The molecular species of diacylglycerol and alkylacylglycerol of Madin-Darby canine Kidney (MDCK) cells were analyzed to determine the sources of diradylglycerols generated during cell growth and phorbol ester stimulation. MDCK cells in log phase growth contained higher levels of diacylglycerol and alkylacylglycerol than confluent cells. Both subclasses of diradylglycerol showed higher levels of saturated and monoenoic species during log phase. Glycerol incorporation into diradylglycerols was increased during growth, consistent with an increase in their synthesis de novo. Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, caused an increase in the level of diacylglycerol but not alkylacylglycerol. Log phase MDCK cells showed a greater response to TPA treatment than confluent cells. The molecular species of diacylglycerol generated during stimulation with either TPA or dioctanoylglycerol closely resembled the species of phosphatidylcholine. These results indicate that TPA and synthetic diacylglycerol stimulate endogenous diacylglycerol production through the hydrolysis of phosphatidylcholine. In contrast, the higher content of diacylglycerol and alkylacylglycerol in replicating MDCK cells is the result of an increase in their synthesis de novo.

No / Electrically assisted skin delivery of liposomal estradiol; phospholipid as damage retardant.

Les fonctions physiologiques spécifiques de la voie de N-méthylation de la phosphatidyléthanolamine (PE), une des deux voies de biosynthèse de la phosphatidylcholine (PC), restent relativement énigmatiques. Il a été démontré chez les poissons euryhalins qu’un stress hyperosmotique induisait une activation de cette voie métabolique au niveau hépatique. L’objectif de notre travail était de vérifier si ce phénomène se produit aussi chez d’autres animaux euryhalins. Les études réalisées in vivo sur deux espèces de crâbes, Eriocheir sinensis et Carcinus maenas, nous ont permis de montrer que l’acclimatation en eau de mer de ces animaux active la synthèse de PC par N-méthylation de la PE dans l’hépatopancréas. Les marquages radioisotopiques montrent aussi que cette PC est échangée avec le plasma et que ce phénomène est amplifié chez les animaux en eau de mer. Ce pool de PC est utilisé comme précurseur de la bétaïne, un osmoeffecteur organique important chez ces animaux. Nous avons ensuite caractérisé la voie de N-méthylation de la PE chez un animal osmoconformeur, la moule Mytilus galloprovincialis. Les résultats, obtenus in vivo et in vitro sur les tissus isolés, démontrent qu’une activité de N-méthylation de la PE en PC est exprimée dans la glande digestive et les hémocytes circulant de M. galloprovincialis. La PC ainsi synthétisée dans ces tissus est échangée avec l’hémolymphe de l’animal. De l’ensemble de ces observations, nous pouvons conclure que la synthèse de PC par N-méthylation est largement exprimée chez les animaux marins euryhalins et qu’une des fonctions physiologiques de cette voie métabolique est de synthétisée des osmolytes organiques comme la bétaïne / The specific physiological functions of the N-methylation of phosphatidylethanolamine (PE), one of the two biosynthetic pathways of phosphatidylcholine (PC), remain relatively mysterious. It has been demonstrated in euryhaline fish that hyperosmotic stress induced activation of this pathway in the liver. The aim of our work was to verify whether this phenomenon also occurs in other euryhaline animals. In vivo studies on two species of crabs, Eriocheir sinensis and Carcinus maenas, showed that seawater acclimation activates PC synthesis by N-methylation of PE in the hepatopancreas. Radioisotopic labelling also showed that PC is exchanged with the plasma and that this phenomenon is amplified in animals in seawater. This pool of PC is used as a precursor of betaine, an important organic osmoeffector in these animals. We then characterized the process of PE N-methylation in an osmoconforming animal, the mussel Mytilus galloprovincialis. The results, obtained in vivo and in vitro on isolated tissues, show that N-methylation of PE to PC is expressed in the digestive gland and circulating haemocytes in M. galloprovincialis. The PC synthesized in these tissues is exchanged with hemolymph of the animal. From all these observations, we conclude that the synthesis of PC by N-methylation is widely expressed in marine euryhaline animals and that a physiological function of this pathway is to provide organic osmolytes such as betaine

La littérature des dernières décennies regorge de références concernant les bienfaits des acides gras oméga 3, tels que l’EPA (C20:5 n-3) et le DHA (C22:6 n-3) qui jouent un rôle essentiel dans la prévention de nombreuses pathologies comme les maladies neurodégénératives (type Alzheimer). Les sources majeures d’EPA et DHA sont celles d’origine marine. C’est dans ce contexte que cette étude a choisi de s’intéresser aux phospholipides provenant de têtes de saumon. L’objectif était de les extraire, de purifier la phosphatidylcholine (PC) issue du mélange de lipides et d’en déterminer ses propriétés d’auto-assemblage en bicouches. Une approche expérimentale par la caractérisation physico-chimique de ce PC a été complétée par une étude théorique du même composé en utilisant les techniques de simulation par dynamique moléculaire qui a permis une caractérisation à l’échelle moléculaire des bicouches lipidiques. La caractérisation a permis d’aboutir au profil détaillé de la composition du mélange PC saumon, d’élaborer le diagramme de phase PC –eau, de déterminer les propriétés d’empaquetage d’hydratation de ce lipide. Les paramètres choisis pour l’étude en dynamique moléculaire ont permis de reproduire de manière fidèle les résultats expérimentaux, validant ainsi le modèle et les conditions de simulations déterminés au préalable. La caractérisation des propriétés structurales de la PC de saumon sous forme de multicouches a permis d’approfondir la compréhension des mécanismes d’interactions à l’échelle moléculaire existant entre les lipides insaturés eux-mêmes. / The literature of recent decades is replete with references regarding the benefits of omega 3 fatty acids such as EPA (C20:5 n-3) and DHA (C22:6 n-3) which play an essential role in preventing many diseases such as neurodegenerative diseases (Alzheimer's type). The major sources of EPA and DHA are those of marine origin. It is within this context that this study chose to deal with phospholipids from salmon heads. The objective was to extract, purify phosphatidylcholine (PC) derived from the mixture of lipids and determine its properties of self-assembly into bilayers. An experimental approach by the physicochemical characterization of this PC was supplemented by a theoretical study of the same compound using the techniques of molecular dynamics simulation that allowed a molecular-scale characterization of lipid bilayers. The characterization resulted in detailed profile of the mixture composition of salmon PC, to draw up the phase diagram of PC-water, to determine the packing and hydration properties of this lipid. The parameters chosen for the study of molecular dynamics have faithfully reproduced the experimental results, thus validating the model and simulation conditions determined in advance. The characterization of structural properties of the PC as a multilayer salmon has deepened the understanding of interaction mechanisms at the molecular level between unsaturated lipids themselves.

Résumé : Le métabolisme monocarboné ou métabolisme 1-C désigne l’ensemble des voies métaboliques permettant la synthèse et / ou le recyclage de molécules donneur de groupement monocarboné au cours des réactions de méthylation. L’objectif de ce travail était d’évaluer l’implication des métabolismes de la choline, de la phosphatidylcholine (PC) et de la bétaïne dans la physiopathologie des désordres impliquant le métabolisme 1-C en période prénatale et postnatale. Nous avons montré une augmentation progressive de l’expression de la majorité des gènes impliqués dans le métabolisme 1-C au cours de l’ontogénèse hépatique murine, tandis que les leur expression était plus faible avec des profils plus variable au niveau cérébral. Chez l’homme, les valeurs normales des concentrations des intermédiaires du métabolisme 1-C dans le liquide amniotique (LA) en fonction du terme gestationnel ont été déterminées pour tous les paramètres et les concentrations de S-adénosyl-homocystéine et de méthionine étaient augmentées dans les LA du groupe affecté par des défauts de fermeture du tube neural (DFTN) suggérant que certains cas de DFTN pourraient être associés à des déséquilibres du métabolisme 1-C. En post natal, nous avons montré à la fois chez l’homme et l’animal, que les hyperhomocystéinémie d’origine nutritionnelles ou génétiques induisaient une déplétion en bétaïne, épargnant uniquement le rein où elle est un osmolyte majeur. Dans un modèle murin de déficit en cystathionine–beta synthase induisant une hyperhomocystéinémie, une technique de lipidomique ciblée a montré au niveau hépatique des modifications qualitatives des phospholipides (PLs) avec une diminution des PC contenant des acides gras insaturés et des phosphatidyléthanolmines contenant de l’acide arachidonique. Ces modifications des PLs pourraient jouer un rôle dans la constitution de la stéatose hépatique observée dans l’histoire naturelle de cette maladie. En conclusion, ce travail a permis de montrer que la choline, la bétaïne et les PC sont des acteurs indissociables du métabolisme 1-C qui pourraient être impliqués dans la physiopathologie des DFTN et dans les hyperhomocystéinémies. Ils pourraient également être impliqués dans la physiopathologie des stéatoses hépatiques non alcooliques ou des déficits cognitifs, dans lesquels des désordres du métabolisme 1-C ont été observés. / Abstract: One carbon metabolism or 1C metabolism includes all metabolic pathways for the synthesis and / or recycling of molecules involved in methylation reactions. The objective of this study was to evaluate the involvement of choline, phosphatidylcholine (PC) and betaine metabolisms in the pathophysiology of diseases with impaired 1-C metabolism in prenatal and postnatal period. We showed a progressive increase of the expression of the majority of genes involved in 1-C metabolism during the mouse liver ontogeny while their gene expression was at lower levels and with more variable patterns during brain ontogeny. In humans, amniotic fluid concentrations of all intermediates of 1-C metabolism according to gestational term were determined and we observed increased concentrations of S-adenosyl-homocysteine and methionine in pregnancies affected by neural tube defects (NTD) suggesting that some NTDs cases could be associated with an imbalance in 1-C metabolism. In the postnatal period we showed that both in animal and humans and both in nutritional and genetic hyperhomocysteinemia, that betaine pools were decreased, only sparing the kidney betaine concentrations, where betaine acts as an essential osmolyte. In a mouse model of cystathionine-beta synthase deficiency inducing hyperhomocysteinemia, a technic of targeted lipidomic revealed qualitative changes in the liver phospholipids composition, in particular a decrease of PC containing unsaturated fatty acids and of phosphatidylethanolamine containing arachidonic acid and an increase of phosphatidylethanolamine containing docosohaexaenoic acid. This phospholipids remodelage may participate in the development of the steatosis observed in the natural history of this disease. In conclusion, this study has shown that choline, betaine and phosphatidylcholine are essential actors of 1-C metabolism that could be involved in the pathogenesis of NTD and hyperhomocystéinemia. They could also be involved in the pathophgysiology of non alcoholic fatty liver disease or cognitive decline, in which disorders of 1-C metabolism were observed.

Cell and organelle membranes consist of a complex mixture of phospholipids that determine their size, shape, and function. Among the distinct types of phospholipids found in membranes of living organisms, phosphatidylcholine (PC) is the most abundant. The rate-limiting step of the predominant pathway for PC synthesis in eukaryotic cells is catalysed by the enzyme, CTP: phosphocholine cytidylyltransferase α (CCTα or PCYT1A). CCTα has a critical role in lipid metabolism and also has direct clinical relevance as mutations in CCTα result in an interesting spectrum of human diseases, such as lipodystrophy with fatty liver, growth plate dysplasia and cone-rod related dystrophy. Numerous biochemical and structural studies on purified CCTα have revealed its membrane-bound activation and suggested that it acts as a lipid compositional sensor, yet the in vivo mechanism of how CCTα senses and regulates PC levels in membranes remains unclear. Here I show that in budding yeast Saccharomyces cerevisiae, Pct1, the yeast homolog of CCTα, is intranuclear and translocates to the nuclear membrane in response to changes in membrane properties and the need for membrane PC synthesis. By aligning imaging with lipidomic analysis and data-driven modelling, Pct1 membrane association is demonstrated to correlate with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous Pct1. These data suggest an ancient mechanism by which CCTα senses lipid packing defects and regulates phospholipid homeostasis from the nucleus. Additionally, I identified the importance of mammalian CCTα in early adipogenesis and investigated the enzymatic function of PCYT1A mutants in fibroblasts from lipodystrophic patients. The allele Val142Met is evaluated to be the main cause of loss-of-function in the compound heterozygous mutations by using yeast survival assay. These results collectively provide preliminary evidence for the pathogenicity of PCYT1A mutations in adipose tissue. From yeast to humans, this study uncovers the critical role of Pct1/CCTα in maintaining the internal membrane environment.

Metabolic energy is most efficiently stored as triacylglycerol (TAG). This neutral lipid accumulates mainly within adipose tissues, but it can be stored and used in all types of cells. Within cells it is packed in organelles called lipid droplets (LDs). They consist of a core of neutral lipids like TAG and cholesterol esters, which is surrounded by a phospholipid monolayer that mainly consists of phosphatidylcholine (PC). Attached to or inserted into this monolayer are various proteins, mainly LD specific structural proteins or lipid metabolic enzymes. Though excess uptake of nutrition leads to lipid accumulation in all kinds of body tissues, which is accompanied by the augmentation of LDs and results in cellular dysfunction and the development of metabolic diseases, relatively little is known about the biogenesis and growth of LDs. This thesis focuses on diacylglycerol acyltransferase 2 (DGAT2), an enzyme of the TAG biosynthetic pathway, and on lyso-phosphatidylcholine acyltransferases 1 and 2 (LPCAT1 and LPCAT2), both enzymes of one of the PC biosynthetic pathways called Lands cycle. The data presented in this thesis show that these enzymes can localize to LDs and that they actively synthesize TAG and PC at the surface of LDs. While the LPCATs reside on LDs independent from the nutrition status of the cell, DGAT2 accumulates on LDs upon excess availability of oleic acid. DGAT2, LPCAT1 and LPCAT2 differ in their structure from other iso-enzymes that catalyze the same reactions. This thesis shows that they exhibit a monotopic conformation and that they contain a hydrophobic stretch that presumably forms a hairpin. This topology enables them to localize to both a phospholipid bilayer like the membrane of the endoplasmic reticulum and to a phospholipid monolayer like the surface of LDs. The different biophysical properties of the structures of iso-enzymes might be responsible for their subcellular localization and the formation of distinct TAG or PC pools that are destined for different purposes. This would explain, why the iso-enzymes are often not able to replace each other. Knock-down and overexpression experiments performed in this thesis show that the activity of LPCAT1, LPCAT2 and DGAT2 influence the packaging of lipids within LDs. Knock-down of LPCAT1 and LPCAT2 leads to an increase in LD size without concomitant increase in the amount of TAG. Combined with the finding that the profile of the PC species of the LD surface reflects the substrate preferences of LPCAT1 and LPCAT2, the results suggest that these enzymes are responsible for the formation of the LD surface. Therefore, the increase in LD size upon LPCAT1 and LPCAT2 knock-down results from an adjustment of the surface-to-volume ratio in response to reduced availability of surface lipids. The connection between LPCATs and LD size was corroborated in the model organism Drosophila melanogaster. Three different knockout fly strains of the Drosophila homologue of LPCAT1 and LPCAT2, CG32699, exhibit enlarged LDs in the fat body of the L3 larvae. Furthermore, the data presented suggest that the morphology of LDs is important for the secretion of stored lipids. The reduction of LPCAT1 in liver cells leads to a reduction in lipoprotein particle release. This was shown by measuring the amount of released apolipoproteinB with two different methods, by measuring the release of lipids and by quantification of the amount of released hepatitis C virus, which is known to rely on LD interaction for replication and on lipoprotein particles for cellular release. DGAT2 is recruited to LDs upon excess availability of oleic acid and its overexpression leads to the formation of many, but relatively small LDs. Here, it is shown that DGAT2 interacts with acyl-CoA synthetase ligase 1 (ACSL1), an enzyme that catalyzes the activation of free fatty acids with Coenzyme A. This interaction does not influence the stability of DGAT2 nor does it seem to affect lipid synthesis. Nevertheless, it shows an influence on lipid packaging in LDs. While overexpression of DGAT2 results in the appearance of smaller LDs, overexpression of ACSL1 leads to an increase in LD size. Coexpression of ACSL1 and DGAT2 reverses the phenotypes obtained by single overexpression and normalizes the mean LD diameter to values observed at normal conditions. In conclusion, this thesis shows that LDs are able to synthesize the components of their core and their surface, which underlines their independent function in metabolism. Additionally, the results show that LDs can grow by local synthesis and that the responsible enzymes exhibit a monotopic membrane topology, which might be crucial for LD localization. Furthermore, the obtained data suggest that the localization and the ratio between different enzyme activities influence the packaging of lipids and affects lipid secretion and therefore impact the whole body lipid metabolism.

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Orientador: Antonio Carlos de Laurentiz / Resumo: O experimento foi conduzido no Setor de Avicultura da Universidade Estadual Paulista – UNESP, Faculdade de Engenharia de Ilha Solteira, com a finalidade de avaliar o efeito da inclusão de níveis crescentes de goma de soja (0, 1, 2, 3, 4 e 5%) na alimentação de poedeiras comercias, e verificar a viabilidade econômica de sua utilização como mais um produto comercial derivado da soja. Foram utilizadas 180 poedeiras comerciais leves da linhagem Lohmann, com 40 semanas de idade, durante o período de 112 dias (quatro ciclos de 28 dias), distribuídas em um delineamento inteiramente casualizado, totalizando 6 tratamentos com 5 repetições (6 aves por parcela). No experimento foram avaliados dados de parâmetros zootécnicos: porcentagem de postura (ave/dia), consumo de ração (g/ave/dia), peso dos ovos (g), conversão alimentar (kg/kg), qualidade interna e externa dos ovos e estabilidade oxidativa dos ovos. No que se refere aos parâmetros de desempenho a inclusão de 5% de goma na dieta aumentou o consumo de ração e a maior produção de ovos foi observada nos tratamentos com a inclusão de 3 e 5% de goma. Quanto ao peso médio dos ovos e massa de ovos a inclusão de goma a partir de 3% favoreceu o aumento dos mesmos. Para os parâmetros de qualidade dos ovos os tratamentos com 4 e 5% de goma foram os que apresentaram os menores valores de unidade Haugh, o aumento da coloração da gema ocorreu a partir da inclusão de 3% de goma. A estabilidade oxidativa dos ovos apresentou diferença (P<0,05) apen... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre