Central white matter and peripheral nerves function by conducting action potentials, which rely on the presence of transmembrane potentials generated by ion gradients. The maintenance of these transmembrane potentials is the main energy-dependent process in the nervous system. In this thesis I investigated the ability of endogenous glycogen to support the energy requirements of nervous tissue and the role of lactate in this process. Glycogen in the CNS is located in astrocytes but is capable of supporting axonal conduction, implying axon-glial metabolic interactions. These interactions were investigated in both the mouse optic nerve (MON), a central white matter tract, and the mouse sciatic nerve (MSN), a mixed peripheral nerve. Electrophysiological techniques were used to record action potential conduction in the nerves as an index of nerve function. Parallel experiments to quantify glycogen content using biochemical assay, or simultaneous real-time measurement of lactate release from the nerves using enzyme-based lactate biosensors, correlated action potential conduction with glycogen content, or lactate release, respectively. Depletion of glycogen leaves the MON vulnerable to irreversible injury to a greater extent than exposure to moderate hyperthermia during aglycemia. Glycogen also greatly enhanced the neuroprotective effects of mild hypothermia during aglycemia. Under resting conditions lactate in the immediate vicinity of the MON was stable at ~0.5 mM, a concentration that increased with axonal activity, dependent upon stimulus intensity. Raising extracellular K+ evoked lactate release, suggesting that increased neuronal activity promotes lactate release. Inhibition of glycogen metabolism, partly reduced lactate release from the MON, implying that glycogen metabolism is important under normal physiological conditions. The relative contribution of glycogen to lactate release increased with axonal activity, consistent with activity-induced glycogenolysis. These studies were then extended to the peripheral nervous system as the role of glycogen in this tissue has not previously been considered. Glycogen, which was present in Schwann cells, supported myelinated, but not un-myelinated axons during aglycemia, suggesting a more complex and selective neuroprotective role than that in central white matter. These results advance our understanding of white matter energy metabolism in relation to both the contributions of glycogen and lactate. The novel functional role of glycogen in supporting peripheral nerve function has also been described.

Molecular Dynamics (MD) and Quartz Crystal Microbalance (QCM) techniques can provide unique insights into what drives protein-ligand association. The major urinary protein (MUP) binds small ligands in a deeply buried hydrophobic pocket. Detailed calorimetric studies have shown that ligand binding is driven by enthalpic effects, not entropic effects [1]. Previous studies have shown that this is due to 'dewetting' of the binding site cavity even in the absence of ligands, and have also characterised the complex changes in molecular flexibility that accompany ligand binding-features that may be correlated with NMR data [2]. Recent MD revealed the hydration effects of apo-MUP and also shown where certain regions of MUP become more flexible upon ligand binding. They have also shown a water molecule remains close to the tyrosine in the binding pocket [2]. In our current MD studies and OCM experiments we have used wild type and 2 different mutants of MUP to study the binding effects of the ligand IBM. The first mutant has an OH group removed from the binding site of MUP (i.e. tyrosine to phenylalanine (Y120F)). The second mutant has an extra OH group in the binding site (i.e. alanine to serine (A103S)). For all three systems the hydration and flexibility upon ligand binding has been analysed. The hydration analysis from MD reveal (from radial distribution curves and hydration density maps) there is a small density of water that remains even without the presence of the ligand for the WT MUP whereas a larger density of water remains in the binding cavity of the A103S hydrophilic MUP simulation. The results are based on the average structure generated from the 1 mus simulations. The Y120F MUP simulations reveal that there is no water molecules present in the binding cavity. However, as protein molecules are very dynamic in nature, water molecules are observed to hop in and out of the binding pockets for both mutant MUP (but not WT MUP) simulations over the 1 mus simulations. On the other hand the experimental QCM results reveal that on ligand binding no water loss is observed for Y120F mutant MUP whereas A103S and WT MUP have about 2 water molecules which are lost in the binding cavity. The flexibility results from the MD simulations reveal that WT MUP have some residues which increase in flexibility whilst other residues which decrease in flexibility on ligand binding. However, the Y120F hydrophobic MUP show an overall decrease in flexibility whereas the A103S MUP shows an overall increase in flexibility on ligand binding. In contrast the experimental OCM and AFM results reveal that there is an increase in flexibility on ligand binding to all 3 different types of MUP molecules. The experimental and the simulation data have shown a variation in results but it is to be noted that the results cannot be directly compared as the analytical experiments are a surface based techniques whereas the MD simulations do not involve a surface. However, the contrast observed between computer simulation and experiments has revealed important information on the ligand binding effects on MUP. [1] Bingham, R.J., J.B.C. Findlay, S.Y. Hsieh, A.P. Kalverda, A. Kjeliberg, C. Perazzolo, S.E.V. Phillips, K. Seshadri, C.H. Trinh, W. B. TurnbulI, G. Bodenhausen, and S.W. Homans. 2004. Thermodynamics of binding of 2-methoxy-3-lsopropylpyrazlne and 2- methoxy-3-lsobutylpyrazine to the major urinary protein. J. Am. Chem. Soc. 126:1675-1681. [2] Barratt, E., R.J. Bingham. D.J. Warner, C.A. Laughton, S.E.V. Phillips, and S.W. Homans. 2005. Van der Waals interactions dominate ligand-protein association in a protein binding site occluded from solvent water. J. Am. Chem. Soc. 127:11827-11834.

An increased understanding of the peripheral cannabinoid receptor (CB2) is required due to the CB2 receptor's emerging involvement in a number of disease states. New fluorescent technologies are capable of generating information about the CB2 receptor systems that has been unachievable using existing pharmacological methods i.e. radioisotope techniques. Our work, to develop fluorescently labelled CB2 receptor ligands, will provide cannabis researchers a unique pharmacological tool to use in conjunction with these emerging fluorescent technologies. This will aid the understanding of cellular actions of cannabinoids and accelerate the discovery of novel CB2 selective drugs. We report on the design, synthesis, and biological evaluation of novel fluorescent ligands targeted towards the CB2 receptor. The fluorescent ligands were designed and synthesised by conjugating recognized high affinity selective CB2 ligands (JTE2-3, JTE2-6 & JWH015) to appropriate fluorescent dyes via a chemical linker. Positioning of the fluorescent dye upon the pharmacophore was guided using established SAR data and supplemented by in-house molecular modelling experiments. Our results showed that modification of JTE2-3 and JTE2-6 with dansyl and BODIPY fluorophores resulted in fluorescent ligands which displayed poor affinity to the CB2 receptor and consequently were unsuccessful when used in fluorescent confocal microscopy experiments. Furthermore, our studies revealed important species selectivity, associated with JTE2-3, which was previously unrecognised. Using de novo drug design on JWH015, we synthesised and tested a 3-naphthyl modified fluorescent conjugate, 3-Gly-NBD-JWH015. The compound retained limited affinity to the CB2 receptor, but fluorescent confocal microscopy did not reveal specific receptor membrane binding. Further experiments using 3-naphthyl precursor compounds, displaying less bulky 3-substituents, demonstrated that limited modification to the 3-naphthyl position of JWH015 was tolerated and provided a first insight to the SAR at this position.

Cytochrome P450 4A1 (CYP4A1) is involved in omega-hydroxylation of fatty acids and eicosanoids. The resulting metabolites may have physiological activities such as regulation of blood pressure. In order to identify structural determinants of substrate binding, site-directed mutagenesis was used. According to a model of CYP4A1, the residues K93, R87 and N116 were predicted to respond to substrate binding. To test the hypothesis, we designed a series of mutants, K93E, R87E, R87E/K93E, R87W/K93E, N116E and N116E/K93E, which would change the substrate specificity of CYP4A1 from a fatty acid to a fatty amine omega-hydroxylase. To reconstitute CYP4A1 activity in vitro, cytochrome b5 and cytochrome P450 reductase were expressed in E.coli and purified. Recombinant CYP4A1 and mutants were expressed in E.coli with an OmpA signal peptide. The conditions of expression were optimised; the enzyme was purified by Ni2+-chelate affinity chromatography. Under optimal conditions, the expression level of CYP4A1 was approximately 60-100nmol/l; mutants were expressed at various levels. The purified enzymes were used in a spectral substrate-binding assay. The K93E mutation did not induce a major change in the substrate specificity from fatty acid to amine; however, K93E showed weak binding to dodecyltrimethylammonium bromide and the Ks (Spectral dissociation constant) value for binding lauric acid increased about three times. R87E mutants had low affinity for lauric acid, but did not show increased affinity for dodecyltrimethylammonium bromide. N116E is similar to wild type CYP4A1 in substrate affinity and specificity. N116/K93E could not be examined owing to the low expression level. These results suggest that K93 is not the principle residue for substrate binding but could be involved in transient contact with substrate; that R87 is crucial for keeping the substrate binding but might not directly contribute to binding of substrate and that N116 does not contribute directly to substrate contact. The residues, H141, R142, R143 and F149, which are located in the conserved Chelix in CYP4A1, were also investigated. We hypothesised that H141 and F149 bind to conserved residues in the I-helix. R142 and R143 may be involved in contacts with electron donors of CYP. Seven mutants including H141R, H141F, H141L, R142A, R143A, F149I and F149Y were constructed. All mutants were expressed and purified as for CYP4A1. R142A was expressed in low level and not further purified. F149I yielded proteins of the expected size, but these proteins did not support a 450nm peak in a reduced CO-difference spectrum, demonstrating an improperly folded enzyme. The enzyme activity of other mutants for lauric acid metabolism is variable; preliminary data showed that the H141L, H141F, R143A and F149Y had very poor enzyme activity, whereas the H141R retained enzyme activity. The results suggest that certain C-helix: I-helix contacts are not required for correct folding of the haem-environment, but are required for function of the P450 enzyme.

The venom of the black widow spider (BWSV) uniquely contains a family of high molecular weight proteins that cause uncontrolled vesicle release in synapses. Two membrane receptors for BWSV have been identified, one of these being latrophilin/CIRL (LPH), a member of the G-protein coupled receptor superfamily of cell-signalling receptors and the other being neurexin. In mammals, LPH and neurexin have been shown to bind BWSV, but their function is unclear. We established C.elegans as a model system for studying the effects of BWSV by microinjection of venom into wild-type (N2) C.elegans, which showed that the venom had an acute lethal effect over a million-fold range of concentrations. BWSV treated with SDS (0.1%) or heat before injection reduced the kill rate in N2 C.elegans to zero, this suggests that the active component of the venom is a protein. FPLC of BWSV demonstrated that the active component of BWSV toxic to C.elegans resembles epsilon- latroinsectotoxin. Identification of a homologue of the latrophilin gene in C.elegans, BO457.1, induced a functional knockout of the latrophilin gene by RNA interference (RNAi). The knockout was examined for a change in phenotype, which occurred in RNAi treated worms, compared to N2, and was extensively characterised. LPH knockout C.elegans were completely resistant to the lethal effects of BWSV over the same concentration range as that used in the N2 worms, whereas RNAi of CYP37A1, BO286.2 and neurexin 1alpha homologue has no effect on BWSV toxicity. We have shown that a C.elegans latrophilin homologue mediates the toxic effects of black widow spider venom in the nematode and identified a high molecular weight latrotoxin that kills C.elegans. Additionally, the data provide evidence for an important role of LPH in nerve cell function.

The capability of the QqQLit hybrid triple quadrupole linear ion trap mass spectrometer to profile endogenous metabolites has been assessed by the analysis of three different families of metabolites; nucleotides from bacteria and N-acyl ethanolamines and N-acyl glycerols from rat tissues. Mass spectrometry methods were developed based on employing a survey scan, either precursor ion or neutral loss, coupled with full product ion spectra. This approach identified families of metabolites with a common structural core and provided the structural information for the reliable identification of known and unknown metabolites. By targeting structural similarities, this approach has opened the window of metabolites that can be profiled beyond the constraints of available references standards. A method to profile phosphate containing endogenous metabolites, particularly nucleotide metabolites, was based on the identification of the phosphate moiety following collision induced dissociation. Employing a precursor ion scan, this approach was successfully applied to the analysis of nucleotides in bacterial samples Escherichia coli MG 1655 and Pseudomonas aeruginosa. A more comprehensive profile of nucleotides was observed compared to targeted approaches. Furthermore, a considerable number of additional analytes were identified which were unlikely to be nucleotides and probably result from other endogenous phosphate containing metabolites, demonstrating the scope of the approach outside nucleotides alone. The use of this methodology was also successful in the profiling ofN-acyl ethanolamines and N-acyl glycerols. Targeting core structures common to each family of metabolite, the ethanolmine and glycerol moiety, precursor ion and neutral loss survey scans were successfully employed in identifying a wider number of these metabolites in various rat tissues than previously reported. The profile of rat testi was notably different from other tissues investigated due to the presence of MAG and NAB C22:5; analytes not detected in other tissues by this method. Furthermore, as far as it can be ascertained, MAG C22:5 has not been previously reported in rat tissues. A quantitative method based on precursor ion - product ion transitions was developed based on the NABs and MAGs identified by the survey scans. By employing this method to analyze various rat tissues harvested immediately after death and five hours post mortem, quantitative data was obtained not only for a broad range of NABs and MAGs at basal levels but also an insight into postmortem changes of these analytes.

The total land available to farm globally is only one quarter of the land available. With the current world population currently rising, standing at over 6.6 billion people in August 2008, a need to produce larger food quantities is an ever increasing pressure to scientists and farmers. The options available to support demands are to produce crops that have higher yields grown on land we currently have available, crops with increased tolerance to abiotic stresses, such as saline toxicity and crops to reclaim land that has been damaged by human use such as heavy metal contaminated land. There are currently over 400 plant species belonging to 45 different families that can tolerate and accumulate excessive amounts of heavy metals, such as nickel, cadmium and zinc. Thlaspi caerulescens a member of the family Brassicaceae (which is therefore closely related to Arabidopsis thaliana), is a well studied model for studying heavy metal accumulation as it accumulates zinc, nickel and sometimes cadmium to high levels without showing signs of toxicity. The primary aim of this research was to identify and confirm potential genes responsible for the hyperaccumulation of zinc, using microarray and qPCR technologies. The second aim was to functionally test any highlighted, potential candidate genes through transgenics, therefore this project aimed to develop a transformation protocol to study potential candidate genes in planta. The microarray successfully identified genes that were differentially expressed in the hyperaccumulator T. caerulescens compared to T. avense, several were confirmed by qPCR. A good candidate gene from this and other studies on Thlaspi caerulescens and Arabidopsis haleri was HMA4 which is a member of the P1B-ATPase family. An RNAi construct was successfully made of the HMA4 gene in an attempt to silence the gene in planta. Attempts were made to transform Thlaspi caerulescens through tissue culture and floral dip methods; however these were unsuccessful due difficulties of T. caerulescens cultivation and transformation. Future strategies would include rapid cycling of plants and heterologous expression of native T. caerulescens genes in Arabidopsis thaliana.

All eukaryotic mRNAs process a cap structure (m7G(5')ppp(5')N) at the S' end of their message and most have an A as the first nucleotide after the cap. However, 30% of messages within eukaryotic cells have a C (m7G(t')ppp(5')C) as the first nucleotide followed by a short polypyrimidine tract. These mRNAs are termed TOP (Tenninal Oligopyrimidine tract) messages and are co-ordinately regulated by mitogenic, growth and nutritional stimuli. This work describes the construction of a reporter vector that encodes mRNA containing the TOP motif, and its use in a series of systematic experiments to further investigate the translational regulation of TOP messages. Given that TOP containing mRNAs are known to encode proteins involved in the translational machinery, these findings have important implications with regard to translational control and translation related disease. In this study, reporter vectors have been used to investigate the role of the mTOR and PI3K signalling pathways, which have previously been implicated in the translational regulation of TOP containing mRNAs. The data obtained suggests that the mTOR signalling pathway may be involved in the regulation of TOP containing mRNAs. The canonical initiation and frans-acting factor requirements of TOP mRNAs were also investigated using a combination of protein over-expression and affinity purification of TOP-containing-mRNA:protein complexes. The data obtained raises the possibility that eIF4E may not be required in the initiation of TOP containing mRNA translation. The candidate trans-acting factors that were identified include La, ILF2 and EBPl, the latter of which has previously been shown to associate with mature ribosomes in the cytoplasm. Finally, affinity purification of TOP-containing-mRNA:microRNA complexes was carried out. Candidate microRNAs which may be involved in the regulation of TOP containing mRNAs were identified. The data obtained was consistent with a previous study, which suggested that microRNA-IOa may bind to the S'UTR of TOP containing mRNAs.

Half of cancers retain wild type p53 but have alterations in the pathways involved in p53 regulation. Murine double minute 2 (Mdm2) regulates p53 by acting as an E3 ubiquitin ligase, which tags p53 for degradation through the proteasome. A small molecule inhibitor, a 5-deazaflavin analogue, has previously been identified by high throughput screening to inhibit Mdm2 E3 ubiquitin ligase activity, thereby reactivating apoptotic function of p53 selectively in cancer cells. Ninety 5-deazaflavin analogues have been synthesised by an optimized existing method and a novel method of synthesis, using the required 6-anilinouracil and 2-p-toluenesulfonyloxybenzaldehyde.The biological ability of the 5-deazaflavin analogues to act as inhibitors of Mdm2 E3 ubiquitin ligase activity to reactivate p53 has been ascertained. A new quantitative biological assay was developed, by scientists based at the Beatson Institute, for 5-deazaflavin compounds, showing excellent inhibition of Mdm2 E3 ubiquitin ligase activity on the previous qualitative biological assay, to yield IC50 data. The biological results have established a clear and logical structure-activity relationship comprising of an electron-withdrawing hydrophobic substituent at the nine position and the N10 phenyl being a prerequisite for activity as a Mdm2 inhibitor. Also meta substitution of the N10 phenyl improves activity against Mdm2 E3 ubiquitin ligase activity. Hit optimization has occurred with 10-(3-chlorophenyl)-9-trifluoromethyl-5-deazaflavin being thirty times more active than the previous identified hit compound, 10-(4-chlorophenyl)-7-nitro-5-deazaflavin. Using the X-ray crystal structure of the Mdm2/MdmX heterodimer, an improved understanding of how Mdm2 acts as an E3 ubiquitin ligase is described and used to form a hypothesis of how 5-deazaflavin analogues function as inhibitors of Mdm2. The work suggests the principle that small molecular weight compounds can inhibit E3 ubiquitin ligases as a possible anti-cancer therapy, and provide the foundation and framework for additional studies and investigation in a new and developing field of medicinal chemistry.

Calpastatin is widely known as an endogenous specific inhibitor to the ubiquitously expressed calpain an enzyme responsible for proteolysis of myofibrillar proteins during post-mortem degradation of muscle. The presence of the calpastatin polypeptide in muscle indicates that the activity of calpain can be potentially down regulated which could result in meat toughness. Asssement of calpastatin activity in meat could be a predictive marker to meat tenderness and variation in the gene has the potential to become a candidate genetic marker which is associated with meat tenderness. The variability and inconsistency produced in meat tenderness post-mortem could be reduced if animals could be selected based on this potential genetic marker prior to slaughter which in turn will reduce the cost in meat processing and ultimately achieve the main objective of producing consistently tender meat. Previous studies have successfully sequenced bovine calpastatin cDNA and found that a series of promoters in the 5’ region are responsible for transcribing Type I, II and III mRNA for calpastatin. The presence and length of CA tandem repeat sequence 5’ to the transcription start site of Type I calpastatin mRNA is believed to play a significant role in regulating the transcriptional activity of this promoter. This thesis investigated the hypothesis that there was a relationship between length polymorphisms of CA repeat located 5’ to the promoter region of Type I bovine calpastatin which altered the level of calpastatin transcripts and ultimately influenced meat shear force value due to the variation in calpain inhibition. Apart from this, transcriptional activity of promoter for Type I, II, and Type III calpastatin were also assessed as well as their response towards agents involved in signalling cascade associated with the agents that stimulate hypertrophic growth. In order to investigate the CA tandem repeat polymorphisms, a PCR based cloning strategy was developed in this study which allowed amplification of this region. Cattle (n=6) of different breed and meat tenderness had their CA tandem repeat sequences amplified which were then cloned into a ZsGreen based reporter construct and transcriptional activity of the promoter were measured using fluorescence imager (Typhoon Trio). From the results, there was no direct correlation (R=0.28) found between the CA tandem repeat length and the shear force value of the meat. However, transcriptional activity for Type I promoter was significantly affected (P<0.05) by changing the length of CA tandem repeat (40-60bp). In general, the calpastatin promoters displayed negative response towards treatment with cAMP(P<0.05) and there were no significant changes to the promoter activity when it was treated with forskolin. Furthermore, a significant reduction in promoter activity (P<0.05) was observed from all calpastatin promoters with calcimycin treatment. The research shows that the type I calpastatin promoter has transcriptional activity and is regulated by secondary messengers which activate cAMP dependent kinases. Although altering the CA tandem repeat length alters promoter activity, there appears to be no simple relationship between its length and toughness, as determined by shear force. However the differential activity of the three calpastatin promoters indicates that there are potentially multiple mechanisms by which its activity can be regulated.