Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains x, 101 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 81-101).

Thesis (Ph. D.)--University of Kentucky, 2004. / Title from document title page (viewed on June 22, 2006). Document formatted into pages; contains viii, 116 p. : ill. (some col.). Includes abstract and vita. Includes bibliographical references (p. 103-114).

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

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Li, Kwun Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 83-88). / Abstract also in Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Acknowledgements --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xiii / List of Figures --- p.xiv / List of Abbreviations --- p.xvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The Plant Secretory and Endocytic Pathways --- p.2 / Chapter 1.2 --- PVC Proteomics Analysis Led to the Identification of AtEMP --- p.5 / Chapter 1.3 --- EMP70 Family Proteins --- p.5 / Chapter 1.3.1 --- General structure of EMP70 proteins --- p.5 / Chapter 1.3.2 --- EMP70 in other organisms --- p.8 / Chapter 1.3.3 --- EMP70 proteins in Arabidopsis --- p.9 / Chapter 1.4 --- Accession Numbers --- p.10 / Chapter 1.5 --- Research Objectives --- p.14 / Chapter Chapter 2 --- Generation and Characterization of Transgenic Tobacco BY-2 Cell Lines Expressing Selective AtEMP-GFP Fusions --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Materials and Methods --- p.17 / Chapter 2.2.1 --- RNA extraction and cDNA generation --- p.17 / Chapter 2.2.2 --- Construct making --- p.18 / Chapter 2.2.3 --- Bacterial strains --- p.21 / Chapter 2.2.4 --- Transformation of BY-2 cells --- p.21 / Chapter 2.2.5 --- Confocal fluorescence screening of tobacco BY-2 cells --- p.23 / Chapter 2.2.6 --- Drug treatments --- p.23 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Western blot analysis of tobacco BY-2 cell lines expressing AtEMP-GFP fusions --- p.25 / Chapter 2.3.2 --- Subcellular localization of AtEMP-GFP fusions to the PVC in transgenic BY-2 cells --- p.27 / Chapter 2.4 --- Summary --- p.30 / Chapter Chapter 3 --- Generation and Characterization of Antibodies Against Various AtEMPs --- p.31 / Chapter 3.1 --- Introduction --- p.32 / Chapter 3.2 --- Materials and Methods --- p.33 / Chapter 3.2.1 --- Generation of antibodies --- p.33 / Chapter 3.2.2 --- Screening of antibodies --- p.36 / Chapter 3.2.2.1 --- SDS-PAGE and western blot analysis --- p.36 / Chapter 3.2.2.2 --- Confocal immunofluorescence studies --- p.38 / Chapter 3.3 --- Results --- p.39 / Chapter 3.3.1 --- AtEMP antibodies recognized EMP70 proteins in plant cells --- p.39 / Chapter 3.3.2 --- Organelles marked by anti-AtEMPs are closely associated with the Golgi apparatus --- p.40 / Chapter 3.4 --- Summary --- p.49 / Chapter Chapter 4 --- Subcellular Localization of GFP-tagged AtEMP Fusions via Transient Expression --- p.50 / Chapter 4.1 --- Introduction --- p.51 / Chapter 4.2 --- Materials and Methods --- p.52 / Chapter 4.2.1 --- Making of transient expression constructs --- p.52 / Chapter 4.2.2 --- Transient expression --- p.57 / Chapter 4.3 --- Results --- p.59 / Chapter 4.3.1 --- PVC localization of AtEMP-GFP fusions --- p.59 / Chapter 4.3.2 --- Golgi localization of GFP-AtEMP and GFP-AtEMP-S fusions --- p.62 / Chapter 4.4 --- Summary --- p.66 / Chapter Chapter 5 --- Immunogold Electron Microscope Localization of AtEMPs --- p.67 / Chapter 5.1 --- Introduction --- p.68 / Chapter 5.2 --- Materials and Methods --- p.68 / Chapter 5.2.1 --- High-pressure freezing / freeze substitution --- p.68 / Chapter 5.2.2 --- Ultra-thin sectioning --- p.69 / Chapter 5.2.3 --- Immunogold labeling --- p.69 / Chapter 5.2.4 --- Post-staining and transmission election microscopy --- p.69 / Chapter 5.3 --- Results and Summary --- p.70 / Chapter Chapter 6 --- Discussion and Future Perspectives --- p.74 / Chapter 6.1 --- Hypothesis --- p.75 / Chapter 6.2 --- Subcellular localization of AtEMPs --- p.76 / Chapter 6.2.1 --- GFP-tagged AtEMP fusions --- p.76 / Chapter 6.2.2 --- Endogenous EMP70 proteins in BY-2 cells --- p.77 / Chapter 6.3 --- Targeting motifs in AtEMPs --- p.79 / Chapter 6.4 --- Conclusions --- p.81 / Chapter 6.5 --- Future perspectives --- p.82 / Chapter 6.5.1 --- Targeting motifs --- p.82 / Chapter 6.5.2 --- Functional studies --- p.82 / References --- p.83

The aim of the study called Creating of constructs for study of function DRM1 in Arabidopsis thaliana was to create a construct harbouring the gene of interest DRM1 (dormancy-associated protein), and other components necessary for successful transgenosis, by which will be transformed Arabidopsis thaliana plants in order to study the function of this gene in the plant organism. DRM1 function has not been identified yet, but the gene was annotated as putative dormancy-associated protein. The final gene cassette, containing DRM1 gene under promoter for the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), was inserted into the vector pGreen II giving the recombinant plasmid pWell17A whose completeness was verified by restriction analysis. The Rubisco promoter ensure DRM1 gene overexpression in transgenic plants and on the basis of this overexpression is determined DRM1 gene function.

Cytokinins are important group of phytohormones regulating many physiological processes ranging from cell division to programmed cell death. This thesis is focused on effects of cytokinin levels in response to heat stress in Arabidopsis thaliana. Analysis of transgenic plants with regulated expression of ipt and HvCKX showed that cytokinins and their optimal levels play important role in the morphological alterations induced by heat stress. Seedlings with increased and decreased levels of cytokinins exhibit inhibition of petioles growth, decreased length of blades of true leaves and reduced leaf area. To obtain insights into molecular events underlying early response to heat stress LC-MS analysis of whole proteom was performed. Analysis revealed 57 differentialy regulated proteins in response to heat stress in Columbia ecotype. On the cellular level, most of the proteins were located in cytosol (47 %) nebo plastids (32 %). Coparative analysis between wild-type seedlings and seedlings with decreased level of cytokinins confirmed 31 proteinInfluencing plant responses to temperature stress modulating cytokinin levels - fenomic and proteomic analysiss regulated by cytokinins in response to heat stress. Among these proteins, desarurase 7 and Tudor SN1 protein were previously found as important factors in response to heat stress.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2017-03-29T11:03:04Z No. of bitstreams: 1 texto completo.pdf: 3774617 bytes, checksum: 197f3ca5359c93c3476e850581110d85 (MD5) / Made available in DSpace on 2017-03-29T11:03:04Z (GMT). No. of bitstreams: 1 texto completo.pdf: 3774617 bytes, checksum: 197f3ca5359c93c3476e850581110d85 (MD5) Previous issue date: 2013-04-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O arsênio (As) é um elemento não só tóxico, mas também altamente genotóxico aos seres vivos. Muitas lacunas precisam ser preenchidas com relação aos processos causadores de toxidade do As em plantas, bem como os mecanismos de tolerância e sensibilidade a este metalóide. Para isso, plantas de Arabidopsis thaliana (WT, mutantes msh2 e transgênicas repórteres em mutações) e Allium cepa foram expostas a 0, 2, 8 e 16 mg As L -1, durante cinco dias, em sistema hidropônico ou em meio de cultura. As plantas acumularam grandes teores de As nas raízes e apresentaram elevado fator de translocação para a parte aérea, e também alterações no acúmulo de nutrientes. Os sintomas visuais se intensificaram com o aumento da concentração de As na solução nutritiva. As raízes adquiriram coloração escura e aspecto gelatinoso, danificado e aumento no comprimento e densidade dos pêlos; a parte aérea apresentou aumento dos teores de antocianinas e sinais de senescência precoce, bem como alterações na espessura de tecidos. O estresse oxidativo e a redução dos teores de fósforo foram apontados como os principais efeitos do As capazes de causar toxidez, evidenciando os danos indiretos deste elemento no organismo. Foram verificadas importantes alterações fotossintéticas, bem como indícios de danos ao processo de respiração celular devido o aumento da expressão de genes codificantes de oxidases alternativas. Também foram observadas alterações nos teores de açúcares em folhas jovens, maduras e raízes. O As promoveu fragmentação do DNA nos ápices radiculares de A. cepa e aumento das taxas de mutação pontual e de recombinação-não homóloga em A. thaliana. O significativo aumento da expressão dos genes msh2 e msh7, codificadores de enzimas-chave do processo mismatch repair, que realiza o reparo de bases danificadas ou erroneamente inseridas no DNA, sugeriu a importância deste mecanismo no combate à genotoxidade do As em A. thaliana. Isso foi confirmado pela maior sensibilidade observada nas plantas mutantes msh2 ao As, detectada visualmente via aumento da peroxidação de lipídios. Observou-se inibição da atividade da protease caspase-3, associada ao processo de morte celular programada, reforçando a capacidade de inibição da atividade enzimática pelo As. / Arsenic (As) is not only a toxic element, but also highly genotoxic to living organisms. Several gaps in our understanding of As toxicity in plants need to be filled, including the mechanisms that result in tolerance and sensitivity to this metalloid. For this reason Arabidopsis thaliana plants (WT, msh2 mutants and transgenic reporters in mutation process) and Allium cepa were exposed to 0, 2, 8 e 16 mg As L -1, for five days, in a hydroponic system or in culture medium. The plants accumulated large amounts of As in roots and presented a high translocation factor to the shoot, and also showed changes in nutrient accumulation. The visual symptoms have intensified with the increasing of As concentration in the nutritive solution. Roots showed dark coloration and a damaged and gelatinous aspect, with increased roots hair length and density. The shoots showed accumulation of anthocyanins and signs of early senescence, as well as changes in tissue thickness. Oxidative stress and reduction of phosphorus concentration in tissues have been implicated as the main cause of toxicity, evidencing the indirect damage from this element in the organism. Important changes in photosynthesis were observed, as signs of damage to respiration, due to increased expression of alternative oxidase genes. Thus, changes in the levels of synthesis and utilization of sugars by plants were observed. As promoted DNA fragmentation in A. cepa and increased rates of point mutation and nonhomologous recombination. The significant increase in the expression pattern of the msh2 and msh7 genes, which encode key enzymes in DNA repair process, suggests the importance of this mechanism in defense against As genotoxicity in A. thaliana. This was confirmed by the greater sensitivity observed in msh2 mutants to As as indicated by visual symptoms and by an increase in lipid peroxidation. Inhibition of the activity of the caspase-3 protease was also observed, evidencing the As capacity of enzyme activity inhibition. / Tese enviada pela secretaria do curso por e-mail, em 28-03-17.