Cold adaptation in stationary phase in Bacillus subtilis One of the most important abiotic factor which influences life of bacterial cells is the ambient temperature. A decrease of this temperature is usually accompanied usually with the loss of the fluidity of bacterial cytoplasmatic membrane. While the mechanisms of the responses to the cold shock during the exponential phase of growth are well known for Bacillus subtilis, the responses of stationary phase cells had not been studied yet (despite the stationary phase is the most common state of microorganism in the nature). There are two independent mechanisms which restores much needed fluidity in Bacillus subtilis - short-term adaptation and long-term adaptation. Short-term adaptation is based on the function of fatty acid desaturase coded by des gene. Long-term adaptation relies on the change in ratio of iso- and anteiso- branched fatty acids. In this work we examinated membrane adaptation during stationary phase under two different conditions, namely under cultivation at stable low temperature and after cold shock. The highest activity of Pdes was observed for cultivation at 25 řC and for the cold shock applied from cultivation in 37 řC to 25 řC. Anisotropy measurements and fatty acids analysis were also performed. Results indicated, that the...

The homeoviscous adaptation hypothesis states that the relative abundance of polyunsaturated fatty acids (PUFAs) in membrane phospholipids of ectothermic organisms decreases with increasing temperatures to maintainvital membrane properties. We reared Daphnia magna at 15°, 20°, and 25°C and increasing dietary concentrations of the long-chain PUFA eicosapentaenoic acid (EPA) to test the hypothesis that the well-documented increase in heat tolerance of high-temperature-reared Daphnia is due to a reduction in body PUFA concentrations. Heat tolerance was assessed by measuring the time to immobility at a lethally high temperature (Timm at 37°C), and whole body lipid fluorescence polarization (FP) was used as an estimate of membrane fluidity. At all rearing temperatures, EPA supplementation resulted in an increase in the relative abundance of EPA in body tissues, but only at 15° and 25°C did this result in a decrease in heat tolerance, and only at 20°C was this associated with an increase in membrane fluidity (i.e., decrease in FP). Overall, however, the degree of tissue fatty acid unsaturation correlated well with heat tolerance and FP. Our results support the homeoviscous adaptation hypothesis by showing that cold-reared Daphnia accumulate PUFAs within their body tissues and thus are more susceptible to heat than hot-reared Daphnia accumulating fewer PUFAs. However, our data also point out that further studies are required that elucidate the complex relationships between PUFA supply, membrane fluidity, and heat tolerance in ectotherms.

No description available.

Proteins are known to form functional clusters in plasma membranes. In order to identify individual proteins within clusters we developed a method to visualize by atomic force microscopy (AFM) the cytoplasmic surface of native plasma membrane, excised from Xenopus laevis oocyte and spread on poly-L-lysine coated glass. After removal of the vitelline membrane intact oocytes were brought in contact with coated glass and then rolled off. Inside-out oriented plasma membrane patches left at the glass surface were first identified with the lipid fluorescent marker FM1-43 and then scanned by AFM. Membrane patches exhibiting the typical phospholipid bilayer height of 5 nm showed multiple proteins, protruding from the inner surface of the membrane, with heights of 5 to 20 nm. Modelling plasma membrane proteins as spherical structures embedded in the lipid bilayer and protruding into the cytoplasm allowed an estimation of the respective molecular masses. Proteins ranged from 35 to 2,000 kDa with a peak value of 280 kDa. The most frequently found membrane protein structure (40/μm2) had a total height of 10 nm and an estimated molecular mass of 280 kDa. Membrane proteins were found firmly attached to the poly-L-lysine coated glass surface while the lipid bilayer was found highly mobile. We detected protein structures with distinguishable subunits of still unknown identity. Since X. laevis oocyte is a generally accepted expression system for foreign proteins, this method could turn out to be useful to structurally identify specific proteins in their native environment at the molecular level. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

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.

A Doença de Alzheimer (DA) é uma desordem neurodegenerativa progressiva que causa comprometimento cognitivo em idosos. O diagnóstico clínico da DA é complexo. Existe uma grande necessidade de técnicas capazes de detectar a doença nos estágios iniciais, tanto para auxiliar o diagnóstico quanto para monitorar a efetividade dos tratamentos disponíveis. As alterações bioquímicas da DA são resultado de processos celulares como o metabolismo da proteína precursora do amilóide (APP), fosforilação da tau, stress oxidativo, inflamação e desregulação lipídica. Até o momento não existem marcadores bioquímicos para auxiliar o diagnóstico da DA. Este trabalho avaliou três possíveis candidatos a marcadores bioquímicos para a DA. Foram investigados a razão da APP (rAPP) de 130/110 kDa, fluidez de membrana e atividade da fosfolipase A2 em plaquetas de pacientes com DA e Comprometimento Cognitivo Leve (CCL), comparando-se seus resultados com controles idosos saudáveis. A fluidez das membranas das plaquetas foi avaliada por meio da anisotropia com a sonda fluorescente DPH (Difenilhexatrieno); a comparação das razões da APP foi realizada por Western Blotting empregando o anticorpo 22C11 e a da atividade da PLA2 foi determinada por ensaio radioenzimático com substratos e concentrações de cálcio específicas para cada um dos três principais grupos da enzima. A rAPP, as atividades da sPLA2 e iPLA2 estavam significantemente reduzidas na DA quando comparadas com controles, enquanto que a cPLA2 e a fluidez de membrana não apresentaram diferenças entre os grupos. A rAPP e a iPLA2 também apresentaram diferenças significativas entre CCL e DA, além de estarem correlacionadas com os parâmetros cognitivos MEEM e CAMCOG. A rAPP também estava correlacionada com a anistropia do DPH. / Alzheimer disease (AD) is a progressive neurodegenerative disorder that causes cognitive impairment in the elderly. The clinical diagnosis of AD is complex. Thus, there is a great need for sensitive techniques to detect neurodegeneration in the early stages to asset in the diagnosis and to follow the effectiveness of therapy. The biochemical alterations in the AD brain result from cellular processes such as amyloid precursor protein (APP) metabolism, tau phosphorylation, oxidative stress, inflammation and lipid dysregulation. So far there are no biochemical markers to help the AD diagnosis. The purpose of this study was to evaluate three possible candidates to biochemical marker of AD. The APP 130/110 kDa ratio, membrane fluidity and phospholipase A2 activity in platelets of patients with AD and mild cognitive impairment (MCI) were investigated compared to their results with healthy elderly controls. The membrane fluidity of platelets was assessed by the fluorescence anisotropy of DPH (diphenyl-hexatriene); the levels of APP isoforms were evaluated by Western Blot analysis using 22C11 antibody and the PLA2 activity was measured by radio-enzymatic assay with enzyme specific substrate and calcium concentrations for each one of the three main groups of the enzyme. The APP ratio (APPr), the sPLA2 and iPLA2 activity were markedly decreased in AD in comparing with controls, whereas a cPLA2 and membrane fluidity didn\'t show any alteration between the groups evaluated. The APPr and iPLA2 also showed significant differences between MCI e AD, and were correlated with cognitive parameters MMSE and CAMCOG. The APPr was also correlated with DPH anisotropy.

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. / 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.

A Doença de Alzheimer (DA) é uma desordem neurodegenerativa progressiva que causa comprometimento cognitivo em idosos. O diagnóstico clínico da DA é complexo. Existe uma grande necessidade de técnicas capazes de detectar a doença nos estágios iniciais, tanto para auxiliar o diagnóstico quanto para monitorar a efetividade dos tratamentos disponíveis. As alterações bioquímicas da DA são resultado de processos celulares como o metabolismo da proteína precursora do amilóide (APP), fosforilação da tau, stress oxidativo, inflamação e desregulação lipídica. Até o momento não existem marcadores bioquímicos para auxiliar o diagnóstico da DA. Este trabalho avaliou três possíveis candidatos a marcadores bioquímicos para a DA. Foram investigados a razão da APP (rAPP) de 130/110 kDa, fluidez de membrana e atividade da fosfolipase A2 em plaquetas de pacientes com DA e Comprometimento Cognitivo Leve (CCL), comparando-se seus resultados com controles idosos saudáveis. A fluidez das membranas das plaquetas foi avaliada por meio da anisotropia com a sonda fluorescente DPH (Difenilhexatrieno); a comparação das razões da APP foi realizada por Western Blotting empregando o anticorpo 22C11 e a da atividade da PLA2 foi determinada por ensaio radioenzimático com substratos e concentrações de cálcio específicas para cada um dos três principais grupos da enzima. A rAPP, as atividades da sPLA2 e iPLA2 estavam significantemente reduzidas na DA quando comparadas com controles, enquanto que a cPLA2 e a fluidez de membrana não apresentaram diferenças entre os grupos. A rAPP e a iPLA2 também apresentaram diferenças significativas entre CCL e DA, além de estarem correlacionadas com os parâmetros cognitivos MEEM e CAMCOG. A rAPP também estava correlacionada com a anistropia do DPH. / Alzheimer disease (AD) is a progressive neurodegenerative disorder that causes cognitive impairment in the elderly. The clinical diagnosis of AD is complex. Thus, there is a great need for sensitive techniques to detect neurodegeneration in the early stages to asset in the diagnosis and to follow the effectiveness of therapy. The biochemical alterations in the AD brain result from cellular processes such as amyloid precursor protein (APP) metabolism, tau phosphorylation, oxidative stress, inflammation and lipid dysregulation. So far there are no biochemical markers to help the AD diagnosis. The purpose of this study was to evaluate three possible candidates to biochemical marker of AD. The APP 130/110 kDa ratio, membrane fluidity and phospholipase A2 activity in platelets of patients with AD and mild cognitive impairment (MCI) were investigated compared to their results with healthy elderly controls. The membrane fluidity of platelets was assessed by the fluorescence anisotropy of DPH (diphenyl-hexatriene); the levels of APP isoforms were evaluated by Western Blot analysis using 22C11 antibody and the PLA2 activity was measured by radio-enzymatic assay with enzyme specific substrate and calcium concentrations for each one of the three main groups of the enzyme. The APP ratio (APPr), the sPLA2 and iPLA2 activity were markedly decreased in AD in comparing with controls, whereas a cPLA2 and membrane fluidity didn\'t show any alteration between the groups evaluated. The APPr and iPLA2 also showed significant differences between MCI e AD, and were correlated with cognitive parameters MMSE and CAMCOG. The APPr was also correlated with DPH anisotropy.

The mechanism of microparticle shedding from the plasma membrane of calcium-loaded cells has been investigated in erythrocytes and platelets. Recent studies have revealed the physiological and clinical importance of microparticle release from nucleated cells such as lymphocytes and endothelium. The experiments of this study were designed to address whether simple mechanisms discovered in platelets and erythrocytes also apply to the more complex nucleated cells. Four such mechanisms were addressed: potassium efflux, transbilayer phosphatidylserine migration, cytoskeleton degradation, and membrane lipid order. The rate and amount of microparticle release in the presence of a calcium ionophore, ionomycin, was assayed by light scatter at 500 nm. To inhibit the calcium-activated potassium current, cells were exposed to 1 mM quinine or a high-potassium buffer. Both interventions substantially attenuated microparticle shedding induced by ionomycin. Microparticle release was also greatly reduced in a lymphocyte cell line deficient in the expression of scramblase, the enzyme responsible for calcium-stimulated phosphatidylserine migration to the cell surface. This result indicated that such phosphatidylserine exposure is also required for microparticle shedding. The importance of cytoskeletal rearrangement was evaluated through the use of E64-d, a calpain inhibitor, which appeared to have no affect on release. Thus, if cytoskeleton degradation is important for microparticle release, a different enzyme or protein must be involved. Finally, the effect of membrane physical properties was addressed by varying the experimental temperature (32–42 °C). A significant positive trend in the rate of microparticle release as a function of temperature was observed. Fluorescence experiments with trimethylammoniumdiphenylhexatriene and patman revealed significant differences in the level of apparent membrane order along that temperature range. Ionomycin treatment appeared to cause further disordering of the membrane, although the magnitude of this change was minimally temperature-sensitive. Thus, it was concluded that microparticle release depends more on the initial level of membrane order than on the change imposed by calcium uptake. In general, mechanisms involved in particle release from platelets and erythrocytes appeared relevant tolymphocytes with the exception of the hydrolytic enzyme involved in cytoskeletal degradation.

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 / 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