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

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-15T14:03:58Z No. of bitstreams: 1 Juliana Stolze Efeito...2015.pdf: 1337321 bytes, checksum: 6fdad9cde6d0cc06bd37fdbe677450ae (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-15T14:04:13Z (GMT) No. of bitstreams: 1 Juliana Stolze Efeito...2015.pdf: 1337321 bytes, checksum: 6fdad9cde6d0cc06bd37fdbe677450ae (MD5) / Made available in DSpace on 2016-02-15T14:04:13Z (GMT). No. of bitstreams: 1 Juliana Stolze Efeito...2015.pdf: 1337321 bytes, checksum: 6fdad9cde6d0cc06bd37fdbe677450ae (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Introdução: as doenças cardiovasculares acometem milhares de pessoas no mundo. Destas, a doença arterosclerótica está entre as de maior morbimortalidade. Para a avaliação da necessidade de intervenções hemodinâmicas e/ou revascularização miocárdica, há a necessidade da realização do cateterismo (CATE), procedimento de imagem indicado para evidenciar pontos de obstrução e determinar a melhor estratégia cirúrgica. Para a realização do CATE utiliza-se heparina sódica (5000 UI) in bolus. Atualmente, sabe-se que a heparina interfere no remodelamento de partículas lipoproteicas por liberação da lipoproteína lipase (LPL) e da lipase hepática (LH), essa ação pode alterar o transporte reverso do colesterol (TRC), em função de modificações no metabolismo das lipoproteínas. Métodos: foram selecionados por conveniência 20 pacientes, 10 do sexo masculino e 10 do sexo feminino, ambos os sexos, entre 45 e 73 anos, admitidos no Hospital Ana Neri, submetidos à cineangiocoronariografia (CATE). Todas as determinações laboratoriais foram realizadas antes e depois do CATE. Resultados: houve aumento significativo da atividade da lipase e diminuição da concentração dos triglicérides depois do CATE na análise geral e estratificada pelo sexo (p<0,05; Teste t pareado). A razão HDL-C/apoA aumentou significativamente depois do CATE, já a razão LDL-C/apoB não aumentou, nem diminuiu nas análises geral e estratificada por sexo. Enquanto a razão de risco cardiovascular TG/HDL-C diminuiu significativamente, a ApoB/apoA aumentou significativamente na análise geral e estratificada por sexo depois do CATE. As análises de correlações tiveram comportamentos diferentes, sendo a significância estatística encontrada dependente do grupo analisado (geral, masculino e feminino). A concentração do não-HDL-C, semelhante à determinação da haptoglobina, tiveram diminuição significativa na análise geral e no sexo masculino depois do CATE (p<0,05; Teste t pareado), o grupo feminino não mostrou significância. As taxas de incorporação de colesterol livre e fosfolípides não foram significativas depois do CATE. Conclusão: A administração in bolus de heparina sódica interfere no remodelamento de partículas lipoproteicas, sendo este fato evidenciado pelas variações das razões de risco, tais como, HDL-C/apoA, TG/HDL-C. O percentual de incorporação dos fosfolípides e colesterol livre na HDL mostrou-se influenciado em relação ao sexo, o que o torna relevante dado aos resultados encontrados. A utilização de razões de risco e ainda suas correlações mostraram-se melhores indicadores de desfecho sugestivo de doença cardiovascular nessa casuística do que quando avaliados apenas os marcadores séricos do perfil lipídico isoladamente. / Introduction: cardiovascular diseases affect thousands of people worldwide. Of these, the atherosclerotic disease is one of the most morbidity and mortality. To evaluate the need for hemodynamic interventions and / or CABG, the catheterization (CATE) is performed, an imaging procedure to evidence obstruction and to determine the best surgical strategy. To perform CATE, is necessary to use in bolus sodium heparin (5000 IU). Currently, it is known that heparin interferes with the remodeling of the lipoprotein particles by releasing lipoprotein lipase (LPL) and hepatic lipase (HL), this action may alter the reverse cholesterol transport (TRC), by changes in lipoprotein metabolism. Methods: were selected by convenience 20 patients, 10 male and 10 female, both gender, between 45 and 73 years old, admitted to the Hospital Ana Neri, who underwent coronary angiography (CATE). All laboratory measurements were performed before and after CATE. Results: were significant increase in lipase activity and decreased concentration of triglycerides after CATE, in the overall analysis and stratified by sex (p<0.05, paired t test). The HDL-C/apoA ratio increased significantly after CATE, since the LDL-C/apoB ratio has not increased or decreased in the general analysis, and stratified by gender. While TG/HDL-C cardiovascular risk ratio decreased significantly, ApoB/apoA increased significantly in the overall analysis, and stratified by sex after CATE. The correlation analysis had different behaviors, and the statistic significance found, were dependent of the group analyzed (generally male and female). The concentration of non-HDL-C, similar to the determination of haptoglobin, had a significant decrease in the overall analysis and in males after CATE (p<0.05, paired t-test), the female group do not show significance. The free cholesterol and phospholipids incorporation rates were not significant after the CATE. Conclusion: The administration of in bolus sodium heparin interferes in lipoprotein particles remodeling, by evidences from risk ratios variations, such as HDL-C/apoA, and TG/HDL-C. The percentage of phospholipids and free cholesterol incorporation in HDL shows sex influences, which makes it relevant to the obtained results. The use of hazard ratios and their correlations were better surrogate markers at these casuistic of cardiovascular disease than when serum markers of lipid profile were evaluated alone.

Parmi les nombreux effets bénéfiques des phospholipides on retient essentiellement leur rôle dans la protection du système cardio-vasculaire, l'amélioration de la mémoire et de l'apprentissage. Ces effets peuvent s'expliquer en partie par le fait que les phospholipides sont riches en acide gras polyinsaturés (acides eicosapentaénoïque et docosahexaénoïque) ou les acides gras essentiels. C’est pourquoi il nous est apparu comme étant important d’explorer des sources naturelles potentielles contenant des phospholipides polyinsaturés destinées au développement de produits alimentaires nouveaux supplémentés en phospholipides.Ce travail a permis de mettre au point des méthodes d’extraction, de purification et de séparation des phospholipides afin de caractèriser les sources naturelles de phospholipides polyinsaturés. Une supplémentation des aliments avec des phospholipides polyinsaturés ne peut être envisagé sans avoir une bonne connaissance de leur stabilité au cours de la transformation et de la conservation des aliments. Nous avons donc développé des méthodes d’identification des produits de dégradation des phospholipides et de détermination de leur cinétique d’oxydation pendant les traitements thermiques. Ainsi, nous avons montré que les phospholipides sont plus stables que les triacylglycérides, ceci étant du probablement à la présence d’un groupe aminé dans la tête polaire. / Among the numerous beneficial effects of phospholipides can highlight their role in protecting the cardiovascular system and improving memory and learning. These effects can be partially explained by the fact that phospholipids are rich in polyunsaturated (eicosapentaenoic and docosahexaenoic acids) or essential fatty acids. Thus, it is very significant to explore potential food sources containing polyunsaturated rich phospholipids for the development new food products supplemented with phospholipids.This work allowed a series method for the extraction, purification, separation and quantification of phospholipids in order to characterize the polyunsaturated phospholipid sources. Food supplementation with polyunsaturated phospholipids rises however the question of their stability during food processing at high temperature. Then we have developed methods to identify phospholipids degradation products and determine their kinetics of the oxidation during thermal treatment which is a widespread food processing.Phospholipids (PE and PC) are more stable than triacylglyceride, this is due to the presence of an amino group in the molecule.

As coberturas de hidroxiapatita (HAp) são utilizadas para aumentar a osteointegração em implantes de titânio (Ti), devido à sua capacidade de promover a biomineralização para corrigir defeitos esqueléticos e craniofaciais. O objetivo desta pesquisa foi avaliar a influência dos revestimentos sobre culturas primárias de osteoblastos. Na primeira fase de estudos, desenvolvemos uma nova abordagem de revestimento baseada em filmes Langmuir-Blodgett (LB) de dihexadecilfosfato (DHP) e ácido octadecilfosfônico (OPA) depositados em discos Ti, e crescimento subsequente de cristais de HAp. Analisamos a viabilidade dos osteoblastos, a atividade da fosfatase alcalina (ALP) e a formação da matriz mineralizada por métodos colorimétricos e a morfologia das culturas por microscopia eletrônica de varredura e microscopia confocal. Os resultados revelaram que o revestimento DHP/HAp aumentou a viabilidade dos osteoblastos até 150% em comparação com o controle em todos os dias testados. O revestimento OPA/HAp promoveu a maior viabilidade ao 14 dias (190%). A atividade de ALP foi aumentada apenas pelo revestimento de DHP/HAp ao 14º dia em comparação com o controle e Ti limpo. A microscopia eletrônica de varredura e as microfotografias confocais revelaram diferenças morfológicas entre os osteoblastos cultivados em ambos os revestimentos, aumentando o seu número e o espalhamento. O revestimento de DHP/HAp aumentou a produção de nódulos biomineralizados. O ensaio de biomineralização pela técnica do Vermelho de Alizarina mostrou que o revestimento de OPA/HAp possuía uma concentração de cálcio (Ca2+) 1,88 vezes superior à cobertura de DHP/HAp. Uma vez que a literatura relata que o Ca2+ pode estimular ou inibir a atividade da ALP e, consequentemente, o processo de biomineralização, as diferenças no comportamento desses dois revestimentos podem estar relacionadas às diferenças de concentração de superfície de Ca2+. O bom desempenho do revestimento de DHP/HAp pode estar relacionado às características da composição química, adicionada à técnica de deposição LB. Na segunda fase da pesquisa, as monocamadas de Langmuir de DHP e dipalmitoil fosfatidilcolina (DPPC) foram testadas e utilizadas para incorporar o paratormônio 1-34 (PTH 1-34) (DHP/Ca+PTH e DPPC/Ca+PTH, respectivamente). Também foram testadas as ações dos revestimentos DHP/HAp com PTH em solução (DHP/HAp+PTH S) e gotejado (DHP/HAp+PTH G) em culturas de osteoblastos. Um potencial zeta negativo em pH 7,4 foi encontrado (-14,9 mV) para o PTH 1-34. A isoterma de DPPC mostrou um aumento da área mínima ocupada por molécula lipídica após a injeção de PTH na subfase de água (50 ?L de solução 0,5 mg/mL) em 10,97 Å2, o que pode ser devido à inserção de PTH neste filme. A área mínima de DHP foi alterada em 2,3 Å2, o que não é estatisticamente significativo. A análise de QCM mostrou um depósito de 72,5 ng de PTH em filme de DPPC e 29,3 ng de PTH em filme de DHP para cada 25 ?g de PTH injetado na cuba de Langmuir. A viabilidade celular e a formação da matriz mineralizada de culturas de osteoblastos crescidas em DHP/Ca+PTH e revestimentos DPPC/Ca+PTH diminuíram quando comparadas com Ti limpo. Os revestimentos DHP/HAp+PTH S e DHP/HAp+PTH G mostraram ser tão eficientes quanto o Ti DHP/HAp para estimular o processo de biomineralização. Mas a cobertura de DHP/HAp+PTH G aumentou a viabilidade dos osteoblastos e a formação de matriz mineralizada quando comparada com Ti DHP/HAp. Esta é uma cobertura inovadora que abre precedentes para o uso da técnica de gotejamento em HAp para outros hormônios e drogas que agem sobre o tecido ósseo. / Due to their ability to promote biomineralization, Hydroxyapatite (HAp) coatings are used to increase the osteointegration in titanium (Ti) implants, in order to correct skeletal and craniofacial defects. The objective of the research was to evaluate the influence of the coatings on osteoblasts primary cultures. In the first phase of the research we developed a new coating approach based on Langmuir-Blodgett (LB) films of dihexadecyl phosphate (DHP) and octadecylphosphonic acid (OPA) deposited on Ti discs and subsequent growth of HAp crystals. We analyzed the osteoblast viability, alkaline phosphatase (ALP) activity and mineralized matrix formation by colorimetric methods, and the morphology of the cultures by scanning electron microscopy and confocal micrographies. The results revealed that the DHP/HAp coating increased osteoblast viability up to 150% compared to the control at all days tested. The OPA/HAp coating promoted the highest viability on the 14th day (190%). The ALP activity was enhanced only by the DHP/HAp coating on the 14th day compared to control, and clean Ti. To explore the morphology of the cells, the scanning electron microscopy and confocal micrographies were obtained, and revealed morphological differences between osteoblasts grown on both coated Ti compared to clean Ti. Both coatings increased the number and spreading of osteoblasts, while the DHP/HAp coating enhanced the production of biomineralized nodules. The Alizarin Red assay showed that OPA/HAp coating has 1.88 times higher calcium (Ca2+) concentration than DHP/HAp. The same test confirmed the increase of mineralization only by DHP/HAp coating compared to clean Ti. Since literature reports that Ca2+ can stimulate or inhibit the ALP activity and consequently, the biomineralization process, the differences on the behavior of these two coatings could be related to the Ca2+ surface concentration differences. The good performance of the DHP/HAp coating can be explained due to the characteristics of the chemical composition, added to the LB deposition technique. In the second phase of the research, Langmuir monolayers of DHP and dipalmitoyl phosphatidylcholine (DPPC) was tested and used to incorporate 1-34 parathyroid hormone (PTH 1-34) (DHP/Ca+PTH, and DPPC/Ca+PTH, respectively). DHP/HAp coatings with PTH in solution (DHP/HAp+PTH S), and dropped (DHP/HAp+PTH G) also were tested on osteoblasts cultures. A negative zeta-potential at pH 7.4 was found (-14.9 mV) to PTH 1-34. The Langmuir isotherm of DPPC showed an increase of the minimum area occupied per lipid molecule after the PTH injection into the water subphase (50 ?L of 0.5 mg/mL solution) by 10.97 Å2, which could be due to the insertion of PTH in this film. The DHP minimum area changed by 2.3 Å2, which is not statistically significant. The QCM analysis showed the deposit of 72.5 ng of PTH on DPPC film, and 29.3 ng of PTH on DHP film for each 25 ?g of PTH injected into the Langmuir trough. The cell viability and matrix mineralization of osteoblasts cultures grown on DHP/Ca+PTH, and DPPC/Ca+PTH coatings decreased when compared to clean Ti. DHP/HAp+PTH S and DHP/HAp+PTH G coatings proved to be as efficient as Ti DHP/HAp to stimulate the biomineralization process. But DHP/HAp+PTH G increased the osteoblast viabilitiy and formation of mineralized matrix when compared to Ti DHP/HAp. This is an innovative coating that sets the precedent for the use of the drip technique on HAp for other hormones and drugs that act on bone tissue.

Os fosfolípides e moléculas relacionadas compreendem 60% da porção não aquosa do cérebro e são os principais constituintes das membranas de células neuronais e gliais. Os fosfolípides são essenciais para todas as células vivas e, portanto, mudanças no seu metabolismo podem influenciar o organismo. Alterações no metabolismo de fosfolípides estão envolvidas em inúmeras doenças neuropsiquiátricas incluindo a doença de Alzheimer, esquizofrenia e o transtorno bipolar. Neste trabalho, tivemos por objetivo compreender a composição lipídica de metabólitos relacionados à membrana de pacientes com diferentes doenças neuropsiquiátricas. Para isto, utilizamos Análise por Injeção em Fluxo (FIA) acoplado à espectrometria de massas, uma metodologia analítica robusta que proporciona um perfil completo das substâncias em matrizes complexas. Para interpretação dos resultados, usamos o método estatístico CART - Classification and Regression Tree. Encontramos 4 metabólitos que são capazes de distinguir pacientes com TB de pacientes com SCZ e outros 3 metabólitos que, juntos, são capazes de diferenciar indivíduos com CCL e DA. Esses resultados evidenciam o potencial dos fosfolípides de membrana como biomarcadores que podem auxiliar na confirmação diagnóstica e elucidação de mecanismos fisiopatológicos das doenças estudadas / Phospholipids and related molecules comprise 60% of the non-aqueous portion of the brain and are the major constituents of neuronal and glial cell membranes. Phospholipids are essential for all living cells and therefore changes in their metabolism can influence the organism. Changes in phospholipid metabolism are known to be involved in numerous neuropsychiatric disorders including Alzheimer\'s disease, schizophrenia and bipolar disorder. In this way, we aimed to understand the lipid composition of membrane-related metabolites of patients with different neuropsychiatric diseases. For this, we use Flow Injection Analysis (FIA) coupled with mass spectrometry, a robust analytical methodology that provides a complete profile of the substances in complex matrices. To interpret the results, we chose to perform the CART method - Classification and Regression Tree. We found 4 metabolites that are able to distinguish TB patients from patients with SCZ and 3 other metabolites that together are able to differentiate individuals with CCL and AD. These results show us the potential of membrane phospholipids as diagnostic biomarkers, which may aid in the diagnostic confirmation and elucidation of pathophysiological mechanisms of the diseases studied

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-20T14:00:33Z No. of bitstreams: 1 marciahelenafaverodesouzatostes.pdf: 1066519 bytes, checksum: 07f8ee85ef6f588253dbd1d696fe04b2 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-02T11:22:19Z (GMT) No. of bitstreams: 1 marciahelenafaverodesouzatostes.pdf: 1066519 bytes, checksum: 07f8ee85ef6f588253dbd1d696fe04b2 (MD5) / Made available in DSpace on 2016-07-02T11:22:19Z (GMT). No. of bitstreams: 1 marciahelenafaverodesouzatostes.pdf: 1066519 bytes, checksum: 07f8ee85ef6f588253dbd1d696fe04b2 (MD5) Previous issue date: 2012-03-02 / O autismo infantil é um transtorno do desenvolvimento, definido pelo déficit na interação social, alterações da comunicação e padrões limitados de comportamentos. Alterações imunológicas, no metabolismo de fosfolípides de membrana e no estresse oxidativo estão subjacentes. Este trabalho avaliou os níveis dos marcadores de ativação imunológica, de fosfolípides de membrana e de óxido nítrico em 24 crianças autistas, pareadas por idade e sexo com crianças saudáveis, e investigou a variação desses níveis em crianças autistas com diferentes perfis de desenvolvimento. Após a avaliação psiquiátrica, as amostras de sangue foram coletadas e submetidas à análise de IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α por citometria de fluxo, à análise de fosfolipase A2 por ensaio fluorimétrico, à análise de fosfolípides por cromatografia gasosa e à determinação do óxido nítrico pelo método de Griess. A escala de desenvolvimento do perfil psicoeducacional revisado (PEP-R) foi aplicada. Os dados foram analisados estatisticamente (ANOVA e teste post hoc de Bonferroni) com auxílio do SPSS versão 13. Os níveis plasmáticos de IFN-γ e óxido nítrico foram significativamente elevados entre as crianças autistas em comparação com o grupo controle. A atividade de fosfolipase A2 e os níveis plasmáticos de ácido araquidônico estavam elevados, enquanto os de ácidos eicosapentaenóico e docosahexaenóico estavam reduzidos entre as crianças autistas. Os escores obtidos no PEP-R diferiram significativamente nos dois grupos, mas não houve correlação com os resultados das dosagens laboratoriais. A análise conjunta de dados sugere uma associação do estresse oxidativo com desregulação imune e alteração do metabolismo lipídico no autismo infantil. / Autism in children is a developmental disorder defined by deficits in social interaction, impaired communication and limitations in the standard behavior. There are also immunological changes, in metabolism of membrane phospholipids and in oxidative stress. This study evaluated the levels of the immune activators markers from membrane phospholipids and nitric oxide in 24 autistic children, matched for age and sex with healthy children. This study also investigated the variation of these levels in children with autism with different development profiles. After psychiatric evaluation, blood samples were collected and subjected to analysis of IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α by flow cytometry, to analysis of phospholipase A2 by fluorimetric analysis, to analysis of phospholipids by gas chromatograph and to determination of nitric oxide by the Griess method. The scale of the development of psychoeducational profile revised (PEP-R) was applied. Data were statistically analyzed (ANOVA and post hoc Bonferroni test) using SPSS version13. Plasma levels of IFN-γ and nitric oxide were significantly higher among children with autism compared with the control group. The activity of phospholipase A2 and plasma levels of arachidonic acid were elevated, whereas eicosapentaenoic acid and docosahexaenoic acid were reduced among children with autism. The scores obtained in the PEP-R differed significantly between the two groups, but no correlation with the results of laboratory measurements was found. The joint analysis of data suggests an association of oxidative stress with immune dysregulation and alteration of lipid metabolism in infantile autism.

The biological membrane surrounding the living cell provides a sealed barrier that tightly regulates the interactions with the outside environment. A large number of integral membrane proteins mediate these interactions and are involved in a wide variety of biological processes. An increasing number of studies have led to the conclusion that lipids provide more than a hydrophobic solvent for membrane proteins, and that interactions between lipids and proteins are required to allow protein function. ABC transporters are one of the most important family of membrane proteins. However, the importance of their lipidic environment is largely unknown. Only a few studies showed that their activity was dependent on the lipidic composition of the surrounding bilayer. The bacterial ABC transporter HorA was used as a model to probe the influence of the lipidic environment on that class of membrane proteins.<p><p> HorA is a multidrug transporter expressed in Lactobacillus brevis, a Gram-positive beer spoilage bacterium. It turned out that phosphatidylethanolamine (PE) was indispensable to maintain both the activity and the structural integrity of HorA.<p> Surprisingly, replacement of PE by the chemically related PC (phosphatidylcholine) did not led to the suppression of HorA activity, but to an unexpected phenotype. Whereas the cytoplasmic domains of HorA were still able to hydrolyze ATP, the membrane parts of the transporter were unable to use that energy to mediate substrate transport. Using several biophysical methods particularly adapted to the study of reconstituted systems, we showed that the structure of HorA is strongly altered by this lipid replacement. In particular, the structural organization of the transmembrane domains of the protein is strongly affected.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Les lipides cationiques sont des molécules amphiphiles chargées positivement et couramment utilisées comme vecteur de transfection tant in vitro qu'in vivo avec une bonne efficacité. <p><p>Cependant, la transfection in vivo par voie intraveineuse à l'aide de complexes lipides cationiques/ADN (lipoplexes) induit une réponse inflammatoire, attribuée à la présence de séquences CpG dans les plasmides transportés, et qui limite l'efficacité de transfection des complexes.<p><p>Il a été montré dans notre laboratoire que la pré-injection de liposomes de diC14-amidine, un lipide cationique mis au point dans notre laboratoire, avant l'injection des lipoplexes diC14-amidine/ADN permettait d'augmenter l'efficacité de transfection tout en diminuant la production de TNF-¦Á dans le sérum [Elouahabi et al. 2003b]. Ce résultat suggérait une nouvelle propriété anti-inflammatoire de la diC14-amidine dans le cas d'une inflammation causée par les lipoplexes de diC14-amidine/ADN. <p><p>Dans le présent travail, nous d¨¦montrons que les macrophages de la rate sont les principales cellules productrices de TNF-¦Á suite à l'injection intraveineuse des lipoplexes à des souris. Ces mêmes cellules sont également la principale cible des liposomes de diC14-amidine après injection intraveineuse. Nous avons dès lors centré notre étude sur ce type cellulaire, en utilisant une lignée établie de macrophages murins. <p><p>Nos résultats ont permis de confirmer la capacité des liposomes de diC14-amidine à inhiber la sécrétion des cytokines pro-inflammatoires induites par des séquences CpG, des lipopolysaccharides ou des poly(I :C). Fait intéressant, la présence de sérum est indispensable à la propriété anti-inflammatoire des liposomes de diC14-amidine. Par fractionnement successifs des composants du sérum, nous avons pu montrer que seuls les phospholipides des lipoprotéines pouvaient conférer cette propriété aux liposomes de diC14-amidine. <p> / Doctorat en sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Phospholipids are an important group of polar lipids constituting the main component of cell membranes. Their proportion may vary depending on many factors of the surrounding environment in which the cell is located. Determination of membrane phospholipids is essential in many scientific, industrial and economic sectors. The aim of this work has been to develop a separation method for determination of membrane phospholipids by which it was possible to analyze phosphorylated parts of phospholipids and fatty acids from one sample. Comprehensive gas chromatography with mass detection (GC×GC-MS) was chosen for the assay. This method allows the separation of the entire sample on two serially connected different columns, among which is the interface called modulator. The preparation of the sample includes a cleavage of the phospholipid molecule by the enzyme phospholipase C, which released the phosphorylated polar headgroups. These polar parts had to be derivatized before analysis. The principle of the chosen derivatization consisted in the use of two different silylation agents (hexamethyldisilazane and N,O-Bis(trimethylsilyl) trifluoracetamide) in two steps. Conditions were selected for efficient separation of the silylderivatives of phosphorylated headgroups using GC×GC-MS using a cryogenic...

Par quel mécanisme les protéines de signalisation sont ciblées à un compartiment membranaire spécifique est une question fondamentale en biologie cellulaire. Les phosphatidylinositol phosphates (PIPs) sont des lipides anioniques qui jouent un rôle majeur dans ce phénomène. Ces lipides sont en très faible quantité dans la bicouche membranaire mais sont des sites d'ancrage spécifiques aux différents compartiments membranaires, et contribuent ainsi à leur identité. Il a été proposé que cette organisation s'apparente à un "code", qui peut être lu par des protéines qui intéragissent spécifiquement avec ces PIPs (Kutateladze 2010). Nous avons établi une carte de la localisation des différents PIPs dans les cellules d'épiderme racinaire chez Arabidopsis, et nous avons trouvé que les PIPs ne s'accumulent pas à un compartiment donné mais plutôt que chaque PIP est distribué dans plusieurs compartiments distincts, et à différentes concentrations (Simon, Platre et al., 2014). Ces données suggèrent que le "code PIP" n'est pas suffisant pour expliquer la sélection des protéines liant les lipides parmi les différents compartiments membranaires de la cellule chez la plante. Les PIPs sont des lipides négativement chargés et nous avons proposé que cette propriété physique pourrait contribuer à l'identité des membranes en régulant leurs charges de surface. Pour vérifier cette hypothèse, nous avons développé une collection de biosenseurs génétiquement codés pour analyser les charges de surface des membranes au niveau subcellulaire, in vivo. Nous avons trouvé que la membrane plasmique a une signature électrostatique spécifique contrôlée par le PI(4)P. De plus, ce champ électrostatique dépendent du PI(4)P contrôle la localisation et la fonction de plusieurs protéines agissant en aval de récepteurs kinase ou impliquées dans certaines voies de signalisation hormonale tel que l’auxine ou les brassinosteroïdes. Ainsi, les PI(4)P sont de véritables marqueurs de la membrane plasmique qui sont essentiels pour l’homéostasie des membranes et le développement des plantes. / How signaling proteins are targeted to specific membrane compartments is a fundamental question in cell and developmental biology. Phosphoinositides, a class of anionic lipids, take center stage in this process. They are minor lipids of the membrane bilayer that provide specific docking sites on membrane compartments and contribute to their identity. As such it has been proposed that there is a “phosphoinositide code” that allows the targeting of lipid binding proteins to specific membrane compartments (Kutateladze 2010). We recently mapped the localization of the different phosphoinositides in Arabidopsis epidermal cell, and found that they do not accumulate to a specific compartment but rather that each phosphoinositide is distributed to several compartments, albeit at different concentration (Simon et al., 2014). These data suggest that, unlike in other eukaryotic cells, the “phosphoinositide code” hypothesis is not sufficient to explain membrane selectivity of lipid binding proteins in plants. Phosphoinositides are negatively charged lipids and we hypothesized that this physical property might also contribute to membrane identity by regulating surface charges. To investigate membrane electrostatic properties at the subcellular level, we designed a set of genetically encoded biosensors able to report membrane surface charges. We found that the plasma membrane (PM) has a specific electrostatic signature that is controlled by the phosphoinositide PI4P. We further show that this PI4P-dependent electrostatic field controls the PM localization and function of several proteins involved in receptor kinase and phytohormone signaling as the auxin or brassinosteroids. Thus, PI(4)P are plasma membrane markers which are essential for the membrane homeostasis and plants development.