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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Annexin A6 involvement in the organization of cholesterol-rich membrane microdomains : evidence from cells of the Niemann-Pick type C disease patients and biomimetic lipid monolayers

Domoń, Magdalena 13 December 2011 (has links) (PDF)
The Niemann-Pick type C (NPC) disease is a lysosomal lipid storage disorder caused by mutations in one of the two genes NPC1 or NPC2 encoding proteins of the late endosome/lysosome compartment (LE/LY). Defect in these proteins alters vesicular transport and leads to abnormal accumulation of cholesterol (Chol) in LE/LY. There are some lines of evidence suggesting that annexin A6 (AnxA6) participates in vesicular transport of Chol and may interact with membrane domains enriched in Chol and bind Chol. In this work we characterized the membrane microdomains resistant to Triton X-100, i.e., detergent-resistant membranes (DRMs) isolated from NPC patient-derived fibroblasts and from control cells. NPC cells contain a significantly higher amount of DRMs than the control cells that is consistent with the defect in Chol turnover in NPC cells. We also studied the mechanism of AnxA6 involvement in the NPC-induced changes in the membrane organization and showed that in the presence of calcium some AnxA6 molecules associate with the DRMs. This suggests that AnxA6 may play a role in the membrane lateral organization, contributing thus to the etiology of NPC disease. We then focused on the interaction of AnxA6-1 with Chol-rich membranes and on the involvement of its flexible region and VAAEIL sequence in these interactions. For this purpose, kinetics of the interfacial adsorption of human recombinant AnxA6 to Langmuir monolayers containing phosphatidylcholine, Chol and/or cholesteryl acetate were measured. Our data suggest that AnxA6 exhibits the highest affinity to Chol-containing monolayers and that the hydroxyl group of Chol plays a pivotal role in the AnxA6-lipid interactions in vitro.
2

Synthesis of Nickel-Chelating Fluorinated Lipids for Membrane Protein Monolayer Crystallisations

Waleed Hussein Unknown Date (has links)
Abstract 3D crystallisation of membrane proteins presents a bottleneck for the determination of the structures of membrane proteins. Obtaining 3D crystals of membrane proteins is made difficult by a number of factors including the poor solubility and instability of membrane proteins outside of their native membrane environment. 2D crystallisation of membrane proteins offers an alternative to preserve the conformational structure and functional activities of membrane proteins within their native bilayer membranes in 2D arrays from which the structure of membrane proteins can be determined. Different techniques exist for obtaining 2D crystals of membrane proteins including surface crystallisation or more commonly 2D crystallisation by detergent removal (using either dilution, dialysis, hydrophobic resin adsorption or cyclodextrin complexation) to promote reconstitution of the protein molecules within bilayer-forming lipids. Another method which has been emerged and is being used increasingly is the lipid monolayer technique for 2D crystallisation of proteins. The use of lipid monolayers to bind and adsorb proteins is an attractive and increasingly important method for generating high localised concentrations of oriented proteins and protein complexes. These bound proteins can be imaged directly, or they may form 2D crystalline arrays that are amenable to structure determination by single particle analysis or 2D electron crystallography. 2D crystals grown by this technique can also be used to initiate the growth of 3D crystals for X-ray diffraction analysis. Many derivatised lipids have been prepared for use with this technique, incorporating a diverse range of ligands to enable binding to specific proteins. Synthetic lipids containing functionalised head groups that chelate Ni2+ or Cu2+ have also been prepared to bind and orient expressed proteins that contain His-tags. Protein-binding monolayer-forming lipids generally consist of two distinct components: (1) a branched hydrocarbon tail to confer fluidity to the monolayer and (2) a functionalised hydrophilic head group to facilitate binding of protein molecules at the air-water interface. Newer examples of these compounds also incorporate perfluorinated hydrocarbon moieties to confer detergent resistance to these lipids. The present work discusses the chemistry of all these functionalised lipids and their contributions to monolayer 2D protein crystallisation. This thesis focuses on the synthesis of novel nickel-chelating fluorinated lipids to be used as a template for 2D crystallisation of His-tagged membrane proteins at the air/water interface. These monolayer-forming lipids have been designed with three distinct components: (i) a branched hydrocarbon tail to confer fluidity of the monolayer, (ii) a perfluorinated central core for detergent resistance, and (iii) a nickel-chelating hydrophilic head group to facilitate binding of recombinant, polyhistidine-tagged fusion proteins. Alkylations of fluorinated alcohols used in these syntheses proceed in good yields only with the application of prolonged sonication and, in some cases, in the presence of phase-transfer catalysts. Biophysical properties of Langmuir monolayers formed by our target synthetic fluorinated lipids were studied, comparing the results obtained with those of DOPC and DOGS Ni-NTA as examples of non fluorinated lipids. The Langmuir films were characterised by surface pressure-area isotherms and X-ray reflectometry to show their fluidity, thickness and packing density. The stability of fluorinated lipid monolayers and their ability to resist the solubilisation effects of a number of detergents were investigated using monolayer and affinity grid techniques. Results showed that fluorinated lipids offer an improved resistance to the solubilisation effects of detergents compared with their non-fluorinated counterparts. A number of trials for 2D crystallisation of both soluble and membrane proteins have been performed using fluorinated lipid monolayers. These new synthetic fluorinated lipids were successfully used to obtain 2D crystals of the His-tagged membrane protein BmrA from Bacillus subtillis by the monolayer technique.
3

Synthesis of Nickel-Chelating Fluorinated Lipids for Membrane Protein Monolayer Crystallisations

Waleed Hussein Unknown Date (has links)
Abstract 3D crystallisation of membrane proteins presents a bottleneck for the determination of the structures of membrane proteins. Obtaining 3D crystals of membrane proteins is made difficult by a number of factors including the poor solubility and instability of membrane proteins outside of their native membrane environment. 2D crystallisation of membrane proteins offers an alternative to preserve the conformational structure and functional activities of membrane proteins within their native bilayer membranes in 2D arrays from which the structure of membrane proteins can be determined. Different techniques exist for obtaining 2D crystals of membrane proteins including surface crystallisation or more commonly 2D crystallisation by detergent removal (using either dilution, dialysis, hydrophobic resin adsorption or cyclodextrin complexation) to promote reconstitution of the protein molecules within bilayer-forming lipids. Another method which has been emerged and is being used increasingly is the lipid monolayer technique for 2D crystallisation of proteins. The use of lipid monolayers to bind and adsorb proteins is an attractive and increasingly important method for generating high localised concentrations of oriented proteins and protein complexes. These bound proteins can be imaged directly, or they may form 2D crystalline arrays that are amenable to structure determination by single particle analysis or 2D electron crystallography. 2D crystals grown by this technique can also be used to initiate the growth of 3D crystals for X-ray diffraction analysis. Many derivatised lipids have been prepared for use with this technique, incorporating a diverse range of ligands to enable binding to specific proteins. Synthetic lipids containing functionalised head groups that chelate Ni2+ or Cu2+ have also been prepared to bind and orient expressed proteins that contain His-tags. Protein-binding monolayer-forming lipids generally consist of two distinct components: (1) a branched hydrocarbon tail to confer fluidity to the monolayer and (2) a functionalised hydrophilic head group to facilitate binding of protein molecules at the air-water interface. Newer examples of these compounds also incorporate perfluorinated hydrocarbon moieties to confer detergent resistance to these lipids. The present work discusses the chemistry of all these functionalised lipids and their contributions to monolayer 2D protein crystallisation. This thesis focuses on the synthesis of novel nickel-chelating fluorinated lipids to be used as a template for 2D crystallisation of His-tagged membrane proteins at the air/water interface. These monolayer-forming lipids have been designed with three distinct components: (i) a branched hydrocarbon tail to confer fluidity of the monolayer, (ii) a perfluorinated central core for detergent resistance, and (iii) a nickel-chelating hydrophilic head group to facilitate binding of recombinant, polyhistidine-tagged fusion proteins. Alkylations of fluorinated alcohols used in these syntheses proceed in good yields only with the application of prolonged sonication and, in some cases, in the presence of phase-transfer catalysts. Biophysical properties of Langmuir monolayers formed by our target synthetic fluorinated lipids were studied, comparing the results obtained with those of DOPC and DOGS Ni-NTA as examples of non fluorinated lipids. The Langmuir films were characterised by surface pressure-area isotherms and X-ray reflectometry to show their fluidity, thickness and packing density. The stability of fluorinated lipid monolayers and their ability to resist the solubilisation effects of a number of detergents were investigated using monolayer and affinity grid techniques. Results showed that fluorinated lipids offer an improved resistance to the solubilisation effects of detergents compared with their non-fluorinated counterparts. A number of trials for 2D crystallisation of both soluble and membrane proteins have been performed using fluorinated lipid monolayers. These new synthetic fluorinated lipids were successfully used to obtain 2D crystals of the His-tagged membrane protein BmrA from Bacillus subtillis by the monolayer technique.
4

Annexin A6 involvement in the organization of cholesterol-rich membrane microdomains : evidence from cells of the Niemann-Pick type C disease patients and biomimetic lipid monolayers / Rôle de l’annexine A6 dans l’organisation des microdomaines membranaires enrichis en cholestérol : mise en évidence sur des cellules atteintes de la maladie de Niemann-Pick et des monocouches lipidiques biomimétiques

Domoń, Magdalena 13 December 2011 (has links)
La maladie de Niemann-Pick de type C (NPC) est une lipidose lysosomale complexe due à une mutation d’un des gènes NPC1 ou NPC2, qui codent pour ces protéines localisées dans les compartiments endo-lysosomaux (LE/LY). Leur absence altère le trafic intracellulaire et induit l’accumulation du cholestérol (Chol) dans les LE/LY. De plus, l’AnxA6 semble participer au transport vésiculaire du Chol en interagissant avec les microdomaines membranaires enrichis en Chol, ou avec le Chol lui-même. Dans ce travail, nous avons isolé des microdomaines membranaires résistant au Triton X-100 (également appelés DRMs pour detergent resistant membranes) à partir de lignée cellulaire NPC L1 ou de cellules saines. Les fibroblastes NPC contiennent plus de DRMs que les fibroblastes sains. Ceci semble être corrélé aux problèmes de transport du Chol dans les cellules NPC. Nous avons aussi montré qu’en présence de calcium, une partie de l’AnxA6 est associé aux DRMs, suggérant que l’AnxA6 participe à l’organisation de la membrane et par ce bias à l’étiologie de la maladie de NPC. Nous avons alors analysé les interactions de l’AnxA6-1 avec les microdomaines riches en Chol ainsi que l’implication de sa région flexible et de la séquence VAAEIL dans ces interactions. Leurs interactions avec des monocouches de Langmuir constituées de phosphatidylcholine, Chol et/ou d’acétate de cholestéryle. Nos résultats montrent que l’AnxA6 a la plus grande affinité pour les monocouches contenant du Chol ainsi que l’implication du groupement hydroxyle du Chol lors de ces interactions. / The Niemann-Pick type C (NPC) disease is a lysosomal lipid storage disorder caused by mutations in one of the two genes NPC1 or NPC2 encoding proteins of the late endosome/lysosome compartment (LE/LY). Defect in these proteins alters vesicular transport and leads to abnormal accumulation of cholesterol (Chol) in LE/LY. There are some lines of evidence suggesting that annexin A6 (AnxA6) participates in vesicular transport of Chol and may interact with membrane domains enriched in Chol and bind Chol. In this work we characterized the membrane microdomains resistant to Triton X-100, i.e., detergent-resistant membranes (DRMs) isolated from NPC patient-derived fibroblasts and from control cells. NPC cells contain a significantly higher amount of DRMs than the control cells that is consistent with the defect in Chol turnover in NPC cells. We also studied the mechanism of AnxA6 involvement in the NPC-induced changes in the membrane organization and showed that in the presence of calcium some AnxA6 molecules associate with the DRMs. This suggests that AnxA6 may play a role in the membrane lateral organization, contributing thus to the etiology of NPC disease. We then focused on the interaction of AnxA6-1 with Chol-rich membranes and on the involvement of its flexible region and VAAEIL sequence in these interactions. For this purpose, kinetics of the interfacial adsorption of human recombinant AnxA6 to Langmuir monolayers containing phosphatidylcholine, Chol and/or cholesteryl acetate were measured. Our data suggest that AnxA6 exhibits the highest affinity to Chol-containing monolayers and that the hydroxyl group of Chol plays a pivotal role in the AnxA6-lipid interactions in vitro.
5

Characterization of Molecular Glycerophospholipids by Quadrupole Time-of-Flight Mass Spectrometry

Ekroos, Kim 10 November 2003 (has links) (PDF)
The physical properties of glycerophospholipids (GPLs) are not only determined by the head group (HG), but also by their fatty acid (FA) chains, which affect their distribution and function within membranes in the cell. Understanding the microheterogenity of lipid membranes on a molecular level requires qualitative and quantitative characterization of individual lipids and identification of their FA moieties. The aim of my study was to introduce the new technology of multiple precursor ion scanning (MPIS) on a QSTAR Pulsar time-of-flight mass spectrometer (QqTOF) to analyze lipids. Detailed information on fatty acid composition of individual GPL molecules could be obtained in parallel with conventional profiling of lipid classes, and this could be done by direct analysis of total lipid extracts. This method was termed Fatty Acid Scanning (FAS) and Head Group Scanning HGS, respectively. In this way the molecular GPL composition of total lipid extracts could be charted in a single analysis accurately and rapidly at a low picomole concentration level. Furthermore, combining FAS and HGS together with ion trap MS3 analysis allowed complete charting of the molecular composition of PCs, including quantification of their positional isomers, thus providing a detailed and comprehensive characterization of molecular composition of the pool of PCs. Development of the Lipid Profiler software allowed full automation and rapid processing of complex data, including identification and quantification of molecular GPLs. This approach was evaluated by preliminary applications. First, the molecular composition of PCs of total lipid extracts of MDCK cells and of human red blood cells (RBC) could accurately be charted. Significant presence of positional isomers was observed increasing the total number of individual PC species close to one hundred. Secondly, the molecular PC and SM species distribution in detergent resistant membranes (DRMs) prepared by Triton X-100 DRMs were analyzed and were found to be enriched in distinct GPLs. The distribution in PCs and SMs of Triton X-100 DRMs of RBC were compared with those of the DRMs of MDCK cells. Finally, combining the use of a 96 well plate and a robotic system demonstrated that these analyses can be automated and analyzed with high throughput. This system we termed Shotgun Lipidomics. Taken together, this mass spectrometric methodology provides rapid and detailed insight into the distribution of the molecular GPLs of membranes and membrane sub-fractions.
6

Characterization of Molecular Glycerophospholipids by Quadrupole Time-of-Flight Mass Spectrometry

Ekroos, Kim 12 December 2003 (has links)
The physical properties of glycerophospholipids (GPLs) are not only determined by the head group (HG), but also by their fatty acid (FA) chains, which affect their distribution and function within membranes in the cell. Understanding the microheterogenity of lipid membranes on a molecular level requires qualitative and quantitative characterization of individual lipids and identification of their FA moieties. The aim of my study was to introduce the new technology of multiple precursor ion scanning (MPIS) on a QSTAR Pulsar time-of-flight mass spectrometer (QqTOF) to analyze lipids. Detailed information on fatty acid composition of individual GPL molecules could be obtained in parallel with conventional profiling of lipid classes, and this could be done by direct analysis of total lipid extracts. This method was termed Fatty Acid Scanning (FAS) and Head Group Scanning HGS, respectively. In this way the molecular GPL composition of total lipid extracts could be charted in a single analysis accurately and rapidly at a low picomole concentration level. Furthermore, combining FAS and HGS together with ion trap MS3 analysis allowed complete charting of the molecular composition of PCs, including quantification of their positional isomers, thus providing a detailed and comprehensive characterization of molecular composition of the pool of PCs. Development of the Lipid Profiler software allowed full automation and rapid processing of complex data, including identification and quantification of molecular GPLs. This approach was evaluated by preliminary applications. First, the molecular composition of PCs of total lipid extracts of MDCK cells and of human red blood cells (RBC) could accurately be charted. Significant presence of positional isomers was observed increasing the total number of individual PC species close to one hundred. Secondly, the molecular PC and SM species distribution in detergent resistant membranes (DRMs) prepared by Triton X-100 DRMs were analyzed and were found to be enriched in distinct GPLs. The distribution in PCs and SMs of Triton X-100 DRMs of RBC were compared with those of the DRMs of MDCK cells. Finally, combining the use of a 96 well plate and a robotic system demonstrated that these analyses can be automated and analyzed with high throughput. This system we termed Shotgun Lipidomics. Taken together, this mass spectrometric methodology provides rapid and detailed insight into the distribution of the molecular GPLs of membranes and membrane sub-fractions.
7

Study of the dynamics of biomolecules by high speed atomic force microscopy and surface enhanced Raman spectroscopy / L'étude dynamique des biomolécules par le microscope à force atomique haute-vitesse (HS-AFM) et la spectroscopie Raman exaltée de surface (SERS)

Aybeke, Ece Neslihan 08 July 2015 (has links)
Ce travail de thèse se focalise sur le couplage du microscope à force atomique haute–vitesse (HS-AFM) et de la spectroscopie Raman exaltée de surface (SERS) pour la détection des biomolécules. Nous avons élaboré un protocole de fabrication pour produire les substrats “SERS-actifs”. L’efficacité des substrats de nanoparticules cristalline d’or, d’argent ou bimétallique argent–or a été évaluée. Nous avons étudié l’impact des propriétés optiques et morphologiques des substrats sur l’intensité Raman en analysant des échantillons tests tels que la bipyridine éthylène et le bleu de méthylène. Nous nous sommes interessés à trois problematiques biologiques distinctes par analyses HS-AFM et SERS. Dans un premier cas, nous avons détecté la signature chimique de protéine cytochrome b5. Ce travail a été suivi par des études sur le changement de conformation de la protéine de choc thermique leuconostoc oenos Lo 18 en fonction de la concentration et du pH. La dernière application consiste en l’analyse des interactions membrane – virus. Afin de réaliser les analyses simultanées Raman/AFM, nous avons adapté notre protocole de fabrication pour couvrir la surface des pointes AFM commerciales par des nanoparticules d’or cristallines. Les études de diffusion Raman exaltée par effet de pointe (TERS) ont été effectuées sur les échantillons de disulfure de molybdène pour évaluer la qualité des pointes TERS. Pour finir, nous présentons une nouvelle configuration de couplage HS-AFM et spectroscopie Raman. Nous discutons des modifications et des défis rencontrés. / This thesis focuses on the coupling of High–Speed Atomic Force Microscopy (HS-AFM) and Surface Enhanced Raman Spectroscopy (SERS) for biomolecule analysis. We have designed a fabrication protocol to manufacture “SERS-active” substrates. The efficacy of gold, silver and gold-silver bimetallic crystalline nanoparticle substrates were evaluated. We have investigated the impact of optical and morphological features of the substrates on Raman signal intensity by analyzing well-known samples such as bipyridine ethylene and methylene blue molecules. We took an interest in three distinct biological problematics with HS-AFM and SERS analyses. First, we have detected the chemical signature of cytochrome b5 protein. This study was followed by the investigation of conformational changes of small heat shock leuconostoc oenos Lo 18 protein in function of pH level and concentrations. The last application consists to the analyse a membrane and a virus interaction. In order to realize simultaneous Raman/AFM analysis, we have adapted our fabrication protocol to cover the surface of commercial AFM probes by crystalline gold nanoparticles. Tip – Enhanced Raman Spectroscopy (TERS) studies were performed on molybdenum disulfide to evaluate the quality of TERS probes. In the last part of this work, we have designed a new setup to combine Ando’s HS-AFM setup with Raman spectroscopy. We present the modifications that have been carried out and the challenges that we have encountered.

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