Electroperméabilisation de systèmes modèles

L'électroperméabilisation est un procédé fondé sur l'application d'impulsions électriques qui peuvent induire une perméabilisation réversible de la membrane plasmique de cellules vivantes. En d'autres termes: si vous soumettez une cellule à des impulsions électriques d'amplitude et de durée judicieusement choisies, vous serez alors en mesure d'introduire dans son cytoplasme des molécules d'intérêt autrement incapables de traverser son enveloppe externe, et ce sans affecter sa viabilité. Cette technique a donné lieu à diverses applications, notamment dans le cadre de la lutte contre le cancer ou des thérapies géniques. Comportant moins de risques que les méthodes de transfection virales ou chimiques, son usage est de plus en plus répandu dans la communauté médicale. Cependant, les processus de réorganisation de la membrane, au niveau microscopique, sont encore méconnus et sujets à débat. Une meilleure description de ces phénomènes permettrait d'améliorer l'efficacité et la sécurité des protocoles de traitement. Une stratégie possible pour accroître notre compréhension de l'électroperméabilisation consiste en la réalisation d'expériences sur des systèmes modèles. Cette thèse aborde l'étude de l'effet d'impulsions électriques perméabilisantes de longue durée (quelques millisecondes) sur des systèmes lipidiques artificiels, des vésicules unilamellaires géantes. Il est décrit comment ce travail sur systèmes modèles a contribué à améliorer notre compréhension fondamentale de l'électroperméabilisation, mais aussi comment il a donné lieu à deux catégories d'applications: le chargement de vésicules avec des macromolécules et la mesure de grandeurs physiques caractéristiques des bicouches lipidiques, les tensions de bord. Ces recherches comportent aussi un aspect de modélisation de l'entrée dans des cellules électroperméabilisées de différentes molécules, via la résolution numérique d'équations aux dérivées partielles gouvernant l'évolution de leur concentration. Cette partie apporte des éléments de réponse visant à expliquer les différences observées expérimentalement entre le transfert de petites et de macro-molécules.Electropermeabilization is a process based on the application of electric pulses which can induce a transient permeabilization of the cell plasma membrane. If you submit a cell to a sequence of electric pulses with appropriate amplitude and duration, you will manage to introduce in the cytoplasm some molecules otherwise unable to cross the external envelope, and to do so without decreasing cell viability. This technique has led to various applications, notably in the fight against cancer and in the field of gene therapy. Being safer than chemical and viral transfection methods, it has become increasingly popular among the medical community. However, membrane reorganization processes at the microscopic level are not yet fully understood, and are still a matter of debate. A better description of these phenomena would allow to improve the efficiency and the safety of the clinical protocols. In this prospect, a possible strategy consists in the study of electric field effects on model systems. This thesis focuses on the influence of long duration (in the millisecond range) electric pulses on artificial lipid systems, giant unilamellar vesicles. It explains how these studies on model systems contributed to our fundamental knowledge of electropermeabilization, and also how they resulted in two practical applications: a method for loading liposomes with macromolecules, and a method for measuring a characteristic physical property of lipid bilayers, the edge tension. It also contains a part where the entry of different molecules into electropermeabilized cells was studied via the numerical resolution of partial differential equations governing the evolution of the molecule concentration. This part of the thesis brings some clues for understanding the experimentally observed different behaviours between the electrotransfer of small and macromolecules

Visualization of membrane loss during the shrinkage of giant vesicles under electropulsation

We study the effect of permeabilizing electric fields applied to two different types of giant unilamellar vesicles, the first formed from EggPC lipids and the second formed from DOPC lipids. Experiments on vesicles of both lipid types show a decrease in vesicle radius which is interpreted as being due to lipid loss during the permeabilization process. We show that the decrease in size can be qualitatively explained as a loss of lipid area which is proportional to the area of the vesicle which is permeabilized. Three possible mechanisms responsible for lipid loss were directly observed: pore formation, vesicle formation and tubule formation.Comment: Final published versio

A New Method for Measuring Edge Tensions and Stability of Lipid Bilayers: Effect of Membrane Composition

We report a new and facile method for measuring edge tensions of lipid membranes. The approach is based on electroporation of giant unilamellar vesicles and analysis of the pore closure dynamics. We applied this method to evaluate the edge tension in membranes with four different compositions: egg phosphatidylcholine (EggPC), dioleoylphosphatidylcholine (DOPC), and mixtures of the latter with cholesterol and dioleoylphosphatidylethanolamine (DOPE). Our data confirm previous results for EggPC and DOPC. The addition of 17 mol % cholesterol to the DOPC membrane causes an increase in the membrane edge tension. On the contrary, when the same fraction of DOPE is added to the membrane, a decrease in the edge tension is observed, which is an unexpected result considering the inverted-cone shape geometry of the molecule. Presumably, interlipid hydrogen bonding lies in the origin of this behavior. Furthermore, cholesterol was found to lower the lysis tension of DOPC bilayers. This behavior differs from that observed on bilayers made of stearoyloleoylphosphatidylcholine, suggesting that cholesterol influences the membrane mechanical stability in a lipid-specific manner

Pharmacology of (2S,4Z)-N-[(2S)-2-hydroxy-2-phenylethyl]-4-(methoxyimino) -1-[(2 -methyl[1,1 -biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxamide, a new potent and selective nonpeptide antagonist of the oxytocin receptor.

ABSTRACT We have discovered a new, potent, selective, and orally active oxytocin receptor antagonist, (2S,4Z)-N-[(2S)-2-hydroxy-2-phenylethyl]-4-(methoxyimino)-1-[(2Ј-methyl[1,1Ј-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxamide (compound 1). We report the biochemical, pharmacological, and pharmacokinetic characterization in vitro and in vivo of this compound. Premature birth is a major problem in obstetrics affecting about 10% of all births and being the largest cause of perinatal morbidity and mortality. The impact on society is significant in terms of costs of neonatal intensive care and for the emotional and social stress to the family. The physiopathology of human preterm labor is complex and multifactorial. Preterm increase of uterine activity is a common complication of pregnancy and accounts for many cases of preterm labor. Pharmacological interventions aimed at maintaining uterine quiescence (tocolysis) have been, and are likely to remain, the cornerstone of pharmaceutical management of preterm labor. However, current tocolytic agents (␤-mimetics, magnesium sulfate, calcium channel blockers, or prostaglandin synthesis inhibitors) suffer from a minimal effectiveness and show important fetal and maternal side effects. Therefore, it is obvious that a safe and effective oral treatment delaying spontaneous preterm birth would have tremendous clinical benefits. The peptide hormone oxytocin (OT) is a potent contractor of the human uterus. OT mediates its effect through activation of the G protein-coupled oxytocin receptor (OT-R) that is expressed in myometrial cells. OT-R is coupled to phospholipase C activation, leading to intracellular synthesis of inositol phosphates and mobilization of calcium. In turn, the rise in intracellular calcium concentration promotes a cascade of events, including phosphorylation of myosin, that then acts on actin and induces uterine muscle cell contraction. Before onset of labor and in the term myometrium, the OT-R density Article, publication date, and citation information can be found a

Électroperméabilisation de systèmes modèles

Electropermeabilization is a process based on the application of electric pulses which can induce a transient permeabilization of the cell plasma membrane. If you submit a cell to a sequence of electric pulses with appropriate amplitude and duration, you will manage to introduce in the cytoplasm some molecules otherwise unable to cross the external envelope, and to do so without decreasing cell viability. This technique has led to various applications, notably in the fight against cancer and in the field of gene therapy. Being safer than chemical and viral transfection methods, it has become increasingly popular among the medical community. However, membrane reorganization processes at the microscopic level are not yet fully understood, and are still a matter of debate. A better description of these phenomena would allow to improve the efficiency and the safety of the clinical protocols. In this prospect, a possible strategy consists in the study of electric field effects on model systems. This thesis focuses on the influence of long duration (in the millisecond range) electric pulses on artificial lipid systems, giant unilamellar vesicles. It explains how these studies on model systems contributed to our fundamental knowledge of electropermeabilization, and also how they resulted in two practical applications: a method for loading liposomes with macromolecules, and a method for measuring a characteristic physical property of lipid bilayers, the edge tension. It also contains a part where the entry of different molecules into electropermeabilized cells was studied via the numerical resolution of partial differential equations governing the evolution of the molecule concentration. This part of the thesis brings some clues for understanding the experimentally observed different behaviours between the electrotransfer of small and macromolecules.L'électroperméabilisation est un procédé fondé sur l'application d'impulsions électriques qui peuvent induire une perméabilisation réversible de la membrane plasmique de cellules vivantes. En d'autres termes: si vous soumettez une cellule à des impulsions électriques d'amplitude et de durée judicieusement choisies, vous serez alors en mesure d'introduire dans son cytoplasme des molécules d'intérêt autrement incapables de traverser son enveloppe externe, et ce sans affecter sa viabilité. Cette technique a donné lieu à diverses applications, notamment dans le cadre de la lutte contre le cancer ou des thérapies géniques. Comportant moins de risques que les méthodes de transfection virales ou chimiques, son usage est de plus en plus répandu dans la communauté médicale. Cependant, les processus de réorganisation de la membrane, au niveau microscopique, sont encore méconnus et sujets à débat. Une meilleure description de ces phénomènes permettrait d'améliorer l'efficacité et la sécurité des protocoles de traitement. Une stratégie possible pour accroître notre compréhension de l'électroperméabilisation consiste en la réalisation d'expériences sur des systèmes modèles. Cette thèse aborde l'étude de l'effet d'impulsions électriques perméabilisantes de longue durée (quelques millisecondes) sur des systèmes lipidiques artificiels, des vésicules unilamellaires géantes. Il est décrit comment ce travail sur systèmes modèles a contribué à améliorer notre compréhension fondamentale de l'électroperméabilisation, mais aussi comment il a donné lieu à deux catégories d'applications: le chargement de vésicules avec des macromolécules et la mesure de grandeurs physiques caractéristiques des bicouches lipidiques, les tensions de bord. Ces recherches comportent aussi un aspect de modélisation de l'entrée dans des cellules électroperméabilisées de différentes molécules, via la résolution numérique d'équations aux dérivées partielles gouvernant l'évolution de leur concentration. Cette partie apporte des éléments de réponse visant à expliquer les différences observées expérimentalement entre le transfert de petites et de macromolécules

Electroperméabilisation de systèmes modèles

TOULOUSE3-SCD-Bib. electronique (315559904) / SudocSudocFranceF

Organochlorine Pesticides (OCPs) in sediment core MAR16-02 from the Eure River, France

We measured organochlorine pesticides (OCPs) in sediment cores (collected in two independant ponds) to study the relationships between sediment deposits characteristics and organic contaminants, and the temporal trends of OCPs were reconstructed since 1940s in the Eure River watershed, major tributary of the Seine estuary (France). The analysis of particulate OCPs from the sediment cores (MAR16-02 and DAM17-02) was performed using microwave-assisted extraction, with toluene:acetone mixture for extraction. Following the microwave-assisted extraction, a gas chromatograph (7890B, Agilent, US) coupled to a mass spectrometer (MS; 7000C) was used to measure the concentrations