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1
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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Dyer, Blake S., and n/a. "The synthesis and characterisation of phosphatidylinositol mannans." University of Otago. Department of Chemistry, 2008. http://adt.otago.ac.nz./public/adt-NZDU20080415.142001.
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Mycobacterial cell wall components have been shown to elicit a range of immunological responses in mammalian hosts. A family of cell wall antigens, the phosphatidylinositol mannans (PIMs), have been shown to reduce allergic response in a murine model of allergic airway disease and have been suggested as potential therapeutic agents. Isolation and characterisation of these compounds is not facile. To confirm the structure of PIMs a number of phosphatidylinositols (PIs), 1a-c, PIM1s 2a, 2d and 2e, and AcPIM1s, 2g and 2f, were prepared to allow assignment of the acylation pattern of natural products and for evaluation in immunological assays. As the natural products include 19:0 acylation in the form of (R)-tuberculostearoyl residues, a source of (R)-tuberculostearic acid was needed. To this end, an efficient synthesis of (R)-tuberculostearic acid from (S)-citronellol, utilising a copper-catalysed cross-coupling reaction and a modified Julia olefination, was developed. This material was incorporated into diacylglycerols prepared from (R)-benzyl glycidol.
A protected myo-inositol derivative, 188, and two protected pseudo-disaccharides, 10 and 241, were prepared from myo-inositol via desymmetrisation utilising a camphylidene acetal. These were coupled with diacylglycerols via a phosphate ester and deprotected to give PIs, PIM1s and AcPIM1s.
Mass spectrometry studies were undertaken on the PIs, 1a-c, PIM1s 2a, 2d and 2e, and AcPIM1s, 2g and 2f which structures that have been established by chemical synthesis. Comparison of these data with those reported for natural PIs and PIMs containing 19:0 ((R)-tuberculostearoyl) and 16:0 (palmitoyl) acyl groups unequivocally established that the 19:0 residue was located at the sn-1 and the 16:0 at the sn-2 position of the glycerol moiety in nature.
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Kawashima, Masahiro. "High-resolution imaging mass spectrometry reveals detailed spatial distribution of phosphatidylinositols in human breast cancer." Kyoto University, 2014. http://hdl.handle.net/2433/188666.
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Riggs, Bridget May. "Phosphatidylinositol synthase of tetrahymena utilization of inositol isomers in the headgroup exchange reaction /." [Pensacola, Fla.] : University of West Florida, 2006. http://purl.fcla.edu/fcla/etd/WFE0000030.
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Hartley, David Alan. "Dissection of Lymphocyte Activation: Defining a Role for PI-3 Kinase." eScholarship@UMMS, 1996. http://escholarship.umassmed.edu/gsbs_diss/212.
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This dissertation was intended to identify potential roles for phosphatidylinositol-3 kinase (PI-3 kinase) in the responses of lymphocytes to activation. To understand what functions PI-3 kinase is performing in lymphocytes, experiments were performed to identify proteins that will stably associate with the p85 subunit of PI-3 kinase. Co-precipitation revealed an activation dependent association of p85 with two different phosphotyrosine containing proteins. One protein, pp36-38, is a membrane protein that interacts with PI-3 kinase, PLCγ1, and Grb2/S0S. The other associated protein was identified as the proto-oncogene c-Cbl. The interaction of p85 with cbl was shown to be mediated through the SH2 domains of p85. More importantly, the interactions of p85 with p36-38 and cbl were found to be specific for p85 isoforms. Although the SH2 domains of the α and β isoforms are highly similar in amino acid sequence, they are shown to establish distinct protein interactions in intact cells. Experiments on the cbl/PI-3 kinase complex revealed a stimulation dependent translocation into membrane and insoluble/cytoskeletal fractions of wild type, but not mutant cells. The movement of cbl did not require tyrosine phosphorylation or PI-3 kinase activity. The cbl/PI-3 kinase complex was greatly enhanced in the membrane fraction in contrast to the cytosol, where the largest concentration of cbl can be found. In addition, these complexes were found to form at the membrane in the absence of the tyrosine kinase, p56lck.
6
Malaby, Andrew W. "An Integrated Structural Mechanism for Relief of Autoinhibition and Membrane Targeting in Cytohesin Family Guanine Nucleotide Exchange Factors: A Dissertation." eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/703.
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Guanine nucleotide exchange factors (GEFs) regulate and organize diverse cellular processes through their role in converting GTPases from the inactive GDP bound state to the active GTP bound state. An increasing number of GEFs undergo autoregulatory mechanisms through complex intramolecular interactions. Relief of autoinhibition involves specific phosphorylation or binding to lipid and/or effector proteins at sites distal from the catalytic domain, and is often coupled to membrane recruitment. In Cytohesin Arf GEFs, the catalytic Sec7 domain is autoinhibited by a linker region and C-terminal helix flanking a Pleckstrin Homology (PH) domain. Upon binding of the PH domain to low abundance phosphoinositides, the GTPase Arf6-GTP can both relieve autoinhibition and recruit Cytohesins to the plasma membrane. This thesis focuses on determining the molecular mechanism underlying both these functions.
The structural mechanisms by which Arf6-GTP binding relieves autoinhibition were studied using biochemical and crystallographic studies. The crystal structure of the Grp1 PH domain in complex with Arf6 revealed that Arf6-GTP binding relieves autoinhibition through competitive sequestration of the inhibitory elements into grooves formed at the periphery of the interface. Importantly, the interaction orients all known membrane targeting components to a common surface. Detailed biochemical studies showed a common mode of binding among Cytohesin family members in which phosphoinositide head group binding primes the interaction with Arf6, and membrane recruitment of both stimulatory and substrate Arf enhances the effect.
To assess changes in the Sec7 domain conformation upon activation, Size Exclusion Chromatography in line with Small Angle X-Ray Scattering (SEC-SAXS) was performed. The unique nature of this data led to the development of a novel data analysis and processing strategy. A graphically based, python-extensible software package was created for data normalization, buffer correction, Guinier Analysis, and constant background subtraction. As an unbiased substitute for traditional buffer subtraction, a method to reconstruct the protein scattering through singular value decomposition (SVD) and linear combination of the basis vectors was developed. These methods produced exceptional data quality and allowed versatility for application to other data collection techniques or systems, especially those lacking confident buffer matching or low signal.
SEC-SAXS confirmed the overall structure of autoinhibited Grp1 in solution and showed only slight overall changes upon activation by deletion of the autoinhibitory Cterminal helix. Fusion of Arf6 with Grp1 produced a consistently elongated shape in the active state that was incompatible with the autoinhibited or theoretical active positions of the Sec7 domain. Monte Carlo and rigid body modeling using known structural domains revealed a requirement for Sec7-PH linker flexibility in addition to Sec7 domain mobility. These data support an integrated structural model whereby phosphoinositides and Arf-GTP support nucleotide exchange at membranes through allosteric activation, membrane recruitment, and large-scale rearrangement of the Sec7 domain. Overall, these findings offer insight into Cytohesin function that can be applied to assess relief of autoinhibition in the context of other GEFs and GTPases.
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Malaby, Andrew W. "An Integrated Structural Mechanism for Relief of Autoinhibition and Membrane Targeting in Cytohesin Family Guanine Nucleotide Exchange Factors: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/703.
Full textAbstract:
Guanine nucleotide exchange factors (GEFs) regulate and organize diverse cellular processes through their role in converting GTPases from the inactive GDP bound state to the active GTP bound state. An increasing number of GEFs undergo autoregulatory mechanisms through complex intramolecular interactions. Relief of autoinhibition involves specific phosphorylation or binding to lipid and/or effector proteins at sites distal from the catalytic domain, and is often coupled to membrane recruitment. In Cytohesin Arf GEFs, the catalytic Sec7 domain is autoinhibited by a linker region and C-terminal helix flanking a Pleckstrin Homology (PH) domain. Upon binding of the PH domain to low abundance phosphoinositides, the GTPase Arf6-GTP can both relieve autoinhibition and recruit Cytohesins to the plasma membrane. This thesis focuses on determining the molecular mechanism underlying both these functions.
The structural mechanisms by which Arf6-GTP binding relieves autoinhibition were studied using biochemical and crystallographic studies. The crystal structure of the Grp1 PH domain in complex with Arf6 revealed that Arf6-GTP binding relieves autoinhibition through competitive sequestration of the inhibitory elements into grooves formed at the periphery of the interface. Importantly, the interaction orients all known membrane targeting components to a common surface. Detailed biochemical studies showed a common mode of binding among Cytohesin family members in which phosphoinositide head group binding primes the interaction with Arf6, and membrane recruitment of both stimulatory and substrate Arf enhances the effect.
To assess changes in the Sec7 domain conformation upon activation, Size Exclusion Chromatography in line with Small Angle X-Ray Scattering (SEC-SAXS) was performed. The unique nature of this data led to the development of a novel data analysis and processing strategy. A graphically based, python-extensible software package was created for data normalization, buffer correction, Guinier Analysis, and constant background subtraction. As an unbiased substitute for traditional buffer subtraction, a method to reconstruct the protein scattering through singular value decomposition (SVD) and linear combination of the basis vectors was developed. These methods produced exceptional data quality and allowed versatility for application to other data collection techniques or systems, especially those lacking confident buffer matching or low signal.
SEC-SAXS confirmed the overall structure of autoinhibited Grp1 in solution and showed only slight overall changes upon activation by deletion of the autoinhibitory Cterminal helix. Fusion of Arf6 with Grp1 produced a consistently elongated shape in the active state that was incompatible with the autoinhibited or theoretical active positions of the Sec7 domain. Monte Carlo and rigid body modeling using known structural domains revealed a requirement for Sec7-PH linker flexibility in addition to Sec7 domain mobility. These data support an integrated structural model whereby phosphoinositides and Arf-GTP support nucleotide exchange at membranes through allosteric activation, membrane recruitment, and large-scale rearrangement of the Sec7 domain. Overall, these findings offer insight into Cytohesin function that can be applied to assess relief of autoinhibition in the context of other GEFs and GTPases.
8
Xiang, Hong. "Alpha₁-adrenoceptor-mediated phosphoinositide breakdown and inotropic responses in right ventricles of streptozotocin-diabetic rats." Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/31036.
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The morbidity of and the mortality from cardiac disease are higher in diabetic patients. Clinical and experimental evidence suggests that diabetes-induced changes at the level of myocardium can, at least partially, contribute to these cardiac problems. The mechanism(s) involved in this diabetic cardiomyopathy is still unclear, but one defect appears to occur in the alpha₁-adrenoceptor system. Altered myocardial sensitivity and responsiveness to alpha₁-adrenoceptor agonists have been reported in experimental diabetes mellitus. Stimulation of alpha₁-adrenoceptors is known to produce a positive inotropic effect and has been recently shown to stimulate the hydrolysis of phosphoinositides. To evaluate the possibility that the changes in the inotropic responsiveness to alpha₁-adrenoceptor stimulation in the diabetic heart could be linked to altered alpha₁-adrenoceptor-stimulated
phosphoinositide turnover and further to the development of diabetic cardiomyopathy, we studied contractility and receptor-stimulated phosphoinositide turnover following norepinephrine (in the presence of propranolol) stimulation in right ventricles from male Wistar rats (200-225 g) which were made diabetic with streptozotocin (55 mg/kg, i.v.). Rats were sacrificed six weeks after the induction of diabetes. Diabetic rats were characterized by decreased body weight gain, hypoinsulinemia, hyperglycemia and hyperlipidemia.
Stimulation of alpha₁-adrenoceptors by norepinephrine (in the presence of propranolol) in right ventricles resulted in the formation of inositol monophosphate (measured with a radioisotope method) and inositol 1,4,5-trisphosphate (measured with an inositol 1,4,5-trisphosphate protein binding assay kit) in a time- and concentration-dependent manner in both control and diabetic rats. The increase in inositol 1,4,5-trisphosphate levels preceded the increase in the alpha₁-adrenoceptor-mediated positive inotropic effect. Diabetic hearts showed a greater maximum inotropic response to norepinephrine stimulation and also had a higher inositol 1,4,5-trisphosphate levels. However, with the radioisotope method, a decreased inositol monophosphate formation was shown in diabetic hearts compared with controls.
Omega-3 fatty acids supplementation (Promega[symbol omitted], 0.5 ml/kg/day) had no significant effect on the changes in norepinephrine-stimulated inositol monophosphate formation in diabetic hearts.
In the presence of the cyclooxygenase inhibitor indomethacin or the thromboxane synthetase inhibitor imidazole, the norepinephrine-stimulated positive inotropic effect and inositol 1,4,5-trisphosphate formation were significantly increased in control hearts, but were unaltered in the hearts from diabetics. The addition of the prostacyclin synthetase inhibitor tranylcypromine reduced the norepinephrine-stimulated positive inotropic effect and
inositol 1,4,5-trisphosphate formation only in diabetic hearts and had no effect in the controls.
While inositol 1,4,5-trisphosphate may be able to mediate only transient inotropic effects produced by alpha₁-adrenoceptor stimulation, diacylglycerol may provoke a sustained positive inotropic effect by activating slow Ca²⁺ channels through stimulation of protein kinase C. Our results showed that the diabetic hearts had a higher protein kinase C activity in the membrane fraction compared with controls and this was accompanied by a decrease in cytosolic protein kinase C activity.
The present study suggests that the increases in inositol 1,4,5-trisphosphate levels and the membrane fraction protein kinase C activity may be implicated in the increased inotropic responsiveness to alpha₁-adrenoceptor stimulation in the hearts of the streptozotocin-diabetic rats. The increases in inositol 1,4,5-trisphosphate level and protein kinase C activity could induce Ca²⁺ overload in the diabetic heart which might be involved in the development of diabetic cardiomyopathy. The results from the omega-3 fatty acid study indicate that the changes in cardiac alpha₁-adrenoceptor-mediated inositol phosphates formation cannot contribute to the previously described improved cardiac function of omega-3 fatty acid-treated streptozotocin-diabetic rats. The nature and physiological significance of the enhanced positive inotropic effect and inositol 1,4,5-trisphosphate formation in the control heart
with the addition of indomethacin and imidazole is still unclear. The effect of tranylcypromine may indicate the participation of prostaglandins in mediating the enhanced alpha₁-inotropic effect of norepinephrine in the diabetic heart.Pharmaceutical Sciences, Faculty of
Graduate
9
Pesesse, Xavier. "Clonage moléculaire et caractérisation de SHIP2, une nouvelle inositols et phosphatidylinositols polyphosphates 5-phosphatase qui contrôle la voie de signalisation de la PI 3-kinase." Doctoral thesis, Universite Libre de Bruxelles, 2000. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/211771.
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Walker, Valerie Glynis. "Pl3-kinase mediates cSrc activation and podosome formation through the adaptor protein, AFAP-110, in response to PKC[alpha] activation." Morgantown, W. Va. : [West Virginia University Libraries], 2007. https://eidr.wvu.edu/etd/documentdata.eTD?documentid=5191.
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Thesis (Ph. D.)--West Virginia University, 2007.Title from document title page. Document formatted into pages; contains viii, 306 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.
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Persson, Anders. "Affinity partitioning of membranes purification of rat liver plasma membranes and localization of phosphatidylinositol 4-kinase /." Lund : Biochemistry, Chemical Center, University of Lund, 1995. http://books.google.com/books?id=BqpqAAAAMAAJ.
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Jäger, Karin. "Glycosyl-phosphatidylinositol-anchored acetyl-cholinesterase and phosphatidylinositol-specific phospholipase C /." Bern, 1990. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Saunders, Alex Michael. "Synthesis of deuterated phosphatidylinositol phosphates." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:65f93fcc-791a-491f-88c5-8ccb5db58592.
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Phosphatidylinositol phosphates (PtdInsPn) are intracellular signalling molecules that are important in many key biological processes, in particular Ca2+ signalling pathways. Dysfunction of these processes has been implicated in numerous diseases including diabetes and many cancers. Some aspects of PtdInsPn signalling have been heavily investigated; PTEN, PKC/Akt, PtdIns3K and PtdIns4K are all important therapeutic targets that have seen much attention in industrial endeavours. Inositol-based probes and tool compounds for these targets typically incorporate a fluorescent tag or photo-crosslinking group, usually at the lipid tails. It is increasingly apparent that the nature of the lipid chains plays a key role in determining the sub-cellular localisation of the PtdInsPn and hence modification of the lipids is potentially detrimental to the biological function of the tool compounds. An additional challenge in the development of inositol-based tools compounds is the difficult and lengthy syntheses that are employed to obtain the target compounds. To address this, we have developed a novel asymmetric route that allows rapid synthesis of PtdIns and PtdIns(4,5)P2. This route has been designed to allow incorporation of multiple deuterium atoms onto the myo-inositol ring (C-perdeuterated). To achieve this, we began with the aromatic compound quinol and built up the myo-inositol ring piecewise, allowing for deuterium incorporation. This methodology utilised a Pd-catalysed dynamic kinetic resolution on a conduritol B derivative to form optically-pure myo-inositol derivatives in high e.e. ( > 99%) toward the synthesis of D6-PtdIns(4,5)P2. The incorporation of deuterium into these compounds should be minimally disruptive to their biological activity, while the difference in molecular mass between the endogenous and tool compounds enables their use in a range of biological assays. In addition, the incorporation of the deuterium onto the myo-inositol ring will allow for the detection of downstream effects relating to the myo-inositol ring post-hydrolysis of PtdIns(4,5)P2 to be observed, which is currently not possible with other probes.
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Roberts, Hilary Frances. "Regulation of phosphatidylinositol 5-phosphate 4-kinase IIα and of its substrate, phosphatidylinositol 5-phosphate." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615129.
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de, Vos Sarah. "Phosphatidylinositol 3-phosphates in plant cell signalling." Thesis, University of East Anglia, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251889.
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Zhao, Li. "Mechanistic Studies on Phosphatidylinositol-specific Phospholitase C." The Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1047485476.
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Zhao, Li. "Mechanistic studies on phosphatidylinositol-specific phospholipase C." Connect to this title online, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1047485476.
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Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xix, 135 p.; also includes graphics (some col.) Includes bibliographical references (p. 128-135). Available online via OhioLINK's ETD Center
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Valet, Colin. "Rôles de la PI3 kinase de classe II alpha et de la PI3K de classe III, vps34, dans la production et les fonctions plaquettaires." Thesis, Toulouse 3, 2017. http://www.theses.fr/2017TOU30032.
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Les mégacaryocytes sont des cellules de la moelle osseuse qui par un processus complexe et encore mal caractérisé, mégacaryopoïèse/thrombopoïèse, donnent naissance, in fine, aux plaquettes sanguines. La différenciation mégacaryocytaire nécessite un intense remodelage nucléaire et cytoplasmique, guidé à la fois par des facteurs intrinsèques mais aussi par des facteurs extrinsèques tel que le microenvironnement médullaire. Les plaquettes sanguines sont des acteurs essentiels du maintien de l'intégrité vasculaire. Elles sont les premiers éléments cellulaires à intervenir dans l'arrêt du saignement lors d'une blessure vasculaire par la formation d'un thrombus via des mécanismes d'adhésion, de sécrétion et d'agrégation, trois étapes majeures de l'hémostase physiologique. Dans un premier temps, mes travaux de thèse visent à déterminer le rôle inconnu de l'isoforme alpha des PI3Ks de classe II (PI3KC2a), de la PI3K de classe III (Vps34) et de leur produit, le phosphatidylinositol 3 monophosphate (PI3P), dans la production et les fonctions plaquettaires. Grâce à un modèle murin présentant une inactivation partielle de la PI3KC2a, j'ai mis en évidence son rôle clé dans la génération d'un pool basal de PI3P dans les plaquettes. L'inactivation de la PI3KC2a affecte la composition du cortex sous-membranaire plaquettaire induisant une morphologie plaquettaire anormale, une accumulation de plaquettes à deux corps appelées " barbell-shaped proplatelets ", un défaut de formation du thrombus ex vivo et un retard d'occlusion de la carotide après lésion in vivo. Ainsi, la PI3KC2a joue un rôle majeur dans le maintien de l'intégrité du squelette membranaire contrôlant la structure et la dynamique membranaire, processus critique à la production de plaquettes fonctionnelles. D'autre part, la délétion de Vps34 spécifiquement dans la lignée mégacaryocyte/plaquette se traduit par une microthrombopénie modérée associée à une migration anormale des mégacaryocytes liées à un défaut de trafic vésiculaire et une diminution du taux de PI3P. De façon intéressante, Vps34 joue aussi un rôle dans l'activation plaquettaire en régulant la production de PI3P sous stimulation, la croissance du thrombus ex vivo et les capacités thrombotiques in vivo. Le rôle de Vps34 dans la plaquette indépendamment de son rôle dans le mégacaryocyte a été confirmé via l'utilisation de nouveaux inhibiteurs spécifiques de Vps34, SAR405 et INH1, ex vivo. Vps34 est donc critique dans la régulation de la production plaquettaire par les mégacaryocytes ainsi que dans l'activation plaquettaire. Dans un deuxième temps, je me suis intéressé à l'impact du microenvironnement médullaire sur la mégacaryopoïèse, et plus spécifiquement sur la communication entre adipocytes médullaires et progéniteurs hématopoïétiques lors de leur différenciation en mégacaryocytes. Grace à un système de coculture in vitro, j'ai montré que les adipocytes améliorent la différenciation mégacaryocytaire via un transfert direct de lipides, dans un but non-énergétique. Dans un contexte d'obésité, nous observons, in vivo, associée à une adiposité médullaire augmentée une maturation mégacaryocytaire exacerbée, une production et une demi-vie plaquettaire défectueuses ayant pour conséquence une macrothrombopénie. Ainsi, le microenvironnement médullaire et plus particulièrement l'adipocyte impacte directement sur la mégacaryopoïèse et la production plaquettaire. En conclusion, ces travaux de thèse contribuent à caractériser les mécanismes de production et de fonction plaquettaire régulés par des facteurs intrinsèques tels que le PI3KC2a et Vps34, ainsi que par des facteurs extrinsèques tels que l'adipocyte médullaireMegakaryopoiesis is a highly specialised and complex process occurring in the bone marrow, by which megakaryocytes give rise to de novo circulating blood platelets. Megakaryocyte differentiation implies cytoplasmic and nuclear rearrangements regulated by intrinsic as well as extrinsic factors such as bone marrow microenvironment. Platelets play a critical role in preventing blood loss after vascular injury by orchestrating clot formation through mechanisms of adhesion, secretion and aggregation. These mechanisms are the three major steps of physiological haemostasis leading to the maintenance of vascular integrity. Firstly, my thesis work focused on characterizing the role of class II PI3K alpha isoform (PI3KC2a), class III PI3K (Vps34) and their common product the phosphatidylinositol 3 monophosphate (PI3P) in platelet production and function. Using a unique mouse model partially inactivated for PI3KC2a, I highlighted its key role in the production of a basal PI3P housekeeping pool in platelets. PI3KC2a partial inactivation affects platelet membrane skeleton composition leading to an abnormal platelet morphology, an enrichment of platelet with two cell bodies recently called "barbell-shaped proplatelets", an ex vivo defective thrombus formation and an in vivo delayed carotid occlusion following injury. Thus, PI3KC2a plays a major role in membrane structure and dynamics by maintaining membrane skeleton integrity, which is crucial for functional platelet production. On the other hand, Vps34 specific deletion in megakaryocyte/platelet lineage induced mild microthombopenia correlated to an abnormal megakaryocyte migration linked to an affected PI3P production as well as vesicular trafficking in megakaryocytes. In platelets, Vps34 plays a role in their activation by regulating PI3P production under stimulation, ex vivo thrombus growth and in vivo thrombotic capacity. Vps34 role in platelet independently from its role in megakaryocyte was confirmed using two recently developed inhibitors, SAR405 and INH1, which reproduced ex vivo thrombus growth defects. Therefore, Vps34 is critical for platelet production by megakaryocyte as well as platelet activation. Secondly, I studied the impact of bone marrow microenvironment on megakaryopoiesis and more specifically the crosstalk between medullar adipocytes and hematopoietic progenitors differentiating towards the megakaryocyte lineage. Using an in vitro coculture assay, I demonstrated that adipocytes enhanced megakaryocyte differentiation through a direct lipid transfer, in a non-energetic aim. In the context of obesity, increased marrow adipocity is associated to enhanced megakaryocyte differentiation and defective platelet production and lifespan leading to macrothrombopenia. Thus, bone marrow microenvironment through adipocytes impact directly on megakaryopoiesis and platelet production. Altogether my thesis work contributes to better understand platelet production and function, mechanisms regulated by intrinsic factors such as PI3KC2a and Vps34 as well as extrinsic factors like medullar adipocytes
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Morris, James Benjamin. "Phosphatidylinositol 5-phosphate 4-kinases in animal cells." Thesis, University of Cambridge, 2001. https://www.repository.cam.ac.uk/handle/1810/265451.
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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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Grove, S. J. A. "The synthesis of D-3 phosphorylated phosphatidylinositol analogues." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360599.
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Prosser, Simon Edward. "Phosphatidylinositol transfer protein : an investigation of its function." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299880.
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Bulley, Simon. "Studies of endogenous phosphatidylinositol 5-phosphate 4-kinases." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709369.
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Whyte, Gillian. "Synthesis and evaluation of receptors for phosphatidylinositol phosphates." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/18967.
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Phosphatidylinositol phosphates (PIPs) are signalling phospholipids with a diverse set of cellular functions. The most important of these PIPs are phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) and phosphatidylinositol (3,4,5) trisphosphate (PI(3,4,5)P3). These two are responsible for a number of cellular events which regulate cell growth, proliferation and apoptosis. Deregulation of PIP levels disrupts these pathways and can therefore lead to uncontrolled cell growth and which can lead to tumourigenesis. Control of PIP levels has been identified as a potential target for diagnosis and intervention in diseases including Alzheimer’s disease, cancer, and the genetic disorder Lowe Syndrome. The use of small molecules that bind PIPs has been shown by our group to interfere with protein-PIP interactions. By preventing proteins from binding to PIPs, the effective concentration of the target PIP is reduced, proteins are not recruited to the membrane for activation and downstream signalling pathways are attenuated. Therefore, in this project a series of small artificial receptors were synthesised and were shown to bind PI(4,5)P2 and PI(3,4,5)P3. The aim of this was to control their effective levels and manipulate their downstream signalling pathways. PI(4,5)P2-binding receptors were developed based on existing lead compounds established within our group. Comparison of a receptor with only one binding motif with the established two motif receptor showed differences in affinity as well as specificity for the phospholipid target PI(4,5)P2 over the headgroup IP3. In cells, the effect of the receptors on the downstream signalling pathways was examined using phosphorylated Akt as an indicator of this pathway’s activation. Two fluorescent receptors were designed and one of these was used as a novel PI(4,5)P2 detection tool on immobilised phospholipid. Preliminary results show that this receptor may also be used to directly image PI(4,5)P2 in fixed cells. Novel PI(3,4,5)P3 receptors were designed with phosphate-binding motifs and a range of spacers. The specificity of each receptor was identified and binding affinities towards PI(3,4,5)P3 were established. Inhibition of protein-lipid interaction and inhibition of PIP-metabolising enzymes were also studied. The effect of these new receptors in attenuating the Akt pathway in cancer cells was also investigated, and they were found to lack the efficacy of PI(4,5)P2-binding receptors. The ability of some of these artificial receptors to bind phospholipids in the cell and affect subsequent signalling pathways indicates that phospholipids are a viable additional drug targets for many diseases.
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Stack, Jeffrey H. Emr Scott. "Protein and phosphatidylinositol kinases in yeast protein sorting /." Diss., Pasadena, Calif. : California Institute of Technology, 1995. http://resolver.caltech.edu/CaltechETD:etd-10262007-081900.
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Liu, Xinyu. "Glycosylphosphatidylinositols and phosphatidylinositol mannosides : chemical syntheses and method development /." Zürich : ETH, 2007. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17093.
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Gülkan, Hülya. "Differenzierung der humanen Phosphatidylinositol-4-Kinasen PI4K230 und PI4K92." [S.l.] : [s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=959538372.
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Siragusa, Mauro. "Role of Phosphatidylinositol 3-Kinasey in Angiogenesis and Vasculogenesis." Thesis, University of Bristol, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.520205.
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Ulrich, Michele Leslie. "Phosphatidylinositol cycle and bipolar disorder, investigation of intracellular calcium." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ60508.pdf.
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Tan, Siow Khoon. "The role of phosphatidylinositol transfer proteins in phosphoinositide signalling." Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.397987.
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Williams, Fay Kathleen. "Phosphatidylinositol (3,5) bisphosphate dependent membrane trafficking in S. cerevisiae." Thesis, University of Birmingham, 2012. http://etheses.bham.ac.uk//id/eprint/3626/.
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Phosphoinositides are lipid signals that control cellular processes and are particularly closely associated with the control of membrane trafficking. PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) is the most recently identified phosphoinositide and was previously recognised as controlling events in the late endocytic system between the late endosome and the vacuole/lysosome. Primarily associated with retrograde trafficking from the vacuole/lysosome to the late endosome/MVB, PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) is generated by the kinase Fab1p (PIKfyve in animals). In mammalian cells, PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) has also been implicated in control of ill-defined trafficking pathways close to the Golgi; for example, the recycling of mannose-6-phosphate receptor (M6R) back to the Golgi and also the trafficking of some types of ion and nutrient channels from the Golgi to the cell surface. This thesis describes attempts to study putative PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) dependent trafficking in the early endocytic system of \(\char{cmmi10}{0x53}\). \(\char{cmmi10}{0x63}\)\(\char{cmmi10}{0x65}\)\(\char{cmmi10}{0x72}\)\(\char{cmmi10}{0x65}\)\(\char{cmmi10}{0x76}\)\(\char{cmmi10}{0x69}\)\(\char{cmmi10}{0x73}\)\(\char{cmmi10}{0x69}\)\(\char{cmmi10}{0x61}\)\(\char{cmmi10}{0x65}\) using two model proteins; the recycling of Vps10p from late endosome to Golgi and of Chs3p from recycling endosome to Golgi.
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Shi, Xiaomeng. "Phosphatidylinositol-specific phospholipase C: Conformational changes upon membrane binding." Thesis, Boston College, 2010. http://hdl.handle.net/2345/1413.
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Thesis advisor: Mary F. RobertsPhosphatidylinositol-specific phospholipase C (PI-PLC) from B. thuringiensis is activated by phosphatidylcholine (PC) surfaces for both phosphatidylinositol (PI) cleavage to inositol 1,2-(cyclic)-phosphate (cIP) and subsequent hydrolysis of cIP to inositol-1-phosphate. These enzyme kinetics strongly suggest that this PI-PLC has two discrete binding sites for phospholipids - the active site binding PI (or substrate competitors) and an activator site specific for PC. However, it is difficult to determine the orientation and conformation of peripheral membrane proteins when docked to target membranes, let alone where sites for these might be on the protein. In this thesis, various biophysical techniques were applied to this bacterial PI-PLC to obtain structural information in the absence and presence of membranes to characterize specific conformational changes that occur when the protein binds to activating membranes. The crystal structures of an interfacially impaired double mutant of PI-PLC, W47A/W242A, was solved and showed the protein as a homodimer. The major interactions came from four clustered surface tyrosine residues from each monomer. This structure suggested the possibility of PI-PLC dimerization on membrane surfaces as part of the mechanism for interfacial activation. Mutations of these tyrosines showed a loss of activity and membrane binding. Crystal structures of these mutant proteins showed no significant change in the proteins, consistent with either disruption of a dimerization interface of a specific PC binding motif. FRET was used to try and monitor oligomerization of PI-PLC, derivatized on a cysteine introduced at residue 280 (W280C) with either a donor or acceptor fluorophore, on vesicle surfaces. The results suggested some specific aggregation could occur on very PC-rich surfaces but not on phospholipid vesicles with at least 50 mol% anionic phospholipids, strongly suggesting that a stable dimer was not forming when the enzyme was bound to vesicles mimicking conditions where enzyme specific activity is high. If dimerization occurs on surfaces, it must be transient. To examine which portions of the PI-PLC are interacting with membrane and to further explore if there is any evidence for PI-PLC dimerization on membrane surface, deuterium exchange coupled by mass spectrometry experiments were carried out with wild type PI-PLC, W47A/W242A and a covalent dimer formed from W242C that is more active than wild type enzyme. Results showed (i) a stable short helix B (containing an exposed tryptophan thought to insert into membranes) in wild type PI-PLC and its complete destabilization in W47A/W242C, (ii) a flexible surface loop (containing another tryptophan thought to partition into the membrane) that became protected when the protein was bound to vesicles, and (iii) reduced deuterium exchange for the peptide containing the tyrosines that either mediate transient dimerization or form a PC binding site.. These observations modify how we envision the protein anchoring to substrate-containing membranes
Thesis (PhD) — Boston College, 2010
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Shim, Hyeseok. "Biology of Type 2 Phosphatidylinositol-5-Phosphate 4-Kinase." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:23845419.
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Type 2 phosphatidylinositol-5-phosphate 4-kinase (PI5P4K) converts phosphatidylinositol-5-phosphate to phosphatidylinositol-4,5-bisphosphate. Mammals have three genes, PIP4K2A, PIP4K2B and PIP4K2C that encode the enzymes PI5P4Kα, PI5P4Kβ and PI5P4Kγ respectively. Studies in mice showed that PI5P4Kβ is a negative regulator of insulin signaling (Lamia et al., 2004) and that co-deletion of Pip4k2b and Trp53 resulted in synthetic embryonic lethality (Emerling et al., 2013). Also, deletion of two alleles of Pip4k2a and one allele of Pip4k2b suppressed the appearance of tumors in Trp53-/- mice. These studies suggest that drugs targeting PI5P4Kα and β could be effective therapies for treating insulin resistance, type 2 diabetes and TP53 mutant cancers.
While less is known about PI5P4Kγ, several genome-wide association studies have revealed a SNP in front of the PIP4K2C at an autoimmunity susceptibility loci (Raychaudhuri et al., 2008). To evaluate the role of PI5P4Kγ, I generated Pip4k2c-/- mice and found an inflammatory phenotype with increased tissue immune infiltrates and pro-inflammatory cytokines, correlating with increased helper T cells and decreased regulatory T cells. Also, Pip4k2c-/- mice exhibited upregulated mammalian target of rapamycin complex 1 (mTORC1) signaling in tissues and rapamycin treatment reduced the inflammation of these mice. These studies support the concept that the SNP identified at the PIP4K2C locus in human patients with autoimmunity contributes to disease by reducing expression of PI5P4Kγ and indicates that inhibition of mTORC1 would be beneficial to these patients.
Finally, in collaboration with Dr. Nathanael Gray’s laboratory we identified small molecules that covalently react with PI5P4Ks and thereby cause irreversible inhibition. These compounds, PIP4Kin1 and PIP4Kin2 mimicked the effect of shRNA mediated knockdown or knockout of PI5P4Kα and PI5P4Kβ, and impaired the growth of several TP53 mutant cancer cell lines, with little effect on most TP53 wild type cell lines. Utilizing the xenograft tumor model with BT474 (TP53 mutant) and MCF7 (TP53 wild type) cells, we showed that daily treatment of the mice with PIP4Kin2 inhibited the growth of the BT474 tumors but not the MCF7 tumors, without causing any obvious toxicity. These results further validate PI5P4Kα and PI5P4Kβ as targets for treating TP53 mutant cancers.Medical Sciences
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Suer, Silke. "Biochemische Charakterisierung der humanen Phosphatidylinositol 4-Kinasen, PI4K92 und PI4K230." [S.l.] : [s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=960683577.
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Scheid, Michael. "Phosphatidylinositol 3-OH kinase, an important element in survival signalling." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0024/NQ38976.pdf.
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Ma, Kewei. "Investigation of the phosphatidylinositol 3-kinase pathway in B cells." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/3818.
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There is hardly a cellular process that is not regulated in some way by phosphoinositides, which makes biochemical and physiological studies of these lipids extremely important. PI 3-kinases are key regulators of phosphoinositide metabolism and have been shown to affect a large variety of cellular responses. The key products of PI 3-kinases that have functional activity in higher eukaryotic cells are PI(3,4,5)P₃ and PI(3,4)P₂. PI(3,4,5)P₃ is universally accepted as one of the most important second messengers in signal transduction. However, our knowledge of the functions of PI(3,4)P₂ as a lipid second messenger is much less precise. In this dissertation, work was undertaken to elucidate the regulation of PI(3,4,5)P₃ and PI(3,4)P₂ production and downstream signaling in B cells. Cells with membrane targeted exogenous SHIP were utilized to manipulate phosphoinositide levels. The relationship of PI(3,4,5)P₃ and PI(3,4)P₂ levels to downstream PKB phosphorylation and activation was studied. PI(3,4,5)P₃ and PI(3,4)P₂ levels were found to closely correlate with PKB phosphorylation levels at Thr308 and Ser473, respectively. In addition, PI(3,4)P₂ levels determine the PKB activity in the cytosol; while PI(3,4,5)P₃ levels determine PKB activity at the plasma membrane. Different doses and different forms of B cell receptor (BCR) agonists were used for stimulation. PI 3-kinase activation was studied carefully following stimulation with low doses of anti-BCR antibody and F(ab')₂ fragments. Low concentrations of F(ab')₂ fragments produced higher levels of PI(3,4,5)P₃ than did a high concentration of F(ab')₂ fragments. Downstream PKB signaling was studied in these models. Similar conclusions were drawn from both SHIP over-expressing BJAB cells and dose-dependent BCR stimulations. We speculated that phosphoinositides’ regulation of the kinetics of PKB phosphorylation at Ser473 and Thr308 might be mediated by additional proteins. Investigation of plasma membrane-associated PKB showed that it formed a protein complex of around 400KD, which we attempted to characterize further with respect to PKB phosphorylation and association with lipids. In conclusion, phosphoinositide production is intricately regulated in vivo to control downstream signaling. The levels of PI(3,4)P₂ and PI(3,4,5)P₃ have precise and profound effects on PKB and other molecules such as TAPP and Bam32. This study has contributed new insight into the PI 3-kinase signaling pathway from the aspect of phosphoinositide lipid function.
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Coghill, Emma Louise. "Mapping the phosphatidylinositol binding site of microtubule associated protein 2." Thesis, Imperial College London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.369061.
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Shore, Angharad Mair. "Study of nuclear targets of phosphatidylinositol-3 kinase in lymphocytes." Thesis, Cardiff University, 2006. http://orca.cf.ac.uk/54332/.
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Pathways regulated by Phosphotidylinositide-3-kinase (PI3K) have emerged as important mediators of cell proliferation and survival. When altered, several components of this pathway have been identified to contribute towards a wide range of human malignancies. PI3K has been implicated in the development of several EBV-associated malignancies of both lymphoid and epithelial origin. These include Burkitt's lymphoma, Hodgkin's disease and nasopharyngeal carcinoma. Although progress has been made in dissecting the pathways regulated by PI3K, the key components contributing to lymphocyte transformation have not been fully characterised. This study sought to investigate downstream targets of PI3K in lymphocytes in order to further our understanding of the contribution of PI3K signalling to lymphocyte proliferation and survival, particularly within the context of EBV-associated B-cell lymphomas. Initial work in this study revealed that a component of the mammalian ribosome, S6-ribosomal protein, is a major target for PI3K activation in transformed lymphocytes. In order to study PI3K and EBV regulated proteins on a larger scale, the technology of two-dimensional electrophoresis (2DE) was employed. The use of 2DE in combination with a PI3K inhibitor did not allow the identification of PI3K regulated proteins. However, three EBV regulated proteins were detected in the B-lymphocyte nucleus using this technology. The technology was further developed to study the post-translational modifications of DNA bound transcription factors. This detected multiple isoforms of the cAMP-response element binding protein (CREB), signal transducers and activators of transcription 1 (STAT1) and forkhead box O (FOXO) transcription factors in the nuclei of EBV immortalized lymphocytes. More detailed analysis of the PI3K regulated pro-apoptotic transcription factor, FOXOl, revealed that this protein is downregulated in EBV positive cells at both the transcriptional and translational levels. This downregulation was shown to directly correlate with the protein expression of a known target gene activated by FOXOl, Bcl-6, and to inversely correlate with protein levels of Cyclin D2, a target transcriptionally repressed by FOXOl. Further investigations into the mechanisms by which EBV downregulates FOXOl implicated a role for two EBV encoded proteins, Latent membrane protein-1 (LMP1) and LMP2A in the downregulation of both FOXOl, and its target gene, Bcl-6. In conclusion, this work has explored the use of antibody detection and proteomic techniques for the identification and analysis of nuclear proteins and transcription factors regulated by PI3K and EBV. Together, these investigations have deepened our understanding of the molecular changes that occur in lymphocytes in response to EBV infection, and how EBV may influence malignancy.
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Whittingham, Jane. "Cellular Roles for Proteins Linked to Phosphatidylinositol (3,5)-bisphosphate Metabolism." Thesis, University of Liverpool, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.502364.
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The phosphoinositide lipids Ptdlns(3)P and Ptdlns(3,5)P2 play important roles on the endocytic pathway. Ptdlns(3)P localises to early endosomes and multivesicular bodies (MVBs) and is proposed to recruit proteins involved in fusion of early endosomes and internalisation of ubiquitinated receptors. Ptdlns(3,5)P2 is proposed to be involved in terminal maturation of Iysosomes and endosome to Golgi transport, but the precise role of this lipid in mammalian cells remains unclear. Studies in yeast have identified various proteins associated with Ptdlns(3,5)P2 metabolism, for which mammalian homologues have been found. These are the Ptdlns(3)P5- kinase Fab1/PIKfyve, Fig4 a 5-phosphatase that dephosphorylates Ptdlns(3,5)P2, Vac14 which has been shown to act as an upstream activator of PIKfyve, and WIPI-2/Svp1 a putative downstream effector of Ptdlns(3,5)P2. In this study I have further characterised three of these proteins and examined the cellular roles of all four with respect to a variety of trafficking pathways in which Ptdlns(3,5)P2 has been implicated. siRNA was used to examine the knockdown phenotypes of each of these proteins. Furthermore, I directly compared for the first time the effects of knockdown of PIKfyve, with acute pharmacological inhibition of its enzymatic activity. Loss of PIKfyve activity caused a failure in the retrieval of a variety of different cargos to the trans-Golgi network (TGN), including mannose-6-phosphate receptors, responsible for efficient delivery of lysosomal enzymes, and the TGN resident protein TGN46, leading to their accumulation in dispersed punctae. A failure in tyrosine kinase receptor downregulation was also observed following combined knockdown of PIKfyve with either Vac14 or Fig4 or following pharmacological inhibition of PIKfyve. PIKfyve knockdown alone had no effect, suggesting a low threshold of Ptdlns(3,5)P2 is necessary and sufficient for this pathway. Svp1 is the best characterised Ptdlns(3,5)P2 effector in yeast and is also an autophagy-related gene (Atg18), thereby implicating Ptdlns(3,5)P2 in this process. A family of putative mammalian Svp1 homologues have been identified, known as the WIPI family. I investigated the role of Ptdlns(3,5)P2 and WIPI-2 in mammalian autophagy. By monitoring formation of the autophagosome marker GFP-LC3 II, PIKfyve and WIPI-2 were found to have opposite effects. Furthermore, WIPI-2 redistributes to punctate structures upon induction of autophagy, which partially colocalise with autophagosome markers, in a manner dependent not on PIKfyve but on PI(3)-kinase activity. The evidence presented suggests that Ptdlns(3,5)P2 may playa role in mediating the maturation of a subset of MVBs, leading to swelling of endosomal compartments and rendering the MVBllate endosome or autophagosomes refractory to fusion with the lysosome in cells depleted of PIKfyve.
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Mills, Ian Geoffrey. "Effects of phosphatidylinositol 3-phosphate binding proteins on endosomal dynamics." Thesis, University of Liverpool, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367253.
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Zeh, Andrea Kauffmann. "Regulation of phosphatidylinositol 4-kinase activity in EGF receptor signalling." Thesis, Open University, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259497.
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Alli-Balogun, Ganiyu Olabanji. "The cellular functions of mammalian type II phosphatidylinositol 4-kinases." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/10039515/.
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Type II phosphatidylinositol 4-kinases (PI4KIIs), PI4KIIα and PI4KIIβ, both catalyse phosphatidylinositol 4-phosphate (PI(4)P) synthesis and are implicated in the control of trafficking from the trans-Golgi network (TGN) and endosomal membranes. It has been suggested that these closely related isoforms perform redundant roles. This study addresses the issue of functional overlap, by studying the location of the PI(4)P pools synthesised by each isoform, the associated membrane trafficking routes and the functional consequences of loss of these PI4P pools. The TGN localisations of PI4KIIα and PI4KIIβ could be distinguished by co-immunostaining with TGN markers syntaxin 6 and TGN46, indicating that the isoforms localise to separate TGN domains. In addition, depletion of PI4KII isoforms using small interfering RNA (siRNA) had differential effects on TGN pools of PI(4)P, with PI4KIIα loss significantly affecting a syntaxin 6 positive PI(4)P pool while PI4KIIβ depletion altered a TGN46 positive pool; thus indicating the synthesis of metabolically separate PI(4)P pools by these two isoforms. Depletion of either PI4KII isoform also impaired post-TGN traffic of cation independent mannose 6-phosphate receptor (CI-M6PR) and the endo-lysosomal traffic and degradation of the EGF receptor which is suggestive of overlapping roles for both isoforms in post-TGN traffic. PI4KII gene silencing also had differential effects on the actin cytoskeleton. Loss of PI4KIIα led to increased stress fibre formation while PI4KIIβ depletion induced the formation of functional invadopodia containing membrane type I matrix metalloproteinase (MT1-MMP). This was accompanied by decreased colocalization of MT1-MMP with the endosomal markers Rab5 and Rab7 that control lysosomal trafficking and regulate surface levels of MT1-MMP. However, MT1-MMP showed increased colocalisation with Rab8, which mediates exocytic trafficking and pro-invasive activity of MT1-MMP. In addition, depletion of PI4KIIβ conferred a migratory phenotype on minimally invasive HeLa and MCF-7 cell lines. This cell phenotype was substantiated by oncogenomic database analyses showing that loss of PI4KIIβ expression was a risk factor for numerous human carcinomas.
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Stamatkin, Christopher W. "PHOSPHATIDYLINOSITOL 3-KINASE (PI3K) AS A THERAPEUTIC TARGET IN NSCLC." UKnowledge, 2014. http://uknowledge.uky.edu/pharmacy_etds/58.
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Deregulated activation of phosphatidylinositol 3-kinase (PI3K) pathway is central to many human malignancies. The functions of this pathway are critical for normal cell metabolism, proliferation, and survival. In lung cancers, the PI3K pathway activity is often aberrantly driven by multiple mutations, including EGFR, KRAS, and PIK3CA. Molecules targeting the PI3K pathway are intensely investigated as potential anti-cancer agents. Although inhibitors of the pathway are currently in clinical trials, rational and targeted use of these compounds, alone or in combination, requires an understanding of isoform-specific activity in context. We sought to identify class IA PI3K enzyme (p110a/PIK3CA, p110b/PIK3CB, p110d/PIK3CD) activities using isoform-specific inhibitors in a lung cancer model system. Treatment of non-small cell lung cancer (NSCLC) cell lines with PIK3CA, PIK3CB, PIK3CD or PIK3CB/D inhibitors resulted in pharmacokinetic and pharmacodynamic responses that frequently tracked with a specific mutation status. Activation of PIK3CA dictated response to the PIK3CA-specific inhibitor while deletion of PTEN phosphatase indicated response to the PIK3CB inhibitor. The PIK3CD isoform-specific inhibitors lacked efficacy in all NSCLC cell lines tested, however treatment at increased concentrations likely provide concurrent inhibition of both PIK3CB/D isoforms improving activity of either agent alone but did not track with a single biomarker. The observed pharmacodynamic and proliferation responses to isoform-specific inhibitors suggested that PI3K isoforms may functionally compensate for loss of another in certain genetic backgrounds. These studies demonstrate unanticipated cellular responses to PI3K isoform inhibition in NSCLC, suggesting that patient populations with specific mutations can benefit from certain isoform-selective inhibitors, or combinations, allowing for rational and targeted clinical use of these agents.
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Johnson, Erin Ellen. "Physiological Role of Vps34 Phosphatidylinositol 3-Kinase in Mammalian Cells." University of Toledo Health Science Campus / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=mco1115925989.
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Conway, Bruce R. "Regulation of Phosphatidylinositol 4-kinase Activity in Rat Pancreatic Acini." VCU Scholars Compass, 1992. http://scholarscompass.vcu.edu/etd/4504.
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The present report describes the characteristics and regulatory properties of phosphatidylinositol (Ptdlns) 4-kinase activity in rat exocrine pancreas. The membrane associated Ptdlns 4-kinase displayed a broad pH profile with optimal activity at neutral to alkaline pH. Carbachol (CCh) elicits a concentration- and time-dependent increase in Ptdlns 4-kinase activity in homogenates derived from agonist-stimulated acini. This effect was blocked by N-methylscopolamine and mimicked by muscarine. The enzyme had an apparent Km for Ptdlns and ATP of 4 and 60 uM, respectively. CCh caused no discernible change in the Km for either Ptdlns or ATP, but did produce a modest increase in the Vmax. The cell permeable diacylglycerol analogues dioctanoylglycerol (DiC8) and oleoyl acetylglycerol (OAG) also produced a concentration-dependent increase in Ptdins 4-kinase activity which was blocked by the protein kinase c inhibitor, staurosporine. Cell permeable monobutyryl cAMP caused an increase in PtdIns 4-kinase activity when added to intact acini. This agent caused a significant decrease in the apparent Km for ATP but had no effect on PtdIns utilization or the Vmax of the reaction. Moreover, pretreatment of the acinar homogenate with the catalytic subunit of protein kinase A (PKA) produced a concentration-dependent increase in enzyme activity suggesting that phosphorylation may be involved in the regulation of Ptdins 4-kinase. Epidermal growth factor (EGF) elicited a concentration-dependent increase in PtdIns 4-kinase activity in homogenates derived from agonist-stimulated pancreatic acini. The combination of CCh and EGF produced a response which was not synergistic or additive. EGF, unlike CCh, failed to cause [32^P]Ptdins(4,5)P2 breakdown, suggesting different mechanisms for the increase in Ptdins 4-kinase activity induced by EGF and CCh. Furthermore, exposure of pancreatic acinar cells to EGF failed to elevate cyclic AMP levels, suggesting that a third pathway exists for the regulation of Ptdins 4-kinase activity in the exocrine pancreas. We conclude that PtdIns 4-kinase represents a key component of the signaling pathways utilized by various receptor agonists and that phosphoinositide synthesis is under direct and tight regulation by PKC- and PKA-dependent pathways.
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Zhang, Yanling. "The roles and regulation of phosphatidylinositol 3,5-bisphosphates in mammals." Diss., University of Iowa, 2008. http://ir.uiowa.edu/etd/1.
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Hermelink, Antje. "Phosphatidylinositol 3-kinase [gamma] characterization of a protein-lipid interaction." Berlin dissertation.de, 2008. http://d-nb.info/994112912/04.
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Furse, Samuel. "The synthetic preparation and physical behaviour of phosphatidylinositol-4-phosphates." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/6402.
Full textAbstract:
The current set of three studies comprises preparative synthetic organic chemistry, membrane biophysics, and enzymology. Specifically, racemic and chiral synthetic organic preparations of distearoylphosphatidylinositol-4-phosphate (DSPIP) are described, along with tangential studies that inform use of protecting group strategies and the limits and behaviour of key myo-inositol-based intermediates. The biophysical behaviour of this lipid as a function of temperature, pressure, and concentration in dioleoyl phosphatidylcholine and hydration has been examined. Unsaturated analogues of DSPIP, comprising oleoyl (SOPIP) and γ-linolenoyl (SGPIP) have also been prepared, both racemically and chirally. The Enzymology study comprised kinetic assays of the three enantio-pure lipids DSPIP, SOPIP and SGPIP prepared in the organic study with Salmonella (bacterial) phosphatase SopB and has established that increasing numbers of unsaturated bonds in the glyceride residue of the inositide has a decreasing effect on activity of the enzyme and are in agreement with previous preliminary results for this enzyme. This body of work provides further evidence that the sensitivity of biological systems to fatty acids is primarily physical.
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Robinson, Kevin Spencer. "The phosphatidylinositol signal transduction system in the yeast Saccharomyces cerevisiae." Thesis, University of Bath, 1992. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316975.
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Qazi, Sanjive. "Acetylcholine muscarinic receptors and phosphatidylinositol turnover in the locust CNS." Thesis, University of Bath, 1992. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.332283.
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