Title page, table of contents and summary only. The complete thesis in print form is available from the University of Adelaide Library. / Assimilable nitrogen, which is typically lacking in grape juice, is an important nutritional requirement of Saccharomyces cerevisiae. As such, fermentations frequently become protracted, terminate prematurely or develop undesirable aroma profiles. Amino acids and ammonium are the main sources of assimilable nitrogen in grape juice. The amino acid proline often predominates. Proline uptake is mediated by a high affinity, proline-specific permease, Put4p, and a low affinity general amino acid permease, Gap1p. The expression and activity of these transporters is subject to nitrogen catabolite repression (NCR) and nitrogen catabolite inactivation (NCI). That is, in the presence of a preferred nitrogen source, the expression of PUT4 and GAP1 is repressed and the permeases are inactivated. For yeast to fully exploit proline, its transport must be derepressed by depletion of other (preferred) amino acids and molecular oxygen must be present to allow proline catabolism by proline oxidase. Consequently, as oxygen is typically depleted well before the other amino acids in grape juice are reduced to non-repressive concentrations, proline is largely un-utilised by yeast during oenological fermentation. This study aims to overcome these metabolic restrictions on proline utilisation. A preliminary study was conducted to determine the potential for proline transport-capable strains to utilise proline during the initial stages of fermentation when oxygen may be present, particularly in red grape must. Initially, the transcriptional regulation of the PUT4 gene was targeted to generate strains capable of proline transport under normally repressive conditions. In the first case, the URE2 gene, encoding a negative regulator involved in nitrogen discrimination, was deleted. In the second case, PUT4 was expressed from the constitutive TEF2 promoter. It was observed that both strains express PUT4 in the presence of a preferred nitrogen source. This expression led to Put4p activity during the initial stages of growth and fermentation, with Put4p activity declining over the course of the growth phase. Proline removal from the media, however, was limited to the initial stages of fermentation while oxygen was available. It seems that the rapid depletion of oxygen limits the amount of proline transported into the yeast cell. The two proline transport-capable mutants were analysed for growth and fermentation characteristics. It was found that the deletion of the URE2 gene led to a slow initial growth and the formation of a larger biomass. The ure2 delete strain also utilised significantly more nitrogen during fermentation than the wild type. Consequently, a ure2 delete strain would not be suitable for industrial use. The expression of PUT4 from a constitutive promoter did lead to an increase in nitrogen assimilation during fermentation when compared with the wild type. However, this observed increase was significantly less than that observed in the ure2 delete strain. In an effort to produce a proline transport-capable strain with potential industrial benefit, strains constitutive for PUT4 specifically were isolated using random, in vitro mutagenesis of the PUT4 promoter region. Four point mutations were identified that, when introduced singly into the PUT4 promoter, led to expression of PUT4 in the presence of a preferred nitrogen source. The rapid depletion of oxygen observed in the preliminary study will limit the potential usefulness of strains capable of proline transport. Micro-oxygenation is rapidly becoming an accepted practice during oenological fermentation. The potential benefit of the controlled addition of oxygen during fermentation is restricted by the timing of any oxygen addition. Oxygen additions made at the onset of the stationary phase are the most beneficial. During the preliminary study, it was noted that Put4p activity decreased during the growth phase to low levels at the onset of the stationary phase. To ensure that sufficient active Put4p is present at the onset of the stationary phase, the post-translational control of the Put4p was investigated. Site-directed mutagenesis was used to target residues in the carboxy-terminal region of Put4p that are potentially involved in the ammonia-induced down-regulation of the permease. The substitution, S605A, lead to the amelioration of ammonia-induced down-regulation of Put4p. The activity of the Put4p S605A variant decreased over the course of the growth phase, but not to the same extent observed in the wild type. Furthermore, a recovery seen after down-regulation restored a greater percentage of the original activity compared with the wild type. To determine whether such a strain proved better able to ferment media in the presence of micro-oxygenation, the fermentation kinetics of a strain constitutively expressing PUT4(S605A) were compared with the wild type. Micro-oxygenation of ferments did not result in an increase in fermentation rate nor a decrease in fermentation time in the mutant. However, the cell viability of the strain capable of proline transport was increased in comparison with the wild type, suggesting a role for proline in stress responses within the yeast cell. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1048932 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 2002

Thesis (M.Ed.)--Kutztown State College, 1972. / Source: Masters Abstracts International, Volume: 45-06, page: 3056. Typescript. Includes bibliographical references (leaves 23-24)

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph.D.) -- University of Rhode Island, 2008. / Typescript. Includes bibliographical references (leaves 132-134).

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13182号 / 論農博第2861号 / 新制||農||1061(附属図書館) / 学位論文||H30||N5104(農学部図書室) / (主査)教授 阪井 康能, 教授 植田 和光, 教授 小川 順 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Winter injury of bermudagrass (Cynodon spp.) continues to be a problem across the transition zone. In an attempt to delay or induce winter dormancy while maintaining cold hardiness, applications of seaweed extract (SWE) (0.54 kg ha-1), ethephon (16 L ha-1), Fe (1 kg ha-1), and N (49 kg ha-1) took place every three weeks during the fall of 2001 and 2002. Cultivars examined included 'Riviera', 'Midiron', 'Princess', and 'Tifway'. Tifway exhibited greatest fall color retention in both years of the study. Ethephon promoted early senescence and turfgrass quality during fall ratings in both years of the study while N, Fe, and SWE increased quality over the control in 2001 and only N showed better quality and color retention over the control in 2002. Samples removed from cold acclimated plots were artificially frozen as a measure of cold hardiness. Treatments did not have an effect on post freeze regrowth, however, cultivar was significant in both years. Midiron showed best regrowth followed by Riviera, Tifway, and Princess. In both years Riviera and Midiron displayed the quickest and greatest amount of spring greenup followed by Tifway and then Princess. Ethephon reduced greenup in both years and SWE, Fe, and N showed no differences from the control in 2001 and Fe showed significantly better greenup in 2002. Proline and Linolenic acid levels were highest in Midiron, followed by Riviera, Tifway, and Princess. Nitrogen, SWE, and Fe generally did not have an effect on linolenic acid and no consistent effects were noted on proline concentration. Ethephon treatments did not have an effect on linolenic acid levels, however, there was a negative effect on proline concentrations. The results of this study indicate that judicial N applications during the fall can promote color retention and do not have a negative effect on bermudagrass cold-tolerance. Linolenic acid and proline findings also help to explain differences in cold-tolerance between different bermudagrass cultivars. / Ph. D.

No description available.

Depuis de nombreuses années, les phosphines chirales multifonctionnelles se sont révélées être des outils synthétiques puissants en organocatalyse asymétrique. Ces catalyseurs qui contiennent un site de base de Lewis et un site d'acide de Bronsted, ont reçu une attention particulière en raison de leur efficacité pour créer des liaisons C-C avec de très bonnes énantiosélectivités. A notre connaissance, la synthèse d'organocatalyseurs de type thiourée-phosphine dérivés de la (L)-proline n'a jamais été décrite dans la littérature. Ce sujet de thèse concerne la synthèse d'une nouvelle famille d'organocatalyseur chiral de type thiourée-phosphine dérivée de (L)-proline, un produit naturel, disponible et peu coûteux. Nous avons mis au point plusieurs méthodes de synthèse efficaces qui nous ont permis de préparer trois familles de phosphines-thiourées à partir de la (L)- proline. Ainsi, sept nouveaux composés énantiopurs ont été préparés au cours de ce travail. Ces composés ont été utilisés comme catalyseurs dans des réactions asymétriques catalysées par des phosphines. Ces réactions incluent la cyclisation [3+2], la réaction de Baylis-Hillman, la réaction de Friedel-Crafts, la réaction de substitution nucléophile. / For many years, multifunctional chiral phosphines have proven to be powerful synthetic tools in asymmetric organocatalysis. These catalysts, containing Lewis basic and Brnsted acidic sites, have received considerable attention owing to their highly efficiency to create C-C bond by asymmetric organocatalysis. To our knowledge, the synthesis of organocatalysts type thiourea-phosphine derivatives (L) -proline have never been described in the literature. In this work, we wish to report the synthesis of new family of bifunctional chiral thiourea-phosphine organocatalyst derived from L-proline, a natural available product. We developed efficient methods to prepare three families of phosphine thiourea derived from L-proline. Thus, Seven new enantiopure compounds were synthesized in this study. They were used as catalyst asymmetric reaction catalyzed by phosphines: [3+2] cyclisation, Baylis-Hillman reaction, Friedel-Crafts reaction and nucleophilic substitution.

Ma Hoi-Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 113-120). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract --- p.II / 摘要 --- p.III / Content --- p.IV / Abbreviations --- p.X / List of Figures --- p.XII / List of Tables --- p.XIV / Chapter Chapter One --- Introduction --- p.1 / Chapter 1.1 --- Interactions that stabilize proteins --- p.1 / Chapter 1.2 --- Some common strategies of protein engineering to improve thermostability --- p.6 / Chapter 1.3 --- Ribosomal protein T. celer L30e as a study model for thermostability --- p.7 / Chapter 1.4 --- Extra proline residue is one of the insights by comparing the two proteins --- p.10 / Chapter Chapter Two --- Materials and Methods --- p.13 / Chapter 2.1 --- General Techniques --- p.13 / Chapter 2.1.1 --- Preparation of Escherichia coli competent cells --- p.13 / Chapter 2.1.2 --- Transformation of Escherichia coli competent cells --- p.14 / Chapter 2.1.3 --- Spectrophotometric quantitation of DNA --- p.14 / Chapter 2.1.4 --- Agarose gel electrophoresis --- p.14 / Chapter 2.1.5 --- DNA extraction from agarose gel electrophoresis using Viogene Gene Clean kit --- p.15 / Chapter 2.1.6 --- Plasmid DNA minipreperation by Wizard® Plus SV Minipreps DNA Purification System from Promega --- p.16 / Chapter 2.1.7 --- Polymerase Chain Reaction (PCR) --- p.17 / Chapter 2.1.8 --- Ligation of DNA fragments --- p.18 / Chapter 2.1.9 --- Sonication of pellet resuspension --- p.18 / Chapter 2.1.10 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.19 / Chapter 2.1.11 --- Native polyacrylamide gel electrophoresis --- p.20 / Chapter 2.1.12 --- Staining of protein in polyacrylamide gel by Coommassie Brillant Blue R250 --- p.22 / Chapter 2.1.13 --- Protein Concentration determination --- p.22 / Chapter 2.2 --- Cloning the Mutant Genes --- p.22 / Chapter 2.2.1 --- Site-directed mutagenesis --- p.22 / Chapter 2.2.1.1 --- Generation of full length mutant gene by megaprimer --- p.23 / Chapter 2.2.1.2 --- Generation of mutant gene by QuikChange® Site-Directed Mutagenesis Kit from Stratagene --- p.26 / Chapter 2.2.2 --- Restriction Digestion of DNA --- p.27 / Chapter 2.2.3 --- Ligation of DNA fragments --- p.27 / Chapter 2.2.4 --- Screening for successful inserted plasmid clones from ligation reactions --- p.28 / Chapter 2.2.4.1 --- By PCR --- p.28 / Chapter 2.2.4.2 --- By restriction digestion --- p.28 / Chapter 2.2.5 --- DNA sequencing --- p.29 / Chapter 2.3 --- Expression and Purification of Protein --- p.29 / Chapter 2.3.1 --- "General bacterial culture, harvesting and lysis" --- p.29 / Chapter 2.3.2 --- Purification of recombinant wild type TRP and mutants --- p.30 / Chapter 2.3.3 --- Purification of recombinant wild type YRP and mutants --- p.32 / Chapter 2.4 --- Thermodynamic Studies by Circular Dichroism (CD) Spectrometry --- p.34 / Chapter 2.4.1 --- Thermodynamic studies by guanidine-induced denaturations --- p.34 / Chapter 2.4.2 --- Themodynamic studies by thermal denaturations --- p.36 / Chapter 2.4.3 --- ACp measurement of the TRP mutants --- p.37 / Chapter 2.4.3.1 --- By Gibbs-Helmholtz analysis --- p.37 / Chapter 2.4.3.2 --- By van't Hoff analysis --- p.37 / Chapter 2.5 --- Crystal Screen for the Mutant T. celer L30e --- p.38 / Chapter 2.5.1 --- T. celer L30e Pro→Ala and Pro→Gly mutants --- p.38 / Chapter 2.5.2 --- Yeast L30e K65P mutant --- p.38 / Chapter 2.6 --- Sequences of Primers --- p.39 / Chapter 2.6.1 --- Primers for TRP and its mutants --- p.39 / Chapter 2.6.2 --- Primers for YRP and its mutantsReagents and buffers --- p.40 / Chapter 2.7 --- Reagents and Buffers --- p.40 / Chapter 2.7.1 --- Reagents for competent cell preparation --- p.40 / Chapter 2.7.2 --- Nucleic acid eletrophoresis buffers --- p.41 / Chapter 2.7.3 --- Media for bacterial culture --- p.41 / Chapter 2.7.4 --- Reagents for SDS-PAGE --- p.42 / Chapter 2.7.5 --- Buffers for TRP purification --- p.44 / Chapter 2.7.6 --- Buffers for YRP purification --- p.45 / Chapter 2.7.7 --- Buffer for Circular Dichroism (CD) Spectrometry --- p.46 / Chapter Chapter Three --- Results --- p.48 / Chapter 3.1 --- "Cloning, expression and purification of the mutant proteins" --- p.48 / Chapter 3.1.1 --- "Mutagenesis, cloning and purification of the thermophilic proteins - T. celer L30e protein and its mutants" --- p.48 / Chapter 3.1.2 --- "Mutagenesis, cloning and purification of the mesophilic proteins - yeast L30e protein and its mutants" --- p.52 / Chapter 3.2 --- Stability of Pro→Ala/Gly mutants of T. celer L30e at 298K --- p.55 / Chapter 3.2.1 --- Design of alanine and glycine mutants from thermophilic homologue --- p.55 / Chapter 3.2.2 --- "Among alanine mutants, only P59A was destabilized" --- p.55 / Chapter 3.2.3 --- Ala→Gly mutations destabilized the protein --- p.59 / Chapter 3.3 --- Stability of Xaa→Pro mutants of yeast L30e at 298K --- p.61 / Chapter 3.3.1 --- Design of proline mutants from mesophilic homologue --- p.61 / Chapter 3.3.2 --- "K65P, corresponding to P59 in T. celer L30e, stabilized yeast L30e" --- p.62 / Chapter 3.3.3 --- Yeast L30e mutated with thermophilic consensus sequence did not give a more stable protein --- p.65 / Chapter 3.4 --- Temperature dependency of the stability of the mutants of T. celer L30e --- p.67 / Chapter 3.4.1 --- The trend of ΔGU was consistence through 25 to 75°C --- p.67 / Chapter 3.4.2 --- Melting temperatures of T. celer mutants determined by thermal denaturations --- p.68 / Chapter 3.5 --- pH dependency of melting temperatures --- p.75 / Chapter 3.5.1 --- ΔCP values of the P59A/G mutants determined by van't HofF's analyses increased significantly --- p.77 / Chapter 3.6 --- No structural change was observed in the crystal structure of P59A --- p.80 / Chapter Chapter Four --- Discussion --- p.84 / Chapter 4.1 --- The trend of stability from guanidine-induced denaturation agreed with that from thermal denaturations --- p.86 / Chapter 4.2 --- The magnitude of destabilization of P59A and Ala→Gly mutation was consistent with the expected destabilization due to entropy --- p.87 / Chapter 4.3 --- Entropic effect had little effect for residues in flexible region --- p.93 / Chapter 4.4 --- Stabilization forces that compensate the entropic effect --- p.96 / Chapter 4.5 --- Compensatory stabilization due to the release of amide group --- p.99 / Chapter 4.5.1 --- Intra-molecular H-bond in P88A --- p.99 / Chapter 4.5.2 --- Solvent-protein H-bond in P43A --- p.103 / Chapter 4.6 --- Consensus concept was not applicable in our model --- p.110 / Chapter 4.7 --- "Pro→Ala mutation destabilized the protein increase the protein's ACP value, however enthalpy and entropy change were difficult to be decomposed" --- p.111 / Chapter 4.8 --- Concluding Remarks --- p.112 / References --- p.113

Au cours de cette thèse, des dyades chromophoriques à base d’oligoprolines ont été développées afin d’étudier les transferts d’énergies au sein de système hélicoïdaux. L’étude préliminaire de ces dyades n’a pas permis d’obtenir l’étude de transfert d’énergie souhaitée. La présence conjointe de deux conformères, ainsi que des problèmes de pureté, semblent en être la cause. C’est pourquoi dans un second temps, plusieurs modèles d’oligoprolines ayant une conformation largement majoritaire ont été conçus. Basés sur un blocage de conformation via des effets stériques cumulés, les modèles présentés conservent une unique conformation dans des solvants favorisant les deux types d’hélice. Pour finir, une propagation de la conformation a été étudiée à partir des composés modèles afin de créer une dyade chromophorique induite. L’étude photo-physique de celle-ci permet d’en étudier le transfert d’énergie. / During this thesis, chromophoric dyads based on oligoprolines have been developed in order to studyEnergy transfer within helical systems. Preliminary studies of these dyads were unsuccessful to study thedesired energy transfer. The presence of two conformers and purity problems seems to be responsible. This iswhy, in a second step, several oligoproline models with a major conformation were designed. Based on aconformation induction via cumulative steric effects, the models presented maintain a unique conformation insolvents favoring both types of helices. Finally, a propagation of the conformation will be studied based on themodel compounds in order to create an induced chromophoric dyad. The photo-physical study show anefficient energy transfer.