Nucleoside analogues have been extensively used in medication. The nucleoside analogue cordycepin is the principal bioactive compound found in the caterpillar fungi (Cordyceps and Ophiocordyceps). It has been shown to have biological activity, including anti-inflammatory, immunomodulatory and anti-proliferative activity in many kinds of malignant cells. Intracellular drug interactions at the nucleotide level can be explained by understanding the intracellular metabolism of nucleoside analogues as well as their plasma metabolism since their efficacy of therapy or toxicity does not associate with the plasma level of nucleoside. Therefore, investigation of the metabolism of nucleoside analogues is required for a full understanding of their pharmacological activity and toxicity. For that reason, here an ion pairing LC-MS/MS method has been developed for quantitative analysis of the nucleoside analogue cordycepin and the metabolites and its application to cell culture, using in vitro and in vivo studies. Several HPLC parameters and extraction techniques have been optimised, followed by optimisation of the mass spectrometry method by examining the fragmentation of nucleotides. The method was then validated and applied to study the metabolism of cordycepin in vitro and in vivo, to investigate the effect of the cordycepin treatment with or without pentostatin on the intracellular level of endogenous nucleotides, and to examine the intracellular metabolism of nucleoside analogue 4-thiouridine and the effect of its metabolite on the metabolic balance of adenine and uridine nucleotides. The study on the intracellular metabolism of cordycepin in MCF7 and HeLa cells shows that cordycepin was rapidly metabolized into the deaminated form by adenosine deaminase (ADA) in culture medium as well as in cancer cells; therefore combination with pentostatin, an ADA inhibitor, resulting in the highly accumulated phosphorylated metabolite intracellularly. In contrast, cordycepin in C. militaris extracts showed much lower degradation in non-heat-treated serum compared with pure cordycepin that indicates a strong evidence of the presence of a deaminase inhibitor in the extract of Cordyceps. Moreover, the determination of concentrations of cordycepin and the metabolites in the plasma and liver of rats dosed with cordycepin proves that the half-life of cordycepin and its metabolites are very short in the plasma; nevertheless they are accumulated in the liver with repeated administration. Treatment using cordycepin initially caused an increase in the intracellular concentrations of nucleoside triphosphate, but in the long term, the active metabolite of cordycepin likely induced a long term change in the cell resulting in a drop in nucleotide levels. Pentostatin on its own reduced nucleoside triphosphates levels in the long term and combination with cordycepin increased the effect of cordycepin on nucleotide concentrations. High levels of the accumulated cordycepin triphosphate led to a massive decline in nucleotide levels. A study on the intracellular metabolism of nucleoside analogue 4-thiouridine has shown that generally the uptake of 4-thiouridine into NIH 3T3 cells was fast and the phosphorylated metabolite rapidly was developed only after two min labelling. However, it was also shown that its phosphorylation was not very efficient, but the level of the phosphorylated metabolite increased in serum-stimulated cells likely because the enzyme was upregulated in the presence of growth factor. Moreover, the present study provides additional evidence that 4-thiouridine and its metabolite have no adverse effect on the metabolic balance of adenine and uridine nucleotides. This study confirms that pharmacological activity of nucleosides analogues and their cytotoxicity highly rely on the accumulation of their phosphorylated metabolites. Consequently, the activity and the level of the enzymes involved in their metabolism are highly influential on their pharmacological action as well as their toxicity.

Basic Helix-Loop-Helix proteins are transcriptional regulators crucial for many development processes. Using gain- and loss-of-function analysis in zebrafish, the functional role of two members of this protein family, lyl1 (Lymphoblastic leukaemia 1) and Id4 (Inhibitor of differentiation 4) in stem cell fate was determined. Ectopic overexpression of lyl1 resulted in the expansion of haematopoietic stem cell pool and its progeny promoting erythrocyte differentiation and suppressing myeloid differentiation. TALEN-mediated lyl1-/¬- embryos developed normally but displayed distinct marker gene expression during primitive and definitive haematopoiesis establishing a role for lyl1 in both waves of haematopoiesis. During primitive haematopoiesis expression of scl/tal1 and gata1 was unaltered but expression of pu.1 was increased suggesting that lyl1 antagonises myeloid differentiation. Lyl1-deficiency resulted in reduction of Gfi1aa expression during primitive and definitive haematopoiesis. In addition, a reduction in the expression of c-myb in the caudal hematopoietic tissue and rag1 in the thymus was observed indicating that lyl1 is required to maintain the definitive haematopoietic stem cell pool and to drive T lymphopoiesis. In adult zebrafish lyl1 regulates lineage choice driving lymphopoiesis and suppressing myelopoiesis. Morpholino-mediated knockdown of Id4 alone or in combination with p53 resulted in reduced cell proliferation, increased cell death and premature neuronal differentiation. Phenotypic analysis of TALEN-mediated Id4 mutants confirmed that Id4 plays a crucial role in the expansion of neural stem cells and timing of neuronal differentiation. Inhibition of p38MAPK in Id4 morphants as well as Id4-/- mutants resulted in a phenotypic rescue establishing that Id4 negatively regulates p38MAPK activity to ensure normal neurogenesis.

Cytochrome P450s (CYPs) are a superfamily of enzymes involved in the phase I metabolism of endogenous and exogenous substances. They are present in almost all forms of life and have been studied extensively, particularly in relation to human medicine, where knowledge of their activities is essential for predicting drug-drug interactions. In the horse, little is currently known about CYP-specific drug metabolism, which holds importance for animal welfare and for doping control within the horseracing industry where drug-specific metabolites are tested for on race days. Recently the first recombinant equine CYPs have been produced, allowing specific data on equine P450 activity to be gathered for the first time. During the current study,46 full-length P450 sequences were identified from the equine genome. RT-PCR analysis was then carried out on equine liver in order to detect hepatic expression of P450s across various families. After this, cold-induction (pCold) E. coli were used for production of recombinant P450 proteins for subsquent functional testing. Four recombinant equine P450s were successfully expressed (CYP1A1, CYP2A13, CYP2C92 and CYP2D50). Due to being the isoforms most likely to be involved in drug metabolism, rCYP2D50 and rCYP2C92 were selected to be screened against ten of the most commonly used horse drugs to identify potential substrates. rCYP2C92 appeared to metabolise all four NSAIDs tested (flunixin, ketoprofen, phenylbutazone and diclofenac), however presence of the known hydroxylated metabolites of diclofenac and phenylbutazone (4-hydroxydiclofenac and oxyphenbutazone, respectively) could not be confirmed despite being present within equine liver microsome and human recombinant CYP2C9 samples. In spite of the apparant acivity displayed by rCYP2C92 towards all four NSAIDs, no conclussions can be made about this enzyme’s role in NSAID metabolism due to a lack of known hydroxylated metabolite production.

Stable telomeres play a key role to the survival of cancer cells; therefore, different cancer chemotherapeutic approaches have been developed in order to disrupt or destabilise telomeres or telomerase. One of the newest methods is the disruption of vital protein–protein interactions in the telomere, such as that between shelterin components TRF1 and TIN2. The principal aim of this project was to obtain a novel peptide-like molecule, an analogue of a key interacting region of TIN2 that could compete effectively for the binding sites on TRF1 and so lead to the destabilisation of telomere structure. Molecular modelling and simulations were undertaken as the starting point of the project. Structure-based drug design was applied, starting from the available crystal structure data. A library of peptide analogues of the TRF1-binding motif in TIN2 was designed using the MM-GBSA simulation method to predict binding affinities. Then, a number of the peptide analogues were selected from the library for further investigations. The secondary goal was to investigate the accuracy of the predicted ΔGbinding values and try to optimise them; the latter aim was set out after finding a significant difference in the predicted binding free energy values after repeating the identical protocol for the same complex system. Therefore, different approaches were applied to optimise the predicted ΔGbinding values. Subsequently, selected TIN2 peptide analogues were synthesised in the laboratory using Fmoc solid-phase peptide synthesis. Then, the hTRF1 protein was expressed and purified in preparation for the development of the in vitro assay. Finally, biophysical evaluations and screening of the peptide analogues were performed using fluorescence polarisation assay. One of the peptide analogues developed in this study was identified as an early lead compound. In addition, the findings of this research showed that the ΔGbinding values of the peptide analogues have significantly improved accuracy after optimisation. As a result of these investigations, suggestions were identified for future research.

Accelerated carbohydrate utilisation and suppressed lipid oxidative metabolism are hallmarks of heart failure (HF). Hypoxia mimics failing heart metabolic reprogramming and has been suggested to play a role in cardiac metabolic switch. One aspect of the regulation of cardiac energy metabolism is the pyruvate dehydrogenase complex (PDC). Hypoxia inducible factor (HIF) signalling is thought regulate hypoxia-induced adaptations. HIF transcriptional activity is controlled by prolyl hydroxylase domain (PHD) protein and factor inhibiting HIF (FIH-1). In chapter 3 revealed that relative to baseline, acute hypoxia increased cardiac lactate efflux and suppressed fatty acid oxidation (FAO) rates in non-treated isolated mouse hearts with final cardiac recovery being 63% of baseline values. Hypoxic and post-hypoxic PDC activation, via dichloroacetate (DCA), decreased cardiac lactate release and FAO during reoxygenation, but failed to improve cardiac recovery relative to control hearts. Chapter 4 sought to establish how chronic hypoxia (11%) upregulates cardiac glycolytic flux, determined via 3H-glucose. Findings of this chapter indicate that of four enzymes considered to set the pace of glycolysis, upregulated pyruvate kinase (PK) flux, appears to explain accelerated hypoxia-induced cardiac glycolytic flux. Western blotting analysis revealed increased PK M2 protein isoform. Sustained hypoxia increased pentose phosphate pathway (PPP) flux, but left lactate accumulation unaltered. Chapter 5 examined the role of sustained in vivo hypoxia in modulating cardiac tolerance to subsequent acute H/R injury and chronic PDC activation in modifying hypoxic heart tolerance to acute injury. Chronic hypoxia reduced cardiac tolerance to H/R injury accompanied by increased glycolytic flux and lactate efflux during reoxygenation injury. Chronic PDC activation improved hypoxic heart tolerance to the acute injury and normalized cardiac metabolic flux and reduced tissue lactate accumulation during reoxygenation, indicative of increased carbohydrate oxidation. Collectively, the data appear to imply that forced carbohydrate oxidation normalizes hypoxic heart recovery from acute injury. In chapter 6 we demonstrated that global FIH-1 deletion increased isolated heart glycolytic flux at baseline and during reoxygenated. FIH-1 KO hearts displayed increased reoxygenated hexokinase (HK) and PK activities, but no changes in PK protein isoforms. Functional analysis revealed that FIH-1 deficiency does not affect isolated heart function at baseline and in response to acute injury. Acute PDC activation does not appear to improve cardiac function during acute hypoxic stress. Conversely, chronic PDC activation normalized, via restored metabolic flux, cardiac tolerance to acute injury following sustained in vivo hypoxia. Furthermore, the present thesis revealed increased PPP flux following sustained in vivo hypoxia, and proposed a pivotal role PKM2 may play in the regulation of hypoxic heart carbohydrate metabolism. In addition, we identified FIH-1 as a novel regulator of cardiac carbohydrate metabolism at baseline and following acute hypoxic injury.

Enzymes from filamentous fungi have a key role in degradation of the most abundant biopolymers found in nature, cellulose and hemicelluloses. For this reason, these enzymes are of great interest in the industrial conversion of lignocellulosic substrates into biofuels. The production of plant cell wall degrading enzymes is regulated mainly at the transcriptional level in filamentous fungi but little is known about the signalling pathways and transcription factors (TFs) involved in this regulation in Aspergillus niger. RNA-sequencing analysis has been previously carried out to investigate the transcriptional changes that occur when A. niger is transferred from the simple carbon source glucose onto the complex lignocellulosic biomass wheat straw. This has highlighted the up-regulation in transcript level of genes encoding some glycosyl hydrolase (GH) enzymes as well as hydrophobic surface interacting proteins (HSIPs) that may be involved in the interface between lignocellulosic biomass and A. niger. Genes encoding the key TFs XlnR, ClrA and ClrB were deleted from A. niger and the resulting strains were assessed for growth on glucose and wheat straw, transcription of genes encoding glycosyl hydrolases and saccharification activity. Growth of all mutant strains, based in straw on measurement of pH and assay of glucosamine, was impaired in relation to the wild-type (WT) strain although deletion of clrA had less effect than deletion of xlnR or clrB. Release of sugars from wheat straw was also lowered when culture filtrates from TF deletion strains were compared with WT culture filtrates. Transcript levels of cbhA, bglB, eglC and xynA were measured in all strains in glucose and wheat straw media in batch culture with and without pH control. Transcript levels from cbhA, bglB and eglC were lowered in all mutant strains compared to WT although the impact of deleting clrA was not pronounced with expression of eglC and had no effect on xynA. The impact on transcription was not related to changes in pH. In addition to impaired growth on wheat straw, the ΔxlnR strain was sensitive to oxidative stress and displayed cell wall defects in the glucose condition suggesting additional roles for XlnR. Phosphorylation is a key reversible modification that regulates protein function, subcellular localization, complex formation, activation of TFs and cell signalling pathways. A phosphoproteomic study was carried out on both the WT and the ΔxlnR deletion strains of A. niger in order to identify key regulators of the signalling pathways involved in the breakdown of a lignocellulosic substrate, wheat straw. The analysis consisted of comparing the phosphoproteome profiles of the strains when grown in glucose with the phosphoproteome profile of the same strains when exposed to wheat straw for 6h, 12h and 24h. The results suggested a difference in the phosphoproteome profiles of the two strains when exposed to both glucose and wheat straw. These data may provide new information on the importance of XlnR in the regulation of expression of GHs but also in controlling the environment to which A. niger is exposed depending on the nutrient availability. To investigate the role of HSIPs in the induction of A. niger response to wheat straw, single gene deletion strains for hfbD, hyp1 and hsbA as well as the double deletion strain for hfbD and hyp1 have been constructed. The expression of some genes encoding GH enzymes was then followed in these strains using qRT-PCR. The results showed that the transcript levels of the GH genes studied were lowered in the HSIPs deletion strains when compared to the wild-type strain, when the cultures were transferred from glucose medium to wheat straw. These results suggest that HSIPs may have a role in the utilisation of lignocellulosic biomass in A. niger. The precise nature of such a role as well as the characterisation of new TFs, such as ClrB, provides new areas of improvement for industrial processes for production of second generation biofuels.

Survivin is an essential protein that is highly overexpressed in most human cancers and is known to be involved in many cellular processes by virtue of its different locations and interacting partners in the cell. At mitosis it is part of the chromosomal passenger complex, which ensures correct temporal and spatial regulation of chromosome movements and cytokinesis. Survivin also acts in apoptosis suppression via distinct mechanisms many of which have been ascribed to the mitochondrial pool of survivin. However, it is still unclear precisely how survivin gains access to this organelle, its other possible functions while there and its effect on mitochondrial morphology. Though mitochondria play an essential role in apoptosis induction and execution, they also provide phospholipids for incorporation into membranes. Of particular interest to this study, they synthesise phosphatidylethanolamine (PE), by decarboxylation of phosphatidylserine, which is catalysed by phosphatidylserine decarboxylase (PSD). The purpose of this thesis was to test the hypothesis that mitochondrial survivin regulates PSD-mediated conversion of PS to PE in the mitochondria. This thesis shows PSD is a novel partner of survivin which interacts with it both in vivo and in vitro. The interaction was mapped to the N-terminal BIR domain of survivin. Furthermore, only mature catalytically active PSD could interact with survivin, suggesting that survivin regulates PSD activity, and thereby could regulate PE availability to the cell. In addition to discovering that survivin is a novel regulator of PSD activity, this thesis also identified a second isoform of human PSD, and discovered that the NH2 terminal ten amino acids of survivin area non-canonical mitochondrial targeting sequence. As PE is an essential phospholipid which is required for maintenance of mitochondrial integrity, successful cytokinesis and lipidation of autophagosomes during autophagy, the identification of mitochondrial survivin as a regulator of PSD activity can be expected to have a significant impact on cellular health. Therefore, this thesis identified a completely novel mechanism by which mitochondrial survivin could be further fuelling tumorigenesis in vivo.

Circular dichroism (CD) spectroscopy is an important technique in studying protein structure, especially for protein secondary structures and conformational changes during biological processes. A fully quantitative theory of the relationship between protein conformation and optical spectroscopy would facilitate deeper interpretation and insight into biophysical and simulation studies of protein dynamics and folding. Vibrational structure in the electronic CD spectra of proteins is an important source of information on protein conformation and can be exploited to study structure and folding. We employ the state-averaged complete active space (CAS) method to calculate the ab initio electronic ground and excited states of N-methylacetamide (NMA), toluene, p-cresol and 3-methylindole (3-MI), which represent chromophores that are significant in the CD spectroscopy of proteins in the far- and near-ultraviolet (UV) regions. The results of these calculations are used to incorporate vibronic levels of the excited states into first principles calculations of CD using an exciton approach. The far-UV CD spectra of a set of 49 proteins, comprising a range of structural types, are calculated to assess the influence of the vibrational structure. The calculated spectra of -helical proteins are better resolved using the vibronic parameters and correlation between the experimental and the calculated intensity of less regular  structure proteins improves over most wavelengths in the far-UV. No obvious improvement is observed in the calculated spectra of regular -sheet proteins. The near-UV CD spectra of 40 proteins are calculated with the new parameter set and the correlation between the computed and the experimental intensity from 270 to 290 nm is much improved. The contribution of individual chromophores to the CD spectra has been calculated for several mutants and in many cases helps rationalize changes in their experimental spectra. Considering conformational flexibility by using families of NMR structures leads to further improvements for some proteins and illustrates an informative level of sensitivity to side chain conformation. In several cases, the near-UV CD calculations can distinguish the native protein structure from a set of computer-generated misfolded decoy structures. CD spectra of proteins are better reproduced in both far- and near-UV by considering vibrational structures in electronic transitions of chromophores. This improvement can provide more details in connecting the spectroscopic data to the conformations of proteins and will encourage a broader use of CD in protein studies. Besides shedding light on the importance of vibronic transition, results in this thesis also show other aspects that may further improve CD calculations, such as developing parameters of disulfide bond, calculating CD using molecular dynamics (MD) trajectories and taking into account the influence of surroundings of chromophores.

Bone repair is not always a spontaneous process. In some cases, intervention is required. This can involve the use of autograft but requires donor tissue. As a consequence there is a potential lack of material and donor site morbidity. Current alternatives are limited. There is a need for synthetic alternatives with a similar efficacy to autograft. Growth factors are currently being explored to address this need. A limiting factor to growth factor approaches are safety concerns and high costs. Both these problems stem from the fact that growth factors have short in vivo half lives and are administered at supraphysiological levels to maximise the duration of effect. There is a strong need for a growth factor delivery system that can maintain therapeutic doses and restrict administration to a specific location. This is currently limited by the fragile nature of growth factors. Microparticles were utilised. Microparticles were formed from poly(DL-lactic-coglycolic acid) with a poly(ethylene glycol) based plasticiser. This provided a method to modulate protein release based on the specific polymer formulation. Protein release was assessed with a model protein. The biological activities of released growth factors were assessed. Microparticles were fabricated for the delivery of vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and bone morphogenetic protein-2 (BMP-2) for release at time points conducive with osteogenic regeneration. A method was developed and validated to combine these microparticles with a suitable scaffold material. These composite scaffolds were developed with the intention of assessing controlled release of growth factors in a bone segmental defect. A method to fabricate microparticles with consistent size distributions and morphologies was developed. Formulations were tailored such that protein release from microparrticles could be from 2 days to 30 days. The biological activity of the released model protein was verified, as was the biological activity of released BMP-2. A method was devised to combine microparticles with a scaffold suitable for osteogenic regeneration of a segmental defect. This composite scaffold maintained a high level of porosity making it suitable for tissue ingress and growth factor diffusion. This study addresses key limitations to growth factor therapies. The sustained release of growth factors has the potential to mitigate dose-induced toxic effects as well as maintain therapeutic concentrations for longer periods. The nature of the delivery system delivers localised growth factors minimising the risk of systemic dosing leading to adverse reactions. This microparticle technology has potential in developmental research research as well as clinical therapies.

This thesis considers the statistical modelling and analysis of proteomic mass spectrometry data. Proteomics is a relatively new field of study and tried and tested methods of analysis do not yet exist. Mass spectrometry output is high-dimensional and so we firstly develop an algorithm to identify peaks in the spectra in order to reduce the dimensionality of the datasets. We use the results along with a variety of classification methods to examine the classification of new spectra based on a training set. Another method to reduce the complexity of the problem is to fit a parametric model to the data. We model the data as a mixture of Gaussian peaks with parameters representing the peak locations, heights and variances, and apply a Bayesian Markov chain Monte Carlo (MCMC) algorithm to obtain their estimates. These resulting estimates are used to identify m/z values where differences are apparent between groups, where the m/z value of an ion is its mass divided by its charge. A multilevel modelling framework is also considered to incorporate the structure in the data and locations exhibiting differences are again obtained. We consider two mass spectrometry datasets in detail. The first consists of mass spectra from breast cancer cells which either have or have not been treated with the chemotherapeutic agent Taxol. The second consists of mass spectra from melanoma cells classified as stage I or stage IV using the TNM system. Using the MCMC and multilevel techniques described above we show that, in both datasets, small subsets of the available m/z values can be identified which exhibit significant differences in protein expression between groups. Also we see that good classification of new data can also be achieved using a small number of m/z values and that the classification rate does not fall greatly when compared with results from the complete spectra. For both datasets we compare our results with those in the literature which use other techniques on the same data. We conclude by discussing potential areas for further research.