This thesis consists of work on the <I>wis1</I> pathway: the analysis of some of the upstream components and the isolation and characterisation of genes that lie downstream of <I>wis1</I>. The <I>mcs4, win1 </I>and <I>wis4</I> genes had already been shown to lie upstream of <I>wis1</I>. Strains were constructed with different combinations of mutations in these genes. <I>fbp1</I> transcription was assayed in these strains. An additive effect was seen in <I>win1 wis4</I> double mutants, suggesting that <I>win1</I> and<I> wis4</I> act in parallel. To identify functionally-related genes downstream of <I>wis1</I>, the stress sensitivity of <I>wis1Δ</I> cells was exploited. A screen for extragenic suppressing mutations was carried out. Several hundred heat resistant mutants were isolated. Some also presented the salt sensitivity and/or cell length defect of <I>wisΔ</I>. Twelve such <I>sow</I> (for suppressor of <I>wis1Δ</I>) mutants, each of which containing a single suppressing mutation, were analysed further. They fell into two linkage groups: <I>sow1</I> (nine strains) and <I>sow2</I> (three strains). When the <I>sow </I>mutations were crossed into a <I>wis1<SUP>+</SUP></I> background, both <I>sow1</I> and <I>sow2</I> were able to grow at temperatures above the usual range for <I>S. pombe</I>. In addition, <I>sow1</I> strains divide at a shorter length than wild type, indicating a mitotic advance, and <I>sow2</I> cells have a slightly aberrant morphology. To determine whether the <I>sow</I> mutations corresponded to any known genes, crosses were carried out between the <I>sow</I> mutants and mutants in the following genes: <I>wis1</I> pathway genes (<I>sty1, atf1</I>, <I>ppa1, ppa2 </I>and <I>ppe1</I>), cAMP pathway genes (<I>cyr1</I>, <I>pka1</I>) cell cycle regulation genes (<I>cdc2, cdc25, wee1, cdc13</I>), a heat shock protein (hsp90) gene (<I>swo1</I>) and a gene required for maintenance of the mitotic cell cycle (<I>pat1</I>).

No description available.

A potential path to the development of small-molecule inhibitors is to identify small-molecules that mimic the interactions of short peptides with proteins. The present study uses Perl scripts and a MySQL database to build a unique dataset of 258 protein-peptide interactions (ProPep) from structures contained in the Protein Data Bank. The physiochemical and structural nature of protein-peptide interfaces were analysed in part using a novel amino acid pictogram analysis alongside accessible surface area, residue pairing and amino acid composition analysis. The results indicate that, for the peptide, proline residues and tyrosine residues play specific roles in protein-peptide interfaces. Furthermore it was observed that the peptide residues are significantly more buried than the residues of the cognate protein surface. The virtual screening program LIDAEUS was used to mine chemical databases to identify novel peptidomimetic compounds that have evincible binding to protein targets of therapeutic interest. Target-based and fragment-based virtual screening identified a series of potential compounds targeting the interaction between p21 and PCNA. Whilst the docking results were promising, results from testing in biological assays were inconclusive. A target-based virtual screening approach to identify small-molecule mimics of the interaction between the GnRH peptide and GnRH receptor yielded two promising compounds that demonstrated weak binding in biological assays. A third study to identify small-molecules binding to the SH3 domain of PSD-95 produced some promising hit compounds that as yet have not been tested in binding assays.

This thesis describes a possible alternative isoprenoid pathway in bacteria by considering some previously unpublished feeding studies in the context of the related background literature. Three synthetic routes to 2,4-dihydroxy-4-methyltetrahydropyran (63) and three synthetic strategies towards the synthesis of 2-carboxy-2,4-dihydroxy-4-methyltetrahydropyran (63) are discussed. These compounds are considered as potential intermediates in the proposed alternative bacterial isoprenoid pathway. Labelled synthesis of (63) and structural analysis of (63) and 4-hydroxy-2-methoxy-4-methyltetrahydropyran (99) by proton nmr are also described. Feeding studies including the <SUP>13</SUP>C isotopically labelled tetrahydropyrans (63) and (99) are described and a revised interpretation of all of the feeding studies considered. HMGCoA synthase is assayed in <i>Rh. capsulata</i> after a description of its assay in bakers yeast.

<i>Shewanella oneidensis</i> MR-1 is a Gram-negative bacterium isolated from anaerobic freshwater lake sediments of Lake Oneida that exhibits remarkable respiratory versatility. In the absence of molecular oxygen, <i>S. oneidensis</i> MR-1 couples anaerobic growth to the reduction of various substrates, including ferric iron (FeIII), thiosulfate (S₂O₃^2-), sulfite (SO₃^2-), trimethylamine <i>N-</i>oxide (TMAO), nitrate (NO₃^-), nitrite (NO₂^-) and organic substrates such was fumarate. The metabolic flexibility of <i>S. oneidensis</i> MR-1 is coupled to a complex and branched anaerobic respiratory chain. The respiratory enzymes of the fumarate reduction pathway have been extensively studied in <i>S. oneidensis</i> MR-1 and the related marine bacterium, <i>S. frigidimarina</i> NCIMB400. The terminal fumarate reductase of <i>Shewanella </i>is a soluble periplasmic flavocytochrome c₃ (Fcc₃) that catalyses the unidirectional production of succinate. The X-ray crystal structure of Fcc₃ solved to high resolution provided the first detailed insight into the catalytic mechanism of fumarate reduction. In this work, the Fcc₃ X-ray crystal structure provided a structural template to construct homology models of related flavoenzymes of unknown structure and function. The novel flavoenzymes were identified by sequence analysis of the <i>S. oneidensis</i> MR-1 genome and were shown to comprise separately encoded flavin (FccA54, FccA56 and FccA342) and cytochrome subunits (FccB54, FccB56 and FccB342), respectively, that were related by sequence to the corresponding domains in Fcc₃. Molecular modelling of the catalytic flavin-binding subunits led to the suggestion that these related enzymes catalyse the reduction of acrylate-like substrates. Several biologically relevant plant metabolites, including phenylacrylates incorporated into lignin, were identified as potential substrates of the Fcc₃-like enzymes. An <i>fccA54</i> and <i>fccB54</i> knockout strain of <i>S. oneidensis</i> MR-1 (MB5415) was constructed and grown anaerobically with each of the candidate acrylates to ascertain the biological function of FccA54.

No description available.

The cultural characteristics of various mucoid strains of Ps. aeruginosa were examined, a wide variation in the time when the polymer was produced, the levels synthesised and the composition of the polymer occurred. The only similarity between these strains is that they produced an acetylated polyuronide. Mucoid strains were found to be highly unstable in ammonia limited chemostat cultures. Small colonies appeared at a high rate which led to a rapid displacement of the parental type indicating that some of these variants possessed a competitive advantage over the wild type. Ultimately selection for a non mucoid strain with a higher yield of biomass on the growth-limiting substrate occurred. The pathway for the biosynthesis of alginate appears to differ very little from the reaction sequences proposed in both Az. vinelandii and Fucus gardneri. In the study of enzymes involved in the biosynthesis by PAO strains similarities occurred to the biosynthetic pathway of colanic acid found in E.coli. The wild type contains all the enzymes necessary to produce alginate but it is not expressed. Synthesis of alginate leads to an increase in the levels of enzymes involved in the formation of the presursor GDP-Mannuronic acid. In non mucoid strains both sup+ and sup 7, GDP-Mannose dehydrogenase was absent. Also in these strains either GDP-Mannose pyrophosphorylase or phosphomannose isomerase was absent. In some sup strains elevated levels of certain carbohydrate enzymes were found. The regulation/repression of the enzymes is complex but the pathway lacks the fine control mechanisms present in other biosynthetic pathways. Synthesis of alginate led to an increase in resistance to carbenicillin and a decrease in resistance to tetracycline. In salts increase in resistance to EDTA and SDS. This effect was greatly magnified in salts deficient media. In salts deficient media both mucoid and sup + and sup strains were more resistant to deoxycholate; in salts media this was not observed.

No description available.

The first part of this work, described in Part I, consisted of experiments on the growth and behaviour of algae in tubs and tanks; this investigation, which was carried out in the Botany Department of the University of Reading, was designed as an experimental approach to some of the problems of the ecology of freshwater algae. Most of the previous work on this subject has been directed along two lines. Much of it may be called descriptive ecology - i.e., observations on the changes in the flora of natural bodies of water, often with data for physical and chemical variables over the same period, and attempts to correlata these with changes. in the algal population. The other type of work has consisted of investigations of the behaviour of algae when isolated in pure culture. In the first type of work it has rarely been attempted to alter the conditions experimentally in order to test hypotheses. In the case of experiments in pure culture, the results obtained would appear to be more directly applicable to algal physiology than to the ecology of algae in the field.

No description available.