free and carrier-based DPI formulations have shown advantages with different challenges for pulmonary administration of insulin and GLP-1. According to an online survey conducted in 2019, patients with diabetes generally accepted the idea of insulin delivery via inhalers as 73.4% of participants were willing to try insulin inhalers. National Health Service availability will have a significant influence on participants' willingness to try insulin inhalers. The successful inhaled insulin and GLP-1 products will provide an alternative treatment option for people with diabetes by reducing the burden of injection related barriers therefore improve patient compliance and adherence to antidiabetic therapy and quality of life affected by injection treatment.

The radical Truce-Smiles rearrangement is an intramolecular aryl migration reaction that allows for the conversion of sulfonamide or sulfonate starting materials into higher value organic products, usually using the extrusion of sulfur dioxide as an entropic driving force for the reaction. Presented herein are three transformations using modern photochemical techniques for the mild generation of radical species. Firstly, an allylamine-based starting material is transformed into an arylsulfonylacetamide starting material. Difluroalkyl radicals could undergo intermolecular addition to this unactivated alkene, triggering a TruceSmiles rearrangement, giving a carboarylated product. The reactions proceeds under metal-free conditions, with a postulated EDA complex mechanism, using N,N,N',N'-tetramethylethylenediamine (TMEDA) as the stoichiometric electron donor. Building on these results, a decarboxylative, desulfonylative Truce-Smiles rearrangements was developed. To achieve this, a ß-amino acid starting material was converted into a sulfonamide starting material and an energy-transfer mechanism was employed for the radical decarboxylation. This methodology was used to synthesise a range of arylethylamine products, relevant to the pharmaceutical industry. Finally a strained 3-aminocyclobutanone starting material was used to realise the first Truce-Smiles rearrangement by strain release using an Eosin Y dye under visible light irradiation at room temperature. The arylated ß-amino nitrile products were precursors to arylated 1,3-diamines or ß-amino acids.

Recognition-driven labelling techniques such as affinity/activity-based probes or photoaffinity labelling (PAL) are invaluable tools for the interrogation and study of biological processes in cellulo. Among those, PAL has proven to be the most powerful method, supplemented by the temporal control of its photoactivation step. However, the UV irradiation required for reagent activation and non-selective reactivity of the generated intermediates often results in low photo cross-linking yields and numerous off-target reactions. Therefore, the development of a more selective strategy using visible-light mediated photocatalysis appears a promising alternative. This thesis describes the efforts towards the development of a novel ligand-mediated strategy using visible light-activated -diazo sulfonium-based probes for the modification of histidine residues. Histidines represent attractive amino acid targets by virtue of their naturally low abundance but recurrent presence in protein active sites and pivotal biological roles. In addition, the design of a histidine-selective strategy constitutes an ideal opportunity to provide a complementary approach to the histidine bioconjugation toolkit given the lack of robust methods. Firstly, the biocompatibility and functional group tolerance of the reaction are demonstrated on short peptidic systems. Subsequent studies towards the application of the reaction on a complex ligand-protein system in vitro are also described. Owing to the modularity of the photoreactive a-diazo sulfonium motif employed, a broad variety of probes were designed. Finally, the research performed towards the development of an analogous affinity-guided catalyst strategy using an iridium-containing ligand and small diazo sulfonium trapping reagents is reported.

Bacteria have evolved sensory pathways which allow the detection and response to changes in the environment which is essential for survival. Some of these pathways rely on eukaryotic-like serine/threonine protein kinases (STPKs). One STPK conserved within Actinobacteria is PknG which is found within ecologically, industrially and medically important bacteria. One such bacteria is Mycobacterium tuberculosis, which causes Tuberculosis in humans. Research has highlighted that PknG, and its phosphorylation target GarA, respond to amino acid availability and are essential for the regulation of glutamate metabolism. Currently, there is no proven mechanism which could explain how cytoplasmic PknG could be activated by external amino acids. A model has been proposed, involving two proteins conserved in the pknG operon: GlnH, a predicted glutamine-binding protein, and GlnX, a membrane protein with unknown function. The main aim of this project was to provide evidence supporting the hypothesis that both GlnH and GlnX are involved in the activation of PknG in response to the presence of amino acids. Recombinant GlnH from M. tuberculosis and Corynebacterium glutamicum was characterised using ligand screens and Kd measurements. This highlighted that GlnH binds, with a high affinity, the amino acids known to trigger GarA phosphorylation in bacteria: aspartate and glutamate. Furthermore, weak ligands were identified for each protein: asparagine, histidine, cysteine and isocitrate. Structure determination of histidine-bound and aspartate-bound M. tuberculosis GlnH allows further understanding of the binding specificity. To test for a putative protein-protein interaction between GlnH and GlnX, attempts were made to produce a soluble form of GlnX for interaction assays. The understanding of the activation mechanism of the PknG/GarA pathway could allow therapeutics to be developed which could successfully target this essential pathway.

Environmental enteropathy (EE) is thought to underlie stunting, a widely prevalent phenomenon in the developing world with lifelong detrimental consequences. EE is characterised by increased intestinal permeability and reduced absorptive capacity in a chronically inflamed gut, and occurs in nutritionally vulnerable populations. Deficits of amino acids and micronutrients may cause intestinal immune and barrier dysfunction, mediated through cellular nutrient sensing pathways. Mechanistic target of rapamycin complex 1 (MTORC1) is the most important of these, and is of particular interest in EE due to its roles in both epithelial and immune cell function. It was hypothesised that supplementation with the amino acids (AA) L-Glutamine, L-Tryptophan and L-Leucine +/- high dose multiple micronutrients (MM) could modify the abnormal small intestinal histology and permeability seen in EE, and would modify lamina propria lymphocyte (LPL) MTORC1 signalling. A randomised controlled trial was therefore undertaken. Assessments of many of the pathophysiological domains of EE (absorptive area; mucosal inflammation; microbial translocation; permeability; systemic immune activation; host-microbiota interactions; enterohumoral signalling), as well as LPL MTORC1 signalling, were performed in 84 healthy adult Zambians with EE before and after 16 weeks' supplementation. AA supplementation improved villus height, whereas AA with MM supplementation improved in vivo barrier permeability. Supplementation did not affect LPL MTORC1 signalling; however, MTORC1 activity correlated with villus height and the size of the LP TH1 population. Metabonomics identified several potentially relevant metabolites which were upregulated by the interventions. Of note, other observed abnormalities (e.g. elevated translocation/activation markers; depressed levels of GLP2) did not change with supplementation and were mainly not correlated with the improvements in histology and permeability, suggesting that one or more pathophysiological domains (e.g. absorptive area; permeability) may be independently modifiable. These results and others explored in this thesis provide new insights into the complex relationship between nutrition, intestinal health, and EE.

The N-methyl-D-aspartate (NMDA) receptors (NMDAR) are a subtype of ionotropic glutamate receptors that are highly expressed in the central nervous system (CNS) and are involved in the excitatory synaptic transmission and synaptic plasticity that underpin many critical CNS functions. NMDAR dysfunction has been implicated in Alzheimer's disease, neuropathic pain, schizophrenia, and depression, among others. Most non-selective NMDAR antagonists (e.g. ketamine) have undesirable side-effects that restrict their clinical utility for relieving symptoms of neuropathic pain and treatment-resistant depression, but indirect modulators of NMDAR function offer the potential to have reduced side-effects relative to non-selective antagonists. One approach is to inhibit the production of the NMDAR co-agonist, D-serine, which is produced endogenously by conversion of L-serine to D-serine by the enzyme serine racemase (SR). Inhibitors of SR that reduce the production of D-serine may therefore have therapeutic benefits in disorders associated with NMDAR hyperfunction. In this thesis, human SR(hSR) was expressed in a bacterial host and isolated to a high degree of purity (>90%). The crystal structure of SR was solved in-house using the purified protein, to a resolution of 2.2Å (in complex with malonate) and 1.9Å (holoenzyme). To measure SR activity, a coupled biochemical assay was optimised, in which produced D-serine is degraded by D-amino acid oxidase (DAO) and the resulting H2O2 is quantified by a chemiluminescence reaction with HRP and luminol to produce light. Assay parameters such as Kmand IC50 were determined, and following further optimisation to enable medium-through put screening, a single-point screen of 3000 fragments was performed to identify novel inhibitory hit matter. A final list of 61 specific hits was collated of fragments that did not exhibit nonspecific inhibition in a DAO counter screen, and demonstrated IC50 values against SR approximately 1mM or below. These specific hits were characterised with biophysical methods adapted for SR (thermal shift, microscale thermophoresis) to determine binding affinities and structure stabilisation. Finally, via crystal soaking, the crystal structure of SR bound to an inhibitory fragment (KD=960μM) was revealed to a resolution of 1.8Å.

The study of pharmaceutical preformulation includes the evaluation of pharmacokinetic, pharmacodynamic and physicochemical properties of the drug molecules that aid the formulation. However, it has a limited role in determining drug dosage optimisation in the formulation. The study of drug-Plasma Protein Binding (PPB), and the lipophilicity, solubility, and ionic behaviours of the desired drug molecules addresses the gap and enhances our undertraining related to the behaviour of the drug molecules in the body. The High-Performance Liquid Chromatography (HPLC) technique was used in the current study to assess drug-PPB interaction. Using Michael Abraham's 'Linear Free Energy Relationship' (LFER) method, two major plasma proteins namely, Human Serum Albumin as HSA and α-1-Acid Glycoprotein as AGP, were used and the structural properties governing drug-plasma protein binding was determined. This is the first time that the effect of ionised species on PPB has been quantitatively evaluated. In addition, the molecular interactions also play a key role in the supramolecular complexes of co-crystals. The project also evaluated the co-crystallisation process and its effect on physicochemical properties of the drug. In the current study, amino acids (AAs) have been observed to be a prominent source of coformers. The AAs showed co-crystals formation with carboxylic acids, nonsteroidal anti-inflammatory drug (NSAID) and citric acid which overcome the hygroscopicity problems and improved the physical stability issues during storage. This study has also identified a new formulation which is helpful for improvement in the stability of effervescent tablets at various relative humidity (RH) conditions which will reduce the manufacturing cost associated with the production of effervescent tablets.

Diabetes mellitus currently affects over 422 million people globally and over 80% of patients with diabetes will develop diabetic retinopathy. Patients with diabetic retinopathy initially develop background retinopathy, which does not cause significant deterioration of visual function; however, background retinopathy may progress and lead to proliferative diabetic retinopathy and diabetic macular oedema, both of which cause severe visual dysfunction if left untreated. Current therapies for diabetic retinopathy include invasive intravitreal injections and laser photocoagulation; however these treatments only attenuate the progression of proliferative diabetic retinopathy and diabetic macular oedema. Aside from prevention by maintaining good blood glucose and blood pressure control, there are currently no treatments to prevent progression to late-stage diabetic retinopathy and new innovations in the field have not significantly progressed. For this reason, we have used untargeted –omics approaches to identify previously unknown pathological pathways in diabetes. In this thesis, I have analysed a range of trace metals in donor retinas and found that total copper was increased in diabetic retinas compared with non-diabetic. This result was replicated in streptozotocin-induced diabetic rat retinas and further evidenced by upregulation of metallothioneins and caeruloplasmin in diabetic rat retinas compared with non-diabetic. Treatment with the copper chelator triethylenetetramine modulated these changes, the downstream effects of which require further study. This is the first description, to our knowledge, of dysregulated copper homeostasis in the diabetic retina. I have also mapped metabolic changes in streptozotocin-induced diabetic rat retinas and found previously undescribed metabolite changes such as diabetes-induced downregulation of scyllo inositol. This coincided with substantial changes to retinal lipids during diabetes and changes to individual lipids were consistent within their respective class. I have also found a pattern whereby regardless of the extent of change to a lipid class in diabetes, lipids containing docosahexaenoic acid (22:6 carbon chain) were consistently downregulated. This is thought to be the first study to describe this range of metabolite changes in the diabetic retina but also the first study to describe this range of metabolite analysis concomitantly within the same tissue sample. The data from this study provides new insights into metallomic and metabolic dysfunction in diabetic retinopathy and shown that these data are reproducible. We suggest that there is plenty of scope for further research to investigate mechanisms behind copper dysregulation, how this affects pathogenesis of diabetic retinopathy along with new insights into dysregulated metabolic pathways.

Alzheimer's disease (AD) is the most common form of dementia, which is characterised by extracellular Aβ plaques and intracellular neurofibrillary tangles, comprised of fibrils of Aβ42 and tau protein, respectively. A species of tau protein localised to the nucleus has been discovered, but its role in AD is still unclear. Glutamate excitotoxicity, oxidative stress, DNA damage, alteration of the chromatin and nucleolar stress are key features of AD. The early stages of the disease are characterised by minimal neurodegeneration and altered protein synthesis machinery. The culprit (s) and molecular link between these changes and the role of nuclear tau are unclear. This work utilised glutamate stress and Aβ42 oligomers to investigate the involvement of nuclear tau in the chromatin alteration, nucleolar dysfunction, and downstream protein synthesis impairment that occurs in AD. This revealed that glutamate stress in SHSY5Y neuroblastoma cells results in oxidative stress, a nuclear upsurge of phosphorylated tau and delocalisation of nucleolar tau, alongside, DNA damage, heterochromatin loss, nucleolar stress and protein synthesis inhibition, partly through eIF2α phosphorylation. Likewise, short incubation of SHSY5Y cells with Aβ42 oligomers led to significant oxidative stress, with gradual accumulation of nucleolar stress, which resulted in altered transcription and processing of 45S pre-rRNA and decrease in protein synthesis, without DNA damage. Although both glutamate and Aβ ultimately decreased protein synthesis, Aβ incubation led to an increase in heterochromatin formation and a reduction in RNA synthesis without DNA damage, pointing to a different mechanism of toxicity by the Aβ and glutamate stress. To characterise a nuclear role for tau, this work localised tau in the nucleolus and heterochromatin in the SHSY5Y cells and the human brain, where it associates with TIP5 - a key player in heterochromatin formation. Accordingly, tau knockdown destabilises the heterochromatin and increases rDNA transcription, indicating that tau is essential for silencing of the rDNA and heterochromatin stability, similar to TIP5. Overall, this thesis provides evidence that implicates glutamate and Aβ toxicity in some of the changes that occur in the disease and specifically implicates Aβ42 as a key culprit that drives changes in the early stage of the disease. It also reveals a new role for tau in the nucleus and points to its pathological involvement in AD.

This thesis is focussed on the investigation of matrices for matrix-assisted diffusion-ordered spectroscopy (MAD). Diffusion-ordered spectroscopy (DOSY) is a family of experiments where the resonances in the chemical shift dimension are further dispersed in an extra dimension according to diffusion coefficient. A typical DOSY spectrum shows one single diffusion coefficient for all the resonances coming from one single species. However, If two or more resonances overlap, the diffusion resolution of the DOSY spectrum is compromised and a spurious diffusion coefficient results, intermediate between the species. In case of signal overlap, the use of more advanced processing methods aids to separate two analytes that differ by at least 30% in diffusion coefficient. In practice, many mixtures contain species of similar diffusion coefficients whose resonances overlap in the chemical shift dimension. The addition of co-solutes can modify the chemical environment (matrix), with which different analytes interact to different extents, and enhance the diffusion resolution of DOSY. However, the addition of co-solutes can risk the benefits of DOSY by increasing the probability of signal overlap. Signal overlap in MAD is avoided by using a 1H NMR-invisible surfactant such as sodium perfluorooctanoate (NaPFO), which has replaced each proton by a fluorine atom. PFO micelles are a tunable matrix which allows the separation of analytes via coulombic interactions by adjusting the pH. Differences in diffusion coefficient in NaPFO solution can be analysed using a modified Lindman's law to model the diffusion coefficient as a function of pH. The model rationalises the binding constants of analytes to PFO micelles with good accuracy, subject to the spectral data quality. Another alternative to resolve diffusion coefficients using the invisible MAD approach is by means of a commercially available alkyl surfactant like cetyltrimethylammonium bromide (CTAB). CTAB in high ionic strength solution forms worm-like micelles whose resonances can be filtered out from the final DOSY spectrum. CTAB worm-like micelles have short transverse relaxation times compared to all of the analytes in the mixture. If a transverse relaxation filter is positioned at the beginning of a standard DOSY pulse sequence, as in PROJECT-Oneshot, the strong CTAB signals vanish and leave behind only the analyte resonances and hence avoid signal overlap. Finally, the use of bovine serum albumin (BSA) as a potential invisible matrix, using a similar approach to CTAB worm-like micelles is investigated, using a relaxation-weighted DOSY pulse sequence to suppress most of the BSA background signal (at a cost in analyte signal to noise ratio). An alternative to suppress most of the BSA background and preserve most of the analyte signal is by means of mild transverse relaxation filtration and spectral editing to obtain an edited DOSY spectrum that shows only the analyte signals. Nonetheless, it is a shame that useful MAD results can only be obtained under a narrow set of conditions: i) different mole ratios BSA: analyte to aid diffusion resolution, ii) mild T2 filtration to improve analyte signal to noise ratio and iii) spectral editing to remove residual BSA background.

Insulin-like growth factor-I (IGF-I) is the only known potentially modifiable risk factor for prostate cancer. Intake of dietary protein, especially from dairy products, might also be associated with risk and with circulating IGF-I, but it is not clear if amino acids play a role in these relationships. Moreover, investigations of circulating concentrations of metabolites might reveal novel risk factors for prostate cancer. This thesis investigates plasma concentrations of amino acids and other metabolites in relation to protein intake, IGF-I, and prostate cancer risk using data from the European Prospective Investigation into Cancer and Nutrition (EPIC). To characterise plasma metabolite profile in men consuming markedly different amounts and types of animal products (meat-eaters, fish-eaters, vegetarians and vegans), cross-sectional analyses of 392 men in the EPIC-Oxford sub-cohort were conducted. Of 21 amino acids, six varied significantly by diet group, and the metabolite profile of vegans was different from those of other diet groups owing to lower concentrations of several glycerophospholipids and sphingolipids. In a case-control study nested within EPIC, with a mean follow-up time of seven years, the relationship of plasma metabolites with risk of prostate cancer overall, by time to diagnosis, by tumour characteristics, and with risk of prostate cancer death, was investigated. Data from 1,077 matched sets suggested that seven metabolites, from various classes, were associated with risk of prostate cancer overall (p < 0.05). After correction for multiple testing, 12 glycerophospholipids were inversely associated with risk of advanced prostate cancer (the strongest OR1SD = 0.54; 95%CI: 0.40-0.72). In multivariate analyses, including data from 1,593 matched sets, principal component analysis (PCA) and treelet transform (TT) were used to identify patterns in metabolite profile, five of which were associated with risk of more aggressive tumour sub-types (high grade, advanced and aggressive disease) and/or prostate cancer death. There was a ≈ 50% lower risk of advanced and high grade prostate cancer in men with metabolite profiles characterised by high glycerophospholipids and sphingolipids (for advanced ORTT, top vs bottom third = 0:48; 95%CI: 0:31-0:74), with similar results for high grade and PCA). To investigate if associations between protein intake and circulating IGF-I may be mediated by plasma amino acid concentrations, cross-sectional analyses of amino acid concentrations with protein intake and IGF-I concentrations were carried out in 1,697 and 1,142 control participants, respectively, from the nested case-control study. Dairy protein intake was positively associated with concentrations of branched-chain amino acids and several other essential amino acids, while plant protein intake was strongly associated only with histidine. Serum IGF-I was positively associated with arginine and inversely with ornithine and certain amino acid ratios. In conclusion, men with different dietary habits with respect to the consumption of protein types have different amino acid and metabolite profiles, and metabolite concentrations may be associated with risk of more high-risk prostate cancer sub-types (high grade, advanced and aggressive disease) and prostate cancer death. Further large-scale studies are needed to determine if metabolites play a role in aetiology or are markers of sub-clinical prostate cancer.

Ligand-observed NMR spectroscopy is frequently employed in early-stage drug discovery, often as an initial screen to narrow the field of potential drug-like molecules. However, its use is limited to this early stage. More information regarding binding mode can be extracted from these experiments via quantification, and this should help extend the remit of these experiments beyond simple screening functions. Initially, it was shown that the amount of signal that could be produced from an STD NMR experiment could be dramatically increased by careful consideration of the selective saturation pulse. By systematically shortening the Gaussian pulse and positioning it at specific offset positions, it was shown that these dramatic increases in signal are genuine and need not result in false positives. Quantitative STD NMR spectroscopy as applied to Hsp90 and a series of small fragment ligands provided evidence to suggest that the precise inter-atomic distances between a protein and ligand within a crystal structure correlate with both initial rates of STD build up, and T1-adjusted STD values. This precise correlation has implications for chemotype clustering and initial binding mode selection, something which should be useful in the absence of a crystal structure. Taking the same quantitative principles and applying to LOGSY experiments elucidated another, discrete property of protein-ligand binding. Examining the ‘LOGSY difference’ signal for protons of a ligand allows us to see what protons are in close proximity to conserved, bound water at the protein-ligand binding interface. This is fundamentally different to the information gained from STD experiments. Applying the insights to a protein of a different nature, Ras, it was shown that quantitative STD can be applied to proteins of both different size and structure. Furthermore, more evidence was acquired to suggest that conserved, bound water in the binding site really is responsible for generating LOGSY signal. In the absence of these molecules, as in Ras, proximity of a proton to an exchangeable tends to dominate. In addition we were able to show that these quantitative methods can be used together to help eliminate incorrect computationally generated docking poses. The work presented in this thesis provides evidence for the advantages of STD and LOGSY NMR spectroscopy in fragment-based drug discovery. The information that can be extracted from relatively simple ligand-observed NMR experiments should be used to provide more evidence at an earlier stage of the drug discovery process, hopefully reducing late-stage attrition and helping us get to the therapeutic drug molecules we need a little more quickly.

This thesis aims to address issues relating to genome architecture and base composition. The first part of this thesis addresses questions relating to codon usage. Initially I will investigate thousands of bacterial species using a detailed analysis of strengths of selection acting upon codons usage while also investigating patterns of optimal codon changes with respect to genomic base composition and tRNA abundance. I report that selection on codon usage increases throughout the length of highly expressed genes, in particular, the first quarter of genes have significantly lower selection. Further, it is clear that factors affecting genomic base composition can eventually lead to changes in optimal codons if the change in base composition is strong enough, however these patterns differ substantially between amino acids. The debate over translational efficiency vs. accuracy was addressed by comparing sites of differing conservation. Differing conservation were defined using a phylogenetic method, allowing sites to change in their extent of conservation throughout the tree. The results show that translational accuracy acts strongly on the top 10% of conserved sites, however is relatively weak when compared to the efficiency for other sites. Also detected is a reduction in apparent selection on codon usage on the bottom 10% of conserved sites which is likely to be caused by conflicting positive selection on amino acids. Finally, although differences in patterns are observed between amino acids, the general relationship to conservation is similar. As much of the variation in codon usage is determined by variation in base composition, this aspect of base composition is investigated in the second part of the thesis. The observed variation in intragenomic base composition in bacteria was found to be far higher than expected for GC-rich bacteria. The non-core part of the genome contributes to this variation to a greater extent than the core part, suggesting that processes such as AT-rich horizontal gene transfer may be involved. Secondly, base composition is modelled under Brownian motion and as an extension, the Ornstein- Uhlenbeck process, which allows for multiple optima throughout the tree. The model including optima fits the data better than standard Brownian motion or Brownian motion with multiple diffusion coefficients. Finally, I investigate a case where a previous codon usage analysis has been seriously confounded by an unusual genome architecture of abnormal regional base composition in two species of eukaryotic parasites in the genus Theileria. In both species, the background G+C content is 37% at most, out of the four syntenic chromosomes. In many orthologous regions however, T.annulata has a decreased G+C content of 28% while T.parva has an increased G+C content of 41%. Various factors coincide with this remarkable divergence: increased divergence at all types of site, recombination hot spots in T.parva, an increased frequency of tandem repeats and DNA sequence motifs in both species. The evolutionary origins of these unusual patterns will be discussed.

This thesis is concerned with the development of methodology for the regioselective ring-opening of aziridines and aziridinium intermediates and its subsequent application to the asymmetric synthesis of β-substituted-α-amino acids and 1,2,3,4-tetrahydroisoquinolines. Chapter 1 introduces methods for the formation of aziridines and aziridinium ions and focusses on their utility as intermediates in synthesis. Chapter 2 describes studies into the synthesis of aziridines from enantiopure α-hydroxy-β-amino esters and their subsequent conversion to the corresponding β-hydroxy-α-amino acids via either a regioselective ring-opening with Cl3CCO2H, or a rearrangement promoted by Cl3CCO2H. Application of this procedure to both syn- and anti-configured substrates enabled the syntheses of (S,S)-allo-threonine, (2R,3S)-threonine, (R,R)-3-hydroxyphenylalanine and (2S,3R)-3-hydroxyphenylalanine. Chapter 3 details attempts to truncate the synthesis described in Chapter 2 by investigating the synthesis of enantiopure anti-β-hydroxy-α-amino acids via the intermediacy of aziridinium ions. These studies culminated in the development of a regioselective and stereospecific one-pot aziridinium formation and ring-opening protocol, leading to the synthesis of a range of C(3)-aryl and C(3)-alkyl substituted anti-β-hydroxy-α-amino acids. Chapter 4 discusses the conversion of enantiopure anti-α-hydroxy-β-amino esters to anti-β-fluoro-α-amino esters via the regioselective and stereospecific ring-opening of an aziridinium intermediates in situ. The subsequent development of a one-pot deprotection strategy leads to a concise and expedient synthesis of anti-β-fluorophenylalanines. The extension of this methodology to access a representative anti-α,β-diamino acid is also demonstrated. Chapter 5 describes the development of a one-pot diastereoselective rearrangement of enantiopure α-hydroxy-β-amino esters to 1,2,3,4-tetrahydroisoquinolines. The substrate scope of this reaction manifold is examined and application to the asymmetric synthesis of enantiopure 1,2,3,4-tetrahydroisoquinolines also discussed. Chapter 6 contains full experimental procedures and characterisation data for all compounds synthesised in Chapters 2, 3, 4 and 5.

NASEM (2021) recently made strides in characterizing effects of 5 individual EAA on milk protein production. However, there are 15 other AA that are incorporated into milk protein, and as such, these AA likely also play significant roles in driving milk protein synthesis, but lack of data prevents their incorporation into current models. A greater supply of AA to the mammary glands does not always mirror AA absorption—the process by which absorbed AA convert into milk protein is variable, and this may be linked to the way the udder regulates AA uptake to preserve intracellular balance. AA transporters housed within the cellular membranes of mammary epithelial cells (MEC), the mammary glands' constituents, are responsible for mediating this intracellular balance. Thus, the objectives of this dissertation were to investigate how AA transport is affected by various AA concentrations using both in vitro and in vivo approaches. In study 1, we evaluated effects of valine and a group of NEAA (AQG; Ala, Gln, and Gly) on exchange transport rates of AA in bovine MEC. High AQG concentrations stimulated Leu, Phe, and Val influx rate parameters, demonstrating that AQG likely increased transport activity for these substrates through exchange transporters. Additionally, high Val concentrations decreased Ile and Leu net uptakes, which occurred via efflux stimulation and transamination downregulation. In study 2, we aimed to identify the effects of 10 EAA and 2 Tyr (CDENSPY) on transport rates and transporter regulation (mRNA expression and protein abundance). Within the physiological AA concentrations used, we were able to measure differential effects of AA on each AA transporter. For example, His stimulated SLC38A2 and SLC38A2 mRNA expression at a decreasing rate; the apex for this curve was reached at a concentration very close to mean plasma concentrations in lactating dairy cows. Therefore, we determined that these transporters may be transcriptionally regulated to regulate intracellular His concentrations. Additionally, all EAA and NEAA groups were involved in significant 2-way interactions on transporter expression and activity. Furthermore, we measured transport rates and rate constants (free of mass influence) of 12 AA to determine important AA on influx, efflux, transamination, irreversible loss, and protein synthesis. We demonstrated competitive inhibition among several AA that share transport systems such as between BCAA. Furthermore, we again demonstrated that NEAA can stimulate transport activity for AA involved in exchange transport. In study 3, we investigated the effects of jugular Lys, Ile, Val, or AQG infusion on mammary AA metabolism and production in lactating dairy cows. Interestingly, Val decreased DMI and milk protein production along with net uptakes of several AA, while the remaining treatments had little metabolic effects. In study 4, we demonstrated that both high protein and starch concentrations independently stimulated milk protein production, but glucose precursor partitioning (lactate, propionate and other) was only affected by starch. In conclusion, we anticipate that nutrition models estimating milk protein production will eventually incorporate up to 20 AA and multiple 2-way interactions; additionally, extremely high concentrations of AA should be prevented to combat negative impacts caused by AA imbalances. However, much more work is required to take steps in this direction.

Urbanization introduces a set of novel stressors for animals in these modified habitats. Developing young, in particular, can be at risk due to increased predation, exposure, and high food intake requirements. Song sparrows (Melospiza melodia) are a model wild songbird system that persist in both urban and rural habitats, and have young that develop very quickly. I investigated how urbanization changes the arthropod community, how that in turn affects nestling diet, and ultimately if there were any differences in nestling amino acid profiles or body condition between habitat types. Prior work found that urban habitats also have far higher rates of brood parasitism, which can additionally restrict food intake for urban nestlings. However, this prior work has also found that urban song sparrow adults avoid consequences for living in urban habitats, so I wanted to see if nestlings were also able to avoid the consequences of urban living. In chapter one, I found our urban arthropod communities had lower average arthropod abundance, biomass, and diversity when compared to rural arthropod communities. Song sparrow nestling diets differed somewhat in composition of arthropod prey items by habitat, but urban and rural nestlings had the same average biomass of stomach contents. In chapter two, I investigated whether different habitat types or brood parasitism altered body size and amino acid concentration of song sparrow nestlings. Only gamma-aminobutyric acid differed, being significantly lower in urban, parasitized nestlings. No groups differed in metrics of body condition. In chapter three, I performed a meta-analysis to investigate how brood-parasitic brown-headed cowbirds (Molothrus ater) affect host clutch size and nest success. Host clutch sizes were lower when parasitized, but surprisingly non-parasitized nests were more likely to fail. Despite lower arthropod food availability and high levels of brood parasitism, urban song sparrow nestlings are not compromised, supporting the conclusion that urbanization does not necessarily have negative consequences for individuals of this species.

The use of stereoselective aminohydrolases (acylases) in kinetic resolutions is a commonly employed industrial route to both L- and D- α-amino acids from Nacetylated- DL-starting materials. However, a flaw in this process is the need for repeated racemisation steps of the non-desired enantiomer to achieve yields >50%. A solution to this drawback would be a dynamic kinetic resolution driven by an in situ racemisation step that would allow the yield to approach 100% A cheap and “green” catalyst for this racemisation would be an enzyme such as N-acyl amino acid racemase (NAAAR) from the actinobacteria Amycolatopsis sp. Ts-1-60. This enzyme requires no organic cofactor, is stable at high temperatures (~60°C) and importantly, shows no racemase activity toward free amino acids. Unfortunately, the activity of NAAAR with N-acetyl substrates is low and reported to suffer from inhibition above substrate concentrations of 50 mM, prohibiting its use in NAAAR/acylase coupled dynamic kinetic resolutions under industrial conditions. In an attempt to remove these limitations, directed evolution has been applied to the Amycolatopsis Ts-1-60 NAAAR to engineer a variant enzyme with increased activity towards N-acetyl substrates. An improved variant NAAAR may allow for a commercial NAAAR/acylase coupled dynamic resolution process. Directed evolution has proven to be a highly versatile and successful tool for protein engineering. However, the ability to screen for improved variants is often technically difficult or time consuming and in the case of NAAAR this is especially true as the substrate and product are simply enantiomers of an N-acetyl amino acid. To overcome this, an enantioselective genetic selection system has been employed to allow the screening of mutagenic NAAAR libraries. After several rounds of selection, a NAAAR variant (NAAAR G291D F323Y) has been isolated with increased racemase activity towards a range of synthetically useful N-acetyl substrates. This enzyme has been over-expressed, purified and its characteristics compared to the wild-type and other variants discovered during the evolution process. NAAAR G291D F323Y has been crystallised to 2.71 Å allowing a molecular basis for the increase in catalytic activity to be proposed. Coupling of this enzyme with a stereoselective acylase has been used to produce enantiopure amino acids in >99% yield, twice the inherent 50% maximum yield of acylase based resolutions. Early results suggest this NAAAR variant has the potential to be employed on a multi kilogram scale for the economical production of enantiopure L- and D- α-amino acids.

Maternal effects are the non-genetic contributions of mothers (or fathers) towards the phenotype of their offspring. Maternal effects are now well recognised as a facilitator for evolutionary change in offspring phenotypes and life history strategies which can have effects on population dynamics, population divergence and even speciation. Furthermore, maternal effects have been shown to have a heritable genetic basis and that they are genetically variable, which suggests that they contribute to maintaining phenotypic variation. Maternal effects may impede or accelerate responses to selection which has implications for adaptive evolution and making predictions about their evolutionary potential. The importance of their contribution to phenotypic variation and life history evolution has made maternal effects an important consideration in fields such as conservation and population biology, evolutionary ecology and evolutionary genetics. The aim of this thesis is to investigate if maternal effects can influence offspring life history traits and fitness parameters through maternal resources via the egg. Main questions that are asked include: can maternal effects help facilitate transition to a novel host-diet (Chapter 2); does maternal diet influence egg composition and, if so, does this have an effect on offspring life-history parameters (Chapter 3); is there a genetic basis to egg composition and is there potential for egg composition to evolve (Chapter 4); and are defensive compounds from the diet transferred into the eggs, if so, are these uni- or biparentally transferred and does this offer protection against predation (Chapter 5)? To address these questions we used a specialist insect herbivore, the large milkweed bug Oncopeltus fasciatus (Hemiptera: Lygaeidae). In the wild, O. fasciatus feed on plants from the genus Asclepias (Apocynacea). However, O. fasciatus can be reared successfully in laboratories on sunflower seeds Helianthus annus. For our experiments we used two populations of O. fasciatus, one population has been maintained on dry seeds of A. syriaca while the other population has been reared and maintained on sunflower seeds. The results of Chapter 2 were suggestive of a maternal host-diet effect on egg mass and hatching success, but we did not find evidence that maternal host-diet was significant in influencing a transition to a novel host. In Chapter 3 we found that there was variation in the free amino acid profiles of the eggs between our treatments suggesting that amino acid profiles may be influenced by maternal diet. The results of our multivariate selection analysis to examine linear and nonlinear (quadratic) relationships between maternal diet and the free amino acid profiles of the eggs suggest that there may be population-specific responses which can influence specific amino acid profiles in relation to hatchling mass. In Chapter 4 we used only the milkweed-adapted population to determine if there was a genetic basis to amino acid profiles in the eggs. We constructed a genetic variance-covariance (G) matrix to determine the strength and direction of the relationships between amino acids and to assess the potential for amino acid profiles to evolve. While we found genetic variation for amino acids, and that there was evidence for positive moderate to strong genetic correlations between many of them, we also found evidence for constraints for the potential for amino acid profiles to evolve as evidenced by the calculation of gmax (which represents the linear combination of components that has the highest genetic variance and which is the most accessible to evolution). In Chapter 5 we found maternal, but not paternal, transmission of cardenolides into the eggs. However, this did not confer protection of all eggs against predation from larvae of the green lacewing Chrysoperla carnea. Overall, results suggest that for our populations of O. fasciatus, maternal effects are significant in influencing early life history traits such as egg mass and hatchling mass. However, we did not find any significant effects on other offspring life history or fitness parameters that we measured. This may be surprising as positive, and negative, effects of non-genetic contributions of females (and males) to their offspring has been widely reported in many taxa. The patterns and implications of maternal resource allocation and their effects on offspring life history evolution are explored and discussed, as are the limitations of our experimental designs. I hope that this research can be used to stimulate further investigations into maternal effects and the relationships between host-plant, maternal allocation strategies and life history evolution.

Biomolecular systems, in particular those involving proteins and their constituents, have been the focus of much research in the last century. The relationship between experiment, development of models and simulation has enabled vast improvements in our knowledge of subjects such as protein folding and the processes by which key biomolecules affect the human body. In particular, vital information can be obtained from understanding the building blocks of polypeptides and proteins involved in these processes. This work focuses on simulating two such building blocks; glutamate, the salt of the proteinogenic amino acid, glutamic acid, and glycine-proline-glutamate, or GPE, a related tripeptide. Both are important in neurotransmission processes in the brain. Glutamate is the most abundant neurotransmitter in the central nervous system and GPE is an important neuroprotective agent. This work aims to elucidate the key structural properties of aqueous solutions of glutamate and GPE, focusing on the solute-solute as well as the solute-solvent interactions. Both systems were considered with classical empirical potentials using the CHARMM22 force-fi eld. The glutamate system has also been studied using Car-Parrinello Molecular Dynamics and classical parallel tempering. In both the aqueous glutamate and GPE systems the molecules formed a large proportion of bifurcated bonding motifs with both carboxyl groups, but not with the amin (N-terminal) of the molecules. Bifurcated bonds form between solute molecules as well as in the solute-solvent interactions. The structure of the glutamate solution was found to be dependent on the initial con figuration and thus the parallel tempering simulations enabled better sampling of the conformational landscape. In addition, in the glutamate system single water molecules form a stable structure by bonding to both the amine (N-terminal) and C -carboxyl within the same glutamate molecule.

The potential of countercurrent chromatography (CCC) as a small footprint bioreactor/separator for manufacture of enantiopure chiral molecules was explored, using as a model reaction the isolation of L-amino butyric acid (L-ABA) from a DL-ABA racemate and the enantioselectivity of D-amino acid oxidase (DAAO). Bioconversion of D-ABA to ketobutyric acid (KBA) by DAAO, immobilised by selective partitioning in the stationary phase of the CCC centrifuge, was accompanied by separation of unreacted L-ABA from KBA by the countercurrent action of the centrifuge. For effective bioreactor/separator action, a high partition of the biocatalyst to the stationary phase was required in order to retain the biocatalyst in the coil, with differing partitions of substrates and products between the stationary phase (SP) and mobile phase (MP) so that these could be separated. Aqueous two-phase systems (ATPS) were the major two-phase systems used to provide SP and MP, as these are well reported to be effective in preserving enzyme activity. The distribution ratios of DL-ABA, KBA and DAAO were measured in a range of phases with polyethylene glycols (PEGs) of different molecular weights, different salts, and different compositions of PEG and salt, using an automated robotic method, developed for the purpose. A system of 14% w/w PEG 1000/ 14% w/w potassium phosphate, pH 7.6, gave the best combination of distributions ratios (CPEG phase/Csalt phase = CSP/CMP) for ABA, KBA and biocatalyst (DAAO) of 0.6, 2.4 and 19.6 respectively. A limited number of aqueous-organic and ionic liquid two-phase systems were also reviewed, but found unsatisfactory. CCC operating conditions such as substrate concentration, biocatalyst concentration, the mobile phase flow rate (residence time in the CCC coil), temperature, rotational speed and operational modes (single flow and multiple-dual flow) and types of mixing (cascade and wave-like) were optimised to produce total conversion of D-ABA to KBA, which was then completely separated from unreacted, enantiomerically pure (>99% ee), LABA. Advantages of the CCC bioreactor over conventional technology include reduced equipment footprint, cheaper running costs, and faster purifications. However, in its current format the drawbacks, such as enzyme instability and excessive optimisation time, reduce its commercial appeal. Additional investigations into the use of whole cell preparations of biocatalyst in the CCC bioreactor showed potential to overcome the problem of enzyme instability and this may in the future give the CCC bioreactor a place in the enantioseparation field.

In this thesis, two potential applications of transdermal iontophoresis in the field of sports medicine were studied: (1) the local delivery of dexamethasone phosphate (Dex-Phos), a corticosteroid used to treat musculoskeletal inflammation, and (2) the extraction of systemic amino acids (AAs), potential biological markers of fatigue in athletes. The iontophoretic delivery of Dex-Phos was studied, in vitro, in order to evaluate the effects of competing ions and electroosmosis, and identify the optimal conditions for its delivery. The iontophoretic extraction of AAs from the skin was first studied in vitro, before evaluating the method in a group of human volunteers. Dex-Phos was best delivered by iontophoresis from the cathode in absence of background electrolyte in the drug solution. In this situation, the delivery of Dex-Phos is limited principally by the competition with counter-ions (mainly Na+) present subdermally and the small mobility of the drug inside the membrane. The accumulation of Cl-, released by the Ag/AgCl cathode in the drug solution during current passage, can also reduce Dex-Phos delivery. The extraction of zwitterionic AAs from the skin during iontophoresis was highly influenced by their presence in the outermost layer of the skin, the stratum corneum (SC). In the pig skin model, the amount of the AAs extracted during a short extraction period (1 hour) correlated with their abundance in the SC. Once this ‘reservoir’ was emptied (after ~3 hours of iontophoresis), the subdermal compartment could be sampled, suggesting that the method could be used to monitor systemic levels of AAs. The experiments in human volunteers revealed, however, that a 4-hour iontophoretic extraction period was insufficient to deplete the AAs SC ‘reservoir’. It follows that the method can be used to evaluate the abundance of AAs in the SC, but is unpractical for the clinical monitoring of their systemic levels.

Nitrogen is requisite for yeast growth and metabolism during wine fermentation, and contributes to wine flavor development. Understanding how yeast assimilable nitrogen (YAN) impacts wine chemistry and sensory properties helps inform nitrogen management decisions in both the vineyard and winery. This study used Virginia-grown Chardonel, obtained from a multi-year viticulture study evaluating the impact of nitrogen fertilizer on grapevines. The objective of this work is to determine if vineyard treatments resulted in sufficient juice YAN profile differences to impart chemical and/or sensory differences in resulting wines. Grapes were combined from each field replication of six vineyard nitrogen treatments and pressed to generate six juice lots. Each juice was divided in two for triplicate fermentation using one of two Saccharomyces cerevisiae x bayanus yeast strains: EC1118 or QA23 to investigate the interactive effects of YAN and yeast strain. Juice and wine samples were frozen until chemical and UPLC-PDA amino acid analysis. Fermentation replications of each wine were combined, bottled, and stored at 4ºC for four months until evaluation via sensory sorting task with untrained panelists (n=63). Juice YAN concentrations ranged from 157-201mg N/L, a small variation in terms of commercial winemaking. Chemical differences between wines produced with the same juice but different yeast strains were larger in wines that had lower juice YAN concentrations. DISTATIS analysis of sorting results showed differences but no pattern in sensory perceptions of wines based on nitrogen profile or yeast strain. This range of nitrogen profiles shows limited impact of YAN profile on final wine quality.

Main-chain conformations where one amino acid is described as gammaR (or alphaR) and an adjacent one is gammaL (or alphaL) generally give rise to a structure where the three main-chain NH groups (one from each of the two residues and the one that follows) form a depression. Due to the electronegativities of the atoms involved, this depression is able to accommodate an atom with a whole or partial negative charge. The negatively charged atom, when present, is also stabilised through hydrogen bonds with the NH groups. We propose the name "nest" for this feature. The nest is a common motif with 8% of residues in an average protein being involved in one. It is also a novel motif even though the situations it occurs in are well known. The negatively charged atom, or anion, that often occupies the nest varies and may be a main chain carbonyl group such as in the paperclip (Schellman motif), it may also be a phosphate group as in the P-loop superfamily that binds ATP and GTP. Overlapping (compound) nests and adjacent (tandem) nests are often observed and encourage the binding of larger more complex anionic groups. Examples of this include the compound nest of the P-loop which has 5 successive NH groups that bind the p phosphate group of nucleotide triphosphate. The longest compound nests are found surrounding cysteine bound [2Fe2S] and [4Fe4S] iron sulphur centres, which are also anionic groups. A totally novel motif occurs where the first residue of the nest is Asp, Asn, Ser or Thr. In any of these cases the sidechain acts as the negatively charged group so that the sidechain is effectively interacting with its own main-chain NH group and the two that follow. These motifs are relatively common and are named depending on the identity of the side chain involved; where the first amino acid is Asn or As the motif is known as an Asx-nest, when it is Ser or Thr the motif is an ST-nest. These are important motifs and are found in the calcium-binding loops of EF-hand proteins. The discovery of the nest prompted an investigation into polypeptides where successive residues have main-chain phi,psi conformations of opposite hand (alternating enantiomeric main-chain dihedral angles). Through the creation of model polypeptides on computer the full range of possible conformations was examined producing a plot not unlike the Ramachandran plot. All alternating enantiomeric structures are ring-shaped or extended, and some are found to occur in proteins, the commonest approximating to the nest. Another conformation related to the nest but more extended is found in the four stretches of polypeptide that line the tetrameric K+ channel, in this case the main-chain CO groups are the important feature and bind the K+ ions in the channel. A different ring-shaped structure where the main-chain CO groups point to the centre of the ring is employed for specific Ca2+ ion binding in the annexin, phospholipase A2 and subtilisin loops, and the regularly arranged beta-roll loops of the serralysin protease family. This feature we propose calling the catgrip.

Two novel lectins were purified from the Moraceae plant Morus nigra or Black Mulberry. These lectins were named Morus nigra agglutinin I (MNA I) and Morus nigra agglutinin II (MNA II). Data obtained from carbohydrate inhibition studies indicated that MNA I specifically bound N-acetylgalactosamine and D-galactose, and in contrast, MNA II selectivity bound α-methyl-D-mannoside and D-mannose. MNA I is composed of two glycosylated α-chains and two β-chains, both of which share a high degree of amino acid sequence identity with the N-terminal regions of the α- and β-chains of the Moraceae lectins, Jacalin and Macluar pomifera agglutinin (MPA). MNA II consists of three dominant subunits, each of which has the same N-terminal amino acid sequence. This sequence shares a degree of N-terminal amino acid sequence identity with the Moraceae D-mannose binding lectin KM+ and the β-chains of Jacalin, MPA and MNA I. Glycosylation of the MNA II subunits was not detected. The binding characteristics of Moraceae lectins to the intestinal brush border were assessed using both in vitro and in vivo approaches. Initially in vitro studies using Brush Border Vesicle preparations were conducted. Thereafter in situ experiments using rat intestinal ligated loops were carried out. The in vitro and in situ binding studies showed that MNA I had a low binding capacity for the rat intestinal brush border, however Western blot studies illustrated that MNA I selectively bound to two rat intestinal brush boarder proteins. Initial in vitro data indicated that MNA II also had a low binding capacity for the rat intestinal brush border. However, additional in vitro and in situ studies showed MNA II bound extensively to the jejunal villi. Furthermore, considerable MNA II uptake into the jejunal enterocytes and lamina propria was detected.

Over the last forty years considerable evidence has been gathered in support of the hypothesis that amino acids are among the most important neurotransmitters within the central nervous system. The development of compounds that inhibit the actions of these recently described neurotransmitters led to the identification of several subtypes of amino acid receptor. Recognition of the neurotoxic effects mediated by excitatory amino acids (EAAs) has prompted investigation of the role of these naturally occurring substances in the processes that culminate in irreversible neuronal injury and death. The observation that EAA receptor antagonists protect neurons from the toxic effects of hypoxia and potent EAA receptor agonists has stimulated interest in the potential therapeutic use of this new class of drug in neurodegenerative disorders. These include stroke, head injury, epilepsy, Parkinson's and Alzheimer's diseases, motorneurone disease, Huntington's Chorea and the neurological sequelae of the acquired immunodeficiency syndrome (AIDS). Surgical procedures performed with the aid of cardiopulmonary bypass (CPB) represent a novel human model of diffuse neuronal injury. Advances in both technology and surgical technique have brought about a steady decline in the mortality and morbidity associated with such cardiac surgery. The fall in the incidence of major perioperative neurological sequelae (brain death, coma, stroke, seizures and altered conscious level) has paralleled that of other complications. Despite this apparent improvement in outcome, formal neuropsychological testing reveals that a significant proportion of patients sustain persistent deficits in cognitive function after otherwise successful surgery. Amongst the many factors implicated in the genesis of this phenomenon, the delivery of microemboli to the cerebral microcirculation during CPB, appears to be one of the most important. This thesis reviews some of the historical landmarks in the evolution of coronary artery bypass surgery (CABS); discusses the neurological consequences of cardiac surgery and reviews the evidence for a role for amino acids in neurotransmission and neurotoxicity. The hypothesis that the incidence and severity of neuropsychological deficits after CPB can be reduced by Remacemide, a selective inhibitor of excitotoxic amino acids, is examined in a detailed study of patients undergoing CABS. Evidence for pharmacological neuroprotection is presented in this first reported use of an EAA antagonist in this clinical setting.

Neisseria meningitidis is an obligate human pathogen responsible for endemic and epidemic disease world-wide. Currently available vaccine preparations are based upon group specific polysaccharides, however the structure of the polysaccharide of group B strains closely resembles that of human glycoproteins and it is non-immimogenic. Traditional epidemiological studies of meningococcal disease have relied on detecting differences in surface antigen expression between strains by serological methods. Sequence variation in the class 1 outer membrane protein (OMP) has been shown to occur in regions not recognised by monoclonal antibodies representing a hidden source of epidemiological information. The variation of two additional OMPs, class 2/3 and Opc in a collection of strains from patients and close contacts following a disease outbreak was investigated. Comparison of the inferred amino acid sequences showed that three distinct variants within serotype 2a were present. Sequencing of the class 3 expressing strains allowed identification of two non-typeable strains. Of the 22 strain only nine were found to possess the opc gene, with four distinct variants present. In order to evaluate the vaccine potential of a particular protein it is advantageous to prepare a source of that protein free from other meningococcal components. The porB gene, which codes for the class 3 protein was amplified by PCR and cloned into two expression systems. The highest levels of recombinant class 3 (rC13) protein expression were achieved in the Xpress™ system in E.coli JMlOl, which inserts the gene downstream of a N-terminal fusion encoding six histidine residues that functions as a metal binding domain, allowing a one-step purification procedure by immobilised metal affinity chromatography. Sequencing of the insert was used to ensure that no mutations had arisen as a result of the original gene amplification. Epitopes on the class 3 protein are of a conformational nature and therefore the rC13 protein was inserted into liposomes and Zwittergent 3-14 and NDSB-195 micelles in an attempt to refold the protein. The adjuvant MPLA was also included in some preparations to boost the immune response. The various immunisation preparations all induced antibodies against the homologous class 3 protein as detected by ELISA and Western blotting. Cross-reactivity against a heterologous class 2 and class 3 protein was also seen in some sera. The ability to kill live meningococci is accepted as the correlate of protection. The highest levels of bactericidal activity were shown by sera raised against rC13 in Zwittergent 3-14 with MPLA and rC13 in NDSB-195 with MPLA. Thus, refolding of the rC13 protein had been achieved and had resulted in significant bactericidal activity.

Thyroid hormones (TH) exert their multitude of effects on body development, growth and metabolism largely via transcriptional regulatory pathways. TH-induced transcription is controlled by receptors present in the cell nucleus, therefore extracellular TH must first cross the plasma membrane to gain entry into the cell. The exact mechanisms of TH transport across the plasma membrane are only beginning to be clarified, but it is likely that transport may be an important control step for the effects of TH on transcription. Members of the recently cloned organic anion transporting polypeptide (OATP) family have been shown to transport TH. Inhibitor studies indicate that both the aromatic amino acid System T-type transporter, and the broad scope neutral amino acid transporter System L are mediators of TH uptake into various cell types. However cloned amino acid transporters have not been studied to demonstrate directly whether they can accept TH as substrates.