Questioning the utility of RNA-DNA chimera in gene repair.

In principle, we should be glad that Eric Kmiec and his colleagues published in Science's STKE (1) a detailed experimental protocol of their gene repair method (2, 3). However, a careful reading of their contribution raises more doubts about the method. The research published in Science five years ago by Kmiec and his colleagues was said to demonstrate that chimeric RNA-DNA oligonucleotides could correct the mutation responsible for sickle cell anemia with 50% efficiency (4). Such a remarkable result prompted many laboratories to attempt to replicate the research or utilize the method on their own systems. However, if the method worked at all, which it rarely did, the achieved efficiency was usually lower by several orders of magnitude. Now, in the Science's STKE protocol, we are given crucial information about the method and why it is so important to utilize these expensive chimeric RNA-DNA constructs. In the introduction we are told that the RNA-DNA duplex is more stable than a DNA-DNA duplex and so extends the half-life of the complexes formed between the targeted DNA and the chimeric RNA-DNA oligonucleotides. This logical explanation, however, conflicts with the statement in the section entitled "Transfection with Oligonucleotides and Plasmid DNA" that Kmiec and colleagues have recently demonstrated that classical single-stranded DNA oligonucleotides with a few protective phosphothioate linkages have a "gene repair conversion frequency rivaling that of the RNA/DNA chimera". Indeed, the research cited for that result actually states that single-stranded DNA oligonucleotides are in fact several-fold more efficient (3.7-fold) than the RNA-DNA chimeric constructs (5). If that is the case, it raises the question of why Kmiec and colleagues emphasize the importance of the RNA in their original chimeric constructs. Their own new results show that modified single-stranded DNA oligonucleotides are more effective than the expensive RNA-DNA hybrids. Moreover, the current efficiency of the gene repair by RNA-DNA hybrids, according to Kmiec and colleagues in their recent paper is only 4×10-4 even after several hours of pre-selection permitting multiplification of bacterial cells with the corrected plasmid (5). This efficiency is much lower than the 50% value reported five years ago, but is assuredly much closer to the reality.

"Pa'que veas que te pido perdón en delante toda España" : pragmalingüística y análisis del discurso en los diálogos del talk show televisivo "Diario de Patricia" (2001-2011) : aproximaciones teóricas y análisis

Cette thèse s'inscrit dans le cadre de la Pragmalinguistique et d'Analyse du discours oral et offre une étude approfondie d'un talk show télévisuel espagnol, Diario de Patricia, format dans lequel la présentatrice du même nom reçoit des invités pour parler avec eux de leurs problèmes personnels. Ce travail propose une relecture de grandes théories pragmatiques (comme la Théorie des actes du langage, le Principe de coopération, la Théorie de la politesse ou encore la Théorie de la pertinence) et leur application à des discours réels. Les principes de la linguistique de l'interaction permettent également de décrire l'organisation des talk shows, les stratégies discursives de la présentatrice, la façon dont elle prépare chaque émission et planifie les interviews avec ses invités afin de provoquer chez ceux-ci des réactions émotionnelles qui l'intéressent en vue du taux d'audience. Ce travail de recherche présente également une analyse approfondie des mécanismes pragmalinguistiques utilisés par la présentatrice pour imposer son autorité et pour maîtriser la situation d'une façon que l'auteure qualifie de manipulation des invités, toujours sous une apparence d'empathie et de compréhension.