La Parenthèse ; suivi de Tensions et enfermement dans les Cent Vingt Journées de Sodome du marquis de Sade

Ce mémoire en recherche-création explore l’enfermement volontaire et les différents types de tensions qu’il provoque. Court roman prenant la forme du journal intime, La Parenthèse met en scène un jeune homme qui décide de s’enfermer chez lui une semaine durant et s’interdit tout contact avec l’extérieur – autant pour prendre un congé temporaire de la vie qu’il mène que pour examiner les raisons de sa détresse quotidienne. Le monologue intérieur se transforme rapidement en dialogue, dès lors qu’un double vindicatif, interrompant la voix principale par des « répliques » entre parenthèses, fait son apparition. Une relation houleuse – sous tension – se tisse entre ces deux facettes du personnage tout au long des sept jours de la réclusion, les passages de dispute alternant avec des récits de souvenirs. En somme, le roman tente de dramatiser la question de l’emprisonnement de soi-même et de la limitation de l’écriture, cette limitation pouvant être à la fois malsaine et libératrice. Quant à l’essai, Tensions et enfermement dans les Cent Vingt Journées de Sodome du marquis de Sade, il part du thème de l’enfermement (en l’occurrence, celui des quatre amis qui exécutent le projet de passer quatre mois dans un château isolé) pour postuler une « architecture du désir » dans Les Cent Vingt Journées de Sodome. L’essai mobilise les ressources de la narratologie en prenant en compte les effets du texte sur le lecteur ; sont ainsi mises en évidence les tensions – sexuelle pour les protagonistes, narrative pour le lecteur – élaborées par cette écriture de l’enfermement et de la contrainte, dans laquelle le désir est toujours maintenu mais rarement satisfait.

Étude structuro-fonctionnelle de SecP43 et caractérisation de ses interactions avec SepSecS et l’ARNtSec in vitro

Selenoproteins are proteins containing selenium in the form of the 21st amino acid, selenocysteine. Selenocysteine (Sec) is directly synthesized onto its cognate tRNA (tRNA[Ser]Sec or tRNASec) and inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. Among those factors, SecP43 has been reported to possibly play an essential role in the methylation at the 2’-hydroxylribosyl moiety in the wobble position (Um34) of Sec-tRNA[Ser]Sec and consequently reduce the expression of glutathione peroxidase 1. SecP43 also called tRNASec-associated protein has also been reported to interact in with SepSecS and tRNASec in vivo and the targeted removal of one of these proteins affected the binding of the other to the Sec-tRNASec. The initial aim of the project was to solve the structure of SecP43 by means of x-ray crystallography. Secondly, we were interested in characterizing the interaction of the latter with some of the components of the selenocysteine insertion machinery. These factors are SepSecS and tRNASec. We were able to optimize the expression and the purification of soluble form of the human homologue of SecP43 and of SepSecS by using an adapted auto-induction protocol. This was a major challenge considering that full length SecP43 has not been expressed and purify to date. We did not succeed in crystallizing SecP43. Our failure to crystallize SecP43 is probably due to the fact that it is a partially folded protein as we were able to demonstrate by SAXS (Small Angle X-ray Scattering). The SecP43 envelope calculated by SAXS displayed a rod-shape like structure. In order to enhance the stability of SecP43 required for crystallization, binding affinity studies were conducted to characterize the interaction between SecP43, tRNASec and SepSecS. We did not detect an interaction between SecP43 and tRNASec by using EMSA (Electrophoretic Mobility Shift Assay) and gel filtration. We also could not detect an interaction between SecP43 and SepSecS using a cross-linking assay. In contrast, the tRNASec/SepSecS interaction was demonstrated by EMSA and the addition of SecP43 seemed to reduce the binding affinity. Therefore, SecP43 might induce a conformational change in SepSecS in the presence of tRNASec.

In Vitro Ketamine CYP3A Mediated Metabolism Study using Mammalian Liver S9 Fractions, cDNA Expressed Enzymes and Liquid Chromatography Tandem Mass Spectrometry

Ketamine is widely used in medicine in combination with several benzodiazepines including midazolam. The objectives of this study were to develop a novel HPLC-MS/SRM method capable of quantifying ketamine and norketamine using an isotopic dilution strategy in biological matrices and study the formation of norketamine, the principal metabolite of ketamine with and without the presence of midazolam, a well-known CYP3A substrate. The chromatographic separation was achieved using a Thermo Betasil Phenyl 100 x 2 mm column combined with an isocratic mobile phase composed of acetonitrile, methanol, water and formic acid (60:20:20:0.4) at a flow rate of 300 μL/min. The mass spectrometer was operating in selected reaction monitoring mode and the analytical range was set at 0.05–50 μM. The precision (%CV) and accuracy (%NOM) observed were ranging from 3.9–7.8 and 95.9.2–111.1% respectively. The initial rate of formation of norketamine was determined using various ketamine concentration and Km values of 18.4 μM, 13.8 μM and 30.8 μM for rat, dog and human liver S9 fractions were observed respectively. The metabolic stability of ketamine on liver S9 fractions was significantly higher in human (T1/2 = 159.4 min) compared with rat (T1/2 = 12.6 min) and dog (T1/2 = 7.3 min) liver S9 fractions. Moreover significantly lower IC50 and Ki values observed in human compared with rat and dog liver S9 fractions. Experiments with cDNA expressed CYP3A enzymes showed the formation of norketamine is mediated by CYP3A but results suggest an important contribution from others isoenzymes, most likely CYP2C particularly in rat.