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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
21

Differential Recruitment of Host Proteins to the Coxiella Burnetii Vacuole in the Absence of the Sterol Reductase CBU1206

Ratnayake, Rochelle Chashmi 08 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Q fever is a heavily underdiagnosed and underreported infection caused by the obligate intracellular pathogen Coxiella burnetii. Following entry into the host cell, Coxiella replicates in the acidic phagolysosome-like parasitophorous vacuole termed the Coxiella Containing Vacuole (CCV). The CCV is a large and highly fusogenic compartment that actively fuses with the host endocytic pathway during maturation of the phagolysosome. Evidence suggests that the development of the CCV is sensitive to increasing cholesterol levels and leads to CCV acidification and bacterial death. Therefore, we hypothesize that CCV cholesterol concentration is carefully modulated through the Coxiella encoded sterol reductases (CBU1206 and CBU1158). A ∆CBU1206 mutant of Coxiella is hypersensitive to cholesterol and displays growth defects in intracellular replication and CCV development. Following fusion with the host endocytic pathway, the Coxiella NMII Phase II (WT) CCVs readily acquire host proteins such as LAMP1, CD63, Rab7, ORP1L, RILP, and LC3. These heterotypic events with the host endosomal cascade are presumed to provide selected subsets of endocytosed cargo and membrane. Therefore, I investigated whether ΔCBU1206 CCV heterotypic fusion events are defective due to altered lipid content on the CCV membrane. I observed increased accumulation of sterols on the ΔCBU1206 CCV membrane. Similar to WT, the mutant readily fuses host lysosomes and readily acquires the host glycoprotein LAMP1 but displays reduced localization of CD63 (LAMP3). Additionally, reduced localization of the late endosomal markers Rab7, ORP1L, and RILP was observed suggesting that late endosome fusion maybe defective in ΔCBU1206. Further, reduced localization of LC3 was also observed suggesting that the mutant may also be defective in fusing with autophagosomes. Finally, the mutant possesses a functional Type 4 Secretion System that secretes a moderate amount of effector proteins relative to WT. Considering the vast array of functions accomplished by the effectors secreted, the moderate effector secretion by the mutant could influence the endocytic pathway fusion processes as well as CCV development. Collectively, this body of work suggests that the lack of sterol reductase CBU1206 in Coxiella results in defective heterotypic fusion events of the CCV membrane that could alter pathogenesis and CCV expansion.
22

Caracterisation physiologique et fonctionnelle du transporteur anionique ATCLC-C chez Arabidopsis Thaliana / Physiological and functional characterization of the anion transporter AtCLC-c in Arabidopsis thaliana

Kroniewicz, Laetitia 25 January 2011 (has links)
Chez les végétaux supérieurs, la régulation des mouvements stomatiques permet de contrôler les échanges de CO2 et la montée de la sève brute tout en limitant les pertes excessives d'eau par transpiration. Ce contrôle est assuré par des variations rapides de la turgescence des deux cellules de garde formant le stomate dues à l'activité de nombreux canaux et transporteurs ioniques. Nous avons identifié un nouveau membre de la famille des CLC chez A. thaliana, AtCLC-c exprimé dans la cellule de garde. L'étude de l'expression d'AtCLC-c et du phénotype de mutants invalidés ont permis de démontrer son rôle dans l'ouverture stomatique à la lumière et la fermeture en réponse à l'ABA. Les mutants clcc accumulent moins d'ions Cl- dans leurs cellules de garde par rapport aux plantes sauvages et sont hypersensibles à un stress salin. Enfin, nous avons confirmé par des études d'électrophysiologie la sélectivité d'AtCLC-c aux ions Cl-. L'ensemble de ce travail montre l'importance du transporteur vacuolaire d'ions Cl- AtCLC-c dans les mouvements stomatiques et la tolérance au stress salin. / In plants, the high turgor is assured by ion transport and involves the creation and maintenance of a large vacuolar volume. In recent years, various chloride channels and transporters have been identified to be involved in specific functions such as plant nutrition, stomatal movements or metal tolerance. We have characterized a new member of the CLC family in A. thaliana, AtCLC-c, highly expressed in guard cell and up-regulated by ABA and salt treatment in the whole plant. Knock-out mutants in AtCLC-c are impaired in light-induced stomatal opening and ABA-induced stomatal closing correlated to a large decrease in guard cell Cl- content. Furthermore, clcc mutants are hypersensitive to salt stress compared to wild-type. Finally, using electrophysiological studies, we demonstrated that AtCLC-c is selective to Cl-. Altogether, this work shows that AtCLC-c is a tonoplastic Cl- transporter involved in stomatal movements and salt tolerance.
23

Toxoplasma gondii : étude du réseau de nanotubes membranaires de la vacuole parasitophore et des protéines GRA associées / Toxoplasma gondii,parasitophorous vacuole,dense granules,PI(4,5) P2,membranous tubules , amphipathic alpha helices

Bittame, Amina 14 January 2011 (has links)
Dans la cellule hôte, Toxoplasma gondii se développe dans une vacuole parasitophore (VP) caractérisée par un réseau de nanotubes membranaires (RNM) dont la composition, le mécanisme de formation et la fonction sont obscures. Quelques protéines GRA, dont GRA2 et GRA6, sont sécrétées dans la VP à partir des granules denses puis ciblées au RNM. Cette localisation s'accorde avec l'hélice alpha-hydrophobe de GRA6 et les hélices alpha-amphipathiques de GRA2. Avant et après sécrétion dans la VP, les protéines GRA sont partiellement solubles. Le phénotype de parasites délétés de leur(s) gène(s) GRA2 et/ou GRA6 révèle que ces 2 protéines sont indispensables à la formation du RNM. J'ai montré 1) qu'avant leur insertion dans les membranes de la VP, la solubilité des protéines GRA est préservée grâce à des interactions hydrophobes avec peut être, des micelles de l'espace vacuolaire ; 2) que GRA12, une nouvelle protéine du RNM, n'interagit pas avec GRA2 dans ces membranes. 3) que l'adressage spécifique de GRA6 au RNM est déterminé par son domaine N-terminal hydrophile. 4) J'ai montré que GRA2 recombinante a une affinité pour le phosphatidyl inositol (4, 5) diphosphate avec lequel elle interagit via ses hélices alpha-amphipathiques. GRA2 déforme des liposomes de courbure membranaire importante pour générer de courts tubules membranaires. La tubulation est accentuée par GRA6 qui s'associe aux liposomes, quelque soit leur diamètre. Ces résultats valident le rôle direct de GRA2 et GRA6 dans la formation du RNM et laissent envisager un modèle de sa formation, dans lequel GRA6 favoriserait l'assemblage de vésicules lipidiques que GRA2 fusionnerait en tubules membranaires. / Within the host cell, Toxoplasma gondii multiplies in a parasitophorous vacuole (PV) characterized by a membranous nanotubular network (MNN). Its components, the mechanism of its formation and its function remain unknown. A few GRA proteins, including GRA2 and GRA6, are secreted from the dense granules into the PV and are targeted to the MNN. This location is in agreement with the hydrophobic alpha-helix predicted in GRA6 and with the GRA2 amphipatic alpha-helices. However, before and after their secretion in the PV, the GRA proteins are partially soluble. The phenotypic analysis of parasites deleted from their GRA2 and/or GRA6 gene(s) had shown that both these proteins are indispensable for MNN formation. During my thesis, I showed that before their insertion into the PV membranes, the GRA proteins solubility is preserved by establishing hydrophobic interactions, likely with micelles in the PV space. I also showed that GRA12, a novel MNN-associated protein, does not interact with GRA2 within these membranes. Using GRA6 as a model of study, I contributed to demonstrate that the GRA6 specific targeting to the MNN relies on its N-terminal hydrophilic domain. I demonstrated that recombinant GRA2 recognizes inositol (4, 5) biphosphate with which it interacts via its amphipatic alpha-helices. GRA2 deforms liposomes of steep membrane curvature into short membranous tubules. The tubulation is increased by GRA6 which associates with liposomes independently of their diameter. These results validate the direct role of both GRA2 and GRA6 in MNN formation and led us to propose a model in which GRA6 would tether vesicles, the fusion of which would be induced by GRA2.
24

Le couplage nitrate/proton au sein de l’échangeur AtClCa est essentiel à la physiologie de la plante en réponse aux fluctuations environnementales / Nitrate/proton coupling in AtClCa exchanger is required for plant physiology in response to environment fluctuations

Hodin, Julie 20 June 2018 (has links)
Chez les plantes, le nitrate est un élément essentiel mais sa disponibilité dans le sol est fluctuante. Il est donc stocké dans la vacuole grâce à un échangeur nitrate/proton appelé AtClCa. La famille de protéines ClCs comporte à la fois des échangeurs mais aussi des canaux suggérés comme issus de l’évolution des échangeurs par une conversion mécanistique. Chez Arabidopsis thaliana, seuls des ClCs échangeurs assurent la gestion du nitrate. Deux glutamates très conservés, E203 et E270 dans AtClCa, sont essentiels pour le transport des protons chez les ClCs échangeurs. La mutation du résidu E203 en une alanine, un acide aminé non protonable (E203A) a permis de produire artificiellement une telle conversion mécanistique. Afin de mieux comprendre l’importance physiologique du mécanisme d’échange, une analyse a été conduite sur des plantes exprimant la forme mutée d’AtClCa pour ce glutamate. Chez ces plantes, le stockage vacuolaire est fortement réduit au profit d’une importante assimilation accroissant la teneur en protéines. En dépit de cela, elles présentent un défaut de production de biomasse résultant en grande partie d’une perturbation de l’homéostasie hydrique. Elles sont également plus sensibles aux stress hydrique et probablement azoté. La conservation d’un échangeur est donc requise pour croitre en dépit des fluctuations environnementales. En parallèle, la mutation E270A a été introduite en plante afin d’étudier son importance sur la physiologie d’Arabidopsis. Une analyse préliminaire de la biomasse et des contenus en nitrate et eau de plantes exprimant la forme mutée de ce glutamate est donc présentée dans la seconde partie de cette thèse. / Nitrate is a major element for plant but its availability is very fluctuant in soils. Then, it is stored in vacuoles thanks to a nitrate/proton exchanger named AtClCa. In ClCs, exchangers but also channels were identified, the latest were suggested to be evolved from exchanger in which a mechanistic switch happened. In Arabidopsis thaliana, only exchangers are involved in nitrate management. Two conserved glutamate, E203 and E270 in AtClCa, are essential for protons transport in ClCs exchangers. The mutation of E203 into an alanine, a non-protonable amino acid (E203A) artificially produces such a mechanistic switch. To better understand the physiological importance of this exchange mechanism, a study was conducted in plants expressing the mutated form of AtClCa for this glutamate. In those plants, the vacuolar storage is highly restricted whereas the assimilation is favoured and the protein content increased. Despite that, the biomass production is decreased mostly because of a hydric homeostasis disruption. Those plants are also more sensitive to hydric and probably nitrogenous stress. The exchanger conservation is then required for plant growth whatever the environmental fluctuations. In parallel, the mutation E270A was introduced in planta to study its physiological importance. A preliminary analysis of plant biomass and nitrate and water contents was then performed in plants expressing the E270A mutated form of AtClCa and the results are presented in the second part of the manuscript.
25

Altérations des entrées synaptiques et origine de la vacuolisation dans les motoneurones de souris sod1g93a, modèle de la sclérose latérale amyotrophique / Alteration of synaptic inputs and origin of vacuolation in SOD1 mice motoneurons, model of amyotrophic lateral sclerosis

Martinot, Clemence 30 June 2017 (has links)
La Sclérose Latérale Amyotrophique (SLA) est une maladie neurodégénérative au cours de laquelle les motoneurones meurent. Le premier dysfonctionnement des motoneurones est la rétractation de leurs jonctions neuromusculaires. La présence de vacuoles a été décrite dans l’axone et les dendrites des motoneurones avant la dénervation dans les souris SOD1G93A, modèle murin de la maladie. L’origine des vacuoles n’est pas connue. On peut toutefois se demander si elle pourrait résulter d’un stress excitotoxique. L’excitotoxicité pourrait provenir soit d’une hyperexcitabilité intrinsèque du motoneurone, soit d’une hyperexcitation (balance des entrées excitatrices et inhibitrices modifiées au profit d’une plus grande excitation). Or il a été montré que si les motoneurones sont hyperexcitables au stade embryonnaire dans les souris SOD1G93A, seuls les motoneurones résistants à la SLA (type S) sont hyperexcitables à la deuxième semaine postnatale tandis que les motoneurones vulnérables (types FF et FR) deviennent hypoexcitables avant leur dégénérescence chez l’adulte. Nous avons donc étudié les entrées synaptiques reçues par les motoneurones, pour savoir si la balance excitation/inhibition est déplacée et s’ils sont ainsi hyperexcités. Pour cela nous avons réalisé des enregistrements électrophysiologiques de motoneurones lors de la stimulation de circuits pré-moteurs, et des marquages intracellulaires de motoneurones combinés avec des marquages immunohistochimiques des boutons VGlut1, VGlut2 et Vgat. Nous avons montré que l’amplitude des PPSE monosynaptiques Ia était diminuée dans les souris SOD1, les PPSI di- et trisynaptiques étaient moins nombreux et les interneurones inhibiteurs moins excitables. Cette modification des entrées synaptiques n’était pas due à un changement du nombre de synapses. En revanche, les synapses sont particulièrement nombreuses aux domaines dendritiques qui se vacuolisent dans les souris SOD1, suggérant un lien entre l’activité synaptique et la vacuolisation. Des marquages intracellulaires de motoneurones de souris SOD1, montrent que les vacuoles grandissent avec l’évolution de la maladie, suggérant leur implication dans le processus de dégénération. Grâce à des révélations immunohistochimiques, nous avons montré que ces vacuoles apparaissent dans l’espace intermitochondrial lors de la dégénérescence des mitochondries. Le réticulum endoplasmique est également impliqué. Enfin, l’autophagie, mécanisme d’élimination des organites cellulaires, est déficient au moment de l’apparition des vacuoles, expliquant pourquoi elles s’accumulent avec le temps. Ces résultats amènent à reconsidérer l’hypothèse de l’excitotoxicité supposée comme mécanisme à l’origine de la mort des motoneurones. / Glutamate excitotoxicity arising from excessive entry of calcium in the cell, has long been suggested to contribute to the degeneration of motoneurons in Amyotrophic Lateral Sclerosis (ALS). This hypothesis is enhanced by the observation of vacuoles on motoneurones dendritic tree. Such vacuoles were previously observed on neurons under excitotoxic stress. Excitotoxicity may stem from an intrinsic hyperexcitability of the motoneurons or from a shift of the balance of excitatory / inhibitory inputs received by the motoneurons toward more excitation. Thanks to an in vivo preparation that allows us to make intracellular recordings of motoneurons in adult mice, it was shown that spinal motoneurons do not display an intrinsic hyperexcitability just prior to their degeneration in SOD1 G93A mice, the standard model of ALS. Thus, to study excitotoxicity hypothesis, we decided to study dendritic vacuoles and undersand their genesis, and then to study synaptic inputs on motoneurons, to decipher if there is a hyperexcitability. We have shown, with intracellular labelling and immunohistochemistry, that vacuoles grow with age, that they appear in the intermembrane space of mitochondria, and that deficiency in autophagy prevent their elimination. With electrophysiological recordings, we have shown that monosynaptic EPSP amplitude is reduced in SOD1 mice. IPSP were less numerous and inhibitory interneurons were less excitable. These alterations were not due to synapses numbers, however synapses are preferentially localised on dendritic places that vacuolate, suggesting a link between synaptic activity and vacuolation. These results suggest that excitotoxicity might not be the mecanism of motoneuron death.
26

Etude de l'adressage des protéines GRAs transmembranaires de Toxoplasma gondii aux granules denses et de leur insertion membranaire post-sécrétoire.

Gendrin, Claire 06 December 2007 (has links) (PDF)
Parmi les mécanismes de survie intracellulaire connus, l'export de protéines solubles ou transmembranaires visant à modifier différents compartiments de la cellule-hôte est une stratégie employée par de nombreux pathogènes. Chez Toxoplasma gondii, il a été montré que les granules denses (GD) constituent la voie de sécrétion par défaut pour les protéines solubles. Par contre, le tri de protéines transmembranaires vers les GD et leur maintien sous forme soluble avant insertion membranaire post-sécrétoire font appel à des mécanismes originaux qui ont fait l'objet de ces travaux.<br />Chez le Toxoplasme, la protéine de GD GRA5 est adressée à la membrane de la vacuole parasitophore (MVP) après sécrétion. Exprimée en cellules de mammifères, GRA5 est adressée à la membrane plasmique avec une topologie de type I, ce qui démontre la particularité des mécanismes de sécrétion chez T. gondii. Par une approche basée sur des protéines chimériques présentant des domaines spécifiques de GRA5 et d'une protéine transmembranaire de la membrane plasmique parasitaire (MPP), nous avons pu identifier les déterminants de l'adressage à la MPP versus à la MVP. Nous avons ainsi pu démontrer que le domaine Nt de GRA5 est impliqué dans l'adressage soluble aux GD et est essentiel pour l'insertion membranaire post-sécrétoire dans la MVP. Ces résultats, qui ont été étendus à une autre protéine GRA transmembranaire (GRA6), divergent de l'idée largement répandue selon laquelle les signaux d'adressage des protéines transmembranaires seraient présents dans la queue C-terminale et/ou dépendraient de la longueur du domaine transmembranaire de ces protéines.
27

Die parasitophore Vakuolenmembran der Mikrosporidienspezies Encephalitozoon cuniculi enthält keine endophagosomalen Markerproteine / The parasitophorous vacuole membrane of Encephalitozoon cuniculi lacks host cell membrane proteins

Fasshauer, Verena 11 December 2009 (has links)
No description available.
28

Effects of Collagen Gel Stiffness on Cdc42 Activities of Endothelial Colony Forming Cells during Early Vacuole Formation

Kim, Seung Joon 14 August 2013 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Recent preclinical reports have provided evidence that endothelial colony forming cells (ECFCs), a subset of endothelial progenitor cells, significantly improve vessel formation, largely due to their robust vasculogenic potential. While it has been known that the Rho family GTPase Cdc42 is involved in this ECFC-driven vessel formation process, the effect of extracellular matrix (ECM) stiffness on its activity during vessel formation is largely unknown. Using a fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor, we examined the spatio-temporal activity of Cdc42 of ECFCs in three-dimensional (3D) collagen matrices with varying stiffness. The result revealed that ECFCs exhibited an increase in Cdc42 activity in a soft (150 Pa) matrix, while they were much less responsive in a rigid (1 kPa) matrix. In both soft and rigid matrices, Cdc42 was highly activated near vacuoles. However, its activity is higher in a soft matrix than that in a rigid matrix. The observed Cdc42 activity was closely associated with vacuole formation. Soft matrices induced higher Cdc42 activity and faster vacuole formation than rigid matrices. However, vacuole area is not dependent on the stiffness of matrices. Time courses of Cdc42 activity and vacuole formation data revealed that Cdc42 activity proceeds vacuole formation. Collectively, these results suggest that matrix stiffness is critical in regulating Cdc42 activity in ECFCs and its activation is an important step in early vacuole formation.
29

Molecular Mechanisms of Notochord Vacuole Formation and Their Role in Zebrafish Development

Ellis, Kathryn Leigh January 2014 (has links)
<p>The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. While these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a, and H+-ATPase-dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role for notochord vacuoles in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.</p> / Dissertation
30

Anaplasma phagocytophilum remodels its host cell-derived vacuole into a protective niche by redecorating the vacuolar membrane with select Rab GTPases and bacterial proteins

Huang, Bernice 11 November 2011 (has links)
Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to cause the emerging tick-transmitted disease, human granulocytic anaplasmosis (HGA). Following entry, the pathogen replicates within a host cell-derived vacuole that fails to mature along the endocytic pathway, does not acidify, and does not fuse with lysosomes. Selective fusogenicity is prototypical of many vacuole-adapted pathogens and has been attributed, at least in part, to pathogen modification of the vacuolar inclusion membrane and/or to selective recruitment or exclusion of host trafficking regulators. As a result, the A. phagocytophilum-occupied vacuolar membrane (AVM) provides a unique interface to study the host-pathogen interactions critical to A. phagocytophilum intracellular survival. Diverse vacuole-adapted pathogens; including Chlamydia, Legionella, and Salmonella; selectively recruit host Rab GTPases to their vacuolar membranes to establish replicative permissive niches within their host cells. Rab GTPases coordinate many aspects of endocytic and exocytic cargo delivery. We determined that the A. phagocytophilum-occupied vacuole (ApV) selectively recruits a subset of fluorescently-tagged Rabs that are predominantly associated with recycling endosomes. Another emerging theme among vacuole-adapted pathogens is the ability to hijack ubiquitin machinery to modulate host cellular processes. Mono- and polyubiquitination differentially dictate the subcellular localization, activity, and fate of protein substrates. Monoubiquitination directs membrane traffic from the plasma membrane to the endosome and has been shown to promote autophagy. We show that monoubiquitinated proteins decorate the AVM during infection of promyelocytic HL-60 cells, endothelial RF/6A cells, and to a lesser extent, embryonic tick ISE6 cells. Importantly, tetracycline treatment concomitantly promotes loss of the recycling endosome-associated GFP-Rabs and ubiquitinated proteins and acquisition of the late endosomal marker, Rab7, and lysosomal marker, LAMP-1, implicating bacterial-derived proteins in the ApV's altered fusogenicity. Therefore, we rationalized that A. phagocytophilum-encoded proteins that associate with the AVM may establish interactions with the host cell that are important for intracellular survival. By focusing on A. phagocytophilum proteins that are induced during host infection, we identified the first two bacterial-encoded proteins -- APH_1387 and APH_0032 -- that modify the AVM. Although functional studies are hindered by the lack of a system to genetically manipulate Anaplasma, the pathobiological roles of APH_1387 and APH_0032 are likely unique, as both proteins exhibit very little or no homology with any previously described protein. APH_1387 and APH_0032 are present at the cytoplasmic face of the AVM, therefore they likely interact with host proteins. We demonstrate that ectopic expression of APH_1387 and APH_0032 inhibits the ApV development in A. phagocytophilum infected cells. The results presented in this dissertation contribute to our understanding of how A. phagocytophilum modifies the vacuolar membrane in which it resides to establish a safe haven and evade lysosomal degradation.

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