I used the C. elegans genetic model to examine the role of ceramide biosynthesis (sphingolipid pathway) and iron regulation and found that each process impacts germline development and function. Using a sphingolipid specific antibody mAb15B4, I found that sphingolipids are associated with germ granules (P granules) within C. elegans and zebrafish; thus, suggesting conservation of macromolecules associated with germ granules. Phenotype analysis of ceramide biosynthesis mutants in C. elegans revealed that this pathway is essential for normal germline function in the aging adult hermaphrodite; specifically, precocious germline senescence was observed. Furthermore, I found that disruption of ceramide biosynthesis, via the hyl-2 deletion mutation, negatively impacts mAb15B4 localization at the P granules. Through genetic suppression analysis, I determined that insulin signaling and lipid biosynthesis can modulate the mAb15B4 localization to P granules. Additional, phenotype analysis showed that ceramide biosynthesis dysfunction decreased fecundity, and led to germline structure defects and uterine tumors. Through suppression analysis, I determined that modulation of the insulin signaling pathway suppressed the precocious germline senescence due to ceramide biosynthesis dysfunction. Since the presence of uterine tumors is associated with reproductive senescence I concluded that ceramide biosynthesis has a role in germline maintenance in the aging of the germline (germline senescence). The other important fate of a germ cell is programmed cell death. Apoptosis, which occurs through a highly conserved molecular pathway, is a normal component of growth and homeostatic processes. I used C. elegans to gain a greater understanding of the cisd gene function. The C. elegans genome has three previously uncharacterized cisd genes which code for CISD-1 (homology to vertebrate mitoNEET/CISD1 and NAF-1/CISD2) and CISD-3.1 and CISD-3.2 (homology to vertebrate Miner2/CISD3). I determined that independent disruption of the cisd genes resulted in a significant increase in the number of cell corpses within the adult hermaphrodite germline. Genetic analysis was used to examine the dysfunction of cisd-1 relative to the cell death canonical pathway genes. The increased gamete cell death in the cisd-1 hermaphrodite is suppressed by the ced-9 (Bcl-2 homolog) gain-of-function and requires functional CED-3 (caspase) and CED-4 (APAF). Additionally, the increased germ cell programmed cell death is facilitated by the pro-apoptotic, CED-9-binding protein, CED-13. Further analysis of the cisd gene family members show that cisd-3.2 dysfunction leads to germline defects and reproductive dysfunction, suggesting defects in germline stem cell proliferation. Expression analysis using the cisd promoters to drive fluorescent protein reporters showed that the cisd gene family is expressed in various tissues including the germline; fusion protein analysis showed that CISD-3 is mitochondrial localized. I propose that cisd-3.2 germline defects are a result of abnormal mitochondrial function. Combined, this work is significant because it identifies sphingolipids as a new component of embryonic P granules, a role for ceramide biosynthesis in reproductive senescence, and places the cisd gene family members as regulators of physiological germline programmed cell death acting through CED-13 and the core apoptotic machinery. Furthermore, it is the first study to show that a CISD3 protein family member is required for normal germline function. These findings support the idea that ceramide biosynthesis and iron regulation are core components in germline development and function.

La prévalence de l'obésité et du diabète de type II est en constante augmentation dans les pays industrialisés. La manifestation hépatique de ces pathologies est la NAFLD (" Non-Alcoholic Fatty Liver Disease "). Celle-ci représente aujourd'hui un réel problème de santé publique et résulte d'atteintes métaboliques et hépatiques. La NAFLD démarre par l'accumulation excessive de lipides dans les hépatocytes nommée " stéatose hépatique ". Ces lipides s'accumulent sous différentes formes comme les triglycérides, les esters de cholestérol, les diglycérides et les céramides. La lipotoxicité induite par l'accumulation de ces espèces lipidiques est à l'origine d'un dysfonctionnement au niveau cellulaire et d'une insulino-résistance. Dans ce contexte, les objectifs de ce travail de thèse ont été d'étudier in vivo le rôle des céramides dans l'apparition et les complications de la NAFLD. Pour cela, nous avons utilisé différentes approches : pharmacologiques, génétiques et nutritionnelles. Par une approche pharmacologique, nous avons montré que la fumonisine B1, un contaminant alimentaire ciblant la synthèse des céramides, est à l'origine d'une toxicité hépatique dépendante d'un facteur de transcription essentiel du métabolisme lipidique, LXR (" Liver X Receptor "). Puis, nous avons combiné différentes approches nutritionnelles et génétiques permettant d'induire ou de protéger de la stéatose hépatique. Pour cela, nous avons utilisé des souris invalidées de façon totale ou hépatocyte-spécifique pour PPARa (" Peroxisome Proliferator-Activated Receptor alpha "), un facteur de transcription essentiel au catabolisme des lipides. Cela nous a permis de confirmer le rôle essentiel de PPARa hépatocytaire dans la réponse au jeûne et l'implication systémique de PPARa dans la régulation du métabolisme des céramides au cours de l'obésité induite par un régime HFD (" High Fat Diet "). Enfin, nous avons utilisé des souris invalidées au niveau hépatocytaire pour la sous-unité catalytique de la PI3 Kinase alpha, p110a. Cela nous a permis de confirmer le rôle majeur de la voie de signalisation à l'insuline dépendante de p110a dans l'apparition de l'insulino-résistance dissociée de la stéatose hépatique induite par un régime HFD. De façon intéressante, grâce à ce modèle, nous avons démontré que la lipolyse adipocytaire (et non l'inhibition de la voie insulinémique) représente le signal dominant de l'activité hépatique de PPARa durant le jeûne. L'ensemble de ces travaux mettent en avant le rôle des céramides dans la lipotoxicité associée à la stéatose hépatique. / Prevalence of obesity and type II diabetes is constantly increasing in industrialized countries. NAFLD (" Non-Alcoholic Fatty Liver Disease ") is the hepatic manifestation of these pathologies. NAFLD represents a significant public health problem and is defined as a nexus of metabolic and hepatic diseases. NAFLD begins with fatty accumulation in the liver named "hepatic steatosis". Lipids can accumulate in different forms like triglycerides, cholesterol esters, diglycerides and ceramides. Lipotoxicity induced by the accumulation of these lipid species leads to cellular dysfunction and insulin resistance. In this context, we studied the role of ceramides in apparition and evolution of NAFLD in vivo. For this purpose, we used pharmacological, genetic and nutritional approaches. By pharmacological approach, we showed that fumonisin b1, a mycotoxin targeting ceramide synthesis, leads to hepatic toxicity, which is dependent from LXR ("Liver X Receptor"), a major transcriptional regulator of lipid metabolism. Then, we combined genetic and nutritional approaches in order to induce or protect from hepatic steatosis. For this, we used mice with hepatic or total deletion for PPARa (" Peroxisome Proliferator-Activated Receptor alpha "), a transcriptional factor essential in fatty acid catabolism. First, this model allow us to confirm the role of hepatocyte PPARa in response to fasting and second, to demonstrate the systemic involvement of PPARa in regulating ceramide metabolism during obesity induced by an HFD ("High Fat Diet"). Last, we used p110a liver-specific knockout mice, the catalytic subunit of PI3Kinase alpha. With this model, we confirmed the critical role of p110a-dependent insulin signaling in insulin resistance dissociated from hepatic steatosis induced by a HFD. Interestingly, we demonstrated with this model that free fatty acid released from adipocyte lipolysis (rather than inhibition by p110a-dependent insulin signaling) determines PPARa activity in the liver. Finally, this work highlights the key role of ceramides in lipotoxicity associated with hepatic steatosis.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Zuzana Jarešová Supervisor: PharmDr. Andrej Kováčik, Ph.D. Consultant: PharmDr. Lukáš Opálka, Ph.D. Title of diploma thesis: STUDY OF FREE SPHINGOID BASES IN SKIN BARRIER The skin barrier, localized in the stratum corneum (SC), consists of corneocytes and an intercellular matrix formed from three types of lipids - ceramides, free fatty acids, and cholesterol, represented in an equimolar ratio. The overall arrangement of lipids is organized and highly specialized. Ceramides are structurally formed from the fatty acid acyl attached to a sphingoid base. In minor but not insignificant amounts, free sphingoid bases can also be found in the skin barrier. Several studies show that there is an increased concentration of free sphingoid bases in skin barrier disorders, such as atopic dermatitis. Although it is assumed that the presence of free sphingoid bases affects the skin barrier, it is not elucidated the way of their participation till today. The lack of studies or their diverse results leads us to the main goal of this thesis - to clarify how free sphingoid bases influence the skin barrier. In this work, the model membranes were prepared by the isolation of human SC ex vivo. Sphingosine (S),...

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Denisa Kubátová Supervisor: PharmDr. Andrej Kováčik, Ph.D. Consultant: PharmDr. Lukáš Opálka, Ph.D. Title of diploma thesis: Study of sphingosine, dihydrosphingosine and phytosphingosine in skin barrier models The stratum corneum (SC), the uppermost layer of the skin, localized in the uppermost part of the epidermis, represents the skin barrier of the organism. SC is composed of corneocytes and an intercellular lipid matrix, which is formed by ceramides (Cer), free fatty acids (FFA), and cholesterol (Chol) in an equimolar ratio. Substances from the group of sphingolipids - Cer, are sphingoid bases (for example, sphingosine (S), dihydrosphingosine (dS), phytosphingosine (P)) acylated with a fatty acid (for example, lignoceric acid (LIG)). In the lipid matrix, the metabolic products of Cer (free sphingoid bases) are also present, but their role in SC barrier functions is not clear. Some studies show that Cer with different sphingoid bases, and increased presence of free sphingoid bases, can lead to a change in the permeability of the skin barrier. This work aimed to study the effect of permeability of sphingoid bases on the model membrane permeability. Nine types of membranes were prepared; they...

Le stratum corneum (SC), couche supérieure de l’épiderme, est composé principalement de cornéocytes entourés d’une matrice lipidique. Cette structure particulière confère au SC son rôle de barrière et protège l’organisme de la perte en eau, de la pénétration de substances exogènes et de l’irradiation ultra-violette (UV). La matrice lipidique du SC est constituée de trois lipides majeurs : les céramides, les acides gras et le cholestérol organisés en phase cristalline. Cette matrice est la principale voie de pénétration des molécules exogènes à travers la peau. L’estimation de l’absorption cutanée pour l’analyse du risque des produits cosmétiques est basée sur les recommandations de l’Organisation de Coopération et de Développement Économiques (OCDE) qui prend en compte les propriétés physicochimiques des molécules i.e. Log Pow (lipophilie) et MW (masse moléculaire). En effet, l’OCDE considère une absorption de 100% pour une molécule ayant une MW inférieure à 500g/mol et un Log Pow compris en -1 et +4. En dehors de ces valeurs, l’OCDE applique une estimation de 10%. Hors, cette estimation est bien souvent loin de la réalité et a besoin d’être affinée. Notre travail s’est focalisé sur le développement d’un critère d’évaluation de la pénétration cutanée afin de moduler les données de l’OCDE par trois approches différentes : chromatographie d’affinité, spectroscopie de fluorescence et microspectroscopie infra-rouge à transformée de Fourier (FTIR) avec une source synchrotron. Etant donné que les propriétés barrières de la peau sont étroitement liées à la composition en céramides du SC, les méthodes développées en chromatographie d’affinité et spectroscopie de fluorescence se sont focalisées sur l’interaction céramide-molécules. Un critère prédictif de la pénétration percutanée a été défini avec chacune de ces méthodes :  et I. La troisième méthodologie a consisté à développer un autre critère (Sindex) par microspectroscopie FTIR avec une source synchrotron. La distribution cutanée des molécules a été suivie sur coupes microtomées de biopsies humaines. A partir de Sindex, une cartographie prédictive de la pénétration percutanée des molécules a été établie. Notre design expérimental comprenait des molécules (filtres UV, conservateurs, actifs cosmétiques) avec des Log Pow et MW variés (cf annexe 1). La pénétration cutanée de ces molécules a été étudiée avec une méthode de référence : cellules de Franz couplées à la chromatographie. Ces données de référence ont servi à valider les modèles et critères prédictifs développés. Ce travail a permis d’explorer de nouvelles pistes pour l’étude prédictive de la pénétration percutanée et de développer ainsi de nouveaux critères. Utilisés en complément des propriétés physicochimiques des molécules, ces nouveaux critères permettent d’affiner l’estimation de la pénétration cutanée de molécules exogènes pour l’analyse du risque. / The stratum corneum (SC) is the upper skin layer and due to its particular composition, corneocytes embedded in a lipidic matrix, it owns a role of barrier function and protects our body against water loss, penetration of exogenous molecules and UV irradiation. Its lipidic matrix is composed of three major lipids: fatty acids, cholesterol and ceramides, organised in liquid crystalline phase. This high cohesion creates cement between corneocytes. This cement is the principal pathway taken by the exogenous molecules to penetrate the skin. Percutaneous penetration estimation of cosmetic products is today based on the Organisation for Economic Co-operation and Development (OECD) recommendations, regarding molecules structural characteristics i.e. Log Pow (polarity) and MW (molecular weight). The OECD claims that 100% dermal absorption may be assumed if the exogenous molecule molecular mass is lower than 500 g/mol and Log Pow ranged between -1 and +4. Besides these values, a 10% coefficient is applied. This approach is sometimes far from reality. Our work focused on developing new evaluation criteria of percutaneous penetration from three different approaches: affinity chromatography, fluorescence spectroscopy and FTIR microspectroscopy with a synchrotron source in order to modulate OECD predictions. Considering that skin barrier properties are closely linked to ceramide composition and conformation within the SC, two methods were developed to study the interaction between ceramides and exogenous molecules by affinity chromatography and fluorescence spectroscopy. A predictive criterion of percutaneous penetration was developed from each of these methods:  and I. The third methodology consisted of developing a predictive criterion, Sindex, by FTIR microspectroscopy with a synchrotron source, on microtomized cuts of human skin biopsies. A predictive cartography was build from Sindex. Our experimental design included exogenous molecules (e.g. UV filters, preservatives, cosmetic actives) with various Log Pow and MW (cf annexe 1). Molecules skin penetration was studied with a Franz cell device coupled to HPLC analysis. These results served as reference data to validate our predictive models and criteria.This work permitted to set up new methods for predicting skin penetration of exogenous molecules and to develop complementary predictive criterion to Log Pow and MW. These new criterion will serve to modulate OECD predictions.

Autosomal dominant epidermolytic ichthyosis (EI) is a rare disease characterized by intra-epidermal blistering due to mutations in either of two keratin genes, KRT1 and KRT10, expressed by suprabasal keratinocytes. Autosomal recessive congenital ichthyosis (ARCI) is a non-blistering, hyperkeratotic disease caused by mutations in one of the following genes: ABCA12, ALOX12B, ALOXE3, TGM1, CYP4F22, NIPAL4 and SLC27A4, which are all essential for skin barrier homeostasis. ARCI and EI often respond well to treatment with retinoids, but the mechanism of action is unclear. The aim of this thesis was to increase the knowledge of pathogenic pathways in ichthyosis and to find new explanations to the effect of retinoids. In vitro studies of immortalized keratinocytes from EI patients showed an abnormal keratin aggregation after heat stress, that could be partially inhibited by pre-treatment with all-trans retinoic acid (ATRA) or retinoic acid receptor α-agonists. ATRA treatment also reduced the relative expression of mutated vs wildtype KRT10. The clearance of ATRA in human keratinocytes was found to be mediated by CYP26B1. In skin biopsies from ARCI patients, immunofluorescence analysis of 12R-LOX, eLOX-3, TGM1, ichthyin and FATP4 showed altered expression, not only of the mutated protein, but also of the other proteins. These observations are consistent with a feedback regulatory mechanism by which the loss of one protein results in an up-regulation of other proteins. Furthermore, 12R-LOX, eLOX-3 and TGM1 were intimately co-localized in stratum corneum, as were ichthyin and FATP4, suggesting that the proteins are linked to the same metabolic pathway. When treated with a CYP26 inhibitor known to raise the endogenous ATRA level of the skin, two patients with NIPAL4 mutations, initially exhibiting increased co-localization signals for 12R-LOX and eLOX-3, displayed normalized lipoxygenase expressions and showed clinical improvement. In conclusion, mechanisms are proposed by which pathogenic keratin aggregations in EI and epidermal protein deficiencies in ARCI patients may be mitigated by retinoids. Furthermore, the vivid crosstalk between proteins incriminated in ARCI suggests that these enzymes operate along a common metabolic pathway essential for producing barrier lipids in stratum corneum. Any abrogation of this production may cause barrier failure, hence resulting in a compensatory hyperkeratosis characteristic of congenital ichthyosis.

N-(4-Hydroxyphenyl) retinamide (4-HPR) is a derivative of all-trans-retinoic acid that induces apoptosis in cancer cell lines and is being tested in clinical trials as a relatively non-toxic anti-cancer agent. 4-HPR induces de novo sphingolipid biosynthesis and production of ceramide has been suggested to contribute to the growth arrest and apoptosis. To characterize the types of ceramides that might be involved, we used liquid chromatography, electrospray ionization tandem mass spectrometry (LC ESI-MS/MS) to analyze the sphingolipids, and found that 4-HPR increased total sphingolipid amounts, but unexpectedly, ceramides (i.e., N-acylsphingosines) changed very little, and in some cases decreased. Instead, dihydroceramides (i.e., N-acylsphinganines) increased as much as 10-fold, both as the free species and as the backbones of dihydrosphingomyelins and dihydrohexosylceramides. To determine if 4-HPR inhibits dihydroceramide desaturase, we synthesized NBD-dihydroceramide and treated Hek293 cells with 4-HPR and analyzed the metabolites by HPLC. These analyses showed that NBD-dihydroceramide was taken up by the cells and converted to NBD-ceramides and more complex NBD-sphingolipids in control cells, however, within one hour of treatment with 10 ~{ and L~}M 4-HPR, the production of NBD-ceramide was blocked. In vitro assays of the desaturase using NBD-dihydroceramide also showed rapid and complete inhibition by 4-HPR. Interestingly, when Hek cells were treated with 4-HPR for one hour then the medium was changed, the recovery of dihydroceramide desaturase activity was very slow (i.e., t1/2 > 66 h); therefore, either 4-HPR is difficult to remove from cells or the inhibition is essentially irreversible. These findings establish that 4-HPR not only induces de novo sphingolipid biosynthesis but also inhibits dihydroceramide desaturase, resulting in production of abnormally high proportions of sphingolipids with dihydroceramide as the backbone. This raises the possibility that some of the effects of 4-HPR on cell behavior may be due to the presence of these abnormal species.

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.

This thesis describes the characterization of ceramide (Cer) biosynthesis by mammalian cells. The possibility that Cer undergo developmental changes was explored using mouse embryonic stem cells versus embryoid bodies by analysis of the Cer subspecies by liquid chromatography, electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS) and of the transcript levels for enzymes involved in Cer biosynthesis by qRT-PCR. Cer of embroid bodies had higher proportions of very-long-chain fatty acids, which correlated with the relative expression of mRNA for the respective Cer synthases (CerS) and fatty acyl-CoA elongases, as well as changes in the fatty acyl-CoA's of the cells. Therefore, it is clear that Cer subspecies change during embryogenesis, possibly for functionally important reasons. One CerS isoform, CerS2, was studied further because it has the broadest tissue distribution and a remarkable fatty acyl-CoA specificity, utilizing longer acyl-chain CoAs (C20-C26) in vitro. The fatty acid chain selectivity was refined by analysis of the Cer from livers from CerS2 null mice, which displayed very little Cer with fatty acyl chains with 24 + 2 carbons. Another interesting structural variation was discovered in studies of cells treated with fumonisin B1 (FB1), which inhibits CerS. Under these conditions, cells in culture and animals accumulate substantial amounts of a novel sphingoid base that was identified as 1-deoxysphinganine. This compound arises from utilization of L-alanine instead of L-serine by serine palmitoyltransferase (SPT) based on the inability of LYB cells, which lack SPT, to make 1-deoxysphinganine. In the absence of FB1, 1-deoxysphinganine is primarily acylated to 1-deoxydihydroceramides. These are an underappreciated category of bioactive sphingoid bases and "ceramides" that might play important roles in cell regulation and disease. In summary, cells contain a wide variety of Cer subspecies that are determined by changes in expression of CerS, enzymes that produce co-substrates (such as fatty acyl-CoAs), and the types of amino acids utilized by SPT, the initial enzyme of de novo sphingolipid biosynthesis. One can envision how these changes might impact membranes structure as well as signaling by this family of highly bioactive compounds.

Thesis (Ph. D.)--Lunds universitet, 2005. / Title from title screen. Description based on contents viewed May 20, 2005. Includes bibliographical references (p. [36]-[48]).