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

In Bakterien werden Phospholipide auf der cytoplasmatischen Seite der Plasmamembran synthetisiert. Damit ein gleichmäßiges Wachstum und somit die Stabilität biogener Membranen, d.h. Membranen, an bzw. in denen Lipidsynthese stattfindet, gewährleistet ist, muss zumindestens die Hälfte neu synthetisierter Lipide auf die entgegengesetzte Membranhälfte gelangen. Aus früheren Untersuchungen ist bereits bekannt, dass dieser transversale Phospholipidaustausch, auch als Flip-Flop bezeichnet, sehr schnell, kopfgruppenunabhängig und möglicherweise proteinabhängig ist. Dennoch sind die genauen Mechanismen dieser Prozesse noch weitgehend unverstanden. Um die oben erwähnten grundlegenden Phospholipidtransportprozesse zwischen beiden Membranhälften genauer untersuchen zu können, wandten wir einen neuartigen, sogenannten stopped-flow BSA back-extraction Assay an. Mit Hilfe dieses Assays, waren wir in der Lage, die transversale Bewegung und die Verteilung von kurzkettigen, fluoreszenzmarkierten Phospholipidanaloga über beide Membranhälften in ex vivo-Membranen zu charakterisieren. Der stopped-flow BSA back-extraction Assay basiert auf der Technik der stopped-flow-Spektroskopie und der Tatsache, dass BSA in der Lage ist, kurzkettige, fluoreszenzmarkierte Lipidanaloga aus der äußeren Leaflet von (biologischen) Membranen zu extrahieren. Wir entschieden uns für invertierte Membranvesikel der Plasmamembran (IIMV) vom E.coli Wildtypstamm MG1655 als Untersuchungsobjekt, einerseits, weil diese Vesikel nur eine Membran besitzen und zum Anderen, weil IIMV sich sehr gut als Modell für den Flip-Flop von Phospholipiden nutzen lassen. Wir beobachteten, dass kurzkettige, fluoreszenzmarkierte Analoga der beiden am häufigsten in E.coli vorkommenden Phospholipide, Phosphatidylethanolamin (PE) und Phosphatidylglycerol (PG), sehr schnell, d.h. mit Halbwertzeiten von weniger als drei Minuten, über die Membran von IIMV verteilten. Weiterhin verhielten sich kurzkettige, fluoreszenzmarkierte Analoga von den E.coli-fremden Phospholipiden, Phosphatidylcholin (PC) und Phosphatidylserin (PS), ähnlich wie die Analoga von PE und PG. Überraschenderweise, fanden wir heraus, dass alle oben genannten Phospholipidanaloga im Gleichgewichtszustand nicht gleichmässig über beide Membranhälften verteilt waren. Inwiefern Proteine an dieser transversalen Bewegung der Phospholipidanaloga beteiligt sind, sollten Messungen des Flip-Flop von Analoga an unbehandelten und mit Proteinase K inkubierten Vesikeln zeigen, die aus einem Detergenzextrakt von IIMV rekonstituiert wurden. Zunächst konnten wir zeigen, dass die schnelle Bewegung der Phospholipidanaloga über die Membran von rekonstituierten, nicht mit Proteinase K behandelten Vesikeln (Proteoliposomen) erhalten blieb. Nach Inkubation mit Proteinase K wurde jedoch der Flip-Flop von PE- und PG-Analoga vollständig inhibiert. Untersuchungen an rekonstituierten Serien von Proteoliposomen mit ansteigendem bakteriellen Proteingehalt zeigten, dass in Proteoliposomen ohne bakterielle Proteine kein Flip-Flop stattfand und somit nur 50% der fluoreszenten Analoga extrahiert wurden. In Proteoliposomen, die bakterielle Proteine enthielten, stieg das Ausmass der Extrahierbarkeit der untersuchten Analoga mit steigendem Proteingehalt. Diese Daten zeigten sehr deutlich, dass die transversale Bewegung von Phospholipiden über die innere Membran von E.coli durch Proteine vermittelt wird. Schlussfolgernd aus unseren Analysen konnten wir zeigen, dass die transversale Bewegung von Phospholipidanaloga über die Membran von IIMV sehr schnell, proteinabhängig, bidirektional und kopfgruppenunbhängig ist. Zur Identifizierung der molekularen Grundlagen der proteinvermittelten, schnellen Transversalbewegung von Phospholipiden über IIMV-Membranen, nutzen wir Ionenaustauschchromatografie. Zur unserer Überraschung mussten wir feststellen, dass in keiner der rekonstituierten Fraktionen eine nennenswerte Anreicherung der Flippaseaktivität auftrat. Möglicherweise sind mehrere Proteine, mit unterschiedlichen Nettoladungen, oder aber auch Untereinheiten, die sich nicht durch Anionenaustauscher trennen liessen, am Flip-Flop von Phospholipiden beteiligt. Weitergehende Analysen mit anderen Proteinfraktionierungsmethoden sind notwendig, um den oder die Flippasekomplex(e) zu identifizieren. / In the plasma membrane of bacteria, phospholipids are synthesized on the cytoplasmic leaflet of the plasma membrane. To ensure balanced growth and thus, stability of biogenic membranes, half of the newly synthesized lipids must move to the opposing leaflet. It is known that this phospholipid transmembrane movement (flip-flop) is rapid, head-group independent and possibly protein mediated. However, the exact mechanism of this process remains elusive. To investigate these fundamental transbilayer phospholipid transport processes in biogenic membranes, a novel stopped-flow BSA back-exchange assay was utilized to characterize the transmembrane movement and transbilayer distribution of fluorescent labeled, short-chain phospholipid analogues in ex vivo membranes. This approach is based on stopped-flow fluorescence spectroscopy, and the fact that BSA is able to extract fluorescent labeled, short-chain phospholipid analogues from the outer leaflet of (bio)membranes. We chose isolated inverted inner membrane vesicles (IIMV) derived from E.coli wild type MG1655, both for their simple membrane organization and for their suitability as a simple model organism for phospholipid flip-flop. We observed that fluorescent-labeled, short-chain analogues of the major phospholipids in E.coli, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), rapidly redistributed across the IIMV bilayer with half-times of less than three minutes. Furthermore, fluorescent, short-chain phospholipid analogues of phosphatidylcholine (PC) and phosphatidylserine (PS), which are not naturally occurring phospholipids in E.coli membranes, behaved similar to the PE and PG analogues. To analyze the relevance of proteins for the transmembrane movement of fluorescent analogues, we measured flip-flop of phospholipid analogues in reconstituted and/or untreated and proteinase K treated vesicles generated from protein detergent extracts of IIMV. The amount of extractable fluorescent phospholipids analogues correlated with the amount of protein reconstituted into the proteoliposomes, strongly indicating, that protein concentrations below 100 µg/ml were not sufficient to equip every vesicle with proteins that facilitate the transmembrane movement of the fluorescent analogues. We found that the rapid transbilayer movement of phospholipid analogues across the membrane was maintained in untreated reconstituted vesicles. However, the flip-flop of fluorescent PG and PE analogues was eliminated in proteinase K treated vesicles. In conclusion, our analysis showed that the transmembrane movement of the phospholipid analogues across the membrane of IIMV was protein-mediated, very rapid, bi-directional and head-group independent. To identify the molecular basis of the protein-mediated, rapid transmembrane movement of phospholipids across IIMV membranes, we used ion exchange chromatography (IEC) to separate the IIMV proteins. To our surprise, we did not observe an enhanced flip-flop activity in any of the fractions, indicating that at least two proteins with possibly opposite net charges or several subunits, which were not separable by AEC, are involved. Further analysis using different protein separation techniques will be necessary to identify the putative flippase complex.

Orientadores: Mirna Lucia Gigante, Rafael Jimenez-Flores / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-09-11T21:12:34Z (GMT). No. of bitstreams: 1 Costa_MarceladeRezende_D.pdf: 1269005 bytes, checksum: be9b972dd7766eba5cc6917dd666c5f0 (MD5) Previous issue date: 2008 / Resumo: O leitelho de soro é um subproduto do processamento de manteiga a partir do creme de soro. Esse subproduto lácteo contém fragmentos da membrana do glóbulo de gordura do leite (MGGL), material rico em componentes com funções nutricionais e efeitos benéficos à saúde, destacando-se os fosfolipídeos. O objetivo desse trabalho foi obter um ingrediente lácteo enriquecido em fosfolipídeos da MGGL a partir do leitelho de soro utilizando a associação de duas tecnologias de fracionamento: ultrafiltração e extração com fluido supercritico (EFS). O leitelho de soro foi submetido à ultrafiltração e a cinco diafiltrações a 25 °C e o retentado obtido foi seco em spray-dryer, obtendo-se o leitelho de soro em pó (LSP), o qual foi submetido a três ciclos de extração a 50 °C com dióxido de carb ono supercrítico. As matériasprimas, produtos finais e uma amostra comercial de leitelho tradicional em pó (LTP) foram analisados quanto a sua composição centesimal, teor de fosfolipídeos, perfis lipídico e protéico. Os leitelhos de soro em pó, submetidos ou não à EFS, e o LTP foram avaliados quanto ao tamanho de partículas e suas propriedades funcionais (solubilidade protéica e capacidade emulsificante) em pH 5 e 7. A filtração em membrana reduziu em 74 e 96% os teores de lactose e cinzas no retentado e aumentou os de proteínas, lipídeos e fosfolipídeos em 91, 190 e 300%, respectivamente. A EFS reduziu em 55,4% o teor de lipídeos, removendo exclusivamente lipídeos apolares, com isso aumentou em 71% o teor de fosfolipídeos no leitelho de soro em pó, resultando em um produto com 73% de proteínas, 21% de lipídeos, 3% de lactose, 3% de cinzas e 12% de fosfolipídeos, em base seca. As propriedades funcionais dos leitelhos de soro em pó, submetidos à extração supercrítica (LSP-EFS) ou não (LSP), foram pouco ou não afetadas pelo pH, enquanto o abaixamento de pH prejudicou as características do LTP, devido ao alto teor de caseínas nesse tipo de pó em relação aos de leitelhos de soro. Os pós em soluções (5% de proteína) apresentaram de 69 a 84% de suas partículas, em % volumétrica, entre 10 e 100 µm. A solução de LTP em pH 5 foi a que apresentou a maior quantidade de partículas acima de 100 µm. LSP e LSP-EFS apresentaram solubilidades protéicas em torno de 86 e 84%, respectivamente, independente do pH. O LTP teve a solubilidade reduzida de 86 para 73% quando o pH foi reduzido de 7 para 5. Em pH 7, as emulsões (20% óleo de canola, 1% proteína) de LSP e de LSPEFS apresentaram melhor estabilidade (IC de 0,2 e 0,4%, respectivamente) do a emulsão de LTP (IC de 3,0%). Em pH 5, a emulsão de LSP-EFS foi a que mostrou melhor estabilidade, apresentando um IC de 7%, valor cerca de 82% menor do que os das emulsões de LSP e LTP. Os leitelhos de soro em pó obtidos podem ser considerados ingredientes com características promissoras, associando propriedades tecnológicas, especialmente para uso em alimentos de baixo pH, e conteúdo de compostos potencialmente benéficos à saúde, principalmente após o tratamento com extração supercrítica / Abstract: OBTENTION OF DAIRY INGREDIENT ENRICHED IN POLAR LIPIDS FROM WHEY BUTTERMILK. Whey buttermilk is a by-product from the whey cream processing into butter. This dairy by-product contains milk fat globule membrane (MFGM) fragments, a material rich in components with nutritional functions and beneficial health effects, especially phospholipids. The objective of this research was obtaining a dairy ingredient enriched in MFGM phospholipids from whey buttermilk using the association of two fractionation technologies: ultrafiltration and supercritical fluid extraction (SFE). Whey buttermilk was submitted to ultrafiltration and five diafiltrations at 25 °C. The retentate was spray-dried and later s ubmitted to three cycles of extraction at 50 °C with supercritical carbon dioxi de. Raw materials, final products and a commercial traditional buttermilk powder sample (BMP) were analyzed for gross composition, lipid and protein profiles, and phospholipids content. Whey buttermilk powders, before and after the SFE, and the BMP had particle size and some functional properties (protein solubility and emulsifying capacity) evaluated in pH 5 and 7. Membrane filtration reduced in 74 and 96% lactose and ash contents in the retentate and increased proteins, lipids and phospholipids in 91, 190 and 300%, respectively. SFE reduced in 55.4% the lipids content, removing exclusively non-polar lipids, while increased in 71% the phospholipids content in whey buttermilk powder, resulting in a product with 73% of proteins, 21% of lipids, 3% of lactose, 3% of ash and 12% of phospholipids, in dry matter basis. Functional properties of the whey buttermilk powders, treated (WBP-SFE) or not through SFE (WBP), were little or not affected by pH, while dropping the pH impaired the BMP features, due the high casein content in this type of powder in relation to the ones from whey buttermilk. The powders in solutions (5% protein) presented from 69 to 84% of the particles, in volume%, between 10 and 100 µm. The BMP solution in pH 5 was the one with the biggest amount of particles above 100 µm. WBP and WBP-SFE showed protein solubilities around 86 and 84%, respectively, independent of pH. BMP had the solubility reduced from 86 to 73% when pH was reduced from 7 to 5. In pH 7, the emulsions (20% canola oil, 1% protein) of the WBP and the WBP showed better stability (CI of 0.2 and 0.4%, respectively) than the BMP emulsion (CI of 3.0%). When in pH 5, WBP-SFE emulsion had the best stability, presenting CI of 7%, value about 82% smaller than the ones of WBP and BMP emulsions. Whey buttermilk powders obtained in this work can be considered ingredients with promising features, combining technological properties, especially for use in low pH foods, and content of components with potential health benefits, mainly after the supercritical extraction treatment / Doutorado / Doutor em Tecnologia de Alimentos

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.

L'emploi des agents antibactériens est un moyen important d'une part dans la lutte contre les infections bactériennes et d'autre part pour conserver les produits alimentaires jusqu'à leur consommation. La perte d'efficacité des antibiotiques par le développement de résistance bactérienne ainsi que la toxicité des conservateurs synthétiques rend nécessaire le développement de nouveaux produits antibactériens naturels. Les protéines et les peptides antibactériens agissant sur les membranes bactériennes paraissent une alternative pour limiter l'instauration de résistances bactériennes. L'ovotransferrine est une protéine du blanc d'œuf ayant des propriétés membranotropes responsable entre autre de son activité antibactérienne. L'objectif de cette thèse est d'étudier les mécanismes membranotropes de l'ovotransferrine vis-à-vis des membranes externe et cytoplasmique d'E. coli en utilisant respectivement des monocouches de LPS (lipopolysaccharides) et de phospholipides comme modèles membranaires expérimentales. L'ovotransferrine possède une capacité d'insertion dans la monocouche de LPS qui dépend de la concentration protéique, de la compacité de la monocouche et de la conformation des molécules de LPS. L'ovotransferrine s'adsorbe faiblement à la monocouche de phospholipides. Ainsi, les monocouches sont perturbées par la désorganisation des lipides. L'analyse comparative de l'ovotransferrine chauffée à sec avec la forme native a montré la conservation des structures secondaire et tertiaire avec une augmentation de l'hydrophobie de surface et probablement de la flexibilité et une affinité plus élevée aux interfaces hydrophiles/hydrophobes (eau/air). L'activité membranaire de l'ovotransferrine est accrue après son chauffage à sec. La capacité d'insertion dans la monocouche de LPS est amplifiée avec une affinité plus importante. Une capacité d'insertion dans la monocouche de phospholipides est générée pour la forme chauffée à sec associée à une adsorption plus élevée. L'ovotransferrine chauffée à sec induit des perturbations plus importantes des monocouches à des concentrations protéiques plus faibles. / The use of antibacterial agents is very important, firstly, on the fight against bacterial infections, and secondly, to keep food products until its consumption. The loss of antibiotics effectiveness through the development of bacterial resistance and the toxicity of synthetic preservatives necessitates the development of new natural antibacterial products. Antibacterial proteins and peptides acting on the bacterial membranes appear as an alternative to limit the introduction of bacterial resistances. Ovotransferrin is an egg-white protein with membranotropic properties responsible among other things for its antibacterial activity. The aim of this thesis is to study the membranotropic mechanisms of ovotransferrin towards the outer and cytoplasmic membranes of E. coli using respectively monolayers of LPS (lipopolysaccharides) and phospholipids as experimental membrane models. Ovotransferrin has an insertion capacity in LPS monolayer that is dependent on protein concentration, monolayer compactness, and LPS molecule conformation. Ovotransferrin weakly adsorbs to the monolayer of phospholipids. Thus, the monolayers are disturbed by the disorganization of the lipids. Comparative analysis of dry-heated ovotransferrin with the native form showed conservation of secondary and tertiary structures with an increase of surface hydrophobicity and probably of flexibility and higher affinity to hydrophilic/hydrophobic interfaces (water/air). The insertion capacity in the LPS monolayer is amplified with greater affinity. Insertion capacity in the phospholipid monolayer is generated for the dry heated form associated with higher adsorption. Dry-heated ovotransferrin induces greater disruption of monolayers at lower protein concentrations.

Les leishmanioses sont des parasitoses en constante évolution, et l’accroissement d’apparitions de résistances vis-à-vis des traitements disponibles en fait une des préoccupations majeures des organismes de santé publique dans le monde. La miltefosine est actuellement le seul antileishmanien actif par voie orale. Son mécanisme d’action implique les lipides et notamment les phospholipides membranaires du parasite.Afin d’évaluer les effets de la miltefosine sur le parasite ainsi que les mécanismes de résistances, une étude lipidomique d’un clone de Leishmania donovani cultivé sous différentes conditions (traité, résistant, résistant-traité) a été réalisée dans le présent travail. Des analyses couplant une séparation des phospholipides en Chromatographie Liquide Haute-Performance à polarité de Phase Normale (NP-HPLC) avec un Spectromètre de Masse (MS) équippé d’une source d’Ionisation ElectroSpray (ESI) ont été traitées par chimiométrie, à l’aide d’une Correction Orthogonale du Signal suivie d’une Analyse Discriminante par Moindre Carrés Partiels (OSC-PLS-DA). Les principales espèces moléculaires permettant de distinguer les différentes cultures ont ensuite fait l’objet d’une identification structurale par spectrométrie de masse en tandem. Des hypothèses métaboliques ont pu être posées.Puis l’étude a été étendue à une plus grande variété de lipides, séparés par NP-HPLC. Pour cela une comparaison des sources d’ionisation à pression atmosphérique (ESI, Ionisation Chimique à Pression Atmosphérique et PhotoIonisation à Pression Atmosphérique) a été nécessaire afin de sélectionner la mieux adaptée pour un tel couplage. Les mécanismes d’action de la miltefosine et de l’amphotéricine B ont alors fait l’objet d’une étude lipidomique. / Leishmaniasis is a more and more spreading disease, and resistance of parasites toward antileishmanial drugs is a concern for public safety organizations troughout the world. Miltefosine is the only oral drug, and its mechanism of action implies membrane lipids, and phospholipids, of parasite cells.In order to assess this mechanism of action, and resistance mechanisms developed, a lipidomic study of Leishmania donovani strains (treated, resistant, treated-resistant) was performed in the present work. A Normal-Phase High-Performance Liquid Chromatography (NP-HPLC) was coupled to an ElectroSpray Ionisation Mass Spectrometer (ESI-MS) to analyze phospholipids, and data were computed using an Orthogonal Signal Correction-Partial Least Squares-Discriminant Analysis (OSC-PLS-DA). Molecular species responsible for the differenciation of strains were then structuraly identified using tandem mass spectrometry. Hypotheses on metabolic pathways implied were then proposed.The study was then extended to a broader range of lipids, also analyzed through NP-HPLC-MS. A comparison of Atmospheric Pressure Ion sources (ESI, Atmospheric Pressure Chemical Ionization and Atmospheric Pressure PhotoIonization) was thus necessary in order to select the most suitable source. A lipidomic study was then performed to assess mechanisms of action and mechanisms of resistance concerning miltefosine and Amphotericin B.

De acordo com a Organização Mundial da Saúde, AIDS,malária e tuberculose são as três maiores doenças infectantes do mundo, atingindo principalmente crianças. Regiões paupérrimas e de clima tropical, como a África sub-saariana, são as mais atingidas. Este quadro agrava-se com a disseminação de cepas do Plasmodium falciparum resistentes à cloroquina e multi-resistentes.Além disso, alguns fármacos utilizados na terapêutica da malária apresentam vários efeitos adversos, comprometendo o tratamento. Trata-se de um grande desafio e o seu enfrentamento requer estratégias. O desenvolvimento de novos quimioterápicos deve fundamentar-se em diferenças bioquímicas e morfológicas entre as células do hospedeiro e do parasita. A biossíntese de fosfolipídeos de membrana em parasitas do grupo Apicomplexa é de extrema importância para a maturação e a reprodução do parasita e constitui-se em bom alvo para novos antimaláricos, uma vez que é encontrada somente em parasitas. Hemácias infectadas têm sua absorção modificada em relação aos eritrócitos não-infectados, conferindo seletividade a substâncias como lipídeos. O trabalho em questão propõe a síntese de antimetabólitos da serina, visando à inibição das enzimas fosfatidilserina síntase e serina descarboxilase, fundamentais para a biossíntese de fosfolipídeos de membrana desses parasitas.. Cinco derivados heterocíclicos da serine foram sintetizados: derivados diidroimidazólico, diidroxazólico, diidroxazínico, diidropirimidínico e diidrooxatiólico. Também, o transportador fosfolipídico com o ácido esteárico foi sintetizado. Os antimetabólitos serão acoplados a esse e outros fosfolipídeos, obtendo-se fármacos dirigidos específicos direcionados seletivamente a eritrócitos infectados. / According to the World Health Organization, Aids, malaria and tuberculosis are the three greatest infectious diseases in the world. Children are the most involved in those diseases. Extremely poor regions, as sub-Saharan, Africa, are the most affected. In the worst case scenario, one of the parasites that causes malaria, Plasmodium falciparum, become resistant to chloroquine and the current therapy. Besides, some drugs used in the mataria chemotherapy are very toxic, showing many side effects, and compromising the treatment. This is a big challenge and facing it requires new strategies.. The development of chemotherapeutic has been inspired in biochemical differences between the parasite and the host. Plasmodíum falciparum needs to biosynthesize phospholipids for their membrane. These phospholipids are very important to the maturation and reproduction of the parasite and occur only in it.. This makes the phospholipids biosynthesis a good target for new and specific antimalarial drug design. Infected red blood cell shows modified permeation, allowing the lipids to be freely transported, what is not usual in the non-infected red blood cells. This said, in the present work the design and synthesis of serine metabolic inhibitors, using the bioisosteric strategy, have been proposed. The inhibition of the phosphatidylserine biosynthesis, an important phospholipid, is expected. These inhibitors will be linked to phospholipids, to promote the selective permeation to the infected red-blood cell.. These inhibitors will be linked to phospholipids, to promote the selective permeation to the infected redblood cell. In the present work five heterocyclic serine inhibitors: diidroimidazolic, diidroxazolic, diidroxazinico, diidropyriminic and diidroxatiolic. Also synthesized a phospholipid to be connected to the heterocyclic inhibitors.

De acordo com a Organização Mundial da Saúde, AIDS,malária e tuberculose são as três maiores doenças infectantes do mundo, atingindo principalmente crianças. Regiões paupérrimas e de clima tropical, como a África sub-saariana, são as mais atingidas. Este quadro agrava-se com a disseminação de cepas do Plasmodium falciparum resistentes à cloroquina e multi-resistentes.Além disso, alguns fármacos utilizados na terapêutica da malária apresentam vários efeitos adversos, comprometendo o tratamento. Trata-se de um grande desafio e o seu enfrentamento requer estratégias. O desenvolvimento de novos quimioterápicos deve fundamentar-se em diferenças bioquímicas e morfológicas entre as células do hospedeiro e do parasita. A biossíntese de fosfolipídeos de membrana em parasitas do grupo Apicomplexa é de extrema importância para a maturação e a reprodução do parasita e constitui-se em bom alvo para novos antimaláricos, uma vez que é encontrada somente em parasitas. Hemácias infectadas têm sua absorção modificada em relação aos eritrócitos não-infectados, conferindo seletividade a substâncias como lipídeos. O trabalho em questão propõe a síntese de antimetabólitos da serina, visando à inibição das enzimas fosfatidilserina síntase e serina descarboxilase, fundamentais para a biossíntese de fosfolipídeos de membrana desses parasitas.. Cinco derivados heterocíclicos da serine foram sintetizados: derivados diidroimidazólico, diidroxazólico, diidroxazínico, diidropirimidínico e diidrooxatiólico. Também, o transportador fosfolipídico com o ácido esteárico foi sintetizado. Os antimetabólitos serão acoplados a esse e outros fosfolipídeos, obtendo-se fármacos dirigidos específicos direcionados seletivamente a eritrócitos infectados. / According to the World Health Organization, Aids, malaria and tuberculosis are the three greatest infectious diseases in the world. Children are the most involved in those diseases. Extremely poor regions, as sub-Saharan, Africa, are the most affected. In the worst case scenario, one of the parasites that causes malaria, Plasmodium falciparum, become resistant to chloroquine and the current therapy. Besides, some drugs used in the mataria chemotherapy are very toxic, showing many side effects, and compromising the treatment. This is a big challenge and facing it requires new strategies.. The development of chemotherapeutic has been inspired in biochemical differences between the parasite and the host. Plasmodíum falciparum needs to biosynthesize phospholipids for their membrane. These phospholipids are very important to the maturation and reproduction of the parasite and occur only in it.. This makes the phospholipids biosynthesis a good target for new and specific antimalarial drug design. Infected red blood cell shows modified permeation, allowing the lipids to be freely transported, what is not usual in the non-infected red blood cells. This said, in the present work the design and synthesis of serine metabolic inhibitors, using the bioisosteric strategy, have been proposed. The inhibition of the phosphatidylserine biosynthesis, an important phospholipid, is expected. These inhibitors will be linked to phospholipids, to promote the selective permeation to the infected red-blood cell.. These inhibitors will be linked to phospholipids, to promote the selective permeation to the infected redblood cell. In the present work five heterocyclic serine inhibitors: diidroimidazolic, diidroxazolic, diidroxazinico, diidropyriminic and diidroxatiolic. Also synthesized a phospholipid to be connected to the heterocyclic inhibitors.

<p>Insulin sensitivity is related to the fatty acid profile of skeletal muscle. The aim of this thesis was to investigate whether physical activity and dietary fat quality, independent of each other, influence the fatty acid composition of the skeletal muscle lipids. In an intervention study where middle-aged men were exercising for six weeks, and in a cross-sectional study comparing sedentary with endurance trained young men, it was demonstrated that the fatty acid composition of skeletal muscle lipids differed between physical active and inactive men. In brief, a lower proportion of palmitic acid (16:0) and total n-6 polyunsaturated fatty acids (PUFA) and a higher proportion of stearic (18:0) and oleic acid (18:1n-9) and total n-3 PUFA in the muscle phospholipids were associated with physical activity, despite similar fatty acid composition of the diet. In the second study, that included a larger training volume, differences in the fatty acid profile were also found in the skeletal muscle triglycerides. </p><p>In contrast, after short-term supra-maximal exercise we found no significant changes in the proportion of the fatty acids in skeletal muscle. </p><p>Furthermore, after a treatment period of three months, with diets with various dietary fat quality, the proportions of saturated fatty acids (14:0, 15:0 and 17:0) were higher and the proportion of 18:1 n-9 lower in subjects with a high intake of saturated fatty acids compared with subjects with a high intake of monounsaturated fatty acids. In addition subjects given n-3 supplementation had a higher proportion of total n-3 PUFA and lower n-6 PUFA in the skeletal muscle phospholipids than controls. Differences similar to those observed in the phospholipids were found in the triglycerides. </p><p>In summary, these results suggest that regular aerobic physical activity and dietary fat quality influence the fatty acid composition of the skeletal muscle lipids, which may affect insulin sensitivity and glucose homeostasis. </p>

Weltweit versuchen Wissenschaftler, künstliche Viren für den Gentransfer zu konstruieren, die nicht reproduktionsfähig sind. Diese sollen die Vorteile der natürlichen Viren besitzen (effizienter Transport von genetischem Material), jedoch keine Antigene auf ihrer Oberfläche tragen, die Immunreaktionen auslösen. <br><br> Ziel dieses Projektes ist es, einen künstlichen Viruspartikel herzustellen, dessen Basis eine Polyelektrolytenhohlkugel bildet, die mit einer Lipiddoppelschicht bedeckt ist. Um intakte Doppelschichten zu erzeugen, muss die Wechselwirkung zwischen Lipid und Polyelektrolyt (z.B. DNA) verstanden und optimiert werden. Dazu ist es notwendig, die strukturelle Grundlage der Interaktion aufzuklären. Positiv geladene Lipide gehen zwar starke Wechselwirkungen mit der negativ geladenen DNA ein, sie wirken jedoch toxisch auf biologische Zellen. In der vorliegenden Arbeit wurde daher die durch zweiwertige Kationen vermittelte Kopplung von genomischer oder Plasmid-DNA an zwitterionische oder negativ geladene Phospholipide an zwei Modellsystemen untersucht. <br><br> 1. Modellsystem: Lipidmonoschicht an der Wasser/Luft-Grenzfläche <br> Methoden:<br> Filmwaagentechnik in Kombination mit IR-Spektroskopie (IRRAS), Röntgenreflexion (XR), Röntgendiffraktion (GIXD), Brewsterwinkel-Mikroskopie (BAM), Röntgenfluoreszenz (XRF) und Oberflächenpotentialmessungen <br> Resultate:<br> A) Die Anwesenheit der zweiwertigen Kationen Ba2+, Mg2+, Ca2+ oder Mn2+ in der Subphase hat keinen nachweisbaren Einfluss auf die Struktur der zwitterionischen DMPE- (1,2-Dimyristoyl-phosphatidyl-ethanolamin) Monoschicht. <br> B) In der Subphase gelöste DNA adsorbiert nur in Gegenwart dieser Kationen an der DMPE-Monoschicht. <br> C) Sowohl die Adsorption genomischer Kalbsthymus-DNA als auch der Plasmid-DNA pGL3 bewirkt eine Reduktion des Neigungswinkels der Alkylketten, die auf einen veränderten Platzbedarf der Kopfgruppe zurückzuführen ist. Durch die Umorientierung der Kopfgruppe wird die elektrostatische Wechselwirkung zwischen den positiv geladenen Stickstoffatomen der Lipidkopfgruppen und den negativ geladenen DNA-Phosphaten erhöht.<br> D) Die adsorbierte DNA weist eine geordnete Struktur auf, wenn sie durch Barium-, Magnesium-, Calcium- oder Manganionen komplexiert ist. Der Abstand zwischen parallelen DNA-Strängen hängt dabei von der Größe der DNA-Fragmente sowie von der Art des Kations ab. Die größten Abstände ergeben sich mit Bariumionen, gefolgt von Magnesium- und Calciumionen. Die kleinsten DNA-Abstände werden durch Komplexierung mit Manganionen erhalten. Diese Ionenreihenfolge stellt sich sowohl für genomische DNA als auch für Plasmid-DNA ein. <br> E) Die DNA-Abstände werden durch die Kompression des Lipidfilms nicht beeinflusst. Zwischen der Lipidmonoschicht und der adsorbierten DNA besteht demnach nur eine schwache Wechselwirkung. Offensichtlich befindet sich die durch zweiwertige Kationen komplexierte DNA als weitgehend eigenständige Schicht unter dem Lipidfilm. <br> <br><br> 2. Modellsystem: Lipiddoppelschicht an der fest/flüssig-Grenzfläche<br> Methoden:<br> Neutronenreflexion (NR) und Quarzmikrowaage (QCM-D)<br> Resultate:<br> A) Das zwitterionische Phospholipid DMPC (1,2-Dimyristoyl-phosphatidylcholin) bildet keine Lipiddoppelschicht auf planaren Polyelektrolytmultischichten aus, deren letzte Lage das positiv geladene PAH (Polyallylamin) ist. <br> B) Hingegen bildet DMPC auf dem negativ geladenen PSS (Polystyrolsulfonat) eine Doppelschicht aus, die jedoch Defekte aufweist. <br> C) Eine Adsorption von genomischer Kalbsthymus-DNA auf dieser Lipidschicht findet nur in Gegenwart von Calciumionen statt. Andere zweiwertige Kationen wurden nicht untersucht.<br> D) Das negativ geladene Phospholipid DLPA (1,2-Dilauryl-phosphatidsäure) bildet auf dem positiv geladenen PAH eine Lipiddoppelschicht aus, die Defekte aufweist. <br> E) DNA adsorbiert ebenfalls erst in Anwesenheit von Calciumionen in der Lösung an die DLPA-Schicht.<br> F) Durch die Zugabe von EDTA (Ethylendiamintetraessigsäure) werden die Calciumionen dem DLPA/DNA-Komplex entzogen, wodurch dieser dissoziiert. Demnach ist die calciuminduzierte Bildung dieser Komplexe reversibel. / All over the world scientists are trying to engineer artificial viruses, which do not replicate, for gene delivery. These artificial viruses should have the advantages of natural viruses such as efficient transport of genetic material, but they should not carry antigens, which cause immune reactions, on their top portion.<br><br> The aim of this project is to develop an artificial virus particle that is based on a polyelectrolyte hollow capsule which is covered by a lipid bilayer. To create intact bilayers, it is crucial to understand and optimize the interaction between lipids and polyelectrolytes (e. g. DNA). Therefore the structural basis of that interaction must be elucidated. Positively charged lipids interact strongly with the negatively charged DNA but they cause toxic reactions in biological cells. Hence the present work used two model systems to study the coupling of genomic or plasmid DNA to zwitterionic or negatively charged phospholipids induced by divalent cations. <br><br> 1. Model system: Lipid monolayer at the air/water-interface <br> Methods: <br> Langmuir filmbalance in combination with IR-spectroscopy (IRRAS), X-ray reflectometry (XR), X-ray diffraction (GIXD), Brewster angle microscopy (BAM), X-ray fluorescence (XRF), and surface potential measurements<br> Results:<br> A) The presence of the divalent cations Ba2+, Mg2+, Ca2+ or Mn2+ in the subphase has no traceable influence on the structure of a zwitterionic DMPE (1,2-dimyristoyl-phosphatidyl-ethanolamine) monolayer.<br> B) DNA which is dissolved in the subphase adsorbs to the DMPE-monolayer only if divalent cations are present.<br> C) The adsorption of genomic calf thymus DNA as well as of the plasmid DNA pGL3 causes a reduction of the tilt angle of the lipid alkyl chains. The tilt reduction can be ascribed to a change in the space required by the lipid head group. This change in head group orientation increases the electrostatic interaction between the positively charged nitrogen atoms in the lipid head and the negatively charged DNA phosphates.<br> D) The adsorbed DNA exhibits an ordered structure if it is complexed by barium, magnesium, calcium or manganese ions. The spacing between parallel DNA strands depends on the size of the DNA fragments as well as on the kind of cation. The largest DNA-spacings are observed with barium ions, followed by magnesium and calcium ions. DNA-complexation with manganese ions causes the smallest spacings. This order of ions is observed for both genomic and plasmid DNA.<br> E) Compression of the monolayer does not influence the DNA spacings. Thus the interaction between the lipid monolayer and adsorbed DNA is only weak. The DNA must exist as a more or less separate layer under the lipid film.<br> <br><br> 2. Model system: Lipid bilayer at the solid/fluid-interface<br> Methods: <br> Neutron reflectometry (NR), and Quartz crystal microbalance (QCM-D)<br> Results:<br> A) The zwitterionic phospholipid DMPC (1,2-dimyristoyl phosphatidylcholine) does not form lipid bilayers on top of planar polyelectrolyte multilayers covered with the positively charged PAH (polyallylamine).<br> B) In contrast, DMPC forms a lipid bilayer with defects on top of the negatively charged PSS (polystyrolsulfonate) terminated polyelectrolyte cushion.<br> C) Genomic calf thymus DNA adsorbs only to the DMPC layer in presence of calcium ions. Different ions were not examined.<br> D) The negatively charged phospholipid DLPA (1,2-dilauryl-phosphatidic acid) also forms a lipid bilayer with defects on top of the PAH-terminated cushion.<br> E) The DNA adsorbs also to the DLPA layer only in the presence of calcium ions in the solution.<br> F) By addition of EDTA (ethylenediaminetretraacetic acid) the calcium cations are removed from the DLPA/DNA-complex and the complex dissociates. Thus the calcium induced formation of that complex is reversible.<br><br>