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This thesis is concerned with the study of membranes, with the aim of developing an innovative targeted vectorization system, as well as a "natural" biomimetic system allowing in situ studies of the cell membrane. The interaction of liposomes covering a wide range of membrane fluidity has been measured with cellular models of prostate cancer. It was shown that the membrane fluidity of the liposomes governs their interaction with the cells, the liposomes having a fluid membrane preferentially interacting with the tumor cells; liposomes having a rigid membrane preferentially interacting with the control cells. Investigation of the liposome internalization mechanism has revealed that it is based on a process of membrane fusion. In parallel, a biomimetic bilayer model (pep-tBLM) previously developed in the laboratory has been characterized by QCM-D and by BALM, a new method of ultra-high resolution real-time microscopy. BALM allowed the visualization of local and dynamic molecular fluxes, leading to new mechanistic insights into the pep-tBLM formation process. Finally, BALM made it possible to monitor the formation of a pep-tBLM from liposomes made of natural extracted lipids. All of this work has led to a better understanding of the physicochemistry of the membrane-membrane interaction. It constitutes a solid starting point for the development of new innovative strategies, both in the field of vectorization applied to cancer, but also in the design of biomimetic models that best reproduce the native environment of the cell membrane, Ces travaux de thèse s’intéressent à l’étude des membranes, dans le but de développer un système innovant de vectorisation ciblée, ainsi qu’un système biomimétique « naturel » permettant des études in situ de la membrane cellulaire. L’interaction de liposomes couvrant une large gamme de fluidité membranaire a été mesurée avec des modèles cellulaires de cancer de la prostate. Il a été observé que la fluidité membranaire des liposomes gouverne leur interaction avec les cellules, les liposomes ayant une membrane fluide interagissant préférentiellement avec les cellules tumorales ; les liposomes ayant une membrane rigide interagissant préférentiellement avec les cellules contrôles. L’étude du mécanisme d’internalisation des liposomes a révélé que ce dernier est basé sur un processus de fusion membranaire. En parallèle, un modèle de bicouche biomimétique (pep-tBLM) précédemment développé au laboratoire a été caractérisé par QCM-D et par BALM, une nouvelle méthode de microscopie optique à ultra-haute résolution en temps réel. La BALM nous a permis de visualiser des flux de matières locaux et dynamiques, et ainsi d’obtenir des informations inédites sur le mécanisme de formation des pep-tBLM. Enfin, la BALM a permis de suivre la formation d’une pep-tBLM à partir de lipides naturels extraits. L’ensemble de ces travaux a permis de mieux comprendre la physicochimie de l’interaction membrane-membrane, et constitue un point de départ solide pour le développement de nouvelles stratégies innovantes, à la fois dans la vectorisation appliquée au cancer, mais également dans la conception de modèles biomimétiques reproduisant au mieux l’environnement natif des membranes cellulaires

Animal pathogenic enterobacteria are major source of foodborne outbreaks. Among them, Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is known to lead to severe diseases like Haemolytic Uremic Syndrome (HUS). The adhesion on tissues, which is the first step initiating the colonisation, is mediated by cell surface proteins and organelles, including flagellum. This present work aims to study the interaction between the bacterial flagellum with lipid membrane into a relevant biological model, the Giant Unilamellar Vesicles (GUVs). GUVs are widely used as a membrane model, because of their similar size and curvature to the cell plasma membrane. A bacterial adhesion enhanced in the presence of flagella was confirmed on GUVs by microscopy, and precisely, by a newly developed assay for this work, allowing bacterial adhesion quantification on liposomes. By testing different head groups, the nature of the fatty acid chains or the liposome curvature, the lipid packing was found to be a key parameter for the adhesion. To test this, the incorporation of polyunsaturated lipids on HT29 cell line was used. The WT strain of EHEC O157:H7 was significantly more adherent than its non-flagellated mutant. The hydrophobicity was identified to influence the flagellum interaction with the lipid bilayer, by using a post-traductionnal modification (methylation) of the flagellin, the flagellum main component. With lipid packing, membrane fluidity leads to a heterogeneous bacterial adhesion on our models, mimicking the phase segregation, potentially existing on plasma membrane. It results that the flagellum is advantageous to reach potential lipid rafts. At the end of this work, a first mechanism of bacterial flagellum adhesion on lipid bilayer has been proposed and highlights the key parameters, but as well new questionings, especially the existence of an interaction with the cytoskeleton. A future precise knowledge of this mechanism represents a step toward understanding pathogen virulence, which is mandatory to develop alternative solutions to antibiotics.; Les entérobactéries pathogènes sont responsables d’épidémies majeures souvent d’origine alimentaire. Parmi elles, Escherichia coli entérohémorragique (EHEC) O157:H7 est connue pour conduire à de graves symptômes, tels que le syndrome hémolytique et urémique (HUS). L’adhésion sur un tissu, qui est la première étape initiant la colonisation, est orchestrée par des organites et protéines situés sur la paroi cellulaire bactérienne, en particulier le flagelle. Ce travail a pour objectif d’étudier l’interaction du flagelle bactérien avec les lipides de la membrane dans un système biologiquement pertinent, à savoir les liposomes unilamellaires géants (GUV, pour Giant Unilamellar Vesicles). Les GUVs sont largement utilisées en tant que modèle membranaire, car elles possèdent une taille et une courbure similaire à la membrane plasmique des cellules eucaryotes. Une meilleure adhésion bactérienne en présence du flagelle a été confirmée sur GUVs par microscopie, puis de manière précise par un essai développé dans le cadre de ce travail, permettant de quantifier l’adhésion bactérienne sur liposomes. En faisant varier la nature des têtes polaires, des chaînes carbonées et la courbure des liposomes, l’ordre lipidique s’est avéré être un paramètre clé de l’adhésion. Ceci a été vérifié à l’échelle cellulaire par l’incorporation de lipides polyinsaturés dans la membrane de cellules HT29, sur lesquelles la souche sauvage O157:H7 adhère significativement plus que son mutant non-flagellé. L’hydrophobicité du flagelle a été identifiée comme un facteur important pour l’interaction avec la membrane, grâce à une modification post-traductionnelle (méthylation) de la flagelline, composante majeure du flagelle. De concert avec l’ordre lipidique, la fluidité membranaire entraîne une adhésion bactérienne hétérogène sur notre modèle mimant la ségrégation de phases, qui existe potentiellement sur la membrane plasmique. Il en résulte que le flagelle est un avantage pour conquérir les potentiels radeaux lipidiques. A l’issue de ce travail, un premier mécanisme d’adhésion du flagelle bactérien sur la bicouche lipidique a été proposé et met en évidence les paramètres clés, mais aussi de nouvelles interrogations, notamment l’existence d’une interaction avec le cytosquelette. Une connaissance précise de ce mécanisme représente une étape vers la compréhension de la virulence des pathogènes, qui est indispensable pour développer des solutions alternatives aux antibiotiques.

The middle-ear reflex (MER) reduces acoustic energy carried by the high intensity noises rich in low frequencies at entering the cochlea. His bilateral trigger thus protects the cochlea. Disruption of this reflex by solvents can increase cochleo-traumatic effects of noise, especially among industrial workers, where noise and solvent are often associated. The main objective of this work was to develop a screening test capable of identifying the volatile substances that could modify the reflex. Moreover, the choice of solvents allowed us to study the mode of action of solvents on the neurons involved in the reflex circuit. For this purpose, Brown Norway rats were anesthetized and then exposed to aromatic solvents selected according to their lipophilicity (log Kow) and/or their structure. The amplitude of the MER is determined by measuring cubic distortion product oto-acoustic emissions. For that, aromatic solvents appear to act directly on the neuronal targets involved in the acoustic reflex circuit, rather than on membrane fluidity. The affinity of this interaction is determined by stereospecific parameters rather than lipophilocity. Additionally, NMR spectra for brain microsomes confirmed that brain lipid fluidity was unaffected by toluene exposure. In conclusion, the MER can be used to detect hazardous volatiles substances for the hearing when co-exposed to noise. Moreover, this study revealed that aromatic solvents have a neuropharmacological and/or cochleotoxic action that can act separately on the hearing of workers exposed to noise and solvents simultaneously.; Le réflexe de l’oreille moyenne (ROM) diminue l’énergie acoustique portée par les bruits riches en basses fréquences et de fortes intensités qui pénètrent dans la cochlée. Son déclenchement bilatéral permet ainsi de protéger la cochlée. La perturbation de ce réflexe par des solvants peut accroître les effets cochléo-traumatisants du bruit, notamment chez les salariés du secteur industriel, où bruit et solvant sont souvent associés. L’objectif principal de ces travaux était d’élaborer un test de criblage capable d’identifier les substances volatiles susceptibles de modifier le réflexe. De plus, le choix des solvants nous a permis d’étudier le mode d’action des solvants sur les neurones impliqués dans l’arc réflexe. Pour cela, des rats Brown Norway anesthésiés ont été exposés par inhalation aux solvants aromatiques choisis selon leur lipophilie (log Kow) et/ou selon leur structure. L’amplitude du ROM a été déterminée grâce à la mesure de l’intensité du produit de distorsion acoustique. Les résultats montrent que les effets des solvants sur le ROM sont conditionnés par les paramètres stéréospécifiques des molécules et non par leur lipophilie. Par ailleurs, l’analyse RMN des microsomes de cerveaux de rats confirme que le toluène n’influence pas la fluidité membranaire. En conclusion, le ROM est un bon outil pour détecter des substances dangereuses pour l’audition en cas de co-exposition avec du bruit. De plus, nous pouvons dire que les solvants aromatiques ont une action neuropharmacologique et/ou cochléotoxique qui peuvent retentir de façon distincte sur l’audition des sujets co-exposés au bruit et à des solvants.

The middle-ear reflex (MER) reduces acoustic energy carried by the high intensity noises rich in low frequencies at entering the cochlea. His bilateral trigger thus protects the cochlea. Disruption of this reflex by solvents can increase cochleo-traumatic effects of noise, especially among industrial workers, where noise and solvent are often associated. The main objective of this work was to develop a screening test capable of identifying the volatile substances that could modify the reflex. Moreover, the choice of solvents allowed us to study the mode of action of solvents on the neurons involved in the reflex circuit. For this purpose, Brown Norway rats were anesthetized and then exposed to aromatic solvents selected according to their lipophilicity (log Kow) and/or their structure. The amplitude of the MER is determined by measuring cubic distortion product oto-acoustic emissions. For that, aromatic solvents appear to act directly on the neuronal targets involved in the acoustic reflex circuit, rather than on membrane fluidity. The affinity of this interaction is determined by stereospecific parameters rather than lipophilocity. Additionally, NMR spectra for brain microsomes confirmed that brain lipid fluidity was unaffected by toluene exposure. In conclusion, the MER can be used to detect hazardous volatiles substances for the hearing when co-exposed to noise. Moreover, this study revealed that aromatic solvents have a neuropharmacological and/or cochleotoxic action that can act separately on the hearing of workers exposed to noise and solvents simultaneously.; Le réflexe de l’oreille moyenne (ROM) diminue l’énergie acoustique portée par les bruits riches en basses fréquences et de fortes intensités qui pénètrent dans la cochlée. Son déclenchement bilatéral permet ainsi de protéger la cochlée. La perturbation de ce réflexe par des solvants peut accroître les effets cochléo-traumatisants du bruit, notamment chez les salariés du secteur industriel, où bruit et solvant sont souvent associés. L’objectif principal de ces travaux était d’élaborer un test de criblage capable d’identifier les substances volatiles susceptibles de modifier le réflexe. De plus, le choix des solvants nous a permis d’étudier le mode d’action des solvants sur les neurones impliqués dans l’arc réflexe. Pour cela, des rats Brown Norway anesthésiés ont été exposés par inhalation aux solvants aromatiques choisis selon leur lipophilie (log Kow) et/ou selon leur structure. L’amplitude du ROM a été déterminée grâce à la mesure de l’intensité du produit de distorsion acoustique. Les résultats montrent que les effets des solvants sur le ROM sont conditionnés par les paramètres stéréospécifiques des molécules et non par leur lipophilie. Par ailleurs, l’analyse RMN des microsomes de cerveaux de rats confirme que le toluène n’influence pas la fluidité membranaire. En conclusion, le ROM est un bon outil pour détecter des substances dangereuses pour l’audition en cas de co-exposition avec du bruit. De plus, nous pouvons dire que les solvants aromatiques ont une action neuropharmacologique et/ou cochléotoxique qui peuvent retentir de façon distincte sur l’audition des sujets co-exposés au bruit et à des solvants.

Lactic acid bacteria, which have a significant industrial importance, are widely distributed in frozen or freeze-dried state for further use in industrial processes such as cheesemaking. However, stabilization processes (freezing or freeze-drying) causes different stresses which can lead to low survival rates and functionality losses of microorganisms. In this context, this thesis aimed at better understanding the impact of the physiological state of L. lactis TOMSC161 cells during fermentation on their freeze-drying and storage resistance, and at developing simple but efficient tools to evaluate the cells physiological state during fermentation for industrials.In the first part of this work, the influence of fermentation parameters (temperature, pH and harvesting time) on the growth and resistance of L. lactis TOMSC161 to each step of the freeze-drying process and storage has been investigated While the strain performance was not deteriorated after freezing, L. lactis was sensitive to the drying step and to ambient temperature storage. Moreover, the fermentation temperature and the harvesting time influenced the drying resistance of this bacterium. L. lactis TOMSC161 cells grown at 32 °C, pH 6.2 and harvested late (at late stationary phase) exhibited therefore both an optimal growth and the highest resistance to freeze-drying and storage at 4 °C. In the second part, a deep characterization of the L. lactis TOMSC161 membrane at a biochemical and biophysical level was analyzed during fermentation at different temperatures and was linked to the freeze-drying and storage resistance of starters. The cyclopropanation of unsaturated fatty acids of L. lactis TOMSC161 during fermentation was correlated with a membrane rigidification and allowed an improvement of the cell tolerance to freeze-drying and storage. Conversely, cultivating cells at lower fermentation temperature than the optimum growth temperature induced as expected a homeoviscous adaptation as evidenced by lowered lipid phase transition temperature but did not induce any improvement of resistance to this preservation process.In the third part, the physiological state characterization of L. lactis TOMSC161 cells was completed by investigating at the transcriptomic and proteomic levels as well as the cellular oxidation state. The results proved that the freeze-drying process caused intracellular reactive oxygen species (ROS) formation, responsible of degradation of the starter performance during storage at 25 °C. Furthermore, the cellular oxidation state decreased during fermentation and was explained, in the stationary phase, by a slowdown of the aerobic energy metabolism and the induction of oxidative stress responses. This initial “pre-adaptation” of L. lactis TOMSC161 during fermentation allowed improving their tolerance to freeze-drying and storage by a limitation of ROS accumulation through the whole preservation process.Finally, this work has been concluded by verifying the functionalities of starter freeze-dried in the optimized production conditions defined in this thesis during cheesemaking. Despite the freezedrying step, the technological properties of L. lactis TOMSC161 were preserved, thus validating the performed optimization.; Les ferments lactiques, d’une importance industrielle considérable, sont très largement commercialisés sous forme concentrée, congelée ou lyophilisée en vue de leur utilisation ultérieure dans les procédés industriels tels que les procédés de production de fromage. Cependant, les procédés de stabilisation (congélation et lyophilisation) engendrent différents stress qui peuvent conduire à de faibles taux de survie et des pertes de fonctionnalités des microorganismes. Dans ce contexte, ce travail de thèse vise à mieux comprendre l’incidence de l’état physiologique des cellules de L. lactis TOMSC161 au moment de la récolte sur leur tolérance à la lyophilisation et au stockage, et de développer des outils simples mais efficaces d’évaluation de l’état physiologique des cellules au cours de la fermentation pour les industriels. Dans une première partie, l’influence de différents paramètres de fermentation (température, pH et phase de croissance) sur la croissance et la résistance de L. lactis TOMSC161 à chaque étape du procédé de lyophilisation et au stockage a été étudiée. Alors que les performances de la souche ne sont pas dégradées après congélation, L. lactis s’est avéré sensible au séchage et au stockage à température ambiante. De plus, la température de fermentation et l’instant de récolte influencent la résistance au séchage de cette bactérie. Ainsi, les cellules de L. lactis TOMSC161 cultivées à 32 °C, pH 6,2 et récoltées tardivement (en phase stationnaire avancée) présentent une croissance optimale et la meilleure résistance à la lyophilisation et au stockage à 4 °C. Dans une seconde partie, une caractérisation approfondie de la membrane de L. lactis TOMSC161 aux niveaux biochimique et biophysique a été réalisée au cours de fermentations à différentes températures (22 et 30 °C) et a été mise en lien avec la résistance des ferments à la lyophilisation et au stockage. La cyclopropanation des acides gras insaturés de L. lactis TOMSC161 au cours de la fermentation est reliée à une rigidification de la membrane et permet d’améliorer la tolérance des cellules à la lyophilisation et au stockage. A l’inverse, la culture des cellules à une température inférieure à la température optimale de croissance induit une adaptation homéovisqueuse de la membrane, mise en évidence par la température de transition lipidique, mais n’a pas induite une amélioration de la résistance à ce procédé de préservation. Dans une troisième partie, la caractérisation de l’état physiologique des cellules de L. lactis TOMSC161 a été complétée au niveau du transcriptome, du protéome et de l’état d’oxydation cellulaire. Le procédé de lyophilisation provoque la formation d’espèces réactives de l’oxygène (ROS) intracellulaires qui dégradent la performance des ferments au cours du stockage. Par ailleurs, l’état d’oxydation des cellules diminue au cours de la fermentation et est expliqué, en phase stationnaire, par un ralentissement du métabolisme énergétique aérobie et une induction des réponses au stress oxydatif. Cette « pré-adaptation » initiale des cellules de L. lactis TOMSC161 au cours de la fermentation permet, là encore, une amélioration de leur tolérance à la lyophilisation et au stockage par une limitation de l’accumulation de ROS lors de ce procédé de préservation. Ce travail se conclut par une vérification des fonctionnalités du ferment lyophilisé, dans les conditions de production optimisées lors de cette thèse, en fabrication fromagère. Malgré l’étape de lyophilisation, les propriétés technologiques de L. lactis TOMSC161 sont conservées, validant ainsi le travail d’optimisation réalisé.

Oenococcus oeni is a lactic acid bacterium which is able to perform malolactic fermentation in wine. The synthesis of the small heat-shock protein (sHsp) Lo18 is one of the mechanisms involved in O. oeni survival in wine. Lo18 possess a chaperone activity on both protein and lipid substrates. pH variations in the range 5-9 were used to modulated Lo18 oligomerization in vitro and indicated that oligomer plasticity is essential for its activities. Electron microscopy studies showed that Lo18 is organised in a double-ring of stacked octamers to form a 16-mer structure at pH 5. The dimer observed at basic pH is thought to be the building block leading to oligomerization.The adaptive response of O. oeni to stress fluidizing its cytoplasmic membrane was also investigated. A transcriptomic study indicated an increase of the transcript level of genes involved in biosynthesis of saturated and unsaturated membrane fatty acids during benzylalcohol stress. On the basis of physiological, molecular and structural approaches, a model describing the first steps of O. oeni response to ethanol stress was proposed. In the early steps of the stress, Lo18 is synthesised and addressed to the membrane under a simplified structure. During the course of adaptation to the presence of ethanol, changes of the phospholipids content occur. This affects Lo18 activities and its affinity for O. oeni membrane.The results allow us to better understand the activities and the role of Lo18 in stress response of O. oeni and highlight the mechanisms involved in the maintenance of membrane integrity, a crucial event for malolactic starter performance in wine; O. oeni est une bactérie lactique responsable de la fermentation malolactique des vins. Un des mécanismes impliqués dans la survie de O. oeni dans ce milieu requière la synthèse de la protéine de stress de faible masse moléculaire (sHsp) Lo18. Cette sHsp exerce une activité de chaperon sur des substrats protéiques et lipidiques.Des variations de pH (5 à 9) ont permis de moduler l'oligomérisation de Lo18 in vitro et de démontrer que sa plasticité oligomérique est un élément clé pour ses activités. Des observations de la sHsp par microscopie électronique ont montré que Lo18 s'organise à pH 5 en un 16-mère composé de deux anneaux superposés ayant comme structure de base probable un dimère.La réponse adaptative de O. oeni a également été caractérisée suite à des stress fluidifiant sa membrane plasmique. Une étude transcriptomique a révélé une augmentation du taux de transcrits pour des gènes dont les produits interviennent dans la biosynthèse des acides gras membranaires saturés et insaturés lors d'un stress à l'alcool benzylique. Des approches physiologique, moléculaire et structurale ont permis de proposer un modèle décrivant l'action chronologique de Lo18 en lien avec ses deux activités de chaperon en réponse à un stress éthanol. Dès l'application du stress, Lo18 est fortement synthétisée et agit préférentiellement à la membrane sous une forme quaternaire simplifiée. O. oeni modifie alors sa composition en acides gras membranaires, affectant ainsi l'affinité de Lo18 pour la membrane ainsi que ses activités.Les résultats obtenus permettent non seulement, de mieux comprendre le fonctionnement et le rôle de Lo18 dans la réponse au stress de O. oeni mais aussi de mettre en exergue les mécanismes d'adaptation préservant l'intégrité de sa membrane cellulaire, élément essentiel dans la survie et la performance des ferments malolactiques dans le vin

Omega-3 and omega-6 poly-unsaturated fatty acids (PUFA) are the major families of PUFA that can be found as components of the human diet. After ingestion, both omega-3 and omega-6 PUFA are distributed to every cell in the body where they are involved in a myriad of physiological processes, including regulation of cardiovascular, immune, hormonal, metabolic, neuronal, and visual functions. At the cellular level, these effects are mediated by changes in membrane phospholipids structure, by interference with eicosanoid intracellular signaling, and by regulation of gene expression. The literature suggests the antiepileptic properties of PUFA, although these evidences emerge from basic science rather than from clinical trials. Several hypotheses have been suggested to explain the anticonvulsive effects of PUFA: modification of the membrane fluidity, direct action of PUFA on cell membrane ionic channels and/or receptors, modulation of inflammatory responses. Regarding the published clinical trials, the data are conflicting. It is currently not known whether different doses or different omega-3: omega-6 ratios would be effective.

The use of fluorescent probes for the appraisal of microviscosity properties of cell membranes, both inside the lipid double layer and/or near the double layer surface, are today an usual tool in cell biology. In the present paper an attempt was made to establish if there are significant changes in the mechanical properties of the host cell membranes.

This communication is based on a preliminary work which emphasized a topological model of biomembranes from Thom's Catastrophe Theory. In this model called swallowtail bifurcation set, the structural state of a biomembrane was within the control of two structural attractors. Then, the physiological act of this biomembrane resulted in a sudden transfer of weight from the hydrophilic attractor to the hydrophobic attractor. In this consecutive work, the physiological act appears to be one of the four stages which permit to describe the larger notion of cyclic membrane function. Two of these stages unfold in the structural axis of the swallowtail model. They prepare the two others (physiological act and refractory stages) which expand in the functional direction. This conceiving of cyclic membrane function is applied to physiological examples such as the action potential and the endocytosis. Then, changes in this function are discussed on the basis of pathological data.

Studies carried out over the last ten years have determined the structure and chemical composition of the stratum corneum and their significance for biologic function; the stratum corneum (SC) is composed of flat layers linked to each other by desmosome plates. Intercellular spaces are composed mainly of double lipid layers which are responsible for the main part of the barrier function of the SC. This barrier between the body and the environment must be effective despite the deformations and various insults (chemical, physicochemical) to which the SC is subjected. The SC fulfills its barrier function through remarkable physical properties of which most originate in an ability to bind water molecules to protein sites whose presence depends on the humidity of the environment in which the SC was produced. Lastly, the SC exhibits some enzyme activities and consequently can no longer be considered as an inert membrane.

A large number of studies have given clear indications that ethanol does affect the physicochemical properties of the membrane. Membrane reorganization and adaptation can develop against the acute disordering effect of ethanol during chronic intoxication. Nevertheless, there has been so far no direct evidence of correlations between functional tolerance or dependence and membrane physical states. Membrane physical state can be assessed by fluorescence polarization of DPH in the absence (measure of membrane 'fluidity') or presence (measured of membrane sensitivity) of ethanol added in vitro. Functional tolerance has been already correlated with a reduced synaptic membrane sensitivity to ethanol (membrane tolerance). Behavioural dependence was shown to be quantifiable by measurement of alcohol intake in a free choice situation (water/alcohol) solution). This dependence model allowed us to define a membrane dependence which consists in an increased membrane rigidity (or decrease in 'fluidity') persistent after withdrawal, and which was correlated to the intensity of the behavioural dependence. This biophysical expression of dependence seems rather independent of the biophysical membrane tolerance (resistance to the acute ethanol fluidizing effect), which was found to be rapidly reversible after withdrawal and re-induced by alcohol re-intake, requiring recent periods of current abuse to be expected.

It was hypothesized that membrane rigidification of tumor cells could enhance the expression of tumor associated antigens (6) For this purpose, we treated in vitro hypernephroma cell with cholesterol hemisuccinate (CHS) or 25-hydroxysterol (250HC) and we evaluated their immunogenicity by skin-tests in 26 patients after radical nephrectomy. The skin-tests were positive in 50% cases with CHS treated cells, and in 35% with 250HC treated cells. The immune responses were characterized as delayed hypersensitivity. However determinations of all membrane fluidity suggested that membrane rigidification was not the unique factor involved. These results could therefore be of clinical interest in the treatment of renal carcinoma by active immunotherapy.

The specific binding of [125I] hCG to ovarian membrane preparations as well as membrane fluidity have been investigated in immature rats during hormonally-induced pseudopregnancy. Membrane fluidity was monitored either by fluorescence polarization analysis of 1,6-diphenyl-1,3,5-hexatriene or by electron spin resonance of 16-, 12-, 5-doxyl stearic acid and CAT 16. A significant positive correlation was found between membrane lipid rigidity and the number of LH/hCG receptors. Luteinization of the ovary induced mobility of molecules in the hydrophobic membrane part at about the C16 carbon level. The changes in rigidity of membrane lipid were the apparent result of alterations in the cholesterol to phospholipids ratio. The results suggest that the increased rigidity of membrane lipid during pseudopregnancy may maximally expose ovarian LH/hCG receptors maintained in a cryptic form.

The main target of local anaesthetics on nervous tissue is the sodium channel. Molecular biology and electrophysiology have shown different mechanisms of action on this sodium channel, which depend on the chemical structure and electrostatic charge of the local anaesthetic molecule. There are two main types of action, shown up on the isolated axon, a direct one on the sodium channel itself and an alteration in the lipids surrounding the channel. These effects have been shown on the isolated axon and explain the anaesthetic effect by an inhibition of the sodium current. Experimental studies have also shown the effects of local anaesthetics on different organelles within the cell, and so on intracellular metabolism. Mitochondrial energetic metabolism, and therefore ATP synthesis, is reduced by local anaesthetics at several levels. The respiratory enzyme chain is inhibited by small concentrations of local anaesthetic, especially NADH dehydrogenase and ubiquinone succinate dehydrogenase. Moreover, local anaesthetics increase the mitochondrial membrane permeability to protons, thus removing the moving force behind ATPase activity in ATP synthesis; this leads to a drastic fall in available energy. This effect is further increased by a direct inhibition of ATPase and ATP/ADP translocation. Other enzyme systems of other organelles are also disturbed by local anaesthetics, such as the endoplasmic reticular Ca++ ATPase, which is inhibited, so altering the calcium concentration within the cytosol. Local anaesthetics also inhibit lipolysis and glycogenesis. Receptors such as the acetylcholine receptors are blocked by local anaesthetics. The mechanism of action of these drugs on all these protein systems is two-fold: an alteration of protein structure, but also of the lipids surrounding them.(ABSTRACT TRUNCATED AT 250 WORDS)

Rats fed a dietary peroxidized fish oil showed an increase in cytochrome P-450 content and ethoxy-coumarin deethylase (ECDE) activity in liver microsomes. Administration of DL-alpha-tocopherol led to different effects according to the extent of the peroxidation in the fish oil. In rats fed a de-peroxidized oil, the inductive effect of phenobarbital on UDP-glucuronosyltransferase (UDGPT) activity was depressed by tocopherol. By the same time, induction of P-450 and ECDE remained unchanged, that of epoxide hydrase slightly increased. By contrast tocopherol strongly potentiated the inductive effect of phenobarbital toward UDPGT activity (group I substrates) in rats fed the peroxidized fish oil. The modification of the inductive effect of phenobarbital in combination with tocopherol on UDPGT activities was concomitant with an increase in seric transaminase activity and with a reverse effect as revealed from the study of the rate of fluorescent probes penetration in microsomes. The possible toxicity of the strong dose of tocopherol is discussed.

Incorporation in vitro of 1,6-diphenyl-1,3,5 hexatriene (DPH) in washed human blood platelets was studied to determine a reference value of fluorescence emission anisotropy of DPH and thus the "microfluidity " of platelet lipid zones. Parameters evaluated were the effect of time of incorporation of the marker, of temperature (25 and 37 degrees C), and of cellular concentration, as well as the effect of platelet aging, enabling determination of valid experimental conditions for studying interaction with drug molecules.

For 9 weeks, 80 male SPF Wistar rats were fed purified diets containing mixtures of vegetable oils (15% by weight) with different linoleic and linolenic contents. Diet (L+) contained large amounts of linoleic acid (53% of the total fatty acids), diet (L+Ln) contained the same amount of linoleic acid but also 10% of alpha-linolenic acid, diet (L-) supplied a low level of linoleic acid (12% of the total fatty acids) and so did diet (L-Ln) which also contained 10% of alpha-linolenic acid. The levels and fatty acid composition of heart phospholipids were determined. Liposomes, prepared from the total phospholipids extracted from rat hearts, were tested at different temperatures (15 to 50 degrees C) for their permeability to urea and fluidity; fluidity was monitored by fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). As already demonstrated, dietary alpha-linolenic acid substituted C22 polyunsaturated fatty acids of the linoleic family (n-6) for those of the linolenic acid family (n-3). This substitution remained high, even though linoleic acid represented more than 50% of the total dietary fatty acids. Diphosphatidylglycerol markedly increased in heart phospholipids of rats fed diets (L+) and (L+Ln). The changes in liposome permeability observed were rather well correlated with the unsaturation index of the phospholipid fatty acids, whereas fluidity changes were not. Fluidity decreased in liposomes of rats fed high levels of linoleic acid. Factors such as diphosphatidylglycerol or (n-3) fatty acid concentration in heart phospholipids could explain these results.

The results presented in this paper establish relationships between structural, morphological and functional properties of cytoplasmic membrane vesicles isolated from an E. coli unsaturated fatty auxotroph. The membranes were isolated from cells grown in the presence of either oleic, linolenic or elaidic acids. High-angle X-ray diffraction studies show that order-disorder transitions induced by temperature variation sand associated with the hydrocarbon chains of the lipids are a function of the fatty acid composition of the membranes. In some cases, "cocrystallization" of various lipid species takes place within a single type of ordered domains. In other cases, there is a segregation of various lipid species into more than one distinct type of ordered domain. The various order-disorder transitions observed, induce morphological changes in the hydrophobic core of the membranes which can be detected by freeze-fracture electron microscopy. A random distribution of particles on the fracture faces is observed when the hydrocarbon chains of the lipids are disordered. Upon ordering of the hydrocarbon chains, particles are excluded from the ordered domains and as a consequence, smooth areas and areas with dense ly packed particles are observed. Discontinuities in the rate of energy-dependent beta-galactoside uptake as a function of temperature correlate with the order-disorder transitions observed. The increased slope of the Arrhenius representation in the temperature region of the transition is associated with the segregation of the carrier proteins excluded from the ordered membrane domains and their subsequent aggregation in the membrane domains containing the disordered lipids.

Oxidized fish oil (OFO) feeding is followed in the rat by the increase of vitamin A plasmatic level, and modifications of microsomal membranes (decrease of fluorophore ANS fixation). This inhibit the microsomal increase of cytochrome P 450, mixed function oxidase (MFO), any UDP-glucuronosyl transferases (group I) induced by phenobarbital treatment. On the other hand, vit A over load modify the microsomal structure of the opposite way (increase of ANS fixation). This effect is enlarged when OFO is added. In this case cyt P 450 is decreased. It seems that the magnification of vit. A effect is similar to increased microsomal membrane lability by oxidized fish oil addition.

Two young girls with type IIa homozygotic familial hypercholesterolemia were treated by plasma exchange to reduce circulating cholesterol levels, values prior to treatment being abnormally elevated (greater than 10 mmol/l). Simultaneous determination of membrane fluidity (by fluorescence polarization) of erythrocytes and lymphocytes showed marked decreases due principally to an increase in the intramembrane cholesterol/phospholipid ratio. Values after treatment showed a tendency to return to physiologic levels without, however, attaining normal values. In vitro studies of cholesterol incorporation in these membranes demonstrated the primordial role of cholesterol in these processes of membrane rigidity, and their reversibility. It is suggested that certain physical membranal properties could provide a useful index to assess need for more or less frequent plasma exchanges.

Chronic alcohol intoxication is able to modify the lipid structure and fluidity of synaptosomal membranes and influence hereby the activity of some membraneous enzymes and receptors. These changes could play a key role in the development of alcohol tolerance and dependence. This "membraneous hypothesis" allows to explain, at least in part, the influence of nutritional factors as well as of differences in genetic or individual susceptibility on alcohol dependence. It suggests furthermore the possibility of prevention of alcohol dependence through dietary and pharmacological means.

The effect of C1-C8 n-alcohols on 3T3 cell growth is studied using flow cytofluorometry. Methanol and ethanol markedly lengthen either the duration of G1, or that of G2+ M when present in relatively higher doses. The effect of longer chains is always to increase G2+M significantly. This may be due to deviations depending on alcoholic chain length in membrane lipid fluidity.

Plasma membranes from erythrocytes were used to study various parameters: phospholipid methylation, viscosity and fragility. These parameters were analysed in 57 normal subjects (52 +/- 3 years old) and 14 patients with idiopathic Parkinson's disease without previous treatment (71 +/- 2 years old). No significant correlations were observed between the various parameters and sex or age in the normal group. No correlation was observed between fragility and methylation or viscosity. A statistically significant correlation was found between viscosity and phospholipid methylation. In the patients with Parkinson's disease, fragility is identical to the normal group but the viscosity is increased by 25 p. 100. The increase of viscosity is more obvious among the patients with a pure or predominantly akinetic form of the disease. These results favour the previously described correlation between phospholipid methylation and viscosity of the plasma membrane and suggest a modification of these parameters in Parkinson's disease. They also suggest that the increased viscosity observed in Parkinson patients could render the receptors inaccessible to their endogenous ligands.

Fluorescence polarization of red blood cell (RBC) membranes evaluated using DPH was measured in patients with uncontrolled insulin-dependent diabetes mellitus and in controls. The effect of in vitro addition of pentoxifylline and propentofylline (10(-5) and 10(-4) M) was studied. The fluorescence polarization parameter (P) was lower in the diabetic patients (p = 0.20 +/- 0.03, n = 8) as compared to the controls (p = 0.28 +/- 0.02), n = 8), reflecting an increase in probe mobility in the hydrophobic environment in the depth of the double lipid layer. In vitro addition of pentoxifylline had no effect on the fluorescence parameter of RBC membranes from controls, whereas both concentrations of pentoxifylline studied (p = 0.24 +/- 0.03) significantly increased the fluorescence parameter of RBC membranes from diabetics. Propentofylline had no effect. These findings suggest that the active site responsible for improved membrane fluidity of RBC from diabetics is located on the methyl radical present in the pentoxifylline molecule but not in the propentofylline molecule.

The olfactory nerve of the garfish, the rabbit vagus nerve and the sciatic nerve of the frog labelled with pyrene or triethylammonium-butyl-pyrene show during the action potential a transient decrease in the Ie/Im ratio which suggest a small transient decrease in nerve membrane fluidity.