Rabbit antisera directed against human milk sialyloligosaccharides were used to detect specific monosialylgangliosides from the lipid fraction of human meconium. Gangliosides of this fraction were detected after thin layer chromatography by immuno-staining with specific anti-oligosaccharide sera. The monosialylganglioside fraction of human meconium was subjected to ozonolysis and alkali-fragmentation and the resulting ganglioside-derived oligosaccharides were reduced with NaB [³H]₄ and partially separated using paper chromatography. The [³H]-oligosaccharide alditols were assayed for binding to specific anti-oligosaccharide sera in a direct-binding radioimmunoassay using nitrocellulose filters to collect immune-complexes. Radiolabeled oligosaccharide alditols which were recognized by specific antisera were affinity purified by eluting nitrocelIulose filters containing antibody-oligosaccharide complexes or using columns of immobilized anti-oligosaccharide antibodies. Structural analyses of two sialyl[³H]tetrasaccharide alditols obtained in this way were carried out with sequential enzymatic degradation using specific exoglycosidases. The products of enzymatic digestions were identified by cochromatography in paper with known standards. Data obtained from these experiments are consistent with the presence of the following, previously unidentified gangliosides in human meconium: NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-cer Galβ1-3[NeuAcα2-6]GlcNAcα1-3Galβ1-4Glc-cer / Ph. D.

Un membre de la famille des tétraspanines, CD82, est une protéine transmembranaire et l'un des rares suppresseurs de métastase identifié jusqu'à présent. Cependant, le mécanisme de suppression de métastase induite par CD82 reste mal compris. Les tétraspanines, y compris CD82, ont la propriété unique de créer un réseau d'interactions protéines-protéines à la membrane plasmique, appelé « tetraspanin web ». Dans ce réseau, CD82 est connu pour interagir avec d’autres tétraspanines, y compris CD9, CD81 et CD151, en plus d’autres protéines membranaires telles que les intégrines, les récepteurs de facteurs de croissance et les protéines de type immunoglobuline. De plus, des travaux antérieurs ont identifié que l'interaction de CD82 avec l’EGFR, d'autres tétraspanines et les intégrines dépend de l'expression des gangliosides au sein de la membrane plasmique.À ce jour, les études dans ce domaine ont utilisé des techniques d'ensemble qui ne peuvent pas tenir compte de la dynamique et de la stochasticité de la membrane, alors qu'il est maintenant bien établi que l'organisation spatio-temporelle de ses composants est cruciale pour certaines fonctions cellulaires.Ainsi, lors de ma thèse de doctorat, j'ai cherché à étudier à la fois la dynamique et la compartimentation de CD82 et de ses partenaires à la membrane plasmique des cellules épithéliales mammaires HB2. Pour ce faire, la technique de pistage en molécule unique basée sur l’utilisation d’un microscope TIRF a été utilisée afin d’obtenir des informations directes à l'échelle nanométrique sur la dynamique de protéines individuelles dans les cellules vivantes. Nos expériences en pistage de molécule unique ont démontré que l'expression de CD82 augmentait la dynamique CD81 à la membrane plasmique des cellules HB2 et modifiait ses interactions au sein du tetraspanin web. En revanche, les dynamiques de CD9 et de l’intégrine α3 n'ont pas été modifiées par l'expression de CD82. De plus, en modifiant enzymatiquement l'expression des gangliosides, nous avons montré que ces lipides sont impliqués à la fois dans la dynamique et la compartimentation des tétraspanines à la membrane plasmique. En effet, la déplétion en gangliosides entraine une augmentation de la dynamique de CD82, CD81 et de l’intégrine α3 ainsi qu'une redistribution des tétraspanines à la membrane plasmique. Nous avons également étudié la migration en 2D des cellules HB2 et montré que CD82 et les gangliosides modifiaient de façon différentielle la migration des cellules HB2.L’ensemble de nos résultats démontrent que CD82 et les gangliosides modulent de manière différente la dynamique et la compartimentation des tétraspanines et de leurs partenaires à la membrane plasmique des cellules HB2. Enfin, ce travail suggère que l'activité de CD82 en tant que suppresseur de métastase pourrait être en partie liée à sa capacité, en coopération avec les gangliosides, à moduler l'organisation spatio-temporelle de ses partenaires au sein du tetraspanin web. / A member of the family of tetraspanins, CD82, is a transmembrane protein and one of the rare metastasis suppressors identified so far. However, the mechanism of CD82-induced metastasis suppression remains not fully revealed. Tetraspanins, including CD82, have the unique property to create a network of protein-protein interactions within the plasma membrane, called tetraspanin web. Within this network, tetraspanins interact with each other (eg. CD82 with CD9, CD81 and CD151) as well as with other proteins, such as: integrins, growth factor receptors and immunoglobulin-like proteins. Additionally previous work has identified that the interaction of CD82 with EGFR, other tetraspanins and integrins depends on the expression of gangliosides at the plasma membrane.To date, studies in this field have employed ensemble-averaging techniques which are unable to account for membrane dynamics and stochasticity. Nevertheless, it is now well established that the spatio-temporal organization of its components is crucial for cellular functions.Thus, during my PhD thesis I aimed to study both the dynamics and partitioning of CD82 and its partners at the plasma membrane of HB2 mammary cells. To achieve this aim, a TIRF-based Single Molecule Tracking (SMT) approach was employed to provide direct nanoscale insights by observing individual proteins in living cells. Our SMT experiments demonstrated that CD82 overexpression increased CD81 dynamics at the plasma membrane of HB2 cells and modified its interaction within the tetraspanin web. In contrast, CD9 and α3 integrin dynamics were not modified by CD82 expression. Moreover, by enzymatically tuning gangliosides expression, we showed that these lipids are involved in both dynamics and partitioning of tetraspanins at the plasma membrane. Indeed, gangliosides depletion resulted in an increase in CD82, CD81 and α3 integrin dynamics as well as a redistribution of tetraspanins at the plasma membrane. We also investigated the 2D migration of HB2 cells showing that CD82 and gangliosides differentially altered the cellular migration of HB2 cells.Taken together, our results demonstrate that both CD82 and gangliosides differentially modulate the dynamics and partitioning of tetraspanins and their partners at the plasma membrane of HB2 cells. Finally, this work suggests that CD82 activity as metastasis suppressor could be in part linked to its ability, in cooperation with gangliosides, to modulate the spatio-temporal organization of its partners within the tetraspanin web.

La maladie d'Alzheimer, maladie neurodégénérative la plus courante, est la cause de50% des cas de démence. La maladie d’Alzheimer est provoquée par l'agrégation d'unamyloïde, le peptide Aβ1-42, dans le cerveau des patients.De nombreuses études relient la toxicité des amyloïdes à l'existence de diversesstructures intermédiaires survenant avant la formation des fibres et / ou leur interactionspécifique avec les membranes.Dans cette étude, nous nous sommes centrés sur l'interaction entre des modèlesmembranaires et le peptide Aβ1-42 (WT et des mutants plus ou moins toxiques) évaluée parplusieurs techniques biophysiques (ellipsométrie, PM-IRRAS, fluorescence de la ThT, fuitede calcéine, PWR, cryo-MET). Nous avons tout d'abord étudié l’interaction avec des modèlesde membrane simples (100% DOPG ou 100% DOPC). Nous avons établi que la force motricede l'interaction entre tous les peptides et la membrane n’est pas régie par des interactionsélectrostatiques, mais est favorisée en présence des têtes polaires PG qui peuvent interagiravec le peptide par l'intermédiaire de liaisons hydrogènes. Nous avons démontré quel'oligomère le plus toxique induit des dommages sur les membranes de PG, ce qui diminue laformation de fibres.Une nouvelle composition lipidique constituée de GM1, cholestérol, sphingomyélineet POPC a été choisie pour mimer les membranes neuronales. Des techniques innovantes : laspectroscopie infrarouge à l'échelle nanométrique et l’AFM haute-vitesse ont été utilisées,respectivement, pour accéder à la morphologie et la structure secondaire des peptides enprésence de membranes et observer la dynamique de cette interaction. Les résultats obtenusmontrent que les gangliosides GM1 et le cholestérol jouent un rôle central dans l'interactiond’Aβ avec les membranes. Nous avons été en mesure de proposer un modèle du mécanismed'interaction : Aβ1-42 s’accumule sur les domaines de GM1 présents dans la membrane via desliaisons hydrogène, puis s’insère dans la membrane par les domaines enrichis en cholestérol.Le cholestérol et les gangliosides sont nécessaires pour l'interaction d’Aβ1-42 avec lamembrane.Afin de suivre la cinétique d'agrégation d’Aβ et de comprendre ses différents étatsd'agrégation par spectroscopie infrarouge, l’élaboration d’une cellule microfluidique adaptée aété entreprise. / Alzheimer’s disease is the most common neurodegenerative disease, leading to 50% ofdementia cases, caused by the aggregation of an amyloid, the Aβ1-42 peptide in patients brain.Many studies link the toxicity of amyloids, as A1-42 involved in Alzheimer disease, tothe existence of various intermediate structures prior to fiber formation and /or their specificinteraction with membranes.In this study we focused on the interaction between membrane models and A1-42peptides and variants more or less toxic with several biophysical techniques (ellipsometry,PM-IRRAS, ThT fluorescence, calceine leakage, PWR, cryo-TEM). First, with simplemembrane models (pure DOPG or pure DOPC), we established that the driving force for theinteraction between all the peptides and membrane is not governed by electrostatic interactionbut is favored in presence of PG headgroups that may interact with peptide via hydrogenbonding. We demonstrated that the most toxic oligomer induces PG membrane damage,decreasing the formation of fibers.New lipid composition GM1, cholesterol, sphingomyelin and POPC has been chosento mimic neuronal membranes. Innovative techniques: as nanoscale infrared spectroscopy andhigh-speed AFM were used to assess to the morphology and the secondary structure of thepeptides in presence of membrane and to observe the dynamic of this interaction,respectively. The results obtained show that the gangliosides GM1 and the cholesterol play acentral role in the interaction of Aβ with membranes. We were able to propose a model of theinteraction mechanism: Aβ1-42 firstly accumulates on the GM1 domains present in themembrane via hydrogen bonding and then inserts the membrane in the cholesterol enricheddomains. Cholesterol and gangliosides are required for the interaction of Aβ1-42 withmembrane.In order to follow the kinetic of Aβ agregation and understand its different agregationstates with infrared spectroscopy, a microfluidic cell fabrication has been investigated.

Accidents Vasculaire Cérébraux (parfois appelés AVC ou attaques cérébrales) sont les 3èmes causes principales de mortalité et les causes de handicap dans les pays industrialisés. Ils représentent un problème de santé publique en raison de leurs fréquences, de leurs mortalités et des handicaps physiques et cognitifs qu'ils entraînent. Malgré d’importants progrès réalisés dans le domaine de la physiopathogénie de l’ischémie cérébrale, on ne dispose pas encore, aujourd’hui, de thérapeutiques efficaces pour traiter un accident vasculaire cérébral lors de sa phase aiguë. Les gangliosides GM1 sont des composants majeurs du feuillet externe de la membrane cellulaire au niveau du système nerveux central. Ces gangliosides ont des effets antineurotoxiques, neuroprotecteurs et les propriétés neurorestoratrices sur les divers neurotransmetteurs du SNC mais pour le moment ils n’ont pas des valeurs thérapeutiques en raison de leurs manques de biodisponibilités et de leurs manques de perméabilité de la barrière hématoencéphalique (BHE). Nous avons synthétisé les structures simplifiées de ces gangliosides GM1, L’idée qui a prévalu pour la conception, des nouvelles structures a été : d’introduire des chaînes grasses saturées ou insaturées sur un motif sérine, tyrosine ou cystéine, de remplacer la fonction carboxylique par des groupements reconnus comme bioisostères classiques ou non classiques de cette fonction ; comme par exemple les fonctions phosphonique, tétrazolique, 2,4-oxadiazolidine-3,5-dione. Nous avons élaboré un nouveau modèle de composé neuroprotecteur dans lequel une chaîne grasse est couplée à une entité acide ascorbique pour améliorer le passage de la BHE. Pour étudier l’activité neuroprotectrice des composés synthétisés nous avons utilisé deux modèles biologiques in vitro : un modèle cellulaire (cellules HT22) et un modèle tissulaire (tranches de cerveaux). / Stroke is the third leading cause of mortality and the primary cause of disability in adults. Therefore, it is critical to identify new, efficacious pharmacological treatments. One pharmacological approach for treatment of stroke is called neuroprotective therapy. It has been reported that the amphiphilic, monosialotetrahexosylganglioside (GM1) (II3 NeuAc-GgOsc4Cer) has antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. Since GM1 is not of therapeutic value because of its lack of bioavailability and its low blood-brain barrier (BBB) permeation. Thus, molecules that mimic GM1 represent a novel approach to neuroprotection. We have synthesized the smalls GM1-like analogues whose simplified structure includes various lipophilic saturated, unsaturated, or cyclic polyunsaturated moieties have been introduced, while in the second series, thecarboxylic acid function was replaced by different hydrophilic groups including bioisosters of the carboxylate, such as a phosphonic acid, a tetrazole, the 1,2,4-oxadiazolidine-3,5-dione or an ascorbic acid moiety. Introduction of ascorbic acid was supported by several reports that showed that ascorbic acid conjugates can improve BBB permeation properties. We report their neuroprotective effects in two distinct models of nerve cell death using hippocampus-derived HT22 cells and an additional neuroprotective assays using cortical slices injured by glutamate confirmed these results.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Os mastócitos são células multifuncionais do sistema imunológico que participam em diversos processos biológicos. As funções dos mastócitos estão diretamente relacionados com a sua ativação e, subsequente, liberação de mediadores químicos. Os eventos iniciais da ativação dos mastócitos e da transdução de sinais ocorrem em microdomínios lipídicos (lipid rafts) da membrana plasmática. Os gangliosídeos derivados do GD1b são constituintes dos lipid rafts de mastócitos de roedores. O intercruzamento destes gangliosídeos pelo mAb AA4, resulta na formação de agregados (caps) na superfície celular e promove uma ativação parcial dos mastócitos, sem que ocorra a desgranulação. A ativação é semelhante a observada quando os FcRIs são intercruzados por antígenos multivalentes ligados a IgEs, mas neste caso ocorre a desgranulação. O presente estudo tem como objetivo caracterizar o papel dos gangliosídeos derivados do GD1b na liberação de mediadores de mastócitos da linhagem RBL-2H3. O intercruzamento dos gangliosídeos derivados do GD1b resulta na ativação dos fatores de transcrição NFAT e NFB e esta ativação é mediada pela proteína quinase Syk. A ativação destes fatores de transcrição resulta na liberação de mediadores neo-sintetizados, tais como: TNF-, interleucina (IL)-4. Por outro lado, o intercruzamento dos gangliosídeos derivados de GD1b não induz a liberação dos mediadores neoformados como o leucotrieno B4 (LTB4) e o leucotrieno C4 (LTC4). A agregação dos gangliosídeos derivados do GD1b resulta na desorganização dos lipid rafts e na redistribuição de seus componentes, como demostrado pela análise proteômica. Estes dados mostraram proteínas capazes de desencadear uma ativação parcial dos mastócitos e proteínas reguladoras negativas da desgranulação estão up reguladas, enquanto que proteínas críticas para a transdução do sinal estão down reguladas. Os resultados obtidos neste trabalho demonstram que os gangliosídeos derivados do GD1b desempenham papel crucial na integridade dos lipid rafts modulando a ativação e liberação de mediadores de mastócitos. / Mast cells are immunoregulatory cells that participate in diverse biological events. The action of mast cells is directly related to their activation and subsequent mediator release. Early signal transduction events occur in lipid rafts in the plasma membrane. GD1b-derived gangliosides are known constituents of lipid rafts in rodent mast cells. The cross-linking of these gangliosides by mAb AA4 results in a partial activation of mast cells similar to that observed when FcRIs are cross-linked, but does not result in the mast cell degranulation. With time, the gangliosides bound to mAb AA4 cap on the cell surface. The present study aims to characterize the role of the rodent mast cell specific gangliosides derived from GD1b in mediator release from RBL-2H3 mast cells. Cross-linking the GD1b-derived gangliosides activated the transcription factors NFAT and NFB and this activation was mediated by Syk. The activation of theses transcription factors by cross-linked GD1b-derived gangliosides results in the release of the neo-synthesized mediators TNF- and interleukin (IL)-4. However, cross-linking GD1b-derived gangliosides did not stimulate release of the newly formed mediators leukotriene B4 (LTB4) and leukotriene C4 (LTC4). Capping of GD1b-derived gangliosides disorganized lipid rafts and resulted in a redistribution of lipid raft components. Proteomic analysis showed that proteins that trigger mast cell activation and negative regulatory proteins of degranulation are up regulated, whereas proteins critical for signal transduction are down regulated in mast cells where the gangliosides are capped. The results of this work demonstrate that the mast cell-specific GD1b-derived gangliosides are crucial in maintaining the functional integrity of the lipid rafts and modulate cell activation and subsequent mediator release from mast cells.

As células tronco mesenquimais de tecido adiposo (MSCs-TA) são células progenitoras que residem entre adipócitos e contribuem para o turnover do tecido adiposo. Gangliosídios são glicoensfigolipídios localizados na membrana das células, envolvidos na regulação do crescimento celular, interação de superfície, sinalização transmembrana e diferenciação celular. O gangliosídio neural GD2 foi relatado como um marcador de superfície de células tronco mesenquimais de medula óssea e cordão umbilical, mas existem poucos dados sobre a expressão do GD2 em MSCs-TA indiferenciadas e nas diferenciadas para adipócito ou osteoblasto. Nosso principal objetivo foi estudar a expressão de gangliosídios nas MSCs-TA, em especial o GD2, durante a diferenciação adipogênica e osteogênica. Para isso, as MSCs-TA foram isoladas de lipoaspirado humano, cultivadas e induzidas para diferenciação adipogênica e osteogênica. As análises foram feitas por HPTLC, microscopia confocal, citometria de fluxo e PCR em tempo real. Por HPTLC, as MSCs-TA indiferenciadas e MSCs-TA diferenciadas para adipócitos e osteoblasto mostraram aumento do perfil de gangliosídios complexos. A microscopia confocal evidenciou os gangliosídios GM3, GM1 e GD2 na superfície das células e, por citometria de fluxo, identificamos uma subpopulação de células GD2 positivas nas MSCs-TA e MSCs-TA diferenciadas para adipócito ou osteoblasto. Entretanto, o percentual de células GD2 positivas decresceu com a diferenciação. A expressão do mRNA da GD2 sintase aumentou na diferenciação adipogênica e diminui na diferenciação osteogênica. O GD2 é um substrato para a biosíntese de gangliosídios complexos e o aumento da expressão da GD2 sintase pode estar relacionado com o aumento de gangliosídios complexos que ocorre durante a diferenciação adipogênica. / Mesenchymal Stem Cells from Adipose Tissue (MSCs-TA) are progenitor cells that reside between adipocytes, and contribute to the turnover of adipose tissue. Gangliosides are glycosphingolipids localized in cell membrane, involved in cell growth regulation, surface interaction, transmembrane signaling and differentiation. The neural ganglioside GD2 has been reported as surface marker for MSCs from bone marrow and umbilical cord, but sparse data exist about the expression of GD2 in MSCs-TA and during the differentiation to adipocytes and osteoblast. Our aim was to study the expression of glangliosides, in special of GD2 in MSCs-TA and during the adipogenic and osteogenic differentiation. Thus MSCs-TA were isolated from lipoaspirate, cultured and induced to adipogenic and osteogenic differentiation. Then, we examined the gangliosides expression by HPTLC, confocal microscopy, flow citometry and real-time PCR. By HPTLC, the MSCs-TA and MSCs-TA differentiated into adipocytes and osteoblast demonstrate an increased complex gangliosides profile. The confocal microscopy showed the presence of GM3, GM1, and GD2 on the cell surface. By the flow cytometry, we identified a GD2 positive subpopulation in MSCs-TA and in MSCs-TA differentiated to adipocytes and osteoblast. However, the percentage of GD2 positive cells decreased with the differentiation. The expression of GD2 synthase mRNA increased during the adipogenic differentiation and decreased in osteogenic differentiation. GD2 is a substrate for the complex gangliosides biosynthesis, and the increase in GD2 synthase expression could be related with the increase in complex gangliosides that occurs during the adipogenic differentiation.

Bovine lactoferrin (BLF) and bovine transferrin (BTF) are major-iron transport and regulation proteins found in bovine whey. BLF and BTF must interact with the eukaryotic cell surface to mediate their biological function of iron delivery and cellular functions of inflammatory and immunological modulation. As common components of the eukaryotic cell surface, gangliosides were used for affinity purification of BLF and BTF. Bovine gangliosides were isolated from fresh buttermilk and covalently immobilized onto controlled-pore glass beads (66 μg/g beads). After the matrix was loaded with whey protein (WPI or WPC), lactoferrin was eluted with 1 M NaCl and lll identified by N-terminal protein sequencing. Pretreated whey isolate (1 % wt/vol) showed the highest lactoferrin purity with 40% among protein sources, and whey protein isolate (10% wt/vol) showed the highest recovery with 105%. Bovine transferrin was eluted with sodium phosphate buffers at pH 7 after the immobilized matrix was loaded with a 2% (wt/vol) whey solution. The ganglioside column resulted in a 74.2% recovery of BTF from whey, and the BTF was enriched to 61% purity after Mono-Q chromatography. Bovine transferrin was identified by SDS-PAGE analysis, Western analysis, and isoelectrofocusing. In conclusion, immobilized gangliosides can be used to purify BLF and BTF from bovine whey.

As células tronco mesenquimais de tecido adiposo (MSCs-TA) são células progenitoras que residem entre adipócitos e contribuem para o turnover do tecido adiposo. Gangliosídios são glicoensfigolipídios localizados na membrana das células, envolvidos na regulação do crescimento celular, interação de superfície, sinalização transmembrana e diferenciação celular. O gangliosídio neural GD2 foi relatado como um marcador de superfície de células tronco mesenquimais de medula óssea e cordão umbilical, mas existem poucos dados sobre a expressão do GD2 em MSCs-TA indiferenciadas e nas diferenciadas para adipócito ou osteoblasto. Nosso principal objetivo foi estudar a expressão de gangliosídios nas MSCs-TA, em especial o GD2, durante a diferenciação adipogênica e osteogênica. Para isso, as MSCs-TA foram isoladas de lipoaspirado humano, cultivadas e induzidas para diferenciação adipogênica e osteogênica. As análises foram feitas por HPTLC, microscopia confocal, citometria de fluxo e PCR em tempo real. Por HPTLC, as MSCs-TA indiferenciadas e MSCs-TA diferenciadas para adipócitos e osteoblasto mostraram aumento do perfil de gangliosídios complexos. A microscopia confocal evidenciou os gangliosídios GM3, GM1 e GD2 na superfície das células e, por citometria de fluxo, identificamos uma subpopulação de células GD2 positivas nas MSCs-TA e MSCs-TA diferenciadas para adipócito ou osteoblasto. Entretanto, o percentual de células GD2 positivas decresceu com a diferenciação. A expressão do mRNA da GD2 sintase aumentou na diferenciação adipogênica e diminui na diferenciação osteogênica. O GD2 é um substrato para a biosíntese de gangliosídios complexos e o aumento da expressão da GD2 sintase pode estar relacionado com o aumento de gangliosídios complexos que ocorre durante a diferenciação adipogênica. / Mesenchymal Stem Cells from Adipose Tissue (MSCs-TA) are progenitor cells that reside between adipocytes, and contribute to the turnover of adipose tissue. Gangliosides are glycosphingolipids localized in cell membrane, involved in cell growth regulation, surface interaction, transmembrane signaling and differentiation. The neural ganglioside GD2 has been reported as surface marker for MSCs from bone marrow and umbilical cord, but sparse data exist about the expression of GD2 in MSCs-TA and during the differentiation to adipocytes and osteoblast. Our aim was to study the expression of glangliosides, in special of GD2 in MSCs-TA and during the adipogenic and osteogenic differentiation. Thus MSCs-TA were isolated from lipoaspirate, cultured and induced to adipogenic and osteogenic differentiation. Then, we examined the gangliosides expression by HPTLC, confocal microscopy, flow citometry and real-time PCR. By HPTLC, the MSCs-TA and MSCs-TA differentiated into adipocytes and osteoblast demonstrate an increased complex gangliosides profile. The confocal microscopy showed the presence of GM3, GM1, and GD2 on the cell surface. By the flow cytometry, we identified a GD2 positive subpopulation in MSCs-TA and in MSCs-TA differentiated to adipocytes and osteoblast. However, the percentage of GD2 positive cells decreased with the differentiation. The expression of GD2 synthase mRNA increased during the adipogenic differentiation and decreased in osteogenic differentiation. GD2 is a substrate for the complex gangliosides biosynthesis, and the increase in GD2 synthase expression could be related with the increase in complex gangliosides that occurs during the adipogenic differentiation.

As células tronco mesenquimais de tecido adiposo (MSCs-TA) são células progenitoras que residem entre adipócitos e contribuem para o turnover do tecido adiposo. Gangliosídios são glicoensfigolipídios localizados na membrana das células, envolvidos na regulação do crescimento celular, interação de superfície, sinalização transmembrana e diferenciação celular. O gangliosídio neural GD2 foi relatado como um marcador de superfície de células tronco mesenquimais de medula óssea e cordão umbilical, mas existem poucos dados sobre a expressão do GD2 em MSCs-TA indiferenciadas e nas diferenciadas para adipócito ou osteoblasto. Nosso principal objetivo foi estudar a expressão de gangliosídios nas MSCs-TA, em especial o GD2, durante a diferenciação adipogênica e osteogênica. Para isso, as MSCs-TA foram isoladas de lipoaspirado humano, cultivadas e induzidas para diferenciação adipogênica e osteogênica. As análises foram feitas por HPTLC, microscopia confocal, citometria de fluxo e PCR em tempo real. Por HPTLC, as MSCs-TA indiferenciadas e MSCs-TA diferenciadas para adipócitos e osteoblasto mostraram aumento do perfil de gangliosídios complexos. A microscopia confocal evidenciou os gangliosídios GM3, GM1 e GD2 na superfície das células e, por citometria de fluxo, identificamos uma subpopulação de células GD2 positivas nas MSCs-TA e MSCs-TA diferenciadas para adipócito ou osteoblasto. Entretanto, o percentual de células GD2 positivas decresceu com a diferenciação. A expressão do mRNA da GD2 sintase aumentou na diferenciação adipogênica e diminui na diferenciação osteogênica. O GD2 é um substrato para a biosíntese de gangliosídios complexos e o aumento da expressão da GD2 sintase pode estar relacionado com o aumento de gangliosídios complexos que ocorre durante a diferenciação adipogênica. / Mesenchymal Stem Cells from Adipose Tissue (MSCs-TA) are progenitor cells that reside between adipocytes, and contribute to the turnover of adipose tissue. Gangliosides are glycosphingolipids localized in cell membrane, involved in cell growth regulation, surface interaction, transmembrane signaling and differentiation. The neural ganglioside GD2 has been reported as surface marker for MSCs from bone marrow and umbilical cord, but sparse data exist about the expression of GD2 in MSCs-TA and during the differentiation to adipocytes and osteoblast. Our aim was to study the expression of glangliosides, in special of GD2 in MSCs-TA and during the adipogenic and osteogenic differentiation. Thus MSCs-TA were isolated from lipoaspirate, cultured and induced to adipogenic and osteogenic differentiation. Then, we examined the gangliosides expression by HPTLC, confocal microscopy, flow citometry and real-time PCR. By HPTLC, the MSCs-TA and MSCs-TA differentiated into adipocytes and osteoblast demonstrate an increased complex gangliosides profile. The confocal microscopy showed the presence of GM3, GM1, and GD2 on the cell surface. By the flow cytometry, we identified a GD2 positive subpopulation in MSCs-TA and in MSCs-TA differentiated to adipocytes and osteoblast. However, the percentage of GD2 positive cells decreased with the differentiation. The expression of GD2 synthase mRNA increased during the adipogenic differentiation and decreased in osteogenic differentiation. GD2 is a substrate for the complex gangliosides biosynthesis, and the increase in GD2 synthase expression could be related with the increase in complex gangliosides that occurs during the adipogenic differentiation.