Sphingolipids are found in essentially all animals, plants and fungi, and some prokaryotic organisms and viruses. Sphingolipids function as structural components of membranes, lipoproteins, and as cell signaling modulators and mediators. To complicate matters further, sphingolipids often vary in type in different regions of tissues, and even in single cells, the subcellular localization of sphingolipids and their metabolic enzymes, transport proteins and targets may influence their functions. It is important to study sphingolipids spatial distribution within living organisms to understand how sphingolipids are involved in complex biochemical processes. As part of this thesis, procedures were optimized for the use of matrix assisted laser desorption/ionization (MALDI) tissue mass spectrometry (TIMS) to visualize the location of several types of lipids including sulfatides (ST), gangliosides and phosphoglycerolipids in brains from a mouse model for Tay-Sachs/Sandhoff disease. MALDI-TIMS was next applied to human ovarian carcinoma tissue to detect sulfatide location and established that ST are associated specifically with the regions of the ovarian tissue that bear the carcinoma. Electrospray ionization tandem mass spectrometry (ESI-MS-MS) was also used to confirm that ST and galactosylceramide (GalCer) are elevated in ovarian cancer. Gene expression data using tumor cells collected using laser capture microdissection revealed greater expression of mRNAs for GalCer synthase, GalCer sulfotransferase (Gal3ST1) and other enzymes of ST biosynthesis in epithelial ovarian carcinoma cells. This is a unique combination of two complementary, profiling technologies--mass spectrometry (metabolomic approach) with analysis of gene expression to study complex cancer pathology. The next study focused on the subcellular location of sphingolipids. In comparison with wild type Hek293 cells, a Hek293 cell line stably overexpressing serine palmitoyltransferase (SPT1/2 cells) was found to have elevated amounts of all subspecies of ceramide (Cer), but produces disproportionately higher amounts of C18-Cer and GalCer. Since Cer is known to inhibit protein ER/Golgi trafficking, these studies found that the higher production of Cer caused impairment of ER/Golgi trafficking of Ceramide synthase 1 (CerS1), thus increased C18-Cer. In addition, since GalCer is only synthesized in the lumen of the ER, this impairement of ER/Golgi trafficking also gave GalCer synthase access to its substrate and increased GalCer biosynthesis. These studies illustrate the complexity of sphingolipid biology and the usefulness of multiple tools to understand sphingolipid complex biological processes.

Using the known sequences for serine palmitoyltransferase (SPT) and ceramide kinase (CERK) from Arabidopsis, candidates for the corresponding genes in Loblolly pine were cloned and examined during embryogenesis. The cloned two cDNA sequences from Loblolly pine, which has similarity of 81% and 88% respectively to two subunits of SPT1 and SPT2 in Arabidopsis, were presumed as the Loblolly pine SPT1 and SPT2 (Pt-SPT1 and Pt-SPT2). A few different versions of Pt-SPT1 mRNAs (2223 nts, 756 nts, 822 nts, and 754 nts respectively), most likely the alternative splicing results, were found. Three of these mRNAs are capable of encoding proteins. The long version (2223 nts) encodes a protein with 484 amino acids (Pt-SPT1); two short versions (822 nts, 756 nts) encode a 90 a.a. protein. Another cDNA sequence of 2396 nts encodes a protein of 493 a.a. (Pt-SPT2). Both predicted Pt-SPT1 and Pt-SPT2 proteins possess highly conserved serine palmitoyltransferase functional domains (E value 5.7e-61). Their expression patterns are different between somatic and zygotic embryogenesis. Two different versions of mRNAs, with 2786 nts (long), and 2320 nts (short) respectively, of ceramide kinases in Loblolly pine (Pt-CERKs) have been cloned. The long version encodes a protein with 721 a.a.; the short version with 560 a.a. The expression patterns for these two CERK mRNAs are different during embryo development. The long version is constitutively expressed, while the short one is only expressed in some stages with much lower expression level. Overexpression Pt-CERKL, Pt-CERKS, and Pt-CERKF in E.coli and function analysis in vitro show that all Pt-CERKs appear to have the same catalytic functions as their homologs in human and Arabidopsis, but with different efficiency. The catalytic efficiency was dramatically lower in the short Pt-CERK protein compared with the long Pt-CERK protein and Pt-CERKF. The membrane system is not necessary for the catalytic reactions of these three Pt-CERKs in vitro and Pt-CERKs were less dependent on the Ca2+ ions. Thus, these studies have provided the first information about SPT- and CERK- like proteins in loblolly pine, and open new avenues of investigation for the roles of sphingolipids in embryonic development.

Type 2 diabetes is a disease characterized by the inability of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normoglycemia. Increased levels of saturated fatty acids such as palmitate are believed to contribute to β-cell failure and the development of the disease. In the present thesis, mechanisms behind palmitate-induced β-cell apoptosis were explored. Palmitate augmented insulin secretion after short exposure to the fatty acid, but attenuated the secretory response after longer exposure. Elevated levels of palmitate increased endoplasmic reticulum (ER) stress and induced apoptosis. When insulin secretion was inhibited by diazoxide, palmitate-induced ER stress and apoptosis were reduced. In comparison to palmitate, the mono-unsaturated fatty acid oleate increased neither ER stress nor apoptosis. Furthermore, shuttling of fatty acids into triglycerides and β-oxidation was favored in cells exposed to oleate compared to palmitate. When the levels of stearoyl-CoA desaturase 1 (SCD1), the enzyme responsible for conversion of saturated to mono-unsaturated fatty acids, were reduced, up-regulation of ER chaperones and components of the proteasome was observed. Cells with reduced levels of SCD1 showed increased sensitivity to palmitate, as exposure to the fatty acid increased levels of ER stress and apoptosis. Palmitate-induced apoptosis of the β-cell has been linked to alterations in sphingolipid metabolism. In cells with reduced levels of sphingosine kinase (SphK) 2, palmitate failed to induce apoptosis, and ER stress was reduced. Furthermore, SphK2 was required for the palmitate-induced activation of c-Jun N-terminal kinase (JNK). In contrast, knockdown of SphK1 sensitized the cell to palmitate-induced apoptosis independently of ER stress. In summary, palmitate induces β-cell apoptosis, which is partly dependent on the induction of ER stress. The mechanisms investigated support the notion that increased protein load on the ER, low degree of triglyceride formation and β-oxidation, and perturbations in sphingolipid metabolism contribute to palmitate-induced apoptosis in insulin-producing β-cells.

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

One of the first embryonic structures generated during early human development is the neural tube. The embryonic process of neurulation, including neural tube closure, is necessary for proper brain and spinal cord development, whereas improper closure leads to neural tube defects including anencephaly, spina bifida, and craniorachischisis. The mechanism by which these defects occur is unknown, but some evidence suggest that redox disruption may play a role. Cellular redox state is important in regulating key processes during neural tube closure, including differentiation, proliferation, gene expression, and apoptosis. This study aims to determine whether redox potential shifts and these key processes are affected similarly or differentially after treatment with three neural tube defect-inducing developmental toxicants: ceramide (C2), valproic acid (VPA), and fumonisin (FB1). Using the P19 cell model of neurogenesis, in both undifferentiated and terminally differentiated cells, we analyzed glutathione (GSH) redox (Eh) potential to evaluate the effect of each toxicant over time. We show that in C2 and VPA treated cultures an oxidizing shift occurs, but interestingly, FB1 treatment results in a reducing shift in embryonic GSH Eh as compared to untreated cultures. Using the chick embryo model, comparable redox shifts were observed as were seen in P19 cells, supporting similarity between the models. To better understand how differential shifts in the redox state can result in similar defects, we then examined potential variances in neuronal differentiation and cellular proliferation, survival, metabolism, adhesion, and gene expression under each treatment. We report changes to cellular and embryonic endpoints that support dysmorphogenesis, likely the result of oxidizing or reducing stress that altered redox state. These results support the need for broad comparative analyses such as this to determine whether toxicants that cause the same types of defects, whether NTDs or others, act through similar or different mechanisms. This can better inform preventative measures used to reduce the risk and occurrence of birth defects.

no / Age is characterized by deficits in synaptic function identified by decreased performance of aged animals in spatial learning tasks and reduced ability of animals to sustain long-term potentiation (LTP). Several cellular and molecular events are correlated with these deficits, many of which are indicative of cell stress. Thus there is evidence of age-related neuroinflammatory stress and oxidative stress and these have been linked with microglial activation which is likely to be primarily responsible for the age-related increase in production of proinflammatory cytokines and reactive oxygen species. It is significant that agents which decrease microglial activation are commonly associated with restoration of function. We set out to examine whether the n-3 polyunsaturated fatty acid docosapentaenoic acid (DPA), which is a metabolite of eicosapentaenoic acid (EPA), could modulate the age-related increase in microglial activation and the associated increase in oxidative changes and therefore impact on synaptic function in aged rats. We demonstrate that DPA possesses neurorestorative effects and is capable of downregulating microglial activation. The data show that it also decreases the coupled activation of sphingomyelinase and caspase 3, probably as a result of its ability to decrease age-related oxidative changes, and consequently attenuates the age-related decrease in spatial learning and LTP.

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.

A obesidade, doença multifatorial, ocasiona inúmeros distúrbios no metabolismo lipídico e energético, provocando disfunção na bioenergética mitocondrial. A partir deste fato, o presente estudo teve como hipóteses que: o desequilíbrio na bioenergética mitocondrial e as alterações metabólicas causadas pela obesidade são terapeuticamente modificados com o treinamento físico. Dessa maneira, o objetivo do estudo foi avaliar a capacidade oxidativa e conteúdo mitocondrial em tecido adiposo branco, marcadores de doenças cardiosculares (esfingolipídios e N-óxido de trimetilamina, TMAO) e as alterações na composição corporal, desempenho físico e taxa metabólica de repouso (TMR) de mulheres com obesidade submetidas a treinamento físico combinado. A casuística do presente trabalho foi composta de 14 mulheres adultas jovens com diagnótico clínico de obesidade (IMC 33±3 kg/m² e idade 35±6 anos). Foram submetidas a um programa de treinamento físico combinado (exercícios aeróbios e força alternadamente, 55 min à 75-90% da frequência cardíaca máxima, 3 vezes por semana, durante 8 semanas). Todas as participantes foram avaliadas antes e após a intervenção com o treinamento, quanto a: composição corporal, TMR, oxidação de substratos (carboidrato e lipídios) e coeficiente respiratório (QR), desempenho físico, capacidade oxidativa (respiração acoplada: VADP/VOLIGO, e respiração desacoplada: VOLIGO/VCCCP) e conteúdo mitocondrial (enzima citrato sintase, CS) em tecido adiposo branco, nível de esfingolípidios, TMAO e precursores plasmáticos. Os dados foram analisados pelo test t pareado ou Wilcoxon (as pacientes foram consideradas controle de si próprio), após determinação da normalidade da amostra, considerado nível de significância p<= 0,05. Após a intervenção (treinamento físico combinado), houve o aumento da TMR, oxidação de lipídios e desempenho físico, com redução da oxidação de carboidratos e QR, mas não houve perda de peso e alteração da composição corporal. Após o treinamento combinado houve, o aumento da atividade da enzima CS (marcador de conteúdo mitocondrial) e redução à respiração desacoplada (VOLIGO/VCCCP). No plasma, o treinamento físico foi capaz de reduzir os níveis de esfingolipídios e TMAO (fatores de risco cardiovasculares). Também foram encontradas correlações positivas entre TMR, oxidação de lipídios e desempenho físico com CS e negativamente correlacionado com respiração desacoplada. Concluindo, o treinamento físico em mulheres com obesidade aumentou o metabolismo energético, com aumento da TMR, conteúdo e grau de acoplamento mitocondrial, aumentou o desempenho físico e reduziu fatores de risco cardiovasculares (TMAO), independente da perda de peso. / The obesity, a multifactorial disease, causes various metabolic disorders in lipid and energy metabolism, may induce mitochondrial bioenergetic dysfunction. From this, the present study hypothesized that: mitochondrial bioenergetics dysfunction and metabolic problems caused by obesity are be therapeutically modified with physical training. Thus, the objective of study was to evaluated: the oxidative capacity and mitochondrial content in white adipose tissue, markers of cardiovascular diseases (sphingolipids and trimethylamine N-oxide, TMAO) and changes of body composition, physical performance and resting metabolic rate (TMR) of obese women submitted to combined physical training. The present study was composed of 14 young women with obesity (BMI 33 ± 3 kg/m² and age 35 ± 6 years old). They underwent a combined physical training program (aerobic exercises and strength alternately, 55 min at 75-90% of maximal heart rate, 3 times a week, for 8 weeks). All participants were evaluated before and after the intervention: body composition, TMR, substrates oxidation (carbohydrate and lipids) and respiratory coefficient (RQ), physical performance, oxidative capacity (by mitochondrial respiration - Couple: VADP/VOLIGO; Uncoupling: VOLIGO/VCCCP) and Citrate Sinthase activity in white adipose tissue, level of sphingolipids, TMAO and precursors from plasma. Data analysis were made by paired t test or Wilcoxon, after normality determination of the sample, with level of significance p <0.05. After intervention with combined physical training, there was an increase in TMR, lipid oxidation and physical performance, reduced carbohydrate oxidation and RQ, but did not cause weight loss and changes of body composition. In adipose tissue, physical activity increased CS activity (mitochondrial content marker) and reduced uncoupling respiration (VOLIGO/VCCCP). In plasma, physical training was able to reduce levels of sphingolipids and TMAO (cardiovascular risk factors). In addition, positive correlations were found between, TMR, lipid oxidation and physical performance with CS and negatively correlation with uncoupling respiration. Therefore, physical training in obese women improve energy metabolism, with increased TMR, content and degree of mitochondrial coupling, increased physical performance and reduced cardiovascular risk factors, regardless of weight loss.

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.

<p>Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with bad prognosis, which predominates in males with advanced age. However, studies of the endocannabinoid system and how it affects tumour behaviour provides the basis for designing innovative therapeutic strategies that could open new opportunities for treatment of patient with MCL. It has earlier been shown that the cannabinoid receptor ligand (R)-(+)-methanandamide (R-MA) induce cell death in MCL by accumulation of ceramide. Ceramide has a pro-apoptotic effect on the cell but could be metabolized by the enzymes glucosylceramide synthase (GCS) and sphingosine kinase 1 (SphK1) to molecules with pro-proliferative effect. Therefore, treatments with R-MA on Jeko-1 MCL cell line were performed in this study to determine interference in the proliferative behaviour as well as in the gene expression of the enzymes GCS and SphK1. In addition, treatments with chemotherapeutic substances, such as doxorubicin or cytarabine (Ara-C), and combinations of R-MA and chemotherapeutic substance, were performed for the same reason. Results showed that the proliferation behaviour of Jeko cells remained unaffected when treated with R-MA, in contrast to the decreased proliferative effects shown when treated with cytostatics or combinations of R-MA and cytostatics. Furthermore, a tendency for up-regulation of GCS and SphK1 expression was recognized when cells were treated with cytostatics or combination of cytostatics and R-MA, in contrast to cells treated with R-MA alone. Although, R-MA alone had a tendency for a small down-regulation of GCS expression, it contributed to a potential elevation of GCS expression when combined with Ara-C or doxorubicin. It is believed that the effect from upregulated levels of the metabolizing enzymes GCS and SphK1 is balanced by, earlier observed, up-regulations of the ceramide synthesis enzymes.</p>