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.

No description available.

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.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2017-03-29T11:17:16Z No. of bitstreams: 1 texto completo.pdf: 905568 bytes, checksum: 94c53779a921ceebf36267e09ef933a1 (MD5) / Made available in DSpace on 2017-03-29T11:17:16Z (GMT). No. of bitstreams: 1 texto completo.pdf: 905568 bytes, checksum: 94c53779a921ceebf36267e09ef933a1 (MD5) Previous issue date: 2013-02-28 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Ao contrário de outros transportadores de ADP/ATP, ADNT1 é o único que medeia um antiporte um de ATP preferencialmente por AMP, e, em menor grau, por ADP. Um trabalho prévio sugere que a expressão ADNT1 é maior em pontas de raízes e em tecidos senescentes. Considerando a elevada expressão de ADNT1 em tecidos senescentes, nos propusemos a investigar o papel do ADNT1 durante o processo de senescência induzida pelo escuro. Sob estas condições, plantas mutantes de Arabidopsis thaliana deficientes na expressão do transportador ADNT1 exibiram uma senescência antecipada em relação ao tipo selvagem, como evidenciado tanto pelo fenótipo visual das plantas após o crescimento em longos períodos de escuridão, como pela perda de clorofilas e da capacidade fotossintética. O tratamento prolongado de escuro levou, em geral, a um declínio mais rápido nos mutantes do que nos do tipo selvagem nos teores de clorofila e nos níveis de sacarose e de proteínas. Por outro lado, os níveis de aminoácidos totais e de alguns intermediários do ciclo TCA, como malato, fumarato e isocitrato geralmente aumentaram significativamente nos mutantes no final do tratamento de escuro. As razões NADH/NAD + e NADPH/NADP+ também se apresentaram maiores nos mutantes em comparação com o tipo selvagem, com a progressão da escuridão. Além disso, as plantas mutantes apresentaram sintomas de senescência precoce em comparação ao tipo selvagem, mesmo sob condições tidas como não estressantes. Estes dados demonstram, assim, que ADNT1 não é funcionalmente redundante aos previamente caracterizados transportadores de ADP/ATP, especialmente durante a falta de carbono, e reforçam a função potencial de ADNT1 no fornecimento da energia necessária para suportar o crescimento de tecidos vegetais heterotróficos. / Unlike other ADP/ATP carriers, ADNT1 is the only one that mediates an antiport of ATP, AMP, and, to a lesser extent, ADP and the corresponding deoxyadenine nucleotides. Previous work observed that ADNT1 expression is much stronger in root tips and senescing tissues. Considering the high expression of ADNT1 in senescing tissues, we have investigated the role of ADNT1 during the process of dark-induced senescence. Under these conditions, Arabidopsis thaliana mutants deficient in the expression of ADNT1 transporter displayed a similar, yet milder, early onset of senescence as evidenced both by the visual phenotype of plants following growth in extended periods of darkness and the loss of chlorophyll and photosynthetic competence. The extended dark treatment led in general to a more rapidly decline in the mutants than in the wild type in the levels of sucrose and protein. By contrast, the levels of total amino acids and TCA cycle intermediates malate, fumarate and isocitrate generally increased significantly in the mutants at the end of dark treatment. The NADH/NAD + and NADPH/NADP+ ratios also increased in mutants in comparison to the wild type with progression of the darkness. Additionally, the mutant plants exhibited symptoms of early senescence in comparison to the wild type even under optimal conditions. Altogether the data obtained demonstrate that ADNT1 is not functionally redundant to the previously characterized ADP/ATP carriers, especially during carbon starvation and reinforce the potential function for ADNT1 in the provision of energy which is required to support growth in heterotrophic plant tissues. / Não foi localizado o cpf do autor. Tese enviada pela secretaria do curso por e-mail, em 28-03-17.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2018-04-19T17:29:27Z No. of bitstreams: 1 texto completo.pdf: 1046673 bytes, checksum: 303cdb874bd37b4e599895a7138301a8 (MD5) / Made available in DSpace on 2018-04-19T17:29:27Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1046673 bytes, checksum: 303cdb874bd37b4e599895a7138301a8 (MD5) Previous issue date: 2017-07-21 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Recentemente, uma nova camada de defesa antiviral foi caracterizada em Arabidopsis thaliana, que é mediada pelo receptor NIK (NSP-Interacting Kinase) e protege a planta contra begomovirus. Quando a planta é infectada por um vírus, NIK1 oligomeriza com outro receptor imune ou com NIK1 para transfosforilar um ao outro e consequentemente, ativar o domínio cinase de NIK1. NIK1 é capaz de se ligar a proteína ribossomal L10 (RPL10), e é capaz de mediar sua fosforilação e subsequente translocação ao núcleo, onde RPL10 interage com LIMYB (L10-interacting Myb domain-containing protein) para regular negativamente a expressão de genes de proteínas ribossomais, levando a supressão global da tradução. Os mRNAs virais não são capazes de escapar ao mecanismo de regulação de tradução do hospedeiro, aumentando assim a resistência contra begomovirus. A proteína viral NSP interage com NIK1 e inibe sua atividade cinase, aumentando a patogenicidade do begomovírus em seu hospedeiro. Foi demonstrado em trabalhos anteriores que o mutante NIK1-T474D é um excelente alvo para engenharia genética, pois é constitutivamente ativada em linhagens transgênicas. Nesse trabalho, primeiramente foi realizado a reconstrução in silico da via de sinalização antiviral mediada por NIK1 em tomateiro, identificando possíveis componentes homólogos dessa via. Em seguida, foi examinada a possibilidade do duplo mutante T474D-T469A de conferir resistência contra begomovírus em linhagens transgênicas de tomateiro. Além disso, a linhagem transgênica NIK1-T474D-T469A foi transformada com o componente a jusante da via de sinalilzação antiviral, LIMYB de Arabidopsis, e examinamos o efeito da reconstituição dessa via de defesa durante a infecção viral. Os resultado dessa investigação indicam que a expressão de NIK1-T474D/T469A em linhagens transgênicas de tomateiro foi efetiva para suprimir a expressão de genes ribossomais, indicando que o duplo mutante é constitutivamente ativado em tomateiro. Além disso, a expressão de LIMYB em combinação com NIK1-T469A/T474D promoveu um efeito aditivo na repressão dos genes de proteínas ribossomais, possivelmente revelando uma melhorestratégia para obtenção de resistência a begomovírus. Para averiguar essa hipótese, as linhagens transgênicas foram desafiadas com os begomovírus de tomateiro ToYSV e ToSRV e a infecção foi monitorada por meio de sintomatologia e quantificação do DNA viral. Os resultados demonstraram que a expressão de NIK1-T469A/T474D em combinação com LIMYB é potencialmente um alvo melhor para modificar geneticamente genótipos suscetíveis. / Recently, a new layer of antiviral defenses was characterized in Arabidopsis thaliana, which is mediated by the immune receptor NIK (NSP-Interacting Kinase) and protects plants against begomoviruses. Upon virus infection, NIK1 oligomerizes with itself or another immune receptor to transphosphorylate one another and to activate the NIK1 kinase domain. Activated NIK1 mediates the phosphorylation of the ribosomal protein L10 (RPL10) and subsequent translocation to the nucleus, where RPL10 interacts with LIMYB (L10-interacting Myb domain-containing protein) to down-regulate the expression of ribosomal protein genes, leading to global translation suppression. The viral mRNAs are not able to escape this host translation regulatory mechanism enhancing host resistance against begomoviruses. The viral protein NSP interacts with NIK1 and inhibits its kinase activity, increasing the pathogenicity of begomoviruses by their hosts. We have previously demonstrated that the NIK1 mutant T474D is an excellent target for engineering begomovirus resistance because it is constitutively activated in transgenic lines. In this investigation, we first reconstructed in silico the NIK1-mediated antiviral signaling in tomato by identifying the tomato homologs of the components of the signaling pathway. Then, we examined the property of the double mutant T474D-T469A to confer resistance against begomoviruses in tomato transgenic lines. Furthermore, we overexpressed the downstream component of NIK1 antiviral signaling, LIMYB from Arabidopsis, in the T474D-T469A transgenic lines and examined the effect of reconstituting the antiviral pathway on begomovirus infection. Our results indicated that expression of T474D/T469A in transgenic lines was effective to suppress the expression of ribosomal protein genes, indicating that this double mutant is constitutively activated in tomato. Furthermore, expression of LIMYB in combination with T469A/T474D promoted an additive effect in ribosomal protein gene repression, possibly uncovering a better strategy to acquire begomovirus resistance. To examine this hypothesis, we challenged the transgenic lines with the tomato-infecting begomoviruses ToYSV and ToSRV and monitored the infection by symptomatology and quantitation of viral DNA.Our results demonstrated that expression of T469A/T474D in combination with LIMYB holds the potential to be a better target for engineering begomovirus resistance in susceptible genotypes. / Ficha catalográfica com erro: Morais de Ávila, Larissa Gabriela.