Thesis (M.S.)--University of Michigan, 1991.

Thesis (M. Sc. Med.)--University of Sydney, 2007. / Title from title screen (viewed 15 October 2008). Submitted in fulfilment of the requirements for the degree of Master of Science in Medicine to the Discipline of Medicine, Faculty of Medicine. Includes bibliographical references. Also available in print form.

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

Wheat (Triticum aestivum and T. Durum) is an extremely important agronomic crop produced worldwide. Wheat consumption has doubled in the last 30 years with approximately 600 million tons consumed per annum. According to the International Maize and Wheat Improvement Center, worldwide wheat demand will increase over 40 percent by 2020, while land as well as resources available for the production will decrease significantly if the current trend prevails. The wheat industry is challenged with abiotic and biotic stressors that lead to reduction in crop yields. Increase knowledge of wheat’s biochemical constitution and functional biology is of paramount importance to improve wheat so as to meet with this demand. Pesticides and fungicides are being used to control biotic stress imposed by insect pest and fungi pathogens but these chemicals pose a risk to the environment and human health. To this effect, there is re-evaluation of pesticides currently in use by the Environmental Protection Agency, via mandates of the 1996 Food Quality Protection Act and those with higher perceived risks are banned. Genetic resistance is now a more environmental friendly and effective method of controlling insect pest and rust diseases of wheat than the costly spraying with pesticides and fungicides. Although, resistant cultivars effectively prevent current prevailing pathotypes of leaf rust and biotypes of Russian wheat aphid from attacking wheat, new pathotypes and biotypes of the pathogen/pest may develop and infect resistant cultivars. Therefore, breeders are continually searching for new sources of resistance. Proteomic approaches can be utilised to ascertain target enzymes and proteins from resistant lines that could be utilised to augment the natural tolerance of agronomically favourable varieties of wheat. With this ultimate goal in mind, the aim of this study was to elucidate the mechanism and synchronicity of wheat resistance to leaf rust (Puccinia triticina) and Russian wheat aphid (Duiraphis noxia) SA1. To determine the resistance mechanism of the wheat cultivars to leaf rust infection and Russian wheat aphid infestation, a proteomics approach using two-dimensional gel electrophoresis was used in order to determine the effect of RWA SA1 on the wheat cultivars proteome. Differentially expressed proteins that were up or down regulated (appearing or disappearing) were identified using PDQuestTM Basic 2-DE Gel analysis software. Proteins bands of interest were in-gel trypsin digested as per the protocol described in Schevchenko et al. (2007) and analysed using a Dionex Ultimate 3000 RSLC system coupled to an AB Sciex 6600 TripleTOF mass spectrometer. Protein pilot v5 using Paragon search engine (AB Sciex) was used for comparison of the obtained MS/MS spectra with a custom database containing sequences of Puccinia triticina (Uniprot Swissprot), Triticum aestivum (Uniprot TrEMBL) and Russian wheat aphid (Uniprot TrEMBL) as well as a list of sequences from common contaminating proteins. Proteins with a threshold of ≥99.9 percent confidence were reported. A total of 72 proteins were putatively identified from the 37 protein spots excised originating from either leaf rust or Russian wheat aphid experiments. Sixty-three of these proteins were associated with wheat response to stress imposed by RWA SA1 feeding while 39 were associated with infection by Puccinia triticina. Several enzymes involved in the Calvin cycle, electron transport and ATP synthesis were observed to be differentially regulated suggesting greater metabolic requirements in the wheat plants following aphid infestation and leaf rust infection. Proteins directly associated with photosynthesis were also differentially regulated following RWA SA1 infestation and P.

Made available in DSpace on 2014-10-09T12:50:00Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:56Z (GMT). No. of bitstreams: 1 10428.pdf: 372288 bytes, checksum: 253ee200ddc2b857d89f9a216cf7f8e6 (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP; Faculdade de Odontologia - USP

Made available in DSpace on 2014-10-09T12:35:30Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:04:37Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:50:00Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:56Z (GMT). No. of bitstreams: 1 10428.pdf: 372288 bytes, checksum: 253ee200ddc2b857d89f9a216cf7f8e6 (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP; Faculdade de Odontologia - USP

Made available in DSpace on 2014-10-09T12:35:30Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:04:37Z (GMT). No. of bitstreams: 0 / Muitas das proteínas de valor biomédico relevante são encontradas em baixas concentrações em suas fontes nativas. O alto nível de expressão de proteínas recombinantes em E. coli, muitas vezes gera o acúmulo de proteínas como agregados insolúveis no citoplasma e/ou periplasma da bactéria, denominados de corpos de inclusão (CI). A alta pressão tem sido amplamente utilizada no estudo da conformação das proteínas,ela modula as interações proteína-proteína e proteína-solvente através de mudanças no volume das mesmas, promovendo a entrada de água nas cavidades não expostas da molécula e promovendo hidratação e solubilização dos agregados. O presente trabalho teve como objetivo a renaturação de proteínas recombinantes expressas como CI em Escherichia coli usando alta pressão hidrostática como condição branda de dissociação dos agregados. As tiorredoxinas TsnC e TrxA, a proteína YbbN e a proteína comigratória com bacterioferritina (Bcp), todas de Xylella fastidiosa, foram estudadas neste trabalho. As condições de renaturação foram otimizadas, utilizando-se diferentes proporções do par redox, concentrações de GdnHCl, presença de aditivos e esquemas de descompressão. Para a quantificação e análise da eficácia do processo de renaturação das proteínas sob pressão foram utilizadas as técnicas de microscopia eletrônica de varredura dos CI e de SDS-PAGE, e ensaios de atividade enzimática das proteínas. A TsnC foi renaturada em Tris HCl 50 mM com proporção de 10GSH:1GSSG em concentração final de 10 mM, 0,75 M GdnHCl, na presença de 0,5 M de Triton-X e a pressão utilizada foi de 2,4 kbar por 1 hora e 30 minutos seguida de descompressão direta e incubação por 16 horas em pressão atmosférica. O rendimento final de obtenção de TsnC solúvel foi altíssimo, de até 89,9%. A renaturação de proteína YbbN, nunca antes descrita, foi obtida em tampão de renaturação Tris HCl 50 mM, na presença de 0,5 M de L-Arginina e a pressão utilizada foi de 2,4 kbar por 1 hora e 30 minutos seguida de descompressão direta e incubação por 16 horas em pressão atmosférica. A proteína YbbN, que apresentou atividade de tioredoxina, foi renaturada com rendimento de até 98% a partir da proteína insolúvel nos CI. Foi possível a solubilização da tiorredoxina TrxA e Bcp sob alta pressão hidrostática em tampão de renaturação Tris HCl 50 mM, utilizando diferentes proporções do par redox na concentração final de 10 mM e 1,5 M de GdnHCl, porém não foi possível obter a atividade biológicas destas proteínas. Mostramos também que a L-Arginina apresenta efeito auxiliar na solubilização dos CI induzida pela alta pressão, e ao mesmo tempo se mostrou altamente protetora contra a inativação da atividade da YbbN promovida pela incubação em altas temperaturas, o que sugere que a presença deste aminoácido pode ter alta aplicabilidade juntamente com a aplicação de altas pressões para elevar os rendimentos de renaturação de proteínas recombinantes a partir de CI. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:54:18Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:09:21Z (GMT). No. of bitstreams: 1 12722.pdf: 558008 bytes, checksum: 01284590d84b5a596a55c0b0b7177695 (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP; Faculdade de Odontologia, Universidade de Sao Paulo, Sao Paulo

Bacterial galls on Douglas-fir (Pseudotsuga menzeisii [Mirb.] Franco), collected from the southern tip of Vancouver Island, the Greater Vancouver area and the Hope region of British Columbia, were generally globose in shape with rough, irregular surfaces and measured between 0.5 and 2.0 cm in diameter. The galls were generally located on the tips of branches or twigs of 10- to 20-year old Douglas-fir trees. The bacterial gall disease appeared to affect few Douglas-fir trees in the collection areas and bacterial galls were not found on any other coniferous species. Furthermore, there have been no reports of serious damage to natural forests in British Columbia due to bacterial gall disease. Young, greenhouse-grown Douglas-fir seedlings occasionally died if the tip of the main stem was artificially inoculated. Often new growing tips would be produced affecting the growth form of the seedlings. Two types of gall-forming Erwinia spp. were isolated from Douglas-fir galls. Typical isolates, tentatively identified by fatty acid analysis as Erwinia salicis, produced galls which were rough and irregular in shape composed of multiple outgrowths marked by a single or cross-shaped fissure. The atypical isolate, tentatively identified by fatty acid analysis as Erwinia herbicola subsp. herbicola, produced galls which were smooth and generally round in shape with the surface cracking as the gall expanded. Colonies of the typical isolates grown on casein-peptone-glucose media were characteristically round, slightly domed with somewhat concentric ridging observed near the margins of the colonies. Three to 4 day old colonies of the atypical isolates grown on casein-peptone-glucose media were characteristically round and concave while older colonies produced an extracellular slime and were more irregular in shape. In Luria Broth, the typical isolates grew at temperatures of up to 32°C while the atypical isolate grew at temperatures of up 34°C. The typical isolate was resistant to a wider range of antibiotics than the atypical isolate. Polyclonal antisera were produced against glutaraldehyde-fixed whole cells of both the typical T-2789 and atypical A-0181 gall-forming Erwinia isolates. The purified antisera were isolate specific as tested by immunodiffusion and an indirect ELISA against several different phytopathogenic bacteria including Pseudomonas syringae pv. syringae, Erwinia herbicola subsp. herbicola, Agrobacterium tumefaciens, Rhizobium leguminosarum and Erwinia carotovora subsp. carotovora. Plasmid profiles of the typical Erwinia isolates contained one band while the atypical isolate characteristically contained 4 to 5 bands which appeared to be different forms of at least one plasmid. Restriction digests of the typical isolates suggested a size of approximately 50 kb while complex digestion profiles were obtained for the atypical isolates because of the difficulty in isolating individual plasmid types. From visual estimates against Hindlll-digested lambda DNA, a size of between 10 and 20 kb was suggested for the fastest moving plasmid band of the atypical isolate. No homology was observed between the different plasmid types characteristic of the two isolates. The role of the plasmid DNA of the atypical isolate in pathogenesis was not determined because curing of the plasmid(s) was not successful using high temperature treatments plus chemical curing agents. Heat treatment experiments, in which the pathogen was selectively killed at various times after inoculation, demonstrated that the bacteria are required to be present for gall induction and continued development of the gall for both of the gall-forming Erwinia isolate types. Pathogenicity of the isolated bacteria was tested on 14 conifer species, other than Douglas-fir, including Abies, Chamaecyparis, Pinus and Thuja spp. The typical isolates were weakly pathogenic on Abies, Larix and Picea spp. The atypical isolate was weakly pathogenic on Abies, Chamaecyparis, Larix, Picea and Pinus spp. Due to the limited damage caused on the conifers tested and to their infrequent occurrence, these gall-forming pathogens do not appear to be of economic importance to the forestry industry. / Land and Food Systems, Faculty of / Graduate