Die Degranulation von Mastzellen soll durch Glycosphingolipide, welche mit der Zellmembran wechselwirken inhibiert werden. Der Sphingosingrundkörper wurde in zehn-stufigen Synthese ausgehend von N-Boc-Serin, aufgebaut. Die anschließende Glycosylierung erfolgte nach der Trichloracetimidatmethode in sehr guten Ausbeuten und stellte den Schlüsselschritt dar. Durch die Variation von unter Anderem der Amidseitenkette, der Glycosylkopfgruppe und des Sphingosingrundkörpers wurde eine Vielzahl an Derivaten für das Screening im Degranulationsassay bereitgestellt. / The present dissertation covers the synthesis of glycosphingolipids which interact with the cell membrane in order to inhibit the degranulation of mast cells. The sphingosin body was synthesized in ten steps starting from N-Boc-Serin. The key step, the glycosylation was achieved using the trichloracetimidat method. The variation of the amid sidechain, the gylcosyl headgroup and the sphingosin body created a number of derivatives that were tested in the degranulation assay.

L’apoptose est une forme de mort cellulaire essentielle au développement et au maintien de l’homéostase chez les animaux multicellulaires. La machinerie apoptotiq ue requiert la participation des caspases, des protéases conservées dans l’évolution et celle des organelles cytoplasmiques. Les lysosomes subissent des ruptures partielles, labilisation de la membrane lysosomale (LML), qui entraînent l’activation des cathepsines dans le cytoplasme de cellules cancéreuses humaines en apoptose induite par la camptothecin (CPT), incluant les histiocytes humains U-937. Ces modifications lysosomales se manifestent tôt durant l’activation de l’apoptose, concomitamment avec la perméabilisation de la mitochondrie et l’activation des caspases. Une étude protéomique quantitative et comparative a permis d’identifier des changements précoces dans l’expression/localisation de protéines lysosomales de cellules U-937 en apoptose. Lors de deux expériences indépendantes, sur plus de 538 protéines lysosomales identifiées et quantifiées grâce au marquage isobarique iTRAQ et LC-ESIMS/ MS, 18 protéines augmentent et 9 diminuent dans les lysosomes purifiés de cellules en cours d’apoptose comparativement aux cellules contrôles. Les candidats validés par immuno-buvardage et microscopie confocale incluent le stérol-4-alpha-carboxylate 3- déhydrogénase, le prosaposin et la protéine kinase C delta (PKC-d). Des expériences fonctionnelles ont démontrées que la translocation de PKC-d aux lysosomes est requise pour la LML puisque la réduction de son expression par ARN interférents ou l’inhibition de son activité à l’aide du rottlerin empêche la LML lors de l’apoptose induite par la CPT. La translocation de PKC-d aux lysosomes conduit à la phosphorylation et l’activation de la sphingomyelinase acide lysosomale (ASM), et à l’accroissement subséquent du contenu en céramide (CER) à la membrane lysosomale. Cette accumulation de CER endogène aux lysosomes est un évènement critique pour la LML induite par la CPT car l’inhibition de l’activité de PKC-d ou de ASM diminue la formation de CER et la LML.Ces résultats révèlent un nouveau mécanisme par lequel la PKC-d active l’ASM qui conduit à son tour à l’accumulation de CER à la membrane lysosomale et déclenche la LML et l’activation de la voie lysosomale de l’apoptose induite par la CPT. En somme, ce mécanisme confirme l’importance du métabolisme des sphingolipides dans l’activation de la voie lysosomale de l’apoptose. / Apoptosis is a distinct form of regulated cell death which is essential for the development and homeostasis maintenance of multicellular animals. Apoptosis is an evolutionary conserved process involving a specific molecular pathway, known as the caspase cascade, and the different cytoplasmic organelles. A lysosomal pathway, characterized by partial rupture, labilization of lysosomal membranes (LML), and cathepsin activation in the cytoplasm, is evoked during camptothecin-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. Comparative and quantitative proteome analyses were performed to identify early changes in lysosomal protein expression/localization from U-937 cells undergoing apoptosis. In two independent experiments, among a total of more than 538 proteins putatively identified and quantitated by iTRAQ isobaric labelling and LC-ESI-MS/MS, 18 proteins were found to be upregulated and 9 downregulated in lysosomes purified from early apoptotic compared to control cells. Protein expression was validated by Western blotting on enriched lysosome fractions, and protein localization confirmed by fluorescence confocal microscopy of representative protein candidates, whose functions are associated with lysosomal membrane fluidity and dynamics. These include sterol-4-alpha-carboxylate 3-dehydrogenase (NSDHL), prosaposin (PSAP) and protein kinase C delta (PKC-d). Functional experiments demonstrate that PKC-d translocation to lysosomes is required for LML, as silencing its expression with RNA interference or suppressing its activity with the inhibitor rottlerin prevents CPT-induced LLM. PKC-d translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase of ceramide (CER) content at lysosomes. The accumulation of endogenous CER at lysosomes is a critical event for CPT-induced LLM as suppressing PKC-d or ASM activity reduces both CPT-mediated CER generation at lysosomes and CPT-induced LLM.These findings reveal a novel mechanism by which PKC-d mediates ASM phosphorylation/activation and CER accumulation at lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment. Taken together, these results confirm the importance of sphingolipid metabolism in the activation of the lysosomal pathway of apoptosis.

L’objectif de cette thèse a été d’optimiser la réponse immunitaire anti-tumorale induite par les cellules dendritiques (DC) et les cellules iNKTs, en réponse à la prise en charge du KRN7000 (a-galactosyl-céramide) par la molécule CD1d située sur les DCs. Le premier axe de travail visait à synthétiser de nouveaux analogues du KRN7000, en fonctionnalisant la position C6 du sucre et en greffant un groupement phényl sur l’une des chaînes grasses. Les études in vitro ont montré que les modifications apportées par rapport au KRN7000 n’ont pas altéré la prise en charge des molécules obtenues par les DCs. Dans tous les cas, une sécrétion de cytokines a pu être observée. Des études complémentaires visant à décrire le profil cytokinique in vivo sont en cours. Le second axe a consisté en la mise au point d’une stratégie de vectorisation du KRN7000 afin de favoriser sa présentation aux DCs, en l’associant à des molécules d’intérêt comme un peptide spécifique d’une tumeur, une molécule de ciblage des DCs ou des ligands des TLRs. Dans les conditions utilisées, le phénomène d’anergie induit classiquement par l’administration répétée du KRN7000 n’a pas pu être levé. Cependant, nous avons montré d’une part que le KRN7000 vectorisé dans les liposomes est toujours pris en charge par les cellules dendritiques, et d’autre part qu’une réponse immunitaire se traduisant par la production de cytokines par les cellules iNKTs est induite. / The aim of this project was to optimize the anti-tumor immune response induced by dendritic and iNKTs cells, in response to KRN7000 (a-galactosyl-ceramide), which interacts with CD1d molecule situated on DCs. At first we synthesized new analogues of KRN7000, by functionalizing the C6 position of the carbohydrate moiety and by grafting a phenyl group on one of the fatty chain. In vitro studies indicated that chemical modifications of KRN7000 did not alter its interaction with CD1d. In all cases, cytokine secretion was observed. Further studies are in progress to describe the in vivo cytokine profile. In a second step, we developed liposomal constructs incorporating KRN7000 to optimize its presentation to DCs. Some constructions containing KRN7000 were able to associate a peptide, a targetting molecule of DCs or TLR ligands. Even if the anergy phenomenon induced by repeated administrations of KRN7000 could not be regulated by the use of liposomes, we have shown that encapsulated KRN7000 is still supported by DCs and that an immune response resulting in cytokines secretion by iNKT cells is induced.

A leishmaniose é uma doença parasitária de ampla distribuição, para a qual se dispõe de um limitado arsenal terapêutico. Trabalhos recentes mostraram que tamoxifeno é eficaz no tratamento de leishmaniose experimental. Nesse trabalho, avaliamos a terapia combinada de tamoxifeno com os fármacos utilizados atualmente no tratamento desta enfermidade. A interação entre os fármacos mostrou-se aditiva, tanto in vitro como in vivo. Em paralelo, analisamos os efeitos de tamoxifeno na biossíntese de esfingolipídios em Leishmania, sendo identificada a redução da síntese de fosfatidilinositol e inositolfosforil ceramida (IPC) e acúmulo de ceramida acilada. A redução na biossíntese de IPC não pode ser atribuída a redução no transporte de inositol, mas provavelmente está relacionada à inibição da enzima IPC sintase. Estes resultados indicam novas estratégias para superar as deficiências encontradas no tratamento de leishmaniose utilizando tamoxifeno, um fármaco clinicamente bem conhecido que exerce ações em múltiplos alvos em Leishmania. / Leishmaniasis is a parasitic disease with wide distribution and limited treatment. Recent reports demonstrate that tamoxifen is an effective drug for experimental leishmaniasis treatment. In this work, we evaluated the combined therapy of tamoxifen with current drugs used in leishmaniasis chemotherapy. The drug interaction was additive both, in vitro and in vivo. We also evaluated tamoxifen effect on in Leishmania sphingolipids biosynthesis. We found a reduction in phosphatidylinositol and inositol phosphorylceramide (IPC) synthesis and an accumulation of acilceramide. The reduction in IPC biosynthesis could not be assigned to the reduction observed in inositol transport, but probably is related to IPC synthase inhibition. These results show new strategies to circumvent shortcomings of leishmaniasis treatment using tamoxifen, a multitarget drug in Leishmania and widely used in the chemotherapy of breast cancer.

Die Degranulation von Mastzellen soll durch Glycosphingolipide, welche mit der Zellmembran wechselwirken inhibiert werden. Der Sphingosingrundkörper wurde in zehn-stufigen Synthese ausgehend von N-Boc-Serin, aufgebaut. Die anschließende Glycosylierung erfolgte nach der Trichloracetimidatmethode in sehr guten Ausbeuten und stellte den Schlüsselschritt dar. Durch die Variation von unter Anderem der Amidseitenkette, der Glycosylkopfgruppe und des Sphingosingrundkörpers wurde eine Vielzahl an Derivaten für das Screening im Degranulationsassay bereitgestellt. / The present dissertation covers the synthesis of glycosphingolipids which interact with the cell membrane in order to inhibit the degranulation of mast cells. The sphingosin body was synthesized in ten steps starting from N-Boc-Serin. The key step, the glycosylation was achieved using the trichloracetimidat method. The variation of the amid sidechain, the gylcosyl headgroup and the sphingosin body created a number of derivatives that were tested in the degranulation assay.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Modulation of sphingolipid-induced apoptosis is a potential mechanism to enhance the effectiveness of chemotherapeutic drugs. Ceramide is a pleiotropic, sphingolipid produced by cells in response to inflammatory cytokines, chemotherapeutic drugs and ionizing radiation. Ceramide is a potent activator of protein phosphatases, including protein phosphatase 2A (PP2A) leading to dephosphorylation of substrates important in regulating mitochondrial dysfunction and apoptosis. Previous studies demonstrated that death associated protein kinase (DAPK) plays a role in ceramide-induced apoptosis via an unknown mechanism. The tumor suppressor DAPK is a calcium/calmodulin regulated serine/threonine kinase with an important role in regulating cytoskeletal dynamics. Auto-phosphorylation within the calmodulin-binding domain at serine308 inhibits DAPK catalytic activity. Dephosphorylation of serine308 by a hitherto unknown phosphatase enhances kinase activity and proteasomal mediated degradation of DAPK. In these studies, using a tandem affinity purification procedure coupled to LC-MS/MS, we have identified two holoenzyme forms of PP2A as DAPK interacting proteins. These phosphatase holoenzymes dephosphorylate DAPK at Serine308 in vitro and in vivo resulting in enhanced kinase activity of DAPK. The enzymatic activity of PP2A also negatively regulates DAPK protein levels by enhancing proteasomal-mediated degradation of the kinase, as a means to attenuate prolonged kinase activation. These studies also demonstrate that ceramide causes a caspase-independent cell detachment in HeLa cells, a human cervical carcinoma cell line. Subsequent to detachment, these cells underwent caspase-dependent apoptosis due to lack of adhesion, termed anoikis. Overexpression of wild type DAPK induced cell rounding and detachment similar to cells treated with ceramide; however, this effect was not observed following expression of a phosphorylation mutant, S308E DAPK. Finally, the endogenous interaction of DAPK and PP2A was determined to be required for ceramide-induced cell detachment and anoikis. Together these studies have provided exciting and essential new data regarding the mechanisms of cell adhesion and anoikis. These results define a novel cellular pathway initiated by ceramide-mediated activation of PP2A and DAPK to regulate inside-out signaling and promote anoikis.

Acquired cellular resistance to traditional chemotherapeutics is a common obstacle in the treatment of most cancer cell types. This resistance occurs as a result of changes in the underlying molecular mechanisms of disease progression. The development of novel chemotherapeutic approaches designed to enhance the efficacy of protypical anti-cancer drugs is important in order to overcome this issue. Such approaches will aid in understanding the biomolecular phenomena responsible for drug resistance and disease progression. Combining signaling pathway inhibitors has become an effective strategy for enhancing tumor cell death by targeting multiple pathways known to regulate cell survival. Pemetrexed, an FDA-approved anti-folate drug, targets thymidylate synthase (TS) and a secondary folate-dependent enzyme, 5’ aminoimidazole-carboximide ribonucleotide formyltransferase (AICART); both important for DNA synthesis. Studies performed by our collaborator demonstrated that TS inhibition causes intracellular accumulation of ZMP+ and activation of AMPK which is known to induce autophagy in mammalian cells. Previous studies from our lab and others showed that sorafenib, a multi-kinase inhibitor of Raf-1 and class III receptor tyrosine kinases, was able to induce a cytotoxic form of autophagy in a variety of tumor cell types. Combination treatment using pemetrexed and sorafenib in these cancer cells resulted in an enhancement of autophagy and cell lethality beyond that of individual drugs alone. Inhibition of autophagy suppressed the toxic interactions of these drugs in all cell types examined. Pemetrexed/sorafenib cotherapy also proved to be an effective treatment for triple negative breast cancer cells having advanced to a stage of estrogen independence. Fulvestrant-resistant MCF7 cells were more sensitive to the drug combination than parental, estrogen-dependent MCF7 cells. Breast cancer cells cotreated with pemetrexed and sorafenib exhibited enhanced MEK/ERK signaling, Src activation that was dependent on platelet-derived growth factor β (PDGFRβ) downregulation, elevated protein phosphatase 2A (PP2A) activity, and increased de novo ceramide synthesis. Studies using a mouse model of experimentally-induced breast cancer validated drug combination effectiveness through inhibition of tumor growth, while no deleterious effects on normal tissues were observed. The data presented demonstrates that pemetrexed/sorafenib cotreatment augments chemosensitivity in both in vitro and in vivo systems. Based upon these findings, a Phase I clinical trial involving pemetrexed and sorafenib in breast cancer patients with solid, recurrent tumors was begun in 2011. In conclusion, this work strongly supports a promising therapeutic utility for the pemetrexed/sorafenib combination in treatment of various cancer cell types.

Les céramides sont des lipides de la classe des sphingolipides issus de la N-acylation d’une base sphingoide par un acide gras. Ces lipides et leurs analogues suscitent un grand intérêt comme composants actifs dans les industries pharmaceutique et cosmétique. Parmi les biocatalyseurs capables de réaliser la synthèse de ce type de lipide, la lipase B de Candida antarctica semble être l’enzyme la plus adaptée à la production de « pseudo-céramides » à partir d’amino-polyols. Dans ce contexte, nous avons abordé l’étude de l’acylation de composés de type « amino-alcool »catalysée par la lipase B de Candida antarctica, en gardant à l'esprit une approche fondamentale afin d’élargir les connaissances actuelles sur ce sujet. La première partie de notre travail a ainsi traité de l’étude cinétique de l’acylation de composés monofonctionnels afin de déterminer les mécanismes réactionnels et l’énantio sélectivité de la lipase B de Candida antarctica pour les réactions de N-acylation et de O-acylation. Les parties suivantes de notre travail ont porté sur une étude structure-réactivité du substrat accepteur d’acyle et sur l’étude de l’influence du solvant utilisé (solvant organique ou liquide ionique) afin de déterminer les facteurs clés influençant la chimio sélectivité et la régio sélectivité de la lipase B de Candida antarctica lors de l’acylation de composés multifonctionnels de type « amino-alcool ». Finalement, à partir des connaissances acquises dans les différentes parties, nous avons développé et optimisé un procédé de synthèse enzymatique de « pseudo-céramides » (O,N-diacyl aminopropanediols) mis en oeuvre en réacteur continu à « lit fixe ». / Ceramides are lipids from the sphingolipide class derived from the N-acylation of a sphingoid base from a fatty acid. These lipids and their analogs are compounds of interest used as active components in pharmaceutical and cosmetic industries. Among biocatalysts able to synthesize this type of lipids, Candida antarctica lipase B appears to be the most appropriate enzyme for the production of "pseudo-ceramides" derived from amino-polyols. In this context, we have studied the acylation of amino-alcohol-like compounds catalyzed by Candida antarctica lipase B, keeping inmind a fundamental approach to expand the current knowledge on this subject. The first part of our work aimed to determine the reaction mechanisms and the enantio selectivity exhibited by Candida antarctica lipase B during O-acylation and N-acylation reactions, by conducting a kinetic study of mono functional compound acylation. In the following parts of our work, we performed a structurere activity study of the acyl acceptor substrate and evaluated the effect of the solvent used (organic solvent or ionic liquid) to determine the key factors influencing the chemo selectivity and the regio selectivity of the Candida antarctica lipase B-catalyzed acylation of polyfunctional amino alcohol compounds. Finally, starting from the knowledge acquired in the previous parts, we have developed and optimized an enzymatic process of “pseudo-ceramide” (O,N-diacylaminopropanediol) synthesis performed in a continuous packed-bed reactor.

The citrulline-nitric oxide (NO) cycle, comprised of the enzymes argininosuccinate synthase (AS), argininosuccinate lyase (AL) and endothelial nitric oxide synthase (eNOS), is responsible for the regulated production of endothelial NO. Although most studies have focused on eNOS to uncover important regulatory mechanisms, we and others have determined that AS is an essential and regulated step in endothelial NO production. AS is rate limiting for endothelial NO production and is the primary source of arginine, the substrate for eNOS-mediated NO production, despite saturating intracellular levels of arginine and available arginine transport systems. AS is essential for endothelial cell viability and its expression is regulated coordinately with eNOS by TNF and thiazolidenediones with concomitant effects on NO production. Given the importance of AS for endothelial health, we explored three independent regulatory mechanisms. In Chapter One, the functional consequences of altered AS expression due to overexpression, insulin, VEGF and ceramide were studied. We demonstrated that overexpression of AS leads to enhanced NO production and that insulin, VEGF and ceramide coordinately regulate the expression of AS and eNOS. In Chapter Two, the first post-translational modifications of AS in the endothelium were characterized. We determined that AS is an endogenous phosphoprotein in the endothelium, described several levels of biological significance of AS phosphorylation, identified 7 sites of AS phosphorylation and began to uncover the direct impact of phosphorylation on AS function. Finally, in Chapter Three, endothelial AS subcellular localization was defined and important protein interactions were identified including caveolin-1 and HSP90. The work presented in this dissertation demonstrates that multiple mechanisms regulate the function of AS, often coordinately with eNOS, and have a direct impact on nitric oxide production. Our findings suggest that the global understanding of the citrulline-NO cycle as a metabolic unit will unravel new paradigms that will re-define our understanding of the regulation of vascular function by NO.