This thesis contains three chapters concerning synthetic studies towards the alkaloids (-)-α-kainic acid; a natural product isolated from the Japanese marine algae Kainin-sou (海人草) or Digenea simplex and the close family member (-)-domoic acid isolated from another Japanese marine algae Doumoi or Chondria armata. Chapter one gives an introduction to the isolation, structure and biological activity of kainic acid, domoic acid and their analogues. Chapter one also contains a discussion of the previous syntheses of both kainic acid, domoic acid and domoic acid C. It is written with the aim of selecting key aspects of each synthesis and in turn gives a critical account of each piece of work. Chapter two is concerned with the results obtained from the experimental section of this thesis. Disclosed is a novel method for the construction of (-)-α-kainic acid via an ene-reaction on a 1,6-diene intermediate. The synthesis comprises of eight linear steps from readily available D-serine and through the use of simple methodology forms the target compound kainic acid in a satisfactory overall yield of 20 %. This thesis also investigates the possibility of installing a variety of side chains to the biologically active kainoid core via a cross-metathesis reaction on an unsaturated carbon appendage. Chapter three contains the experimental procedures carried out for the synthesis of the compounds discussed in chapter two.

Human exonuclease1 (hExo1) is a member of the eukaryotic nuclease family that includes Rad2/Xeroderma pigmentosum complementation group G (XPG), flap endonuclease1 (FEN1) and gap endonuclease1 (GEN1). Human exonuclease1 is involved in multiple DNA metabolism processes, including DNA repair and replication. Most of the fundamental roles of Exo1 have been described in yeast. In this study, hExo1 protein was over expressed from both insect cells and bacteria and over expressed protein was purified to near homogeneity. In this research project, a biochemical characterization of full-length hExo1 is reported. As well as assaying hExo1 on different dsDNA substrates, the factors essential for the thermodynamic stability of hExo1 were determined. It is shown in this study that resection activity and stability of hExo1 on dsDNA is modulated by temperature, pH and salt concentration. The DNA end resection process is a guiding principle to cellular response during DNA double strand break lesion and is pivotal for genome maintenance. Even though insufficient DNA resection restrains homology-directed repair mechanisms and the activation of ATR (ataxia telangiectasia and Rad3 related)-dependent checkpoint, over-resection results in production of an excessive single-stranded DNA that could lead to genomic instability. Nonetheless, the control mechanisms for DNA end resection are not yet understood fully. In this study it is shown that the major resection nuclease hExo1 is both positively and negatively controlled by protein-protein interactions to enable a proper DNA end resection mechanism. This report show that 14-3-3ζ 14-3-3ε proteins interact with the C-terminus region of hExo1 and allosterically control hExo1 DNA end-resection activity while PCNA sliding clamp increases the DNA end resection activity by hExo1. Circular dichroism shows that the C-terminus region of hExo1 is intrinsically disordered with significant polyproline type II conformations. Dynamic Light Scattering and Sedimentation Velocity Analytical Ultracentrifugation results show that a monomeric, partly intrinsically disordered, form persists for hExo1 in solution with an expanded hydrodynamic radius of 118 Å. Taking into consideration the structural propensity of hExo1 and the fact that, more often the binding sites for the 14-3-3 proteins are found within the unstructured regions, this study propose that the disorder-to-order transition of the ligand molecule’s structure might be a model of how hExo1 is negatively control by the 14-3-3 proteins. Results of this project work provide crucial insights into a pioneering process of DNA end resection regulation a critical event in genome maintenance and may be implicative in cancer treatment.

Polymorphic epithelial mucins are large complex glycoproteins which consist of a single polypeptide backbone, a large domain of which is usually made up of degenerate tandem repeats. One such molecule MUC1 is expressed at the surface of human mammary cells and is developmentally regulated and aberrantly expressed in tumours. These mucins have been identified as the target antigens for a number of murine monoclonal antibodies raised against a variety of immunogens including human milk products and breast tumour extracts. The anti-MUC1 antibodies have been shown to display tumour reactivity and have been used both as agents for imaging and in immunoassays to assess tumour burden and response to therapy. A number of these antibodies have been found to define epitopes within the tandem repeat of the protein core of the MUC1. Hydropathicity calculations and secondary structure predictions on the twenty amino acid tandem repeat of the MUC1 core protein have identified a hydrophilic domain Pro-Asp-Thr-Arg-Pro-Ala-Pro, which has a high probability of turn formation. It is within this domain that the epitopes of the anti-MUC1 antibodies are found. High field N.M.R. studies undertaken on an antigenic twenty amino acid peptide corresponding to the tandem repeat sequence of MUC1 in dimethyl sulphoxide have identified the presence of a type-I beta-turn in the region Pro-Asp-Thr-Arg. This turn overlaps the epitopes of all of the MUC1 antibodies characterised to date. In an attempt to identify more precisely the conformational requirements for binding to two anti-MUC1 antibodies, HMFG1 and HMFG2, the solution structures of several MUC1 core related peptides in dimethyl sulphoxide have been investigated. All of the peptides studied have been found to contain either beta-turns or modified turns which overlap their hydrophilic epitope domains. While these observations may provide some explanation for the observed reactivity of the different peptides, they give little insight into the precise structural requirements for antibody binding. Due to the flexibility of linear peptides in solution it is not possible to define the side chain conformations which are crucial to the processes of antibody recognition. In order to define the bound conformations of antigenic peptides using N.M.R. it is necessary to undertake experiments in the presence of antibody. Initial experiments have been performed in order to determine the conformation of the MUC 1 core related twenty amino acid peptide when bound to the anti-mucin antibody C595. In addition DNA coding for the variable domains of C595 has been cloned and sequenced in order to facilitate both expression of recombinant antibody binding fragments and the modelling of the binding site. These studies should provide a clearer understanding of the structural basis of antibody recognition of the peptides, and may give an insight into the specificity of anti-MUC1 antibodies for malignant cells.

Feruloyl esterases (E.C. 3.1.1.73), a subclass of the carboxylic acid esterases (E.C. 3.1.1.1), are able to hydrolyse the ester bond between the hydroxycinnamic acids and sugars present in the plant cell walls, and have been classified as Types A or B based on their substrate specificity for aromatic moieties. They constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri-food industries. In order to expand the range of available enzymes, we have examined the production of feruloyl esterases by the filamentous fungi Talaromyces slipitalus and Neurospora crassa. Neurospora crassa has been shown to produce multiple feruloyl esterase activities depending upon the time of fermentation with either sugar beet pulp or wheat bran substrates. A gene identified on the basis of its expression on sugar beet pulp has been cloned and over-expressed in Pichia pasloris. The gene encodes a single domain feruloyl esterase (NcFae-l), which represents the first report of a nonmodular Type-B enzyme and the purified recombinant protein has been shown to exhibit concentration dependent substrate inhibition. The kinetic behaviour of the non-modular enzyme is discussed in terms of the diversity in the roles of the feruloyl esterases in the mobilisation of plant cell wall materials and their respective modes of action. A novel feruloyl esterase (TsF AEC, Type-C feruloyl esterase) that exhibits broad substrate specificity in culture supernatants of Talaromyces slipitalus when grown on sugar beet pulp, has been cloned and over-expressed in Pichiapasloris. Various gene fusions have been constructed to investigate the use of alternative signal peptides by P. pastoris and to produce an authentic feruloyl esterase featuring the N-terminal sequence determined for the native enzyme. It has been demonstrated that additional amino acids at the N-terminus of the FAEC sequence do not influence the catalytic capacity of the enzyme, and that the nature of the signal sequence does not appreciably alter the yield of the secreted enzyme. NcFae-l and TsF AEC contain internal peptide sequences that correspond with the consensus motif G-X-S-X-G that contains the catalytic serine nucleophile conserved in the esterase enzyme supcrfamily. The serine residues at the centre of these peptide motifs have been independently mutated and the corresponding enzymes overexpressed in P. pastoris to identify essential serine residues as candidate nucleophiles responsible for catalysing the enzymatic reaction. Based on activity profile data and supported by the characterisation of a recombinant Type-D feruloyl esterase from N. crassa, a feruloyl esterase sub-classification is proposed and discussed in terms of the evolutionary relationships existing between carbohydrate esterases.

Bioactive lipids or lipids that activate specific signalling pathways are involved in the regulation and maintenance of normal bodily functions. Furthermore, bioactive lipid targets have been implicated in a number of conditions such as cancer, asthma and arthritis, all of which contain an inflammatory element. Additionally, a number of bioactive lipids are also known to target several different types of receptors. These include the cannabinoid receptors, peroxisome proliferator activated receptor alpha (PPARα) and transient receptor potential vanilloid-1 (TRPV1) ion channels. The assessment of bioactive lipid levels in biological systems is important for understanding their role in cell function and pathological events. The aims of this thesis were to develop an analytical method that allows the simultaneous identification and measurement of lipid mediators involved in inflammatory responses, including cyclooxygenase, lipoxygenase and cytochrome P450 metabolites, along with the endocannabinoids to determine the role of bioactive lipids in models of acute and chronic inflammatory pain. A validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows the simultaneous identification and measurement of 42 bioactive lipids was developed, capable of measuring COX metabolites; PGD[subscript]2/PGE[subscript]2, TXB[subscript]2, LOX metabolites; 5-, 12-, 15-HETE, HODEs, oxoODEs, CYP metabolites; 8- and 11-HETE, EETs and the endocannabinoids; AEA and 2-AG and endocannabinoid-like compounds PEA and OEA. Levels of these analytes were quantified in rat hindpaw, dorsal root ganglia, knee joint, plasma, spinal cord and brain. This LC-MS/MS method was then used to investigate how levels of these bioactive lipids were altered in inflammation and pain. Experiments in this thesis using a PPARα competitive binding assay showed that 8-HETE and PEA bind to PPARα, along with other lipids such as fatty acids and EETs. levels of PPARα ligands were altered in the carrageenan model of inflammatory pain, suggesting that changes in bioactive lipid metabolism may influence the contribution of PPARα in inflammatory pain states. Additionally, in the same model of inflammatory pain, levels of TRPV1 ligands were altered, supporting the known role of TRPV1 in inflammatory pain states. Inhibition of 15- lipoxygenase or blockade of the metabolites resulted in attenuation of hyperalgesia, which was supported by alterations in bioactive lipid levels in vitro but not in vivo. The role of bioactive lipids in a rat model of osteoarthritis was also investigated. Levels of TRPV1 ligands were altered in this model of osteoarthritis, accompanied by the time-dependent development of pain behaviour, supporting a contribution of TRPV1 to OA-induced pain. In conclusion, the development of a novel LC-MS/MS analytical method capable of measuring a large number of bioactive lipids in vitro and in vivo, have provided novel findings to support the involvement of these lipids in inflammation and pain. Overall, these data provide evidence for the involvement of PPARα and TRPV1 ligands in inflammatory pain states.

An introductory chapter discusses the steric block, RNase H and RNA interference antisense mechanisms and the application of antisense nucleic acids as therapeutic agents. Examples of existing chemical modifications of the sugar and backbone regions of nucleic acids are given, followed by the introduction of the vinylphosphonate modification. The vinylphosphonate has previously been examined in DNA and has been synthesised by either Pd(0) catalysed cross-coupling of an H-phosphonate with a vinyl bromide, or by the cross-metathesis of a vinylphosphonate with a terminal olefin. This thesis details the first examples of the vinylphosphonate modification in RNA. The initial aim of this project was the synthesis of a range of nucleosides where the 5'-C-O was replaced by a vinyl bromide carbon-carbon double bond. Starting from alpha-D-glucose, acid catalysed formation of the 1,2:5,6-diisopropylidene alpha-D-glucofuranose was carried out followed by protection of the 3-OH as an acetate. The 5,6-isopropylidene was then subjected to H5IO6 mediated one-pot hydrolysis-oxidative cleavage to obtain the 5-aldehyde. Wittig olefination using CBr4 and Ph3P led to the dibromo olefin which was then stereoselectively reduced using dimethyl phosphite and diisopropylamine to obtain the pure trans-vinyl bromide. Following hydrolysis of the acetate, the stereochemistry of the 3-OH was then inverted by sequential oxidation and reduction. With the correct stereochemistry, the 3-OH was protected as the 2-methylnaphthyl ether. The 1,2-isopropylidene moiety was then hydrolysed and acetylated to the bis-acetate which was subjected to Vorbruggen conditions obtaining the uridine (93%), adenosine (77%), cytidine (30) and guanosine (63%) vinyl bromide nucleosides. The 2'-OAc of the nucleosides were hydrolysed to the 2'-OH in yields of 74-92%. The uridine 2'-OH was protected as the 2'-OTBS ether (98%), analogous to the commercially available phosphoramidites used in automated oligonucleotide synthesis. Similarly, the adenosine and uridine nucleosides could also be blocked as the 2'-OMe (59% and 73% respectively). In the case of the uridine vinyl bromide, the 3'-O-(2-methylnaphthyl) protecting group was cleaved using DDQ, this then enabled the vinylphosphonate-linked uridine dinucleotides to be functionalised at the 3'-OH as the cyanoethyl phosphoramidite using N,N-diisopropyl-2-cyanoethyl-chlorophosphoramidite, DIPEA and DMAP in dichloromethane (2'-OTBS 74%, 2'-OMe 41%). These could then be used in automated solid phase oligonucleotide synthesis. The H-phosphonates were prepared in a single step form the commercially available phosphoramidites using a tetrazole. These were then coupled to the vinyl bromide nucleosides using standard conditions of Pd(OAc)2 (0.2 eq.), dppf (0.4 eq.) and propylene oxide (20 eq.) in THF at 70 oC in a sealed vial for 6 hours. A range of vinylphosphonate-linked dinucleotides were accessed in yields of 61-99%. A detailed experimental section at the end of this thesis describes the procedures used in the synthesis and the analysis of the structures obtained.

The first main objective of this project was to isolate cellular fibronectin from cultures of skin fibroblasts derived from tuberous sclerosis (TS) patients and normals and to compare their structures, paying particular attention to the content and pattern of carbohydrate (glycosylation). The second main objective of this project was to establish the expression and localisation of glycoproteins fibronectin, laminin and tenascin of the extracellular matrix (ECM) in cultures of skin fibroblasts derived from patients with TS and normal individuals. In order to achieve these objectives, fibroblasts were established from primary cultures of skin explants of patients with TS. Control cells were cultured from skin explants donated by people not known to be suffering from any disorder. The purification of cellular fibronectin was achieved from conditioned medium of skin fibroblasts of TS patients and control fibroblasts using Prosep-gelatin affinity chromatography and gel filtration chromatography techniques. Analysis of purified cellular fibronectin by high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) revealed that the carbohydrate portion of the fibronectin molecule was made up of galactose, mannose, glucosamine, galactosamine, sialic acid and fucose. An increased concentration of sialic acid, galactosamine, glucosamine, galactose and mannose was observed in purified fibronectin derived from neck and ungual fibromas of patients with TS. To provide a total increase of carbohydrates more than two fold in comparison to normal fibroblastsderived fibronectin. Purified cellular fibronectin from conditioned medium of fibroblasts grown from skin lesions of different TS patients and from normal skin fibroblasts did not express HNK-1 (anti-leu 7) carbohydrate epitope. Normal skin fibroblasts showed an altered morphology and less confluence when grown on cell culture plates coated with cellular fibronectin derived from TS fibroblasts compared with control fibronectin. This may be a consequence of an altered glycosylation of this protein. The amino acid composition of the purified fibronectin from TS fibroblasts was very similar to that purified fibronectins from normal fibroblasts and to standard commercial plasma and cellular fibronectins. Laminin and tenascin were partially purified from conditioned cell culture medium demonstrating their synthesis and secretion into the cell culture medium by dermal skin fibroblasts. Expression and distribution of fibronectin, tenascin and laminin by established TS and normal skin fibroblasts using immunofluorescence, ELISA, and flow cytometry techniques were analysed and presented qualitative and quantitatively in this thesis. Increased expression and altered distribution of fibronectin and tenascin were observed in the fibroblasts derived from ungual fibroma lesion of a TS patient, but not in fibroblasts of neck fibroma, forehead plaque lesion or unaffacted skin of TS patients in comparison to control fibroblasts. However, increased expression and altered distribution of laminin were observed in neck fibroma-derived fibroblasts in contrast to fibronectin and tenascin. Laminin expression was not changed in ungual fibroma and forehead plaque lesion-derived fibroblasts in comparison to control fibroblasts. Altered distribution of fibronectin was well observed by immunofluorescence particularly in large cells of ungual fibroma. Similar differences were observed with laminin of cells from neck fibroma of TS patients. These results suggest the abnormal assembly of ECM in different TS skin lesions. Abnormal migration of cells during early embryonic development and the hardening of tissues associated with TS may result from abnormal assembly of the ECM. Alterations in distribution and structure of these adhesive glycoproteins may cause functional disruption in their binding and interactions with cells and ECM macromolecules. Studies of these changes in the ECM components may contribute to the understanding of the mechanisms involved in the aetiology of hardened tissues of TS.

Glutamine synthetase (GS), the enzyme responsible for the first step in the assimilation of ammonium in higher plants, generally exists in a number of isoforms associated with different tissues or cellular compartments within the plant. This study has investigated the GS isoenzyme composition of roots and N-fixing root nodules of the temperate legume, Lotus comiculatus, and has used a novel transgenic approach to manipulate the spatial distribution of the enzyme within these tissues. The isoforms of GS were studied using ion-exchange chromatography and western blotting, and a nodule-specific isoform was identified. Nitrate treatment of the N2-fixing plants had a marked effect on the nodule isoform, converting it to a form that was indistinguishable by ion-exchange chromatography from the root isoenzyme. To allow tissue-specific manipulation of GS activity, a chimaeric gene was constructed consisting of a translational fusion between the pat and uidA genes coding for phosphinothricin acetyl transferase (PAT) and β-glucuronidase (GUS), respectively. The PAT enzyme detoxifies the GS inhibitor, phosphinothricin, while GUS is a readily assayable marker enzyme that allowed the localisation of PAT activity within the plant tissues to be inferred. The pat::uidA gene was shown to encode a bifunctional enzyme when expressed in E. coli and in transgenic L. comiculatus plants. Transgenic lines carrying a nodule-specific promoter fused to pat::uidA were resistant to PPT only when nodulated. In some of these lines, nodule GS activity was completely resistant to soil applications of PPT under conditions where root GS was 100% inhibited. After long-term PPT treatment, one line (12E) showed a two-fold increase in nitrogenase activity, a four-fold increase in GS activity, and a 50% increase in dry matter production. Possible explanations for how specific inhibition of root GS led to these effects are discussed.

Trichodiene and [14C]trichodiene have been produced in high yields by treatment of Fusarium culmorum CMI 14764 cultures with the furanocoumarin xanthotoxin. Smaller amounts of isotrichodermin (ITD) and the unsubstituted trichothecene 12,13-epoxytrichothec-9-ene (EPT) were obtained in the same way. EPT was also produced by semi-synthesis from ITD. Trichodiene (TDN) was shown to be a precursor of the trichothecene mycotoxins in F. culmorum, including EPT, ITD, calonectrin (CAL), 7a-hydroxycalonectrin (7-hydroxyCAL), 15-deacetylcalonectrin, 3-acetyldeoxynivalenol (3-AcDON) and 7,8-dihydroxycalonectrin (DHC). When large amounts of TDN were supplied, a new trichodiene metabolite was found to accumulate which was fully characterised as 12,13-epoxy-2a, 11 adihydroxytrichodiene, and given the trivial name isotrichodiol. A method for the production of 14C-labelled isotrichodiol (ITdiol) was developed, and the incorporation of ["C]ITdiol into 3-AcDON, DHC and 7-hydroxyCAL was demonstrated. Slow, acid-catalysed cyclisation of ITdiol to EPT and pre-sambucoin was demonstrated, and allylic isomerisation to both 9a- and 9p-trichodiol was also detected. Labelled pre-sambucoin was incorporated into sambucoin by F. culmorwn, and ITdiol is thus proposed as a precursor to both sambucoin and sambucinol, aswel as to the trichothecenes. A range of semi-synthetic derivatives of TDN were prepared and tested as possible inhibitors of the post-TDN biosynthesic pathway to trichothecenes in F. culmorum. In whole-cell systems all the derivatives inhibited the incorporation of labelled TDN into trichothecenes, and also initiated the production of ITdiol. One derivative, 9P, 10ß-epoxytrichodiene, was shown to be biotransformed by the fungus, undergoing 12,13-epoxidation with subsequent hydroxylation at C-3 producing 3a-hydroxy-9(3,10(3; 12,13-diepoxytrichodiene. 9ß-Trichodiol was isolated from Trichothecium roseum, and its slow, acidcatalysed cyclisation to EPT was demonstrated. 9a-Trichotriol, 9ß-trichotriol and isotrichotriol were isolated from F. culmorum for the first time, and literature assignments for the stereochemistry of the C-9 hydroxyl in trichodiol and trichotriol are reassessed. The incorporation of [`4C]ITdiol into trichothecenes in F. culmorum was found to be approximately 5 times greater than the incorporation of [14C]-913-trichotriol, and was shown to be inhibited by isotrichotriol but not by 9ß-trichodiol and 9ß-trichotriol. It is proposed that trichodiol and trichotriol are not biosynthetic intermediates in the pathway to the trichothecenes, and that they are non-enzymic metabolites produced from ITdiol and isotrichotriol, respectively, by acid-catalysed isomerisations. A new scheme for the biosynthesis of trichothecenes is proposed in which ITdiol and isotrichotriol are intermediates in the production of isotrichodermol from TDN. Two novel compounds, 15-deacetyl-7,8-dihydroxycalonectrin (15-deacetylDHC) and 8a-hydroxyisotrichodiol were isolated from F. culmorum, and 15-deacetylDHC and DHC were shown to be precursors to 3-AcDON. It is proposed that the post-cyclisation biosynthesis of 3-AcDON involves sequential oxygenation of isotrichodermol at C-15, C-7 and C-8 producing DHC, which then undergoes deacylation to 15-deacetylDHC followed by oxidation at C-8 to 3-AcDON.

Colicins are a family of bacterial toxins, which kill Escherichia coli cells and other closely related species. Their mode of action requires binding to an outer membrane receptor, translocation across the outer membrane and periplasm and cytotoxic action on a specific target. Colicins usually kill cells either by attacking the bacterial RNA or DNA or by forming pores in the inner membrane of the cell. Their cytotoxic activity can be inhibited by the high affinity binding of an immunity protein. For Group A colicins, translocation requires interaction between the N-terminal domain of the colicin and a series of membrane bound and periplasmic proteins called the Tol system (TolB, TolR, TolA, TolQ and Pal). Three residues of colicin E9 have previously been shown to be essential for an interaction with TolB. This study suggests that these residues play differing roles in the interaction with TolB. Other residues surrounding these previously identified residues are also shown to be involved in the interaction with TolB. In order to allow cytotoxicity, the immunity protein of colicins E3 and E9 must be lost on entry of the colicin to a target cell. This work has demonstrated by Surface Plasmon Resonance and Atomic Force Microscopy that the affinity of colicins E3 and E9 for TolB is increased when the immunity protein is removed. This observation has implications for the mechanism by which the immunity protein dissociates from the colicin. Finally this study has used Surface Plasmon Resonance to explore differences between pore-forming and enzymatic colicins in their interactions with Tol proteins. Although the pore-former colicin A interacts with TolR, TolA and TolB, the endonuclease colicins E3 and E9 were shown only to interact with TolB. This suggests that pore-forming and endonuclease colicins use the Tol system in different ways in order to translocate across the periplasm.