The main aim of this thesis was to characterize membrane-solute interactions using artificial membranes in immobilized liposome chromatography or capillary electrophoresis. The partitioning of a solute into a cell membrane is an essential step in diffusion across the membrane. It is a valid parameter in drug research and can be linked to the permeability as well as the absorption of drugs. Immobilized liposome chromatography was also used to study partitioning of lignin precursor models. Lignin precursors are synthesized within plant cells and need to pass the membrane to be incorporated into lignin in the cell wall. In immobilized liposome chromatography, liposomes or lipid bilayer disks were immobilized in gel beads and the partitioning of solutes was determined. Capillary electrophoresis using disks as a pseudostationary phase was introduced as a new approach in drug partitioning studies. In addition, octanol/water partitioning was used to determine the hydrophobicity of the lignin precursor models. Electrostatic interactions occurred between bilayers and charged drugs, whereas neutral drugs were less affected. However, neutral lignin precursor models exhibited polar interactions. Moreover, upon changing the buffer ionic strength or the buffer ions, the interactions between charged drugs and neutral liposomes were affected. Hydrophobic interactions were also revealed by including a fatty acid or a neutral detergent into the bilayer or by using a buffer with a high salt concentration. The bilayer manipulation had only a moderate effect on drug partitioning, but the high salt concentration had a large impact on partitioning of lignin precursor models. Upon comparing the partitioning into liposomes and disks, the latter showed a more pronounced partitioning due to the larger fraction of lipids readily available for interaction. Finally, bilayer disk capillary electrophoresis was successfully introduced for partitioning studies of charged drugs. This application will be evaluated further as an analytical partitioning method and separation technique.

No description available.

Diese Arbeit konzentriert sich auf die Anwendbarkeit eines hochmodernen 100 kHz BB-VSFG-Spektrometers, das kürzlich im SALSA Photonics Lab entwickelt wurde, für die Analyse der Grenzflächenstruktur von Alkylketten, des sie umgebenden Wassers und der Phosphatkopfgruppen von Phospholipidschichten. Zunächst wurden Phospholipid-Doppelschichten, die mehrere Komponenten enthalten, bei Laserwiederholraten von 5, 10, 50 und 100 kHz mit konstanter Pulsenergie untersucht. Die BB-VSFG-Spektren legen nahe, dass die Phospholipid-Doppelschichten während der Messungen ohne wärmeinduzierte Veränderungen stabil waren. Darüber hinaus bot die Erhöhung der Laserwiederholungsrate eine praktikable Möglichkeit, Spektren in kurzen Datenerfassungszeiten zu erhalten, ohne dass das Signal-Rausch-Verhältnis beeinträchtigt wurde. Die extrem kurze Aufnahmezeit von 500 ms, die hohe spektrale Auflösung und alle verwendeten Pulsparameter sorgen dafür, dass bei Messungen unter Umgebungsbedingungen keine thermisch bedingten Photoschäden auftreten. Es wurde eine systematische Untersuchung von ein- und zweikomponentigen Phospholipid-Monoschichten in Abhängigkeit von der Oberflächenspannung und dem Mischungsverhältnis für verschiedene Kombinationen an Polarisationen durchgeführt und die Abhängigkeit der Schwingungsspektren untersucht. Die Struktur von Alkylketten und umgebendem Wasser wurde anhand derselben Modellsystemen analysiert. Bislang nicht beobachtete Schwingungsbanden und Spektren von Monolagen mit geringer Oberflächenbedeckung wurden mit einem bisher nicht erreichten Signal-Rausch-Verhältnis gemessen und beschrieben. Die Struktur von Phospholipid-Monolagen mit identischen Kopfgruppen und unterschiedlichen Ketten wurde analysiert und verglichen. Die Spektren bestätigten die Anwesenheit von Wassermolekülen in der Nähe der Phosphat- und Cholingruppen der Phospholipid-Monolagen. / This thesis focuses on the applicability of a state-of-the-art 100 kHz BB-VSFG spectrometer recently developed at the SALSA Photonics Lab and on the analysis of the interfacial structure of alkyl chains, surrounding water, and the phosphate head groups of phospholipid layers. First, multi-component phospholipid bilayers were studied at laser repetition rates of 5, 10, 50, and 100 kHz at constant pulse energy. The spectra suggest that the phospholipid bilayers were stable during the measurements with no heat-induced distortions. Moreover, an increase in the laser repetition rate provided a feasible route to obtain spectra in short data acquisition times without compromising the signal-to-noise ratio. The extremely short acquisition time of 500 ms, the high spectral resolution, and all applied pulse parameters ensured no thermal induced photodamages occur during the measurements. A systematic study of one- and two-component phospholipid monolayers as a function of surface tension and mixture ratio at different polarization combinations was performed and the dependence of the vibrational spectra was explored. The structure of alkyl chains and surrounding water was analyzed using the same model systems. Vibrational modes that were previously unseen and spectra of monolayers at low surface coverage were reported for the first time with an unprecedented signal-to-noise ratio. The structure of phospholipid monolayers containing identical head groups and different chains was analyzed and compared. The order of the phospholipid molecules as a function of the composition of the monolayers was inferred from the spectral data. The influence of the hydration and/or changes in the orientation of the phosphate group was visible from the spectra as well.

Pre-treatment with polyunsaturated fatty acids or bioactive lipid mediators has been shown to reduce neuronal injury in rodent models of focal ischaemia, but the molecular mechanisms underlying this neuroprotection are unclear. In this study, we aimed to investigate whether systemic administration of alpha linolenic acid (ALA) leads to changes in the profile of mouse brain phospholipid and bioactive lipid mediators in both mouse brain and plasma within the previously determined neuroprotection time window. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) allowed us to detect and identify 47 phospholipids in mouse cerebral cortex, including several phospholipid species not previously reported in brain lipidomic studies. These included a phosphatidylethanolamine species with m/z 720 that has been associated with retinal stem cells. No widespread changes in cerebral cortex phospholipid composition were observed following intravenous ALA. Several significant changes in lipid mediators (P<0.05 with two-way ANOVA and post hoc Dunnett's t test) were detected in ALA-treated animals compared to untreated and vehicle-injected animals. Many of the affected lipid mediators are ligands for prostanoid receptors which have been demonstrated to play a role in the development of brain injury following cerebral ischaemia, implying that changes in bioactive lipid mediators or modulation of prostanoid receptors may occur following ALA pre-treatment in mice. This study illustrates the potential of advanced lipidomic analysis as a novel tool for neurochemists.

Les lipides polaires (LP) laitiers (~2% des lipides du lait) présentent un potentiel bioactif élevé, notamment lié à leur richesse en sphingomyéline (SM, ~25% des LP). Nos hypothèses sont que les LP laitiers peuvent exercer certains de leurs effets bénéfiques par l'intermédiaire de la SM, notamment sur l'intégrité de la barrière intestinale et le microbiote, ce qui pourrait contribuer à réduire l'inflammation métabolique. Nous avons testé à long terme in vivo l'impact de régimes hyperlipidiques (HF) supplémentés en LP laitiers. In vitro, nous avons étudié l'effet des LP laitiers et de la SM (laitière ou d'oeuf) sur l'expression génique des protéines de jonctions serrées. Nos travaux in vitro ont également permis de tester que l'interleurkine-8 (IL-8), impliquée dans la maturation de l'épithélium intestinal, serait un acteur des modifications intestinales en réponse aux LP laitiers et/ou à la SM. L'impact à court terme d'un gavage chez la souris avec des LP laitiers ou de la SM laitière a également été étudié. Après 8 semaines de régime HF supplémenté en LP laitiers (1,6%) les souris présentent un moindre gain de poids en comparaison au régime HF. Nous observons une augmentation de Bifidobacterium animalis pour le groupe contenant 1,1% de LP laitiers. Le groupe nourri avec une supplémentation de 1,6% de LP laitiers présente une diminution de Lactobacillus reuteri et des cryptes coliques plus profondes. Nous retrouvons également une plus forte teneur en acide gras spécifiques des LP laitiers (C23:0, C24:0 et C24:1, présents dans la SM laitière) dans les lipides fécaux. Ces acides gras sont corrélés à la teneur en Lactobacillus spp. Parmi les protéines de jonctions serrées impliquées dans la perméabilité paracellulaire, seule l'expression de ZO-1 tend à être augmentée dans le duodénum. In vitro, lorsque les cellules Caco-2/TC7 sont incubées avec des micelles mixtes supplémentées en SM pure, une augmentation de l'expression génique des protéines de jonctions serrées, ainsi qu'une augmentation de la concentration d'IL-8 en apicale et en basolatérale, sont observées. Ces effets sont également retrouvés avec la SM d'oeuf, contrairement aux LP laitiers totaux. L'incubation d'IL-8 recombinante humaine conduit à une augmentation de l'expression génique des protéines de jonctions serrées. Un gavage avec de la SM laitière pure chez la souris induit une augmentation de l'expression des homologues murins de l'IL-8 (KC et Mip-2). Cette étude suggère que les LP laitiers peuvent limiter la prise de poids induite par un régime HF et moduler le microbiote intestinal. La présence de produits d'hydrolyse spécifiques de la SM pourrait expliquer les effets sur le côlon et le microbiote intestinal. Les résultats in vitro, suggèrent un impact spécifique de la SM sur la barrière intestinale. L'IL-8 semble impliquée dans la régulation de l'expression des protéines de jonctions serrées. Ces résultats contribuent à expliquer les effets bénéfiques démontrés des LP laitiers. L'exploration mécanistique des effets directs et/ou indirects de la SM et de l'IL-8 sur la barrière intestinale reste à élucider / Interest is growing for the metabolic impact of milk polar lipids (MPL, ~2% of dairy lipids), which present a high bioactive potential, particularly related to their content in sphingomyelin (SM, ~ 25% of MPL). Our hypotheses are that MPL can exert some of their beneficial effects through SM, including the integrity of the intestinal barrier and the microbiota, which could contribute to reduce metabolic inflammation. We tested the metabolic impact of the addition of MPL in a high-fat (HF) diet in mice on the modulation of the intestinal barrier. In vitro, we studied the effect of SM (milk or egg) on tight junction protein We also tested in vitro, that interleurkin-8 (IL-8), which is involved in the maturation of the intestinal epithelium, is an actor of intestinal changes in response to MPL and/or MSM. The short-term impact in mice of MPL or milk SM was also studied. After 8 weeks of diet, the supplementation with 1.6% of MPL prevented the HF-diet-induced body weight gain. In caecal microbiota, addition of 1.1% of MPL induced a specific increase in Bifidobacterium spp., in particular B. animalis. The group fed with a 1.6% MPL-supplementation showed a specific decrease in Lactobacteria reuteri and colonic crypt depth were greatest. We also found a higher content of fatty acids specific of MPL (C23:0, C24:0 and C24:1, found in milk SM) in fecal lipids of mice. These fatty acids are correlated with Lactobacillus spp. Among the tight junction proteins involved in paracellular permeability, only the expression of ZO-1 tended to be increased in the duodenum. In vitro, when Caco-2/TC7 cells were incubated with mixed micelles supplemented with pure SM, an increase in the gene expression of tight junction proteins (ZO-1, occludin, JAM-1, claudin-1) and an increase in apical and basolateral IL-8 concentration were observed. These effects were also found with egg SM, unlike total MPL. Incubation of recombinant human IL-8 led to an increase in gene expression of tight junction proteins. Gavage with pure milk- SM in mice induced an increase in the expression of murine homologs of IL-8 (KC and Mip-2). Our results show that MPL can limit HF-induced body weight gain and modulate the abundance of beneficial bacteria of the gut microbiota. The presence of SM-specific hydrolysis products may explain the effects on the colon and gut microbiota. In vitro results suggest a specific impact of pure SM on the intestinal barrier. IL-8 appears to be involved in the regulation of tight junction protein expression. This can contribute to explain reported beneficial effects of MPL in mice regarding HF induced metabolic disorders. The mechanistic exploration of direct and / or indirect effects of SM and IL-8 on the intestinal barrier remains to be elucidated

Foram examinadas as propriedades conformacionais de um peptídeo (YRWPFGNHL-NH2) presente na primeira alça extra-celular do receptor AT1 do hormônio angiotensina II. Espectros de dicroísmo circular (CD) e fluorescência foram obtidos em solução aquosa (em função do pH e da temperatura), na presença de um solvente indutor de estrutura secundária (trifluoroetanol, TFE), e na presença de micelas carregadas negativamente (dodecil sulfato de sódio, SDS) ou zwitteriônicas (N-hexadecil-N,N-dimetil-3-amônio-1-propano sulfonato, HPS e lisofosfatidilcolina, liso-PC), e de bicamadas contendo um fosfolipídio zwitteriônico (1-palmitoil-2-oleoil fosfatidilcolina, POPC) ou uma mistura de POPC e um fosfolipídio carregado negativamente (ácido l-palmitoil-2-oleoil fosfatídico, POPA). O estudo por calorimetria de titulação permitiu analisar a termodinâmica da ligação. Espectros de CD indicaram uma estrutura flexível em solução aquosa, modulada pelo pH e pela temperatura. Na presença de TFE, de micelas, e de vesículas de POPC:POPA o peptídeo adquire estrutura secundária, sugerindo a presença de uma dobra beta. Espectros de fluorescência intrínseca (W3) mostraram um deslocamento do comprimento de onda máximo de emissão (&#955;max) para o azul na presença de micelas e de vesículas de POPC:POPA. Observou-se também um aumento da intensidade de fluorescência, exceto no caso de SDS. Esses resultados indicaram que o peptídeo interagiu com os agregados. Não se observou alteração da fluorescência na presença de POPC. Medidas de anisotropia de fluorescência mostraram que, quando ligado, o peptídeo toma-se mais imobilizado. Estudos de supressão de fluorescência empregando agentes supressores aquossolúveis e de membrana sugeriram que o W3 localiza-se próximo à interface bicamada-água. Os resultados mostraram que o peptídeo se liga a micelas zwitteriônicas e negativas, enquanto que, no caso de bicamadas (mais empacotadas), a ligação depende da presença de cargas negativas. Experimentos de calorimetria mostraram que o &#916;H de ligação do peptídeo a vesículas é negativo, compatível com a ocorrência de interações eletrostáticas. Foram observadas diferenças qualitativas e quantitativas na ligação do peptídeo às micelas de HPS e liso-PC. A fim de examinar se essas diferenças eram devidas ao empacotamento molecular, foram obtidos espectros de ressonância paramagnética eletrônica de marcadores de spin intercalados nos dois sistemas. Diferenças foram observadas principalmente na região da cabeça polar, as quais poderiam ser responsáveis pela diferença de comportamento do peptídeo em ambos os agregados. Os resultados obtidos para a primeira alça extra-celular do receptor AT1 indicam que a sua conformação pode ser modulada pelo pH e pela polaridade do ambiente, que ela pode interagir com a membrana através de interações hidrofóbicas e eletrostáticas, e ainda que essa interação depende do grau de empacotamento da fase lipídica. Esses resultados estão de acordo com a visão de que domínios extra-membranares de GPCRs situam-se na interface membrana-água. As alterações conformacionais induzidas pelo meio, bem como pela interação entre domínios extra-membranares de GPCRs e a fase lipídica ou pela ligação do agonista, poderiam ter um papel no mecanismo molecular de transdução de sinal. / In this work the conformational properties of a peptide (YRWPFGNHL-NH2) whose sequence is present in the first extra-cellular loop of the angiotensin II AT1 receptor were examined. Circular dichroism (CD) and fluorescence spectra were obtained in aqueous solution (as a function of pH and temperature), in the presence of a secondary structure inducing solvent (trifluoroethanol, TFE), and in the presence of negatively charged (sodium dodecyl sulfate, SDS) or zwitterionic (N-hexadecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, HPS and lysophosphatidylcholine, liso-PC) micelles, and of bilayers containing a zwitterionic phospholipid (l-palmitoyl-2-oleoyl phosphatidylcholine, POPC) or a mixture of POPC and a negatively charged phospholipid (l-palmitoyl-2-oleoyl phosphatidic acid, POPA). Studies of titration calorimetry allowed the analysis of the binding thermodynamics. CD spectra indicated a flexible structure in aqueous solution, which was modulated by pH and temperature. In the presence of TFE, micelles and of POPC:POPA vesicles, the peptide acquired secondary structure, which is suggestive of a &#946; turn. The intrinsic fluorescence spectra (W3) showed a blue-shift of the maximum emission wavelenght (&#955;max) in the presence of micelles and of POPC:POPA vesicles. An increase in fluorescence intensity was also observed, except in the case of SDS. These results indicated that the peptide interacted with the aggregates. No fluorescence changes were observed in the presence of POPC. Fluorescence anisotropy measurements showed that, when bound to the aggregates, the peptide becomes more immobilized. Fluorescence quenching studies using water soluble and membrane-bound quenchers suggested that W3 is located close to the water-bilayer interface. The results showed that the peptide binds to negativelly charged and zwitterionic micelles, while in the case of bilayers (which are more tightly packed) the binding depends on the presence of negative charges. Titration calorimetry showed that &#916;H of peptide binding to the vesicles is negative, which is compatible with the ocurrence of eletrostatic interactions. Qualitative and quantitative differences in binding of the peptide to HPS and liso-PC micelles were observed. In order to examine whether these differences were due to molecular packing, electron paramagnetic resonance spectra of spin labels intercalated in both systems were obtained. Differences were observed, mainly in the polar head group region, that could be responsible for the different behaviour displayed by the peptide in both aggregates. The results obtained for the first extra-cellular loop of the AT1 receptor indicated that its conformation can be modulated by pH and by the polarity of the medium, and that it can interact with the membrane through hydrophobic and electrostatic interactions, and also that this interaction depends on the molecular packing of the lipid phase. These results are in aggrement with the idea that the GPCRs extra-membrane domains are located at the water-membrane interface. Conformational changes induced by the medium, as well as by the interaction between extra-cellular segments and the membrane or by ligand binding, could play a role in the molecular mechanism of signal transduction.

Actively proliferating cells constantly monitor and re-adjust their metabolic pathways to ensure the replenishment of phospholipids necessary for membrane biogenesis and intracellular trafficking. In Saccharomyces cerevisiae, multiple studies have suggested that lysine acetylation has a role in coordinating phospholipid metabolism, yet its contribution towards phospholipid homeostasis remains uncharacterized. In this study we undertook a genetic screen to explore the connection between lysine acetylation and phospholipid homeostasis. We found that mutants of the lysine acetyltransferase complex, NuA4, shared a negative genetic interaction with a mutant of Sec14, a lipid-binding protein that regulates Golgi phospholipid composition. Through transcriptome, genetic, cell biology, and chemical analysis, we discovered that the growth defects between NuA4 and Sec14 mutants is likely derived from impaired fatty acid biosynthesis suggesting a role for NuA4 as a positive regulator of fatty acid biosynthesis. Secondly, we discovered that acetylation on the conserved lysine residue K109 inhibits the localization and function of the Oxysterol-Binding Protein Osh4- a lipid-binding protein that antagonizes the function of Sec14 at the Golgi. Furthermore, regulation of Oxysterol-Binding Proteins by acetylation may be a conserved mechanism as we found that Osh1, a homologue of Osh4, was also acetylated on the equivalent lysine residue. Altogether, we have demonstrated that lysine acetylation can target multiple different phospholipid metabolic pathways which implies that it has a very important role for the regulation of phospholipid homeostasis.

Cette thèse concerne l’auto-assemblage d'alcanes semi-fluorés (FnHm) et de fluorocarbures (FCs) aux interfaces et leurs interactions avec des phospholipides (PLs) en 2D et 3D. Nous avons étudié les sphérulites formées dans des films de diblocs FnHm. La morphologie de ces sphérulites, concentrique ou radiale, est contrôlée par la longueur des blocs Fn et Hm et la vitesse de refroidissement. Les nanodomaines de diblocs FnHm dans les monocouches de Langmuir sont incompressibles et forment des gels physiques 2D, même à pression nulle. Les blocs Hm sont cristallisés et inclinés d’ ~30°C par rapport à la normale à la surface. La réflectivité de neutrons a montré que l’albumine adsorbée sur des monocouches de PL est désorbée par un FC gazeux. Ce résultat pourrait permettre de lutter contre l’inactivation du surfactant pulmonaire par les protéines sériques. L’ajout de diblocs à des films de PLs accroit l'élasticité des monocouches. Nous avons préparé des microbulles stables à parois de PLs/FnHm, les diblocs agissant en co-surfactants. / This thesis focuses on the self-assembly of semi-fluorinated alkanes (FnHm) and fluorocarbons (FCs) at interfaces and their interactions with phospholipids (PLs) in 2D and 3D. 2D Spherulites were identified in FnHm films for the first time. Their morphology, ring-banded or radial, was controlled by varying block lengths and cooling rate. Nanodomains of FnHm in monolayers formed incompressible 2D physical gels, even at zero surface pressure. The Hm segments are crystalline and titled by 30°C to the normal to the surface. Neutron reflectivity showed that albumin adsorbed on PLs monolayers is desorbed by exposure to FC gas, which opens the potential use of FCs to treat the inactivation of the lung surfactant by serum proteins. Incorporating FnHm into PL monolayers increases their elasticity. Small, stable microbubbles of PLs/FnHm were obtained. FnHm diblocks function as co-surfactants for stabilizing microbubbles.

Les bases fondamentales de la biologie suggèrent que tous les organismes actuels partagent un dernier ancêtre commun, le cenancêtre. Dès que la comparaison moléculaire des organismes des trois domaines du vivant (archées, bactéries et eucaryotes) est devenue possible, d’importants débats ont émergé sur l’habitat du cenancêtre, son rapprochement des origines de la vie, sa nature unique ou communautaire et ses relations avec les trois domaines du vivant. Cependant, jusqu’à il y a peu les informations disponibles sur les organismes modernes n’étaient pas suffisantes pour décrire précisément sa biologie. Notamment, la découverte chez les archées de membranes dont les composants principaux, les phospholipides, sont synthétisés par des mécanismes très différents de ceux des bactéries et les eucaryotes a conduit à proposer que chaque mécanisme de synthèse des phospholipides soit apparu indépendamment dans les lignées modernes. Dans ces hypothèses le cenancêtre aurait été dépourvu de phospholipides et, donc, de membranes. Cela met en cause la nature cellulaire du cenancêtre, qui semblait pourtant soutenue par d’autres indices indirects. Ces contradictions posent la question de l’existence de traces dans les organismes modernes d’une synthèse des phospholipides chez le cenancêtre. Dans cette thèse j’ai profité de l’explosion récente des données génomiques pour répondre à cette question. Il avait déjà montré que des membres de deux superfamilles protéiques universelles pouvaient avoir synthétisé de façon non spécifique chez le cenancêtre les énantiomères de glycérol phosphate servant d’ossature aux phospholipides. Les phospholipides archéens sont composés d’isoprénoïdes et les bactériens et eucaryotes d’acides gras. J’ai donc étudié l’évolution des voies de synthèse de ces molécules ainsi que celle de l’assemblage de tous les composants dans des phospholipides. Mes résultats montrent que la voie de synthèse des isoprénoïdes des eucaryotes et une voie hypothétique de synthèse des acides gras chez les archées avaient probablement des ancêtres moins spécifiques chez le cenancêtre. Une partie au moins de la machinerie d’assemblage des phospholipides semble aussi avoir été présente chez le cenancêtre.Ceci suggère que le cenancêtre avait probablement des mécanismes peu spécifiques de synthèse des phospholipides et que les différences entre les membranes actuelles sont dues à la spécialisation de la machinerie ancestrale dans chaque lignée. Mes observations soulignent aussi l’importance d’étudier le cenancêtre à partir des informations issues des organismes actuels pour éviter toute confusion avec les origines de la vie. / The main bases of Biology suggest that all extant organisms share a last common ancestor, namely the cenancestor. As soon as the comparison of molecular characters of organisms representative of the whole diversity of life became possible, hot debates emerged about the environmental conditions in which the cenancestor lived, its closeness to the origins of life, its single or community nature and its relationships with the three domains of life (Archaea, Bacteria and Eucarya). However, available information about current organisms was for a long time inadequate to precisely describe the biology of this organism. For instance, the observation that the main archaeal membrane components, called phospholipids, are synthesized by different means than their bacterial/eukaryotic counterparts was proposed to reveal that modern phospholipid biosynthesis pathways emerged late in independent lineages and were, therefore, absent in the cenancestor. This hypothesis argued that the cenancestor had no lipid membranes, so it could not be a cellular organism although other indirect clues indicated the opposite. These contradictions raise the question of the presence in modern organisms of traces that the cenancestor had a phospholipid biosynthesis machinery.In this dissertation, I took advantage from the recent accumulation of genomic data to address this issue. Previous work had shown that the members of two universal protein superfamilies could be present in the cenancestor to carry out the non-specific synthesis of the glycerol phosphate enantiomers that are the backbones of modern phospholipids. Bacterial and eukaryotic phospholipids use fatty acids whereas archaeal phospholipids are made up of isoprenoids. Thus, I studied the evolution of the metabolic pathways that synthesize these molecules and build up the phospholipids from their components. My results show that the eukaryotic isoprenoid biosynthesis pathway and a hypothetical archaeal fatty acid biosynthesis pathway are likely to have had less specific ancestors in the cenancestor. In addition, the phospholipid assembly machinery was also probably present in the cenancestor.These results suggest that the cenancestor was likely able to enzymatically synthesize its phospholipids by means less specific than modern ones. Dissimilarities in modern membrane phospholipids would result from the specialization of each biosynthesis system in each lineage. My work also stresses the fact that the cenancestor should be described on the basis of the comparison of modern organisms to avoid frequent confusions between the cenancestor and the origins of life.

Neste trabalho foram estudadas as interações entre os corantes ciânicos com dois cromóforos (BCD) e monocamadas de Langmuir de fosfolipídio (DPPC). Devido ao alto coeficiente de absorção molar da luz na região ? > 600 nm, ao alto rendimento quântico do estado tripleto, à alta fotocitotoxicidade e à alta afinidade com estruturas celulares os BCD são promissores para serem utilizados em terapia fotodinâmica. Os estudos das características da interação de BCD com a monocamada são importantes tanto para esclarecer os efeitos estruturais na interação de monocamadas com outras moléculas quanto para suas aplicações em terapia fotodinâmica e em outras áreas tecnológicas tais como nanoeletrônica, fotônica etc. As isotermas de pressão superficial (?-A) obtidas mostram que os BCD e as monocamadas interagem, pois as isotermas (?-A) se expandem conforme é aumentado à porcentagem de BCD. Esta interação é dependente tanto da estrutura do BCD quanto da força iônica da subfase. A partir das isotermas (?-A) foi obtido o módulo de compressibilidade (Cs-1 = -A (??/?A) das monocamadas, do qual foram analisadas, de maneira mais detalhada, as modificações da monocamada de DPPC devido à presença dos BCD. Estas modificações compreendem o aparecimento da fase líquida expandida (LE) e formação dos domínios antecipada em comparação com a monocamada de DPPC puro. Foi observado que a área mínima por molécula e a elasticidade da monocamada na fase condensada (C) aumentam juntos com a porcentagem dos BCD. Na presença de BCD 180º as isotermas de potencial superficial (?V-A) sofrem uma expansão, no entanto, o valor máximo do potencial permanece nalterado. Isto indica que os BCD estimulam (antecipam) a orientação das moléculas de DPPC na interface ar-água e, consequentemente, a co-orientação das cabeças polares do DPPC, mas não contribuem no potencial superficial. Foi observado que a presença de NaCl na subfase aumenta o efeito dos BCD na formação da monocamada DPPC. Acreditamos que este efeito sinérgico é devido às interações mútuas dos ânions Cl- com a carga positiva do grupo trimetilamônio da cabeça polar do DPPC e das cargas positivas do BCD com a carga negativa do grupo fosfato do DPPC. As imagens obtidas pela microscopia de ângulo de Brewster mostram, em concordância com a análise das isotermas de pressão (?-A) e de potencial (?V- A), que a formação dos domínios, e conseqüentemente da monocamada, é antecipada na presença de BCD. Entretanto, a presença do BCD não altera o formato dos domínios, mas diminui seu tamanho. A análise realizada sobre os espectros de absorção óptica mostrou que os BCD podem formar agregados tipos H na monocamada e não foram observados agregados tipo J. Baseando-se na análise do efeito da estrutura dos BCD, na interação com a monocamada, e dos espectros de absorção propusemos dois modelos de interação dos BCD com a monocamada e acreditamos que o seguinte modelo é a melhor modelagem do sistema: - BCD se inseri na monocamada, paralelamente ao eixo da molécula de DPPC, interagindo tanto com a cauda hidrofóbica quanto com a cabeça polar. Entretanto, os resultados obtidos não permitem excluir completamente o segundo modelo proposto, no qual o BCD se localiza na parte polar da monocamada, perpendicular ao eixo da molécula de DPPC. / In this work the interaction of cyanine dyes with two chromophores with the DPPC phospholipid Langmuir monolayers was studied. Due to their high optical absorption in the region ? > 600 nm, high triplet state quantum yields, high photocytotoxicity and high affinity with cell structures the BCD are promising for application in photodynamic therapy. The study of the interaction of BCD with monolayers is important to clarify the structural effects on the monolayer interaction with other molecules as well as for application in photodynamic therapy and in other fields of technology, such as nanoelectronics, photonics, etc. The superficial pressure isotherms (?-A) demonstrate that BCD do interact with monolayers, the (?-A) isotherms being expanded when the BCD relative content increases. This interaction depends on the BCD structure and the subphase ionic strength, as well. The curves of the compressibility module (Cs-1 = -A (??/?A) of the monolayers were obtained from the (?-A) isotherms, which were used for detailed analysis of the DPPC monolayer formation in the BCD presence. The analysis demonstrated that the monolayer expanded liquid phase and the domain were formed earlier in the BCD presence than for pure DPPC. It was observed that both the minimum area per molecule and the condensed phase elasticity increased when the BCD relative content increased. In the presence of BCD 1800 the superficial potential isotherms (?V-A) were expanded, but the maximum potential value was unchanged. This indicates that BCD stimulated the DPPC molecule orientation on the air-water interphase and, consequently, the co-orientation of the DPPC polar heads, but did not contribute itself in the surface potential. It was observed that NaCl in the subphase increased the BCD effects on the DPPC monolayer formation. We suppose that this synergetic effect is due to the mutual interaction of Cl- anions with positive charges of the trimethylammonium group of the DPPC polar head and that of positive BCD charges with its phosphate group negative charges. Images obtained with the Brewster microscopy confirmed that BCD stimulated the domain and, consequently, the monolayer formation in accordance with the (?-A) and (?V-A) analysis. At the same time the BCD did not change the domain geometry, but reduced their sizes. The analysis of the BCD optical absorption spectra demonstrate that the BCD molecules in the monolayer form H aggregates and no J aggregates were observed. Basing on the analysis of the BCD structure effects on the monolayer formation we can propose two models of the BCD - monolayer interaction and we believe that the follow model is better: - the BCD molecule is inserted in the monolayer interior being parallel with the DPPC molecule axis and interacting with both hydrophobic and polar DPPC parts; However, our data are not sufficient to exclude completely the possibility for the BCD molecule to be localized just in the polar head monolayer part being perpendicular to the DPPC molecule axis.