Dissertations / Theses on the topic 'Membrane contact site'

La communication intercellulaire est essentielle pour le développement et la survie d'organismes multicellulaires. Dans le règne végétale, une des voies privilégiée pour la communication intercellulaire est la voie symplastique qui implique des canaux aux dimensions nanométriques connectant les cellules entre elles, leur permettant d'échanger directement photo-assimilats, miARN, protéines, oligoéléments etc. Observés pour la première fois en 1880 par le botaniste autrichien Eduard Tangl (Tangl 1880; Kohler & Carr 2006), ils ont longtemps été considérés comme de simples trous perméables permettant la diffusion de matériel cellulaire (Lee & Lu 2011; Oparka & Roberts 2001). Etant donné leurs taille nanoscopique, ce n'est que dans les années 1960, avec la démocratisation de la Microscopie Électronique en Transmission (MET) qui permet d'atteindre , que les premiers modèles ultrastructuraux sont établis (Lopez-Saez 1965; Robards 1970). Ils font état d'un canal d'environ 30 à 40 nm de diamètre avec un élément central cylindrique traversant le pore, appelé le desmotubule, connecté au Réticulum Endoplasmique des deux cellules (Figure 1 of our review Tilsner et al. 2016). Dans les années 1980 notre compréhension des plasmodesmes a quelque peu évolué et nous savons maintenant que ces structures ne sont pas de simples trous mais des structures membranaires très spécialisées et régulées (Lucas & Lee 2004; Faulkner & Maule 2011; Furuta et al. 2012). Le modèle ultrastructural actuel découle de la congrégation d'études ultrastructurale, physiologiques et pharmacologiques plus ou moins anciennes dépeignant une structure morphologiquement très souple et changeant de conformation au cours du développement. Les plasmodesmes peuvent réguler leur ouverture/fermeture par la constriction de leurs extrémités grâce à l'accumulation entre la membrane plasmique et la paroi végétale d'un polymère de sucre, la callose qui va pousser la membrane plasmique contre le desmotubule et en obstruer les entrées. Cette modulation permettrait majoritairement de réguler les flux intercellulaires qui impliquent les plasmodesmes. Cependant nos connaissances sur les remaniements membranaires prenant place durant le développement des plasmodesmes et sur la régulation de leur perméabilité sont encore imparfaites.La microscopie électronique en transmission, malgré l'ancienneté de la technique, est l'une des plus résolutive, largement utilisée en biologie. Avec l'amélioration des techniques de préservation d'échantillons, notamment les cryo- méthodes, elle permet d'atteindre à l'heure actuelle des résolutions inférieures à 5 nm en condition contrastée et inclus en résine et peut descendre en dessous du nanomètre pour la cryo-microscopie. Ce potentiel permet aisément l'étude des sous-compartiments cellulaires de l'ordre du µm tel que mitochondries, chloroplastes, noyaux etc. (Frey et al. 2002) mais permet également l'étude ultrastructurale précise de structures de l'ordre de la dizaine de nm (Beck et al. 2007; Al-Amoudi et al. 2007).En revanche, dans son utilisation classique, la microscopie électronique ne permet pas d'accéder à la troisième dimension de l'espace, rendant difficile l'interprétation de structure à l'architecture quelque peu compliquée. En effet, les images produites ne sont que des projections en deux dimensions d'objets en trois dimensions. Cela a mené au développement de la tomographie électronique en transmission (Crowther et al. 1970), méthode basée sur un concept mathématiques formulé par Johann Radon au XIXe siècle. Ce n'est que dans les années 2000 que la tomographie électronique a pris un essor significatif grâce au couplage avec des méthodes d'automatisation informatiques
Plasmodesmata were first observed by Austrian botanist Eduard Tangl in 1880. He devoted himself to studying the anatomy and cytology of plants and his greatest discovery, of course, was the observation and first characterization of plasmodesmata (Tangl 1880, 1884 and 1885). Despite not having access to their ultrastructure, he observed thin striations (see front page engraving) between cotyledon cells of Strychnos nuxvomica and in the endosperm of seeds and described them as being conductive ducts. Already at the time, he was evoking the idea that these strands "unite them [the cells] to an entity of higher order", in other words formulating the first definition of a symplastic domain. lt is only in 1901 that Strasburger finally names these canals "plasmodesmata". His discovery led to a radical change in our conception of the plant entity and brought in new concepts such as the symplasm (Munch 1930) and transmembrane fluxes between cells, which are now being tackled with great interest by numerous research teams around the globe.Because of their size, plasmodesmata ultrastructure was not accessible until the advent of electron microscopy and they were long thought to be simple holes connecting plant cells one-another with no specific regulation. lt is only with the advent of electron microscopy and chemical fixation that botanists started to gain interest in this structure again. And even with these methods allowing the observation of structures down to several nanometers in size, there are still debates on the nature of the canal, its constituents and physiology (Lopez-Saez J. 1965, Robards A. 1970, Ding et al. 1992, Tilney et al. 1991, Overall and Gunning 1982, Schulz et al. 1995).Nowadays, with the advent of modern cryopreservation and three-dimensional electron tomography methods, great improvements are to be done in the understanding of the ultrastructure and physiology of these mysterious canals. More particularly by understanding the link between the membranous rearrangements taking place in these pores and the molecular transit regulation.My work has led us to view plasmodesmata as specialised Membrane Contact Sites (MCS). Hence, by analogy with MCS found in mammals, yeast and plants, this work embraces an original angle on the speculation of the composition and role of the desmotubule-plasma-membrane tethering complex. The work produced during my thesis allowed me to contribute to the publication of one review and two articles, which will constitute the introduction and two main sub-sections of the results chapter, respectively. The introductory review has been published in 2016 in Annual Review of Plant Biology. The first one is still under reviewing at Nature Plant and the other has been published in The Plant Cell journal in April 2015

La protéine OSBP est un transporteur de lipides qui régule la distribution cellulaire du cholestérol. OSBP comprend un domaine PH, deux séquences « coiled coil », un motif FFAT (deux phénylalanines dans un environement acide), et un domaine de liaison de lipides (ORD) à son extrémité C-terminale. Le domaine PH interagit avec le PI(4)P et la petite protéine G Arf1-GTP au niveau du Golgi, alors que le motif FFAT interagit avec la protéine VAP-A, résidente du réticulum endoplasmique (RE). En liant simultanément tous ces déterminants, OSBP stabilise des sites de contact membranaire entre RE et Golgi, permettant ainsi un contre-échange cholestérol / PI(4)P par l'ORD. OSBP contient également une longue séquence N-terminale d’environ 80 aa, intrinsèquement désordonnée, composée principalement de glycine, proline et d'alanine. Nous démontrons que la présence de ce N-terminus désordonné augmente le rayon de Stoke de OSBP tronquée du domaine ORD, et limite sa densité d’association sur la membrane portant le PI(4)P. La protéine dépourvue du N terminus favorise l'agrégation symétrique des liposomes PI(4)P (mimant la membrane du Golgi) par les deux domaines PH du dimère OSBP, alors que la présence de la séquence désordonnée empêche cette association symétrique. De même, nous observons que la distribution d’OSBP sur la membrane de vésicules unilamellaires géantes (GUV) varie selon la présence ou l'absence du N-terminus. En présence de la séquence désordonnée, la protéine est répartie de manière homogène sur toute la surface du GUV, alors que la protéine sans N-terminal a tendance à s'accumuler à l'interface entre deux GUV de type Golgi. Cette accumulation locale ralentit fortement la mobilité de la protéine à l’interface. Un effet similaire du N-terminal sur la dynamique des protéines est observé lorsque l’association de membranes de type ER et Golgi est assuré par des protéines monomériques (dépourvue du coiled coil) en présence de Vap-A. Les résultats de nos expériences in vitro ont été confirmés en cellules vivantes, où la séquence intrinsèquement désordonnée contrôle le recrutement d’OSBP sur les membranes Golgiennes, sa mobilité et sa dynamique d’activité au cours des cycles de transfert de lipides. La plupart des protéines de la famille d’OSBP contiennent des séquences N-terminales de faible complexité, suggérant un mécanisme général de régulation
Oxysterol binding protein (OSBP) is a lipid transfer protein that regulates cholesterol distribution in cell membranes. OSBP consists of a pleckstrin homology (PH) domain, two coiled-coils, a “two phenylalanines in acidic tract” (FFAT) motif and a C-terminal lipid binding OSBP-Related Domain (ORD). The PH domain recognizes PI(4)P and small G protein Arf1-GTP at the Golgi, whereas the FFAT motif interacts with the ER-resident protein VAP-A. By binding all these determinants simultaneously, OSBP creates membrane contact sites between ER and Golgi, allowing the counter-transport of cholesterol and PI(4)P by the ORD. OSBP also contains an intrinsically disordered ~80 aa long N-terminal sequence, composed mostly of glycine, proline and alanine. We demonstrate that the presence of disordered N-terminus increases the Stoke’s radius of OSBP truncated proteins and limits their density and saturation level on PI(4)P-containing membrane. The N-terminus also prevents the two PH domains of OSBP dimer to symmetrically tether two PI(4)P-containing (Golgi-like) liposomes, whereas protein lacking the disordered sequence promotes symmetrical liposome aggregation. Similarly, we observe a difference in OSBP membrane distribution on tethered giant unilamellar vesicles (GUVs), based on the presence/absence of N-terminus. Protein with disordered sequence is homogeneously distributed all over the GUV surface, whereas protein without N-terminus tends to accumulate at the interface between two PI(4)P-containing GUVs. This protein accumulation leads to local overcrowding, which is reflected by slow in-plane diffusion. The effect of N-terminus is also manifested in monomeric OSBPderived proteins that tether ER-like and Golgi-like membranes in the presence of VAP-A. Findings from our in vitro experiments are confirmed in living cells, where N-terminus controls the recruitment of OSBP on Golgi membranes, its motility and the on-and-off dynamics during lipid transfer cycles. Most OSBP-related proteins contain low complexity N-terminal sequences, suggesting a general effect

La protéine OSBP est un transporteur de lipides qui régule la distribution cellulaire du cholestérol. OSBP comprend un domaine PH, deux séquences « coiled coil », un motif FFAT (deux phénylalanines dans un environement acide), et un domaine de liaison de lipides (ORD) à son extrémité C-terminale. Le domaine PH interagit avec le PI(4)P et la petite protéine G Arf1-GTP au niveau du Golgi, alors que le motif FFAT interagit avec la protéine VAP-A, résidente du réticulum endoplasmique (RE). En liant simultanément tous ces déterminants, OSBP stabilise des sites de contact membranaire entre RE et Golgi, permettant ainsi un contre-échange cholestérol / PI(4)P par l'ORD. OSBP contient également une longue séquence N-terminale d’environ 80 aa, intrinsèquement désordonnée, composée principalement de glycine, proline et d'alanine. Nous démontrons que la présence de ce N-terminus désordonné augmente le rayon de Stoke de OSBP tronquée du domaine ORD, et limite sa densité d’association sur la membrane portant le PI(4)P. La protéine dépourvue du N terminus favorise l'agrégation symétrique des liposomes PI(4)P (mimant la membrane du Golgi) par les deux domaines PH du dimère OSBP, alors que la présence de la séquence désordonnée empêche cette association symétrique. De même, nous observons que la distribution d’OSBP sur la membrane de vésicules unilamellaires géantes (GUV) varie selon la présence ou l'absence du N-terminus. En présence de la séquence désordonnée, la protéine est répartie de manière homogène sur toute la surface du GUV, alors que la protéine sans N-terminal a tendance à s'accumuler à l'interface entre deux GUV de type Golgi. Cette accumulation locale ralentit fortement la mobilité de la protéine à l’interface. Un effet similaire du N-terminal sur la dynamique des protéines est observé lorsque l’association de membranes de type ER et Golgi est assuré par des protéines monomériques (dépourvue du coiled coil) en présence de Vap-A. Les résultats de nos expériences in vitro ont été confirmés en cellules vivantes, où la séquence intrinsèquement désordonnée contrôle le recrutement d’OSBP sur les membranes Golgiennes, sa mobilité et sa dynamique d’activité au cours des cycles de transfert de lipides. La plupart des protéines de la famille d’OSBP contiennent des séquences N-terminales de faible complexité, suggérant un mécanisme général de régulation
Oxysterol binding protein (OSBP) is a lipid transfer protein that regulates cholesterol distribution in cell membranes. OSBP consists of a pleckstrin homology (PH) domain, two coiled-coils, a “two phenylalanines in acidic tract” (FFAT) motif and a C-terminal lipid binding OSBP-Related Domain (ORD). The PH domain recognizes PI(4)P and small G protein Arf1-GTP at the Golgi, whereas the FFAT motif interacts with the ER-resident protein VAP-A. By binding all these determinants simultaneously, OSBP creates membrane contact sites between ER and Golgi, allowing the counter-transport of cholesterol and PI(4)P by the ORD. OSBP also contains an intrinsically disordered ~80 aa long N-terminal sequence, composed mostly of glycine, proline and alanine. We demonstrate that the presence of disordered N-terminus increases the Stoke’s radius of OSBP truncated proteins and limits their density and saturation level on PI(4)P-containing membrane. The N-terminus also prevents the two PH domains of OSBP dimer to symmetrically tether two PI(4)P-containing (Golgi-like) liposomes, whereas protein lacking the disordered sequence promotes symmetrical liposome aggregation. Similarly, we observe a difference in OSBP membrane distribution on tethered giant unilamellar vesicles (GUVs), based on the presence/absence of N-terminus. Protein with disordered sequence is homogeneously distributed all over the GUV surface, whereas protein without N-terminus tends to accumulate at the interface between two PI(4)P-containing GUVs. This protein accumulation leads to local overcrowding, which is reflected by slow in-plane diffusion. The effect of N-terminus is also manifested in monomeric OSBPderived proteins that tether ER-like and Golgi-like membranes in the presence of VAP-A. Findings from our in vitro experiments are confirmed in living cells, where N-terminus controls the recruitment of OSBP on Golgi membranes, its motility and the on-and-off dynamics during lipid transfer cycles. Most OSBP-related proteins contain low complexity N-terminal sequences, suggesting a general effect

La multicellularité chez les plantes repose sur une communication intercellulaire qui permette le transfert d'informations à travers l'entièreté de l'organisme. Chez les plantes terrestres, la route principale de ces “conversations cellulaires” est assurée par les plasmodesmes (PD), des canaux nanoscopiques qui traversent la paroi pecto-cellulosique. En effet, ces pores sont impliqués dans la circulation d'une très grande variété de molécules, comme des facteurs de transcription, de l'ARN, des hormones et des métabolites et ceci à tous les stades de la vie végétale, permettant réponses et adaptations à l'environnement. Les PD sont particuliers dans le sens où ils forment une continuité du réticulum endoplasmic (RE), de la membrane plasmique (MP) et du cytoplasme entre les cellules adjacentes. Leur architecture est singulière et consiste en un filament de RE, appelé desmotubule, entouré d'un tube de MP qui, lui, longe la paroi. Les PD sont actuellement décrits comme des sites de contact membranaire, du fait du fort accolement des membranes du RE et de la MP (2 à 10 nm) et de la présence de protéines de jonction qui connectent les deux organelles. Dans la présente étude, nous décrivons au niveau structural et fonctionnel plusieurs membres de la famille des MCTPs (protéine avec de multiples domaines C2 et une région transmembranaire) comme protéines assurant la jonction du RE et de la MP dans les PD. Nous démontrons que ces protéines possèdent les caractéristiques moléculaires nécessaires à l'interaction transitoire avec les lipides anioniques de la MP, via leurs domaines C2, ainsi qu'à l'induction de courbure membranaire au RE, via la région transmembranaire qui agit comme un domaine homologue aux protéines réticulons. Ces données nous ont permis de corréler la fonction des MCTPs à l'architecture et la biogenèse des PD et de réfléchir au rôle du RE à l'intérieur des PD. En conclusion, ce travail a fourni des résultats originaux qui placent les MCTPs comme des protéines centrales dans l'établissement de la structure fine des PD et des fonctions qui y sont associées
Plant multicellularity relies on intercellular communication in order to transmit information from cell to cell and throughout the entire plant body. In land plants, the major line for such cellular conversations is through plasmodesmata (PD) pores, which are nanoscopic membranous tunnels spanning the pecto-cellulosic cell wall. These pores are indeed involved in the transfer of a wide variety of molecules such as transcription factors, RNAs, hormones and metabolites during all stages of plant life, adaptation and responses to their environment. PD are singular amongst other types of intercellular junctions as they provide a direct continuity of the endoplasmic reticulum (ER), the plasma membrane (PM) and the cytosol between neighboring cells. Their architectural organization can be summarized as followed: a thin strand of constricted ER, called desmotubule, is encased in a tube of PM lining the cell wall. PD are seen as a specialized ER-PM membrane contact sites from the very close apposition (2 to 10 nm) of the ER and PM membranes and the presence of tethering elements bridging the two organelles. In this study, we describe the structural organization and function of several members of the MCTP (Multiple C2 domains and Transmembrane region Protein) family which act as ER-PM tethering elements at PD. We show that these proteins possess molecular features capable of transient interaction with anionic lipids of the PM, through their C2 domains, as well as ER membrane shaping, through their transmembrane region which presents homology to a reticulon domain. We further correlate MCTP function with PD architecture and biogenesis, and investigate on the role of the ER inside PD. Altogether, this work provides original data placing MCTPs as core PD proteins that appear to be crucial in the establishment of PD ultrastructure and associated functions

VAP-A est un récepteur ancré à la surface du réticulum endoplasmique (RE) pour des centaines de protéines contenant un motif FFAT. Ses partenaires possèdent des structures et des fonctions très variées. VAP-A participe à la formation des sites de contact membranaire (MCSs) entre le RE et les autres organelles et ceci permet notamment le trafic non vésiculaire des lipides entre les membranes. Par exemple, la protéine de transfert de lipide OSBP interagit avec VAP aux MCSs RE/Golgi afin de transporter le cholestérol contre son gradient de concentration par contre-échange et hydrolyse de phosphatidylinositol-4-phoshate (PI4P). L'interaction entre le domaine Major-Sperm-Protein (MSP) de VAP-A et le motif FFAT de ses partenaires était déjà caractérisé. Cependant, la façon dont ce récepteur universel peut s'adapter à toutes ses cibles dans tous les MCSs, qui sont très différents en terme de géométrie et de stabilité était inconnue.Dans cette étude nous avons utilisé une approche pluridisciplinaire afin de démontrer que VAP-A contient deux régions intrinsèquement désordonnées (IDRs) qui fournissent à la protéine une flexibilité indispensable à son organisation fonctionnelle dans les MCSs. Nous avons montré qu'un mutant de VAP-A sans ses linkers flexibles possède une localisation subcellulaire restreinte aux MCSs RE/mitochondrie. Ce mutant ne peut donc pas soutenir l'activité d'OSBP et CERT aux MCSs RE/Golgi. En revanche, il interagit avec VPS13A et PTPIP51 à la mitochondrie et permet ainsi le transport de lipides qui contribuent au métabolisme des cardiolipines et à la fusion mitochondriale.Ces résultats indiquent que la flexibilité de VAP-A fournie par ses IDRs, joue un rôle clé pour assurer son adaptabilité à différents contextes et plus précisément aux MCSs à durée de vie courte comme les MCSs RE/Golgi, cette étude démontre également l'implication de VAP-A dans la fusion mitochondriale
VAP-A is a receptor at the surface of the endoplasmic reticulum (ER) for hundreds of proteins containing a FFAT motif and having a wide range of structures and functions. VAP-A is also required for creating multiple membrane contact sites (MCSs) between the ER and other compartments, which notably enable non-vesicular lipid exchanges between membranes. For example, the lipid-transfer protein (LTP) OSBP interacts with VAP at ER/Golgi MCS to transport cholesterol through coupled counter-exchange and hydrolysis of PI4P. It is well known that VAP-A partners contain a FFAT motif specifically recognized by the Major-Sperm-Protein (MSP) domain of VAP, however, how this receptor adapts to its different targets in MCSs that are so different in geometry and lifetime is not understood.In this study, we used a multidisciplinary approach to demonstrate that VAP-A contains two intrinsically disordered linkers that provide it with a high degree of flexibility to enable functional organization of different MCSs. A VAP-A mutant without flexible linkers is restricted in its subcellular localization, and does not support lipid transport by OSBP and CERT at ER/Golgi MCS. However, this mutant is present at ER/mitochondria MCS by interacting with VPS13A and PTPIP51, and thus facilitates lipid transport contributing to cardiolipin metabolism and mitochondrial fusion.In conclusion, this work indicates that VAP-A conformational flexibility mediated by its intrinsically disordered regions is key to ensure membrane tethering especially at short-lived MCSs; it also demonstrates the implication of VAP-A in mitochondrial fusion

Les sites de contact membranaire (SCM) sont des régions subcellulaires où deux organites sont physiquement connectés. Ces micro-domaines, moléculairement définis par des interactions protéine-protéine et/ou protéine-membrane, sont impliqués dans la dynamique des organites et la communication inter-organites. Le champ disciplinaire des SCM s’enrichit constamment grâce à la découverte de nouveaux acteurs moléculaires impliqués dans l'attachement des organites entre eux. Dans ce contexte de recherche, nous avons identifié un nouvel acteur impliqué dans la formation de SCM, appelé MOSPD2 (motile sperm domain-containing protein 2). Cette protéine est ancrée dans la membrane du réticulum endoplasmique (RE) ; elle peut interagir grâce à son domaine MSP avec d’autres protéines (associées à d’autres organites cellulaires) dont la caractéristique commune est de posséder un court motif protéique nommé FFAT. Par ces interactions, MOSPD2 permet l’établissement de SCM entre le RE et les endosomes, les mitochondries et l’appareil de Golgi. Ces résultats montrent une nouvelle façon de piéger des organites dans le grand filet cytoplasmique qu’est le RE
Membrane contact sites (MCS) are specific subcellular regions where two organelles are physically connected. Such micro-domains - molecularly defined by protein-protein and/or protein membrane interactions - are involved in organelle dynamic and inter-organelle communication. The field of MCS is constantly expanding thanks to the discovery of new molecular actors involved in organelle tethering. In this context of research, we identified MOSPD2 (motile sperm domain-containing protein 2) as a new factor involved in the formation of MCS. The MOSPD2 protein is anchored to the membrane of the endoplasmic reticulum (ER); it is able to interact thanks to its MSP domain with other organelle-associated proteins which common feature is to have a short protein motif called FFAT. By binding with its protein partners, MOSPD2 establishes MCS between the ER and endosomes, mitochondria and the Golgi apparatus. These results show how a large net covering the entire cytoplasm made by the ER can trap a large variety of cellular organelles

Les cellules eucaryotes sont caractérisées par le cloisonnement des organelles par des membranes. La communication entre les différents compartiments cellulaires est assurée par deux voies de transport : le transport vésiculaire et transport non-vésiculaire. Le transport vésiculaire permet à la fois le trafic des protéines et des lipides d'un compartiment à un autre, alors que le transport non-vésiculaire permet uniquement le trafic des lipides. En effet, les lipides jouent un rôle essentiel dans l'organisation cellulaire. Au cours de ma thèse, je me suis intéressé au rôle des lipides dans le trafic intracellulaire, en utilisant la levure comme organisme modèle. Dans une première partie de ma thèse, j'ai étudié les hélices amphipathiques qui permettent le ciblage des protéines vers des compartiments cellulaires spécifiques. Dans une étude précédente, réalisé au laboratoire a montré que ces hélices amphipathiques interagissaient directement avec les lipides membranaires, ce qui permet un adressage spécifique des protéines en fonction des environnements lipidiques dans la cellule. Deux hélices amphipatiques ont fait l’objet de cette étude : le motif ALPS qui cible les vésicules de la voie sécrétoire précoce, et alpha-synucléine qui reconnaît et fixe les vésicules du compartiment trans-Golgi-membrane plasmique. Dans cette première partie de la thèse j’ai cherché à identifier des motifs similaires à celui d’alpha-synucléine dans les protéines de levure, et de déterminer leurs rôles dans la cellule. Dans une seconde partie de ma thèse, en collaboration avec le laboratoire du Dr Thierry Galli, j'ai étudié de nouveaux composants impliqués dans le métabolisme lipidique aux sites de contact entre le réticulum endoplasmique et la membrane plasmique. Les sites de contact membranaires sont des régions de proches appositions (de l'ordre de 10 à 30 nm) entre deux membranes, généralement entre la membrane du réticulum endoplasmique (RE) et une autre organelle. Ce sont principalement des sites de transfert des lipides et d'ions. Maja Petkovic dans le laboratoire de Thierry Galli a fait la découverte que la protéine SNARE du RE, Sec22, interagit avec une syntaxine (Stx1) de la membrane plasmique dans les neurones, ce qui permet un nouveau mécanisme de contact entre ces deux membranes. J’ai donc essayé de voir si ce mécanisme est conservé chez la levure. Les résultats que j'ai obtenus ont confirmé que la levure Sec22 est capable d'interagir avec une protéine SNARE SSO1 localisée à la membrane plasmatique et homologue de Stx1. J'ai trouvé par co-immunoprecipitation que Sec22 et SSO1 deux interagissent avec les protéines de transfert des lipides localisées aux sites de contact. L'utilisation d'une sonde spécifique au Phosphatidylinositol-4 phosphate (PI4P), nous a permis de montrer que Sec22 est impliquée dans la régulation du niveau de PI4P à la membrane plasmique. Pour disséquer les deux fonctions de Sec22, dans la voie sécrétoire et aux sites de contact, nous avons utilisé l'approche des suppresseurs multicopies dans la levure. Parmi les suppresseurs identifiés, nous avons trouvé le Sfh1, une protéine qui a un rôle potentiel dans le transfert des lipides. Ces résultats confirment bien ceux obtenus par l’équipe de Thierry Galli, montrant que Sec22 a un nouveau rôle aux sites de contact entre le RE et la membrane plasmique et suggèrent que ce complexe SNARE pourrait être impliqué dans transfert de lipides chez la levure
Eukaryotic cells are characterized by their internal membrane compartmentalization, with the various specialized organelles of the cell bounded by lipid membranes. Communication between different cellular compartments occurs via two transport pathways: vesicular transport and non-vesicular transport. Vesicular transport carries both proteins and lipids from one compartment to another in cells, whereas non-vesicular transport carries only lipids. An emerging idea is the important role that lipids play in cellular organization. Lipid binding amphipathic helices such as the ALPS (amphipathic lipid packing sensor) motif are targeted to membranes of a specific lipid composition, and hence act to transfer information encoded in membrane lipids to the vesicle trafficking machinery. The lipid composition of the membranes of different organelles is therefore of great importance. One mechanism that cells use to maintain the distinct lipid compositions of organelles is lipid transport, which occurs preferentially at membrane contact sites (MCS). MCS are regions of close appositions, on the order of 10 to 30 nm, between two membranes, generally between the membrane of the endoplasmic reticulum (ER) and another organelle. In my thesis, I addressed two aspects of how lipids and their transport function in intracellular trafficking, using yeast as a model system. First, I studied amphipathic motifs that mediate targeting of proteins to specific compartments in cells. Lipid binding amphipathic helices were shown in a previous study in the laboratory to mediate specific targeting to distinct lipid environments via direct protein-lipid interactions, both in vitro and in cells. One of these, the ALPS motif, targets vesicles of the early secretory pathway. The other, alpha-synuclein, targets vesicles travelling between the late Golgi, the plasma membrane and endosomes. I studied new potential alpha-synuclein-like motifs in yeast proteins, and their roles in cells. In a second project, in collaboration with the laboratory of Dr. Thierry Galli, I studied new compenents involved in lipid metabolism at contact sites between the endoplasmic reticulum and the plasma membrane. Maja Petkovic in the laboratory of Thierry Galli made the important discovery that the ER-localized SNARE protein Sec22 interacts with a plasma membrane syntaxin in neurons, thus providing a novel mechanism for mediating close contact between these two membranes. I addressed the question of whether this mechanism is conserved in yeast. The results I obtained confirmed that yeast Sec22 is able to interact with a SNARE protein localized to the plasma membrane, Sso1. I found by co-immunoprecitation that Sec22 and Sso1 both interact with lipid transfer proteins localized to ER-plasma membrane contact sites. Using a specific probe for phosphatidylinositol-4 phosphate (PI4P), we showed that Sec22 was involved in regulating the level of PI4P at the plasma membrane. These results extend to yeast those obtained by Maja Petkovic, Thierry Galli and colleauges showing that Sec22 has a novel role at ER-plasma membrane contact sites, and suggest that this SNARE complex might be implicated in lipid transfer at these sites in yeast

The epidermal growth factor receptor (EGFR) can be internalized through different routes. While clathrin-mediated endocytosis destines EGFR for recycling and signaling, internalization through non-clathrin endocytosis (NCE) targets the receptor for degradation. Since NCE appears to be a major negative regulator of EGFR levels, a more complete picture of this pathway would likely reveal new insights into aberrant EGFR signaling observed in many types of cancer. By combining a candidate gene approach with an unbiased proteomic approach, we have defined EGFR-NCE as molecularly distinct from other NCE pathways, relying on functional regulators not previously implicated in endocytosis. We found that reticulon 3 (RTN3), an endoplasmic reticulum (ER)-resident protein, is fundamental for NCE-mediated EGFR internalization, and that its ablation delays EGFR degradation, demonstrating that the NCE pathway is a critical regulator of the EGF-dependent cellular response. We show that, upon stimulation with high dose of EGF, RTN3 is localized in close proximity to EGFR and that it is crucial for the formation of contact sites between the ER and the plasma membrane (PM), which are needed for NCE to proceed efficiently. We also show that ER contact sites are involved in local Ca2+ release: high EGF doses induce a release of Ca2+ from the ER to the PM, which is strongly inhibited upon knockdown of RTN3. This calcium release depends on the inositol trisphosphate (IP3) cascade and is essential for the internalization of EGFR via NCE. In conclusion, we have discovered a new clathrin-independent endocytic pathway that relies on the action of RTN3. RTN3 is necessary for the formation of contact sites between the ER and EGFR-NCE sites at the PM, which are required for IP3R-dependent local calcium release and the completion of EGFR internalization through NCE.

Non-vesicular intracellular lipid traffic is mediated by lipid transfer proteins (LTPs), which contain domains with an internal cavity that can solubilise and transfer lipids. One of the most widespread LTP folds is the Steroidogenic Acute Regulatory Transfer (StART) domain, which forms a hydrophobic pocket, and appears in proteins with different localisations and lipid specificities. The aim of this study was to characterise a new StART-like domain family, which we identified by a bioinformatics approach. I studied aspects of the localisations, functions and structural properties of six StART-like proteins in S. cerevisiae. The yeast StART-like proteins were endoplasmic reticulum (ER)-integral membrane proteins with transmembrane domains, and they localised at membrane contact sites: Lam1p/Lam3p, and Lam2p/Lam4p at junctions between ER and plasma membrane (PM); Lam5p/Lam6p at junctions between the ER and the vacuolar membrane, at nucleus-vacuole junction (NVJ) and at ER-mitochondria contacts. To study their functions, I purified the second StART-like domain of Lam4p, and I identified sterol as its lipid ligand from in vitro binding assays and in a spectroscopy approach with fluorescent ergosterol. We named the whole family LAM for Lipid transfer proteins Anchored at Membrane contact sites. The sterol binding property of the domains was related to a phenotype shared by LAM1, LAM2 and LAM3 delete strains, which showed an increased sensitivity to the sterol-sequestering polyene antifungal drug Amphotericin B (AmB). The two most sensitive strains (lam1∆ and lam3∆), displayed low sphingolipid levels, which is as yet unexplained. All AmB phenotypes were rescued by StART-like domains from the human LAMa, Lam2/4p and Lam5/6p, suggesting that these domains bind sterol. Simultaneous deletion of LAM1, LAM2, and LAM3 significantly reduced the extent of cortical ER-PM contacts, implying that they create the structure of the particularly punctate contact site they target. Finally, I started structural analysis of Lam4S2 to study the mechanism of sterol binding and to confirm our structural model.

Membrane contact sites are dynamic structures where two organelles come into close proximity to regulate and facilitate the flow of material and information between them. One type of inter-organelle communication is lipid exchange, which is essential for membrane maintenance and in response to environmental and cellular stimuli. We recently discovered a new family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs) that is present in all eukaryotes. LAM proteins are integral Endoplasmic Reticulum (ER) proteins containing at least one domain that is structurally similar to the StARkin domain superfamily, a specialised fold that can bind amphipathic ligands such as lipids. The budding yeast, Saccharomyces cerevisiae, has six such proteins: Lam1p-6p. Lam1p-4p are located at contacts between the ER and the plasma membrane (PM), and Lam1p-3p are implicated in retrograde sterol traffic between the ER and PM. The PM contains a high concentration of sterol where it increases rigidity by altering the packing characteristics of the phospholipids in the bilayer. Sterol is also important in the ER, where its levels are low but it is both synthesised and sensed. However, the mechanism by which sterol traffics between the ER and the PM is unknown. This investigation characterises the phenotype of yeast delete LAM strains on Amphotericin B, a sterol sequestering antifungal agent and shows that the conserved StARkin domain of LAM proteins is responsible for resistance against Amphotericin B. Aspergillus fumigatus, a filamentous fungus, has two LAM proteins and the removal of AfLamA causes a severe growth phenotype. Also, in vitro studies indicate that LAM StARkin domains have a clear sterol transfer activity and a mutation that can diminish the function in vivo and in vitro has been identified. These findings present a new candidate protein family for intracellular sterol trafficking.

Initially considered to be a precursor for highly phosphorylated phosphatidylinositol species, phosphatidylinositol 4-phosphate (PI4P) turned out to be "active" in its own right as a pivotal regulator of multiple cellular functions including membrane trafficking, sphingolipid metabolism, autophagy, and cell migration. PI4P levels at the Golgi in control conditions are the result of the balanced activity of Golgi-localized PI4-kinases (PI4KIIIβ and to a lesser extent PI4KIIα) and a single, highly conserved 4-phosphatase known as SAC1, localized in the ER. To coordinate such a variety of functions, multiple layers of regulation are required to modulate PI4P levels in time and space, by directing both PI4P enzymes localization and their activity. Here I show that the phosphatidylinositol 4-phosphate adaptor protein 1 (FAPP1) acts as a PI4P sensor, binds to PI4P and regulates its levels by interacting with and stimulating SAC1 at the level of ER-TGN membrane contact sites (ERTGoCS). At these ERTGoCS, FAPP1 acts as an adaptor that directs and stabilizes SAC1 towards PI4P-rich domains to promote its activity in trans to dephosphorylate PI4P at the TGN. Consequently, FAPP1 depletion leads to a huge increase in PI4P levels at the Golgi. I found that the FAPP1-regulated PI4P pool controls the post-Golgi trafficking of specific cargoes. One of these is the β-lipoprotein ApoB-100, which is more secreted upon FAPP1 depletion, highlighting a possible physiological role of this pool of PI4P controlled at the level of ER-TGN membrane contact sites. Intriguingly, another affected cargo is the autophagy protein ATG9, whose trafficking from the Golgi is increased in FAPP1 KD cells, thus resulting in a strong induction of autophagy. Besides the regulation of specific trafficking events, I found that the pool of PI4P controlled by FAPP1 recruits and promotes the activation of the PI4P-binding oncogene GOLPH3. When PI4P levels increase in FAPP1-KD cells, GOLPH3 is hyperactivated and promotes uncontrolled cell migration and invasion. My thesis work presents new information on the molecular mechanisms underlying the regulation of PI4P at the Golgi complex, identifying FAPP1 as a negative regulator of PI4P levels. It also sheds light on the need to keep PI4P levels within a certain range since an uncontrolled increase in PI4P can result in oncogenic potential and mis-trafficking of selective cargoes. Overall, this work provides novel insights into the homeostatic control of PI4P with an important contribution in understanding the complex picture of PI4P control and function in cell biology.

The Bcl-2 family of proteins regulates the mitochondrial apoptotic pathway and consists of both pro- and anti-apoptotic members. Bax-cc is present in the cytosol of healthy cells. Apoptotic stimuli induce the translocation of Bax-oc to the mitochondria leading to the permeabilisation of the outer mitochondrial membrane (OMM) and the release of downstream pro-apoptotic proteins from the intermembrane space (IMS). The mechanism by which Bax permeabilises the OMM remains unclear. Recent evidence suggests Bax alone might be sufficient to permeabilise the OMM. However, other models indicate the involvement of the mitochondrial permeability transition pore complex or the voltage-dependent anion channel (VDAC) present in the OMM. In this study Bax and C-terminally truncated Bax were expressed as GST-fusion proteins and were immobilised on agarose-GSH. The binding of mitochondrial membrane proteins that might be involved in the Bax mediated release of proteins from the IMS was investigated. The results showed that VDAC and the adenine nucleotide translocase (ANT) were retained by GST-Bax. Exogenous cyclophilin D (Cyp D) was added in the presence of VDAC and ANT and was also retained indicating that Bax interacts with the components of the permeability transition pore complex. The anti-apoptotic Bcl-2 protein Bcl-XL was expressed with a hexahistidine tag at its N-terminus. This was used to investigate its effects on Bax interaction with VDAC and ANT. The ANT ligands atractyloside and bongkrekic acid which promote and inhibit apoptosis respectively were shown to change the relative amounts of VDAC and ANT that bind GST-Bax. Apoptotic cell death has been identified in cardiomyocytes subjected to ischaemia. In this investigation cardiomyocytes transfected with GFP-Bax were treated with cyanide to simulate ischaemia and GFP-Bax translocation was observed using fluorescence microscopy. GFP-Bax co-immunoprecipitated with VDAC and ANT after translocation to mitochondria but not with Cyp D. The implications of these findings are discussed in this thesis.

STARD3 est une protéine endosomale de la famille START (Steroidogenic Acute Regulatory (StAR) Related lipid Transfer), qui lie le cholestérol. STARD3 module l’organisation de la cellule en formant des sites de contact membranaire entre les endosomes et le réticulum endoplasmique (RE). Le lien entre les sites de contact membranaire et le transport du cholestérol n’était pas compris. Dans ce travail, nous montrons que STARD3 en interagissant avec les protéines VAPs (VAMP–Associated Proteins) bâtit une machine moléculaire autonome qui transporte le cholestérol au niveau des contacts RE–endosomes. Ce transport permet la formation de membranes internes dans les endosomes et est potentiellement impliqué dans le fonctionnement de ces organites. De plus, nous avons étudié la fonction de STARD3 dans la maladie Niemann Pick type C, qui est caractérisée par une anomalie du transport de cholestérol dans les endosomes
STARD3 is an endosomal sterol-binding protein which belongs to the START protein family. Remarkably, STARD3 modulates the cellular organization by creating membrane contact sites between the endoplasmic reticulum (ER) and endosomes. The link between ER-endosome contact sites and cholesterol transport was not understood. In this work, we showed that STARD3 and its ER–resident partner, VAMP–associated protein (VAP), assemble into a machine that allows a highly efficient transport of cholesterol within ER–endosome contacts. This cholesterol transport provides building blocks for endosome inner membranes formation, and is probably involved in endosome dynamics. Furthermore, we studied STARD3 function in Niemann Pick type C disease, a condition characterized by an impairment of endosomal cholesterol export

La croissance des neurites au cours du développement neuronal nécessite une expansion de la membrane plasmique (MP) via l’insertion de nouveaux lipides et protéines. Cet événement se produit à la suite de la fusion des vésicules de sécrétion avec la MP. Cependant, plusieurs études ont montré que le transfert non-vésiculaire de lipides au niveau des sites de contact entre le réticulum endoplasmique (RE) et la MP joue aussi un rôle dans la croissance des cellules. Des membres de la famille de synaptotagmines étendues (E-Syts) ont été identifiés comme protéines de transfert des lipides dépendantes du Ca2+ au niveau des jonctions RE-MP.Nous avons découvert qu’un nouveau complexe SNARE aux sites de contact RE-MP, composé par Sec22b et Stx1, est impliqué dans la croissance des neurites bien qu’il soit incapable de favoriser la fusion membranaire. Cependant, la manière dont ce complexe participe à l’extension des neurites reste à élucider. Chez la levure, Sec22 interagit avec les protéines de transfert des lipides de la famille OSH, enrichis aux sites de contact RE-MP.Sur la base de ces observations, notre hypothèse est que le transfert non-vésiculaire de lipides induit par E-Syts au niveau des jonctions RE-MP contenant Sec22b pourrait contribuer à la croissance neuronale. L’objectif de ma thèse était d’explorer cette hypothèse. Nous montrons que Sec22b interagit avec E-Syt2 et Stx1 dans les cellules PC12 et avec E-Syt2, E-Syt3 et Stx3 dans les cellules HeLa. L’interaction Sec22b/E-Syt2 dépend du domaine Longin de Sec22b. La surexpression des E-Syts stabilise l’association Sec22b/Stx1, alors que l’inactivation des E-Syts provoque l’effet inverse. La surexpression de E-Syt2 de type sauvage, mais pas des mutants incapables de transférer les lipides ou non fixés au RE, augmentent la formation de filopodes axonaux et la ramification de neurites dans les neurones en développement. Cet effet est inhibé par une neurotoxine clostridiale clivant Stx1, par l’expression du domaine Sec22b Longin et par un mutant Sec22b ayant une extension entre les domaines SNARE et transmembranaire.En conclusion, mes résultats soutiennent l’idée que les sites de contact Sec22b/Stx1 contribuent à l’expansion de la MP via une interaction avec des protéines de transfert de phospholipides comme E-Syts
The growth of neurites during neuronal development requires a massive increase of surface area via the insertion of new proteins and lipids. This event occurs through the fusion of secretory vesicles with the plasma membrane (PM), the final step of the secretory pathway. Recently, non-vesicular transfer of lipids at contacts between endoplasmic reticulum (ER) and PM was shown to contribute to membrane expansion. Members of the ER-integral membrane protein Extended-Synaptotagmin (E-Syt) family have been identified as Ca2+-dependent lipid transfer proteins at ER-PM contact sites, and shown to transfer glycerophospholipids via their lipid binding domains. The laboratory previously found that a novel ER-PM SNARE complex, composed of the ER-resident Sec22b and the neuronal plasmalemmal Stx1, is involved in neurite growth despite being unable to mediate membrane fusion. However, how this complex participates to neurite extension remained to be elucidated. In yeast, Sec22 interacts with lipid transfer proteins of the OSH family, enriched at the ER- PM contacts, supporting a role for Sec22b-populated ER- PM junctions in non-vesicular lipid transport between these bilayers. Based on these observations, our starting hypothesis was that E-Syts-mediated non-vesicular lipid transfer at Sec22b-populated ER-PM contacts, might contribute to neurite growth. The goal of my PhD was to explore this hypothesis with two specific questions: 1-What are the partners of Sec22b complexes which might be involved in the unconventional mechanisms of membrane expansion? 2-What is the mechanism whereby the non-fusogenic SNARE Sec22b/Stx1 complex acts in neuronal development?Here we show that Sec22b interacts with E-Syt2 and Stx1 in PC12 cells and with E-Syt2, E-Syt3 and Stx3 in HeLa cells. Overexpression of E-Syt2 stabilized Sec22b-Stx3 association, whereas silencing of E-Syt2 had the opposite effect. Overexpression of E-Syt2 full length, but not the mutant forms which are unable to transfer lipids or attach to the ER, increased the formation of filopodia particularly in the growing axon. Finally, this effect was inhibited by a clostridial neurotoxin cleaving Stx1, by the expression of Sec22b Longin domain and a by a Sec22b mutant with extended linker between SNARE and transmembrane domains.In conclusion, these results support the hypothesis that Sec22b/Stx1 junctions may contribute to membrane expansion via an interaction with phospholipid transfer proteins like E-Syts

The topic of this thesis is the degradation of fuel cell electrodes in proton exchange membrane fuel cells (PEMFCs). In particular, the degradation associated with localized fuel starvation, which is often encountered during start-ups and shut-downs (SUs/SDs) of PEMFCs. At SU/SD, O2 and H2 usually coexist in the anode compartment. This situation forces the opposite electrode, i.e. the cathode, to very high potentials, resulting in the corrosion of the carbon supporting the catalyst, referred to as carbon corrosion. The aim of this thesis has been to develop methods, materials and strategies to address the issues associated to carbon corrosion in PEMFC.The extent of catalyst degradation is commonly evaluated determining the electrochemically active surface area (ECSA) of fuel cell electrode. Therefore, it was considered important to study the effect of RH, temperature and type of accelerated degradation test (ADT) on the ECSA. Low RH decreases the ECSA of the electrode, attributed to re-structuring the ionomer and loss of contact with the catalyst.In the search for more durable supports, we evaluated different accelerated degradation tests (ADTs) for carbon corrosion. Potentiostatic holds at 1.2 V vs. RHE were found to be too mild. Potentiostatic holds at 1.4 V vs. RHE were found to induce a large degree of reversibility, also attributed to ionomer re-structuring. Triangle-wave potential cycling was found to irreversibly degrade the electrode within a reasonable amount of time, closely simulating SU/SD conditions.Corrosion of carbon-based supports not only degrades the catalyst by lowering the ECSA, but also has a profound effect on the electrode morphology. Decreased electrode porosity, increased agglomerate size and ionomer enrichment all contribute to the degradation of the mass-transport properties of the cathode. Graphitized carbon fibers were found to be 5 times more corrosion resistant than conventional carbons, primarily attributed to their lower surface area. Furthermore, fibers were found to better maintain the integrity of the electrode morphology, generally showing less degradation of the mass-transport losses. Different system strategies for shut-down were evaluated. Not doing anything to the fuel cell during shut-downs is detrimental for the fuel cell. O2 consumption with a load and H2 purge of the cathode were found to give around 100 times lower degradation rates compared to not doing anything and almost 10 times lower degradation rate than a simple air purge of the anode. Finally, in-situ measurements of contact resistance showed that the contact resistance between GDL and BPP is highly dynamic and changes with operating conditions.
Denna doktorsavhandling behandlar degraderingen av polymerelektrolytbränslecellselektroder. polymerelektrolytbränslecellselektroder. Den handlar särskilt om nedbrytningen av elektroden kopplad till en degraderingsmekanism som heter ”localized fuel starvation” oftast närvarande vid uppstart och nedstängning av bränslecellen. Vid start och stopp kan syrgas och vätgas förekomma samtidigt i anoden. Detta leder till väldigt höga elektrodpotentialer i katoden. Resultatet av detta är att kolbaserade katalysatorbärare korroderar och att bränslecellens livslängd förkortas. Målet med avhandlingen har varit att utveckla metoder, material och strategier för att både öka förståelsen av denna degraderingsmekanism och för att maximera katalysatorbärarens livslängd.Ett vanligt tillvägagångsätt för att bestämma graden av katalysatorns degradering är genom mätning av den elektrokemiskt aktiva ytan hos bränslecellselektroderna. I denna avhandling har dessutom effekten av temperatur och relativ fukthalt studerats. Låga fukthalter minskar den aktiva ytan hos elektroden, vilket sannolikt orsakas av en omstrukturering av jonomeren och av kontaktförlust mellan jonomer och katalysator.Olika accelererade degraderingstester för kolkorrosion har använts. Potentiostatiska tester vid 1.2 V mot RHE visade sig vara för milda. Potentiostatiska tester vid 1.4 V mot RHE visade sig däremot medföra en hög grad av reversibilitet, som också den tros vara orsakad av en omstrukturering av jonomeren. Cykling av elektrodpotentialen degraderade istället elektroden irreversibelt, inom rimlig tid och kunde väldigt nära simulera förhållandena vid uppstart och nedstängning.Korrosionen av katalysatorbäraren medför degradering av katalysatorn och har också en stor inverkan på elektrodens morfologi. En minskad elektrodporositet, en ökad agglomeratstorlek och en anrikning av jonomeren gör att elektrodens masstransportegenskaper försämras. Grafitiska kolfibrer visade sig vara mer resistenta mot kolkorrosion än konventionella kol, främst p.g.a. deras låga ytarea. Grafitiska kolfibrer visade också en förmåga att bättre bibehålla elektrodens morfologi efter accelererade tester, vilket resulterade i lägre masstransportförluster.Olika systemstrategier för nedstängning jämfördes. Att inte göra något under nedstängning är mycket skadligt för bränslecellen. Förbrukning av syre med en last och spolning av katoden med vätgas visade 100 gånger lägre degraderingshastighet av bränslecellsprestanda jämfört med att inte göra något alls och 10 gånger lägre degraderingshastighet jämfört med spolning av anoden med luft. In-situ kontaktresistansmätningar visade att kontaktresistansen mellan bipolära plattor och GDL är dynamisk och kan ändras beroende på driftförhållandena.

QC 20131104

L'objectif de ces travaux vise à évaluer les performances d'un contacteur membranaire à fibres creuses utilisé pour réaliser l'absorption chimique du CO2 dans une solution ammoniacale ainsi que la régénération de cette dernière. Les membranes utilisées sont composites, c'est-à-dire composées d'une fine couche dense recouverte sur un support microporeux, la couche dense permettant d'éviter le mouillage par pénétration de liquide dans la membrane. Pour réaliser ces études, une approche combinant expérimentation et modélisation a été adoptée. Lors de la réalisation de l'absorption chimique avec un contacteur membranaire, des chutes importantes d’efficacité de captage du CO2 au cours du temps ont été observées et confirment les résultats obtenus lors de travaux ultérieurs. Cette baisse des performances est attribuée à la précipitation de sels d’ammonium en phase gaz. Lors de l'utilisation d’un gaz saturé en vapeur d'eau, comme le seraient les fumées industrielles, les performances du procédé se sont révélées stables. Un modèle 1D multi-composant adiabatique du contacteur a été développé sur Aspen Custom Modeler® et validé à partir des résultats expérimentaux. Les simulations réalisées avec ce modèle ont confirmé le potentiel d'intensification volumique de la technologie, toutefois, la réduction des pertes de NH3, grâce à l'utilisation d’une couche dense sélective moins perméable à NH3 qu’au CO2, n’a pas été satisfaisante. Les phénomènes de condensation dans les contacteurs membranaires ont été étudiés par expérimentation et modélisation. Il a ainsi été montré que le mouillage par condensation de la membrane ne devrait pas survenir, par contre, la condensation dans le lumen des fibres creuses entraîne une augmentation importante de la perte de charge pouvant conduire à des coûts de compression des gaz à traiter plus élevés. Des expériences et des simulations sur la régénération de solutions ammoniacales chargées avec des contacteurs membranaires ont été effectuées et des disparités importantes ont été trouvées entre les flux de CO2 mesurés et simulés. Une réduction volumique de trois par rapport à la colonne à garnissage a pu être calculée laissant entrevoir un potentiel intéressant de la technologie pour l’étape de régénération. En collaboration avec les partenaires du projet C2B, dans lequel s’intègre cette thèse, des essais d’absorption de CO2 ont été réalisés sur site avec un contacteur de taille industrielle. Les résultats de ce pilote sont conformes aux résultats obtenus au laboratoire et encourageants quant au transfert de la technologie vers l’échelle industrielle
This work aims to evaluate the performances of hollow fiber membrane contactors used for the CO2 absorption in aqueous ammonia and the regeneration of the latter within the frame of post-combustion CO2 capture. Fibers are made of a thin dense layer coated on a microporous support, the dense layer prevent membrane wetting by liquid penetration. Both experiment and modelling were done. During absorption experiments, important decrease of the CO2 capture efficiency was observed due to ammonium salts precipitation in the gas-side corroborating results from previous works. Experiments with CO2/N2 mixture saturated with water vapor, as would be the case for flue gas, interestingly, showed stable performances of the process. A one-dimensional multi-component adiabatic transfer model for CO2 absorption in NH3 has been implemented in Aspen Custom Modeler® and validated with experimental results. The simulations performed with the model confirmed the volumetric intensification potential of the technology, however, the NH3 slip reduction expected, because of the use of a dense layer more permeable to CO2 than NH3, wasn’t satisfying. Water condensation phenomenon in membrane contactors were studied with both experiments and simulations. It was thus showed that membrane pore wetting by condensation should not happened but gas-side condensation led to an important increase of the pressure drop with the potential of increasing compression costs. Experiments and simulations of the desorption of CO2 from a loaded aqueous ammonia solution with a membrane contactor were performed and important disparities were found between CO2 flux measured and simulated. A volumetric reduction of the membrane contactor when compared to the packed column was calculated highlighting the potential of the technology for the stripping step. In collaboration with the partners of the C2B project, in which this thesis is integrated, CO2 absorption essays were carried out on site with an industrial scale membrane contactor. The results of this pilot are consistent with laboratory results and encourages the transfer of the technology to the industrial scale

Ca2+ is a universal and versatile intracellular messenger, regulating a vast array of biological processes due to variations in the frequency, amplitude, spatial and temporal dynamics of Ca2+ signals. Increases in cytosolic free Ca2+ concentration ([Ca2+]c) are due to influx from either an infinite extracellular Ca2+ pool or from the more limited intracellular Ca2+ stores. Stimulation of the endogenous muscarinic (M3) receptors of human embryonic kidney (HEK) cells with carbachol results in the activation of phospholipase C (PLC) and formation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3Rs), release of Ca2+ from the endoplasmic reticulum (ER), and activation of store-operated Ca2+ entry (SOCE). Lysosomes are the core digestive compartments of the cell, but their importance as signalling organelles is also now widely appreciated. Accumulating evidence indicates that lysosomal Ca2+ is important for their physiological functions. Lysosomal Ca2+ release triggers fusion during membrane trafficking and, through calmodulin, it regulates lysosome size. Luminal Ca2+ is critical for regulation of lysosomal biogenesis and autophagy during starvation through the transcription factor, TFEB. Furthermore, aberrant lysosomal Ca2+ is associated with some lysosomal storage diseases. Lysosomes in mammalian cells have long been suggested to accumulate Ca2+ via a low-affinity Ca2+-H+ exchanger (CAX). This is consistent with evidence that dissipating the lysosomal H+ gradient increased [Ca2+]c and decreased lysosomal free [Ca2+], and with the observation that lysosomal Ca2+ uptake was followed by an increase in pHly. Furthermore, heterologous expression of Xenopus CAX in mammalian cells attenuated carbachol-evoked Ca2+ signals. However, there is no known CAX in mammalian cells, and so the identity of the lysosomal Ca2+ uptake pathway in mammalian cells is unresolved. Using mammalian cells loaded with a fluorescent Ca2+ indicator, I show that dissipating the pHly gradient pharmacologically or by siRNA-mediated knockdown of an essential subunit of the H+ pump, increases the amplitude of IP3-evoked cytosolic Ca2+ signals without affecting those evoked by SOCE. A genetically encoded low-affinity Ca2+ sensor expressed on the lysosome surface reports larger increases in [Ca2+]c than the cytosolic sensor, but only when the Ca2+ signals are evoked by IP3R rather than SOCE. Using cells expressing single IP3R subtypes, I demonstrate that each of the three IP3R subtypes can deliver Ca2+ to lysosomes. I conclude that IP3Rs release Ca2+ within near-lysosome microdomains that fuel a low-affinity lysosomal Ca2+ uptake system. The temporal relationship between the increase in pHly and reduced Ca2+ sequestration suggests that pHly affects the organization of the microdomain rather than the Ca2+ uptake mechanism. I show that abrogation of the lysosome H+ gradient does not acutely prevent uptake of Ca2+ into lysosomes, but disrupts junctions with the ER where the exchange of Ca2+ occurs. The dipeptide, glycyl-L-phenylalanine 2-naphthylamide (L-GPN), is much used to disrupt lysosomes and release Ca2+ from them. The mechanism is widely assumed to require cleavage of GPN by cathepsin C, causing accumulation of amino acid residues, and osmotic lysis of lysosomal membranes. I show, using LysoTracker Red and Oregon Green-dextran to report pHly, that L-GPN is effective in HEK cells lacking functional cathepsin C, following CRISPR-Cas9-mediated gene disruption. Furthermore, D-GPN, which is resistant to cleavage by cathepsin C, is as effective as L-GPN at increasing pHly, and it is similarly effective in cells with and without cathepsin C. L-GPN and D-GPN increase cytosolic pH, and the effect is similar when the lysosomal V-ATPase is inhibited with bafilomycin A1. This is not consistent with GPN releasing the acidic contents of lysosomes. I conclude that the effects of GPN on lysosomes are not mediated by cathepsin C. Both L-GPN and D-GPN evoke Ca2+ release, the response is unaffected by inhibition or knock-out of cathepsin C, but it requires Ca2+ within the ER. GPN-evoked increases in [Ca2+]c require Ca2+ within the ER, but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. I conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes. I conclude that all three IP3R subtypes selectively deliver Ca2+ to lysosomes, and that the low pH within lysosomes is required to maintain the junctions between ER and lysosomes, but not for lysosomal Ca2+ uptake. I suggest that GPN lacks the specificity required to allow selective release of Ca2+ from lysosomes.

Les piles à combustible à membrane échangeuse de protons (PEMFC) doivent répondre à des exigences élevées de coût, performance, et durabilité. Cette thèse propose ainsi d'optimiser les propriétés de conduction électrique et de passivité d'aciers inoxydables pour plaques bipolaires de PEMFC. Ce travail expose la problématique de l'emploi d'aciers inoxydables et des solutions correspondantes, les propriétés des films passifs sur aciers ainsi que leur modification par des procédés bas coût. Des caractérisations des matériaux ont été effectuées, permettant d'étudier le comportement de deux alliages dans différents états et conditions représentatives d'un milieu PEMFC. L'état industriel des tôles ne convient pas à un emploi direct en pile. Une modification de surface étudiée améliore nettement la conduction électrique initiale, maintenant au vieillissement un niveau supérieur à l'état industriel. Ce traitement augmente également la résistance à la corrosion, particulièrement côté anodique
PEM fuel cells have to comply with stringent cost, performance, and durability criteria. Thus, the goal of this PhD was to optimize electrical conduction properties and passivity of stainless steels (SS) for PEMFC bipolar plates. This work presents the possible problems when using SS plates and corresponding solutions, SS passive film properties, as weIl as their modifications by low cost surface treatments. Material characterizations were performed, allowing to study the behaviour of two alloys in different states and conditions representative of a PEMFC media. The plate industrial state is not convenient for direct use in fuel cell. A surface modification studied improves widely electrical conduction at initial state. The performance is degraded with ageing, but maintaining a level higher than the initial industrial state. This treatment increases also corrosion resistance, particularly on the anode side

Indiana University-Purdue University Indianapolis (IUPUI)
Coxiella burnetii, the zoonotic agent of human Q fever and chronic endocarditis, is an obligate intracellular bacterial pathogen. The Coxiella intracellular niche, a large, lysosome-like parasitophorous vacuole (PV), is essential for bacterial survival and replication. There is growing evidence that host cell cholesterol trafficking plays a critical role in PV development and maintenance, prompting an examination of the role of cholesterol-binding host protein ORP1L (Oxysterol binding protein-Related Protein 1, Long) during infection. ORP1L is a multi-functional cholesterol-binding protein involved in late endosome/lysosome (LEL) trafficking, formation of membrane contact sites between LEL and the endoplasmic reticulum (ER), and cholesterol transfer from LEL to the ER. ORP1L localizes to the PV at novel membrane contact sites between the ER and the PV membrane. Ectopically expressed ORP1L in Coxiella-infected cells localizes to the PV membrane early during infection, before significant PV expansion and independent of other PV-localized proteins. Further, the N-terminal ORP1L Ankyrin repeats are both necessary and sufficient for PV localization, suggesting that protein-protein interactions, and not protein-lipid interactions, are primarily involved in PV association. Coxiella employs a Type IVB Secretion System (T4BSS) to translocate effector proteins into the host cytoplasm and manipulate various cellular functions. ORP1L is not found on the PV of a Coxiella mutant lacking a functional T4BSS, indicating a secreted bacterial protein is likely responsible for ORP1L recruitment. We identified a Coxiella mutant with a transposon insertion in CBU_0352 that exhibits a 50% decrease in ORP1L recruitment, suggesting that Coxiella CBU_0352 interacts directly or indirectly with ORP1L. Finally, we found that ORP1L depletion using siRNA alters PV dynamics, resulting in smaller yet more fusogenic Coxiella PVs. Together, these data suggest that ORP1L is specifically recruited to the PV, where it plays a novel role in Coxiella PV development and interactions between the PV and the host cell.