José Luis Abad

Institut Marqués, Spain, Barcelona, Barcino, Catalonia, Spain

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Publications (41)189.13 Total impact

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    ABSTRACT: Several diseases involve alterations in sphingolipid metabolism, so the development of tools for the analysis of sphingolipid metabolic fluxes is of interest. In this work, ω-azidosphingolipids 1-3 have been synthesized and tested as tracers in live cells. The synthesis starts from (S)-Garner's aldehyde and uses bromide or tosyloxy precursors for the introduction of the azido group into the sphingoid base. Studies in HGC-27 cells showed that probes 1-3 compete with the natural metabolites and are incorporated into sphingolipid pathways without affecting cell viability. The reactivity and bioorthogonality of the terminal azido group have been exploited by means of click reactions with different azadibenzocyclooctyne tags. This allows the mass spectrometric characterization of azidosphingolipidomes in pooled samples from different cell populations after independent treatments, providing proof of concept of the applicability of this technology in sphingolipid metabolic flux analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    ChemBioChem 02/2015; 16(4). DOI:10.1002/cbic.201402649 · 3.06 Impact Factor
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    ABSTRACT: Three ceramide analogues have been synthesized, with sphingosine-like chains containing five conjugated double bonds. Pentaene I has an N-palmitoyl acyl chain, while the other two pentaenes contain also a doxyl radical, respectively at C5 (Penta5dox) and at C16 (Penta16 dox) positions of the Nacyl chain. Pentaene I maximum excitation and emission wavelengths in a phospholipid bilayer are 353 nm and 478 nm respectively. Pentaene I does not segregate from the other lipids in the way natural ceramide does, but rather mixes with them in a selective way according to the lipid phases involved. Fluorescence confocal microscopy studies show that, when lipid domains in different physical states coexist, Pentaene I emission is higher in gel than in fluid domains, and in liquid-ordered than in liquid-disordered areas. Electron paramagnetic resonance of the pentaene doxyl probes confirms that these molecules are sensitive to the physical state of the bilayer. Calorimetric and fluorescence quenching experiments suggest that the lipids under study orient themselves in lipid bilayers with their polar moieties located at the lipid-water interface. The doxyl radical in the N-acyl chain quenches the fluorescence of the pentaene group when in close proximity. Because of this property, Penta16dox can detect gel-fluid transitions in phospholipids. The availability of probes for lipids in the gel phase is important in view of novel evidence for the existence of gel microdomains in cell membranes.
    Langmuir 02/2015; DOI:10.1021/la505017x · 4.38 Impact Factor
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    ABSTRACT: A stereoselective synthesis of spisulosine (ES285) and 4,5-dehydrospisulosine stereoisomers is described. Hydrozirconation of 1-pentadecyne with Schwartz reagent, followed by diastereocontrolled addition to L- or D-alaninal afforded the required 2-amino 1,3-diol framework. The resulting sphingoid bases revealed as excellent probes for the profiling of ceramide synthase activity in intact cells. Among the sphingoid bases described in this work, spisulosine (ES285), RBM1-77 and RBM1-73 were the most suitable ones due to their highest acylation rates. These molecules should prove useful to study the role of the different ceramide synthases and the resulting N acyl (dihydro)ceramides in cell fate.
    The Journal of Organic Chemistry 05/2013; 78(12). DOI:10.1021/jo400440z · 4.64 Impact Factor
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    ABSTRACT: Acid ceramidase (AC) catalyzes the hydrolysis of ceramide into sphingosine, in turn a substrate of sphingosine kinases that catalyze its conversion into the mitogenic sphingosine-1-phosphate. AC is expressed at high levels in several tumor types and has been proposed as a cancer therapeutic target. Using a model derived from PC-3 prostate cancer cells, the highly tumorigenic, metastatic and chemoresistant clone PC-3/Mc expressed higher levels of the AC ASAH1 than the non-metastatic clone PC-3/S. Stable knockdown of ASAH1 in PC-3/Mc cells caused an accumulation of ceramides, inhibition of clonogenic potential, increased requirement for growth factors, and inhibition of tumorigenesis and lung metastases. We developed de novo ASAH1 inhibitors, which also caused a dose-dependent accumulation of ceramides in PC-3/Mc cells and inhibited their growth and clonogenicity. Finally, immunohistochemical analysis of primary prostate cancer samples showed that higher levels of ASAH1 were associated with more advanced stages of this neoplasia. These observations confirm ASAH1 as a therapeutic target in advanced and chemoresistant forms of prostate cancer, and suggest that our new potent and specific AC inhibitors could act by counteracting critical growth properties of these highly aggressive tumor cells.
    The Journal of Lipid Research 02/2013; DOI:10.1194/jlr.M032375 · 4.73 Impact Factor
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    ABSTRACT: Modulation of sphingolipid metabolism is a promising strategy for cancer therapy that has already opened innovative approaches for the development of pharmacological tools and rationally designed new drugs. On the other hand, natural products represent a classical and well-established source of chemical diversity that has guided medicinal chemists on the development of new chemical entities with potential therapeutic use. Based on these premises, the aim of this chapter is to provide the reader with a general overview of some of the most representative families of sphingolipid-related natural products that have been described in the recent literature as lead compounds for the design of new modulators of sphingolipid metabolism. Special emphasis is placed on the structural aspects of natural sphingoids and synthetic analogs that have found application as anticancer agents. In addition, their cellular targets and/or their mode of action are also considered.
    Advances in Cancer Research 01/2013; 117C:237-281. DOI:10.1016/B978-0-12-394274-6.00008-X · 4.26 Impact Factor
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    ABSTRACT: Dihydroceramides, the precursors of ceramides in the de novo sphingolipid synthesis, have been recently implicated in active signalling. We previously demonstrated that dihydroceramide accumulation, in response to treatment with the dihydroceramide desaturase inhibitor XM462, induced autophagy with no sign of cell death in the gastric carcinoma HCG27 cell line. Here we show that XM462 treatment induces a transient early increase in dihydroceramides that are successively metabolized into other sphingolipids. Dihydroceramides accumulation is associated with cyclin D1 expression modulation, delayed G1/S transition of cell cycle and increased autophagy. Moreover, XM462 treatment induces ER stress via the activation of the translation inhibitor eIF2α and the pro-survival transcriptional factor Xbp1. Exogenous addition of a short chain dihydroceramide analog reproduces the effects of endogenous accumulation of dihydroceramides, causing cell cycle delay of the G1/S transition, autophagy enhancement, eIF2α activation and Xbp1 splicing. Blocking autophagy with 3-methyladenine abrogates the effect of XM462 on cell cycle and reduces cell survival to XM462 treatment. Furthermore, the XM462-induced survival response is able to reduce etoposide toxicity in HCG27 and HCT116 cancer cells. Our data suggest a role of dihydroceramide in regulating cell proliferation and survival.
    The international journal of biochemistry & cell biology 09/2012; 44(12):2135-2143. DOI:10.1016/j.biocel.2012.08.025 · 4.24 Impact Factor
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    ABSTRACT: Ceramide analogues containing azide groups either in the polar head or in the hydrocarbon chains are non-fluorescent. When incorporated into phospholipid bilayers, they can react in situ with a non-fluorescent 1,8-naphthalimide using click chemistry giving rise to fluorescent ceramide derivatives emitting at ≈440 nm. When incorporated into giant unilamellar vesicles, two-photon excitation at 760 nm allows visualization of the ceramide-containing bilayers. This kind of method may be of general applicability in the study of model and cell membranes.
    Journal of Chemical Biology 07/2012; 5(3):119-23. DOI:10.1007/s12154-012-0075-0
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    ABSTRACT: Corneocyte desquamation has been ascribed to the following: 1) proteolytic degradation of corneodesmosomes (CDs); 2) disorganization of extracellular lamellar bilayers; and/or 3) "swell-shrinkage-slough" from hydration/dehydration. To address the cellular basis for normal exfoliation, we compared changes in lamellar bilayer architecture and CD structure in D-Squame strips from the first versus fifth stripping ("outer" vs. "mid"-stratum corneum (SC), respectively) from nine normal adult forearms. Strippings were either processed for standard electron microscopy (EM) or for ruthenium-, or osmium-tetroxide vapor fixation, followed by immediate epoxy embedment, an artifact-free protocol, which, to our knowledge, is previously unreported. CDs are largely intact in the mid-SC, but replaced by electron-dense (hydrophilic) clefts (lacunae) that expand laterally, splitting lamellar arrays in the outer SC. Some undegraded desmoglein 1/desmocollin 1 redistribute uniformly into corneocyte envelopes (CEs) in the outer SC (shown by proteomics, Z-stack confocal imaging, and immunoEM). CEs then thicken, likely facilitating exfoliation by increasing corneocyte rigidity. In vapor-fixed images, hydration only altered the volume of the extracellular compartment, expanding lacunae, further separating membrane arrays. During dehydration, air replaced water, maintaining the expanded extracellular compartment. Hydration also provoked degradation of membranes by activating contiguous acidic ceramidase activity. Together, these studies identify several parallel mechanisms that orchestrate exfoliation from the surface of normal human skin.
    Journal of Investigative Dermatology 06/2012; 132(10):2430-9. DOI:10.1038/jid.2012.173 · 6.37 Impact Factor
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    ABSTRACT: Three analogs of the dihydroceramide desaturase inhibitor XM462 are reported. The compounds inhibit both dihydroceramide desaturase and acid ceramidase, but with different potencies depending on the N-acyl moiety. Other enzymes of sphingolipid metabolism, such as neutral ceramidase, acid sphingomyelinase, acid glucosylceramide hydrolase, sphingomyelin synthase and glucosylceramide synthase, are not affected. The effect on the sphingolipidome of the two best inhibitors, namely (R,E)-N-(1-hydroxy-4-(tridecylthio)but-3-en-2-yl)octanamide (RBM2-1B) and (R,E)-N-(1-hydroxy-4-(tridecylthio)but-3-en-2-yl)pivalamide (RBM2-1D), is in accordance with the results obtained in the enzyme assays. These two compounds reduce cell viability in A549 and HCT116 cell lines with similar potencies and both induced apoptotic cell death to similar levels than C8-Cer in HCT116 cells. The possible therapeutic implications of the activities of these compounds are discussed.
    Bioorganic & medicinal chemistry 04/2012; 20(10):3173-9. DOI:10.1016/j.bmc.2012.03.073 · 2.95 Impact Factor
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    ABSTRACT: The sphingolipid ceramide is known to play a central role in chemo- and radiation-induced cell death. Acid ceramidase (AC) hydrolyzes ceramide, and thus reduces intracellular levels of this proapoptotic lipid. The role of AC as a putative anticancer target is supported by reports of upregulation in prostate cancer and in some breast tumors. In this study, we determined whether the introduction of an AC inhibitor would enhance the apoptosis-inducing effects of C6-ceramide (C6-cer) in breast cancer cells. Cultured breast cancer cells were treated with DM102 [(2R,3Z)-N-(1-hydroxyoctadec-3-en-2-yl)pivalamide, C6-cer, or the combination. Cell viability and cytotoxic synergy were assessed. Activation of apoptotic pathways, generation of reactive oxygen species, and mitochondrial transmembrane potential were determined. DM102 was a more effective AC inhibitor than N-oleoylethanolamine (NOE) and (1R,2R)-2-N-(tetradecanoylamino)-1-(4'-nitrophenyl)-1,3-propandiol (B-13) in MDA-MB-231, MCF-7, and BT-474 cells. As single agents, C6-cer (IC(50) 5-10 μM) and DM102 (IC(50) 20 μM) were only moderately cytotoxic in MDA-MB-231, MCF-7, and SK-BR-3 cells. Co-administration, however, produced synergistic decreases in viability (combination index <0.5) in all cell lines. Apoptosis was confirmed in MDA-MB-231 cells by detection of caspase 3 cleavage and a >3-fold increase in caspase 3/7 activation, PARP cleavage, and a >70% increase in Annexin-V positive cells. C6-cer/DM102 increased ROS levels 4-fold in MDA-MB-231 cells, shifted the ratio of Bax:Bcl-2 to >9-fold that of control cells, and resulted in mitochondrial membrane depolarization. DM102 also increased the synthesis of (3)H-palmitate-labeled long-chain ceramides by 2-fold when C6-cer was present. These data support the effectiveness of targeting AC in combination with exogenous short-chain ceramide as an anticancer strategy, and warrant continued investigation into the utility of the C6-cer/DM102 drug duo in human breast cancer.
    Breast Cancer Research and Treatment 09/2011; 133(2):447-58. DOI:10.1007/s10549-011-1768-8 · 4.20 Impact Factor
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    ABSTRACT: Sphingolipids are membrane lipids that play important roles in the regulation of cell functions and homeostasis. Alterations in their metabolism have been associated with several pathologies. For this reason, therapeutic strategies based on the design of small molecules to restore sphingolipid levels to their physiological condition have rapidly emerged. In addition, some of these new chemical entities, even if they fail to succeed along the pipeline, can become valuable pharmacological tools for the study of sphingolipid function. Implications of altered sphingolipid metabolism in cancer progression have allowed the identification of new targets for the development of potential anticancer agents. Based on these premises, this review is focused on the most recent achievements in the field, with special attention to the development of small molecules, mainly enzyme inhibitors, able to disrupt some of the key sphingolipid metabolic pathways implicated in cancer progression. On the other hand, metabolic dysregulation can also be modulated by the use of sphingolipid analogs, which can alter the sphingolipid balance driving cells to death or survival and thus becoming useful candidates for subsequent drug development.
    Anti-cancer agents in medicinal chemistry 08/2011; 12(4):285-302. DOI:10.2174/187152012800228643 · 2.94 Impact Factor
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    ABSTRACT: Ceramidases are ubiquitous amidohydrolases that catalyze the cleavage of ceramides into sphingosine and fatty acids. This reaction exerts a cytoprotective role in physiological conditions, while altered ceramidase activities favour a number of human diseases. Among these diseases, several reports point to important roles of ceramidases, mainly the acid ceramidase, in the initiation and progression of cancer, and the response of tumors to radio- or chemotherapy. Multiple reports confirm the interest of acid ceramidase inhibitors as anticancer drugs, either alone or in combination with other therapies. Sphingolipid metabolism plays a role in hematological malignancies and appears as an interesting target for therapeutic intervention. Although the use of ceramidase inhibitors in chemotherapy of hematologic cancers has not been widely investigated, a number of indirect evidence suggest that inhibition of specific ceramidases could potentiate the effect of drugs in clinical use to treat hematologic malignancies and may afford strategies to combat relapses. The arsenal of ceramidase inhibitors so far available is wide and hopefully, upcoming research will assess the feasibility of this approach.
    Anti-cancer agents in medicinal chemistry 06/2011; 11(9):830-43. DOI:10.2174/187152011797655104 · 2.94 Impact Factor
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    ABSTRACT: The T-box brain 1 (Tbr1) gene encodes a transcription factor necessary for the maintenance and/or differentiation of glutamatergic cells in the olfactory bulb (OB) and cortex, although its precise function in the development of glutamatergic neurons is not known. Furthermore, Tbr1 has not been reported to regulate the formation of glial cells. We show that Tbr1 is expressed during the initial stages in the generation of glutamatergic mitral neurons from dividing progenitors in the E12.5 mouse OB. Retroviral-mediated overexpression of Tbr1 in cultured embryonic and adult OB stem cells (OBSC) produces a marked increase in the number of TuJ1(+) neurons (including VGLUT1(+) glutamatergic and GABA(+) neurons) and O4(+) oligodendrocytes. Moreover, transduction of Tbr1 inhibits the production of GFAP(+) astrocytes from both cultured OBSC and dividing progenitor cells in vivo. These results show that the expression of Tbr1 in neural stem and progenitor cells prevents them from following an astrocyte fate during OB development. Our findings suggest that the transduction of Tbr1 into neural stem cells could be useful to increase the production of neurons and oligodendrocytes in studies of neuroregeneration.
    Molecular and Cellular Neuroscience 01/2011; 46(1):108-21. DOI:10.1016/j.mcn.2010.08.011 · 3.73 Impact Factor
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    ABSTRACT: Acid ceramidase (aCDase) is one of several enzymes responsible for ceramide degradation within mammalian cells. As such, aCDase regulates the intracellular levels of the bioactive lipid ceramide. An inherited deficiency of aCDase activity results in Farber disease (FD), also called lipogranulomatosis, which is characterized by ceramide accumulation in the tissues of patients. Diagnosis of FD is confirmed by demonstration of a deficient aCDase activity and the subsequent storage of ceramide. Existing methods include extremely complex assays, many of them using radiolabeled compounds. Therefore, the aCDase assay and the in vitro enzymatic diagnosis of FD are still performed in only a very limited number of specialized laboratories. Here, the new fluorogenic substrate Rbm14-12 was synthesized and characterized as a new tool to determine aCDase activity. The resulting optimized assay was performed in 96-well plates, and different fibroblast and lymphoid cell lines derived from FD patients and controls were tested to measure aCDase activity. As a result, the activity in cells of FD patients was found to be very low or even null. This new fluorogenic method offers a very easy and rapid way for specific and accurate determination of aCDase activity and, consequently, for diagnosis of FD.
    The Journal of Lipid Research 12/2010; 51(12):3542-7. DOI:10.1194/jlr.D010033 · 4.73 Impact Factor
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    ABSTRACT: An unexpected access to a new sphingoid base containing a vinyl sulfide unit is described. The process involves the 'one-pot' regioselective opening of an epoxide with a thiolate, followed by intramolecular acyl transfer, base-promoted elimination, and final hydrolysis. Excess sodium hydride and high dilution conditions are required for optimal yields. The process is amenable to a variety of thiolates. The resulting compounds can be regarded as new hybrid sphingoid bases that combine some structural motifs present in other reported sphingolipid analogues.
    Synlett 12/2010; 2010(19):2950-2952. DOI:10.1055/s-0030-1259047 · 2.46 Impact Factor
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    ABSTRACT: The lateral organization of lipids in cell membranes is thought to regulate numerous cell processes. Most studies focus on the coexistence of two fluid phases, the liquid crystalline (l(d)) and the liquid-ordered (l(o)); the putative presence of gel domains (s(o)) is not usually taken into account. We show that in phospholipid:sphingolipid:cholesterol mixtures, in which sphingomyelin (SM) promoted fluid l(o) domains, dihydrosphingomyelin (DHSM) tended to form rigid domains. Genetic and pharmacological blockade of the dihydroceramide desaturase (Des1), which replaced SM with DHSM in cultured cells, inhibited cell infection by replication-competent and -deficient HIV-1. Increased DHSM levels gave rise to more rigid membranes, resistant to the insertion of the gp41 fusion peptide, thus inhibiting viral-cell membrane fusion. These results clarify the function of dihydrosphingolipids in biological membranes and identify Des1 as a potential target in HIV-1 infection.
    Chemistry & biology 07/2010; 17(7):766-75. DOI:10.1016/j.chembiol.2010.05.023 · 6.59 Impact Factor
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    ABSTRACT: Simple bioactive sphingolipids include ceramide, sphingosine and their phosphorylated forms sphingosine 1-phosphate and ceramide 1-phosphate. These molecules are crucial regulators of cell functions. In particular, they play important roles in the regulation of angiogenesis, apoptosis, cell proliferation, differentiation, migration, and inflammation. Decoding the mechanisms by which these cellular functions are regulated requires detailed understanding of the signaling pathways that are implicated in these processes. Most importantly, the development of inhibitors of the enzymes involved in their metabolism may be crucial for establishing new therapeutic strategies for treatment of disease.
    Progress in lipid research 02/2010; 49(4):316-34. DOI:10.1016/j.plipres.2010.02.004 · 12.96 Impact Factor
  • ChemBioChem 03/2009; 10(5):820-2. DOI:10.1002/cbic.200800809 · 3.06 Impact Factor
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    ABSTRACT: The sex pheromone of the female processionary moth, Thaumetopoea pityocampa, is a unique C16 enyne acetate that is biosynthesized from palmitic acid. Three consecutive desaturation reactions transform this saturated precursor into the triunsaturated fatty acyl intermediate: formation of (Z)-11-hexadecenoic acid, acetylenation to 11-hexadecynoic acid, and final Delta(13) desaturation to (Z)-13-hexadecen-11-ynoic acid. By using degenerate primers common to all reported insect desaturases, a single cDNA sequence was isolated from total RNA of T. pityocampa female pheromone glands. The full-length transcript of this putative desaturase was expressed in elo1Delta/ole1Delta yeast mutants (both elongase 1 and Delta(9) desaturase-deficient) for functional assays. The construct fully rescued the Deltaole1 yeast phenotype, confirming its desaturase activity. Analysis of the unsaturated products from transformed yeast extracts demonstrated that the cloned enzyme showed Delta(11) desaturase, Delta(11) acetylenase, and Delta(13) desaturase activities. Therefore, this single desaturase may account for the three desaturation steps involved in the sex pheromone biosynthetic pathway of the processionary moth.
    Proceedings of the National Academy of Sciences 11/2007; 104(42):16444-9. DOI:10.1073/pnas.0705385104 · 9.81 Impact Factor
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    ABSTRACT: Sphingolipids comprise an important group of biomolecules, some of which have been shown to play important roles in the regulation of many cell functions. From a structural standpoint, they all share a long 2-amino-1,3-diol chain, which can be either saturated (sphinganine), hydroxylated at C4 (phytosphingosine), or unsaturated at C4 (sphingosine) as in most mammalian cells. N-acylation of sphingosine leads to ceramide, a key intermediate in sphingolipid metabolism that can be enzymatically modified at the C1-OH position to other biologically important sphingolipids, such as sphingomyelin or glycosphingolipids. In addition, both ceramide and sphingosine can be phosphorylated at C1-OH to give ceramide-1-phosphate and sphingosine-1-phosphate, respectively. To better understand the biological and biophysical roles of sphingolipids, many efforts have been made to design synthetic analogues as chemical tools able to unravel their structure-activity relationships, and to alter their cellular levels. This last approach has been thoroughly studied by the development of specific inhibitors of some key enzymes that play an important role in biosynthesis or metabolism of these intriguing lipids. With the above premises in mind, the aim of this review is to collect, in a systematic way, the recent efforts described in the literature leading to the development of new chemical entities specifically designed to achieve the above goals.
    ChemMedChem 07/2007; 2(5):580-606. DOI:10.1002/cmdc.200600195 · 3.05 Impact Factor

Publication Stats

1k Citations
189.13 Total Impact Points

Institutions

  • 2009–2015
    • Institut Marqués, Spain, Barcelona
      Barcino, Catalonia, Spain
  • 2007–2013
    • Spanish National Research Council
      • • Department of Biomedicinal Chemistry
      • • Institute of Advanced Chemistry of Catalonia
      Madrid, Madrid, Spain
  • 2012
    • Institut d'Arquitectura Avançada de Catalunya
      Barcino, Catalonia, Spain
  • 2011
    • Parque Científico de Madrid
      Madrid, Madrid, Spain
  • 1998–2002
    • Universidad Autónoma de Madrid
      • Department of Immunology
      Madrid, Madrid, Spain