Indiana University-Purdue University Indianapolis (IUPUI) / Each biological cell is surrounded by a membrane that consists of many different kinds of lipids. The lipids are mainly composed of phospholipids, which form a fluid bilayer that serves as the platform for the function of membrane bound proteins regulating cellular activity. In the research described in this thesis we employed solid state 2H NMR, complemented by DSC (differential scanning calorimetry) and MD (molecular dynamics) simulations, to study the effect of PUFA (polyunsaturated fatty acids) and TFA (trans fatty acids) on molecular organization in protein-free model membranes of controlled composition. These two classes of unsaturated fatty acid incorporate into membrane lipids and have, respectively, a beneficial and harmful impact on health. The aim is to gain insight into the molecular origin of this behavior. DHA (docosahexaenoic acid), which with 6 "natural" cis double bonds is the most highly unsaturated PUFA found in fish oils, and EA (elaidic acid), which with only a single "unnatural" trans double bond is the simplest manmade TFA often found in commercially produced food, were the focus. 2H NMR spectra for [2H31]-N-palmitoylsphingomyelin ([2H31]16:0SM) in SM/16:0-22:6PE (1-palmitoyl-2-docosahexaenoylphosphatidylethanolamine)/cholesterol (1:1:1 mol) mixed membranes were recorded. This system served as our PUFA-containing model. The spectra are consistent with lateral separation into nano-sized (< 20 nm) domains that are SM-rich/cholesterol-rich (raft), characterized by higher chain order, and DHA-rich/cholesterol-poor (non-raft), characterized by lower chain order. The aversion cholesterol has for DHA, as opposed to the affinity cholesterol has for predominantly saturated SM, excludes the sterol from DHA-containing PE-rich domains and DHA from SM-rich/cholesterol-rich domains. It is the formation of highly disordered membrane domains that we hypothesize is responsible, in part, for the diverse health benefits associated with dietary consumption of DHA. 2H NMR spectra for 1-elaidoyl-2-[2H35]stearoylphosphatidylcholine (t18:1-[2H35]18:0PC) and 1-oleoyl-2-[2H35]stearoylphosphatidylcholine (c18:1-[2H35]18:0PC) were recorded to compare membranes with respect to a trans vs. cis ("natural") double bond. The spectra indicate that while a trans double bond produces a smaller deviation from linear conformation than a cis double bond, membrane order is decreased by a comparable amount because the energy barrier to rotation about the C-C single bonds either side of a <italic>trans</italic> or <italic>cis</italic> double bond is reduced. Although EA adopts a conformation somewhat resembling a saturated fatty acid, the TFA is almost as disordered as its <italic>cis</italic> counterpart oleic acid (OA). We speculate that EA could be mistaken for a saturated fatty acid and infiltrate lipid rafts to disrupt the high order therein that is necessary for the function of signaling proteins.

No description available.

Introdução: Devido a diversos fatores, recém-nascidos prematuros, em sua maioria, necessitam de nutrição parenteral e uma fonte lipídica que possua um equilíbrio entre os variados tipos de ácidos graxos. SMOFlipid® 20%, uma nova emulsão lipídica pode ser mais adequada para esse equilíbrio. Objetivo: Avaliar o perfil de incorporação de ácidos graxos em eritrócitos de prematuros recebendo essa nova emulsão lipídica, comparada com outra emulsão baseada em óleo de soja. Métodos: Em um ensaio clinico controlado randomizado duplo cego avaliou-se 47 recém-nascidos pré-termo que receberam nutrição parenteral SMOFlipid® 20% (n=25) ou LIPOVENOS® MCT 20% (n=22). Foram avaliados parâmetros laboratoriais, clínicos, demográficos e o perfil de incorporação de ácidos graxos na membrana de eritrócitos. Resultados: Os parâmetros clínicos e demográficos como peso, perímetro cefálico, comprimento, idade gestacional e índice de Apgar não diferiram entre os grupos. Os valores de triglicerídeos e da lipoproteína de muito baixa densidade (VLDL) foram estatisticamente maiores no grupo SMOFLIPID® 20%. Níveis de Aspartato aminotransferase (AST) foram menores em ambos os grupos e os níveis de bilirrubina total e frações não tiveram diferenças. A emulsão SMOFlipid® 20% aumentou os níveis dos ácidos docosa-hexaenoico DHA (C 22:6 w3) e Eicosapentaenoico EPA (C 20:5 w3) na membrana dos eritrócitos. Conclusões: Neste grupo de recém-nascidos pré-termos, essa nova emulsão lipídica, além de mostrar segurança, contribuiu para uma mudança benéfica no perfil de incorporação de ácidos graxos nas membranas celulares, principalmente DHA e EPA / Introduction: Due to several factors, premature newborn infants, in most cases, require parenteral nutrition and a lipid source with balance among the different types of fatty acids. SMOFlipid® 20%, a new lipid emulsion may be more appropriate for this balance. Objectives: To evaluate the profile of fatty acids incorporation in erythrocytes of premature newborn infants receiving this new lipid emulsion compared with an emulsion based on soybean oil. Methods: In a randomized, controlled, double-blind clinical trial, 47 preterm newborn who received parenteral nutrition SMOFlipid® 20% (n=25) or Lipovenos MCT® 20% (n=22) were evaluated. Laboratorial, clinical and demographic parameters and the profile of incorporation of fatty acids in the erythrocyte membrane were evaluated. Results: The clinical and demographic parameters such as weight, head circumference, length, gestational age, and Apgar scores did not differ between the groups. The values of triglycerides and lipoprotein of very low density (VLDL) were statistically higher in the SMOFlipid® 20% group. Levels of aspartate aminotransferase (AST) were lower in both groups and levels of total bilirubin and fractions had no differences. The SMOFlipid® 20% emulsion increased the levels of the docosahexaenoic acid (DHA) and eicosapentaenoic (EPA) acid in the erythrocytes membrane. Conclusions: In this group of preterm newborn infants, this new lipid emulsion, besides showing security, contributed to a beneficial change in the incorporation profile of fatty acids cell membranes, especially DHA and EPA

Made available in DSpace on 2017-07-21T20:00:00Z (GMT). No. of bitstreams: 1 Lilian Prione.pdf: 1165937 bytes, checksum: d73d6916708640fa9af6db139db8b8c2 (MD5) Previous issue date: 2014-02-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Herbicides are widely used to increase crop production and account for 35% of all agrochemicals applied annually. After application, the herbicides can remain in the soil as hazardous residues. Mesotrione, (2-[4-methylsulfonyl-2-nitrobenzoyl]1,3-cyclohenanedione), is the active ingredient of Callisto, an herbicide commonly used in corn. Pantoea ananatis CCT 7673, an Enterobacteria isolated from water, has been previously cited as mesotrione-degrading strain. The aim of this study was to evaluate the influence of mesotrione and Callisto as oxidative stress-inducing agents for cellular metabolism of Pantoea ananatis CCT7673 and identify possible mechanisms of tolerance. SOD, CAT and GR activities were evaluated in non-denaturing PAGE and CAT, GR and GST in spectrophotometer. Also, the rates of malonaldehyde (MDA), superoxid and peroxide hydrogen (H2O2) were measured in a spectrophotometer. Minimal medium with no herbicide (MM) was used as control. Lipid peroxidation, superoxide and peroxide hydrogen quantification and SOD, CAT, GR and GST activities were analyzed before and after degradation of mesotrione. The herbicide proved to be the cause of oxidative stress, according to peroxide hydrogen data. Unexpectedly, the rates of lipid peroxidation (MDA) and GR showed to be lower in the presence of the herbicide when compared to the control, with no changes in bacterial growth. The activity of GST was higher in mesotrione treatment in comparison to control and Callisto, during and after degradation. These results suggest that this enzyme may be related to the mesotrione degradation, probably by cometabolism. The rates of lipid peroxidation were shown to be lower in the presence of the herbicide compared to the control, with no changes in growth rates when exposed to herbicide. P. ananatis CCT 7673 showed changes in the saturation of membrane lipids. These changes may interfere with herbicide entry into the cell. These characteristics may be associated with a level of phenotypic plasticity in P. ananatis CCT 7673, making this an interesting bacterium for studies of herbicide tolerance and evolution of microbiota in environments subjected to different degrees of selective pressure model. / Herbicidas são amplamente utilizados para aumentar a produção agrícola e são responsáveis por 35% de todos os agrotóxicos aplicados anualmente. Após a aplicação, os herbicidas podem permanecer no solo como resíduos perigosos. O mesotrione (2 - [ 4 - metilsulfonil - 2 - nitrobenzoil ] 1,3- cyclohenanedione ) é o ingrediente ativo de Callisto, um herbicida utilizado no milho . Pantoea ananatis CCT 7673, é uma enterobactéria isolada de água e foi previamente caracterizada como linhagem degradadora do mesotrione. Os objetivos deste trabalho foram o avaliar a influência do mesotrione e Callisto como agentes indutores de estresse oxidativo para o metabolismo celular de Pantoea ananatis CCT7673, bem como identificar possíveis mecanismos de tolerância a estes herbicidas. As atividades enzimáticas de SOD, CAT e GR foram avaliadas em PAGE não desnaturante e de CAT, GR e GST em espectrofotômetro. Além disto, as taxas de malondialdeído (MDA), radical superóxido e peróxido de hidrogênio (H2O2) foram medidas em espectrofotômetro. Todas as análises foram realizadas antes e após a degradação do mesotrione. Meio mínimo sem herbicida (MM) foi utilizado como controle. O herbicida provou ser agente causal do estresse oxidativo pelos dados de peróxido de hidrogênio. Inesperadamente, as taxas de MDA e GR mostraram-se inferiores nos tratamentos com herbicida em relação ao controle, sem alteração no crecimento bacteriano. A atividade de glutationa-S-transferase foi maior no tratamento com mesotrione em comparação com o controle e Callisto, durante e após a degradação. Estes resultados sugerem que essa enzima pode estar relacionada com o processo de degradação do mesotrione, provavelmente por cometabolismo. As taxas de peroxidação lipídica mostraram ser menores na presença do herbicida em comparação com o controle, sem mudanças na taxa de crescimento Quando exposta ao herbicida, P. ananatis CCT 7673 apresentou alterações na saturação de lipídios de membrana. Estas mudanças podem interferir na entrada do herbicida na célula. Tais características podem estar associadas a um nível de plasticidade fenotípica em P. ananatis CCT 7673, tornando essa bactéria um modelo interessante para estudos de tolerância a herbicidas e evolução de microbiotas em ambientes submetidos a diferentes graus de pressão seletiva.

Introdução: Devido a diversos fatores, recém-nascidos prematuros, em sua maioria, necessitam de nutrição parenteral e uma fonte lipídica que possua um equilíbrio entre os variados tipos de ácidos graxos. SMOFlipid® 20%, uma nova emulsão lipídica pode ser mais adequada para esse equilíbrio. Objetivo: Avaliar o perfil de incorporação de ácidos graxos em eritrócitos de prematuros recebendo essa nova emulsão lipídica, comparada com outra emulsão baseada em óleo de soja. Métodos: Em um ensaio clinico controlado randomizado duplo cego avaliou-se 47 recém-nascidos pré-termo que receberam nutrição parenteral SMOFlipid® 20% (n=25) ou LIPOVENOS® MCT 20% (n=22). Foram avaliados parâmetros laboratoriais, clínicos, demográficos e o perfil de incorporação de ácidos graxos na membrana de eritrócitos. Resultados: Os parâmetros clínicos e demográficos como peso, perímetro cefálico, comprimento, idade gestacional e índice de Apgar não diferiram entre os grupos. Os valores de triglicerídeos e da lipoproteína de muito baixa densidade (VLDL) foram estatisticamente maiores no grupo SMOFLIPID® 20%. Níveis de Aspartato aminotransferase (AST) foram menores em ambos os grupos e os níveis de bilirrubina total e frações não tiveram diferenças. A emulsão SMOFlipid® 20% aumentou os níveis dos ácidos docosa-hexaenoico DHA (C 22:6 w3) e Eicosapentaenoico EPA (C 20:5 w3) na membrana dos eritrócitos. Conclusões: Neste grupo de recém-nascidos pré-termos, essa nova emulsão lipídica, além de mostrar segurança, contribuiu para uma mudança benéfica no perfil de incorporação de ácidos graxos nas membranas celulares, principalmente DHA e EPA / Introduction: Due to several factors, premature newborn infants, in most cases, require parenteral nutrition and a lipid source with balance among the different types of fatty acids. SMOFlipid® 20%, a new lipid emulsion may be more appropriate for this balance. Objectives: To evaluate the profile of fatty acids incorporation in erythrocytes of premature newborn infants receiving this new lipid emulsion compared with an emulsion based on soybean oil. Methods: In a randomized, controlled, double-blind clinical trial, 47 preterm newborn who received parenteral nutrition SMOFlipid® 20% (n=25) or Lipovenos MCT® 20% (n=22) were evaluated. Laboratorial, clinical and demographic parameters and the profile of incorporation of fatty acids in the erythrocyte membrane were evaluated. Results: The clinical and demographic parameters such as weight, head circumference, length, gestational age, and Apgar scores did not differ between the groups. The values of triglycerides and lipoprotein of very low density (VLDL) were statistically higher in the SMOFlipid® 20% group. Levels of aspartate aminotransferase (AST) were lower in both groups and levels of total bilirubin and fractions had no differences. The SMOFlipid® 20% emulsion increased the levels of the docosahexaenoic acid (DHA) and eicosapentaenoic (EPA) acid in the erythrocytes membrane. Conclusions: In this group of preterm newborn infants, this new lipid emulsion, besides showing security, contributed to a beneficial change in the incorporation profile of fatty acids cell membranes, especially DHA and EPA

Lors de la dégustation d’un vin, les tannins sont responsables de deux propriétés gustatives, l’amertume et l’astringence, respectivement dues à des associations avec les protéines salivaires et les récepteurs au goût amer. Néanmoins, leurs intensités perçues en bouche vont dépendre de multiples facteurs, notamment la présence de molécules externes aux complexes tannin-protéine tel que les lipides, qu’ils soient situés au niveau des membranes buccales ou issus de l’alimentation. L’objectif de cette thèse a ainsi été d’examiner l’impact des lipides sur ces sensations organoleptiques. Pour ce faire, cette étude, réalisée principalement par RMN, s’est intéressée aux interactions tannin-lipide sur des modèles de membranes buccales et d’émulsions de gouttelettes lipidiques. Nous avons pu alors étudier l’interaction tannin-lipide en termes de localisation, d’affinité et de dynamique. Nos résultats montrent dans un premier temps une localisation du tannin à l’interface de tous les modèles utilisés. En outre, l’insertion de tannins au niveau des vésicules multilamellaires, modèle utilisé pour mimer les membranes buccales, entraîne une fluidification de ce système lipidique. Il a été montré que cet effet fluidifiant dépend de la structure du tannin, de la présence d’éthanol et de la teneur en cholestérol du système lipidique. Enfin, un protocole permettant d’obtenir les constantes d’associations tannin-lipide par RMN a été établi. Ces dernières se sont révélées du même ordre de grandeur que celles relatives aux interactions tannin-protéine salivaire. Ces résultats montrent que les lipides auraient une influence d’une part sur l’astringence via une compétition entre les interactions tannin-lipide et les interactions tannin-protéines salivaires et d’autre part sur l’amertume en perturbant la dynamique de la membrane, ce qui pourrait induire une perturbation des récepteurs gustatifs. / When tasting a wine, tannins are responsible of two gustative properties, bitterness and astringency, respectively due to association between tannins and salivary proteins or bitter receptors. However, perceived intensities depend on several factors, including the presence of external molecules such as lipids, either located in the buccal membranes or from food. The main objective of this thesis was to study the effect of lipids on these two organoleptic properties. For that, this study, carried out mainly by NMR, is interested in tannin-lipid interaction using several models of buccal membranes and lipid droplets. We have studied these interactions in terms of localization, affinity and dynamics. Our results show a localization of tannins at the interface of all studied lipid models. Then, the insertion of tannins in multilamellar vesicles, used to mimic buccal membranes, causes a fluidification effect on these systems. This effect depends on the structure of the tannin, the presence of ethanol and the cholesterol content of the lipid system. Finally, a protocol to determine the tannin-lipid association constants was developed. The latter have proved to be in the same order of magnitude as those for tannin-salivary protein interaction. These results show that lipids could have an influence on the one hand on astringency, due to the competition between tannin-lipid interaction and tannin-salivary protein interaction, and on the other hand on bitterness due to the disturbance of the buccal membrane dynamics, which could induce a disturbance of the gustative receptors.

The interplay between lipids and membrane proteins is known to affect membrane protein topology and thus have significant effect (control) on their functions. In this PhD thesis, the influence of lipids on the membrane protein function was studied using three different membrane protein models. A monotopic membrane protein, monoglucosyldiacylglyecerol synthase (MGS) from Acholeplasma laidlawii is known to induce intracellular vesicles when expressed in Escherichia coli. The mechanism leading to this unusual phenomenon was investigated by various biochemical and biophysical techniques. The results indicated a doubling of lipid synthesis in the cell, which was triggered by the selective binding of MGS to anionic lipids. Multivariate data analysis revealed a good correlation with MGS production. Furthermore, preferential anionic lipid sequestering by MGS was shown to induce a different fatty acid modeling of E. coli membranes. The roles of specific lipid binding and the probable mechanism leading to intracellular vesicle formation were also investigated. As a second model, a MGS homolog from Synechocystis sp. PCC6803 was selected. MgdA is an integral membrane protein with multiple transmembrane helices and a unique membrane topology. The influence of different type of lipids on MgdA activity was tested with different membrane fractions of Synechocystis. Results indicated a very distinct profile compared to Acholeplasma laidlawii MGS. SQDG, an anionic lipid was found to be the species of the membrane that increased the MgdA activity 7-fold whereas two other lipids (PG and PE) had only minor effects on MgdA. Additionally, a working model of MgdA for the biosynthesis and flow of sugar lipids between Synechocystis membranes was proposed. The last model system was another integral membrane protein with a distinct structure but also a different function. The envelope stress sensor, CpxA and its interaction with E. coli membranes were studied. CpxA autophosphorylation activity was found to be positively regulated by phosphatidylethanolamine and negatively by anionic lipids. In contrast, phosphorylation of CpxR by CpxA revealed to be increased with PG but inhibited by CL. Non-bilayer lipids had a negative impact on CpxA phosphotransfer activity. Taken together, these studies provide a better understanding of the significance of the interplay of lipids and model membrane proteins discussed here.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O efeito da L-alanil-glutamina foi testado numa situaÃÃo de estresse oxidativo induzida por um modelo experimental de isquemia/reperfusÃo testicular atravÃs de torÃÃo do cordÃo espermÃtico. Oitenta e quatro ratos Wistar jovens foram distribuÃdos aleatoriamente em 6 grupos da seguinte forma: Grupo Salina 1h (GSa1) e Grupo Ala-Gln 1h (GAg1), tendo sido os animais submetidos à 1 hora de isquemia e 6 horas de reperfusÃo, tratados 30 minutos antes da torÃÃo, por via intravenosa, com soluÃÃo salina e L-alanil-glutamina (0,75g/Kg) respectivamente; Grupo Salina 3h (GSa3) e Grupo Ala-Gln 3h (GAg3), tendo sido os animais submetidos a 3 horas de isquemia e 6 horas de reperfusÃo, tratados 30 minutos antes da torÃÃo, por via intravenosa, com soluÃÃo salina ou L-alanil-glutamina (0,75g/Kg) respectivamente; cada grupo, constituÃdo por 18 ratos, foi distribuÃdo equitativamente, , em 3 subgrupos (T-0, T-2, T-6), cada um com 6 animais, os quais representavam os tempos de coleta do testÃculo, sendo T-0 o tempo mÃximo de isquemia, T-2 apÃs 2 horas de reperfusÃo e T-6 apÃs 6 horas de reperfusÃo. Dois grupos simulados (Sham), cada um com 6 animais, mimetizava o procedimento de induÃÃo de isquemia por 1 ou 3 horas, seguido de um perÃodo de pÃs-trauma de 2 horas, sendo a coleta realizada neste momento. Foram determinadas as concentraÃÃes das substÃncias reativas ao Ãcido tiobarbitÃrico (TBARS) e glutationa reduzida (GSH) nas amostras de tecido (testÃculo) obtidas nos diversos subgrupos. As comparaÃÃes entre os grupos foram realizadas utilizando-se o teste de Mann-Whitney e Kruskal-Wallis com comparaÃÃes temporais pelo teste de Dunn. A significÃncia estatÃstica foi com valores de p<0,05. Houve aumento significante das concentraÃÃes de GSH, no testÃculo dos ratos submetidos à 1 hora de isquemia seguida de 6 horas reperfusÃo, tratados com L-Ala-Gln em todos os tempos estudados, quando comparados ao grupo controle; nos animais submetidos à 3 horas de isquemia, demonstrou-se aumento significativo de GSH no tempo mÃximo de isquemia e apÃs 6 horas de reperfusÃo. Houve reduÃÃo significante nas concentraÃÃes de TBARS, no testÃculo dos ratos submetidos à 3 horas de isquemia seguida de reperfusÃo, nos animais tratados L-Ala-Gln no tempo mÃximo de isquemia e apÃs 2 horas de reperfusÃo. Estes resultados demonstram um efeito protetor que a glutamina exerce no testÃculo durante a isquemia/reperfusÃo, mostrando-se eficaz na manutenÃÃo dos nÃveis teciduais de GSH nos dois modelos de torÃÃo do cordÃo espermÃtico, reduzindo a magnitude do estresse oxidativo e tambÃm reduzindo a peroxidaÃÃo lipÃdica nos animais submetidos à 3 horas de isquemia, atà a segunda hora de reperfusÃo. / The effect of L-alanil-glutamine was tested in a situation of oxidative stress induced by torsion of the spermatic cord. Eighty four male young Wistar rats were distributed randomly in 6 groups: Saline group 1h (GSa1) and Ala-Gln Group 1h (GAg1), animals of these groups were submitted to 1 hour of ischemia and 6 hours of reperfusion and were treated 30 minutes before the torsion, by intravenous way, with saline solution and L-alanil-glutamine (0,75g/Kg) respectively; Saline group 3h (Gsa3) and Ala-Gln Group 3h (Gag3), animals of these groups were submitted to 3 hours of ischemia and 6 hours of reperfusion, had been treated, 30 minutes before the torsion, by intravenous way, with saline solution and L-alanil-glutamine (0,75g/Kg) respectively; each group, consisting of 18 rats, was distributed equitable, in 3 sub-groups (T-0, T-2, T-6), each one with 6 animals, which represented the times that testis were colected, being T-0 the maximum time of ischemia, T-2 after 2 hours of reperfusion and T-6 after 6 hours of reperfusion. Two groups (Sham 1h and Sham 3h) were submitted to sham operation, being the testis colected two hours after simulated trauma. Thiobarbituric acid reactive substances (TBARS) and reduced glutathione levels were assayed in testis. Comparasion between groups were made using Mann-Whitney e Kruskal-Wallis tests, and temporal comparations were made using Dunn test. P values of <0,05 were considered to indicate statistical significance. GSH concentration was significantly increased in testis of the rats submitted to 1 hour of ischemia, followed by 6 hours of reperfusion, treated with L-Ala-Gln in all the studied times, when compared with the control group; in the animals submitted to the 3 hours of ischemia, significant increase of GSH was demonstrated in the maximum time of ischemia and 6 hours of reperfusion time. Significant decrease of TBARS levels were seen in rats submitted to 3 hours of ischemia followed by reperfusion, treated with L-alanil-glutamine, in maximum time of ischemia and after 6 hours of reperfusion. These results demonstrates that glutamine may play a role in the maintenance of the tecidual levels of GSH in two models of ischemia/reperfusion of the testis studied, by an antioxidant effect, and may promote cell membrane protection during the ischemia (3h)/reperfusion by decreasing lipid peroxidation

Les cellules neuroendocrines possèdent d’une part la voie de sécrétion constitutive, existant dans tous les types cellulaires, qui permet le renouvellement continu de la membrane plasmique et de la matrice extracellulaire, et d’autre part la voie de sécrétion régulée, spécifique aux cellules sécrétrices, qui permet la sécrétion d’hormones suite à la stimulation de la cellule. Les organites impliqués dans cette dernière voie sont des granules de sécrétion à cœur dense (GS), sui stockent les hormones ainsi que les glycoprotéines solubles, les granines. Parmi ces dernières, la chromogranine A (CgA) joue un rôle majeur dans la biogénèse des GS mais les mécanismes moléculaires ne sont pas clairement définis. Dans une lignée de cellules non-endocrines COS7 (dépourvues de granines et donc de voie de sécrétion régulée), mon équipe d’accueil a démontré que l’expression de la CgA induit la formation de vésicules présentant une structure et des fonctions caractéristiques des GS. L’analyse du protéome des GS purifiés à partir d’une lignée de cellules COS7 exprimant de manière stable la CgA (COS7-CgA) a révélé la présence de protéines liant les éléments du cytosquelette et le calcium. Durant ma thèse, nous avons focalisé notre attention sur la myosine 1b (myo1b), l’actine et le complexe de nucléation de l’actine Arp2/3 du fait de leur capacité à induire le bourgeonnement de compartiments post-golgiens dans des cellules non-endocrines. Nous avons montré (i) que la myo1b contrôle la formation des GS ainsi que la sécrétion régulée au sein des cellules COS7-CgA et des cellules neuroendocriniennes PC12, et (ii) que la myo1b et le complexe Arp2/3 permettent le recrutement d’actine fibrillaire dans la région golgienne et la formation des GS. Ces travaux montrent pour la première fois l’implication du complexe actomyosine dans la formation des GS. Afin d’identifier le lien moléculaire entre la CgA luminale et la myo1b cytosolique, nous avons recherché les interactions potentielles de la CgA avec les lipides de la membrane du réseau trans-golgien (TGN). Nous avons montré (i) que la CgA interagit avec l’acide phosphatidique (PA), (ii) que les espèces de PA prédominantes sont communes dans les membranes golgienne et granulaire, (iii) que la CgA est capable d’interagir spécifiquement avec des espèces de PA intégrées avec des membranes artificielles et (iv) que l’inhibition de la production du PA au niveau golgien altère significativement la formation des GS et la sécrétion régulée dans les cellules neuroendocrines. L’ensemble des résultats obtenus dans le cadre de ma thèse suggère que l’interaction entre la CgA et le PA est cruciale pour la biogenèse de GS à partir de la membrane du TGN. Nous émettons l’hypothèse que cette interaction est à l’origine de la formation de microdomaines enrichis en PA qui contrôleraient la courbure de la membrane du TGN et le recrutement du complexe actomyosine. / Neuroendocrine cells exhibit the constitutive secretory pathway which is common all cell types and allows the continuous renewal of the plasma membrane and the extracellular matrix, and the regulated secretory pathway, which is specific to secretory cells and allows hormone secretion following cell stimulation. The organelles supporting the latter pathway are dense-core secretory granules (SG), which store hormones and soluble glycoproteins, called granins. Among these, chromogranin A (CgA) plays a major role in the biogenesis of SG but the molecular mechanisms underlying this process are not clearly understood. Using non-endocrine COS7 cell line (which are devoid of granins and regulated secretory pathway), my host team has demonstrated that the CgA expression induces the formation of vesicles with structural and functional characteristic of SG. The proteome analysis of purified SG from a COS7 cell line stably expressing CgA (COS7-CgA) revealed the presence of cytoskeleton- and calcium-binding proteins. During my thesis, we focused our attention on myosin 1b (myo1b), actin and actin nucleation complex Arp2/3 due to their ability to induce the budding of post-Golgi compartments in non-endocrine cells. We have shown (i) that myo1b controls SG formation as welle as the regulated secretion in COS7-CgA and PC12 neuroendocrine cells, (ii) that myo1b and Arp2/3 complex are required to recruit fibrillar actin (F-actin) to the Golgi region and to SG formation. These results highlight for the first time the involvement of the actomyosin complex in SG formation. In order to identify the molecular link between luminal CgA and Cytosolic myo1b, we investigated the potential interactions of CgA with lipids of the trans-Golgi network (TGN) membrane. We showed (i) that CgA interacts with phosphatidic acid (PA), (ii) that the predominant PA species are common in Golgi and granular membranes, (iii) that Cg Ais able to interact specifically with these PA species included in artificial membranes, and (iv) that inhibition of PA production at the Golgi level significantly alters SG formation and regulated secretion in neuroendocrine cells. All these results obtained during my thesis suggest that the interaction between CgA and PA is crucial for SG biogenesis from the TGN membrane. We suggest that this interaction is at the origin of the formation of PA-enriched microdomains that could control the curvature of the TGN membrane and the recruitment of the actomyosin complex.

Indiana University-Purdue University Indianapolis (IUPUI) / Cell membranes have remarkable properties both at the microscopic level and the molecular level. The current research describes the use of physisorbed polymer-grafted lipids in model membranes to investigate some of these properties on both of these length scales. On the microscopic scale, plasma membranes can be thought of as heterogenous thin films. Cell membranes adhered to elastic substrates are capable of sensing substrate/film mismatches and modulating their membrane stiffness to more closely match the substrate. Membrane/substrate mismatch can be modeled by constructing lipopolymer-enriched lipid monolayers with different bending stiffnesses and physisorbing them to rigid substrates which causes buckling. This report describes the use of atomic force microscopy and epimicroscopy to characterize these buckled structures and to illustrate the use of the buckled structures as diffusion barriers in lipid bilayers. In addition, a series of monolayers with varying bending stiffnesses and thicknesses are constructed on rigid substrates to analyze changes in buckling patterns and relate the experimental results to thin film buckling theory. On the molecular scale, plasma membranes can also be thought of as heterogeneous mixtures of lipids where the specific lipid environment is a crucial factor affecting membrane protein function. Unfortunately, heterogeneities involving cholesterol, labeled lipid rafts, are small and transient in live cells. To address this difficulty, the present work describes a model platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains into which transmembrane proteins are incorporated (αvβ3, and α5β1integrins). This flexible platform enables the use of confocal fluorescence fluctuation spectroscopy to quantitatively probe the effect of cholesterol concentrations and the binding of native ligands (vitronectin and fibronectin for αvβ3, and α5β1) on protein oligomerization state and on domain-specific protein sequestration. In particular, the report shows significant ligand-induced integrin sequestration with a low level of dimerization. Cholesterol concentration increases rate of dimerization, but only moderately. Ligand addition does not affect rate of dimerization in either system. The combined results strongly suggest that ligands induce changes to integrin conformation and/or dynamics without inducing changes in integrin oligomerization state, and in fact these ligand-induce conformational changes impact protein-lipid interactions.