No description available.

Intermediate lipid structures such as inverted micelles and interlamellar attachment are thought to play a crucial role in different biological processes like viral infection. Lysophosphatidylcholine has been shown to inhibit membrane fusion at stabilizing concentrations (between 1 and 10% with respect to membrane lipids). Studies in this thesis looked at the effects of Lysophosphatidylcholine (LPC) properties on the inhibition of Sendai viral fusion. The effects of bilayer sidedness preference as well as flip-flop of Lysophosphatidylcholine (lyso PC) were examined. Octadecylrhodamine (R₁₈) lipid mixing assays were used to measure the fusion of Sendai virus with different biological, erythrocyte ghosts, and artificial systems consisting of different lipids and different viral receptor compositions. The data showed that external addition of LPC exhibits a dependency between the incubation time of the lysolipids and the inhibition of viral fusion. The results also demonstrate a relationship between the location of LPC in the bilayer and its ability to inhibit lipid mixing. LPC present only on the outer monolayer plays a role in the inhibition of viral fusion. Reorientation of LPC was also measured for the same incubation periods. A method using Bovine Serum Albumin (BSA) and radioactivity labelled LPC, was applied to measure the flip-flop. Significant transbilayer reorientation of Lyso PC in the bilayer was shown to take place. The rate of flip-flop was measured at 0.32 ± 0.08% LPC /min. Such reorientation can explain the time dependency observed earlier. The conclusions of this thesis lend support to stalk intermediate mechanism of viral membrane fusion. The ability of LPC to inhibit only when present on one side of the bilayer supports the idea of a negatively curved stalk intermediate. Moreover, it showed that the shape and curvature tendencies of the bilayer stabilizer determine its effects on viral fusion. / Thesis / Master of Science (MS)

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Lysophosphatidylcholine (LPC) is a by product of phospholipid metabolism, that under physiological conditions is maintained at a low level. However, through an enhanced degradation of phospholipids and/or a reduced catabolism, LPC accumulates in the plasma and fluids of patients with disorders underscored by inflammation - such as atherosclerosis, diabetes, ischaemia and epilepsy. Previous studies have demonstrated LPC to possess vasoactive properties, able to both induce and inhibit vasodilation. Furthermore, a variety of proteins are sensitive to LPC, including non-selective cation (NSC) channels and Ca2+-activated K+ (KCa) channels. These channels are intimately associated with the maintenance and regulation of vascular tone. The aim of this study was to elucidate the mechanisms underlying the vascular effect of LPC.Aortic segments were constricted with phenylephrine and exposed to cumulative concentrations of LPC, with an ensuing endothelium-dependent, concentration-dependent vasodilation. Inhibitors of nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) abolished LPC-induced responses, implicating nitric oxide (NO) as the mediator. Two cation fluxes were implicated in the dilator activity of LPC - Ca2+ and K+. NSC channel antagonists and reduced extracellular Ca2+ concentration attenuated dilation and reduced the Ca2+ signal activated in isolated rat aortic endothelial cells (RAEC) by LPC, implicating endothelial Ca2+ influx in the response. In addition, LPC also evoked a robust hyperpolarisation of isolated RAEC membrane potential. The K+ channel antagonists TEA+, TRAM-34 and apamin, inhibitors of KCa channels, attenuated both the LPC-induced dilation and RAEC membrane hyperpolarisation, highlighting their potential role in mediating both these processes. HEK293 cells, which lack many of the channels and signalling pathways possessed by other cells, mimicked RAEC in their sensitivity to LPC, generating robust elevations of intracellular Ca2+ when exposed to this lysolipid. Likewise, membrane hyperpolarisations were also observed in HEK293 cells, however, these only occurred when cells expressed recombinant KCa channels. This suggests that KCa channel activation is dependent upon Ca2+ influx, not vice versa. Phospholipase C (PLC) inhibitor U73122, attenuated LPC-induced hyperpolarisation, raising the question as to the possible involvement of G-protein coupled receptors in the bioactivity of LPC. Alternately, LPC might initiate PLC activity, and subsequent NSC channel opening and Ca2+ influx via a perturbation of membrane integrity, like certain local anaesthetics. It is proposed that endothelial NSC-channel activation by LPC initiates endothelial cell signalling, with concomitant activation of Ca2+-sensitive proteins such as NOS, to bring about vasodilation, and KCa channels, which modulate membrane potential and in turn the driving force for Ca2+ entry.

No description available.

It is well established that cytosolic caspase-1 activation, mediated by inflammasome after pathogens-associated molecular patterns (PAMP) and metabolic danger-associated molecular patterns (DAMPs), mediates the initiation of inflammation in endothelial cells by its downstream targets such as Interleukin-1β (IL-1β), Interleukin-18 (IL-18), and Sirtuin-1. However, it remains unknown whether proatherogenic lipids lysophosphatidylcholine (LPC) and reactive oxygen species (ROS) can promote nuclear localization of caspase-1. Using biochemical, bioinformatic, and immunologic approaches, we made the following findings: (1) DNA damage was found in atherosclerotic mice. (2) A nuclear exportation signal was mapped in the CARD domain of pro-caspase-1. LPC promotes nuclear localization of pro-caspase-1 in human aortic endothelial cells (HAECs), which may interrupt DNA damage and repair pathways. (3) Blockage of caspase-1 nuclear cytosol trafficking in HAEC activated by LPC may mediate inflammation and interrupt cell cycle regulation. (4) Pro-caspase-1 in the nucleus inhibits inflammation but promotes interferon pathways. Activation of caspase-1 in the nucleus promotes aging- and fos-related antigen 2 (FRA2) mediated DNA damage and apoptosis. (5) Inhibition of SUMOylation decreases pro-caspase-1 translocation into the cytosol from the nucleus. (6) Blockage of caspase-1 cytosol nuclear trafficking in HAEC activated by H2O2 may decrease caspase-1 activity and increase cell viability. Our results demonstrate, for the first time, that caspase-1 patrols in the cell, senses danger signals and interrupts the balance between DNA damage and DNA repair pathways. It is a novel insight that not only should we suppress the inflammation in the cytosol but also in the nucleus, which is important for the future development of therapeutics for cardiovascular diseases and other inflammatory diseases. / Biomedical Sciences

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

Peripheral nerve damage affects hundreds of thousands of people every year. This study tested the effectiveness of using lysophosphatidylcholine (LPC) in combination with nerve growth factor (NGF) to increase the healing rate of damaged left sciatic nerves in female rats. The rats were randomly divided into eight groups: Sham, Right Sciatic, Crush, LPC, LPC-NGF, Crush- LPC, Crush-NGF, and Crush-LPC-NGF. The healing of the nerves was measured by monitoring gait, electrophysiological parameters (compound muscle action potential amplitudes and nerve conductance velocities) and morphological parameters (total fascicular area, total myelinated fiber counts, fiber densities, fiber diameters, and g-ratio). Gait and electrophysiological parameters were measured three times a week. Morphological parameters were measured at three weeks and at six weeks. The LPC and LPC-NGF groups were not statistically different from the controls (Sham and Right Sciatic) at either of the morphological time points but were statistically different from the controls for the first three weeks for the electrophysiological parameters and gait. The LPC-NGF group did not differ from the LPC group at any time point for any of the parameters. Crush, Crush-LPC, Crush-NGF, and Crush-LPC-NGF groups statistically differed from the controls at week 3 for all parameters and only in the electrophysiological parameters at week 6. Crush-LPC, Crush-NGF, and Crush-LPC-NGF did not differ from each other or from the Crush group. The combination of LPC and NGF did not prove to be an effective treatment for peripheral nerve damage. Future work is recommended to test multiple injections of LPC and NGF.

Increased expression of autotaxin has been shown to promote metastasis formation and cancer proliferation. These actions could be related to the catalytic activity of autotaxin which converts lysophosphatidylcholine into lysophosphatidate extracellularly or non-catalytic functions of autotaxin may be responsible. Also both LPC and LPA have been reported to stimulate migration through their respective receptors. This work investigates the role of autotaxin in controlling the motility of two cancer cell lines. With the use of autotaxin inhibitors we were able to block LPC-induced migration. Knocking-down autotaxin secretion also blocked stimulation of migration by LPC. Autotaxin inhibitors abolished any migratory effects from media collected from autotaxin secreting cells. We determined that LPC alone is unable to stimulate migration. Also we did not observe non-catalytic effects of autotaxin on migration. This thesis provides strong evidence that the inhibition of autotaxin production or activity would provide a beneficial therapy in the prevention of tumour growth or metastasis in patients with autotaxin expressing tumours.

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.