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La maladie d’Alzheimer (MA) est un problème majeur de santé publique. Elle se traduit par des atteintes de la mémoire reposant sur des dysfonctionnements synaptiques induits par les oligomères de peptide ß-amyloïde (Aß). Ceux-ci activent la phospholipase A2 cytosolique (cPLA2) qui libère l’acide arachidonique (ARA) des phospholipides (PL) membranaires neuronaux. L’acyl-CoA synthétase 4 (ACSL4) peut limiter cette libération en favorisant la réincorporation d’ARA dans les PL. Dans l’alimentation occidentale, il constitue une part croissante des apports lipidiques. Contrairement à l’acide docosahexaénoïque (DHA), l’influence de l’ARA dans la MA a été peu étudiée. C’est pourquoi nous avons étudié la mobilisation de l’ARA et son effet sur la sensibilité au peptide Aß. Nous avons montré dans un premier temps qu’un apport alimentaire en ARA affecte la mémoire à court terme et sensibilise les capacités d’apprentissage au peptide Aß. Ces altérations sont associées à des diminutions d’expression des récepteurs AMPA et de l’ACSL4, une prolifération astrocytaire et une incorporation accrue en ARA dans les espèces de PL phosphatidylsérine et phosphatidyléthanolamine (PE). D’autre part, la différenciation des cellules HT22 nous a permis de montrer que l’ACSL4 intervient dans l’incorporation de l’ARA et dans l’équilibre ARA/DHA, notamment dans les espèces PE. Cela indique qu’un excès en ARA dans l’alimentation peut constituer un facteur d’aggravation de la MA et que les enzymes assurant sa mobilisation, comme la cPLA2 et l’ACSL4, peuvent moduler ce risque. La caractérisation de leurs niveaux d’expression pourrait permettre de définir des groupes d’individus à risque vis-à-vis de la MA / Alzheimer’s disease (AD) is a major public health problem. This disease is characterized by memory impairments which are caused by synaptic dysfunctions induced by the oligomers of amyloid-ß peptide (Aß). These activate the cytosolic phospholipase A2 (cPLA2) which releases arachidonic acid (ARA) from neuronal membrane phospholipids (PL) whereas acyl-CoA synthetase 4 (ACSL4) potentially counteracts this release by favoring ARA reincorporation into PL. Western diets contain growing amount of ARA. Contrary to docosahexaenoic acid (DHA), a few studies were devoted to the influence of ARA in AD. This is why we decided to study the mobilization of ARA and its effects on the sensitivity to Aß oligomers. First we showed that dietary ARA reduces short-term memory abilities and increases the deleterious effects of Aß on learning abilities. These alterations of cognitive abilities are associated to reductions of expression levels of AMPA receptors and ACSL4, an astrocyte proliferation, and greater incorporation of ARA in PL species phosphatidylethanolamine (PE) and phosphatidylserine. Secondly, we used differentiated HT22 to show that ACSL4 modulate ARA incorporation and ARA / DHA balance in the PE species. These results indicate that excessive dietary intake of ARA may be a worsening factor in AD and the enzymes regulating ARA mobilization, such as cPLA2 and ACSL4, can modulate this risk. The characterization of their enzymatic activities could allow the identification of groups of individuals at AD risk.

PIZARRO, Juan Carlos Alvarez. Caracteres fisiológicos e bioquímicos da tolerância à salinidade em clones de cajueiro anão precoce. 2006. 135 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2006. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-05-30T14:16:32Z No. of bitstreams: 1 2006_dis_jcapizarro.pdf: 935692 bytes, checksum: c709b8b0747f8a929112611a9c584729 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-07-12T23:10:54Z (GMT) No. of bitstreams: 1 2006_dis_jcapizarro.pdf: 935692 bytes, checksum: c709b8b0747f8a929112611a9c584729 (MD5) / Made available in DSpace on 2016-07-12T23:10:54Z (GMT). No. of bitstreams: 1 2006_dis_jcapizarro.pdf: 935692 bytes, checksum: c709b8b0747f8a929112611a9c584729 (MD5) Previous issue date: 2006 / Early-dwarf cashew seedlings (Anacardium occidentale L.) were used in order to investigate the physiological and biochemical changes induced by salt stress. The seeds (nuts) were sown in plastics pots containing vermiculite moistened with either distilled water (control treatment) or NaCl solutions at 8 and 16 dS.m-1 of electrical conductivity (saline treatment), and kept in greenhouse throughout the experimental period. Uniform 28-day-old seedlings were used for the analyses. The first experiment aimed to select, among five clones (CCP 06, CCP 09, CCP 76, Embrapa 51 and BRS 189), the ones showing contrasting salt-tolerance. The effect of salinity on the growth, gas exchange, water content, leaf succulence, osmotic potential and inorganic (Na+, Cl-, K+) and organic (proline, soluble carbohydrates, quaternary ammonium compounds) solute concentration for both salt-sensitive and salt-tolerant clones was studied. Salinity inhibited the growth of all clones studied, being the inhibitory effect on shoot growth more conspicuous than in root growth. Clone CCP 06 leaf area was the most inhibited by salt stress, while clones BRS 189 and CCP 09 leaf areas were the least affected by salinity. Salt stress caused a great decrease in the cotyledon reserve mobilization especially at 16 dS.m-1. Growth reduction was correlated to the reduction in net photosynthetic rate. CCP 06 and BRS 189 showed the greatest and the lowest reduction in photosynthetic rate at 8 dS.m-1, respectively. Although, salinity reduced stomatal conductance, this reduction was not followed by changes in CO2 internal concentration. The water status, expressed as water content in relation to dry mass, was not changed by salt-stress. Salinity induced the lowering of osmotic potential both in leaves and roots of all clones studied. This osmotic adjustment might have lead to turgor maintenance of those tissues. The concentrations of Cl- and Na+ increased with increasing salt stress. Clones BRS 189 and CCP 09 accumulated more Na+ in the roots, and this could explain their efficiency in maintaining a lower ion concentration in shoots, i.e. they regulated more efficiently the transport of Na+ from roots to shoots. The regulation of Cl- transport to shoots was more efficient in clone CCP 09 than in the others. Salinity did not induce significant changes in leaves and stems K+ concentration, but it induced a reduction of K+ concentration in roots. Salinity also induced increases of quaternary ammonium compounds and proline concentration in BRS 189 root at 8 dS.m-1. In addition, this level of salinity increased soluble carbohydrates in the root sap especially in clones BRS 189 and CCP 06. During the second experiment, the effect of salt stress (NaCl at 8 dS.m-1) on the activity of H+-ATPase, lipid composition and peroxidation of root plasma membrane of both salt-tolerant (BRS 189) and salt- sensitive (CCP 06) clones were studied. The vanadate-sensitive H+-ATPase activity was studied in plasma membrane-enriched vesicles isolated by discontinuous sucrose gradient centrifugation from roots. ATP hidrolizing activity in this fraction was mostly inhibited by vanadate and scarcely, by azide and molybdate, indicating that it was essentially enriched in plasma membrane vesicles. Salinity induced a 1.3-fold increase in the H+-ATPase specific activity in roots of BRS 189 seedlings. Salinity had no appreciable effect on the hydrolytic activity of this enzyme during the growth of CCP 06 seedlings. Likewise, clone BRS 189 roots plasma membrane showed higher sterol content and lower phospholipids/total sterol ratio than clone CCP 06. Both properties could contribute to the decrease in Na+ influx or increase in Na+ efflux or “exclusion” from roots. This could result in less Na+ being transported to the shoot, and thus explaining the higher salt-tolerance of clone BRS 189. The higher degree of root plasma membrane lipid peroxidation of clone, and the lower proline and ammonium quaternary compounds contents of CCP 06 when compared to BRS 189 could also explain the differences in salt-tolerance between the two clones. These organic solutes could protect and stabilize plasma membrane against oxidative stress. Phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phosphatidylserine (PS) were the major phospholipids in the plasma membrane from BRS 189 roots. Salinity induced increases in the relative proportions of PE and phosphatidylinositol (PI), while PG and PA were reduced. No changes were detected in PS in relation to control plant. The importance of lipid composition changes on H+-ATPase activity must be more studied. / O presente trabalho teve por objetivo estudar as respostas fisiológicas e bioquímicas de clones de cajueiro anão-precoce (Anacardium occidentale L.) ao estresse salino. Os experimentos foram conduzidos em casa de vegetação, sendo as plântulas cultivadas em vasos plásticos contendo vermiculita. No primeiro experimento, cinco clones de cajueiro anão-precoce foram submetidos aos tratamentos com NaCl a 0 (controle), 8 e 16 dS.m-1 de condutividade elétrica e objetivou selecionar clones com tolerâncias diferenciadas ao estresse salino. Para isso, foram estudados os efeitos da salinidade no crescimento, nas trocas gasosas, no teor de água, na suculência foliar, no potencial osmótico, nas concentrações de prolina, N-aminossolúveis e carboidratos solúveis e nos teores dos íons inorgânicos (Na+, Cl- e K+). A salinidade reduziu o crescimento das plântulas de todos os clones estudados. Os efeitos inibitórios do NaCl foram mais conspícuos na parte aérea do que nas raízes. O clone CCP 06 foi aquele que apresentou maior redução no crescimento foliar, enquanto os clones BRS 189 e CCP 09 foram os que apresentaram as menores reduções. A salinidade inibiu a mobilização das reservas cotiledonárias, principalmente, na dose mais elevada de sal. A redução no crescimento, pela salinidade, correlacionou-se com a redução na taxa de fotossíntese líquida. Os clones CCP 06 e BRS 189 apresentaram, respectivamente, a maior e a menor redução na taxa fotossintética a 8 dS.m-1. Embora a salinidade tenha reduzido a condutância estomática dos clones de cajueiro anão-precoce, essa redução não foi acompanhada por mudanças nas concentrações internas de CO2. Os clones estudados não apresentaram alterações, em função da salinidade, no estado hídrico das folhas e raízes, porém, apresentaram reduções no potencial osmótico, favorecendo o ajustamento osmótico e, consequentemente, a manutenção da turgescência dos tecidos. Sob condições de estresse salino, os clones BRS 189 e CCP 09 foram os mais eficientes na regulação do transporte do íon Na+ para a parte aérea da plântula, acumulando-o nas raízes. Em relação ao Cl-, o clone CCP 09 mostrou-se o mais eficiente no controle do transporte desse íon. Porém, CCP 06 foi o clone que mais acumulou ambos os íons tóxicos na parte aérea da planta. Com o aumento da salinidade, os teores de potássio dos clones estudados tiveram seus valores reduzidos apenas nas raízes. Na dose de 8 dS.m-1, o BRS 189 foi o clone que mais aumento suas concentrações de N-aminosolúveis e prolina no suco radicular. Nesse mesmo nível de sal, a salinidade aumentou a concentração de carboidratos apenas nos clones CCP 06 e BRS 189. De posse destes resultados, o segundo experimento foi realizado com os clones CCP 06 e BRS 189 que foram os que se mostraram, respectivamente, o menos e o mais tolerante à salinidade. Esse experimento teve por objetivo estudar os efeitos da salinidade (NaCl a 8 dS.m-1) na atividade da H+-ATPase e na composição e peroxidação dos lipídios de membrana plasmática isoladas de raízes das plântulas dos dois clones contrastantes. A salinidade estimulou a atividade da H+-ATPase apenas no clone tolerante, o BRS 189, sendo esse clone o que apresentou maior conteúdo de esteróis totais e menor relação fosfolipídios totais (PLt)/ esteróis totais (Et), tanto em condições controle como de estresse. Esses resultados foram concordantes com o fato de ter sido o BRS 189 o clone que melhor excluiu o Na+ da parte aérea. Nesse clone não foram observadas alterações nos teores de malondialdeído, diferentemente do que ocorreu com o CCP 06, cujos teores aumentaram com o estresse salino. A maior proteção da membrana plasmática do clone BRS 189 ao dano oxidativo está de acordo com os maiores acúmulos de prolina e N-aminossolúveis observados nesse clone. Os principais fosfolipídios da membrana plasmática isolada de raízes do clone BRS 189 foram fosfatilglicerol (PG), fosfatidiletalonamina (PE) e fosfatilserina (PS). A salinidade provocou alterações nas proporções relativas dos fosfolipídios, sendo PE e fosfatidilinositol (PI) os que apresentaram maiores aumentos em relação ao total, enquanto que fosfatidilglicerol (PG) e ácido fosfatídico (PA) foram os que apresentaram maiores reduções. A percentagem de PS, em relação ao total, não foi afetada pela salinidade. No entanto, a relação entre essas mudanças na composição lipídica do BRS 189 pela salinidade e o aumento na atividade da H+-ATPase necessita ser melhor investigada.

O objetivo deste trabalho foi desenvolver biosensores de membranas baseados na técnica de Ressonância Plasmônica de Superfície (SPR) e aplicá-los no estudo da interação do citocromo c (cit c) com modelos de membranas. SPR é uma técnica ótica, que através de medidas de variações de índice de refração (n) próximas a uma interface mensura com alta sensibilidade a adsorção ou ligação de moléculas. Inicialmente desenvolvemos um sistema de gradiente de fluxo acoplado ao SPR, denominado FIG-SPR, e demonstramos a determinação automatizada da variação de n em função da concentração (dn/dC) de diferentes compostos e biopolímeros. O desenvolvimento dos biosensores de membranas iniciou-se com o estudo dos fatores que afetam a formação de uma membrana de bicamada híbrida (HBM). HBMs são compostas de uma monocamada de alcanotiol adsorvida sobre o ouro, e sobre esta uma camada fosfolipídica. A formação da HBM depende da fusão de vesículas em superfícies hidrofóbicas e que não é bem compreendido no nível molecular. Nossos estudos mostraram que na presença de cálcio e espermina a formação da HBM é favorecida, de tal forma que a monocamada de fosfolipídio alcança valores de espessura próximos àqueles previstos, cerca de 20 Å\'. Além disso, mostramos que em soluções de baixa força iônica a camada lipídica não é homogênea. Demonstramos também que a presença de cálcio na concentração 150 mM diminui o tempo de formação da monocamada lipídica cerca de 14 vezes quando comparado ao tempo indicado na literatura. A homogeneidade da HBM e a carga superficial da mesma foram verificadas com a adsorção e a dissociação de cit c e de albumina bovina (BSA). Utilizando HBMs de composição lipídica variada demonstramos a adsorção e a dissociação de cit c induzida por cálcio em HBMs mistas, incluindo um modelo mimético da membrana mitocondrial interna (IMM) constituído de fosfatidilcolina, fosfatidiletanolamina e cardiolipina (PC/PE/CL) na proporção (4,5:3,5:2,0). Demonstramos que a adsorção de cit c nativo segue um perfil cooperativo e padrões esperados de variação de afinidade e cooperatividade em pHs 6,8, 7,4 e 8,0. Um modelo matemático foi desenvolvido para tratar as curvas de ligação de cit c, que é uma adaptação do modelo de Hill para adsorção de proteínas em superfícies. Os resultados de SPR juntamente com dados obtidos por Microscopia de Força Atômica (AFM) sugerem que a ligação cooperativa de cit c com HBM ocorre devido à reorganização das moléculas de CL e formação de domínios fosfolipídicos. O tratamento dos resultados cinéticos da dissociação de cit c por cálcio indica a existência de duas constantes de velocidade de dissociação (kd), sendo a primeira constante (kd1) relacionada à perda das interações eletrostáticas entre a proteína e a HBM, e a segunda (kd2) à perda das interações hidrofóbicas. Além disso, a dissociação do cit c do modelo estudado requer uma concentração mínima de cálcio de 30 µM para se tornar significativa. O estudo da interação entre moléculas de cit c foto-oxidadas (citc405) e a HBM de PC/PE/CL sugerem que ela ocorre com menor afinidade, nos três pHs estudados, se comparados aos resultados com cit c nativo. Além disso, nossos resultados sugerem que o citc405 não é facilmente dissociado por cálcio devido à perda da cooperatividade na interação. Possíveis implicações em eventos celulares destas descobertas, como a liberação do cit c da IMM e a iniciação da apoptose, são discutidas / The aim of this work was to develop membrane biosensors based on Surface Plasmon Resonance (SPR) and to apply them to study the interactions between cytochrome c (cyt c) and model membranes. SPR is an optical technique that provides high-sensitivity measurements of refractive index (n), allowing the characterization of the adsorption and desorption of molecules near interfaces. Initially we developed a flow gradient system connected to SPR, which was called FIG-SPR, and demonstrated the automated determination of the concentration gradient of refractive index (dn/dC) of different materials and biopolymers. The development of the membrane biosensors was initiated by studying the factors that affect the formation of a hybrid bilayer membrane (HBM). HBMs are composed of two monolayers: an alcanethiol monolayer adsorbed on gold over which is adsorbed a layer of phospholipids. The formation of an HBM depends on the fusion of phospholipid vesicles on hydrophobic surfaces, a process that is not well understood at the molecular level. Our results showed that in the presence of calcium and spermine the complete formation of an HBM is facilitated, i.e., the phospholipid monolayer reaches the expected thickness of about 20Å\'. However, in low ionic strength solutions the lipid layer that is formed is not homogeneous. We have also demonstrated that in the presence of 150 mM of calcium the time necessary for the formation of the lipid monolayer is reduced 14 times when compared to the times suggested in the literature. The homogeneity of the HBM and its superficial charge were verified with the adsorption and desorption of cyt c and bovine serum albumine (BSA). The adsorption and desorption of cyt c in different HBMs were studied including a model of the internal mitochondrial membrane (IMM), which is made of phosphatidylcholine, phosphatidylethanolamine and cardiolipin (PC/PE/CL) in the ratio (4,5: 3,5: 2,0). We demonstrated that the adsorption of native cyt c follows a cooperative profile showing expected changes in affinity and cooperativity in different solution pHs of 6,8, 7,4 and 8,0. A mathematical model, which is an adaptation of the Hill model for adsorption of proteins in surfaces, was developed to treat the binding curves of cyt c. The results of SPR together with those obtained by Atomic Force Microscopy (AFM) suggested that the cooperative binding of cyt c in HBMs occurs due to the reorganization of CL molecules and formation of phospholipid domains. The kinetic results of the dissociation of cyt c induced by calcium indicates the existence of two velocity constants (kd), being the larger (kd1) related to the dissociation of cyt c interacting electrostatically with the HBM, and the smaller (kd2) related to the dissociation of cyt c interacting hydrophobically with the HBM. Moreover, the dissociation of cyt c from the HBM requires a minimum calcium concentration of 30 µM. The study of the interaction between photo-oxidized cyt c molecules (cytc405) and the PC/PE/CL HBM suggests that it occurs with smaller affinity when compared with the results obtained with the native cyt c. Moreover, cytc405 is not easily dissociated by calcium due to the loss of the interaction cooperativity with the HBM. Possible implications of these discoveries in cellular events, such as the release of cyt c from the IMM and the initiation of apoptosis, are discussed

Les acides gras saturés (AGS) altèrent la fonctionnalité des organites dans de nombreux types cellulaires. Il a été proposé que ce processus, également nommé lipointoxication, puisse être responsable de plusieurs pathologies humaines telles que le diabète de Type 2.Au niveau cellulaire, l'accumulation d'AGS est associée à une augmentation du taux de saturation des phospholipides (PL) membranaires, les composants majoritaires des membranes des organites, mais également du taux de céramides, impliqués dans l'induction de l'apoptose.Dans une première partie de ce travail, nous avons étudié, chez le modèle cellulaire simple Saccharomyces cerevisiae, la contribution relative des PL saturés et des céramides à la cytotoxicité des AGS. Nous avons pu démontrer que les céramides agissaient à des étapes précoces de la voie de sécrétion, alors que les PL saturés impactaient des étapes plus tardives en altérant en particulier la formation de vésicules de sécrétion.Parallèlement, nous avons également constaté que le taux d'AGS était significativement augmenté dans les PL membranaires des patients atteints d'une maladie génétique, la mucoviscidose. La mutation la plus fréquente responsable de cette maladie, résulte en la rétention de la protéine correspondante dans le réticulum endoplasmique. Des molécules pharmacologiques, capables de corriger le trafic de la protéine à sa destination finale ont été isolées in vitro, mais des limitations importantes ont pu être observées lors des tests cliniques. Nous proposons dans le présent manuscrit que la lipointoxication liée aux AGS pourrait être un écueil important à l'utilisation des correcteurs actuels pour le traitement de la mucoviscidose. / Saturated fatty acids (SFA) have been reported to alter organelle integrity in many cell types. This process, also known as lipotoxicity, has been proposed to be responsible for several human pathologies such as type 2 diabetes.At the cellular level, SFA accumulation is associated with an increase of the saturation rate of membrane phospholipids (PL), the major components of organelle membranes, and an increase of ceramides levels, implicated in apoptosis induction.In the first part of this work, we took advantage of a simple yeast-based model to study the relative contributions of saturated PL and ceramides to SFA cytotoxicity. We demonstrated that ceramides act early in the secretory pathway, while saturated PL impact the later steps, and particularly the formation of secretory vesicles.In parallel, we observed that SFA amounts were significantly increased in the membrane PL of cystic fibrosis (CF) patient cells. The most common mutation responsible for this genetic disease results in the retention of the corresponding protein in the endoplasmic reticulum. Pharmacological agents, which correct the mistrafficking of the protein, have been isolated in vitro, but they did not show significant improvements in clinical trials. We propose in the present manuscript, that SFA-related lipointoxication could be an important bottleneck for the use of these pharmacological agents in clinical trials.

The multidrug transporter LmrP, member of the major facilitator superfamily (MFS), confers L. lactis and recombinant E. coli cells resistance to an array of cytotoxic compounds including antibiotics. LmrP mediates drug extrusion from the plasma membrane by an electrogenic proton/drug exchange reaction, whereby a positively charged substrate may move towards the external medium in exchange for two or more protons moving towards the cytoplasm. Recent studies have suggested that MFS transporters require phosphatidylethanolamine (PE) for function and proper topology. However, the specificity of the PE requirement, as well as the contribution of the electrochemical gradient (the driving force of the substrate transport) to this lipid requirement was not addressed. Here we report a new approach for addressing PE specific requirement for the function and the structure of membranes transporters. We used methyl-PE and dimethyl-PE analogs of PE to show that only replacement of the three hydrogens by methyl moieties leads to changes in the biochemical and biophysical properties of the reconstituted protein. This suggests that LmrP does not depend on the bulk properties of the phospholipids tested but solely on the hydrogen bonding ability of the headgroup. We then show that a single point mutation in LmrP, D68C, is sufficient to recapitulate precisely every biochemical and biophysical effect observed when PE is replaced by phosphatidylcholine (PC) ( including energy transfer between the protein tryptophan residues and the lipid headgroups). We conclude that the negatively charged Asp-68 is likely to participate in the interaction with PE and that such interaction is required for proton gradient sensing, substrate binding, and transport. Because Asp-68 belongs to a highly conserved motif in the Major Facilitator Superfamily (which includes LacY and EmrD), this interaction might be a general feature of these transporters that is involved in proton gradient sensing and lipid dependence.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Les liposomes, vésicules à base de phospholipides, sont des systèmes colloïdaux utilisés en recherche et dans différents domaines industriels (pharmaceutique, cosmétique, nutrition). Néanmoins, leur développement se heurte au manque de diversité des sources de phospholipides (soja et jaune d’oeuf). Parallèlement, les méthodes industrielles d’extraction des phospholipides sont basées essentiellement sur l’utilisation de solvants organiques. Dans ce contexte, mon sujet de thèse a eu pour but d’étudier l’extraction de PL, par le CO2 supercritique, à partir de différentes sources avec comme objectif final la formulation de ces PL en liposomes. La démarche a consisté à explorer différents sous-produits de l’huilerie (tourteau d’oléagineux) et de la pêche (sous-produit de la coquille Saint Jacques), potentiellement riches en phospholipides, puis à mettre en place différentes techniques analytiques pour discriminer et quantifier les types de lipides. Parallèlement, l’influence de différents paramètres de l’extraction par CO2 supercritique sur la pureté des fractions obtenues est abordée ainsi qu’une première approche de formulation de PL en liposomes et la caractérisation de ces vésicules. / Liposomes, phospholipids vesicles, are colloidal systems used in search and different industrial fields (pharmaceutical, cosmetic, nutrition). Nevertheless their development face lack of phospholipid sources (soya and egg yolk). At the same time, industrial methods to extract phospholipids use organic solvents. In this context, the phospholipid extraction were studied using a green technology from new different sources in order to formulate liposomes. Oil mill and fishery by-products (seed cake and scallop) were studied to know phospholipid quantities. Analytical techniques were established to discriminate and quantify lipid types. Simultaneously, varying the operating conditions of CO2 supercritical extraction allowed obtaining extracts with different purities and contents in phospholipids and a first study of liposome formulation was carried out.

La BSP1, principale protéine du plasma séminal bovin, interagit avec les membranes des spermatozoïdes et joue un rôle crucial dans les événements qui conduisent à la fécondité des spermatozoïdes, lors du phénomène de la capacitation. Le but de cette recherche est d’investiguer la nature de ces interactions. Ce travail vise à démontrer l’influence des lipides qui composent les membranes sur l’action de la protéine BSP1. À l’aide de la fluorescence intrinsèque de la protéine, l’affinité de la protéine a été caractérisée pour quatre systèmes lipidiques. Les résultats montrent que la composition lipidique affecte significativement l'affinité de la protéine pour les membranes. Nous avons observé l'ordre suivant : 1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphocholine (POPC) > POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphoéthanolamine (POPE) ≈ POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3-phospho-L-sérine (POPS) > POPC/cholestérol. La protéine interagit préférentiellement avec POPC. La présence de POPE, POPS, ou cholestérol dans la membrane diminue systématiquement l’affinité. Il est connu que la présence de POPE ou cholestérol augmente l’empilement des lipides dans les membranes. Cet effet de condensation des chaînes pourrait être défavorable à l’insertion de la partie hydrophobe de la protéine dans les membranes et réduire ainsi l'affinité. La diminution de l’affinité de la protéine induite par la présence de POPS, un lipide chargé négativement, pourrait être associée aux interactions électrostatiques répulsives car la protéine porte une charge globale négative. La littérature mentionne que la BSP1 extrait sélectivement les phospholipides de type choline et le cholestérol lors de son association avec les membranes de spermatozoïdes. Un efflux lipidique est aussi observé avec des membranes modèles. Nous avons désiré caractériser la « solubilisation » des membranes par la BSP1, par diffusion dynamique de la lumière. Comme étape préliminaire, nous avons étudié comment le détergent Triton X-100 solubilise les membranes en utilisant cette technique. Les mesures démontrent que la composition lipidique des membranes (POPC, POPC/POPE, POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3- [phospho-rac-(1-glycérol)] (POPG)) n’affecte pas le mécanisme général de solubilisation/reconstitution des membranes modèles. Il a été montré qu'il existe trois régions lors des processus de solubilisation pour les différents systèmes lipidiques : i) le détergent se distribue dans les membranes, ii) une coexistence de membranes saturées en détergents et de micelles mixtes de phospholipides/Triton X-100 et iii) exclusivement des micelles mixtes de phospholipides/Triton X-100. Nos résultats montrent que la forme conique de POPE augmente la résistance des membranes à la solubilisation. La présence de POPG, apportant une charge négative à l’interface des membranes, n’induit aucun changement aux processus de solubilisation/reconstitution des membranes par Triton X-100. La diffusion dynamique de la lumière a également permis d’observer si la protéine BSP1 induit des modifications morphologiques des membranes suite à son interaction avec les membranes de POPC. Nos observations n'ont montré aucune variation significative de la taille des particules lors du titrage des vésicules de POPC par la protéine, sur une gamme de rapport molaire de POPC/BSP1 variant de 20 à 0.6. Avec des compositions aussi différentes, on suppose une transition des vésicules saturées en protéine à des complexes de protéine avec un peu de lipides. Cependant, il semble impossible avec la diffusion dynamique de la lumière de différencier ces particules. / BSP1, the main protein in bovine seminal plasma, interacts with sperm membranes and plays a crucial role in events that lead to sperm fertility, during the capacitation. The purpose of this research is to investigate the nature of these interactions. This work aims to demonstrate the influence of the lipids that compose membranes on the action of the BSP1 protein. Using the intrinsic fluorescence of the protein, the affinity of the protein was characterized for four lipid systems. The results show that the lipid composition significantly affects the affinity of the protein for membranes. We observed the following order: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) > POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) ≈ POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) > POPC/cholesterol. The protein interacts preferentially with POPC. The presence of POPE, POPS, or cholesterol in membranes decreases systematically the affinity. It is established that the presence of POPE or cholesterol increases the packing of lipids in membranes. This condensation effect could be detrimental to the insertion of the hydrophobic part of the protein into the membranes and reduces, as a consequence, the affinity. The decrease in protein affinity induced by the presence of POPS, a negatively charged lipid, could be associated with repulsive electrostatic interactions as the protein global charge is negative. The literature mentions that BSP1 selectively extracts choline phospholipids and cholesterol when combined with sperm membranes. A lipid efflux is also observed with model membranes. We characterized membrane "solubilisation" by BSP1, using dynamic light scattering. As a preliminary step, we studied how Triton X-100 detergent solubilizes membranes using this technique. The measurements showed that the lipid composition of the membranes does not affect the general solubilization/reconstitution mechanism of the model membranes (POPC, POPC/POPE, POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1-rac-glycerol) (POPG)). It is known that three different regions exist during the solubilization process for the different lipid systems: i) the detergent is distributed in the membranes, ii) a coexistence of membranes saturated with detergents and mixed phospholipid/Triton X-100 micelles and iii) exclusively mixed phospholipid/Triton X-100 micelles. Our results show that the conical shape of POPE increases the resistance of the membranes to solubilization. The presence of POPG, bringing a negative charge at the membrane interface, does not induce any change in solubilization/reconstitution processes. Dynamic light scattering also made it possible to observe if the BSP1 protein induces morphological changes in the membranes following its interaction with POPC membranes. Our observations showed no significant variation in particle size during the titration of POPC vesicles by the protein, over a molar ratio range of POPC/BSP1 from 20 to 0.6. Considering such different compositions, a transition from vesicles saturated with protein to protein complexes with some lipids is assumed. However, it appeared impossible with dynamic light scattering to differentiate these particles.

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Neuronal membrane disruption and mechanoporation are nanoscale damage mechanisms that critically affect brain cell viability during traumatic brain injury (TBI). These nanoscale cellular impairments are elusive in experiments and necessitate in silico approaches such as molecular dynamics (MD) simulations. Implementing MD, this research aims to investigate the effects of different key factors related to membrane deformation and damage, including force field resolutions, lipid compositions, and loading conditions. To examine the impact of force field resolution, MD deformation simulations were conducted on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayer membranes, using all-atom (AA), united-atom (UA), and coarse-grained Martini (CG-M) force fields. The mechanical responses of the three models progressively changed based on the coarse-graining level. The coarser systems exhibited lower yield stresses and failure strains, and higher mechanoporation damage. To study the influence of lipid components, tensile deformation was applied on seven lipid bilayers, each of which contained a different lipid type commonly found in human brain membrane. Larger headgroup structure, greater degree of unsaturation, and tail-length asymmetry decreased lipid packing, increased the area per lipid (APL), and decreased the failure strain of membrane. Lastly, the deformation behavior of a complex multicomponent MD bilayer (realistically representing human neuronal plasma membrane) under different strain rates and strain states was inspected. The yield stress increased with increasing strain rates and more equibiaxial strain states. Meanwhile, lower strain rates resulted in fewer but larger pores, as well as lower strain and APL at failure. Besides, more equibiaxial strain states exhibited more and larger pores, and lower failure strain. Similar failure APL was obtained regardless of strain states, suggesting that the membrane failed when reaching a critical APL value. In addition, the inclusion of cholesterol was shown to decrease the critical APL. The strain-state dependence results were then used to update the Membrane Failure Limit Diagram (MFLD) that indicates the planar strains for potential membrane failure. Overall, the study provides a non-invasive approach that aids in the current understanding of nanoscale neuronal damage dynamics and essential aspects affecting membrane mechanical responses, and furthermore lays the groundwork for future studies on brain injury biomechanics under various TBI scenarios.