The covalent bond formed between a N-heterocyclic carbene and an aryl-isothiocyanate was discovered to be thermally-reversible. This bond was incorporated into the backbone of an aromatic polymer which, when subjected to heat and excess monomer, would depolymerize to smaller oligomers. In addition these small molecules contain active chain ends and could be repolymerized to reform the original polymer. The high molecular weight material was made into freestanding sheets with desirable mechanical properties and could be made conductive by treatment with iodine. A new poly(triazene) was formed from the reaction of a facially opposed, annulated, bis-N-heterocyclic carbene (NHC) and an organic bis-azide. The NHC as well as the azide were varied and combined to produce a series of polymers which were characterized by GPC, TGA, and NMR. These thermally robust polymers were also coated onto glass slides and rendered electrically conductive by exposure to iodine vapor. A new reagent for Reversible Addition Fragmentation Chain Transfer Polymerization (RAFT) is described. This imidazolium based reagent shows unusually fast kinetics which allows it to control polymerizations at significantly reduced loadings compared to the more traditional neutral dithiocarbamates or dithioesters. The fast kinetics is explained by the rapid rotation of the dithioester about the plane of the cationic N-heterocycle. Sulfonated poly(ether ether ketone) (sPEEK) membranes were blended with imidazoles with varying pKas. The proton conductivity of the membranes was evaluated as a function of pKa and temperature. Interestingly, the conductivity of the dry membranes showed a non-monotonous profile over a temperature range of 25 – 150 C. We use a theoretical model to better understand the mechanistic origins of the observed temperature–conductivity profiles. This model is based on the reaction equilibria between sPEEK’s sulfonic acid groups and the basic sites of the added heterocycles. Using the copper-catalyzed 1,3-dipolar “click” cycloaddition reaction, poly(sulfone)s containing pendant azide moieties were functionalized with various amounts of sodium 3-(prop-2-ynyloxy)propane-1-sulfonate and crosslinked with 1,7-octadiyne. The degree of sulfonation as well as the degree of cross-linking was systematically varied by changing the ratios of the aforementioned reagents. The polymers were cast into membranes, acidified, and then tested for proton conductivity, methanol permeability, and membrane-electrode assembly (MEA) performance. / text

Although spinal cord injury (SCI) is a debilitating condition that presents a large socioeconomic problem in the United States, there is currently no treatment that reliably reduces morbidity and mortality. Current research is aimed at identifying mechanisms involved in the pathophysiology of SCI and using this knowledge to develop rational treatments. We have observed plasma membrane compromise in the acute (within 10 minutes), sub-acute (3 days), and chronic phases (5 weeks) in a rat model of contusion SCI and postulate that it negatively affects neurological outcome. Holes/tears in the plasma membrane were assessed with a dye exclusion assay, in which a fluorescent cell-impermeant dye was injected into the cerebrospinal fluid prior to sacrifice; therefore, cellular uptake of the dye is indicative of plasma membrane compromise. As early as 10 minutes after SCI, widespread uptake of permeability markers was evident in neuronal cell bodies as well as axonal projections. The number of permeable cells and the size of the membrane breaches (measured by using permeability markers of various sizes) varied with distance from the injury site, with larger disruptions located closer to the epicenter. Greater cellular uptake was observed when the impact force was increased (200 > 150 > 100 kdyn > sham). At longer time points (3 days and 5 weeks), substantial permeability marker uptake was observed in axons but not in cell bodies. Cells with increased permeability displayed a variety of pathomorphological alterations, including swelling, blebbing, retraction bulb formation, neurofilament loss, and fragmentation, suggesting that increased plasma membrane permeability is detrimental to cell survival and function. We therefore investigated a clinically-relevant treatment strategy designed to restore plasma membrane integrity. Animals were treated with citicoline, a molecule utilized in the endogenous synthesis of phosphatidylcholine (the major membrane component in mammalian cells). Citicoline has been shown to be beneficial in numerous studies of neurological disease, improving overall outcome by increasing phospholipid synthesis and attenuating phospholipid destruction (by reducing phospholipase A2 activity). However, these mechanisms have not been explored in a model of SCI. When compared to injured animals receiving vehicle (saline) injections, citicoline treatment after SCI did not have a statistically significant effect on cytoplasmic PLA2 activity (at 24h post-injury), the density of permeable axons (at 3 days post-injury), or the lesion volume (at 3 days post-injury). Since citicoline may improve neurological outcome after SCI through mechanisms we did not directly assess, we then conducted a longer-term study to evaluate the overall efficacy of citicoline treatment in terms of longer-term functional and histological consequences. Citicoline did not have a biologically significant effect on behavioral recovery (evaluated during open field locomotion, grid walk and hyperalgesia testing weekly for up to 5 weeks post-injury) or lesion volume (at 5 weeks post-injury). The lack of citicoline-mediated effect may be attributed to experimental parameters (e.g., dosing or sensitivity of outcome measures) or biological inefficacy. Although we were not able to demonstrate that citicoline improves outcome after SCI, the finding that plasma membrane damage occurs in a persistent fashion and is associated with pathophysiological cellular alterations may provide fundamental knowledge necessary for developing treatments targeted at membrane repair. Future work examining the complex mechanisms causing prolonged membrane damage after SCI and evaluating strategies for manipulating these pathways (potentially using citicoline in combination with other pharmacological agents) may lead to a clinically effective therapy.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 166-179). Also issued on the Internet.

Thesis (Ph. D.)--University of Lund, 1995. / Published dissertation. Includes bibliographical references.

Thesis (Ph. D.)--University of Lund, 1995. / Published dissertation. Includes bibliographical references.

Les membranes cellulaires sont impliquées dans de nombreux processus cellulaires : la diffusion des médicaments et des ions, la transduction des signaux, la génération d'énergie, le développement cellulaire (fusion et fission). Les bicouches phospholipides sont les principaux composants des membranes cellulaires, elles constituent une barrière dynamique protégeant les réactions biochimiques cellulaires. La détermination des propriétés biochimiques et mécaniques des bicouches lipidiques et leur évolution avec les conditions environnementales est nécessaire pour étudier la nature des processus cellulaires et l'influence des agents externes (résistance mécanique, perméabilité et réponse biologique). Pour mener de telles caractérisations, des modèles simplifiés de membrane biomimétique, tels que des bicouches lipidiques supportées (SLB), ont été développés. Parmi les techniques de caractérisation disponibles, la microscopie à force atomique (AFM) a été largement utilisée pour étudier l'organisation nanométrique des SLB dans des conditions physiologiques. AFM peut produire des images à la haute résolution et peut également être utilisé pour quantifier la résistance mécanique des SLB au moyen d'expériences de perforation. Pendant 30 ans, AFM a traversé de nombreux développements. Très récemment, le Mode circulaire AFM (CM-AFM) a été développé à l'Université de Technologie de Compiègne. CM-AFM est capable de générer un mouvement de glissement de la pointe AFM sur l'échantillon à une vitesse élevée, constante et continue et de mesurer les forces de frottement latéral rapidement et exactement simultanément avec les forces verticales. Pour la première fois, le CM-AFM sert à caractériser les échantillons biologiques dans des conditions physiologiques, ce qui permet de mesurer simultanément les forces de poinçonnage et de frottement en fonction de la vitesse de glissement. Il offre pour la première fois la capacité de décrire le comportement de friction des SLB en complément de la force de perforation. En raison du besoin important de mesure quantitative, l'optimisation du protocole CM-AFM a été effectuée en premier. Le protocole d'étalonnage du scanner a été établi avec succès pour assurer la précision de la vitesse de glissement. En outre, le protocole d'étalonnage des pointes, basé sur la méthode de Wedge et un échantillon rayé, est également conçu pour déterminer la constante d'étalonnage de la force latérale. Nous avons utilisé CM-AFM pour mesurer les propriétés tribologiques des échantillons solides pour améliorer l'équipement sous milieu liquide. Ensuite, les propriétés mécaniques (forces de poinçonnage et de frottement) des SLB ont été mesurées en fonction de la vitesse de glissement. Les SLB purs et mixtes ont été préparés par la méthode de fusion des vésicules. Différents médias ont également été utilisés pour étudier l'effet des cations monovalents sur les propriétés mécaniques des SLB. Dans tous les cas, la force de frottement augmente linéairement avec la vitesse de glissement, ce qui nous permet de déduire le coefficient visqueux de frottement. Comme prévu, la force de poinçonnage et le coefficient visqueux de frottement sont influencés par la composition des mélanges de lipides, par la nature des cations en milieu liquide et par la longueur des chaînes hydrocarbonées mais pas de manière similaire. L'interprétation de l'évolution du coefficient de force de frottement visqueux avec le système étudié est particulièrement délicate car la force de frottement pourrait être influencée par les propriétés d'interface ou de volume. Cette problématique sera le défi pour les prochaines études. Néanmoins, nos résultats illustrent la puissance de la technique CM-AFM et ouvre de nombreuses possibilités pour caractériser d'autres échantillons biologiques (cellules et tissus) afin de mieux comprendre les mécanismes élémentaires de friction. / Cell membranes are involved in many cellular processes: drugs and ions diffusion, signal transduction, energy generation, cell development (fusion and fission). Phospholipid bilayers are the main components of cell membranes, they act as a dynamic barrier protecting cellular biochemical reactions. The determination of biochemical and mechanical properties of lipid bilayers and their evolution with environmental conditions is necessary to study the nature of cellular processes and the influence of external agents (mechanical resistance, permeability, and biological response). To conduct such characterizations, simplified biomimetic membrane models, such as supported lipid bilayers (SLBs), were developed. Among the available characterization techniques, atomic force microscopy (AFM) has been widely used to study the nanoscale organization of SLBs under physiological conditions. AFM can yield high resolution images and it can also be used to quantify the mechanical resistance of SLBs by means of punch through experiments. For 30 years, AFM has been through many developments. Very recently, the Circular Mode AFM (CM-AFM) has been developed at the Université de Technologie de Compiègne. CM-AFM is able to generate a sliding movement of the AFM tip on the sample at high, constant and continuous velocity and to measure the lateral friction forces fast and accurately simultaneously with the vertical forces. For the first time CM-AFM is used to characterize biological samples under physiological conditions, allowing the simultaneous measurement of both the punch-through and the friction forces as a function of the sliding velocity. It offers for the first time the ability to describe the friction behavior of SLBs in complement of the punch-through force. Due to the important need for quantitative measurement, optimization of the CM-AFM protocol has been done first. Protocol of scanner calibration has been successfully established to ensure the accuracy of sliding velocity. Besides, the protocol for tip calibration, based on wedge method and a scratched sample, is also made to determine the lateral force calibration constant. We have employed CM-AFM to measure the tribological properties of solid samples to improve the equipment under liquid medium. Then, the mechanical properties (punchthrough and friction forces) of SLBs were measured as function of the sliding velocity. Pure and mixed SLBs were prepared by the vesicle fusion method. Various media were also used to study the effect of monovalent cations to the mechanical properties of SLBs. In all cases, the friction force increases linearly with the sliding velocity allowing us to deduce the friction viscous coefficient. As expected both the punchthrough force and the friction viscous coefficient are influenced by the composition of lipid mixtures, by the nature of cations in liquid medium, and by the length of hydrocarbon chains but not in a similar fashion. The interpretation of the evolution of the viscous friction force coefficient with the studied system is particularly tricky as the friction force could be influenced by interface or volume properties. This problematic will be the challenge for the next studies. Nevertheless, our results illustrate how powerful the CM-AFM technique is and it opens wide opportunities to characterize other biological samples (cells and tissues) to gain a better understanding of the elementary mechanisms of friction.

La transfection est le processus de transfert de gènes (ADN) dans des cellules. L'utilisation des plasmas froids à la pression atmosphérique est un excellent vecteur pour la transfection de gènes. Cela peut conduire à une perméabilisation temporaire de la membrane cellulaire permettant ainsi le processus de transfection de gènes, dans lequel l'ADN et les cellules sont exposées aux flux des espèces actives du plasma (électrons, ions et radicaux neutres). Cependant beaucoup de questions restent sans réponse notamment sur les mécanismes de transfection par plasma, en particulier de formation de pores et de perméabilisation de la membrane par interactions avec les espèces actives du plasma. Ainsi, nous avons développé un modèle Monte Carlo simulant la formation de pores de quelques nm de largeur sous l'effet d'un microplasma d'air. Ce model nécessite a priori des données d'entrées sur la densité des espèces chargées et la température du gaz et des électrons. C'est pourquoi nous avons aussi effectué une caractérisation expérimentale par spectroscopie d'émission optique OES de la micro décharge couronne. On a estimé les températures rotationnelles de plusieurs espèces variant entre (700K-2350K) même si dans nos conditions de plasma hors équilibre la température du gaz demeure ~300K. Les variations spatiales de la température vibrationnelle Tvib et des électrons Te le long de l'espace inter-électrode (de la pointe vers l'électrode de masse) ont aussi été estimées (Tvib:3000K-6500K et Te:6.75 eV-3.4eV). Les densités des ions et des électrons ont été déterminées et valent environ 1015 cm-3. Par ailleurs, sachant qu'il n'existe dans la littérature aucune modélisation consacrée à la perméabilisation de la membrane et la formation de pore par interactions avec les espèces actives du plasma, nous avons développé pour la première fois dans la littérature un modèle spécifique de simulation Monte Carlo pour la poration. Chaque espèce du plasma (électrons, ions, neutres radicaux) est considérée comme une macro-espèce (ou super-particule) représentant un grand nombre de particules. La proportion des espèces du plasma arrivant sur la membrane est estimée à partir de leurs flux, calculés à l'aide d'un modèle de cinétique réactionnelle et par mesures spectroscopiques. La membrane est supposée comme une simple structure multicouche de phospholipides et protéines. Les interactions avec les couches membranaires sont considérées comme étant des super-processus (recombinaison, réflexion, activation, ouverture). Une probabilité d'occurrence de chacun de ces super-processus est assignée à chaque super-particule sur la base d'une étude paramétrique. Le but est d'évaluer les effets des paramètres de simulation initiaux ainsi que l'effet des probabilités d'occurrence de chaque processus sur la formation de pores. Plusieurs résultats importants ont été obtenus. Les électrons jouent un rôle principal sur l'activation et l'ouverture des sites dus à leur forte anisotropie dans la direction avant. Malgré les faibles énergies, proche de celle du gaz, des ions et des radicaux, leur processus de réflexion est déterminant pour élargir et approfondir les dimensions des pores. Il a été montré que le nombre initial de particules NP est le paramètre qui contrôle le plus efficacement la formation de pores. De plus, nous avons observé une corrélation directe entre NP et la durée d'exposition de la membrane cellulaire au plasma. Dans les conditions actuelles de simulation, on a obtenu une dynamique de formation de pores avec des dimensions (diamètres~10nm) compatibles pour la transfection de gènes. Les résultats de simulation Monte Carlo ont été qualitativement validés par une comparaison préliminaire avec les mesures des taux de transfection d'ADN et de survie de cellules fibroblaste de souries. La méthode de Monte Carlo développée dans ce travail représente un outil très prometteur pour une meilleure compréhension des mécanismes de transfection de gènes par plasma. / Gene transfection is a technique of deliberately introducing DNA into cells through the membrane. The cold atmospheric plasma CAP is potentially a new alternative, safe and damage-free technique. It can lead to a transient permeabilization of the cell membrane allowing processes of gene transfection in which DNA and cells are both exposed to fluxes of active plasma species (electrons, ions, and neutral radicals). The mechanisms of more particularly membrane poration are far to be clear and controlled. Therefore, the aim of this thesis is to numerically study the mechanisms of plasma-induced membrane permeabilization using a specific micro-air plasma. More precisely, is to develop and exploit a specific Monte Carlo poration model. This model is aimed to simulate the pore formation of few nm of width through cell membranes when irradiated by micro-air plasma. This developed model requires a prior input data on the density of charged particles and the temperature of gas and electrons. Thus, an experimental characterisation by OES of the micro-air corona discharge is performed. Rotation temperature was determined (between 700K to 2350K) even though under our non-equilibrium conditions Tg remains ~300K. OES also has given the space variation from the high voltage tip to the grounded plate of vibration temperatures (between 3000K up to about 6500K) and Te (about 6.75 eV down to 3.4 eV near the plate). A magnitude around 1015cm-3 for the electron and ion densities have been also determined. Moreover, knowing that there are no literature simulations devoted to membrane permeabilization and pore formation when impacted by plasma actives species, we developed for the first time in literature a specific Monte Carlo poration model. In this framework, we assumed each plasma species (electrons, ions, and neutral radicals) as a super-particle grouping a large number of particles. The species fluxes were estimated from a plasma reaction kinetic model and OES study. The membrane layers were assumed as a simple membrane model superposing four layers of phospholipids and proteins. Each layer was constituted by a succession of super-sites subjected to specific super-processes (recombination, reflection, activation of a site, opening, etc). For an accurate exploitation of our model, the estimation of the probability of occurrence of the whole considered super-processes is absolutely necessary. Thus, a large parametric study is conducted. The aim is to evaluate the effects of the initial simulation parameters as well as the magnitude of the occurrence probabilities of each reaction process on pore formation.

The biological membrane surrounding the living cell provides a sealed barrier that tightly regulates the interactions with the outside environment. A large number of integral membrane proteins mediate these interactions and are involved in a wide variety of biological processes. An increasing number of studies have led to the conclusion that lipids provide more than a hydrophobic solvent for membrane proteins, and that interactions between lipids and proteins are required to allow protein function. ABC transporters are one of the most important family of membrane proteins. However, the importance of their lipidic environment is largely unknown. Only a few studies showed that their activity was dependent on the lipidic composition of the surrounding bilayer. The bacterial ABC transporter HorA was used as a model to probe the influence of the lipidic environment on that class of membrane proteins.<p><p> HorA is a multidrug transporter expressed in Lactobacillus brevis, a Gram-positive beer spoilage bacterium. It turned out that phosphatidylethanolamine (PE) was indispensable to maintain both the activity and the structural integrity of HorA.<p> Surprisingly, replacement of PE by the chemically related PC (phosphatidylcholine) did not led to the suppression of HorA activity, but to an unexpected phenotype. Whereas the cytoplasmic domains of HorA were still able to hydrolyze ATP, the membrane parts of the transporter were unable to use that energy to mediate substrate transport. Using several biophysical methods particularly adapted to the study of reconstituted systems, we showed that the structure of HorA is strongly altered by this lipid replacement. In particular, the structural organization of the transmembrane domains of the protein is strongly affected.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

No description available.

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.