No description available.

Pretreatment of recalcitrant lignocellulosic biomass to release sugars for bioconversion into ethanol produces fermentation inhibitors. Increasing yeast inhibitor tolerance should reduce production time and cost. UV mutagenesis followed by genome shuffling using cross mating was performed on Scheffersomyces stipitis strain GS301, a genome shuffled strain with increased tolerance to spent sulphite liquor (SSL). The main fermentation inhibitors in SSL are acetic acid, hydroxymethylfurfural (HMF), and various phenolics. UV mutagenesis resulted in acetic acid tolerant mutants, but they were phenotypically unstable. However, two rounds of UV mutagenesis followed by five rounds of genome shuffling resulted in strains EVB105, EVB205 and EVB505 with increased SSL tolerance and improved acetic acid and HMF tolerance. When fermenting undiluted SSL at pH 5.5, the three strains utilized sugars faster producing higher maximum ethanol than GS301. This study demonstrates that UV mutagenesis with genome shuffling can significantly improve inhibitor tolerance and fermentation performance of yeast. / NSERC Bioconversion Network

Of the organisms with sequenced genomes, plants appear to possess the most rhomboid protease-encoding genes. However, our knowledge of processes in plants that involve Regulated Intramembrane Proteolysis (RIP) and rhomboid proteases remains low. As expressed recently by other researchers, finding a natural substrate for a rhomboid protease represents the biggest experimental challenge. Using yeast mitochondria-based assays, a potential link between the plastid translocon component Tic40 and organellar rhomboid proteases was recently uncovered. In this particular link, rhomboid proteases appear capable of influencing the pattern of imported Tic40 in yeast mitochondria. Tic40 may thus represent a natural plant target of organellar rhomboid proteases. Here, we obtained further motif-oriented evidence supporting Tic40 as a natural plant rhomboid substrate. A comparative analysis of sequences revealed that Tic40 may also possess similar TMD motifs found in the model substrate, Spitz. Rhomboid proteases often require these motifs to cleave substrates within intramembrane environments. Using site-directed mutagenesis and yeast mitochondria assays, the impact of mutations occurring in the motifs ASISS, GV, QP, and GVGVG of Tic40 was assessed. In terms of cleavage and changing the pattern of imported Tic40, some of the mutations showed decreased activities and a few showed enhancements. More importantly, the overall observed pattern associated with select Tic40 mutations resembled the characteristics reported for the model substrates. In particular, mutations in the Tic40 GV motif produced similar results as that observed with Spitz, by drastically decreasing or increasing cleavage as a function of amino acid sequence. / Thesis (Master, Biology) -- Queen's University, 2010-05-30 10:22:07.72

No description available.

Addendum pasted onto back end-paper. / Bibliography: leaves 146-179. / Bibliography: leaves 146-179. / ix, 179, [66] leaves, [2] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis investigates the effect of mutations and an N-terminal extension on the oxidative folding pathway of IGF-I, analyses the structure of the stable mis-folded molecule in terms of its biological interactions, examines the kinetics of the late stages of oxidative folding and finally attempts to dissect the folding pathway of a mutant of IGF-I. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1996?

The bacterium Helicobacter pylori is a human pathogen that infects a large proportion of the world's population and is associated with serious diseases such as gastric ulcers and adenocarcinoma. The motility of this organism, by virtue of sheathed polar flagella is essential to colonisation and persistence in the human host. The sequencing of the H. pylori genome in 1996 identified homologues of the majority of the flagellar genes found in S. enterica serovai typhimurium. These included genes encoding the flagellum ATPase, FliI and FliH a presumptive inhibitor, the primary focus of this study. Sequencing did not originally identify an H. pylori homologue of the flagellar chaperone FliJ, and this is also considered in this study. Bioinformatic analysis and modeling suggests a structural and functional relationship between FliI and homologues such as F1-ATPase α- and β-subunit. In particular, residues 2-91 of FliI resemble the N-terminal domain of the F1-ATPase α- and β-subunits. Biochemical analyses reported in this thesis showed that a truncated FliI-(2- 91) protein was folded, although the N-terminal 18 residues were likely unstructured. Furthermore, deletion mutagenesis showed that this disordered segment of the protein mediates interaction with FliH and very likely forms an amphipathic α-helix upon forming of the FliI-FliH complex. The scanning mutagenesis of this interaction segment of FliI identified a cluster of conserved hydrophobic residues that was critical for the interaction with FliH. Thus, the interaction between FliI and FliH has similarities to the interaction between the N-terminal α-helix of the α-subunit and the globular domain of the δ-subunit of the F1-ATPase. This similarity suggests that FliH, by analogy with the δ-subunit of the F1-ATPase, may function as a molecular stator of the flagellum. The findings presented above have been published (96). The function of a putative H. pylori FliJ homologue, HP0256, was also investigated by knock-out mutagenesis. Disruption of this gene does not abolish flagellar assembly, however further research continued beyond this thesis showed that the knock-out mutant results in impaired motility.

Abstract The broad aim of this thesis is to structurally and functionally explore two α-conotoxins, from venomous sea snails, Vc1.1 and RgIA, in the hope of improving their journey to becoming analgesic therapeutics (introduction to conotoxins in Chapter 1). Vc1.1 is a two-disulfide peptide that is of interest as a potential therapeutic for the treatment of neuropathic pain. Despite investigations, limited structure-activity relationships have been conducted on this α-conotoxin. Consequently there is restricted insight into the interaction of this peptide with one of its analgesic targets, the α9α10 nicotinic acetylcholine receptor (nAChR). Late in this PhD project, the GABAB receptor was implicated as the possible target for conotoxins in neuropathic pain relief. However, there is still debate in the literature with regard to the true target of Vc1.1 and the α9α10 nAChR is still believed to be the target by some groups. This thesis predominantly focuses on the α9α10 nicotinic acetylcholine receptor. Chapter 4 of this thesis presents an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp or Lys (materials and methods described in Chapter 3) and examined using NMR spectroscopy (theory of NMR presented in Chapter 2), to determine the effects of the mutations on the structure of Vc1.1. The structural fold was found to be preserved in all peptides except where Pro was substituted. Chapter 5 explores the effect of these mutations on the blocking of acetylcholine (ACh)-evoked membrane currents at the α9α10 nAChR. Electrophysiological studies showed that the key residues for Vc1.1’s activity are Asp5-Arg7 and Asp11-Ile15, as changes at these positions resulted in the loss of activity at the α9α10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. Hence, Chapter 6 describes a second generation of mutants that was synthesized, namely N9G, N9I, N9L, S4R and S4K+N9A, all of which were more potent than Vc1.1 at both the rat α9α10 and the human α9/rat α10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the α3β2, α3β4 and α7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the α9α10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the α9α10 nAChR subtype and will help in the further development of Vc1.1 or its analogs as drugs. However, despite peptides exhibiting high degrees of potency and selectivity, such as Vc1.1 and RgIA, they are potentially hindered in their development as drugs due to their stability and bioavailability limitations, leading to invasive delivery techniques. Chapter 7 presents a range of cyclic RgIA analogs, tested at their targets the α9α10 nAChR and the GABAB receptor, that retain their activity and increase their stability in human serum relative to non-cyclic RgIA. NMR spectroscopy was used to determine the structure of the non-cyclic peptide and the cyclic peptide to confirm similarities in the global fold of the peptide. Structural perturbations and reduced activities were observed for cyclic RgIA analogs cyclized via linkers composed of three and four residues. Analogs with five, six and seven residues showed no structural perturbations, but differences in their activities at the different receptors. Because cRgIA-6 showed high potency for the GABAB receptor and lower potency for the α9α10 nAChR, this study has identified a GABAB selective peptide. Additionally, because the cRgIA-7 showed high potency for the α9α10 nAChR and low potency for the GABAB receptor, a α9α10 nAChR selective analog has also been identified. With improvements in these peptides against enzymatic attack, they show great potential on their path to becoming orally available analgesics as they may be able to withstand enzymatic conditions in the stomach.

Thesis (M.S.)--Duquesne University, 2007. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 76-80) and index.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves [122]-134).

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / "May 2004." Typescript. Vita. Includes bibliographical references (leaves 156-180). Also issued on the Internet.