In order to achieve efficient gene delivery, we have designed p\(K_a\) modulatable oligopeptides (2COPs) by combining with lysine, histidine and cysteine residues which will bind DNA extracellularly, internalize via endocytosis, provide a tunable endosomal release mechanism, and provide a degradable backbone in order that the DNA can be released once in the cytoplasm. The reducible polycations (RPCs) were synthesized from 2COPs. The sizes, surface charges and the stability of RPC polyplexes under the simulated physiological conditions extra- and intracellularly suggested that these RPCs are promising vectors. In addition, the transfections revealed that the RPCs can facilitate endosomal buffering and intracellular reduction and are non-toxic to cells. The nuclear targeting signal (TAT) was incorporated into these vectors. The reducible copolycations (RcPCs) were synthesized via oxidative polymerisation between 2COPs and TAT. The RcPC polyplexes are ~100 nm, and are positively charged. Gel shift assay revealed that RcPCs have less potential than RPCs to be used as vectors as they are less stable extracellularly than the RPCs. In addition, chloroquine was required to enhance the transfection of RcPCs. Furthermore, there is no improvement in transfection of RcPC compared to RPCs. Therefore, this suggests that the incorporation of TAT does not improve the transfection.

Anions including nucleic acids and lipids have been found to promote amyloid formation in diseases including neurodegenerative conditions such as Alzheimer’s and Creutzfeldt-Jakob disease. However, the direct effects of these close charge-based interactions are not well understood. It is unclear what effect amyloidogenic peptides would have on nucleic acid integrity. Similarly, the direct effects of amyloidogenic polypeptides on liposomes are not well understood. Here I have used a simplified system of short basic peptides with alternating hydrophobic and hydrophilic amino acid residues to study their interactions with polyanionic nucleic acids and fatty acid liposomes. Employing biophysical techniques including X-ray fibre diffraction, circular dichroism spectroscopy and electron microscopy I showed that the polymerized charges of nucleic acids and pseudo-polymerised charges of lipid membranes concentrated and enhanced the formation of amyloid from short basic peptides, many of which would not otherwise form fibres under the conditions explored. In turn, the same peptides bound nucleic acids and promoted their hybridisation at concentrations below their solution Kd, as shown by time-resolved FRET studies. The mutual interactions between peptides and nucleic acids lead to the formation of amyloid nucleic acid (ANA) fibres, which in addition to their importance in disease might have a potential in nano-engineering of biomaterials.

The elucidation of the biochemistry of oxidative phosphorylation is a problem capable of no easy solution. Combined genetic and biochemical investigation is a more recent approach in this direction. Mutants of the yeast Saccharomyces cerevisiae resistant to inhibitors or to uncouplers of oxidative phosphorylation have been isolated in this Laboratory. In part, this Thesis is an investigation into their further properties. Growth characteristics of selected strains are described together with differences in their cytochrome profiles. The phosphorylation capabilities of isolated mitochondria have also been assayed. The effects of the various agents on cellular and mitochondrial respiration are measured. Resistance to uncouplers may be manifest at the mitochondrial level; but in any case this does not lead to a more efficient energy conservation process. The metabolism of hydrocarbons by microorganisms is now becoming of greater importance. Saccharomycopsis lipolytica is a yeast which is able to grow on n-alkanes of medium chain length. Growth curves and cytochrome contents of S. lipolytica cultured on various substrates, Including n-alkanes, are compared. Isolated mitochondria are also examined. Biological membranes contain characteristic fatty acids. The fatty acid profiles of cells and mitochondria of S. cerevisiae and of S. lipolytica after growth on various substrates are illustrated. The kinetics of membrane bound respiratory enzymes are affected by these lipid constitutents as evidenced by Arrhenius plots.

The biogeochemical transformations related to methanesulfonic acid (MSA) formation and degradation are discussed, with reference to the role of marine bacteria and the phylogeny and biochemistry of methylotrophic bacteria are briefly reviewed. The aims of the work presented were [i] to isolate novel MSA utilising bacteria from both seawater and freshwater samples, [ii] to characterise these isolates and [iii] to elucidate the mechanisms by which MSA is metabolised in these isolates. Isolation procedures for the enrichment of MSA-oxidizing bacterial from a wide range of seawater and freshwater sites, are described. Four methylotrophic bacterial strains, TR3, PSCB4 (marine isolates), FW2 and FW6 (freshwater isolates), capable of growth on MSA as a sole carbon source were isolated from the environment. MSA metabolism in strains TR3 and PSCB4 was initiated by an inducible NADB-dependant monooxygenase, which cleaved MSA into formaldehyde and sulfite. Formaldehyde was assimilated via the serine pathway. Cell suspensions of bacteria grown on MSA completely oxidized MSA to carbon dioxide and sulfite with a MSA: Oxygen stoichiometry of 1.0: 2.0. Oxygen electrode-substrate studies indicated the dissimilation of formaldehyde to formate and C02 for energy generation. Methanol was not an intermediate in MSA metabolism, although the strains could grow on methanol and other one-carbon compounds, as well as a variety of heterotrophic substrates. Initial studies of strains FW2 and FW6 indicated that they probably metabolised MSA in a similar way to the marine strains. Carbon dioxide was not fixed by ribulose bisphosphate carboxylase in strains TR3 and PSCB4. These novel facultative methylotrophs have the ability to mineralize MSA and may play an important role in the cycling of global sulfur, since MSA can be a major product from the oxidation of DMS, the principal biogeochemical organosulfur gas in the environment.

In Escherichia coli, and other prokaryotes, thiamine (vitamin B1) is assembled by coupling 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (Hmp-PP) and 4-methyl-5-(β-hydroxyethyl)thiazole phosphate (Thz-P). The thiazole moiety is biosynthesised from 1-deoxyxylulose-5-phosphate, tyrosine, and cysteine, and at least six genes are required including thiH, thiG, thiS, and thiF. Whilst in aerobes, the C2-N3 fragment of Thz-P derives from glycine in a reaction catalysed by the flavoenzyme ThiO, in anaerobes such as E. coli, dehydroglycine is formed from tyrosine in a ThiH dependent reaction. This biosynthetic step requires the cleavage of the Cα-Cβ bond of tyrosine and a release of an aromatic side chain. ThiH shows sequence similarity with the ‘radical S-adenosylmethionine’ (AdoMet) family of proteins, including conserved cysteine ligands to the essential [4Fe-4S] cluster and has been shown to form a complex with ThiG. With the purpose of studying the mechanistic enzymology by which Thz-P is assembled it was crucial to isolate ThiH in the holo-form. Several expression systems and purification methodologies were investigated. The optimisation of the purification method, together with in vitro chemical reconstitution with exogenous iron and sulfide allowed the successful isolation of holo-ThiH. To facilitate the mechanistic investigation of Thz-P biosynthesis, an in vitro assay was developed, and the reaction products formed in vitro were elucidated and quantified. The aromatic by-product derived from the side chain of tyrosine is p-cresol and the remaining fragment yields glyoxylate, a product of hydrolysis of dehydroglycine

No description available.

No description available.

The ability to detect and interrogate DNA sequences allows further understanding and diagnosis of genetic disease. The ability to perform such analysis of genetic material requires highly selective and reliable technologies. Furthermore techniques which can use simple and cheap equipment allow the use of such technologies for point of care analysis. Described in this thesis are two novel DNA probe systems designed for mutation discrimination and sequence recognition of PCR products. A homogenous PCR system using HyBeacons® which utilise FRET to produce a three probe multiplex system and surface enhanced Raman detection method. Both of these systems allow multiplex detection of PCR products and mutation discrimination by melting temperature analysis. The research reported includes investigations into the effects of different modifications to improve the performance of HyBeacon® probes as well as the effect of different dyes in a FRET system, including unique changes in the optical properties of such dyes. Also a novel method of performing melting temperature analysis using an electrochemical potential is reported. In addition to the detection methods described this thesis includes initial work into the stabilisation of quantum dot nanoparticles for their use in aqueous systems as a potential alternative to fluorescent organic molecules.

No description available.

Triplex-forming oligonucleotides (TFOs) have been the subject of extensive research in recent years. They have potential applications in many areas; such as gene-based therapies, site-directed mutation and as biochemical tools. However, triplex technology has been hampered by several problems, including low stability due to electrostatic repulsion between strands. This thesis has investigated combinations of four methods for stabilising triplex DNA; these include incorporation of the positively charged thymine analogues bis-amino-U and propargylamino-dU in TFOs. Also modified TFO’s containing anthraquinone derivatives have been tested. Further, the free-intercalating agent naphthylquinoline has been used to modulate TFO binding. A TFO containing six consecutive BAU molecules has previously been shown to interact with non-target sites. The pH dependence of this TFO was investigated. These experiments showed that considerably higher TFO concentrations were needed to generate a footprint as the pH was increased. The TFO had a high affinity for the exact template (tyrT) at pH 5.0 and 6.0 and showed some evidence of binding even at 30 μM at pH 7.0. These gels also showed evidence of the secondary binding seen in previous studies; this was considerably more evident at pH 5.0, however, suggesting that the secondary binding may be more sensitive to pH than the primary binding. Secondary binding sites for TFOs were examined by ‘Restriction Endonuclease Protection, Selection and Amplification’ or REPSA. REPSA has been used to select for DNA templates that are bound by the 9mer TFO containing six bis-amino-U residues. Fourteen of the sequences which emerged from REPSA were chosen for footprinting with TFOs containing BAU, propargylaminodU or T. The BAU-TFO produced clear footprints on all but one of the REPSA templates tested, indicating that the REPSA process was successful in selecting for sequences which are bound by the TFO. Significantly higher concentrations of the P-TFO were required, and magnesium chloride and / or the triplex binding ligand naphthylquinoline were needed to promote binding. Despite the differences in template sequence there does not appear to be a strong pattern in the binding intensities of the TFOs on the different templates. However, all templates do contain a run of four to eight A’s. Surprisingly it appears from these data that the BAU TFO discriminates better than the P-TFO against non-exact binding sites The selectivity of TFOs containing anthraquinone modifications was also investigated. Anthraquinone intercalates between DNA bases in duplex DNA and can be tethered to the end of a TFO to increase stability. The specificity of five TFOs with different anthraquinone modifications was examined by footprinting against fragments containing mismatches. A doubly modified TFO bound with the highest affinity and was most tolerant of mismatches. Mismatches at the centre of the template had a lesser effect on binding affinity than mismatches at the 3’ end. The effect of a 3’ mismatch was also greater if the anthraquinone was at this end. The presence of an S-base at the 3’ end allowing intercalation of the anthraquinone at a YpR step increased the binding affinity on the exact template in comparison to TFO 3 which did not contain the S-base. The TFO containing the S base did not bind quite as well as the doubly modified TFO however.