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Functional analysis of GPI-anchored and truncated forms of HLA-A2.1Huang, Jui-Han January 1994 (has links)
No description available.
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Dynamique conformationnelle chez les protéines d'adhésion de Babesia : mythe ou réalité ? / Conformational dynamics in the adhesion proteins of Babesia : myth or reality ?Murciano, Brice 07 June 2013 (has links)
L'une des infections parasitaires les plus courantes chez les animaux à travers le monde est la babésiose ou piroplasmose. Causée par le développement intraérythrocytaire d'un parasite du genre Babesia, elle présente de nombreux signes cliniques semblables à ceux du paludisme. Ce parasite, du phylum des Apicomplexes, est transmis via le vecteur tique et effectue son cycle de reproduction dans les cellules rouges du sang de l'hôte vertébré. En Europe B. divergens et B. canis sont les espèces majoritairement responsables respectivement de la babésiose bovine et la babésiose canine. Dans une stratégie de recherche vaccinale, l'étude de protéines parasitaires en contact avec la circulation sanguine est primordiale pour comprendre les interactions hôte-parasite et identifier des candidats vaccins à haut potentiel. Les protéines à ancrage GPI (glycosylphosphatidylinositol) font partie de ces protéines. La première protéine à ancrage GPI décrite chez B. divergens est Bd37.1. Elle induit une protection totale contre une infection à B. divergens à la condition qu'une séquence hydrophobe soit ajoutée en C-terminale. La résolution de la structure RMN de cette protéine a permis de mettre en évidence un probable mécanisme de changement conformationnel en fonction du pH. La structure composée de 3 sous domaines montre que celle-ci n'est maintenue que par des ponts salins qui peuvent se rompre en milieu acide. Or l'environnement membranaire dans lequel évolue Bd37.1 ancrée à la surface du parasite et/ou à l'approche du globule rouge lors de l'invasion est acide. Cette dynamique conformationnelle de la protéine Δ-Bd37, liée à l'environnement membranaire, pourrait être à l'origine du mécanisme qui confère une immunité en fonction de la présence ou non de la séquence hydrophobe en C-terminale de Bd37.1. Nous avons cherché à estimer les implications d'une telle dynamique dans les interactions hôtes-parasites à travers l'étude structurale de 2 protéines parasitaires (Bd37.1 et Bc28.1). Dans le premier cas nous étudions la dynamique conformationnelle de la protéine d'adhésion Bd37.1. Nous avons exploré les différentes conformations que pourrait adopter la protéine Bd37.1 par une approche de biophysique et nous avons stabilisé ces différentes conformations en solution par le biais de mutations pour les étudier. Parmi ces mutants, le mutant EDK-Δ-Bd37 dont les ponts salins ont été rompus montre des caractéristiques différentes de Δ-Bd37. Les données enregistrées sur ce mutant nous ont amené à résoudre sa structure et à tester son pouvoir vaccinant. Dans une seconde partie, nous caractérisons biochimiquement et fonctionnellement une autre protéine Bc28.1, l'orthologue de Bd37.1. chez B. canis, accompagnée de la résolution de sa structure. Nous montrons que Bc28.1 est une protéine d'adhésion localisée à la surface du parasite et nous comparons les structures de Bd37.1 et Bc28.1. Ces deux structures sont finalement très différentes tandis que localisation et fonction sont similaires. / One of the most common parasitic infections in animals worldwide is babesiosis or piroplasmosis. Caused by the intraerythrocytic development of Babesia parasite, it has many clinical signs similar to those of malaria. This parasite of the phylum Apicomplexa, is transmitted via the tick vector and performs its reproductive cycle in red blood cells of the vertebrate host. B. In Europe divergens and B. canis species are mainly responsible respectively for bovine babesiosis and canine babesiosis. A strategy of vaccine research, the study of parasite proteins in contact with the bloodstream is essential for understanding host-parasite interactions and identify vaccine candidates with high potential. Anchored protein GPI (glycosylphosphatidylinositol) are part of these proteins. The first protein GPI anchors described in B. divergens is Bd37.1. It induces complete protection against infection with B. divergens provided a hydrophobic sequence is added at the C-terminus. Resolution NMR structure of this protein has highlighted a probable mechanism of conformational change as a function of pH. The structure consists of three sub areas shows that it is only maintained by salt bridges which can break in acidic medium. However, the environment within which Bd37.1 membrane anchored to the surface of the parasite and / or approach the red blood cell during the invasion is acidic. This conformational dynamics of the protein-Δ Bd37 linked to the membrane environment, could be at the origin of the mechanism that confers immunity depending on the presence or absence of the hydrophobic sequence at the C-terminus of Bd37.1. We sought to assess the implications of such dynamics in host-parasite interactions through structural study of two parasite proteins (Bd37.1 and Bc28.1). In the first case we study the conformational dynamics of the adhesion protein Bd37.1. We explored the different conformations that may be adopted by a protein Bd37.1 biophysical approach and we have stabilized in different conformations in solution through mutations to study. Among these mutants, the mutant Δ-Bd37-EDK including salt bridges were broken shows different characteristics Δ-Bd37. The data on this mutant led us to solve the structure and to test its power vaccinating. In a second part, we characterize biochemically and functionally Bc28.1 another protein, the ortholog Bd37.1. in B. canis, accompanied with the resolution of its structure. We show that Bc28.1 is an adhesion protein localized to the parasite surface and compare the structures and Bd37.1 Bc28.1. These two structures are ultimately very different while location and function are similar.
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Clathrin Independent Carriers: Molecular characterisation of a novel clathrin-independent endocytic pathwayMark Howes Unknown Date (has links)
Endocytosis effectuates a critical interface between the eukaryotic cell and its apposing environment. It is, subsequently, paramount for many physiologically important processes and encompasses a diverse array of mechanisms and pathways. The classical endocytic routes mediated by clathrin and caveolin are the best understood and the molecular roles of their major regulators, such as dynamin, adaptor proteins and various lipid species, are the most comprehensively described. Recent identification of an assortment of constitutive, noncaveolar, clathrin-independent endocytic (CIE) pathways has expanded the endocytic system. Unlike the classical endocytic pathways, little is known about the guiding parameters of CIE routes. Consequently, it is not possible to understand the important cellular roles these pathways may be fulfilling. This study has begun to characterise the very basic parameters governing the morphologically striking Clathrin-Independent Carrier (CLIC) pathway. Development of a diverse molecular toolkit has now allowed the quantitation of endocytic capacity provided by CLICs, the visualisation of subtle sorting components of the CLIC pathway, the isolation of novel CLIC cargo and regulators, and has linked this mechanism to the critical cellular processes of cellular migration and membrane repair. Calculation of the individual capacity of endocytic routes provides important information about the contribution of each pathway to total plasma membrane (PM) uptake and turnover. Quantitation of the volume, surface area and number of structures forming per minute in this study shows that CLICs provide the vast majority of constitutive endocytosis, up to four times the capacity of the clathrin mediated endocytic (CME) pathway. As the equivalent of the entire PM area could pass through the CLIC pathway within 12 minutes it is evident that CLICs are fundamental housekeepers of bulk membrane internalisation. Thus, they are likely to be central regulators of PM homeostasis and turnover. High-resolution tomography, in conjunction with analysis of CLIC cargo trafficking, identifies these carriers as complex, pleiomorphic structures that sort the bulk of membrane to early endosomes and recycle cargo back to the cell surface. Such vast internalisation combined with an ability to rapidly recycle components quickly attributes the CLIC pathway as a complex sorting station. Isolation of novel cargo and regulators has identified a striking array of proteins now associated with the CLIC pathway for the first time. A significant proportion of identified targets localise to lipid-rafts and recycle from the PM, facets consistent with association to the CLIC pathway. Numerous targets have also been directly implicated in clathrin-independent endocytosis by independent groups. Verification of selected cargo, such as CD44, Thy-1 and myoferlin, showing specific internalisation through the CLIC pathway, has provided insight into the sorting ability of the CLIC pathway and links to adhesion turnover and membrane recycling. Consistent with a role in cellular adhesion turnover, it was found that CLICs become polarised within migrating cells. This has shown the first instance of spatial separation between three major endocytic routes, CLICs, caveolae and CME and highlights the important and coordinated roles of multiple endocytic pathways during physiologically significant processes. The specific internalisation of paxillin, Thy-1 and CD44 through CLICs at the leading edge of migrating cells suggests that CLICs rapidly turnover adhesion components for dynamic extracellular sensation during directional cell migration. Indeed, specific ablation of the CLIC pathway significantly impedes cellular migration, implying coordination with CME at the leading edge. This study has defined numerous parameters of the CLIC pathway, developing the current understanding of this poorly defined route and places the CLIC pathway as a unique player during critical cellular processes.
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Clathrin Independent Carriers: Molecular characterisation of a novel clathrin-independent endocytic pathwayMark Howes Unknown Date (has links)
Endocytosis effectuates a critical interface between the eukaryotic cell and its apposing environment. It is, subsequently, paramount for many physiologically important processes and encompasses a diverse array of mechanisms and pathways. The classical endocytic routes mediated by clathrin and caveolin are the best understood and the molecular roles of their major regulators, such as dynamin, adaptor proteins and various lipid species, are the most comprehensively described. Recent identification of an assortment of constitutive, noncaveolar, clathrin-independent endocytic (CIE) pathways has expanded the endocytic system. Unlike the classical endocytic pathways, little is known about the guiding parameters of CIE routes. Consequently, it is not possible to understand the important cellular roles these pathways may be fulfilling. This study has begun to characterise the very basic parameters governing the morphologically striking Clathrin-Independent Carrier (CLIC) pathway. Development of a diverse molecular toolkit has now allowed the quantitation of endocytic capacity provided by CLICs, the visualisation of subtle sorting components of the CLIC pathway, the isolation of novel CLIC cargo and regulators, and has linked this mechanism to the critical cellular processes of cellular migration and membrane repair. Calculation of the individual capacity of endocytic routes provides important information about the contribution of each pathway to total plasma membrane (PM) uptake and turnover. Quantitation of the volume, surface area and number of structures forming per minute in this study shows that CLICs provide the vast majority of constitutive endocytosis, up to four times the capacity of the clathrin mediated endocytic (CME) pathway. As the equivalent of the entire PM area could pass through the CLIC pathway within 12 minutes it is evident that CLICs are fundamental housekeepers of bulk membrane internalisation. Thus, they are likely to be central regulators of PM homeostasis and turnover. High-resolution tomography, in conjunction with analysis of CLIC cargo trafficking, identifies these carriers as complex, pleiomorphic structures that sort the bulk of membrane to early endosomes and recycle cargo back to the cell surface. Such vast internalisation combined with an ability to rapidly recycle components quickly attributes the CLIC pathway as a complex sorting station. Isolation of novel cargo and regulators has identified a striking array of proteins now associated with the CLIC pathway for the first time. A significant proportion of identified targets localise to lipid-rafts and recycle from the PM, facets consistent with association to the CLIC pathway. Numerous targets have also been directly implicated in clathrin-independent endocytosis by independent groups. Verification of selected cargo, such as CD44, Thy-1 and myoferlin, showing specific internalisation through the CLIC pathway, has provided insight into the sorting ability of the CLIC pathway and links to adhesion turnover and membrane recycling. Consistent with a role in cellular adhesion turnover, it was found that CLICs become polarised within migrating cells. This has shown the first instance of spatial separation between three major endocytic routes, CLICs, caveolae and CME and highlights the important and coordinated roles of multiple endocytic pathways during physiologically significant processes. The specific internalisation of paxillin, Thy-1 and CD44 through CLICs at the leading edge of migrating cells suggests that CLICs rapidly turnover adhesion components for dynamic extracellular sensation during directional cell migration. Indeed, specific ablation of the CLIC pathway significantly impedes cellular migration, implying coordination with CME at the leading edge. This study has defined numerous parameters of the CLIC pathway, developing the current understanding of this poorly defined route and places the CLIC pathway as a unique player during critical cellular processes.
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Clathrin Independent Carriers: Molecular characterisation of a novel clathrin-independent endocytic pathwayMark Howes Unknown Date (has links)
Endocytosis effectuates a critical interface between the eukaryotic cell and its apposing environment. It is, subsequently, paramount for many physiologically important processes and encompasses a diverse array of mechanisms and pathways. The classical endocytic routes mediated by clathrin and caveolin are the best understood and the molecular roles of their major regulators, such as dynamin, adaptor proteins and various lipid species, are the most comprehensively described. Recent identification of an assortment of constitutive, noncaveolar, clathrin-independent endocytic (CIE) pathways has expanded the endocytic system. Unlike the classical endocytic pathways, little is known about the guiding parameters of CIE routes. Consequently, it is not possible to understand the important cellular roles these pathways may be fulfilling. This study has begun to characterise the very basic parameters governing the morphologically striking Clathrin-Independent Carrier (CLIC) pathway. Development of a diverse molecular toolkit has now allowed the quantitation of endocytic capacity provided by CLICs, the visualisation of subtle sorting components of the CLIC pathway, the isolation of novel CLIC cargo and regulators, and has linked this mechanism to the critical cellular processes of cellular migration and membrane repair. Calculation of the individual capacity of endocytic routes provides important information about the contribution of each pathway to total plasma membrane (PM) uptake and turnover. Quantitation of the volume, surface area and number of structures forming per minute in this study shows that CLICs provide the vast majority of constitutive endocytosis, up to four times the capacity of the clathrin mediated endocytic (CME) pathway. As the equivalent of the entire PM area could pass through the CLIC pathway within 12 minutes it is evident that CLICs are fundamental housekeepers of bulk membrane internalisation. Thus, they are likely to be central regulators of PM homeostasis and turnover. High-resolution tomography, in conjunction with analysis of CLIC cargo trafficking, identifies these carriers as complex, pleiomorphic structures that sort the bulk of membrane to early endosomes and recycle cargo back to the cell surface. Such vast internalisation combined with an ability to rapidly recycle components quickly attributes the CLIC pathway as a complex sorting station. Isolation of novel cargo and regulators has identified a striking array of proteins now associated with the CLIC pathway for the first time. A significant proportion of identified targets localise to lipid-rafts and recycle from the PM, facets consistent with association to the CLIC pathway. Numerous targets have also been directly implicated in clathrin-independent endocytosis by independent groups. Verification of selected cargo, such as CD44, Thy-1 and myoferlin, showing specific internalisation through the CLIC pathway, has provided insight into the sorting ability of the CLIC pathway and links to adhesion turnover and membrane recycling. Consistent with a role in cellular adhesion turnover, it was found that CLICs become polarised within migrating cells. This has shown the first instance of spatial separation between three major endocytic routes, CLICs, caveolae and CME and highlights the important and coordinated roles of multiple endocytic pathways during physiologically significant processes. The specific internalisation of paxillin, Thy-1 and CD44 through CLICs at the leading edge of migrating cells suggests that CLICs rapidly turnover adhesion components for dynamic extracellular sensation during directional cell migration. Indeed, specific ablation of the CLIC pathway significantly impedes cellular migration, implying coordination with CME at the leading edge. This study has defined numerous parameters of the CLIC pathway, developing the current understanding of this poorly defined route and places the CLIC pathway as a unique player during critical cellular processes.
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Preliminary Steps to Isolate a Novel Receptor for Mac-1Zou, Xiaoyan 12 December 2003 (has links)
No description available.
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Dynamic Meso-Scale Anchorage of GPI-Anchored Receptors in the Plasma Membrane: Prion Protein vs. Thy1 / 細胞膜上のGPIアンカー型受容体のダイナミックなメゾスケール領域への結合と閉じ込め:プリオンタンパク質 vs. Thy1Nemoto, Yuri 23 March 2022 (has links)
京都大学 / 新制・論文博士 / 博士(医科学) / 乙第13482号 / 論医科博第8号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 岩田 想, 教授 秋山 芳展, 教授 近藤 玄 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Modifications de la paroi au cours de la maturation et de la germination des conidies de Scedosporium boydii / A multifaceted study of the cell wall changes during maturation and germination of the conidia in Scedosporium boydiiGhamrawi, Sarah 17 November 2014 (has links)
Les espèces du complexe Scedosporium apiospermum sont des agents pathogènes émergents qui se situent au deuxième rang parmi les champignons filamenteux rencontrés au cours de la mucoviscidose. Ils sont omniprésents et particulièrement rencontrés dans les zones polluées. En dépit de leur importance clinique, nos connaissances sur leur biologie moléculaire et leur physiologie restent limitées. Chez les champignons, la paroi constitue un bouclier protecteur face à des conditions environnementales défavorables, et joue un rôle essentiel dans la pathogénicité. Ici, nous avons étudié les changements dynamiques de la paroi des conidies de S. boydii, l’une des deux espèces majeures de ce complexe avec S. apiospermum, avec pour objectif d'identifier des facteurs de virulence potentiels. En utilisant une large variété de techniques, allant de la microscopie électronique à balayage ou à transmission à l’analyse protéomique des protéines à ancre glycosylphosphatidylinositol (GPI) en passant par la microélectrophorèse et la partition de phase, la cytométrie en flux, la microscopie de force atomique, la résonance paramagnétique électronique, ou encore des techniques moléculaires, nous avons mis en évidence diverses modifications qui se produisent dans la paroi pendant la maturation et la germination des conidies de S. boydii et nous avons identifié la DHN-mélanine ainsi qu'un nombre important de protéines à ancre GPI. Enfin, nous avons fourni la première séquence complète du génome de S. apiospermum qui appuierait les différents domaines de la recherche sur ces champignons que ce soit pour l’étude des mécanismes pathogènes ou pour des applications biotechnologiques. / Species of the Scedosporium apiospermum complex are emerging human pathogens which rank the second, after Aspergillus fumigatus, among the filamentous fungi colonizing the airways of patients with cystic fibrosis. These fungi are ubiquitous in nature and particularly encountered in polluted areas. Despite their clinical relevance, our knowledge about their molecular biology and physiology remains rather limited. In fungi, the cell wall forms a protective shield against adverse environmental conditions, and therefore plays a key role in pathogenesis, which makes it an interesting target for antifungal drug development. Here, in an attempt to identify potential virulence factors, we investigated the dynamic changes of the cell wall of conidia in S. boydii, one of the main pathogenic species within this species complex with Scedosporium apiospermum. Using various techniques, ranging from scanning and transmission electron microscopy to proteomic analysis of glycosylphosphatidylinositol (GPI)- anchored proteins, through two-phase partitioning and microelectrophoresis, atomic force microscopy and chemical force spectroscopy, flow 5 cytometry, electron paramagnetic resonance and molecular techniques, we highlighted various modifications occurring in the cell wall during maturation and germination of S. boydii and we identified DHN-melanin as well as a substantial number of GPI-anchored proteins in the cell wall. Finally, we provided the first publicly available genome sequence of S. apiospermum that would support various research fields on these fungi whether for understanding their pathogenic mechanisms or for various biotechnological applications.
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Internalization and survival mechanisms of human ehrlichiosis agents ehrlichia chaffeensis and anaplasma phagocytophilum in host cellsLin, Mingqun 06 August 2003 (has links)
No description available.
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Functional characterization of GPI-anchored proteins of the SKU5/SKS gene familyZhou, Ke 21 June 2013 (has links) (PDF)
ABP1 (Auxin Binding Protein1), who can bind auxin, is essential for the development of plants. It was proved to have the ability to bind auxin and transduce auxin signal into the cells. It is supposed to be localized and functions at the outer surface of plasma membrane through unknown component. In my thesis, we tried to invesitgate the interaction between ABP1 and the candidate of the unknown component, CBP1 (From maize), which is GPI-acnhored and already identified as the binding ability to synthesized C-terminus peptide of ABP1 in 2006. The orthologous of CBP1 in arabidopsis belongs to a gene family with 19 members, in which only three of them were prediceted to be GPI anchored. We did the functional characterisation of these three GPI-anchored members. Data suggested that GPI-anchored SKS were involved in cell orientation, gametophyte and embryo development.
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