La membrane plasmique est un assemblage supramoléculaire qui délimite la cellule. C’est une structure fine, complexe et dynamique assurant des fonctions multiples et vitales pour la cellule. Sa rupture est un évènement physiologique pour les cellules soumises à des stress mécaniques fréquents et/ou importants, comme les cellules épithéliales, les cellules endothéliales ou les cellules musculaires. Dans des conditions physiopathologiques, la membrane plasmique peut également être endommagée par l’insertion de toxines bactériennes formant des pores (PFTs, pour « pore forming toxins »). Le processus de réparation membranaire et la machinerie protéique associée sont encore mal connus. Connaître les partenaires protéiques et comprendre les mécanismes mis en jeu durant le processus de réparation de la membrane plasmique sont deux enjeux fondamentaux majeurs. En effet, il a été établi qu’une défaillance du processus de réparation membranaire pour les fibres musculaires est la cause principale de certaines dystrophies musculaires. La machinerie protéique de réparation comprend des protéines comme la dysferline, la cavéoline-3 et certaines Annexines (Anx). Les Anx appartiennent à une superfamille de protéines répandue chez la plupart des eucaryotes, qui ont la propriété commune de se lier aux membranes biologiques en présence de calcium (Ca2+). Certaines Anx, comme l’AnxA5, une fois liées aux membranes biologiques s’auto-assemblent spontanément en réseau-2D. Lors de ce travail de thèse, nous avons étudié le rôle de l’AnxA5 dans la réparation membranaire des trophoblastes placentaires et des cellules du muscle squelettique humain. Pour les deux types cellulaires, nous avons montré que l’AnxA5 est un acteur indispensable du processus de réparation membranaire dans le cas de ruptures mécaniques. En associant des approches de microscopie de fluorescence et de microscopie électronique à transmission (MET), nous avons mis en évidence que dans ces cellules, le mécanisme de réparation est principalement basé sur la formation d’un « patch » lipidique. Dans les cellules musculaires, les expériences de MET ont mis en évidence qu’un pool d’AnxA5 endogène se lie aux bords du site de rupture quelques secondes après la lésion du sarcolemme. Ceci suggère qu’après rupture de la membrane plasmique, l’augmentation locale de la concentration calcique intracellulaire provoque la liaison de l’AnxA5 spécifiquement aux bords de la région membranaire lésée où elle forme un réseau-2D. Le réseau-2D stabiliserait localement la membrane et préviendrait sa déchirure, induite par les forces de tensions exercées par le cytosquelette cortical. Nous avons également montré que l’AnxA5 ne semble pas impliquée dans la réparation de la membrane plasmique après insertion de PFTs. Ceci suggère que différents mécanismes de réparation existent et que leur mise en place dépend probablement du type ou de l’importance des dommages. Finalement nous avons étendu notre étude à des lignées cellulaires établies à partir de patients diagnostiqués comme souffrant de dystrophies des ceintures de type 2B (déficience en dysferline) et 1C (déficience en cavéoline-3), respectivement. Nous avons montré, pour ces lignées, que la déficience en dysferline ou cavéoline-3 provoque un défaut de réparation dans le cas des ruptures mécaniques de la membrane plasmique. Dans ces cellules musculaires pathologiques intactes ou endommagées, l’AnxA5 a le même comportement, ce qui suggère que l’action de l’AnxA5 est indépendante de ces protéines. A la différence des cellules déficientes en dysferline, nous avons observé que les cellules déficientes en cavéoline-3 sont capables de réparer efficacement des lésions créées par l’insertion de PFTs dans le sarcolemme. Ce résultat supporte l’hypothèse de l’existence de plusieurs mécanismes de réparation. En conclusion, ce travail montre que l’AnxA5 est un composant clé de la machinerie de réparation dans le cas des ruptures mécaniques. / Plasma membrane is the supramolecular assembly that delimits the cell. It is a thin, dynamic and complex structure, ensuring multiple and vital cell functions. Its disruption is a physiological event occurring in cells submitted to frequent mechanical stresses, such as endothelial cells, epithelial cells and muscle cells. It is also a physiological event for cells exposed to pore forming bacterial toxins (PFTs). Membrane repair mechanisms and associated protein machinery are still poorly understood. This knowledge is, however, essential for obvious physiopathological issues. Indeed, a defect of membrane repair in muscle cells leads to some muscular dystrophies. Membrane repair machinery includes proteins such as dysferlin, MG-53, caveolin-3 and some Annexins (Anx). Anx belong to a superfamily of proteins widely spread in most of eukaryotes, which share the property of binding to biological membranes in the presence of calcium (Ca2+). Here, we investigated the role of AnxA5 in cell membrane repair of human trophoblastic and skeletal muscle cells. We showed that AnxA5 is required for membrane repair of mechanical damages in the two cell types. By combining fluorescence and transmission electron microscopy approaches, we evidenced that membrane repair mechanism in these cells is based on the formation of a lipid “patch”. In human muscle cells, TEM experiments revealed that a pool of endogenous AnxA5 binds to the edges of the torn sarcolemma as soon as a few seconds after membrane disruption. Our results suggest the following mechanism: triggered by the local increase in Ca2+ concentration, AnxA5 molecules bind to PS exposed at the edges of the torn membrane, where they self-assemble into 2D arrays. The formation of 2D arrays strengthens the damaged sarcolemma, counteracts the tensions exerted by the cortical cytoskeleton and thus prevents the expansion of the tear. We showed also that a pool of endogenous AnxA5 binds to intracellular vesicles that obstruct the wounding site. It is likely these vesicles, once associated one to each other, ensure membrane resealing. Our results suggest that sarcolemma repair of damages caused by PFTs is independent of AnxA5. Therefore, different membrane repair mechanisms may exist, their occurrence probably depending on the type and/or the size of damages. Finally, we performed studies on muscle cells established from patients diagnosed with limb girdle muscular dystrophies type 2B (dysferlin-deficient) and 1C (caveolin-3-deficient), respectively. We found that dysferlin or caveolin-3 deficiency leads to a defect of membrane repair, in the case of mechanical damages. AnxA5 behaved similarly in these damaged cells and wild-type cells, suggesting that its function is independent of dysferlin or caveolin-3. Unlike dysferlin-deficient cells, damages created by PFTs are efficiently repaired in caveolin- 3-deficient cells. This result supports the hypothesis that different mechanisms occur in muscle cells, depending on the type of damage. In conclusion, this work indicates that AnxA5 is a key component of the membrane repair machinery, in the case of mechanical disruptions. Our results enable to propose a detailed mode of action for AnxA5.

No description available.

Hydrogen and oxygen or air polymer electrolyte membrane fuel cell is one of the most promising electrical energy conversion devices for a sustainable future due to its high efficiency and zero emission. Membrane electrode assembly (MEA), in which electrochemical reactions occur, is stated to be the heart of the fuel cell. The aim of this study was to develop methods for preparation of MEA with alternative polymer electrolyte membranes and compare their performances with the conventional Nafion&reg / membrane. The alternative membranes were sulphonated polyether-etherketone (SPEEK), composite, blend with sulphonated polyethersulphone (SPES), and polybenzimidazole (PBI). Several powder type MEA preparation techniques were employed by using Nafion&reg / membrane. These were GDL Spraying, Membrane Spraying, and Decal methods. GDL Spraying and Decal were determined as the most efficient and proper MEA preparation methods. These methods were tried to improve further by changing catalyst loading, introducing pore forming agents, and treating membrane and GDL. The highest performance, which was 0.53 W/cm2, for Nafion&reg / membrane was obtained at 70 0C cell temperature. In comparison, it was about 0.68 W/cm2 for a commercial MEA at the same temperature. MEA prepared with SPEEK membrane resulted in lower performance. Moreover, it was found that SPEEK membrane was not suitable for high temperature operation. It was stable up to 80 0C under the cell operating conditions. However, with the blend of 10 wt% SPES to SPEEK, the operating temperature was raised up to 90 0C without any membrane deformation. The highest power outputs were 0.29 W/cm2 (at 70 0C) and 0.27 W/cm2 (at 80 0C) for SPEEK and SPEEK-PES blend membrane based MEAs. The highest temperature, which was 150 0C, was attained with PBI based MEA during fuel cell tests.

No description available.

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.

The thesis research was designed to study the characteristics of two important physiological processes in smooth muscle: Ca2+ transport mediated by the plasmalemmal Ca2+-ATPase and muscarinic receptor-G protein interactions. In resting smooth muscle, several Ca2+ extrusion or sequestration processes offset the passive inward leak of Ca2+. Although biochemical evidence suggests that the plasmalemmal Ca2+ pump plays a key role in this process, the precise role of this enzyme could not be proven until a reliable estimate of the inward Ca2+ leak was measured. Recent studies using dispersed smooth muscle cells from the toad stomach provided an estimate of the basal transmembrane Ca2+ flux rate; thus, we examined the transport capacity of the plasmalemmal Ca2+pump in this tissue. Gastric smooth muscle tissue was disrupted by homogenization and nitrogen cavitation. Membranes enriched 20 fold for plasma membrane markers were obtained using differential centrifugation and purification by flotation on discontinuous sucrose gradients. The membrane vesicles exhibited an ATP-dependent 45Ca uptake that was insensitive to azide or oxalate but sensitive to stimulation by calmodulin or inhibition by orthovanadate and the calmodulin antagonists trifluoperazine (TFP) or calmidazolium (CMZ). 45Ca accumulated in the presence of ATP was rapidly released by Ca2+ ionophore but not by agents that stimulate Ca2+ release from the sarcoplasmic rettculum (caffeine, inositol trisphosphate, GTP). However, both CMZ and TFP evoked a Ca2+ release that was comparable to that observed in the presence of Ca2+ ionophore, suggesting that these compounds have profound effects on membrane Ca2+permeability. 45Ca transport exhibited a high affinity for Ca2+ (KD 0.2 μM) and a high transport capacity, producing a > 12,000-fold gradient for Ca2+and a transmembrane flux rate at least 3-fold greater than that observed in resting smooth muscle cells. As a first step toward understanding the biochemical basis for the diversity of muscarinic cholinergic actions on smooth muscle, we examined the distribution of muscarinic receptor subtypes and coupling to guantne nucleotide-binding (G) proteins in airway and gastric smooth muscle. Receptor subtypes were classified in membranes prepared from bovine trachea and toad stomach based on the relative abilities of the selective antagonists pirenzepine (M1), AF-DX 116 (M2) and 4-DAMP (M3) to displace the binding of nonselective antagonist [3H]QNB (quinuclidinyl benzilate). Based on the binding profiles for these antagonists, it was concluded that both smooth muscle types contain a mixture of M2 and M3 subtypes. In trachea the majority of receptors (86%) were M2, whereas in stomach the majority of receptors (88%) were M3. The displacement of [3H]QNB binding by the agonist oxotremorine indicated a mixed population of high affinity (KD = 4 nM) and low affinity (KD = 2-4 μM) binding sites. The addition of GTPγS abolished all high affinity agonist binding, suggesting that coupling of the receptors to G proteins may confer high affinity. Reaction of membranes with pertussis toxin in the presence of [32P]NAD caused the [32P]-labelling of a ~ 41 kD protein in both gastric and tracheal smooth musc1e. Pretreatment of the membranes with pertussis toxin and NAD completely abolished high affinity agonist binding in gastric smooth muscle, but produced little if any decrease in high affinity agonist binding in trachea. We conclude that, although muscarinic receptor activation leads to the elevation of intracellular Ca2+ and to contraction of both airway and gastric smooth muscle, the dissimilar distributions of receptor subtypes and distinct patterns of coupling to G proteins may indicate that each smooth muscle type uses different receptor-G protein interactions to regulate intracellular signalling pathways.

In response to external stimuli, many intracellular signaling proteins undergo dynamic changes in localization to the plasma membrane. Using the Saccharomyces cerevisiaemating pathway as a model, I investigated the molecular interactions that govern plasma membrane localization of signaling proteins, and how the plasma membrane compartmentalization of a signaling complex influences the overall signaling behavior of the pathway. Signaling proteins often consist of multiple interaction domains that collectively dictate their localization and function. Ste20 is a p21-activated kinase (PAK) that functions downstream of the Rho-type GTPase Cdc42 to activate several mitogen-activated protein (MAP) kinase pathways in budding yeast, including the mating pathway. I identified a short domain in Ste20 that directly binds to membrane lipids via electrostatic interaction. A mutation in this domain abolishes both the localization and function of Ste20. Thus, the previously known Cdc42 binding is necessary but not sufficient; instead, direct membrane binding by Ste20 is also critical. By replacing this domain with heterologous membranebinding domains, I demonstrated that phospholipid specificity is not essential in vivo. Functionally important short membrane-binding domains were also found in the Cdc42 effectors Gic1 and Gic2, indicating that generic membrane binding can work in concert with the CRIB domain to regulate activation of Cdc42 targets. These results underscore the importance of cooperation between protein-protein and protein-membrane interaction in achieving proper localization of signaling proteins at the cell cortex. At the system level, MAP kinase cascades can be graded or switch-like. The budding yeast mating pathway exhibits a graded response to increasing levels of pheromone. Previously the scaffold protein Ste5 was hypothesized to contribute to this graded response. To test this idea, I activated the pathway in a variety of ways and measured the response at the single cell level. I found that the graded response is not perturbed by the deletion of negative regulators of the pathway whereas the response became switch-like when the pathway was activated by a crosstalk stimulus that bypasses the upstream components. Interestingly, activation of the pathway in the cytoplasm using the graded expression of MAPKKK resulted in an ultrasensitive response. In contrast, activation of the pathway at the plasma membrane using the graded expression of membranetargeted active pathway components remained graded. In these settings, the scaffold protein Ste5 increased ultrasensitivity when limited to the cytosol; however, if Ste5 was allowed to function at the plasma membrane, signaling was graded. The results suggest that, in the mating pathway, the inherently ultrasensitive MAPK cascade is converted to a graded system by the scaffoldmediated assembly of signaling complexes at the plasma membrane. Therefore, the plasma membrane localization of Ste5 helps shape the input-output properties of the mating MAPK pathway in a manner that is suitable for the biology of mating. Taken together, this thesis underscores the importance of plasma membrane localization during mating pathway signaling in yeast. The examples described here provide further appreciation of how multiple interaction domains can function together to achieve specific targeting of the signaling proteins, as well as advances in understanding the role of scaffold proteins in modulating signaling behavior to promote graded signaling at the plasma membrane.

Le travail reporté dans ce manuscrit concerne l’élaboration de matériaux conducteurs protoniques destinés à une application en tant que membrane de PEMFC. L’approche considérée, relativement récente dans ce domaine, consiste à élaborer des réseaux seminterpénétrés(semi-IPN). Pour ce faire, un polymère linéaire conducteur protonique (unPEEK sulfoné) a été associé à un réseau réticulé fluoré de type poly(aryl étherperfluorocyclobutane) (PFCB). Ces macromolécules sont obtenues par cyclodimérisationthermique de monomères bis et tris-trifluorovinyléther (TFVE). Différentes séries de semi-IPN ont été élaborées en faisant varier la nature du PFCB, le taux de réticulation, le procédéde mise en oeuvre et le taux de réseau incorporé au sPEEK. L’ensemble des résultats montrequ’il existe une composition pour laquelle les propriétés de conductivité, de gonflement et detenue mécanique sont optimales. Une membrane ayant une conductivité de 155 mS.cm-1 et ungonflement à l’eau deux fois inférieur à celui du sPEEK (dont la conductivité s’élève à127 mS.cm-1) a été obtenue grâce à un ajout de 10 % en masse en réseau fluoré. Par ailleurs,l’incorporation dans le réseau PFCB de monomères contenant des fonctions sulfoniques a étéenvisagée. Un travail important de chimie organique a permis d’obtenir des molécules bis-TFVE comportant des fonctions sulfonées protégées sous forme d’ester sulfonique. Lapolycondensation directe de ces molécules a permis la synthèse de polymères PFCB linéairesconducteurs protoniques d’une CEI préalablement déterminée. / This thesis deals with the conception of proton conducting materials used as PEMFCmembrane. The proposed approach is quite new in this application field and is based on thedevelopment of semi-interpenetrating networks (semi-IPN). A linear conducting polymer(sulfonated PEEK) was combined with a crosslinked fluorinated network, a poly(aryl etherperfluorocyclobutane) (PFCB). These macromolecules are obtained by thermalcyclodimerization of bis and tris trifluorovinylether monomers (TFVE). Different series ofsemi-IPN were prepared by changing the PFCB nature, the crosslinking degree, the synthesisprocess and the proportion of the network added to the sPEEK. The overall results show aspecific semi-IPN composition for which the conductivity, the swelling and mechanicalstrength properties are optimal. A membrane with a proton conductivity of 155 mS.cm-1 and alimited water swelling (50 % lower than for a sPEEK membrane which exhibits a protonconductivity of 127 mS.cm-1) is obtained by adding 10 wt-% of fluorinated network. Inaddition, the incorporation of sulfonated TFVE monomers into the network PFCB has beenconsidered. A significant effort in organic chemistry enabled the synthesis of bis-TFVEmolecules containing protected sulfonated functions in a sulfonate ester form. Linearconducting PFCB polymers with a predeterminated IEC were obtained by directcopolycondensation of these monomers.