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Os óleos e as gorduras, assim denominados em função de sua origem, são ingredientes internacionalmente indispensáveis na formulação de rações para que haja sucesso na produção de frangos de corte. Os lipídios advindos da dieta são grandes fornecedores de energia e de ácidos graxos essenciais ao metabolismo. O aproveitamento nutricional depende da digestibilidade, que, por sua vez, é influenciada pela composição química das moléculas lipídicas. Características ligadas ao comprimento da cadeia carbônica, presença ou não de duplas ligações, configuração das ligações (cis ou trans), presença de ácidos graxos na forma de triglicerídio ou livre e posição do ácido graxo na molécula de glicerol são fatores que alteram não só a digestibilidade dos lipídios, mas também de compostos lipossolúveis. No entanto, em ensaios biológicos é preciso abordar uma ótica multifacetada, considerando, dentro dos limites técnicos da experimentação animal, as diversas interações que se apresentam. Os fatores fisiológicos da ave, principalmente aqueles ligados à digestão, são de extrema importância na condução de estudos de nutrição. Existe uma infinidade de interações físico-químicas durante o processo digestivo, no qual a microbiota intestinal exerce importantes funções. A modulação dos microrganismos presentes no trato gastrintestinal do frango tem influência direta e indireta no aproveitamento da dieta, com mérito em destaque dobrado, especialmente diante dos desafios enfrentados após a União Europeia vetar o uso de antibióticos em rações para animais. Ao longo dos anos, foram realizadas pesquisas que demonstraram o efeito negativo da proliferação indesejada de microrganismos sobre a digestão dos lipídios, afetando principalmente fontes lipídicas com grandes quantidades de ácidos graxos saturados, que são mais susceptíveis às condições inadequadas da digestão. Esse efeito ocorre pela ação da coliltaurina hidrolase bacteriana, que causa desconjugação dos sais biliares endógenos das aves, prejudicando a emulsificação dos lipídios e a consequente formação de micelas. Portanto, este trabalho tem o objetivo de avaliar dietas contendo óleo de soja ou sebo bovino suplementadas com lisofosfolipídios e ácidos orgânicos, explorando as possíveis interações e benefícios que podem haver na associação desses dois aditivos / Oils and fats, so named because of their origin, are internationally indispensable ingredients in feed formulation for success in broilers production. The lipids coming from the diet are major suppliers of energy and essential fatty acids for metabolism. The nutrient utilization is dependent on digestibility, which, in turn, is influenced by the chemical composition of the lipid molecules. Characteristics related to the length of the carbon chain, presence or not of double bonds, links configuration (cis or trans), presence of fatty acids in the form of triglycerides or free fatty acids and the position of fatty acids in the glycerol molecule are factors that alter not only the digestibility of lipids, but also fat-soluble compounds. However, in biological assays, a multifaceted vision approach is required, considering, within the technical limits of animal experimentation, the various existing interactions. The physiological factors of the bird, especially those related to digestion, are extremely important in the conduction of nutrition studies. There is an infinity of physical and chemical interactions during the digestive process, in which the intestinal microbiota plays important roles. Modulation of microorganisms in the gastrointestinal tract of the chicken has direct and indirect influence on the diet utilization, with merit in featured folded, especially considering the challenges faced after the European Union prohibit the use of antibiotics in animal feeds. Over the years, researches have shown the negative effect of undesired proliferation of microorganisms on the lipid digestion, mainly affecting lipid sources with large amounts of saturated fatty acids, which are more susceptible to poor digestion conditions. Such effect occurs by the bacterial cholyltaurine hydrolase activity, causing deconjugation of endogenous bile salts of birds, damaging the emulsification of lipids and the consequent micelles formation. Therefore, this study aims to evaluate diets containing soybean oil or beef tallow supplemented with lysophospholipids and organic acids, exploring the possible interactions and benefits that can occur in the combination of these two additives

The effects of oxidatively modified low density lipoprotein (oxLDL) on atherogenesis may be partly mediated by alterations in the production of nitric oxide (NO) by vascular cells. Lipid hydroperoxides (LOOH) and lysophosphatidylcholine (lysoPC) are the major primary products of LDL oxidation. The purpose of this study was to characterize the effects of oxLDL, LOOH and lysoPC on NO production and the expression of inducible nitric oxide synthase (iNOS) gene in lipopolysaccharide (LPS) stimulated macrophages. LDL was oxidized using an azo-initiator 2,2'-azobis (2- amidinopropane) HCl (ABAP) and octadecadienoic acid was oxidized by lipoxygenase to generate 13-hydroperoxyl octadecadienoic acid (13-HPODE). Our study showed that oxLDL markedly decreased the production of NO, the levels of iNOS protein and iNOS mRNA in LPS stimulated macrophages. The inhibition potential of oxLDL on NO production and iNOS gene expression depended on the levels of LOOH formed in oxLDL and was not due to oxLDL cytotoxicity. Furthermore, 13-HPODE markedly reduced NO production and iNOS protein levels, whereas lysoPC showed only slight reduction. The effects of 13-HPODE and lysoPC did not require an acetylated LDL carrier. Our results suggest that 13-HPODE is a much more potent inhibitor of NO production and iNOS gene expression than lysoPC in LPS stimulated RAW264.7 macrophages.

Lysophosphatidylcholines (LPCs) are a class of pro-inflammatory lipids that play important roles in atherogenesis. LPC activates endothelial cells (ECs) to upregulate adhesion molecules, cytokines and chemokines, which is the initiation step of atherogenesis. However, the mechanisms underlying LPC-triggered EC activation are not fully understood. Previously considered as the toxic by-products of cellular metabolism, mitochondrial reactive oxygen species (mtROS) are recently found to directly contribute to both the innate and adaptive immune responses. Here we tested a novel hypothesis that mtROS serve as signaling mediators for LPC-induced EC activation. Using electron spin resonance and flow cytometry with mtROS-specific fluorescence probe MitoSOX, we found that several LPC species including LPC 16:0, 18:0, and 18:1 induced mtROS in human primary aortic ECs (HAECs). Mechanistically, our analysis using confocal microscopy and Seahorse XF96 mitochondrial function analyzer showed that LPC induced mtROS via increasing mitochondrial calcium-mediated increase of mitochondrial respiration. In addition, we found that mtROS scavenger MitoTEMPO abolished LPC-induced EC activation by downregulating Intercellular adhesion molecule 1 (ICAM-1) in HAECs. Moreover, our analysis with mass spectrometer analysis of histone H3 lysine acetylation and electrophoretic mobility shift assay (EMSA) showed that MitoTEMPO acts by blocking LPC-induced histone H3 lysine 14 acetylation (H3K14ac) and nuclear translocation of pro-inflammatory transcription factor activator protein-1 (AP-1). Remarkably, all the above effects can be inhibited by anti-inflammatory cytokines interleukin (IL-35) and IL-10. Our results indicate that mtROS are responsible for LPC-induced EC activation, which can be inhibited by anti-inflammatory cytokines. MtROS targeting therapies and anti-inflammatory cytokines such as IL-35 may serve as novel therapeutic targets for vascular inflammation and cardiovascular diseases. The studies in this dissertation were supported by grants from the National Institutes of Health (NIH) funded to Dr. Xiao-Feng Yang. / Pharmacology

L’infection par le virus de l’hépatite C (VHC) perturbe le métabolisme lipidique de son hôte. En effet, les particules virales sont hétérogènes mais les plus infectieuses sont celles retrouvées aux plus basses densités du fait de leur association avec des composants des lipoprotéines de très basse densité (VLDL), les lipoprotéines riches en triglycérides (TG) et contenant l’apolipoprotéine B (ApoB) produites par les hépatocytes ; ces complexes sont appelés lipo-viro-particules (LVP). De plus, les malades présentent fréquemment une stéatose hépatique, c'est-à-dire une accumulation de TG dans les gouttelettes lipidiques (GL) des hépatocytes, qui stockent les lipides neutres au sein d’une monocouche de phospholipides essentiellement constitués de phosphatidylcholine. Notre hypothèse de travail est que le VHC usurpe le métabolisme lipidique des hépatocytes au profit de la production de LVP. Une étude publiée au début de mon travail de thèse a révélé que la lysophosphatidylcholine acyltransférase 1 (LPCAT1) catalyse la synthèse de phosphatidylcholine directement à la surface des GL, avec pour conséquence un remodelage de ces dernières. Cela nous a incités à examiner si cette voie est détournée par le VHC, puis à déterminer le rôle de LPCAT1 à la fois dans le métabolisme lipidique des hépatocytes et dans le cycle infectieux du VHC. Lors de l’infection de novo par le VHC, les cellules de la lignée hépatocytaire Huh-7.5.1 et les hépatocytes humains en culture primaire (HHP) présentaient une diminution de l’expression de l’ARNm et de la protéine LPCAT1, suggérant une régulation transcriptionnelle de cette enzyme par le VHC. L’extinction de LPCAT1 en cellules Huh- 7.5.1, infectées ou non, induisait une diminution du nombre des GL accompagnée d’une augmentation de leur taille, suggérant une fusion des GL, ainsi qu’une accumulation intracellulaire de TG à l’état d’équilibre, donc une stéatose. La sécrétion des TG néosynthétisés et de l’ApoB était également augmentée, témoignant d’une augmentation de la production de VLDL. Dans les cellules Huh-7.5.1 et les HHP infectés par le VHC, l’extinction de LPCAT1 n’affectait pas la réplication du génome viral mais augmentait la production de virus infectieux, indiquant un effet sur la morphogenèse du VHC. De plus, les particules virales produites avaient une infectiosité spécifique supérieure corrélant avec une densité plus basse, des propriétés caractéristiques des LVP. En conclusion, le VHC diminue l’expression de LPCAT1, une enzyme associée aux GL des hépatocytes, ce qui apparaît comme une stratégie virale permettant d’augmenter le contenu en TG, et de là l’infectiosité spécifique des particules virales néoformées. Cibler la voie du métabolisme lipidique contrôlée par LPCAT1 représenterait une approche thérapeutique intéressante, car susceptible de réduire à la fois le titre viral et la stéatose hépatique. / No abstract

The enzyme phospholipase A2 (PLA2) specifically hydrolyzes the 2-acyl ester bond of 1,2-diacyl-3-sn-phosphoglycerides releasing fatty acids and lysophospholipids in the presence of Ca2+. Both products represent precursors for signaling molecules that can exert a multitude of biological functions including phospholipid metabolism, exocytosis and inflammation. Consequently, PLA2 not only plays a role in regulating physiological processes, but also exhibits pharmacological effects in inflammatory diseases. Nevertheless, the signaling pathway leading to cell death still remains elusive. In the present study, the cytotoxicity of Naja naja atra PLA2 toward human neuroblastoma SK-N-SH cells and leukemia K562 cells were respectively evaluated to explore the signaling pathway of PLA2-induced cell death. Upon exposure to PLA2, p38 mitogen-activated protein kinase (p38 MAPK) or c-Jun N-terminal kinase (JNK) activation, extracellularsignal-regulated protein kinase (ERK) inactivation, reactive oxygen species (ROS) generation, increase in intracellular Ca2+ concentration, the loss of mitochondrial membrane potential (£G£Zm), cytochrome c release and upregulation of Fas/FasL were found in SK-N-SH or K562 cells. N-Acetylcysteine (ROS scavenger), BAPTA-AM (Ca2+ chelator), SB202190 (p38 MAPK inhibitor) or SP600125 (JNK inhibitor) abrogated p38 MAPK or JNK activation and rescued cell viability, £G£Zm, cytochrome c release and suppressed Fas/FasL upregulation of PLA2-treated cells, but restored phosphorylation of ERK. Activated ERK was found to attenuate p38 MAPK-mediated upregulation of Fas/FasL. Besides, sustained JNK activation was also observed in SB202190/PLA2-treated K562 cells after exterminating p38 MAPK activation, but also retained the cytotoxicity of PLA2. Knockdown of p38 MAPK or JNK1 by siRNA proved that PLA2 induced Fas/FasL upregulation through p38 MAPK/ATF-2 or JNK1/c-Jun pathways in K562 cells. Furthermore, deprivation of catalytic activity could not diminish PLA2-induced cell death and Fas/FasL upregulation. The cytotoxicity of arachidonic acid (AA) and lysophosphatidylcholine (LPC) was not related to the expression of Fas/FasL. The results showed that the cytotoxicity of AA is mediated through mitochondria-dependent death pathway, eliciting by AA-induced ROS generation and Ca2+-evoked activation of p38 MAPK and JNK. Besides, ERK activation abrogated by U0126 improved the ability of AA-mediated Fas/FasL upregulation in K562 cells. Taken together, our results indicate that PLA2-induced cell death is through Ca2+- and ROS evoked p38 MAPK or JNK activation. Upregulation of Fas/FasL partially involves in cytotoxicity of PLA2.

Phospholipids (PL) are converted into lipid biomarkers by the action of phospholipases and reactive oxygen species (ROS), which are activated or released under certain physiological and pathophysiological conditions. Therefore, the in vivo concentration of such lipid biomarkers [e.g., lysophospholipids (LPLs)] is altered in humans and animals under different conditions such as inflammation, stress, medication, and nutrition. LPLs are particularly interesting because they are known to possess proand anti-inflammatory properties and may be generated by two different pathways: either by the influence of phospholipase A2 or by different reactive oxygen species that are generated in significant amounts under inflammatory conditions. Both lead to the cleavage of unsaturated acyl residues. This review provides a short summary of the mechanisms by which lipid biomarkers are generated under in vitro and in vivo conditions. The focus will be on lysophosphatidylcholine (LPC) because usually, this is the LPL species which occurs in the highest concentration and is, thus, easily detectable by chromatographic and spectroscopic methods. Finally, the effects of lipid biomarkers as signaling molecules and their roles in different human and animal pathologies such as infertility, cancer, atherosclerosis, and aging will be shortly discussed.

L’acide docosahexaénoïque (DHA, 22:6n-3) est le principal acide gras oméga-3 des tissus cérébraux et est essentiel au développement et aux fonctions du cerveau. Une diminution de la concentration cérébrale du DHA est observée chez les patients souffrant de maladies neurodégénératives telles que les maladies d'Alzheimer et de Parkinson. Un apport ciblé du DHA au cerveau pourrait compenser ces carences. Le DHA sanguin est transporté à travers la barrière hémato-encéphalique (BHE) plus efficacement lorsqu’il est estérifié en position sn-2 de la lysophosphatidylcholine (lysoPC). Nous produisons au laboratoire une phosphatidylcholine structurée pour mimer la 2-docosahexaénoyl-lysoPC (lysoPC-DHA), nommée AceDoPC (1-acétyl,2-docosahexaénoyl-glycérophosphocholine), qui peut être considérée comme une forme stabilisée de la lysoPC-DHA physiologique et qui est neuroprotectrice dans l’accident ischémique cérébral. Le premier objectif de ce travail a été de comparer le passage du DHA marqué non estérifié ou estérifié dans l’AceDoPC ou dans une phosphatidylcholine (PC-DHA), lié au plasma, à travers un modèle in vitro de la BHE. Nous montrons un passage préférentiel à travers la monocouche endothéliale et une captation préférentielle par les cellules gliales de l’AceDoPC comparativement au DHA non estérifié et à la PC-DHA. Le deuxième objectif de ce travail a été de confirmer si cette préférence pour la forme AceDoPC était également observée in vivo. Nous avons donc étudié, chez des rats âgés de 20 jours, la captation cérébrale des différentes formes d’apport du DHA précédemment utilisées (DHA, AceDoPC, PC-DHA). Nous démontrons que l’AceDoPC apporte le DHA au cerveau plus efficacement que les autres formes d’apport de DHA et que cette préférence pour l’AceDoPC est spécifique au cerveau puisqu’elle n’est pas observée pour les autres organes étudiés. L’AceDoPC est trouvée, en partie, sous forme intacte dans le cerveau. L’autoradiographie ex vivo du cerveau de rat révèle que le DHA provenant de l’AceDoPC est localisé dans des régions cérébrales spécifiques jouant un rôle important dans la mémoire et les fonctions cognitives. Enfin, en utilisant des approches de modélisation moléculaire, nous démontrons que les potentiels électrostatiques et hydrophobes sont distribués de manière très similaire au niveau des surfaces de l’AceDoPC et de la lysoPC-DHA. En conclusion, nos études montrent que l’AceDoPC est un transporteur privilégié et spécifique du DHA au cerveau. En considérant les rôles essentiels du DHA pour le cerveau, cette nouvelle approche de ciblage cérébral du DHA offre des perspectives prometteuses dans le développement de stratégies préventives et thérapeutiques pour les maladies neurologiques. / Docosahexaenoic acid (DHA, 22:6n-3) is the main essential omega-3 fatty acid in brain tissues required for normal brain development and function. A decrease in the cerebral concentration of DHA is observed in patients suffering from neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Targeted intake of DHA to the brain could compensate for these deficiencies. Blood DHA is transported across the blood-brain barrier (BBB) more efficiently when esterified at the sn-2 position of lysophosphatidylcholine (lysoPC). We produce in the laboratory a structured phosphatidylcholine to mimic 2-docosahexaenoyl-lysoPC (lysoPC-DHA), named AceDoPC (1-acetyl,2-docosahexaenoyl-glycerophosphocholine), that may be considered as a stabilized form of the physiological lysoPC-DHA and that is neuroprotective in experimental ischemic stroke. The first objective of this work was to compare the passage of either labeled unesterified DHA or DHA esterified in AceDoPC or in phosphatidylcholine (PC-DHA), bound to plasma, through an in vitro model of the BBB. This model is constituted of a co-culture of bovine brain capillary endothelial cells and glial cells from newborn rats. We show a preferential passage through the endothelial monolayer and a preferential uptake by glial cells of AceDoPC compared to unesterified DHA and PC-DHA. We also show that AceDoPC is hydrolyzed, partly, into lysoPC-DHA and that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most labeled lipid classes in endothelial cells and glial cells. AceDoPC is found, partly, as a whole molecule in the cells. The second objective of this work was to confirm whether this preference for AceDoPC was also observed in vivo. We studied, in 20 days old rats, the brain uptake of different forms of DHA previously used (DHA, AceDoPC, PC-DHA). We demonstrate that AceDoPC provided the brain with DHA more efficiently than the other forms of DHA and that this preference for AceDoPC is specific for the brain because it is not observed for other studied organs. AceDoPC is found, partly, intact in the brain. Ex vivo autoradiography of rat brain reveals that DHA provided from AceDoPC is localized in specific brain regions playing key roles in memory and cognitive functions. Finally, by using molecular modelling approaches, we demonstrate that electrostatic and hydrophobic potentials are distributed very similarly at the surfaces of AceDoPC and lysoPC-DHA. In conclusion, our studies demonstrate that AceDoPC is a privileged and specific carrier of DHA to the brain. Considering the essential roles of DHA for the brain, this new approach to target the brain with DHA offers promising perspectives in the development of preventive and therapeutic strategies for neurological diseases.

La découverte d’adjuvants de vaccination est en pleine expansion dans un contexte règlementaire de plus en plus strict. L’objectif de ce travail de thèse a été de proposer un adjuvant hautement caractérisé de la famille de la lysophosphatidylcholine (LPC) pour un développement clinique. Il avait été précédemment montré que la LPC d’oeuf de poule présentait des propriétés adjuvantes in vitro et in Vivo. Cependant, il s’agit d’un mélange hétérogène d’espèces moléculaires de LPC peu exploitable en clinique. Nous avons donc cherché à 1/ cribler in vitro 5 différentes espèces moléculaires de LPC ayant des activités adjuvantes, et 2/ évaluer chez la souris l’aptitude des molécules sélectionnées à induire des réponses humorales et cellulaires.Deux candidats, les LPC C16 :0 et C18 :0, induisent in vitro la maturation de cellules dendritiques humaines caractérisée par l’augmentation de l’expression des marqueurs de surface, la production de chimiokines pro-Th1 et l’engagement vers un profil Th1 de lymphocytes T CD4+. Chez la souris, l’injection i. v. des candidats induit une réponse inflammatoire transitoire et modérée au profil Th2 (IL-5, IL-6). De plus, ces espèces induisen une réponse humorale spécifique contre 3 antigènes viraux après injection s. c. ou i. m. (NS3du VHC, HBsAg du VHB et gp120 du VIH), proche de celle obtenue avec l’alun et à des doses faibles supposées non toxiques. Dans ces modèles, en revanche, les LPC individuelles ne semblent pas induire de réponses cellulaires spécifiques.L’ensemble de ces résultats ouvre d’intéressantes perspectives pour l’utilisation des LPC C16 :0 et C18 :0 en tant qu’adjuvants de vaccination de réponses immunitaires humorales. / The evaluation of new vaccine adjuvants is growing fast in a stringent regulatory environment. The aim of this thesis project was to propose a highly defined adjuvant derived from the lysophosphatidylcholine (LPC) for clinical development. It has been previously shown that an egg-derived LPC has in vitro and in Vivo adjuvant features. However, it is composed of a heterogeneous mixture of molecular species of LPC, thus precluding itsclinical evaluation. Therefore, we decided to 1/ screen in vitro 5 single species of LPC fortheir adjuvant properties, and 2/ evaluate in the mouse model the ability of selected compounds to induce humoral and cellular responses.Two candidates, the C16:0- and C18:0-LPC, induce in vitro maturation of human dendritic cells as defined by an up regulation of the expression of surface markers, production of pro-Th1 chemokines, and engagement of CD4+ T lymphocytes towards a Th1 profile. Inmice, i. v. injection of both candidates triggers a transient and moderate inflammatory response with a Th2 profile (IL-5, IL-6). In addition, these species initiate specific humoral responses against 3 viral antigens following s. c. or i. m. injection (NS3 from HCV, HBsAgfrom HBV and gp120 from HIV), close to what is achieved with alum and at low doses expected to be safe in humans. In these models, however, single LPC do not mediatespecific cellular responses. Over all, these results open interesting perspectives for the single

Generation of foam cells, an essential step for reverse cholesterol transport (RCT) studies, uses the technique of receptor dependent macrophage loading with radiolabeled acetylated Low Density Lipoprotein (Ac-LDL). In this study, we used the ability of a biologically relevant detergent molecule, Lysophosphatidylcholine (Lyso PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabelled cholesterol / Lyso PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4°C and retained the solubilized cholesterol during one month storage. Macrophages incubated with cholesterol or CE (unlabeled, fluorescently labeled or radiolabeled) / Lyso PtdCho mixed micelles accumulated CE as documented by microscopy, lipid staining, labeled oleate incorporation, and by thin layer chromatography (TLC). Such foam cells unloaded cholesterol when incubated with high density lipoprotein (HDL) and not with oxidized HDL (Ox-HDL). We propose that stable cholesterol or CE / Lyso PtdCho micelles would offer advantages over existing methods. Oxidative stress is associated with heart failure (HF). Previously our research group observed that the patients with low left-ventricular ejection fraction showed accumulation of high level of oxidized LDL (Ox-LDL) when compared with the heart failure patients with normal range of ejection fraction (EF). HDL is known to be atheroprotective and one of its important antioxidative functions is to protect LDL from oxidative modifications. However, HDL itself undergoes oxidation and Ox-HDL becomes functionally poor. It is expected to have a diminished ability to promote reverse cholesterol transport. Therefore, it was hypothesized that the quality of HDL present in the patients with EF would more compromised than those present in the patients with normal EF. Functionality of HDL was evaluated by measuring its cholesterol efflux capacity from foam cells generated in vitro. Functionality of HDL, which is strongly related to the oxidative modifications of HDL was further estimated by measuring paraoxonase 1 (PON1) enzyme activity associated with HDL. Higher the PON1 activity and RCT ability, better is the functionality of HDL.