Dissertations / Theses on the topic 'Phosphoinositides'
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1
Coghlan, Samuel William. "Phosphoinositides : methodology, synthesis, application." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612939.
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Bothmer, John. "Phosphoinositides, aging and Alzheimer's disease." Maastricht : Maastricht : Universitaire Pers Maastricht ; University Library, Maastricht University [Host], 1992. http://arno.unimaas.nl/show.cgi?fid=6504.
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Fang, Xiaoming. "Phosphoinositides in ciliary transport and ciliopathies." Thesis, University of Sheffield, 2018. http://etheses.whiterose.ac.uk/19194/.
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Cilia are finger-like protrusions that function in the detection and processing of a wide variety of signals. In vertebrates, they are intimately involved in early embryonic patterning, the differentiation and function of sensory neurons, morphogenesis and physiology of duct epithelia, cell motility and metabolism. Lipid composition of the ciliary membrane and its contribution to cilia function is one of the least studied areas of cilia biology. Here I use a collection of transgenic lines to determine the phosphoinositide content of the ciliary membrane and to test the role of phosphoinositides in cilia morphogenesis and function. My studies reveal that specific types of phosphoinositides are found in the ciliary membrane and that their content plays an important role in cilia morphology and function. Moreover, I generated a zebrafish model of a human ciliopathy to test whether some aspects of its mutant phenotype can be alleviated by manipulating the phosphoinositide content in the ciliary membrane. Finally, my studies developed a method that is generally applicable to the manipulation of ciliary content of other PIs in a living vertebrate using other enzymes.
4
Blank, Jonathan Louis. "The phospholipase C specific for the phosphoinositides." Thesis, University of Nottingham, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252943.
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Zhang, Xuxiao. "How are class I phosphoinositide 3-kinases regulated?" Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609245.
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Olsson, Henric. "Phosphatidylinositol 4-kinases in rat liver characterization of two forms with different subcellular distribution /." Lund : Biochemistry, Chemical Centre, University of Lund, 1994. http://books.google.com/books?id=L_xqAAAAMAAJ.
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Loovers, Harriët Maria. "Phosphoinositides and inositol 5-phosphatases in Dictyostelium discoideum /." Enschede : PrintPartners Ipskamp, 2005. http://www.gbv.de/dms/goettingen/479206880.pdf.
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Bernier, Louis-Philippe. "Functional regulation of P2X receptor channels by phosphoinositides." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110361.
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Long after its initial discovery as part of DNA and as the main source of energy in the cell, the nucleotide adenosine-5'-triphosphate (ATP) is now rightfully considered as an important extracellular signalling molecule. It is now known that purinergic transmission plays significant physiological roles, notably in neuro- and gliotransmission, in the modulation of the innate and adaptive immune response, in smooth muscle constriction and in regulating blood clotting. It is also actively involved in the generation and maintenance of various pathological states, including chronic neuropathic and inflammatory pain.How purinergic signalling can be implicated in such a diverse array of mechanisms via only one signalling molecule comes from the variety of receptors that are activated by ATP. Two major types of ATP-sensitive receptors exist: the P2X ionotropic receptor channels and the metabotropic P2Y receptors. The P2X family of ATP-gated ion channels is composed of seven subunits that assemble as trimers to form receptors with various functional and pharmacological profiles. Like all ion channels, the activity of P2X receptor channels is tightly regulated by orthosteric and allosteric regulation mechanisms. This thesis provides evidence of a novel type of post-translational regulation mechanism where the levels of intracellular phosphoinositides (PIPn) modulate the channel activity of P2X receptors. The first article included in my thesis focuses on the P2X1 subtype, which is mainly involved in smooth muscle constriction in blood vessels and vas deferens, as well as in the control of platelet aggregation and blood clotting. We demonstrate that the activity of the P2X1 receptor channel is positively regulated by membrane PI(4,5)P2 (PIP2). Depleting the intracellular levels of PIP2 decreased the ATP-activated current carried through the P2X1 channel and had an inhibitory effect on ATP-mediated mesenteric artery constriction, a P2X1-dependent process. Direct binding between the P2X1 cytosolic tail and PIP2 is shown to be necessary for the full expression of P2X1 activity.The next article in this thesis describes the PIPn-dependent regulation of the P2X4 receptor channel, which is highly involved in the generation of neuropathic and inflammatory pain through its expression in spinal cord microglia. Results obtained show that complete P2X4 function is dependent on the levels of PIP2 and PI(3,4,5)P3 (PIP3). Both species of phospholipids potentiate the P2X4-mediated ionic current and calcium entry by directly binding to the C-terminal domain of P2X4 subunits. Activation of co-expressed microglial metabotropic receptors can trigger changes in PIP2 or PIP3 levels, which could affect the contribution of P2X4 in pain-inducing mechanisms.In the third report, we investigate the molecular characteristics of the interaction between membrane PIPn and the various P2X receptors that were shown to be directly modulated by the phospholipids. By analyzing the functional and PIPn-binding effects of various mutations performed on the C-terminal domain of the PIPn-sensitive P2X1, P2X4 and P2X7 subtypes as well as on the PIPn-insensitive P2X5 subtype, we identify the PIPn-binding regulatory motif of P2X receptors. The last article included in this thesis examines the dynamic changes in channel permeability brought by sustained activation of the microglial P2X4 receptor. We show that upon sustained ATP application, P2X4 channels form large conductance pores allowing the flux of large organic molecules. The large pore formation of co-expressed microglial P2X7 receptors has been extensively studied; we show here that P2X4-mediated permeation is mechanistically distinct and, in contrast with P2X7, does not induce membrane blebbing or cell death. Furthermore, we demonstrate that membrane PIPn potentiate the formation of this high-conductance P2X4 pore, suggesting that this property can be regulated by metabotropic changes in PIPn levels.Longtemps après la découverte de sa présence dans l'ADN et de son rôle en tant que principale source d'énergie chimique dans la cellule, le nucléotide adenosine-5'-triphosphate (ATP) est maintenant considéré comme une importante molécule de signalisation extracellulaire. La transmission purinergique est activement impliquée dans plusieurs processus physiologiques, notamment dans la neuro- et glio-transmission, la modulation de la réponse immunitaire innée et adaptative, la constriction vasculaire et la coagulation sanguine. Elle participe aussi à la génération et au maintien de divers états pathologiques, tels la douleur chronique neuropathique et inflammatoire.La signalisation purinergique est impliquée dans des processus aussi diversifiés par l'entremise d'une seule molécule de signalisation grâce à une grande variété de récepteurs activés par l'ATP. Les deux principaux types de récepteurs sensibles à l'ATP sont les récepteurs ionotropiques P2X et les récepteurs métabotropiques P2Y. La famille P2X de canaux ioniques activés par l'ATP est composée de sept sous-unités qui s'assemblent comme trimères pour former des récepteurs possédant divers profils fonctionnels et pharmacologiques. Comme tous les canaux ioniques, l'activité des P2X est étroitement régulée par des mécanismes de régulation orthostériques et allostériques. Cette thèse démontre l'existence d'un nouveau type de mécanisme de régulation post-traductionnel, où les niveaux de phosphoinositides (PIPn) intracellulaires modulent l'activité de canal ionique des récepteurs P2X.Le premier article porte sur le sous-type P2X1, qui contribue à la contraction des muscles lisses dans les vaisseaux sanguins et le canal déférent, ainsi qu'à l'agrégation plaquettaire. Nous démontrons que l'activité du récepteur canal P2X1 est positivement régulée par les PI(4,5)P2 (PIP2) membranaires. La déplétion des niveaux intracellulaires de PIP2 diminue l'amplitude du courant ionique induit par P2X1. Nous montrons que le couplage direct entre le domaine C-terminal cytosolique de P2X1 et PIP2 est nécéssaire pour l'expression complète de l'activité de P2X1.Le second article de cette thèse décrit la régulation PIPn-dépendante du récepteur canal P2X4, qui joue un rôle majeur dans la génération et le maintien de la douleur neuropathique et inflammatoire par son expression dans les microglies de la moelle épinière. Nous démontrons que le fonctionnement de P2X4 dépend des niveaux de PIP2 et de PI(3,4,5)P3 (PIP3). Les deux types de phospholipides potentialisent le courant ionique ainsi que l'entrée de calcium par le canal P2X4 en se liant directement au domaine C-terminal des sous-unités P2X4. Dans le troisième rapport, nous étudions les caractéristiques moléculaires de l'interaction entre les PIPn membranaires et les récepteurs P2X directement modulés par ces phospholipides. En analysant les effets fonctionnels de diverses mutations effectuées sur le domaine C-terminal des sous-types PIPn-dépendant P2X1, P2X4 et P2X7 et sur le sous-type PIPn-indépendant P2X5, nous identifions le motif nécessaire à la liaison P2X-PIPn et à la régulation fonctionnelle du canal par les PIPn.Le dernier article de cette thèse examine les changements dynamiques de la perméabilité du canal ionique apportés par une activation soutenue des récepteurs microgliaux P2X4. Nous montrons que, lors d'une application soutenue d'ATP, les canaux P2X4 forment des pores à haute conductance permettant le flux de molécules organiques à haut poids moléculaire. La formation de larges pores par les récepteurs P2X7 a été étudiée intensivement; nous démontrons ici que la perméation induite par P2X4 est mécanistiquement distincte et, à l'opposé de P2X7, ne mène à aucun réarrangement de la structure membranaire ni à la mort cellulaire. Les PIPn membranaires potentialisent la formation de ces pores à haute conductance par P2X4, suggérant que cette propriété peut être régulée par des changements intracellulaires des niveaux de PIPn.
9
Rutherford, Anna. "3-phosphoinositides and their effectors in endosomal sorting." Thesis, University of Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.434740.
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Hammond, Gerald Raymond Vere. "The role of phosphoinositides in mast cell exocytosis." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1445567/.
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The phosphoinositides (Pin) are a family of phospholipids that contain myo inositol as their headgroup. Despite being present in eukaryotic membranes with low abundance, their high rates of metabolic turnover allow them to control a plethora of cellular functions. In particular, phosphatidylinositol 4,5-&wphosphate (PtdIns(4,5)P2) regulates several processes, including preparing secretory organelles to undergo fusion with the plasma membrane in response to a stimulus (regulated exocytosis), budding and fission of vesicular cargo from the plasma membrane (endocytosis), and controlling the cortical actin cytoskeleton. In this thesis, the role of Pin in regulated exocytosis is examined in mast cells, since these undergo an acute, massive and rapid exocytosis, without any immediate endocytosis. Using a reconstitution approach, it was not possible to define which Pin are involved in exocytosis, although it was concluded that at least one Pin that is not PtdIns(4,5)P2 is required. In order to study PtdIns(4,5)P2 dynamics in primary mast cells, a novel quantitative immunofluoescence technique for Pin was established. Using this technique, PtdIns(4,5)P2 was identified at the plasma membrane of mast cells, but was depleted almost entirely during exocytosis the latter observation was confirmed using biochemical approaches. This depletion was blocked by inhibitors of phospholipase C (PLC), an enzyme that breaks down PtdIns(4,5)P2 into diacylglycerol (DAG) and the calcium mobilising messenger, inositol 1,4,5-frisphosphate (Ins(l,4,5)P3). Although PLC activity was required for initiation of calcium signalling in mast cells, experiments whereby the Ins(l,4,5)P3/calcium pathway was bypassed demonstrated further requirements for PLC activity. These were not precisely defined, but simple elimination of plasma membrane PtdIns(4,5)P2 or production of DAG were not sufficient. Both events may be required in conjunction, however. A model is proposed whereby elimination of plasma membrane PtdIns(4,5)P2 together with production of DAG may activate the protein machinery for membrane fusion during exocytosis.
11
Juss, Jatinder. "Regulation of neutrophil apoptosis by phosphoinositide 3-kinases." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610300.
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Toscano, Sarah. "Functional characterisation of PIP4K in Drosophila melanogaster." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609822.
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Pearce, Verity Quintina. "The role of the PI3K p110δ in innate and adaptive immune responses to Listeria monocytogenes." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607720.
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曾業勤 and Yip-kan Kent Tsang. "Effect of genistein on the vascular actions of lysophosphatidylcholinein the porcine coronary artery: role ofphosphatidylinositol-3-kinase." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B42903208.
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Masson, Glenn Robert. "New insights into the dynamics of phosphoinositide signalling through hydrogen deuterium exchange mass spectrometry." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709108.
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Tsang, Yip-kan Kent. "Effect of genistein on the vascular actions of lysophosphatidylcholine in the porcine coronary artery role of phosphatidylinositol-3-kinase /." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B42903208.
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Bryant, Anne-Marie M. "Physiochemical Characterization of Phosphatidylinositol-4,5-Bisphophate and its Interaction with PTEN-Long." Digital WPI, 2019. https://digitalcommons.wpi.edu/etd-dissertations/578.
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The focus of this dissertation is to understand the physicochemical factors that affect the spatiotemporal control of phosphoinositide signaling events. Despite their low abundance in cellular membranes ( ~ 1% of total lipids) phosphoinositides are assuming major roles in the spatiotemporal regulation of cellular signaling, therefore making this group of lipids an attractive area of study, especially for identifying drug targets. The main phosphoinositide studied in this dissertation is phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which regulates various intracellular signaling pathways, notably the PI3K/AKT pathway. The PI3K/AKT pathway plays a critical role in regulating diverse cellular functions including metabolism, growth, proliferation, and survival. Thus, dysregulation of the PI3K/AKT pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. PI(4,5)P2 regulates phosphoinositide signaling in the PI3K/AKT pathway through interaction of its highly anionic headgroup with polybasic proteins. The highly specific manner that allows hundreds of structurally diverse proteins to interact with lipid species found in such low supply may require the local formation of PI(4,5)P2 clusters (domains). Although a significant amount of evidence has accumulated over the past decade that supports the notion of PI(4,5)P2-rich clusters, our understanding regarding the structural determinants required for cluster formation remains limited. Studies have shown that PI(4,5)P2 clustering is induced by cellular cations interacting with PI(4,5)P2 via electrostatic interactions, suggesting that non-clustering/clustering transitions are particularly sensitive to ionic conditions. However, why some ions are more effectively cluster PI(4,5)P2 than others remains to be understood. For our first research aim, we investigated the effects of divalent (Ca2+) and monovalent cations (Na+, K+ ) on PI(4,5)P2 clustering to understand the ionic environment required for electrostatic PI(4,5)P2 cluster formation. We used monolayers at the air/water interface (Langmuir films) to monitor PI(4,5)P2 molecular packing in the presence of each cation. Our results indicated that Ca2+ individually and Ca2+ along with K+ had a greater effects on PI(4,5)P2 cluster formation than Na+ and K+, individually and combined. We hypothesize that the cations shield the negatively charged headgroups, allowing adjacent PI(4,5)P2 molecules to interact via H- bonding networks. The analysis of the electrostatic environment required for stable PI(4,5)P2 clustering will help us understand important aspects of PI(4,5)P2 mediated signaling events, such as the temporal control of protein binding to PI(4,5)P2 clusters to enhance their function. Another important spatiotemporal modulator that affects the local concentration of PI(4,5)P2 clusters is cholesterol, a steroid present in large quantities (30-40 mole%) in the plasma membrane. Cholesterol has been shown to induce the formation of liquid-ordered domains when interacting with an otherwise gel phase forming lipid, however, the interaction of cholesterol with an inner leaflet lipid species that favors more of a disordered environment to form clusters is poorly understood. We hypothesize that cations along with cholesterol work synergistically to induce PI(4,5)P2 clustering. Thus, our second research aim was to investigate the role of cholesterol on PI(4,5)P2 clustering by monitoring the molecular packing of PI(4,5)P2 in the presence of both cholesterol and cations. This aim was investigated similarly to the first aim with Langmuir trough monolayer film experiments. Our results showed that cholesterol in the presence of Ca2+ had an additive effect leading to the strongest condensation of the monolayer (increase in PI(4,5)P2 packing). Our hypothesis is that Ca2+ significantly reduces the negative electron density of the phosphate groups, allowing the cholesterol hydroxyl group to interact with PI(4,5)P2 headgroup through hydrogen-bond formation. To confirm our hypothesis, we collaborated with a computational group at the NIH that performed all-atom molecular dynamics (MD) simulations that closely agreed with our experimental data. Thus we were able to determine that the cholesterol hydroxyl group directly interacts via hydrogen-bonding with the phosphodiester group as well as the PI(4,5)P2 hydroxyl groups in the 2- and 6-position. The insight into the structural positioning of cholesterol moving closer to the PI(4,5)P2 headgroup region suggests this unique interaction is important for PI(4,5)P2 cluster formation. Other anionic lipid species are suspected to interact with PI(4,5)P2 and strengthen PI(4,5)P2 clustering. We were particularly interested in the interaction of PI(4,5)P2 with phosphatidylinositol (PI) and phosphatidylserine (PS) because both are abundant in the plasma membrane, ~6-10% and ~10-20% respectively, and both electrostatically bind to peripheral proteins. Therefore, the third research aim analyzed the capacity of PI and PS to form stable clusters with PI(4,5)P2. We hypothesize that a mixed PI/PI(4,5)P2 or PS/PI(4,5)P2 domains are ideal for protein binding, since in combination PI or PS with PI(4,5)P2 would provide the necessary negative electrostatic environment, while PI(4,5)P2 would provide the high specificity and additional electrostatics for protein binding. Langmuir trough monolayer films were used to investigate the stabilization of PI/PI(4,5)P2 and PS/PI(4,5)P2 monolayers in the presence of Ca2+. Our results showed a condensation of the monolayer for both PI/PI(4,5)P2 and PS/PI(4,5)P2 with an increase in Ca2+concentrations, which suggests that Ca2+ shields the highly negatively charged phosphomonoester groups of PI(4,5)P2 allowing PI and PS to participate in PI(4,5)P2’s hydrogen-bond network. Interestingly, both PI and PS equally stabilized PI(4,5)P2 cluster formation, therefore it is highly likely that these lipids interact in vivo to form large stable electrostatic domains required for protein binding. The first three aims provided us with information about the physiological relevant environments required for PI(4,5)P2 cluster formation, while the last aim was geared towards understanding the temporal control of protein association with phosphoinositides in the plasma membrane. Specifically, we analyzed the plasma membrane association of PTEN-L, a translation variant protein of PTEN, that has the ability to exit and enter back into cells, unlike classical PTEN. The ability of PTEN-L to facilitate entry across the anionic and hydrophobic layers of the plasma membrane (in the case of direct transport of PTEN-L across the membrane) or into phospholipid transport vesicles (in the case of vesicular transport of PTEN-L across cells) is likely due to the addition of the 173 N-terminal amino acids, the alternative translated region (ATR-domain). Thus, our fourth research aim focused on the biophysical role of the ATR-domain to associate with inner leaflet plasma membrane lipids. Using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to monitor secondary structural changes of the ATR-domain upon lipid binding, it was revealed that both PS and PI(4,5)P2 induced conformational change towards a slight increase in β-sheet content in an otherwise unstructured domain suggesting these lipids are required for ATR-domain interaction with the PM. Further studies revealed that the ATR-domain affects the integrity of PS lipid vesicles, further indicating the presence of PS is required to drive ATR-domain across the membrane. This aim provides information on ATR-domain lipid binding preferences aiding in our understanding of the biological and functional role of PTEN-L as a deliverable tumor suppressor protein. The overall goal of the research in this dissertation is to understand factors that fine-tune PI(4,5)P2 cluster formation in space and time. Our first three research aims were designed to understand the synergistic effects of spatiotemporal modulators (cations, cholesterol, and anionic lipids) on local concentration of PI(4,5)P2 clusters. Our results indicate that Ca2+, cholesterol, and the presence of anionic lipids PI and PS all induce stable domains, thus it is highly likely this is part of the biological environment required in vivo for cationic proteins to bind. The last aim, the association of the ATR-domain with phospholipids in the plasma membrane, provided evidence that PS is likely required to drive the ATR-domain across the plasma membrane. This dissertation unifies nearly two decades worth of research by shedding light on synergistic modulators of PI(4,5)P2 cluster formation (Figure 1). Thus, this work has potentially far reaching consequences for understanding temporal control of the spatially resolved protein activity.
18
Bryant, Anne-Marie M. "Physiochemical Characterization of Phosphatidylinositol-4,5-Bisphophate and its Interaction with PTEN-Long." Digital WPI, 2020. https://digitalcommons.wpi.edu/etd-dissertations/617.
Full textAbstract:
The focus of this dissertation is to understand the physicochemical factors that affect the spatiotemporal control of phosphoinositide signaling events. Despite their low abundance in cellular membranes ( ~ 1% of total lipids) phosphoinositides are assuming major roles in the spatiotemporal regulation of cellular signaling, therefore making this group of lipids an attractive area of study, especially for identifying drug targets. The main phosphoinositide studied in this dissertation is phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which regulates various intracellular signaling pathways, notably the PI3K/AKT pathway. The PI3K/AKT pathway plays a critical role in regulating diverse cellular functions including metabolism, growth, proliferation, and survival. Thus, dysregulation of the PI3K/AKT pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. PI(4,5)P2 regulates phosphoinositide signaling in the PI3K/AKT pathway through interaction of its highly anionic headgroup with polybasic proteins. The highly specific manner that allows hundreds of structurally diverse proteins to interact with lipid species found in such low supply may require the local formation of PI(4,5)P2 clusters (domains). Although a significant amount of evidence has accumulated over the past decade that supports the notion of PI(4,5)P2-rich clusters, our understanding regarding the structural determinants required for cluster formation remains limited. Studies have shown that PI(4,5)P2 clustering is induced by cellular cations interacting with PI(4,5)P2 via electrostatic interactions, suggesting that non-clustering/clustering transitions are particularly sensitive to ionic conditions. However, why some ions are more effectively cluster PI(4,5)P2 than others remains to be understood. For our first research aim, we investigated the effects of divalent (Ca2+) and monovalent cations (Na+, K+ ) on PI(4,5)P2 clustering to understand the ionic environment required for electrostatic PI(4,5)P2 cluster formation. We used monolayers at the air/water interface (Langmuir films) to monitor PI(4,5)P2 molecular packing in the presence of each cation. Our results indicated that Ca2+ individually and Ca2+ along with K+ had a greater effects on PI(4,5)P2 cluster formation than Na+ and K+, individually and combined. We hypothesize that the cations shield the negatively charged headgroups, allowing adjacent PI(4,5)P2 molecules to interact via H- bonding networks. The analysis of the electrostatic environment required for stable PI(4,5)P2 clustering will help us understand important aspects of PI(4,5)P2 mediated signaling events, such as the temporal control of protein binding to PI(4,5)P2 clusters to enhance their function. Another important spatiotemporal modulator that affects the local concentration of PI(4,5)P2 clusters is cholesterol, a steroid present in large quantities (30-40 mole%) in the plasma membrane. Cholesterol has been shown to induce the formation of liquid-ordered domains when interacting with an otherwise gel phase forming lipid, however, the interaction of cholesterol with an inner leaflet lipid species that favors more of a disordered environment to form clusters is poorly understood. We hypothesize that cations along with cholesterol work synergistically to induce PI(4,5)P2 clustering. Thus, our second research aim was to investigate the role of cholesterol on PI(4,5)P2 clustering by monitoring the molecular packing of PI(4,5)P2 in the presence of both cholesterol and cations. This aim was investigated similarly to the first aim with Langmuir trough monolayer film experiments. Our results showed that cholesterol in the presence of Ca2+ had an additive effect leading to the strongest condensation of the monolayer (increase in PI(4,5)P2 packing). Our hypothesis is that Ca2+ significantly reduces the negative electron density of the phosphate groups, allowing the cholesterol hydroxyl group to interact with PI(4,5)P2 headgroup through hydrogen-bond formation. To confirm our hypothesis, we collaborated with a computational group at the NIH that performed all-atom molecular dynamics (MD) simulations that closely agreed with our experimental data. Thus we were able to determine that the cholesterol hydroxyl group directly interacts via hydrogen-bonding with the phosphodiester group as well as the PI(4,5)P2 hydroxyl groups in the 2- and 6-position. The insight into the structural positioning of cholesterol moving closer to the PI(4,5)P2 headgroup region suggests this unique interaction is important for PI(4,5)P2 cluster formation. Other anionic lipid species are suspected to interact with PI(4,5)P2 and strengthen PI(4,5)P2 clustering. We were particularly interested in the interaction of PI(4,5)P2 with phosphatidylinositol (PI) and phosphatidylserine (PS) because both are abundant in the plasma membrane, ~6-10% and ~10-20% respectively, and both electrostatically bind to peripheral proteins. Therefore, the third research aim analyzed the capacity of PI and PS to form stable clusters with PI(4,5)P2. We hypothesize that a mixed PI/PI(4,5)P2 or PS/PI(4,5)P2 domains are ideal for protein binding, since in combination PI or PS with PI(4,5)P2 would provide the necessary negative electrostatic environment, while PI(4,5)P2 would provide the high specificity and additional electrostatics for protein binding. Langmuir trough monolayer films were used to investigate the stabilization of PI/PI(4,5)P2 and PS/PI(4,5)P2 monolayers in the presence of Ca2+. Our results showed a condensation of the monolayer for both PI/PI(4,5)P2 and PS/PI(4,5)P2 with an increase in Ca2+concentrations, which suggests that Ca2+ shields the highly negatively charged phosphomonoester groups of PI(4,5)P2 allowing PI and PS to participate in PI(4,5)P2’s hydrogen-bond network. Interestingly, both PI and PS equally stabilized PI(4,5)P2 cluster formation, therefore it is highly likely that these lipids interact in vivo to form large stable electrostatic domains required for protein binding. The first three aims provided us with information about the physiological relevant environments required for PI(4,5)P2 cluster formation, while the last aim was geared towards understanding the temporal control of protein association with phosphoinositides in the plasma membrane. Specifically, we analyzed the plasma membrane association of PTEN-L, a translation variant protein of PTEN, that has the ability to exit and enter back into cells, unlike classical PTEN. The ability of PTEN-L to facilitate entry across the anionic and hydrophobic layers of the plasma membrane (in the case of direct transport of PTEN-L across the membrane) or into phospholipid transport vesicles (in the case of vesicular transport of PTEN-L across cells) is likely due to the addition of the 173 N-terminal amino acids, the alternative translated region (ATR-domain). Thus, our fourth research aim focused on the biophysical role of the ATR-domain to associate with inner leaflet plasma membrane lipids. Using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to monitor secondary structural changes of the ATR-domain upon lipid binding, it was revealed that both PS and PI(4,5)P2 induced conformational change towards a slight increase in β-sheet content in an otherwise unstructured domain suggesting these lipids are required for ATR-domain interaction with the PM. Further studies revealed that the ATR-domain affects the integrity of PS lipid vesicles, further indicating the presence of PS is required to drive ATR-domain across the membrane. This aim provides information on ATR-domain lipid binding preferences aiding in our understanding of the biological and functional role of PTEN-L as a deliverable tumor suppressor protein. The overall goal of the research in this dissertation is to understand factors that fine-tune PI(4,5)P2 cluster formation in space and time. Our first three research aims were designed to understand the synergistic effects of spatiotemporal modulators (cations, cholesterol, and anionic lipids) on local concentration of PI(4,5)P2 clusters. Our results indicate that Ca2+, cholesterol, and the presence of anionic lipids PI and PS all induce stable domains, thus it is highly likely this is part of the biological environment required in vivo for cationic proteins to bind. The last aim, the association of the ATR-domain with phospholipids in the plasma membrane, provided evidence that PS is likely required to drive the ATR-domain across the plasma membrane. This dissertation unifies nearly two decades worth of research by shedding light on synergistic modulators of PI(4,5)P2 cluster formation (Figure 1). Thus, this work has potentially far reaching consequences for understanding temporal control of the spatially resolved protein activity.
19
Isler, Yasmin Salah Blaih. "Infrared Spectroscopic Characterization of Phosphoinositide Signaling Pathway Components." Kent State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=kent1310586304.
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Pickering, Karen. "Regulation of actin dynamics by phosphoinositides during epithelial closure." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/regulation-of-actin-dynamics-by-phosphoinositides-during-epithelial-closure(88625556-04dd-403c-8a68-8c3344af1f81).html.
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Epithelia act as protective barriers and it is therefore essential that wounded epithelia are rapidly repaired to maintain barrier function. Cells surrounding epithelial wounds become motile following wounding, which involves generating dynamic actin structures that drive closure of the wound. These actin structures include filopodia which are important in the final stage of epithelial closure in which the opposing epithelial edges are joined together. The molecular mechanisms that trigger wound edge cells to become motile are not well understood. Using Drosophila wound healing and the morphogenetic process dorsal closure as models, we find that phosphatidylinositol 3,4,5-triphosphate (PIP3) regulates epithelial closure by promoting the formation of filopodia at epithelial edges. PIP3 accumulates at epithelial edges and genetically depleting PIP3 results in reduced filopodia and defects in epithelial closure. We demonstrate that the GTPase Rac and guanine nucleotide exchange factor Myoblast City function downstream of PIP3 to promote filopodia formation. We also demonstrated that the scaffolding protein Par3/Bazooka and the lipid phosphatase PTEN are responsible for restricting the localisation of PIP3 and consequently the downstream signals to the epithelial leading edge, so acting to determine the location of filopodia formation. This project reveals a novel mechanism by which actin protrusions, required for epithelial closure, are formed in response to epithelial damage. Additionally, we have identified an additional role for PIP3 in regulating the extrusion of cells from epithelial sheets in the Drosophila embryo. This finding implicates PIP3 in the regulation of tissue homoeostasis, and could contribute to our understanding of tumour initiation as unregulated tissue growth can result in the formation of tumours.
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Lin, Thibault. "Mécanismes du transfert intercellulaire des homéoprotéines." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066346/document.
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Les homéoprotéines constituent une famille de facteurs de transcription dont le point commun est la présence en leur sein d’un domaine de liaison à l’ADN particulier, l’homéodomaine. Des travaux menés au laboratoire ont permis de mettre en avant la capacité de nombreux membres de cette famille protéique à pouvoir transférer entre cellules, ouvrant la voie à un nouveau mode d’action de ces protéines, en plus de leur action sur la transcription. L’homéodomaine joue un rôle central dans ce transfert intercellulaire car il contient des séquences requises pour les deux étapes principales d’un tel transfert intercellulaire, sécrétion et internalisation. D’un point de vue mécaniste, ces deux étapes successives du transfert intercellulaire des homéoprotéines font appel à des mécanismes désignés comme non-conventionnels car propres à ce processus. Suite à de précédentes études menées dans le laboratoire mettant en évidence un rôle crucial de la localisation subcellulaire de l’homéoprotéine ENGRAILED-2 (EN-2) dans sa sécrétion, nous avons pu mettre en avant l’existence d’une interaction directe entre EN-2 et certains lipides appartenant à la classe des phosphoinositides [notamment les PI(4,5)P2]. Nous avons ensuite montré que cette interaction est à l’origine de l’association d’une fraction de EN-2 intracellulaire avec les compartiments membranaires. Ces travaux ont également permis de mieux comprendre le rôle de certains protéoglycanes dans l’accumulation de EN-2 à la surface des cellules suite à sa sécrétion et au cours de son internalisation. Enfin nous avons pu mettre en évidence l’implication du domaine d’interaction de EN-2 avec PBX dans le transfert intercellulaire de EN-2Homeoproteins belong to a family of transcription factors which all share a common DNA binding domain, the homeodomain. Beside their action as transcription factors, homeoproteins are also able to be transferred between cells by unconventional means. The two consecutive steps of this intercellular transfer (secretion then internalisation) require sequences located in the homeodomain. Thanks to studies previously lead by our laboratory, we know that subcellular localisation of ENGRAILED-2 (EN-2) homeoprotein is a crucial step in its subsequent secretion. On this basis, we showed that EN-2 interacts directly with a certain subtype of phospholipids, phosphoinositides [e.g. PI(4,5)P2]. Then, we demonstrated than these lipids are involved in the association of EN-2 protein with membraneous compartments within the cell. PI(4,5)P2 located in the inner leaflet of the plasma membrane are also involved in the direct translocation of EN-2 across plasma membrane. This work is also focused on the role of proteoglycans, and more precisely syndecans, in EN-2 cell surface accumulation after both secretion and internalisation. At last, we also established the implication of EN-2 interaction domain with PBX in EN-2 intercellular transfer
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Dukes, Joseph Donaldson. "Investigating the link between phosphoinositides, endosomal trafficking and ESCRT function." Thesis, University of Bath, 2008. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.512257.
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The maturation of early endosomes into multivesicular bodies (MVBs) and subsequent trafficking to lysosomes is an important event for the control and silencing of endocytosed membrane receptors. The endosomal-sorting complex required for transport (ESCRT) proteins appear to play a key role in this event. Phosphatidylinositol lipids including PtdIns(3,5)P2 have been implicated in the MVB-lysosomal pathway and an ESCRT-III component CHMP3 binds to this lipid in vitro. The purpose of this thesis was to investigate the link between ESCRT proteins, PtdIns(3,5)P2 and endo-lysosomal trafficking. Firstly, a protein expressed by Salmonella, which is a phosphatase that acts on PtdIns(3,5)P2, was investigated as a potential tool for manipulating cellular PtdIns(3,5)P2 levels. Our results suggest that it is potentially a useful tool for this purpose and that expression of SopB perturbs endosome to lysosome trafficking. These findings provide further evidence for a role of PtdIns(3,5)P2 in endo-lysosomal trafficking.
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Abbott, Belinda Maree 1976. "Synthesis and structure-activity studies of antiplatelet 2-morpholinochromones." Monash University, Dept. of Medicine, 2003. http://arrow.monash.edu.au/hdl/1959.1/7606.
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Fets, Louise Victoria. "The role of PI(4,5)P₂ signalling in Dictyostelium chemotaxis." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609753.
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Mitra, Sharmistha. "Ubiquitin Modulates Tollip's PtdIns(3)P Binding and Dissociates the Dimeric State of C-Terminal Cue Domain." Diss., Virginia Tech, 2013. http://hdl.handle.net/10919/51151.
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Ubiquitylation is a highly controlled post-translational modification of proteins, in which proteins are conjugated either with monoubiquitin or polyubiquitin chains. Ubiquitin modifications on target proteins are recognized by ubiquitin-binding domains, which are found in several effector proteins. In this study, we describe for the first time how ubiquitin controls the function of the Toll-interacting protein (Tollip), which is an effector protein in the innate immune signaling pathway and an adaptor protein for endosomal trafficking. We have demonstrated that the central C2 domain of Tollip preferentially interacts with phosphoinositides with moderate affinity. Remarkably, we found that ubiquitin modulates Tollip's lipid binding. We have observed an ubiquitin dose-dependent inhibition of binding of Tollip to phosphoinositides and it does so specifically by blocking Tollip C2 domain-phosphoinositide interactions. This led us to discover that the Tollip C2 domain is a novel ubiquitin-binding domain. In addition, we have biophysically characterized the association of the Tollip CUE domain to ubiquitin and compared it with Tollip C2 domain-ubiquitin binding. The Tollip CUE domain reversibly binds ubiquitin with affinity higher than C2 domain and at a site that overlaps with that corresponding to the Tollip C2 domain. We have also found that ubiquitin binding to dimeric Tollip CUE domain induces a drastic conformational change in the protein, leading to the formation of a heterodimeric Tollip CUE-ubiquitin complex. These data suggest that ubiquitin binding to the Tollip C2 and CUE domains and ubiquitin-mediated dissociation of CUE dimer reduces the affinity of the Tollip protein for endosomal phosphoinositides, allowing Tollip's cytoplasmic sequestration. Overall, our findings will provide the structural and molecular basis to understand how Tollip works inside the cell and commit itself to cytosolic signalling or endosomal trafficking in a ligand dependent manner.Ph. D.
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Racaud-Sultan, Claire. "Etude sur l'activité de synthèse des phosphoinositides des plaquettes sanguines activées." Toulouse 3, 1989. http://www.theses.fr/1989TOU31259.
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Peng, Juan. "Étude de la septine 9 et des phosphoinositides dans la cancérogénèse hépatique." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS376.
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Le carcinome hépatocellulaire (CHC) et le cholangiocarcinome (CCA) sont 2 types de cancer primitif du foie. Le CHC est le plus fréquent, cependant l’incidence du CCA augmente partout dans le monde avec un diagnostic difficile, un mauvais pronostic et des thérapies très limitées. Ce travail avait pour objectif d'identifier des cibles pour le diagnostic et la thérapeutique du CCA. Il est basé sur l'étude de la septine 9 et des phosphoinositides (PIs). La septine 9 appartient à une famille de GTPases qui participent à l’organisation des microtubules et du cytosquelette d’actine. Les septines sont impliquées dans la cytokinèse, le trafic vésiculaire et la polarité cellulaire, elles sont aussi des partenaires importantes des PIs. Pour déterminer le rôle de la septine 9 dans le CCA nous nous sommes intéressés à son interaction avec les PIs et avec l’inhibiteur de l’inducteur et activateur de la transcription 1 (PIAS1) qui a été décrite comme une protéine pouvant agir comme une SUMO ligase pour les septines. Nous avons étudié l’expression de la septine 9 et de PIAS1 dans le CCA et le CHC. Nous avons mis en évidence un mécanisme original par lequel, la production du PtdIns5P (Phosphatidylinositol -5-phosphate) permet un recrutement de la septine 9, la stabilisation des microtubules et le transport de PIAS1 du cytoplasme vers le noyau. Il démontre un rôle important des septines en association avec les PIs dans le trafic. De plus, nous avons montré que la septine 9 est un régulateur de la signalisation de l’interféron γ qui agit au niveau de la phosphorylation de STAT1 et l’entrée de PIAS1 dans le noyau. Ce travail peut constituer une nouvelle piste pour la recherche des thérapies ciblées en immunothérapie dans le traitement de ce cancerHepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are two types of primary liver cancer. HCC is the most frequent, however the incidence of CCA increases throughout the world with a difficult diagnosis, poor prognosis and very limited therapies. The objective of this work was to identify targets for the diagnosis and treatment of CCA. It is based on the study of septin 9 and phosphoinositides (PIs). Septin 9 belongs to a family of GTPases that participate in the organization of microtubules and the actin cytoskeleton. Septins are involved in cytokinesis, vesicular trafficking and cellular polarity and are also important partners of PIs. To determine the role of septin 9 in the CCA, we investigated its interaction with PIs and with Protein inhibitory of activated STAT1 (PIAS1), which has been described as a SUMO ligase for septins. We studied the expression of septin 9 and PIAS1 in CCA and CHC. We have demonstrated an original mechanism by which la production of PtdIns5P allows the recruitment of septin 9, the stabilization of microtubules and the transport of PIAS1 from the cytoplasm to the nucleus. It demonstrates an important role of the septins in association with the PIs in trafficking. Besides, we have shown that septin 9 is a regulator of interferon γ signaling which acts at the level of the phosphorylation of STAT1 and the entry of PIAS1 into the nucleus. This work can constitute a new avenue for the research of targeted immunotherapy for this cancer
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Hobday, T. M. C. "The involvement of phosphoinositides and their derivatives in nuclear envelope assembly." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1348479/.
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Nuclear envelope (NE) reassembly during telophase and cytokinesis is an essential process for the completion of mitosis. NE reassembly is a proteolipid process that requires specific signalling and membrane compositions in order to conclude successfully. Previous research into NE reassembly has focused on the protein aspect, however the role of lipids is less well understood. There are currently two models for NE reassembly, one suggests that the endoplasmic reticulum (ER) is targeted to the chromosomes and consequently envelops them with the gaps between the tubules and sheets filled by nuclear pore complexes. The role of lipids in this model has not been investigated however it is suggested that membrane fusion is not required for NE reassembly. An alternative model based in a non-somatic system, has shown that diacylglycerol (DAG), a fusogenic lipid, is able and required to trigger localised membrane fusion in NE reassembly. DAG is a negative curvature lipid and its localised production can trigger fusion due to changes in the structure of the membrane. However, it is possible that these models are not mutually exclusive. While the fusion model has shown an important role for lipids and lipid-modifying enzymes in NE reassembly and membrane fusion, these discoveries have yet to be translated into mammalian systems. This study aims to determine the involvement of phospholipids and their derivatives in mammalian NE assembly, based on previous work from the fusion model, which has shown that DAG and PtdIns(4,5)P2 are involved in the membrane fusion which forms a complete and functional NE. The localisation of these lipids during mitosis has not previously been investigated in somatic cells. This study uses the C1 domains from protein kinase C epsilon (PKCε), and the pleckstrin homology (PH) domain from phospholipase C delta (PLCδ1), which bind directly to DAG and PtdIns(4,5)P2 respectively. These probes indicate the presence of DAG in the NE and PtdIns(4,5)P2 in the peri-nuclear region of interphase cells. For the first time we observe the localisation of the C1 domain to the newly forming NE during telophase suggesting a role for DAG in NE reassembly. The specific role of DAG has been investigated using the inducible dimerisation system, with which DAG kinase ε kinase domain (DGKεK) was inducibly targeted to the NE using the nuclear targeting sequence of lamin B receptor (LBR). Using this chemical biology tool we have been able to show the localised role of fusogenic lipids such as DAG in somatic cells. These unprecedented results have opened a new area of research in the field of the regulation of NE assembly and offer novel mechanisms in the previously described models.
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Zhendre, Vanessa. "Étude de l’implication des phosphoinositides dans la formation de l’enveloppe nucléaire." Thesis, Bordeaux 1, 2010. http://www.theses.fr/2010BOR14133/document.
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Des pathologies telles que la myopathie et certains types de cancer, peuvent être causées par une mauvaise formation de l’enveloppe nucléaire (EN), processus se produisant lors de chaque division cellulaire mais aussi lors de la formation du pronoyau mâle. Un modèle in vitro, dérivé de gamètes d’oursins, a été utilisé afin d’étudier les différentes étapes de formation de l’EN et a permis de révéler plusieurs informations essentielles. Un des points critiques est que des membranes fortement enrichies en phosphoinositides polyphosphorylés (PPIs) sont essentielles à la formation de l’EN, notamment lors des étapes de fusion membranaire. Ces membranes proviennent du cytoplasme de l’ovocyte fécondé (MV1) et des noyaux de spermatozoïdes (NERs). Nous avons construit des modèles membranaires mimant les compositions lipidiques de ces membranes, puis étudié leur structure, leur dynamique ainsi que leur morphologie par spectroscopie de RMN des solides et microscopie électronique. Nous avons montré que les PPIs induisent une courbure membranaire positive, conduisant à la formation de petites vésicules ou de micelles allongées. Plus important encore, dans le modèle « MV1», les membranes sont très fluides. Le modèle « NERs » est constitué de membranes globalement ordonnées, semblables aux phases dites « liquides ordonnées » avec une modulation apportée par la PPIs. Nous avons également construit un modèle membranaire minant la composition lipidique des vésicules MV2, membranes non-enrichies en PPIs mais représentant 90 % des vésicules participant à la formation de l’EN. Ce modèle membranaire présente une dynamique intermédiaire à celle observée pour les modèles MV1 et NERs. Ces propriétés nouvelles ont permis de proposer un mécanisme décrivant le rôle des PPIs lors de la fusion membranaire conduisant à la formation de l’enveloppe nucléaireDiseases, such as myopathies and some types of cancer, can be caused by abnormal nuclear envelope (NE) assembly, a process that takes place at each cell division and during male pronuclear formation. A cell-free assay from sea urchin gametes, that mimics the in vivo male pronucleus formation, has been used to dissect the various stages of NE assembly. This in vitro assay has revealed several novel features. One of the critical aspects is that membranes highly enriched in polyphosphorylated phosphoinositides (PPIs), are essential for NE formation, especially during the stage of membrane fusion. Theses membranes are extracted from the cytoplasm of the fertilised oocyte (MV1) and sperm nuclei (NERs). We made model membranes with similar lipid composition to MV1 and NERs and studied their structure, dynamics and morphologies by solid-state NMR spectroscopy and electron microscopy. We show that PPIs have a positive membrane curvature, inducing small vesicles and elongated micelles. More importantly, we illustrate that “MV1-like” membranes are very fluid. “NERs-like” membranes are globally ordered and belong to the family of liquid ordered phases. We also evidenced that PPIs can counterbalance in part the ordering effect of cholesterol. Moreover we made model membranes with similar lipid composition to MV2, non-enriched in PPIs membranes which constitute 90% of the vesicles forming the NE. This model membrane shows an in-between dynamics compared to MV1 and NERs. We therefore propose a mechanism describing the role of PPIs during membrane fusion leading to nuclear membrane assembly
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Song, Zhimin. "Le rôle des phosphoinositides dans la régulation de l’activation de la NADPH oxydase des neutrophiles." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS152.
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Les neutrophiles participent à la défense de l'hôte en phagocytant les agents pathogènes et en les détruisant via notamment la production de formes réactives de l'oxygène (FRO). Les FRO sont produites par un complexe multi- protéique :la NADPH oxydase (NOX2). Celle-ci peut s’assembler à la membrane du phagosome lors de la phagocytose mais aussi à la membrane plasmique lors de la stimulation des neutrophiles par des agents bactériens ou des médiateurs de l’inflammation. La NADPH oxydase est une arme à double tranchant; une activation excessive ou inappropriée de la NADPH oxydase génère un stress oxydant, facteur aggravant des nombreuses pathologies. Cette enzyme doit donc être finement régulée. La NADPH oxydase est activée lorsque les sous-unités cytosoliques de NOX2 (p67phox, p47phox, p40phox) et la petite GTPase Rac s’assemblent avec les sous-unités membranaires (p22phox et gp91phox) à la membrane phagosomale ou plasmique. P67phox régule le flux d'électrons qui transite via gp91phox du NADPH à O2.-. Des travaux récents indiquent que les phospholipides anioniques contribueraient à la régulation de la NADPH oxydase. De plus, Les protéines organisatrices p40phox et p47phox possèdent des domaines de liaison à ces phosphoinositides : p40phox peut se lier au phosphatidylinositol 3-phosphate (PI(3)P) et p47phox au phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Nous avons donc voulu comprendre le rôle des ces phospholipides dans la régulation de la NADPH oxydase. Dans un premier temps nous nous sommes intéressés au rôle du PI(3)P, présent au phagosome après la fermeture de celui-ci, dans l’activation de la NADPH oxydase. Nos données indiquent que p40phox fonctionne comme un adaptateur, PI(3)P dépendant, permettant de maintenir p67phox dans le complexe de la NADPH oxydase. Le PI(3)P agit comme un « timer » pour l'activation de la NADPH oxydase au phagosome. Nous avons ensuite voulu examiner le rôle du PI(3,4)P2 dans la régulation de la NADPH oxydase à la membrane plasmique. Ce lipide est formé à la membrane plasmique par phosphorylation du PI(4)P par la PI3K de classe I lors de l’activation des neutrophiles. Nous avons montré que, l'activité PI3K de classe I est nécessaire pour maintenir l’activation, intégrine-dépendante, de la NADPH oxydase à la membrane plasmiqueThe NADPH oxidase of the professional phagocyte is essential for the immune system. The phagocyte NADPH oxidase, NOX2, catalyze the reduction of molecular oxygen to superoxide. Superoxide is transformed rapidly into other reactive oxygen species (ROS) which play a critical role in the killing of pathogens in host defense. Indeed neutrophils, the first cells that arrive at the site of infections, engulf pathogens in a process called phagocytosis. The production of reactive oxygen species is then triggered by the NADPH oxidase in the phagosome. The importance of ROS production is demonstrated by the recurrent bacterial and fungal infections that face patients who lack functional NADPH oxidase as in the rare genetic disorder known as the chronic granulomatous disease (CGD). Upon stimulation by bacterial peptide or in some pathological conditions, NADPH oxidase can also be activated at the phagocyte plasma membrane producing ROS in the extracellular medium. So, an excessive or inappropriate NADPH oxidase activation generates oxidative stress involve in chronic inflammation, cardiovascular disease and neurodegenerative disease. The NADPH oxidase activity should be tightly regulated. The activity of the enzyme is the result of the assembly of cytosolic subunits (p47phox, p67phox, p40phox and Rac2) with membranous subunits (gp91phox and p22phox). P67phox regulates the electron flow through gp91phox from NADPH to oxygen leading to the formation of superoxide. Recent data indicate that the anionic phospholipids are important for the NADPH oxidase regulation. Moreover, p40phox and p47phox bear a PX domain that binds respectively phosphatidylinositol3-phosphate (PI3P) and phosphatidylinositol (3,4)-bisphosphate(PI(3,4)P2). Our objective was to decipher the importance of these phosphoinositides on the NADPH oxidase activity. We first examined the role of PI3P, which is present on the cytosolic leaflet of phagosome after its sealing, in NADPH oxidase activation. Our data indicate that p40phox works as a late adaptor controlled by PI3P to maintain p67phox in the NADPH oxidase complex. Thus, PI3P acts as a timer for NADPH oxidase assembly. We then examined the role of PI(3,4)P2 in the activation of the NADPH oxidase assembled at the plasma membrane. PI(3,4)P2 and PI(3,4,5)P3 are formed at the plasma membrane, upon neutrophil activation, by phosphorylation by Class I PI3K of respectively PI4P and PI(4,5)P2. We found that class I PI3K activity is required to maintain the integrin-dependent activation of NADPH oxidase at the plasma membrane
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Laurent, Pierre-Alexandre. "Rôles des phosphoinositides 3-kinases (PI3Ks) α et β de classe IA dans les processus de l'activation plaquettaire et de la thrombose." Thesis, Toulouse 3, 2015. http://www.theses.fr/2015TOU30296.
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Les plaquettes jouent un rôle majeur dans l'hémostase mais également dans les maladies cardiovasculaires qui représentent une des principales causes de mortalité dans les pays industrialisés. Au cours de l'activation plaquettaire, les phosphoinositides 3-kinases (PI3Ks) de classe I génèrent des seconds messagers lipidiques (D3-phosphoinositides) participant activement à la transmission des signaux en aval des principaux récepteurs plaquettaires. Comme l'inhibition de la PI3Kbeta protège des thromboses occlusives sans augmenter le risque de saignement, cette kinase a été proposée comme nouvelle cible antithrombotique. Toutefois, le rôle de la PI3Kbeta dans des modèles de thrombose in vivo et ex vivo, dans des conditions de forces de cisaillement élevées, restait mal documenté au début de ma thèse. Le rôle de la PI3Kalpha?était également mal connu bien que l'utilisation d'inhibiteurs peu spécifiques suggérait son implication en aval du récepteur GPVI. L'objectif de ma thèse a été d'étudier le rôle de la PI3Kbeta dans la formation du thrombus in vivo et ex vivo, et de caractériser la fonction de la PI3Kalpha plaquettaire. Grâce à des souris présentant une invalidation de la PI3Kbeta ou alpha spécifiquement dans la lignée mégacaryocytaire, j'ai montré que la PI3Kbeta plaquettaire jouait un rôle essentiel dans la stabilité du thrombus dans des conditions de forces de cisaillement élevées. Son inhibition affecte l'activation d'Akt et empêche l'inhibition de son effecteur GSK3 au sein même du thrombus. De façon intéressante, l'inhibition de GSK3 restaure la stabilité du thrombus. Ainsi, la PI3Kbeta joue un rôle critique dans le maintien de l'intégrité du thrombus exposé à des forces de cisaillement élevées et cet effet ne peut être compensé par la PI3Kalpha. D'autre part, l'absence de la PI3Kalpha plaquettaire se traduit par un défaut d'agrégation et d'activation d'Akt lors d'une stimulation de faible intensité de GPVI. In vivo, le thrombus formé par les plaquettes invalidées pour la PI3Kalpha consécutivement à une lésion superficielle des artères, est de plus petite taille que chez les souris sauvages. En condition de flux ex vivo, il est noté un retard de formation du thrombus sur les fibres de collagène suggérant l'implication de la PI3Kalpha dans la phase d'adhésion des plaquettes à la matrice. En effet, des études d'adhésion en flux sur une matrice de facteur de von Willebrand (vWF) montrent que la PI3Kalpha est essentielle pour l'adhésion stable des plaquettes à cette matrice via la signalisation outside-in de l'intégrine alphaIIbbeta3. En conclusion, mes travaux de thèse mettent à jour un rôle spécifique des PI3Ks alpha et beta de classe I dans les plaquettes. La PI3Kbeta? est cruciale dans la régulation de la croissance et de la stabilité du thrombus dans des conditions de forces de cisaillement élevées, tandis que la PI3Kalpha?est requise dans la phase initiale d'adhésion des plaquettes sur une matrice de vWF. Ce travail apporte des nouvelles données qui seront utiles notamment dans le contexte du développement d'inhibiteurs sélectifs de PI3Ks en thérapie anticancéreuse et antithrombotiquePlatelets play a major role in cardiovascular diseases which is one principal cause of worldwide death. Class I phosphoinositide 3-kinases (PI3Ks) are important signaling enzymes in the process of blood platelet activation, producing lipid second messengers (D3-phosphoinositides) that are actively involved downstream of major platelet receptors. PI3Kbeta has been proposed as a potential drug target to treat arterial thrombosis. Indeed, inhibition of this lipid kinase leads to protection against occlusive thrombosis without bleeding risk. Nevertheless, nothing is known regarding the role of this kinase in vivo and ex vivo during growth, stabilization, and resistance upon elevation of shear rate. One study, using pharmacological inhibitors, has suggested that both PI3Kalpha and beta are required, in a non redundant way, for full platelet activation through collagen receptor GPVI, but the role of PI3Kalpha still remains elusive in platelets. The aim of my thesis was to study the role of PI3Kbeta during thrombus formation in vivo and ex vivo, and to characterize the role of PI3Kalpha in platelets. For that, I used pharmacological approach and mice with selective deletion of PI3Kbeta?or?PI3Kalpha in the megakaryocyte lineage (PF4-Cre/p110betaflox/flox and PF4-Cre/p110alphaflox/flox). I showed that PI3Kbeta is essential for thrombus growth and stability at high shear rates. Within the growing platelet thrombus, PI3Kbeta inactivation impairs the activating phosphorylations of Akt and the inhibitory phosphorylation of GSK3. In line with these data, pharmacological inhibition of GSK3 restores thrombus stability. Thus, platelet PI3Kbeta has a critical role in maintaining the integrity of the formed thrombus upon elevation of shear rate. In this condition, I showed that PI3Kbeta absence cannot be compensated by PI3Kalpha. In vitro, PI3Kalpha depletion in platelets leads to a light defect of aggregation and Akt activation in response to CRP showing implication of PI3Kalpha downstream GPVI. I observed that, in vivo, thrombi formed by PI3Kalpha depleted platelets after superficial lesion of mesenteric arterial are smaller, and ex vivo, thrombus formation under flow conditions on collagen matrix is delayed. Furthermore, perfusion of these platelets on von Willebrand factor matrix (vWF) shows that PI3Kalpha is required for stable adhesion of platelets through "outside in" signaling. Altogether, these results show an involvement of PI3Kalpha in the course of early step of platelet adhesion. In conclusion, my thesis work highlights an isoform specific role of PI3Ks in platelets. PI3Kbeta is crucial in the regulation of thrombus growth and stability at a high shear rate and PI3Kalpha is required in initial stages of platelet adhesion. Since class I PI3Ks selective inhibitors are under development as cancer treatment, these results may help to anticipate the potential side effects of such treatment on haemostasis
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Ainge, Gary D., and n/a. "The synthesis of phosphatidylinositol mannans and their analogues." University of Otago. Department of Chemistry, 2008. http://adt.otago.ac.nz./public/adt-NZDU20090113.101325.
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Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of glycolipids that possess significant immune modulating properties. To provide discrete synthetic compounds for biological assay, this thesis describes the syntheses of three PIM molecules, namely dipalmitoyl PIM2 (12), PIM4 (84), and PIM6 (108), and two PIM2 analogues designed for increased stability, PIM2ME (147) and PIM2MA (148).
The synthesis of all of these molecules involved mannosylation of 1-O-allyl-3,4,5-tri-O-benzyl-D-myo-inositol (22), which was prepared from methyl α-D-glucopyranoside in 8% yield over 8 steps, using a Ferrier reaction strategy.
A common intermediate, 3,4,5-tri-O-benzyl-2,6-di-O-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-D-myo-inositol (9), was used for the syntheses of 12, 147, and 148. This compound was prepared by bis-mannosylation of the C-1 and C-6 hydroxyl groups of 22 with 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl trichloroacetimidate (63) to give, after protecting group manipulations, the α,α-pseudo-trisaccharide 9 in 37% over 4 steps. The selectivity of the desired α,α-product was found to be increased by the selection of Et₂O as the solvent for the glycosylation reaction.
The C-1 hydroxyl group of 9 was coupled to benzyl (1,2-di-O-palmitoyl-sn-glycero)-diisopropylphosphoramidite (28) using 1H-tetrazole. Global debenzylation of the resulting product gave PIM2 (12) in 23% yield over 6 steps from 22. In a similar fashion 9 was coupled to 1-O-hexadeconyl-2-O-hexadecyl-sn-glycero-3-O-benzyl-(N,N-diisopropyl)-phosphoramidite (156), and subsequent deprotection gave PIM2ME (147) in 30% yield over 2 steps from 9. Coupling of 9 with 2-deoxy-1-O-hexadeconyl-2-O-hexadeconylamino-sn-glycero-3-O-benzyl-(N,N-diisopropyl)-phosphoramidite (172) and subsequent deprotection gave PIM2MA (148) in 47% yield over 2 steps from 9.
A modified approach was required for the syntheses of PIM4 (84) and PIM6 (108). A selective glycosylation of the C-6 hydroxyl of 22 with an orthogonally protected mannose donor would allow extension of the manno-oligosaccharide in a 2+3 or 4+3 glycosylation strategy required to build the pseudo-pentasaccharide or pseudo-heptasaccharide core of 84 or 108 respectively.
Sequential mannosylation of 22, firstly at the more reactive C-6 hydroxyl, with 2-O-acetyl-3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-α-D-mannopyranosyl trichloroacetimidate (85), was followed by mannosylation at the C-2 hydroxyl with 63. Removal of the silyl protecting group followed by a 2+3 coupling with the dimannoside donor, 2-O-acetyl-6-O-(2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-3,4-di-O-benzyl-α-D-mannopyranosyl trichloroacetimidate (95), gave a pseudo-pentasaccharide intermediate. Protecting group manipulations followed by coupling of the of the C-1 hydroxyl group of the inositol ring to phosphoramidite 28, and a global debenzylation, gave PIM4 (84) in 6% yield over 9 steps from 22.
During the synthesis of PIM6 (108), thioglycosylation chemistry was explored and found to be comparable to reactions with trichloroacetimidate donors. Similar methodology was used for the synthesis of PIM6 (108) as had previously been carried out for the synthesis of PIM4 (84). Mannosylation at the more reactive C-6 hydroxyl of 22 with either phenyl 2-O-benzoyl-3,4-di-O-benzyl-6-O-triisopropylsilyl-1-thio-α-D-mannopyranoside (112) or 2-O-benzoyl-3,4-di-O-benzyl-6-O-triisopropylsilyl-α-D-mannopyranosyl trichloroacetimidate (113), was followed by mannosylation at the C-2 hydroxyl with 63. Removal of the silyl group followed by a 4+3 coupling with either of the tetramannoside donors, phenyl (2-O-benzoyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]2)-(3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]2)-(3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]6)-2-O-benzoyl-3,4-di-O-benzyl-1-thio-α-D-mannopyranoside (109) or (2-O-benzoyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]2)- (3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1[to]2)-(3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1[to]6)-2-O-benzoyl-3,4-di-O-benzyl-α-D-marmopyranosyl trichloroacetimidate (131) gave a gave a pseudo-heptasaccharide intermediate. Protecting group manipulations followed by coupling of the of the C-1 hydroxyl group of the inositol ring to phosphoramidite 28, and a global debenzylation, gave PIM6 (108) in 9% yield over 9 steps from 22. To aid characterisation of 108, a sample was deacylated to afford dPIM6 (144) which gave the same spectral data as a sample from a natural source.
The compounds PIM2 (12), PIM4 (84), PIM2ME (147), and PIM2MA (148) were assayed for adjuvant activity and were found to have comparable activity to fractions isolated from natural sources. The analogue PIM2ME (147) gave the best results and is currently undergoing further development.
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King, Katrice. "Phosphoinositide Phase Behavior in Complex Lipid Monolayer Systems." Digital WPI, 2016. https://digitalcommons.wpi.edu/etd-dissertations/129.
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Although phosphatidylinositol (PI) and phosphoinositides (PIPs) only comprise a small percentage of the inner leaflet of the plasma membrane, they mediate a large variety of signaling events. In previous studies, we have observed the absence of macroscopically discernible domains in mixtures of PI/PC and PI(4,5)P2/PC. The addition of cholesterol to these mixtures results in condensation of the monolayer and hence domain formation. To better mimic the ionic conditions and hydrogen bonding properties of the inner leaflet plasma membrane, we investigated in this study the effect of common inner leaflet plasma membrane lipids like phosphatidylethanolamine (PE), phosphatidylserine (PS) and PI, on phosphoinositide domain behavior in the presence of cholesterol and/or bivalent cations.
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Fili, Natalia. "Investigation of the role of phosphoinositides in membrane dynamics : a novel approach." Thesis, Imperial College London, 2006. http://hdl.handle.net/10044/1/12015.
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Pernot, Eileen. "Etude in vivo du rôle de la 5-phosphatase de phosphoinositides SKIP." Doctoral thesis, Universite Libre de Bruxelles, 2008. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210473.
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Les membres de la famille des 5-phosphatases d’inositols polyphosphates et de phosphoinositides sont des enzymes caractérisées par la présence de deux domaines catalytiques conservés qui hydrolysent un phosphate en position 5 sur un noyau inositol. SKIP (Skeletal Muscle and Kidney enriched Inositol Phosphatase), également appelée Pps (Putative PI 5-phosphatase) est un des derniers membres de la famille des 5-phosphatases à avoir été découvert à ce jour. Cette enzyme hydrolyse majoritairement le phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) et le phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Les phosphoinositides (PtdIns) représentent environ 10% des lipides membranaires et sont impliqués dans de nombreuses cascades de signalisation cellulaire conduisant, entre autres, à la prolifération, l’apoptose, la différenciation, la sécrétion, le trafic vésiculaire et la mobilité cellulaire.Des études de surexpression de SKIP en cellules tendent à montrer que cette protéine pourrait jouer un rôle de régulateur négatif dans la formation du cytosquelette d’actine et/ou dans la voie de signalisation de l’insuline.
Afin d’étudier in vivo la fonction de la protéine SKIP chez la souris, nous avons décidé de générer des souris transgéniques surexprimant cette protéine de manière conditionnelle. Dans ce but, nous avons infecté des embryons murins par des lentivirus porteurs d’un transgène SKIP et avons obtenu, après réimplantation des embryons infectés dans des femelles pseudogestantes, deux lignées de souris transgéniques. Celles-ci ont ensuite été croisées avec des souris exprimant la recombinase Cre de manière ubiquitaire afin de pouvoir activer la transcription de SKIP dans l’ensemble des organes. Des expériences de Western blot, de dosage d’activité 5-phosphatase ainsi que des PCR en temps réel sont venus confirmer la présence de la protéine transgénique et de son activité catalytique.
L’ensemble des expériences qui ont été menées du point de vue phénotypique tend à montrer que dans notre modèle, la surexpression de SKIP ne provoque aucune anomalie évidente du point de vue anatomique, glycémique ou immunologique. Toutefois, des expériences concernant la physiologie rénale ont été réalisées sur base des résultats d’immunohistochimie et nous ont permis de détecter une anomalie dans les mécanismes de réabsorption d’eau ainsi que dans l’expression et la phosphorylation des canaux hydriques AQP2.
Doctorat en Sciences
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Bunney, Tom David. "Nuclear Caâ†2â†+-fluxes and 3-phosphorylated phosphoinositides in plant signal transduction." Thesis, University of East Anglia, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389277.
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Emond, Audrey. "Étude du rôle du general receptor for phosphoinositides 1 (GRP1) dans l'adipogenèse." Mémoire, Université de Sherbrooke, 2011. http://savoirs.usherbrooke.ca/handle/11143/4075.
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Many studies have shown that peroxisome-proliferator-activated receptor [gamma] (PPAR[gamma]) plays an important role in adipose tissue formation by activating genes implicated in adipogenesis. PPAR[gamma] heterodimerizes with retinoid X receptor [alpha] (RXR[alpha]), in the presence of ligand, on PPAR response elements (PPREs) in the promoter of target genes involved in adipocyte differentiation. General receptor for phosphoinositides 1 (GRP1) is a corepressor of thyroid hormone receptors (TRs), a nuclear receptor like PPAR[gamma]. GRP1 decreases TRs' transcriptional activity by lowering dimerisation on DNA. Since PPARs and TRs have important structural similarities and that GRP1 interacts with PPARs in vitro, we hypothesized that GRP1 could be a coregulator of PPARs and, thus be implicated in adipogenesis. To better understand GRP1's effect on PPAR[gamma]2, transcriptional activity assays have been done and show that increasing concentrations of GRP1 decrease the transcriptional activity of PPAR[gamma]2. We also studied GRP1 expression by Western blots of total protein extracts from 3T3-L1 cells at different times during differentiation: GRP1 is present in 3T3-L1 preadipocytes and its expression decreases during adipogenesis. According to those results, GRP1 may be a PPAR[gamma] corepressor. After those observations, GRP1 effects on adipogenesis were studied by modulating its expression with lentiviral particles. Interestingly, GRP1 knock-down before inducing 3T3-L1 differentiation, almost abrogates adipogenesis and adipocytes markers, PPAR[gamma] and aP2, while its overexpression increases lipid storage without affecting PPAR[gamma] expression. On the opposite, GRP1 modulation after differentiation induction shows that expression knock-down slightly promotes adipogenesis by increasing PPAR[gamma], aP2 and lipid accumulation and that overexpression weakly decreases lipid storage. Our results suggest that GRP1 implication during adipogenesis occurs at two distinct and precise moments. It seems to be a key factor in the early stages of adipocyte differentiation and to be implicated as a PPAR[gamma] transcriptional activity modulator as a corepressor. Future experiments will help detail modulation of protein expression and underlying mechanisms to better understand the role of GRP1 in adipogenesis and, eventually, comorbidities linked to obesity like cardiovascular diseases and type 2 diabetes mellitus.
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Sladeczek, Fritz. "Étude à différents niveaux du système de second messagers dérivés des phosphoinositides." Montpellier 2, 1987. http://www.theses.fr/1987MON20224.
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Le systeme de second messager aux phosphoinositides est presente a trois niveaux: 1) recepteur alpha 1-adrenergique, son metabolisme in vitro dans la lignee de cellules musculaires non fusionnantes bc::(3)h::(1) et in vivo dans les glandes sous maxillaires de rat, caracterisation du site de reconnaissance des drogues avec la **(3)h-prazosin. 2) etude de l'action de la maitotoxine (mxt) qui permet l'intervention directe au niveau du mecanisme de couplage entre les recepteurs et la phospholipase c, la stimulation des bc::(3)h::(1) est la consequence d'une entree massive d'ions ca**(2+) stimulee par mxt qui induisait une forte stimulation de la production inositol phosphate (ip2). Exclusion que la phospholipase c des cellules bc::(3)h::(1) soit activee par le ca**(2) apres stimulation du recepteur alpha 1 adrenergique. 3) etude de la reponse aux ips dans les neurones striataux en culture, l'effet stimulateur etait medie par un recepteur du type quisqualate et l'effet inhibiteur par des recepteurs nmda et kainate
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Mansat, Melanie. "Signalisation et implication des phosphoinositides dans la myopathie myotubulaire liée à l'X." Thesis, Toulouse 3, 2022. http://www.theses.fr/2022TOU30039.
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Les phosphoinositides (PIs) appartiennent à une famille de lipides membranaires qui jouent un rôle essentiel dans diverses fonctions cellulaires, comme en témoigne l’implication directe de nombreuses enzymes de leur métabolisme dans diverses pathologies humaines telles que des maladies génétiques ou des cancers. Leur métabolisme est extrêmement actif via l’action de PI-kinases et PI-phosphatases spécifiques. Sous l’effet de divers stimuli, la relocalisation de ces enzymes permet une génération ou un appauvrissement rapide et plus ou moins transitoire de certains PIs, permettant une coordination dans le recrutement de protéines impliquées dans divers mécanismes cellulaires tels que la migration, la différentiation, ou encore la prolifération. La protéine MTM1, membre de la famille des myotubularines, est une PI 3-phosphatase qui, in vitro, déphosphoryle le phosphatidylinositol 3-phosphate (PI3P) et le PI(3,5)P2 en PI et PI5P, respectivement. Cette enzyme est mutée dans la myopathie myotubulaire liée à l’X (XLMTM), une maladie rare congénitale grave caractérisée dès la naissance par une hypotonie, une importante faiblesse musculaire et une détresse respiratoire conduisant à la mort précoce des nourrissons. Dans la majorité des cas, une absence de la protéine est constatée. Durant ma thèse, je me suis intéressée à l’étude des rôles des produits et substrats de MTM1, et leurs impacts dans l’étiologie de la pathologie. Pour cela, j’ai utilisé la lignée de myoblastes murins C2C12, lignée ayant la capacité de se différentier en myotubes contractiles et reproduisant les différentes étapes de la myogenèse. Nous avons créé et caractérisé une lignée knockout pour Mtm1 via la technique CRISPR/Cas9 et montré que ces cellules reproduisent les défauts observés dans la pathologie tels que des noyaux mal positionnés, et des myotubes plus fins et petits. A l’aide de ce modèle, nous avons montré que MTM1 est une enzyme majeure pour la production de PI5P, et de façon surprenante (alors que MTM1 s’exprime au cours de la différentiation) mesuré une diminution en PI5P au cours de la différentiation. Nous mettons en évidence un mécanisme original où la coordination entre une PI-phosphatase et PI-kinase est impliquée dans la formation de structures importantes pour la fusion des myoblastes. L’ensemble de ces résultats correspondent à notre premier article. En parallèle, nous avons initié une étude sur les partenaires de MTM1 via l’approche de BioID et les résultats obtenus nous orientent vers un rôle important de MTM1 dans le trafic des intégrines, corrélant avec les défauts observés de localisation de l’intégrine-β1 dans la pathologie. Ces résultats sont intégrés au sein d’un deuxième article qui met en évidence que les altérations épigénétiques sont un mécanisme physiopathologique de la XLMTM, et que l’inhibition des histones désacétylases est une stratégie thérapeutique prometteuse pour cette maladie. En particulier, nous montrons que le traitement des cellules C2C12 knockout pour Mtm1 par l’acide valproïque permet de restaurer un phénotype normal avec un niveau d’expression et une localisation normale de l’intégrine-β1, corrélé avec une restauration des défauts d’adhésion observés. Le détail des protéines identifiées avec l’approche de BioID sont présentés en résultats complémentaires. D’autre part, au vu des rôles décrits de MTM1 et de ses produits et substrats dans le trafic vésiculaire, des travaux ont été réalisés sur sa perturbation en absence de MTM1 notamment via le suivi des protéines Rab et du PI3P et sont également présentés en résultats complémentaires. Ainsi, ce travail de thèse a permis de mettre en place un modèle cellulaire original pour l’étude de MTM1 et de poser les bases moléculaires du rôle de MTM1 et des PIs qu’il métabolise, mais il permet également d’apporter un volet mécanistique à une étude plus large sur la découverte de nouvelles pistes thérapeutiquesPhosphoinositides (PIs) are a minor class of phospholipids that play an essential role in diverse cellular functions highlighted by the direct involvement of their metabolizing enzymes in human pathologies such as genetic diseases or cancers. Their metabolism is extremely active through the action of specific PI-kinases and PI-phosphatases. Under various stimuli, the relocalization of these enzymes allows a rapid and more or less transient generation or depletion of certain PIs, allowing the recruitment of proteins involved in various cellular mechanisms such as migration, differentiation, or proliferation. MTM1, a member of the myotubularin family, is a PI 3-phosphatase that in vitro dephosphorylates phosphatidylinositol 3-phosphate (PI3P) and PI(3,5)P2 into PI and PI5P respectively. This enzyme is mutated in X-linked myotubular myopathy (XLMTM), a rare severe congenital disease characterized at birth by hypotonia, severe muscle weakness and respiratory distress leading to early infant death. In the majority of cases of XLMTM, the expression of MTM1 is strongly reduced or absent. My thesis work focused on the roles of MTM1 products and substrates, and their impact in the etiology of the pathology. For this purpose, I used the C2C12 myoblastic cell line which has the ability to differentiate into contractile myotubes and to reproduce the different stages of myogenesis. We have created and characterized a knockout cell line for Mtm1 using the CRISPR/Cas9 strategy and shown that these cells reproduce the defects observed in the pathology such as nuclei misorganization, and thinner and smaller myotubes. Using this model, we showed that MTM1 is a major enzyme for PI5P production, and surprisingly (while MTM1 is expressed during differentiation), measured a decrease in PI5P level during differentiation. Thus, we hypothesized that PI5P could be metabolized to PI(4,5)P2 by PI5P 4-Kinases (PI5P4Ks), the only conversion pathway for PI5P known to date. Using biochemical, cell biology and microscopy approaches, we demonstrated the involvement of PI5P4Kα in the production of a minor pool of PI(4,5)P2 in particular membrane structures called "podosome-like" essential for myoblast fusion. These results reveal a coordination between a PI-phosphatase and a PI-kinase in the formation of cell structures important for myoblast fusion. In parallel, we have initiated a study on the identification of MTM1 partners through the BioID approach and the results point to an important role of MTM1 in integrin trafficking, correlating with the observed defects in β1-integrin localization in XLMTM. These results are integrated in a second paper which highlights epigenetic alterations as a pathophysiological mechanism of XLMTM, and that histone deacetylase inhibition is a promising therapeutic strategy for this disease. In particular, we show that treatment of Mtm1 knockout C2C12 cells with valproic acid restores a normal phenotype with a normal expression level and localization of integrin-β1, rescuing the observed adhesion defects. Details of the proteins identified with the BioID approach are presented in supplemental results. Moreover, in view of the described roles of MTM1 and its products and substrates, work has been carried out on the disruption of vesicular trafficking in the absence of MTM1. Thus, this thesis work allowed to set up an original cellular model to study MTM1 functions and underlined novel insights into the role of MTM1 and its products and substrates in membrane dynamic during muscle differentiation
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Sladeczek, Fritz. "Etude à différents niveaux du système de seconds messagers dérivés des phosphoinositides." Grenoble 2 : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb37609972w.
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Paulhe, Frédérique. "Régulation différentielle du métabolisme des phosphoinositides par les intégrines αvβ3 et αvβ5 lors de la migration des cellules musculaires lisses." Paris 7, 2002. http://www.theses.fr/2002PA077140.
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Elong, Edimo William's. "Etude du rôle de la phosphatase SHIP2 dans un modèle d'astrocytomes humains." Doctoral thesis, Universite Libre de Bruxelles, 2013. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/218007.
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Le metabolisme des phosphoinositides est constitue dfun reseau complexe dfenzymes et de seconds messagers participant a la regulation de nombreux processus cellulaires :la proliferation, la croissance, la survie et la migration. Le phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), est un second messager intracellulaire tres important dont le taux est controle a la fois au niveau de sa synthese par des kinases et au niveau de sa degradation par des phosphatases. La phosphatase PTEN (phosphatase and tensin homolog deleted on chromosomes 10), gene suppresseur de tumeurs frequemment mutee dans des cancers (comme dans des glioblastomes), le dephosphoryle en position 3. Il existe egalement une activite á inositol 5-phosphatase â pour de nombreux derives du myo-inositol. Cfest la proteine SHIP2 (SH2-containing Inositol 5-phosphatase 2), une phosphatase membre de la famille des phosphatidylinositol polyphosphate 5-phosphatases qui est, entre autre, responsable de cette activite.Le but principal de ce travail de these a ete de mettre en evidence le role pro ou anti-oncogenique de SHIP2 dans un modele cellulaire humain de glioblastomes :les cellules 1321 N1. Ce modele cellulaire a comme particularite lfabsence dfexpression de PTEN au niveau proteique.La caracterisation des clones cellulaires deficients pour SHIP2 nous a permis de mettre en evidence une augmentation du taux du PtdIns(3,4,5)P3 par rapport a des cellules qui expriment un taux normal de SHIP2. Par un simple comptage, nous avons observe une augmentation du nombre de cellules en culture par rapport aux cellules controles. Lfimmunomarquage de la F-actine nous a permis de montrer une difference de morphologie entre les cellules N1 shSHIP2 et les cellules controles. Au travers de Western blots, nous avons observe que la phosphorylation de la PKB ainsi que celle de Erk1/2 etait augmentee dans les cellules deficientes pour SHIP2 en comparaison aux cellules qui expriment SHIP2.Dans les cellules COS-7 transfectees par SHIP2 sauvage, nous avons identifie par spectrometrie de masse 8 sites de phosphorylation sur Tyr, Ser et Thr. En systeme endogene dans les cellules N1, notre etude a demontre la presence de 3 sites de phosphorylations sur Ser (position 132 et 1258) et Thr (position 1254). Nous avons genere deux anticorps specifiques contre la proteine SHIP2 phosphorylee sur la Ser132 et Thr1254. Lfanticorps contre la Ser 132 nous a permis de mettre en evidence une colocalisation entre pSHIP2 Ser132 et le facteur dfepissage SC35 present dans les speckles nucleaires. Cette donnee nous suggere un role nucleaire nouveau pour SHIP2.De maniere originale nous avons mis en evidence dans les cellules dfastrocytomes N1 trois localisations subcellulaires de SHIP2 :i) a la membrane plasmique au niveau des adherences focales ou SHIP2 serait impliquee dans le controle du taux de PtdIns(3,4,5)P3/ PtdIns(4,3)P2, et de la migration ii) au niveau perinucleaire ou SHIP2 ainsi que la forme phosphorylee sur Ser132 seraient impliquees dans des interactions proteiques via ses proprietes de á docking protein â et enfin iii) dans le noyau specifiquement dans les speckles ou la forme phosphorylee de SHIP2 sur Ser132 serait impliquee dans le controle du taux de PtdIns(4,5)P2, mais egalement dans la signalisation nucleaire impliquant les phosphoinositides.La presence de SHIP2 dans les adherences focales nous a amene a rechercher par quel mecanisme SHIP2 pouvait se retrouver a cette localisation. Nous avons par immunoprecipitation suivie dfune analyse par spectrometrie de masse identifie la myosine 1c (Myo1c) comme nouveau partenaire de SHIP2. Les cellules deficientes pour la Myo1c presentent une modification de la morphologie cellulaire, ainsi qufune modification de la localisation intracellulaire de SHIP2. Les cellules N1 deficientes pour la Myo1c montrent aussi une diminution de lfexpression de la sous unite regulatrice de la PI 3-kinase (p85ƒ¿) ainsi qufune absence de la phosphorylation de la PKB sur la Ser473 et la Thr308. En outre, ces cellules N1 shMyo1c presentent un taux eleve dfapoptose compare aux cellules controles.En conclusion, ces travaux ont permis de montrer que dans un modele humain de glioblastomes N1, SHIP2 etait phosphorylee sur la Ser 132 et que cette phosphorylation semblait etre requise pour son activite phosphatase. SHIP2 possedait trois localisations subcellulaires probablement associees a des fonctions differentes, qufelle interagissait au minimum avec les proteines partenaires nouvelles lamin A/C et Myo1c. Enfin SHIP2 exercait un controle negatif sur la proliferation et la migration de ces cellules. Ces resultats mettent en evidence plusieurs fonctions originales de SHIP2 comme son role dans la migration et la proliferation qui ouvrent des perspectives nouvelles dans lfetude de cette phosphatase dans le contexte general et surtout physiopathogique des fonctions attribuees aux phosphoinositides.Doctorat en Sciences biomédicales et pharmaceutiques
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Smith, Sally Jane. "A study of gibberellin signalling in wild oat (Avena fatua) aleurone." Thesis, University of Bristol, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.388030.
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Blaser, Julian. "The utilisation of shRNA screens to investigate the role of phosphoinositide modulator genes in actue myeloid leukaemia." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/the-utilisation-of-shrna-screens-to-investigate-the-role-of-phosphoinositide-modulator-genes-in-actue-myeloid-leukaemia(87b33955-d4e9-4398-bd1d-58e4c5142eb3).html.
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Phosphoinositides (PIs) are pivotal lipid molecules with both scaffolding and signalling functions regulating key aspects of cellular physiology. For example, phosphatidylinositol (3,4,5)-trisphosphate, generated by phosphoinositide 3-kinase (PI3K), is an essential mediator of the PI3K/AKT signalling pathway, which is crucial for cell proliferation, survival and apoptosis. Constitutive activation of this signalling cascade has been identified in acute myeloid leukaemia (AML), the most common haematopoietic malignancy in adults, and experimental deletion of the PI3K antagonists PTEN and SHIP cause leukaemia in mice. However, little is known regarding the role of other PI modulator proteins in AML. Thus, in this thesis, a lentivirally delivered small hairpin RNA (shRNA) library targeting 103 genes (345 pLKO knockdown constructs) with presumed or established roles in PI metabolism was utilised to screen for genes required for AML blast cell viability/proliferation and differentiation. First, knockdown constructs were tested for their impact on proliferation/viability in seven human AML cell lines by measuring fold change in fluorescence of the cell viability dye alamarBlue relative to controls (cells transduced with a non-targeting control hairpin) over three days. This identified 13 candidate genes selected with the criterion that two or more knockdown constructs per gene reduce cell viability/proliferation relative to control by greater than or equal to50 % across all cell lines. From these candidate genes, PIP4K2A, INPP5B and IMPAD1 were selected for downstream validation experiments, which reproduced the observation from the primary screen. For INPP5B and IMPAD1, knockdown constructs also reduced clonogenic potential of primary human AML samples but only showed a modest effect on normal CD34+ haematopoietic stem or progenitor cells (HSPCs) in a methylcellulose based assay. This could be recapitulated in a murine setting where knockdown constructs targeting both genes reduced clonogenic potential of murine MLL- AF9 AML cells with little effect on normal KIT+ HSPCs. In line with this, Inpp5b knockout KIT+ BM cells either failed to immortalise or weakly immortalised, following forced expression of the powerful MLL-AF9 oncogene. A further screen was performed to identify regulators of THP-1 blast cell differentiation, by seeding knockdown construct transduced cells into methylcellulose based semisolid media. After ten days of incubation the degree of macrophage differentiation was evaluated by light microscopy and an arbitrary differentiation score was given. With the criterion that greater than or equal to2 knockdown constructs per gene received the highest differentiation score, reflecting terminal macrophage differentiation of all seeded cells, SBF2 was identified as the top-scoring hit. Validation experiments have confirmed macrophage differentiation based on cytospin preparations of SBF2 knockdown THP-1 cells. Moreover, xenograft assays have shown that knockdown constructs targeting PIP4K2A and SBF2 delayed or abrogated in vivo leukaemogenesis. Thus this work has identified novel roles for PI modulator genes in human AML with possible therapeutic potential.
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Redfern, Roberta E. "Characterization of Binding of PTEN and its Disease Related Mutants to Phospholipid Model Membranes." Kent State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=kent1216671104.
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Esnay, Nicolas. "La protéomique de sous-domaines du trans-Golgi Network révèle un lien entre les sphingolipides et les phosphoinositides chez la plante." Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0416.
Full textAbstract:
La polarité cellulaire est une caractéristique commune à tous les organismes. Jusqu’à récemment, il était assumé que la sécrétion de protéines vers des domaines polaires de la cellule végétale se faisait de façon non polarisée, mais ce point de vue a été re-étudié, la sécrétion est polarisée mais la dynamique, les voies de traficempruntées et les mécanismes sont toujours inconnus. Précédemment, mon laboratoire d’accueil a caractérisé un enrichissement en sphingolipides contenant des acides gras à très longues chaines (VLCFAs) au niveau d’un sous-domaine du trans-Golgi Network (TGN) appelé Vésicules de Sécrétions (SVs). Plus précisément, il a été montré que la longueur des acides gras des sphingolipides jouait un rôle critique dans la sécrétion du transporteur d’auxine PIN2 des SVs vers des domaines polaires de la membrane plasmique. Pendant ma thèse, je me suis intéressé à la question suivante : comment les sphingolipides agissent-t-ils au TGN? En identifiant le protéome des SVs, ainsi qu'en utilisant des outils génétiques et pharmacologiques en combinaison avec la visualisation de marqueurs lipidiques, j'ai pu identifier que les sphingolipides agissent sur l’homéostasie des phosphoinositides en mettant en avant un lien fonctionnel entre ces deux classes de lipides au sein de la cellule végétale. En utilisant un set de marqueurs des phosphoinositides (PIPs), j’ai pu montrer que les sphingolipides ciblent principalement le phosphatidyl-inositol-3-phosphate, PI(3)P et le phosphatidylinositol- 4-phosphate, PI(4)P. De plus, mon analyse protéomique a montré que la localisation d'un ensemble de protéines liées aux PIPs était diminuée dans les SVs/TGN immunopurifiées quand la composition des sphingolipides est altérée. Mes résultats nous forcent à revoir notre vision de la dynamique des lipides au niveau des membranes, et suggère l’idée que la dynamique de remodelage de la composition d’une classe de lipide, les phosphoinositides, peut être modulée par une autre classe de lipide, les sphingolipidesCell polarity is a defining feature of all organisms. Until very recently, it was thought that delivery of proteins to polar domains of root epidermal cells plasma membrane was non-polar, but this view has been re-examined, the delivery is polar but the dynamics, the paths taken, and the mechanisms are unknown. My host team previously characterised an enrichment of Very-Long-Chain-Fatty-Acids (VLCFAs)-containing sphingolipids at the site of secretory vesicles (SVs) sub-domain of the trans-Golgi Network (TGN). Moreover, the length of sphingolipids acyl-chain was found to play a critical role in secretory sorting of the auxin carrier PIN2 from SVsassociated TGN to apical polar domain of the plasma membrane (PM). During my PhD, I addressed the following question: how sphingolipids act at SVs/TGN? Using proteomics of SVs, genetics and pharmacological tools in combination with visualisation of lipid probes we could identify that sphingolipids act on phosphoinositides (PIPs) homeostasis establishing a new functional link between these two lipids in plant cells. Using a set of multi-affinity fluorescent PIPs probes I could show that sphingolipids target phosphatidylinositol-3-phosphate (PI3P) and phosphatidylinositol-4-phosphate (PI4P). Moreover, my proteomic analyses show that several PIPs-related proteins are downregulated in immuno-purified TGN-associated SVs when the sphingolipid composition is altered pharmacologically. My results force the reassessment of our view of lipid membranes dynamics and highlight the idea that dynamic remodelling of the composition of one lipid class, the phosphoinositides, can be modulated by another lipid class, the sphingolipids
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Oueslati, Habib. "Métabolisme des phosphoinositides membranaires et aspects fonctionnels du muscle EDL chez le rat." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq26805.pdf.
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Kischnick, Christian [Verfasser], and Steeve [Akademischer Betreuer] Boulant. "Role of Phosphoinositides in Cellular Polarity and Immunity / Christian Kischnick ; Betreuer: Steeve Boulant." Heidelberg : Universitätsbibliothek Heidelberg, 2018. http://d-nb.info/1177253089/34.
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Lin, Feng [Verfasser]. "Molecular functions of cell plate-associated phosphoinositides during plant somatic cytokinesis / Feng Lin." Halle, 2018. http://d-nb.info/1169132782/34.
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Gaumond, David. "Caractérisation d'un effecteur de phosphoinositides chez le parasite de la malaria Plasmodium falciparum." Master's thesis, Université Laval, 2016. http://hdl.handle.net/20.500.11794/27231.
Full textAbstract:
La malaria est une maladie infectieuse causant plus de 500 000 morts chaque année. La maladie est causée par un protozoaire de la famille Plasmodium. L’apparition de souches résistantes aux traitements actuels et l’absence de vaccin efficace rendent la découverte de nouvelles cibles thérapeutiques urgente. Le parasite possède un complexe apical, un groupement de vacuoles sécrétoires spécialisées contenant les protéines responsables de l’invasion du globule rouge. Nous nous intéressons aux mécanismes gouvernant le transport intracellulaire de ces protéines et à la biogenèse du complexe apical lors de la formation des nouveaux parasites. Plus particulièrement, nous nous intéressons au rôle des phosphoinositides dans le recrutement des protéines à la membrane de l’appareil de Golgi. Par analyse bio-informatique du génome de P. falciparum, nous avons identifié plusieurs protéines effectrices liant potentiellement les phosphoinositides. Les travaux présentés dans ce mémoire concernent Mal13P1.188, une protéine possédant un domaine Pleckstrin homology. Nous proposons que Mal13P1.188 ait un rôle dans la génération du complexe apical en recrutant les protéines le constituant à la membrane du Golgi par la liaison avec les phosphoinositides. Afin de vérifier nos hypothèses, nous avons généré une lignée de parasite dont le gène de Mal13P1.188 est fusionné avec une GFP et une hémagglutinine. À l’aide de cette lignée de parasite, nous avons pu identifier Mal13P1.188 à proximité de l’appareil de Golgi lorsque les parasites étaient sous la forme schizont du cycle érythrocytaire. D’autres expériences ont permis de confirmer que le domaine Pleckstrin homology de Mal13P1.188 était capable de reconnaître les différentes formes de phosphoinositides. Finalement, d’autres travaux devront être faits sur Mal13P1.188 afin de déterminer si elle est essentielle à la survie du parasite.Malaria is a deadly infectious disease taking more than 500,000 lives each year. The disease is caused by a protozoan of the Plasmodium family. Resistant strains beginning to spread and the inexistence of an efficient vaccine make the discovery of new targets urgent. The parasite secretes proteins to invade the red blood cell. Those proteins are regrouped in the apical complex, a group of organelles used for the invasion. Our research team focus on the transport mechanisms that drive the formation of the apical complex during the cellular division of new parasite. In other terms, we are interested on the role of phosphoinositide in the recruitment of protein inside the Golgi apparatus. After a bioinformatics analyse the P. falciparum genome, we identified many effectors protein that can bind phosphoinositides. Among them, we focused our work on Mal13P1.188, a protein with a Pleckstrin homology domain. We propose that Mal13P1.188 has a role in the recruitment of the apical proteins to the Golgi membrane using phosphoinositide as a marker on the membrane. To verify that hypothesis, we generated a strain of parasite with endogenous Mal13P1.188 tagged to a GFP and a hemagglutinin. With those parasites, we identified Mal13P1.188 near the Golgi apparatus during the Schizont stage of the blood cycle. Other experiment confirmed that the Pleckstrin homology domain of Mal13P1.188 is able to bind different form of phosphoinositides. Finally, more work has to be done to confirm if Mal13P1.188 is essential to the parasite survival.