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RNA --- ARN --- RNA. --- General biochemistry --- Molecular biology

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Survivin is an anti-apoptotic protein that belongs to the IAPs (Inhibitor of Apoptosis Proteins) family and controls mitosis.
By alternative splicing, the survivin gene generates two variants : survivin 2B and survivin 3. According to the literature, surviving and its isoforms are strongly expressed during foetal development and also in tumor cells, but they are not expressed in healthy tissues except in thymus and placenta. It has been published that the surviving and its variants produce antigens recognized by cytolitic T lymphocytes that could be used in cancer immunotherapy.
The goal of this work was to measure by quantitative RT-PCR the level of expression of surviving and its variants in normal tissues, tumor lines and tumor clinical sample to evaluate the feasibility of an immunotherapy directed against antigens derived from surviving or its variants. Our results show a high level of expression of surviving and its isoforms in normal cells with a high mitotic index, a level of expression that is similar to that observed in tumor cells.
The second part of this work is an attempt to study the methylation of the promoter of the surviving gene. The methylation status of this promoter in healthy and tumor cells is debated in the literature. In order to clarify this point, we quantified transcripts of surviving and its isoforms in B and T lymphocytes before and after treatment with azadeoxycytidin, a demethylating agent. The results obtained did not allow us to conclude if the promoter was methylated or not in these cells La survivine est une protéine anti-apoptotique appartenant à la famille des IAPs (inhibitors of apoptosis proteins) et régulant la mitose. Grâce à un épissage alternatif, le gène de la survivine génère deux autres formes de la survivine : la survivine 2B et la survivine 3. Selon la littérature, la survivine et ses isoformes seraient fortement exprimées au cours du développement fœtal ainsi que dans les cellules des tissus sains. On a publié que la survivine et ses variantes produisent des antigènes reconnus par des lymphocytes T cytolytiques qui pourraient être utilisés en immunothérapie anti-tumorale.
L’objet de ce mémoire est de mesurer par RT-PDR quantitative le niveau d’expression de la survivine et de ses variants au sein des tissus normaux, des lignées tumorales et des échantillons cliniques de tumeurs pour évaluer les danger d’une immunothérapie dirigée contre les antigènes provenant de la survivine et de ses variants. Les résultats de cette étude montrent un haut niveau d’expression de la survivine et de ses isoformes dans les cellules normales à index mitotique élevé, un niveau d’expression qui est souvent comparable à celui des cellules tumorales.
Une seconde partie du mémoire est consacrée à l’étude de la méthylation du promoteur du gène de la survivine. En effet, l’état de méthylation du gène de la survivine dans les cellules saines et les cellules tumorales est controversé dans la littérature. Pour clarifier l’état de méthylation du promoteur, on a quantifié l’abondance des ARNm de la survivine et de ses isoformes dans des lymphocytes B et T avant et après traitement avec un agent déméthylant, l’azadéoxycytidine. Les résultats obtenus ne nous permettent pas de conclure que le promoteur et méthylé ou pas

RNA, Messenger --- Methylation --- Cells

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Biological techniques --- Enzymology --- Molecular biology --- Ribonucleic acid --- Metabolism --- ARN --- Messenger rna --- Ribonucleoproteins --- Rna precursors --- Rna splicing --- Rna, ribosomal --- Rna, transfer --- Amino acid sequence --- Nucleic acid conformation --- Rna

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Since the discovery regarding interference phenomenon, 20 years ago, RNA interference became standard method to suppress a gene expression into in vitro tests. Nowadays, the point of interest lies in vivo studies. The RNAi could be used as strong tool in specific therapy going from viral infection to cancer, if only the RNAi would have a proper structure. The main obstacle to RNAi potential therapeutic action is due to a difficulty small interfering RNA (siRNA) at the level of its action in vivo. An effective delivery strategy of siRNA must consider some limitations: (i) poor stability, (ii) undesirable non-specific immune response, (iii) a distribution in non-targeted tissues. The development of such a strategy requires a careful understanding of all the mechanisms of action of RNAi, engineering developed to overcome barriers to the delivery of siRNA, the site of action, and finally a clinical trial. This analysis allows us to offer a perspective on the therapeutic use of siRNA in human.

RNA Interference --- Pharmacology --- Gene Expression

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General biochemistry --- Molecular biology --- ARN --- RNA --- Code génétique --- genetic code --- Biosynthèse --- Biosynthesis --- Synthesis. --- Biosynthesis. --- Rna --- biosynthesis --- Synthesis --- biosynthesis. --- RNA. --- RNA - biosynthesis --- RNA - Synthesis