Les discontinuités granulaires dans les ouvrages hydrauliques ou dans leur fondation constituent une source majeure d’instabilités à l’origine des phénomènes d’érosion. L’érosion interne est ainsi définie comme une migration de particules engendrée par un écoulement interne parasite, dans un sol ou dans un ouvrage en terre. A long terme, les conséquences de cette migration peut affecter la stabilité des ouvrages et peut même conduire à leur rupture. La sécurité des ouvrages en terre dépend principalement de la performance de leurs filtres, c’est-à-dire de la capacité du filtre, mis en interne en phase de conception ou en externe en phase de réparation, à retenir les particules fines. L’espace poral d’un filtre granulaire est divisé en volumes élémentaires, appelés pores, reliés entre eux par des étranglements plus étroits appelés constrictions. Des recherches récentes ont montré que la distribution des tailles de constriction (CSD) joue un rôle fondamental dans la compréhension des propriétés de filtration des sols granulaires. Ce travail vise à étudier la CSD et son impact sur les mécanismes de filtration dans les matériaux granulaires. Pour atteindre cet objectif, deux approches ont été suivies dans ce travail: l’une numérique et l’autre analytique. Dans le cas des matériaux de forme sphérique, la méthode des éléments discrets (DEM) permet de calculer la CSD en s’appuyant sur une partition de Delaunay en tétraèdres. Cependant, une CSD plus réaliste peut être obtenue par association des tétraèdres voisins selon un critère basé sur le chevauchement de leurs sphères de vide inscrites. A partir de cette considération et en se basant sur les modèles analytiques existants, un modèle révisé est proposé pour obtenir rapidement la CSD. Les échantillons DEM générés sont ensuite utilisés pour examiner le potentiel de transport des particules fines à travers un filtre d’épaisseur donnée. Les résultats des essais de filtration numériques menés ont montré une corrélation entre la CSD et la possibilité de migration des particules fines. En conséquence, une formule analytique a été proposée pour calculer le diamètre d’ouverture de contrôle des filtres granulaires. Cette taille caractéristique qui prend en compte la granulométrie et la densité du matériau granulaire, a été introduite dans un critère de filtre construit sur la base de la CSD, en vue de le représenter de manière plus physique. Le critère proposé reproduit correctement des résultats expérimentaux rapportés dans la littérature. / The granular discontinuities in hydraulic structures or in their foundation constitute a major source of instabilities causing erosion phenomena, process by which finer soil particles are transported through the voids between coarser particles, under seepage flow. In the long term, the microstructure of the soil will change and the excessive migration become prejudicial to the stability of the structures and may also induce their failure. The safety of earth structures is mainly dependent on the reliability of their filter performance, i.e. the ability of the filter placed inside the structure during construction or outside during repair, to retain fine particles. Indeed, the void space of a granular filter is divided into larger volumes, called pores, connected together by throats or constrictions. Recent researches showed that the distribution of throats (Constriction Size Distribution or CSD) between pores plays a key role to understand the filtration properties of a granular soil. This research is devoted to investigate the constriction sizes and their impact on the mechanisms of filtration in granular spherical materials. To achieve this objective, two approaches were followed in this work: numerical and analytical approaches. In the case of spherical materials, the Discrete Element Method (DEM) can help to compute the CSD using the Delaunay tessellation method. However, a more realistic CSD can be obtained by merging adjacent Delaunay cells based on the concept of the overlap of their maximal inscribed void spheres. Following this consideration and by extending the previously developed analytical models of CSD, a revised model is proposed to quickly obtain the CSD. The DEM data generated are then used to explore the potential of transport of fine particles through a filter of a given thickness by means of numerical filtration tests. A correlation has been found between the CSD and the possibility of migration of fine grains. Accordingly, an analytical formula has been proposed to calculate the controlling constriction size of a filter material. This characteristic size, which takes into account the particle size distribution (PSD) and the density of the material, has been used to reformulate a constriction-based criterion in a more physical manner. The proposed filter design criterion is verified based on experimental data from past studies and a good agreement has been found.

Pseudomonas aeruginosa est un pathogène opportuniste responsable d’infections nosocomiales sévères associées à un taux élevé de mortalité. Le système de sécrétion de Type III (SST3) et les effecteurs qu’il injecte sont considérés comme des facteurs de virulence prépondérants de P. aeruginosa. Récemment nous avons caractérisé, un groupe de souches ne possédant pas les gènes du SST3, mais dont la virulence repose sur la sécrétion d’une nouvelle toxine de 172 kDa, nommée Exolysine (ExlA) qui provoque la perméabilisation de la membrane des cellules hôtes. ExlA est sécrétée dans le milieu par une porine de la membrane externe, nommée ExlB, formant ainsi un nouveau système de sécrétion à deux partenaires (TPS), ExlBA. Outre le domaine TPS du coté N-terminal de la protéine, impliqué dans sa sécrétion, ExlA possède différents domaines ; des répétitions hémagglutinines, cinq motifs Arginine-Glycine-Acide Aspartique (RGD) et un domaine C-Terminal faiblement conservé. Des tests de cytotoxicité sur des cellules eucaryotes ont montrés que la délétion du domaine C-terminal abolissait l’activité toxique d’ExlA. En utilisant un modèle de liposomes et différents types de cellules eucaryotes, comme les globules rouges, nous avons démontré qu’ExlA forme des pores membranaires de 1.6 nm. De plus, par un criblage cellulaire à haut-débit d’une banque de mutants obtenus par une mutagenèse de transposition, nous avons montré qu’un facteur bactérien additionnel était requis dans la toxicité d’ExlA. En effet, parmi les 7 400 mutants, nous avons identifiés 3 transposons insérés dans des gènes codant pour le pili de type IV, démontrant ainsi que cet appendice impliqué dans l’adhésion des bactéries participe à la toxicité d’ExlA, en permettant un contact rapproché entre la bactérie et les cellules hôtes. Un criblage de macrophages primaires de souris KO pour différentes protéines impliquées dans la voie de l’activation de l’inflammasome, nous a permis de démontrer que le pore formé par ExlA est responsable de l’activation de la Caspase-1 par l’inflammasome NLRP3 conduisant à la maturation de l’interleukine-1ß. Une étude bio-informatique a révélé la présence de gènes homologues à exlA chez d’autres espèces de Pseudomonas non pathogènes, comme P. putida, P. protegens, P. entomophila. Nous avons montré que ces bactéries environnementales sont aussi capables de provoquer une mort cellulaire dépendante de la Caspase-1. Finalement, un criblage d’une banque de macrophages dont les gènes ont été invalidés par la technologie CRISPR/cas9 a révélé que plusieurs protéines du système immunitaire, indirectement liées à l’activation de la Caspase-1 sont impliquées dans la mort cellulaire médiée par ExlA. De plus, nous avons montré que plusieurs sgRNAs ciblant un microARN, mir-741, était grandement enrichi dans les macrophages ayant résisté à une infection avec ExlA. Mir-741 régule l’expression d’enzymes (St8sIa1 et Agpat5) impliquées dans la voie de biosynthèse des sphingolipides et des glycérophospholipides, suggérant ainsi que l’activité d’ExlA requiert un environnement lipidique particulier. / Pseudomonas aeruginosa is a human opportunistic pathogen responsible for nosocomial infections associated with high mortality. The type III secretion system (T3SS) and T3SS-exported toxins have been considered as key infectivity virulence factors. Our team recently characterized a group of strains lacking T3SS, but employing a new pore-forming toxin of 172 kDa, named Exolysin (ExlA) that provokes cell membrane disruption. In this work we demonstrated that the ExlA secretion requires ExlB, a predicted outer membrane protein encoded in the same operon, showing that ExlA-ExlB define a new active Two-Partner Secretion (TPS) system. In addition to the TPS secretion signals, ExlA harbors several distinct domains, which comprise hemagglutinin domains, five Arginine-Glycine-Aspartic acid (RGD) motifs and a non-conserved C-terminal region lacking any identifiable sequence motifs. Cytotoxic assays showed that the deletion of the C-terminal region abolishes host-cell cytolysis. Using liposomes and eukaryotic cells, including red blood cells, we demonstrated that ExlA forms membrane pores of 1.6 nm. Based on a transposon mutagenesis strategy and a high throughput cellular live-dead screen, we identified additional bacterial factors required for ExlA-mediated cell lysis. Among 7 400 mutants, we identified three transposons inserted in genes encoding components of the Type IV pili, which are adhesive extracellular appendices. Type IV pili probably mediate close contact between bacteria and host cells and facilitate ExlA cytotoxic activity. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages to achieve host cell intoxication. Using mice primary bone marrow macrophages we showed that ExlA pores provoke activation of Caspase-1 via the NLRP3-inflamasomme followed by the maturation of the pro-interleukin-1ß. Mining of microbial genomic databases revealed the presence of exlA-like genes in other Pseudomonas species rarely associated with human infections P. putida, P. protegens and P. entomophila. Interestingly, we showed that these environmental bacteria are also able to provoke Caspase-1 cleavage and pro-inflammatory cell death of macrophages. Finally, genome-wide loss-of-function CRISPR/cas9 RAW library screen revealed that several components of the immune system response, indirectly linked to Caspase-1 are involved in the ExlA-mediated cell lysis. Moreover, we found at least three sgRNAs targeting miRNA, mir-741 were highly enriched in resistant macrophages challenged by ExlA. This miRNA regulates enzymes (St8sIa1 and Agpat5) in the sphingolipids and glycerophololipids biosynthesis pathways, suggesting that ExlA activity may require proper lipid environment.

[pt] A formação e o acúmulo de condensado em reservatórios de gás retrógrado, especialmente na vizinhança de poços de produção, obstruem parcialmente o fluxo de gás e afetam negativamente a composição dos fluidos produzidos. Entretanto, a previsão de bloqueio por condensado é comumente imprecisa, visto que experimentos raramente reproduzem as condições extremas e composições complexas dos fluidos dos reservatórios, enquanto a maioria dos modelos em escala de poros simplificam demasiadamente os fenômenos físicos associados à transição de fases entre gás e condensado. Para corrigir essa lacuna, um modelo de rede de poros isotérmico composicional e totalmente implícito é apresentado. As redes de poros propostas consistem em estruturas tridimensionais de capilares constritos circulares. Modos de condensação e padrões de escoamento são atrubuídos aos capilares de acordo com a molhabilidade do meio, as saturações locais e a influência de forças viscosas e capilares. Nos nós da rede, pressão e conteúdo molar são determinados através da solução acoplada de equações de balanço molar e consistênc ia de volumes. Concomitantemente, um cálculo de flash à pressão e à temperatura constantes, baseado na equação de estado de Peng e Robinson, é realizado em cada nó, atualizando as saturações e composições das fases. Para a validação do modelo proposto, análises de escoamento foram executadas baseadas em experimentos de escoamento em testemunho reportados na literatura, usando composição dos fluidos e condições de escoamento correspondentes, e geometria do meio poroso aproximada. Curvas de permeabilidade relativa medidas nos experimentos e previstas pelo modelo mostraram boa concordância quantitativa, para dois valores de tensão interfacial e três valores de velocidade de escoamento de gás. Após a validação, o modelo foi usado para avaliar alteração de molhabilidade e injeção de gás como possíveis métodos de recuperação avançada para reservatórios de gás retrógrado. Os resultados exibiram tendências similares àquelas observadas em experimentos de escoamento em testemunhos, e condições ótimas para melhoramento do escoamento foram identificadas. / [en] Liquid dropout and accumulation in gas-condensate reservoirs, especially in the near wellbore region, hinder gas flow and affect negatively the produced fluid composition. Yet, condensate banking forecasting is commonly inaccurate, as experiments seldom reproduce reservoir extreme conditions and complex fluid composition, while most pore-scale models oversimplify the physical phenomena associated with phase transitions between gas and condensate. To address this gap, a fully implicit isothermal compositional pore-network model for gas and condensate flow is presented. The proposed pore-networks consist of 3D structures of constricted circular capillaries. Condensation modes and flow patterns are attributed to the capillaries according to the medium s wettability, local saturations and influence of viscous and capillary forces. At the network nodes, pressure and molar contents are determined via the coupled solution of molar balance and volume consistency equations. Concomitantly, a PT-flash based on the Peng-Robinson equation of state is performed for each node, updating the local phases saturations and compositions. For the proposed model validation, flow analyses were carried out based on coreflooding experiments reported in the literature, with matching fluid composition and flow conditions, and approximated pore-space geometry. Predicted and measured relative permeability curves showed good quantitative agreement, for two values of interfacial tension and three values of gas flow velocity. Following the validation, the model was used to evaluate wettability alteration and gas injection as prospect enhanced recovery methods for gas-condensate reservoirs. Results exhibited similar trends observed in coreflooding experiments and conditions for optimal flow enhancement were identified.

Le système immunitaire est divisé en deux réponses : innée et adaptative. Dans la réponse adaptative, les principaux acteurs sont les cellules T CD8 et CD4, dont l'activation est médiée par le complexe antigène-récepteur (TCR) et la génération de signaux intracellulaires. L'intensité du signal est contrôlée par l'affinité du ligand impliquant la kinase p56lck et la protéine adaptatrice SLP-76. Les souris dépourvues de SLP-76 sont bloquées dans leur développement thymique, ce qui rend difficile l'évaluation de l'importance de l'adaptateur, dans la fonction des cellules T périphériques. Récemment, le laboratoire Rudd a généré une souris knock-in (KI) avec une forme de SLP-76 mutée au niveau d'un seul résidu, K56, ayant des cellules T périphériques normales. Cette mutation empêche SLP-76 de se lier au complexe de pore nucléaire (CPN). L'objectif de ce mémoire est de comprendre le rôle de SLP-76, plus particulièrement du mutant K56E dans le contrôle de certains aspects de la fonction des cellules T périphériques. K56E sur un fond transgénique OT1, a montré une déficience partielle de la fonction et du métabolisme des cellules T en réponse à des ligands peptidiques d'ovalbumine de poulet, de différentes affinités. Plus précisément, les voies de la glycolyse et de la phosphorylation oxydative en ont été altérées. Dans l'ensemble, l'altération des fonctions et du métabolisme des lymphocytes T chez le mutant K56E confirme l'existence d'un lien entre le SLP-76 et le métabolisme des lymphocytes T, ce qui pourrait avoir des implications importantes dans le développement de thérapies ciblant la fonction des lymphocytes T. / The immune system is divided into two responses: innate and adaptive. In the adaptive response, the main players are CD8 and CD4 T-cells whose activation is mediated by ligation of the antigen-receptor complex (TCR) and its generation of intracellular signals. The strength of signal is controlled by the affinity of the ligand in a process that involves upstream kinases such as p56lck and downstream targets such as the adaptor protein SLP-76. Mice lacking SLP-76 are blocked in thymic development, making it difficult to assess the importance of the adaptor in peripheral T-cell function. Recently, the Rudd lab generated a knock-in (KI) mouse with a form of SLP-76 mutated at a single residue K56 which shows a normal peripheral T-cell compartment. The mutant prevents SLP-76 binding to the nuclear pore complex (NPC). The object of this dissertation is to understand role of SLP-76 and specifically the K56E mutant in the control of aspects of peripheral Tcell function. The K56E mutant on an OT1 TCR transgenic background showed a partial impairment of T-cell function and metabolism in response to chicken ovalbumin peptide ligands of different affinities. Specifically, both glycolysis and oxidative phosphorylation pathways were impaired in response to peptide ligand activation. Overall, the impairment of T-cell function and metabolism in the K56E mutant supports a link between SLP-76 and T-cell metabolism which may have important implications in the development of therapies targeting T-cell function.

Coal mine impoundment failures have been well documented to occur due to an increase in excess pore pressure from sustained monotonic loads. Very few failures have ever occurred from dynamic loading events, such as earthquakes, and research has been done regarding the stability of these impoundment structures under such natural seismic loading events. To date no failures or damage have been reported from dynamic loading events caused by near-by production blasting, however little research has been done considering these conditions. Taking into account that current environmental restrictions oblige to increase the capacity of coal impoundments, thus increasing the hazard of such structures, it is necessary to evaluate the effects of near-by blasting on the stability of the impoundment structures. To study the behavior of excess pore pressure under blasting conditions, scaled simulations of blasting events were set inside a controlled sand tank. Simulated blasts were duplicated in both saturated and unsaturated conditions. Explosive charges were detonated within the sand tank at various distances to simulate different scaled distances. Information was collected from geophones for dry and saturated scenarios and additionally from pressure sensors under saturated conditions to assess the behavior of the material under blasting conditions.

Scanning electron microscopy of Ar-ion milled samples from the Eagle Ford Formation, South Texas shows that the character and abundance of porosity changes significantly across burial conditions as a result of compaction, cementation, bitumen generation, and generation of secondary porosity within organic matter (OM). Samples displaying a range of compositions and maturities are imaged and quantified to provide insight into the effects of these processes. Porosity in low-maturity samples (Ro~0.5%) is volumetrically dominated (0.1% -12.5% bulk volume, average 6.2%) by relatively large, mostly interparticle, primary mineral-associated pores (median sizes range 35.9-52.7 nm). Larger pores are generally associated with coccolith debris that is commonly aggregated into pellets. Porosity and pore size correlate directly with calcite abundance and inversely with OM volumes. OM is dominantly detrital kerogen "stringers" that range in size and have spatial distributions and character suggestive of detrital origin. Destruction of primary porosity in low-maturity samples has occurred due to compaction of ductile kerogen and clays and, to a minor degree, as a result of cementation and infill of early bitumen. Smaller, secondary OM-hosted pores (median size range 11.1-14.9 nm) volumetrically dominate porosity (0.02%-3.6% bulk volume, average of 1.36%), in most high-maturity samples (Ro~1.2%-1.3%). Mineral-associated pores are present, but are typically smaller (median size range from 20.3-40.6 nm) and less abundant (0.0%-10.0% bulk volume, average of 2.5%) than at low maturity. Abundant mineral-associated porosity is present locally in samples where incursion of primary pore space by bitumen has not occurred. OM within high-maturity samples is distributed more evenly throughout the rock fabric, occupying spaces similar in size and morphology to primary interparticle pores, coating euhedral crystals (probable cements), and filling intraparticle porosity. These observations, and positive correlation between calcite and OM volumes (OM-hosted pore volume included) in samples with dominantly OM-hosted pore networks, suggests that a large portion of OM within high-maturity samples is diagenetic in origin and has filled primary pore space. Destruction of primary porosity in high-maturity samples has occurred through cementation, bitumen infill, and, possibly greater compaction. Additional porosity, however, has been generated through maturation of OM. / text

A new method is presented here for petrophysical interpretation of heterogeneous carbonates using well logs and core data. Developing this new method was necessary because conventional evaluation methods tend to yield inaccurate predictions of pore connectivity and permeability in the studied field. Difficulties in the petrophysical evaluation of this field are related to shoulder-bed effects, presence of non-connected porosity, rock layers that are thinner than the vertical resolution of well-logging tools, and the effect of oil-base mud (OBM) invasion in the measurements. These problems give rise to uncommon measurements and rock properties, such as: (a) reservoir units contained within thinly bedded and laminated sequences, (b) very high apparent resistivity readings in the oil-bearing zone, (c) separation of apparent resistivity logs with different depths of investigation, (d) complex unimodal and bimodal transverse relaxation distributions of nuclear magnetic resonance (NMR) measurements, (e) reservoir units having total porosity of 0.02 to 0.26 and permeability between 0.001mD to 4.2D, (f) significant differences between total and sonic porosity, and (g) low and constant gamma-ray values. The interpretation method introduced in this thesis is based on the detection of layer boundaries and rock types from high-resolution well logs and on the estimation of layer-by-layer properties using numerical simulation of resistivity, nuclear, and NMR logs. Layer properties were iteratively adjusted until the available well logs were reproduced by numerical simulations. This method honors the reservoir geology and physics of the measurements while adjusting the layer properties; it reduces shoulder-bed effects on well logs, especially across thinly bedded and laminated sequences, thereby yielding improved estimates of interconnected porosity and permeability in rocks that have null mobile water saturation and that were invaded with OBM. Additionally, dynamic simulations of OBM invasion in free-water depth intervals were necessary to estimate permeability. It is found that NMR transverse relaxation measurements are effective for determining rock and fluid properties but are unreliable in the accurate calculation of porosity and permeability in thinly bedded and highly laminated depth sections. In addition, this thesis shows that low resistivity values are associated with the presence of microporosity, and high resistivity values are associated with the presence of interconnected and vuggy porosity. In some layers, a fraction of the vuggy porosity is associated with isolated pores, which does not contribute to fluid flow. An integrated evaluation using multiple measurements, including sonic logs, is therefore necessary to detect isolated porosity. After the correction and simulation, results show, on average, a 34% improvement between estimated and core-measured permeability. Closer agreement was not possible because of limitations in tool resolution and difficulty in obtaining a precise depth match between core and well-log measurements. / text

La maladie coronaire d'origine ischémique reste l'une des principales causes de mortalité dans le monde industrialisé. Le traitement de l'ischémie aiguë du myocarde est cependant entré dans une nouvelle ère où la mortalité peut être diminuée de moitié en utilisant des procédures qui permettent un retour rapide du débit sanguin dans la zone ischémique du myocarde, c'est-à-dire la revascularisation. Toutefois, cette reperfusion entraîne par elle-même des complications appelées lésions de la reperfusion qui ont été décrites pour la première fois par Jennings et al., en 1960. Par conséquent, le développement de stratégies cardioprotectrices associées à la reperfusion constitue un besoin majeur en clinique afin d'améliorer la fonction myocardique, de diminuer l'incidence des arythmies, de retarder l'apparition de la mort cardiomyocytaire et de limiter la taille de l'infarctus du myocarde lors de l'ischémie/reperfusion (I/R). La découverte de deux formes principales de mécanismes cardioprotecteurs endogènes, qui consistent en la réalisation de brefs cycles d'I/R appliqués avant l'ischémie (pré-conditionnement ischémique) ou après une longue période d'ischémie au début de la reperfusion (post-conditionnement ischémique), a encouragé la recherche de nouveaux moyens pharmacologiques capables de protéger le myocarde ischémié/reperfusé et a développé nos connaissances sur les bases moléculaires des lésions et de la survie cellulaire au cours des processus d'I/R.L'étude des mécanismes responsables de l'induction de la mort cellulaire a permis de mettre en évidence le rôle prépondérant joué par la mitochondrie et l'augmentation de la perméabilité de ses membranes, induite notamment par la formation/ouverture d'un pore au niveau des points de contacts entre les membranes mitochondriales ; ce pore a été appelé " pore de transition de la perméabilité mitochondriale " (mPTP). L'inhibition de l'ouverture de ce pore apparaît comme une stratégie privilégiée pour protéger le myocarde.De nombreuses études ont montré que les espèces réactives d'oxygène (EROs) jouent un rôle majeur dans les lésions de l'I/R et dans l'ouverture du mPTP. En revanche, il existe peu d'informations claires sur le seuil et la période de production (ischémie et/ou reperfusion) des EROs qui conduisent à l'ouverture du mPTP.Par conséquent, nous avons mis au point un modèle cellulaire d'hypoxie/réoxygénation (H/R) afin d'établir une relation causale entre la production d'EROs, l'ouverture du mPTP et la mort cellulaire tout en explorant le rôle de différents types d'EROs.Ce modèle d'H/R développé sur des cardiomyocytes de rats adultes fraîchement isolés nous a permis de mesurer en temps réel et simultanément la production des EROs, l'ouverture du mPTP et la mort cellulaire. Nous avons montré que la production des EROs débute pendant la période d'hypoxie et que cette production est directement liée à l'augmentation du temps d'hypoxie. Cette production d'EROs à l'hypoxie, plus particulièrement de radicaux hydroxyles et de peroxyde d'hydrogène, a été directement relié, et ceci pour la première fois, à l'ouverture du mPTP et à la mort cellulaire lors de l'H/R.Nous avons utilisé ce modèle pour étudier le mécanisme d'action de deux stratégies pharmacologiques cardioprotectrices, un nouveau ligand de la protéine translocatrice mitochondriale (TSPO), le TRO 40303, et l'activation de la voie RISK (Reperfusion Injury Salvage Kinase) par la morphine. Nous avons ainsi montré que (1) les propriétés cardioprotectrices du TRO40303 sont associées à une inhibition de l'ouverture du mPTP, ce qui n'avait pas pu être démontré au moyen d'expériences réalisées ex vivo et (2) l'activation de la voie RISK par la morphine, qui aboutit à une limitation de la taille d'infarctus associée à une amélioration des fonctions respiratoires mitochondriales, entraîne également une inhibition de l'ouverture du mPTP et un retard de la mort cellulaire des cardiomyocytes isolés soumis à une H/R.La suite logique de ce travail sera de rechercher si l'inhibition du stress oxydant peut constituer un mécanisme commun aux deux stratégies pharmacologiques cardioprotectrices que nous avons décrites en utilisant notre modèle d'H/R. Pour cela, il serait possible d'étendre notre modèle à des animaux génétiquement modifiés pour appréhender plus précisément les phénomènes impliqués dans cette activité antioxydante.A plus long terme, il sera nécessaire d'approfondir nos connaissances sur la production d'EROs pendant l'I/R en recherchant plus spécifiquement l'origine de cette production, notamment le rôle joué par la mitochondrie et l'effet d'autres espèces réactives dans le but de cibler le traitement et de développer de nouvelles stratégies cardioprotectrices.

A P56S mutation in the VAPB MSP domain is linked to adult-onset amyotrophic lateral sclerosis 8. The objective of this study is to characterize the functional role of VAPB in transport of NE and NPC proteins from the ER to the NE. Over-expression of VAPB-P56S blocked the transport of nucleoporins (Nups) and NE proteins, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanines in an acidic track) antagonizes mutant VAPB effects and restores transport to the NE. VAPB function is required for transport to the NE because knockdown of endogenous VAPB recapitulates this phenotype. Moreover, the compartment in which Nups and NE proteins are sequestered and retained was identified as ER-Golgi intermediate compartment (ERGIC). Moreover, a defect in the transport of NE and NPC proteins attenuates nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Further, VAPB-P56S which is only soluble in SDS was solubilized in the Triton-X-100 fraction similar to VAPB-WT upon co-transfection with the FFAT motif suggesting that FFAT interacts with the insoluble VAPB-P56S protein changing its biophysical properties.

Recent studies have identified Two-Pore Channels (TPCs) as the channels activated by NAADP. To date, most studies that characterized these channels have employed heterologous expression or overexpression systems. The research reported here has focused principally on endogenous TPC activity by using single and dual gene knockout (KO) in a mouse system and has yielded insights into TPC expression levels, subcellular localisation, NAADP binding, and channel function. Mouse models that had been generated by both the “gene-trapping” and the “genetargeting” techniques were obtained and validated. These included a knock-down strain (“hypomorph”). Surprisingly, all TPC mutant mice showed no gross phenotypes. In addition to the two known isoforms in mouse, TPC1 and TPC2, the expression of a shorter variant of TPC1 was discovered; this has an alternative (truncated) N-terminus, and has been termed (DELTA)N-TPC1. All TPC variants/isoforms were widely expressed in all mouse tissue types tested. Overexpression of mouse TPCs in mouse embryonic fibroblasts showed that (DELTA)N-TPC1 and TPC2 were expressed primarily in late endosomes/lysosomes while TPC1 was expressed in both endosomes and lysosomes. Dileucine sorting motifs target TPCs to late endosomes/lysosomes; it was shown that truncation or mutation of dileucine motifs significantly reduced localization in late endosomes/lysosomes. Furthermore, TPCs were shown not to be the direct binding target of NAADP, as the high affinity NAADP binding was retained in hepatic membranes from TPC double KO (DKO) mice. It is concluded that NAADP binds to an (as yet, unidentified) accessory protein. The functional role of TPCs was studied in depth using mouse pancreatic acinar cells. NAADP is known to release Ca²⁺ from the acidic stores in response to the stimulation by the hormone cholecystokinin (CCK). In all TPC mutant mice, CCK was still able to evoke Ca²⁺ oscillations, but with slower and attenuated oscillations in the TPC1 hypomorph, and with slower oscillations in TPC DKO. In all TPC KOs, oscillations were disrupted by known inhibitors of the NAADP-signalling pathway (Ned-19, GPN and bafilomycin A1), indistinguishable from the responses with wild-type cells. This suggests that TPCs are not involved in CCK signalling, although it is possible that functional compensation masked the phenotype arising from the impaired signalling.