20 resultados para Electrophoresis, Polyacrylamide Gel


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Biochemistry, 2011, 50 (20), pp 4251–4262 DOI: 10.1021/bi101605p

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Periodic drought is the primary limitation of plant growth and crop yield. The rise of water demand caused by the increase in world population and climate change, leads to one of the biggest challenges of modern agriculture: to increase food and feed production. De novo DNA methylation is a process regulated by small interfering RNA (siRNAs), which play a role in plant response and adaptation to abiotic stress. In the particular case of water deficit, growing evidences suggest a link between the siRNA pathways and drought response in the model legume Medicago truncatula. As a first step to understand the role of DNA methylation under water stress, we have set up several bioinformatics and molecular methodologies allowing the design of Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems and the assembly of TALENs (transcription activator-like effector nucleases), to target both dicer-like 3 (MtDCL3) and RNA-Dependent RNA polymerase (MtRDR2), enzymes of the RNA-directed DNA methylation pathway. TALENs efficiency was evaluated prior to plant transformation by a yeast-based assay using two different strategies to test TALENs activity: Polyacrylamide gel electrophoresis (PAGE) and Single strand conformation polymorphisms (SSCP). In this assay, yeast cells triple transformation emerged as good and rapid alternative to laborious yeast mating strategies. PAGE analysis might be a valuable tool to test TALENs efficacy in vivo if we could increase TALENs activity. SSCP-based approach proved to be ineffective due to the generation of several false positives. TALENs and CRISPR/Cas9 system constructed and designed in this work will in the future certainly enable the successful disruption of DCL3 and RDR2 genes and shed the light on the relationship between plant stress resistance and epigenetic regulation mediated by siRNAs in M.truncatula.

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Dissertação apresentada à Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Biotecnologia

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International Biodeterioration & Biodegradation,xxx (2009) 1–8

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Science of the total environment 405(2008) 278-285

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Journal of Proteome Research (2006)5: 2720-2726

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Thesis submitted to the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia, for the degree of Doctor of Philosophy in Biochemistry

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Work presented in the context of the European Master in Computational Logics, as partial requisit for the graduation as Master in Computational Logics

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Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Engenharia Química e Bioquímica

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Thesis for the Master degree in Structural and Functional Biochemistry

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Dissertação para obtenção do Grau de Doutor em Biologia

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Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

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Dissertation for the Master’s Degree in Structural and Functional Biochemistry

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Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica