Dissertations / Theses on the topic 'Phospholipids/biological membranes'

Biological membranes mainly constituent lipid molecules along with some proteins and steroles. The properties of the pure lipid bilayers as well as in the presence of other constituents (in case of two or three component systems) are very important to be studied carefully to model these systems and compare them with the realistic systems. Molecular dynamic simulations provide a good opportunity to model such systems and to study them at microscopic level where experiments fail to do. In this thesis we study the structural and dynamic properties of the pure phospholipid bilayers and the phase behavior of phospholipid bilayers when other constituents are present in them. Material and structural properties like area per lipid and area compressibility of the phospholipids show a big scatter in experiments. These properties are studied for different system sizes and it was found that the increasing undulations in large systems effect these properties. A correction was applied to area per lipid and area compressibility using the Helfrich theory in Fourier space. Other structural properties like order of the lipid chains, electron density and radial distribution functions are calculated which give the structure of the lipid bilayer along the normal and in the lateral direction. These properties are compared to the X-ray and neutron scattering experiments after Fourier transform. Thermodynamic properties like heat capacity and heat of melting are also calculated from derivatives of energies available in molecular dynamics. Heat capacity on the other hand include quantum effect and are corrected for that by applying quantum correction using normal mode analysis for a simple as well as ambiguous system like water. Here it is done for SPC/E water model. The purpose of this study is to further apply the quantum corrections on macromolecules like lipids by using this technique. Furthermore the phase behavior of two component systems (phospholipids/cholesterol) is also studied. Phase transition in these systems is observed at different cholesterol concentrations as a function of temperature by looking at different quantities (as an order parameter) like the order of chains, area per molecule and partial specific area. Radial distribution functions are used to look at the in plane structure for different phases having a different lateral or positional order. Adding more cholesterol orders the lipid chains changing a liquid disordered system into a liquid ordered one and turning a solid ordered system into a liquid ordered one. Further more the free energy of domain formation is calculated to investigate the two phasecoexistence in binary systems. Free energy contains two terms. One is bulk freeenergy which was calculated by the chemical potential of cholesterol moleculein a homogeneous system which is favorable for segregation. Second is thefree energy of having an interface which is calculated from the line tension of the interface of two systems with different cholesterol concentration which in unfavorable for domain formation. The size of the domains calculated from these two contributions to the free energy gives the domains of a few nm in size. Though we could not find any such domains by directly looking at our simulations.

QC 20120913

The aim of the thesis project was to examine what form(s) of Amyloid beta (Aβ) (25-­‐35) peptide interact with phospholipids in vitro and the implications of this for the mechanism of Alzheimer’s Diseases (AD). The mechanism of AD is thought to involve protein folding and misfolding. An increasing amount of evidence has shown that protein misfolding plays an important role in the biological and pathological processes of AD. Although seen as the biomedical markers of those diseases, the roles of amyloid aggregates themselves are still not fully understood. Whether the aggregates, or the monomer, or some other intermediates of Aβ cause AD is still unknown. In order to investigate the membrane-­‐interaction of Aβ and its implications for AD, two forms of Aβ, namely levorotary and dextrorotary (L-­‐ and D-­‐) Aβ isomers were used. Evidence has shown that L-­‐ and D-­‐ peptide can each form aggregates in a humid environment. However, when mixed together, L-­‐ and D-­‐ peptides tend not to form any aggregates. Using the mixtures of L-­‐ and D-­‐ peptides at different proportions and as well as using L-­‐ and D-­‐ alone can help us to determine the toxic form of Aβ. Phospholipids have been used to mimic membrane bilayers. Biological membranes in vivo are a complicated system. They contain three types of lipids, namely phospholipids, glycolipids, and steroids. Different types of cells and different membranes have different proportions of those lipids. Studying the interaction between Aβ and membranes in vivo can be extremely difficult. Artificial membranes, which only contains one kind of lipids, on the other hand, are a useful tool for the study of molecular interactions. Phospholipids are the most abundant type of membrane lipid and thus that can be seen as representative of cell membranes. The interactions of Aβ and different kinds of phospholipids have been investigated in this project. This thesis discusses the secondary structure of Aβ in different environment, the interaction between Aβ and phospholipids at the air-­‐water surface, and the location of Aβ in membranes during the interaction. The study provides useful information of the mechanisms and the origin of AD. At the end of the thesis, a discussion chapter analyses the difficulties of studying Aβ and AD and the potentials and inadequacies of this research.

Phospholipid bilayers constitute the primary structural element of biological membranes, and as such, they play a central role in biochemical and biophysical processes at the cellular level, including cell protection, intercellular interactions, trans-membrane transport, cell morphology, and protein function, to name a few. The properties of phospholipid bilayers are thus of great interest from both experimental and theoretical standpoints. Although experiments have provided much of the macroscopic functions and properties of biological membranes, insight into specific mechanisms at the molecular level are seldom accessible by conventional methods. To obtain a better understanding of biochemical and biophysical processes at the molecular level involving phospholipid bilayers, we apply molecular simulation methods to investigate the complexity of the membrane matrix using atomistic models. Here, we discuss three specific biological processes that are associated with biological membranes: 1) membrane stabilization, 2) membrane phase behavior, and 3) fatty acid-induced toxicity in cell membranes. For membrane stabilization, molecular dynamics studies were performed for mixed phospholipid bilayers containing two of the most prevalent phospholipids (phosphatidylcholine and phosphatidylethanolamime) in biological membranes. We presented structural and dynamics properties of these systems, as well as the effect of stabilizing agents, such as trehalose, on their properties. Furthermore, we performed a comprehensive analysis of the phase transition of lipid bilayers and investigated the interactions of stabilizing agents (glucose or trehalose) with lipid bilayers under dehydrated conditions to understand the mechanisms for preservation of cellular systems. For membrane phase behavior, a comprehensive study of the structural properties of saturated and monounsaturated lipid bilayers near the main phase transition were investigated using molecular dynamics simulations. In this study, we demonstrated that atomistic simulations are capable of capturing the phase transformation process of lipid bilayers, providing a valuable set of molecular and structural information at and near its transition state. Lastly, the third study investigated the mechanism for fatty acid-induced toxicity by integrating in vitro and in silico experiments to reveal the biophysical interactions of saturated fatty acid (palmitate) with the cellular membranes and the role of trehalose and unsaturated fatty acids (oleate and linoleate) in preventing changes to the membrane structure. Knowledge gained from this study is essential in the prevention and treatment of obesity-associated cirrhosis diseases.
Ph. D.

A temperatura é o fator ambiental mais importante que afeta a atividade de animais ectotérmicos, como peixes. Ajustes compensatórios à temperatura ocorrem em diferentes cursos temporais, que variam de menos de um minuto a mais de um mês, e as membranas são os primeiros alvos afetados pelas mudanças de temperatura, com resposta imediata dos componentes lipídicos a este desafio. Este trabalho teve como objetivo estimar a capacidade alostática (na estrutura e funções de membrana) no contexto das variáveis climáticas relevantes e caracterizar o âmbito e os mecanismos de mudança, incluindo os mecanismos que concedem tolerância a mudanças de temperatura agudas e crônicas. Juvenis de Steindachneridion parahybae uma espécie de peixe nativa ameaçada de extinção, foram progressivamente resfriados de 30° C a 24, 17 e 12 ° C, nas quais foram mantidas por até 5 dias no tratamento agudo e por até 30 dias no tratamento crônico. Os tecidos hepático, encefálico e branquial foram amostrados, com análises subsequentes das principais frações fosfolipídicas (fosfatidilcolina (FC) e fosfatidiletanolamina (FE) e análises posicionais de cada fração), atividade da Na+/ K+-ATPase e histomorfologia branquial. Os animais mantidos na temperatura mais baixa mostraram uma elevada taxa de mortalidade, provavelmente devido à proximidade desta temperatura ao limite térmico inferior para esta espécie. A atividade da Na+/ K+-ATPase se mostrou aumentada nas temperaturas mais baixas, corroborando o aumento das lesões morfológicas branquiais e massa de fígado para estas temperaturas. Em geral o perfil de ácidos graxos de FC mantiveram-se mais estáveis do que o observado para FE. O teste agudo aparentemente afetou consideravelmente C20-22n3 (FC hepática e sn-1 ; FE encefálica e hepática), enquanto que no teste crônico, C20-22n6 foi o grupamento mais afetado (FC e FE hepático em sn-2 e sn-1). O ensaio agudo mostrou um padrão de manutenção da estrutura de membrana cerebral, com uma diminuição de C20-22n3 hepática e aumento destes ácidos graxos no encéfalo durante o tratamento. Em ambos os tecidos e frações analisados foi possível detectar evidências significativas de reestruturação da membrana, mostrando que o Surubim do Paraíba foi capaz de proporcionar ajustes compensatórios em respostas de aclimatação.
Temperature is the most important environmental factor affecting the activity of ectothermic animals such as fish. Compensatory adjustments to temperature occur with time courses ranging from less than a minute to more than a month, and membranes are the first targets affected by change of temperature, and their lipid components respond immediately to this challenge. This project aimed to estimate the allostatic capacity (in membrane structure and function) in the context of relevant climate variables, and to characterize the scope and the defense mechanisms available, including those yielding tolerance to acute and chronic temperature shifts. Steindachneridion parahybae juveniles, an endangered native fish species, were progressively cooled from 30°C to 24, 17 and 12°C, in which they were maintained for up to 5 days in the acute trial and for up 30 days in the chronic trial. Brain, liver and branchial tissues were sampled, with subsequent analyses of the main phospholipids fractions (phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and the positional analyses of each fraction), Na+/K+-ATPase activity and histomorphology of gills. The animals maintained atlower temperature showed a high rate of mortality, probably because this temperature is near the lower thermal limit for this species. The activity of Na+ K+ATPase increased at lower temperatures, the same pattern observed for morphological injuries in gills and increased liver mass. Generally the fatty acid profiles of PC remained more stable than those in PE. The acute test apparently had affected considerably C20-22n3 (liver PC and sn-1 PC; PE in brain and liver), while for the chronic test, C20-22n6 was more affected (PC and PE liver on sn-2 and sn-1). The acute trial showed a pattern of maintenance of brain membrane structure, with a decrease of PE-associated C20-22n3 in the liver and an increase of these fatty acids in brain during the test. In both tissues and fractions analyzed it was possible to detect significant evidences of membrane restructuring, showing that the Surubim do Paraiba was able to provide compensatory adjustments in acclimation responses

Les solvants eutectiques profonds (DES) sont récemment apparus comme une nouvelle classe de solvants verts présentant un potentiel élevé pour remplacer les solvants organiques usuels. Bien que découverts récemment, les DES ont fait l'objet de nombreuses recherches au cours des dernières années en raison de leurs propriétés intéressantes. Cependant, il reste encore beaucoup à découvrir étant donné le nombre quasiment illimité de DES potentiels et de leur polyvalence. Notre étude vise à examiner l'effet des DES sur les liposomes, adoptés comme modèles membranaires, et sur les membranes cellulaires. Elle a également cherché à évaluer la capacité de solubilisation des DES envers des composés bioactifs volatils. Ainsi, une sélection de DES ainsi que de nouveaux solvants ont été tout d'abord préparés et caractérisés. Des mesures de densité, de viscosité et de polarité ont été effectuées et ont montrées que les propriétés des DES pouvaient être ajustées en fonction de leur composition. L'organisation des phospholipides et des liposomes au sein des DES a ensuite été étudiée à l'aide de microscopies optique et à force atomique. Les phospholipides s'auto-assemblent en vésicules dans les DES à base de chlorure de choline tandis que les liposomes se convertissent en bicouches lipidiques avant leur reconstitution en vésicules. De plus, des études de cytotoxicité et des examens morphologiques ont été combinés afin d'évaluer l'impact de quelques DES sur MDA-MB-231, une lignée cellulaire de cancer du sein humain. Les résultats ont montrés que l'effet dépendait fortement de la composition du DES. D'autre part, la capacité de solubilisation des DES envers des composés bioactifs volatils a été testée par chromatographie en phase gazeuse couplée à un espace de tête. L'influence de la présence d'eau et de certains systèmes d'encapsulation tels que les liposomes et les cyclodextrines sur la capacité de solubilisation des DES ont été analysés. Enfin, la libération du trans-anéthole à partir des DES a été suivie par extraction multiple de l'espace de tête. Les DES ont été capables de mieux solubiliser les composés bioactifs volatils et de contrôler leur libération par rapport à l'eau. Dans l'ensemble, ces travaux mettent en évidence l'utilisation potentielle des systèmes à base de DES comme véhicules de solubilisation de composés bioactifs
Deep eutectic solvents (DES) recently emerged as a novel class of green solvents with a high potential to replace common organic solvents. Despite their novelty, DES were extensively explored in the past years owing to their remarkably interesting properties. Yet, a lot remains to be uncovered given the limitless number of possible DES and their versatility. The current sudy aimed to examine the effect of DES on liposomes, adopted as model membranes, and on cell membranes. It also sought to evaluate the solubilizing ability of DES toward bbioactive volatile compounds. Therefore, a group of selected DES along with new solvents were first prepared and characterized. Density, viscosity and polarity measurements were mainly carried out and showed that DES' properties can be tuned depending on their composition. The organization of phospholipids and liposomes within the DES was then investigated using optical- and atomical force microscopies. Phospholipids self-assembled into vesicles in choline chloride-based DES while liposomes converted to lipid bilayers before their reconstitution into vesicles. Moreover, cytotoxicity studies and morphological examinations were combined to evaluate the impact of some DES on MDA-MB-231, a human breast cancer cell line. Results showed that the effect is highly dependent on the DES' composition. On the other hand, the solubilizing ability of the DES toward bioactive volatile compounds was tested using static headspace-gas chromatography. The influence of the presence of water and some encapsulation systems such as liposomes and cyclodextrins on the overall DES' solubilization efficiency was further analyzed. At last, the release of trans-anethole from the DES was monitored via multiple headspace extraction. DES were able to greatly solubilize the bioactive volatile compounds and to control their release when compared with water. Altogether, this work highlights the potential use of the DES-based systems as solubilization vehicles for bioactive compounds

Les vésicules géantes unilamellaires (GUV) permettent d'étudier efficacement les interactions entre les lipides et les peptides. Dans ce manuscrit, il a été montré que les interactions attractives lipides-peptides sont supprimées par l'attachement de polypeptides de type élastine (ELP) sur des peptides riches en arginine et peuvent être modulées par l'auto-assemblage en micelles ainsi que par le nombre de groupements arginine dans la séquence des peptides capables de pénétrer les cellules. De plus, une nouvelle méthode pour former des GUV à partir de systèmes complexes en seulement quelques minutes a été développée. Cette méthode est basée sur le gonflement d'un film de PVA sous une bicouche lipidique. Elle supprime la dégradation des molécules pendant la formation des GUV de lipides synthétiques, tels que des glycolipides et des phospholipides portant des groupements amides, où les méthodes traditionnelles ne réussissent pas à produire des vésicules non endommagées.

El present treball està dedicat a les aplicacions pràctiques de diversos mètodes teòrics i de simulació incloent la dinàmica molecular, Montecarlo i la mitjana dels càlculs del camp per entendre les propietats físiques del sistema bicapa lipídica, així com la interacció dels objectes a escala nanomètrica en contacte amb lípids desenvolupament i bicapes. En particular, el treball es tracten els següents temes: 1. Optimització i equilibri de propietats bicapes lipídiques utilitzant única cadena de la teoria de camp mig. 2. El desenvolupament del model i estudiar les propietats d'equilibri de bicapes de lípids oxidats amb camp mitjà i els mètodes de dinàmica molecular 3. Estudi de les propietats d'equilibri de doble capa amb nanopartícules utilitzant camp mitjà i els mètodes de Montecarlo. Optimització de polímer translocacional través de la tècnica 5. Mètodes estadístics GPU membrana utilitzats per a les propietats superficials de micro fulles d'alteració bicapes lipídiques.
El presente trabajo está dedicado a las aplicaciones prácticas de varios métodos teóricos y de simulación incluyendo la dinámica molecular, Monte Carlo y la media de los cálculos del campo para entender las propiedades físicas del sistema bicapa lipídica, así como la interacción de los objetos a escala nanométrica en contacto con lípidos desarrollo y bicapas. En particular, el trabajo se tratan los siguientes temas: 1. Optimización y equilibrio de propiedades bicapas lipídicas utilizando única cadena de la teoría de campo medio. 2. El desarrollo del modelo y estudiar las propiedades de equilibrio de bicapas de lípidos oxidados con campo medio y los métodos de dinámica molecular 3. Estudio de las propiedades de equilibrio de bicapa con nanopartículas utilizando campo medio y los métodos de Monte Carlo. Optimización de polímero translocacional través de la técnica 5. Métodos estadísticos GPU membrana utilizados para las propiedades superficiales de micro cuchillas de alteración bicapas lipídicas.
Present work is devoted to the development and practical applications of several theoretical and simulation methods including Molecular dynamics, Monte Carlo and Mean field calculations to understand the physical properties of the lipid bilayer system as well as the interaction of nano-scale object in contact with lipid bilayers. In partcular, the work covers the following topics: 1. Optimization and equilibrium properties of lipid bilayers using Single Chain Mean Field theory. 2. Developing the model and study the equilibrium properties of bilayers with oxidized lipids with mean field and molecular dynamics methods 3. Study the equilibrium properties of bilayer with nanoparticles using mean field and Monte Carlo methods4. Optimization of translocational polymer through membrane GPU technique 5. Statistical methods used to the surface properties of micro blades disrupting lipid bilayers.

The interaction between a membrane protein and its surrounding phospholipids is thought to be crucial for the correct folding and function of the protein. This thesis is focused on the investigation of the interplay between Lactose permease (LacY), a paradigm for secondary transporters present in the inner membrane of Escherichia coli and model systems mimicking its natural lipid environment. Since the role of phospholipids in LacY’s activity is currently being refined, this work represents a contribution to the field by studying the interaction at two different levels: (i) the LacY interplay with the phospholipids present at the annular region in the vicinity of the protein was studied through FRET measurements between a single-tryptophan LacY mutant and diverse pyrene-marked phospholipids, and (ii) the LacY interaction with the more distanced bulk phospholipids was studied through supported proteo-lipid sheets that were analysed using topography, force-spectroscopy and force-volume Atomic Force Microscopy modes. First, after validating LacY preference for phospholipid fluid (Lα) phases in the studied two-component model systems, a different composition between bulk and annular regions was confirmed. Hence, bulk lipids, which were assimilated to the phospholipids in Lα phase, were mainly formed by PG, while PE was the main component of the annular region. This points to a direct annular phospholipid-LacY selectivity because it discards a random phospholipid distribution near the protein. Second, the LacY selectivity for precise phospholipid species at the annular region was found to be related to: (i) a neutral charged phosholipid (PE or PC, with preference for the former), and (ii) phospholipids with large negative spontaneous curvature (C0) (DOPE > POPE). In addition, D68 was revealed as an important amino acid for the protein annular lipid selectivity. Third, the interaction between LacY and the bulk lipids was described as reciprocal. Accordingly, the presence of the protein largely modified the topography and the nanomechanics of the lipid system, especially for the Lα phase, whilst the nanomechanics of LacY itself were different depending on the surrounding lipid matrix: more force was needed to pull LacY form the DPPE:POPG (3:1, mol/mol) system than from the POPE:POPG (3:1, mol/mol) one. Therefore, the bilayer lipid composition seems to determine the forces governing the LacY tight interaction with the membrane and can be thus decisive for the protein correct insertion and activity.
La interacció entre una proteïna de membrana i els fosfolípids que l’envolten és crucial pel bon plegament i la correcta funció de la proteïna. Aquesta tesi està centrada en la investigació de la interacció entre la Lactosa permeasa (LacY), un paradigma dels transportadors secundaris situat a la membrana interna d’Escherichia coli, i sistemes models que mimetitzen el seu entorn lipídic. Aquest treball representa una contribució al camp a través de l’estudi de la interacció a dos nivells: (i) la interacció entre LacY i els fosfolípids presents a la regió anular propera a la proteïna ha estat estudiada a través de mesures de FRET entre un mutant de LacY amb un únic triptòfan i diversos fosfolípids marcats i (ii) la interacció entre LacY amb els fosfolípids més llunyans o bulk s’ha investigat a través de làmines de lípid i proteïna sobre un suport, les quals s’han analitzat a partir de diversos modes de microscòpia de força atòmica (topografia, espectroscòpia de força i force-volume). En primer lloc, s’ha validat la preferència de LacY pels fosfolípids en fases fluïdes (Lα). A més, s’ha confirmat una composició lipídica entre la regió anulars i el bulk. Així, els fosfolípids bulk, considerats com a fosfolípids en fase Lα, tenen PG com a principal component, mentre que PE és el major component de la regió anular. Això sembla indicar una selectivitat entre LacY i els fosfolípids anulars. En segon lloc, s’ha descrit que la selectivitat de LacY per fosfolípid determinat a la regió anular està relacionada amb (i) càrrega neutra i (ii) curvatura espontània (C0) negativa. A més, D68 s’ha assenyalat com un aminoàcid important per la selectivitat de la proteïna envers els lípids anulars. Finalment, s’ha descrit una interacció recíproca entre LacY i els lípids bulk. Així, la presencia de la proteïna modifica la topografia i la nanomecànica del sistema lipídic, especialment de la fase Lα, i, alhora, la nanomecànica de la pròpia LacY varia segons la matriu lipídica que l’envolta. En conseqüència, la composició lipídica de la bicapa sembla determinar les forces que governen l’estreta interacció de LacY amb la membrana i, per tant, aquesta composició és decisiva per la correcta inserció i activitat de la proteïna.

The very fundamental properties of biological membranes can be understood by studying their formation. This sets a good foundation for research related to how the membranes interact with organic molecules and ions; something of great value in the quest of explaining transport phenomena through cell membranes. It is furthermore of growing interest within the pharmacological research and contributes to the apprehension of life at the molecular level. In this thesis Molecular Dynamics has been used to simulate how evenly distributed phospholipids solvated in water leads to the formation of monolayers. An automation program has been written in Python for performing these simulations and is to be used as the foundation for performing simulations in further studies. The program was used to simulate model systems of high- and low concentrations of DPPC lipids. The DPPC lipid, like most other lipids, consist of a hydrophilic "head" part and two lipophilic "tails", which is the main cause of the lipids interacting in such a manner that forms membranes. The low concentration system was simulated for a total of 3 ns with all lipids having reached the surface at 1.5 ns, and the all lipids in the high concentration system had risen at 41 ns with a total simulation time of 43 ns.
De mest grundläggande egenskaperna hos cellmembran kan förstås genom att studera hur dessa bildas. Detta skapar en bra grund för forskning relaterad till hur membranen interagerar med organiska molekyler och joner; något av stort värde i bemödandet att förklara transportfenomen genom cellmembran. Dessutom är det av växande intresse inom den farmakologiska forskningen och bidrar till kunskapen om liv på den molekylära nivån. I denna avhandling har Molekylär Dynamik använts för att simulera hur jämnt fördelade fosfolipider lösta i vatten leder till bildandet av monoskiktade membran. Ett automatiseringsprogram har skrivits i Python för att utföra dessa simuleringar och ska komma att användas som grund för genomförandet av simuleringar i vidare studier. Programmet användes för att simulera modellsystem med höga och låga koncentrationer av DPPC lipider. DPPC lipiden, liksom de flesta andra lipider, består av en hydrofil ''huvud'' -del och två lipofila ''svansar'', vilket är den huvudsakliga orsaken till att lipiderna interagerar på ett sådant sätt som driver bildandet av ett membran. Lågkoncentrationssystemet simulerades i totalt 3 ns, varav 1,5 ns behövdes för att alla lipider skulle nå vattenytan. Alla lipider i högkoncentrationssystemet hade kommit upp till ytan efter 41 ns och för detta system utfördes simuleringen under en total tid på 43 ns.

Les membranes cellulaires sont impliquées dans de nombreux processus cellulaires : la diffusion des médicaments et des ions, la transduction des signaux, la génération d'énergie, le développement cellulaire (fusion et fission). Les bicouches phospholipides sont les principaux composants des membranes cellulaires, elles constituent une barrière dynamique protégeant les réactions biochimiques cellulaires. La détermination des propriétés biochimiques et mécaniques des bicouches lipidiques et leur évolution avec les conditions environnementales est nécessaire pour étudier la nature des processus cellulaires et l'influence des agents externes (résistance mécanique, perméabilité et réponse biologique). Pour mener de telles caractérisations, des modèles simplifiés de membrane biomimétique, tels que des bicouches lipidiques supportées (SLB), ont été développés. Parmi les techniques de caractérisation disponibles, la microscopie à force atomique (AFM) a été largement utilisée pour étudier l'organisation nanométrique des SLB dans des conditions physiologiques. AFM peut produire des images à la haute résolution et peut également être utilisé pour quantifier la résistance mécanique des SLB au moyen d'expériences de perforation. Pendant 30 ans, AFM a traversé de nombreux développements. Très récemment, le Mode circulaire AFM (CM-AFM) a été développé à l'Université de Technologie de Compiègne. CM-AFM est capable de générer un mouvement de glissement de la pointe AFM sur l'échantillon à une vitesse élevée, constante et continue et de mesurer les forces de frottement latéral rapidement et exactement simultanément avec les forces verticales. Pour la première fois, le CM-AFM sert à caractériser les échantillons biologiques dans des conditions physiologiques, ce qui permet de mesurer simultanément les forces de poinçonnage et de frottement en fonction de la vitesse de glissement. Il offre pour la première fois la capacité de décrire le comportement de friction des SLB en complément de la force de perforation. En raison du besoin important de mesure quantitative, l'optimisation du protocole CM-AFM a été effectuée en premier. Le protocole d'étalonnage du scanner a été établi avec succès pour assurer la précision de la vitesse de glissement. En outre, le protocole d'étalonnage des pointes, basé sur la méthode de Wedge et un échantillon rayé, est également conçu pour déterminer la constante d'étalonnage de la force latérale. Nous avons utilisé CM-AFM pour mesurer les propriétés tribologiques des échantillons solides pour améliorer l'équipement sous milieu liquide. Ensuite, les propriétés mécaniques (forces de poinçonnage et de frottement) des SLB ont été mesurées en fonction de la vitesse de glissement. Les SLB purs et mixtes ont été préparés par la méthode de fusion des vésicules. Différents médias ont également été utilisés pour étudier l'effet des cations monovalents sur les propriétés mécaniques des SLB. Dans tous les cas, la force de frottement augmente linéairement avec la vitesse de glissement, ce qui nous permet de déduire le coefficient visqueux de frottement. Comme prévu, la force de poinçonnage et le coefficient visqueux de frottement sont influencés par la composition des mélanges de lipides, par la nature des cations en milieu liquide et par la longueur des chaînes hydrocarbonées mais pas de manière similaire. L'interprétation de l'évolution du coefficient de force de frottement visqueux avec le système étudié est particulièrement délicate car la force de frottement pourrait être influencée par les propriétés d'interface ou de volume. Cette problématique sera le défi pour les prochaines études. Néanmoins, nos résultats illustrent la puissance de la technique CM-AFM et ouvre de nombreuses possibilités pour caractériser d'autres échantillons biologiques (cellules et tissus) afin de mieux comprendre les mécanismes élémentaires de friction
Cell membranes are involved in many cellular processes: drugs and ions diffusion, signal transduction, energy generation, cell development (fusion and fission). Phospholipid bilayers are the main components of cell membranes, they act as a dynamic barrier protecting cellular biochemical reactions. The determination of biochemical and mechanical properties of lipid bilayers and their evolution with environmental conditions is necessary to study the nature of cellular processes and the influence of external agents (mechanical resistance, permeability, and biological response). To conduct such characterizations, simplified biomimetic membrane models, such as supported lipid bilayers (SLBs), were developed. Among the available characterization techniques, atomic force microscopy (AFM) has been widely used to study the nanoscale organization of SLBs under physiological conditions. AFM can yield high resolution images and it can also be used to quantify the mechanical resistance of SLBs by means of punch through experiments. For 30 years, AFM has been through many developments. Very recently, the Circular Mode AFM (CM-AFM) has been developed at the Université de Technologie de Compiègne. CM-AFM is able to generate a sliding movement of the AFM tip on the sample at high, constant and continuous velocity and to measure the lateral friction forces fast and accurately simultaneously with the vertical forces. For the first time CM-AFM is used to characterize biological samples under physiological conditions, allowing the simultaneous measurement of both the punch-through and the friction forces as a function of the sliding velocity. It offers for the first time the ability to describe the friction behavior of SLBs in complement of the punch-through force. Due to the important need for quantitative measurement, optimization of the CM-AFM protocol has been done first. Protocol of scanner calibration has been successfully established to ensure the accuracy of sliding velocity. Besides, the protocol for tip calibration, based on wedge method and a scratched sample, is also made to determine the lateral force calibration constant. We have employed CM-AFM to measure the tribological properties of solid samples to improve the equipment under liquid medium. Then, the mechanical properties (punchthrough and friction forces) of SLBs were measured as function of the sliding velocity. Pure and mixed SLBs were prepared by the vesicle fusion method. Various media were also used to study the effect of monovalent cations to the mechanical properties of SLBs. In all cases, the friction force increases linearly with the sliding velocity allowing us to deduce the friction viscous coefficient. As expected both the punchthrough force and the friction viscous coefficient are influenced by the composition of lipid mixtures, by the nature of cations in liquid medium, and by the length of hydrocarbon chains but not in a similar fashion. The interpretation of the evolution of the viscous friction force coefficient with the studied system is particularly tricky as the friction force could be influenced by interface or volume properties. This problematic will be the challenge for the next studies. Nevertheless, our results illustrate how powerful the CM-AFM technique is and it opens wide opportunities to characterize other biological samples (cells and tissues) to gain a better understanding of the elementary mechanisms of friction

Diacylglycerol (DAG) is required for the generation of transport carriers at the Golgi complex. Several enzymatic reactions have been found to contribute to the maintenance of DAG levels at this organelle. However, the specific metabolic pathways that, under physiological conditions, are regulated to produce the DAG required for the membrane trafficking at the Golgi complex are not known. In the first part of this work we worked on the hypothesis that phospholipid synthesis can control DAG levels at the Golgi complex for the generation of transport carriers at the Golgi complex. To study this, we altered phosphatidylcholine (PC) and phosphatidylinositol synthesis for a short period of time in CHO cells to evaluate the changes in DAG and its effects in membrane trafficking at the Golgi. We found that cellular DAG rapidly increased when PC synthesis was inhibited at the non-permissive temperature for the rate-limiting step of PC synthesis in CHO-MT58 cells. DAG also increased when choline and inositol were not supplied. The major phospholipid classes and triacylglycerol remained unaltered for both experimental approaches. The analysis of Golgi ultrastructure and membrane trafficking showed that 1) the accumulation of the budding vesicular profiles induced by propanolol was prevented by inhibition of PC synthesis, 2) the density of KDEL receptor-containing punctated structures at the endoplasmic reticulum-Golgi interface correlated with the amount of DAG, and 3) the post-Golgi transport of the yellow fluorescent temperature-sensitive G protein of stomatitis virus and the secretion of a secretory form of HRP were both reduced when DAG was lowered. We confirmed that DAG-consuming reactions of lipid synthesis were present in Golgi-enriched fractions. We conclude that phospholipid synthesis pathways play a significant role to regulate the DAG required in Golgi-dependent membrane trafficking. In the second part of this work we investigate the role of phospholipase Cγ1 (PLCγ1), a DAG-producing signalling enzyme, on the membrane traffic at the Golgi complex and in the maintenance of its structure in HeLa cells. We based our work on the effects of the silencing and overexpression of PLCγ1. We found that PLCγ1 is required for post-Golgi trafficking of transmembrane and soluble proteins, that the catalytic activity of PLCγ1 is necessary to maintain the morphology of the Golgi complex, in particular the trans-Golgi compartments, and that PLCγ1 contributes to DAG homeostasis at the Golgi, measured by the localization of the DAG-sensing construct C1-PKCθ-GFP to the Golgi complex. Finally we show that cargo arrival at the Golgi complex increases the DAG production and that PLCγ1 is required for this increase of DAG localized at the Golgi. Our results show for the first time that a physiologic event along the secretory pathway, such as cargo arrival at the Golgi complex, triggers DAG production at this organelle for membrane traffic. We also provide evidence for a pivotal role of PLCγ1 at the Golgi: PLCγ1 mediates the DAG production triggered by cargo arrival and is required for the Golgi structure and membrane traffic at the trans-Golgi compartment. The main conclusions of this work are that: 1- Metabolic pathways for the synthesis of phospholipids that consume DAG regulate its levels at the Golgi complex. 2.- Phospholipid synthesis controls the levels of DAG needed for both retrograde and anterograde trafficking at the Golgi complex. 3.- Cargo arrival at the Golgi complex promotes DAG production. 4.- PLCγ1 is involved in the production of DAG triggered by cargo arrival at the Golgi complex. 5.- PLCγ1 is needed for post-Golgi transport and maintenance of the structure of the Golgi complex.

La composición lipídica de las membranas celulares determina en última instancia sus propiedades biofísicas, afectando así las dinámicas y organización de proteinas transmembrana claves como los receptores acoplados a proteínas G (GPCRs). El objetivo de esta tesis es avanzar en la comprensión y el alcance de la interacción entre lípidos de membrana y GPCRs. Para ello, hemos utilizado simulaciones de dinámicas moleculares para estudiar la complejidad de las membrana biológicas y su efecto en la organización de las GPCRs. Así, hemos desarrollado on marco computacional para analizar con detalle las simulaciones de bicapas lipídicas y de sistemas proteina-membrana. Además, combinando simulaciones computacionales y experimentos con células vivas, demostramos por primera vez que los lípidos de membrana poliinsaturados pueden modular la organización de las GPCRs. Estos resultados podrían abrir en el futuro nuevas puertas al tratamiento de enfermedades como la esquizofrenia o la enfermedad de Parkinson, donde se ha demostrado que las GPCRs juegan un papel vital.
The lipid composition of cell membranes ultimately determines their biophysical properties, thereby affecting the dynamics and organization of key transmembrane proteins like G protein-couple receptors (GPCRs). The aim of this thesis is to make a step forward towards a better understanding of the nature and extent of the interplay between membrane lipids and GPCRs. We have used molecular dynamics simulations to study the complexity of biological membranes and the effect of the membrane environment on the organization of GPCRs. Thus, we developed a computational framework to comprehensively analyze the properties of lipid bilayers and membrane--protein simulations. In addition, by combining computer simulations and experiments in living cells we demonstrate for the first time that membrane polyunsaturated lipids can modulate the organization of GPCRs. These findings could open new doors to the treatment of several conditions like schizophrenia or Parkinson's disease, where GPCRs have been shown to play an vital role.

Cette thèse expérimentale s’inscrit dans le domaine des sciences séparatives et se base sur la technique de SPLITT (SPLIT-flow Thin fractionation). Son objectif consiste en l’étude des mécanismes qui sont à l’origine de la séparation, en continu et sans membrane, d’objets de taille micrométrique dans des mini-séparateurs fluidiques (Step-SPLITT). Les expériences menées, en laboratoire et lors de vols paraboliques, ont révélé le couplage complexe comme l’influence des effets hydrodynamiques et du champ gravitationnel sur la migration transverse des espèces en écoulement. Des visualisations tridimensionnelles par holographie digitale ont corroboré nos résultats et dévoilé des comportements inattendus. Les capacités séparatives des Step-SPLITT ont rendu possible l’analyse et la séparation d’objets biologiques et biomimétiques. Enfin, cette étude complétée par une modélisation tridimensionnelle de l’écoulement nous a permis de mettre au point un nouveau prototype de séparateur.

This experimental thesis belongs to the field of separative sciences and is based on the SPLITT technique (SPLIT-flow Thin fractionation). The objective is to study the mechanisms that are at the origin of continuous and membraneless separation of micron-size species in mini fluidic separators (Step-SPLITT). Experiments undertaken in laboratory and during parabolic flights revealed the complex coupling of the hydrodynamic effects and the gravitational field influencing the transverse migration of the flowing species. Three-dimensional visualizations performed by digital holography confirmed our results and disclosed unexpected behaviours. The separation capacities of Step-SPLITT made the analysis and the separation of biological and biomimetic species possible. In addition this study in conjunction with a three-dimensional flow modelling enabled us to develop a new prototype of separator.
Doctorat en sciences appliquées
info:eu-repo/semantics/nonPublished

Advanced fluorescence techniques were used to explore tree distinct topics concerning biological membrane and their interactions. Following thesis is according to the topic divided into three parts: 1) Ionic effects were studied employing time dependent fluorescence shift experiments and molecular dynamic simulations. Combination of these two approaches are suitable to reveal characteristic like mobility and hydration of particular bilayer segment, lipid packing or ion binding sites. Halide anions were reported to adsorb to the cationic lipid bilayer specifically, altering membrane mobility and organization. Changes in observed parameters follows Hofmeister order. Their effect is mediated either by direct ionic interaction (soft, polarizable ions) as well as via alteration of water structure (hard, non-polarizable ions) in proximity of ion molecule. Further, divalent calcium was shown to bind strongly to neutral and negatively charged lipid bilayers. Several types of binding sites depending on calcium concentration were identified. 2) Two complementary lipopeptides, CPK and CPE, incorporated into distinct lipid bilayers serve as a minimal model inducing membrane fusion. Effectiveness of fusion event might be influenced by lipopeptide-membrane and lipopeptide-lipopeptide interaction. To reveal...

No
Chemical means of manipulating the distribution of lipids across biological membranes is of considerable interest for many biomedical applications as a characteristic lipid distribution is vital for numerous cellular functions. Here we employ atomic-scale molecular simulations to shed light on the ability of certain amphiphilic compounds to promote lipid translocation (flip-flops) across membranes. We show that chemically induced lipid flip-flops are most likely pore-mediated: the actual flip-flop event is a very fast process (time scales of tens of nanoseconds) once a transient water defect has been induced by the amphiphilic chemical (dimethylsulfoxide in this instance). Our findings are consistent with available experimental observations and further emphasize the importance of transient membrane defects for chemical control of lipid distribution across cell membranes

This work was focused on the preparation of model stabilized phospholipid membranes formed on porous polycarbonate carrier. 1,2-dipalmitoyl-sn-glycero-3-phosphocholin was used for their formation in hydrophilic pores of polycarbonate carrier. For characterization of the formation of phospholipid layers, their changes and a study of transport processes, electrochemical impedance spectroscopy and voltammetry were used. Transport of cadmium and copper ions was studied in the presence and in the absence of ionophore calcimycin which was incorporated into the formed of phospholipid membrane. Because these ions are often bound in complexes with various substances, such as low molecular weight organic acids (LMWOAs), this work was also focused on the transport of copper and cadmium ions across the model phospholipid membranes in the presence of malic acid, citric acid and oxalic acid at different pH. Besides the use of ionophore, some pilot experiments were performed to realize the transfer of copper ions using two peptides, nisin and transportan 10. Formation of phospholipid membranes and the transport processes were characterized by two proposed electric equivalent circuits which correspond to the covered and to the uncovered polycarbobate carrier. Keywords: Phospholipids, Membranes, Ionophore, Peptid....

Simulation of processes in cellular membranes Abstract Many important processes in cells involve ions, e.g., fusion of synaptic vesi- cles with neuronal cell membranes is controlled by a divalent cation Ca2+ ; and the exchange of Na+ and K+ drives the the fast electrical signal transmis- sion in neurons. We have investigated model phospholipid membranes and their interactions with these biologically relevant ions. Using state-of-the-art molecular dynamics simulations, we accurately quantified their respective affinites towards neutral and negatively charged phospholipid bilayers. In order to achieve that, we developed a new model of phospholipids termed ECC-lipids, which accounts for the electronic polarization via the electronic continuum correction implemented as charge rescaling. Our simulations with this new force field reach for the first time a quantitative agreement with the experimental lipid electrometer concept for POPC as well as for POPS with all the studied cations. We have also examined the effects of transmembrane voltage on phospholipid bilayers. The electric field induced by the voltage exists exclusively in the hydrophobic region of the membrane, where it has an almost constant strength. This field affects the structure of nearby water molecules highlighting its importance in electroporation. 1