Jan Tytgat

Universitair Psychiatrisch Centrum KU Leuven, Cortenberg, Flemish, Belgium

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Publications (252)898.04 Total impact

  • 18th World IST Congress on Animal, Plant and Microbial Toxins, Oxford, United Kingdom; 08/2015
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    ABSTRACT: We designed a peptide, PnPP-19, comprising the potential active core of Phoneutria nigriventer native toxin PnTx2-6, and investigated its role on EF, its toxicity and immunogenicity. EF was evaluated through ICP/MAP ratio during EFS on rat pelvic ganglion. Corpus cavernous strips were PE-contracted and relaxation was induced by EFS with or without PnPP-19 (10(-8)M). The activity on sodium channels was evaluated by electrophysiological screening of transfected channels on Xenopus oocytes and DRG cells. Antibodies were detected by indirect ELISA in mice previously treated with the peptide. Histopathological studies were performed with mice organs receiving different doses of PnPP-19. PnPP-19 was able to potentiate erection at 4 and 8 Hz, in vivo and ex vivo. It showed no toxicity and low immunogenicity to mice and did not affect sodium channels or rat hearts. PnPP-19 increased cGMP levels at 8 Hz and this effect was inhibited by L-NAME (10(-4)M). EF was partially inhibited by 7-nitroindazole (7-NI, 10(-5)M), a selective inhibitor of nNOS. PnPP-19 potentiates erection, in vivo and ex vivo, via NO/cGMP pathway, does not affect sodium channels or rat heart, and shows no toxicity and low immunogenicity, what makes it a very promising candidate as a novel drug in the therapy of erectile dysfunction. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.
    The Journal of urology 06/2015; DOI:10.1016/j.juro.2015.06.081 · 3.75 Impact Factor
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    ABSTRACT: Ts19 Fragment II (Ts19 Frag-II) was first isolated from the venom of the scorpion Tityus serrulatus (Ts). It is a protein presenting 49 amino acid residues, three disulfide bridges, Mr 5,534Da and was classified as a new member of class (subfamily) 2 of the β-KTxs, the second one described for Ts scorpion. The β-KTx family is composed by two-domain peptides: N-terminal helical domain (NHD), with cytolytic activity, and a C-terminal CSαβ domain (CCD), with Kv blocking activity. The extensive electrophysiological screening (16 Kv channels and 5 Nav channels) showed that Ts19 Frag-II presents a specific and significant blocking effect on Kv1.2 (IC50 value of 544±32nM). However, no cytolytic activity was observed with this toxin. We conclude that the absence of 9 amino acid residues from the N-terminal sequence (compared to Ts19 Frag-I) is responsible for the absence of cytolytic activity. In order to prove this hypothesis, we synthesized the peptide with these 9 amino acid residues, called Ts19 Frag-III. As expected, Ts19 Frag-III showed to be cytolytic and did not block the Kv1.2 channel. The post-translational modifications of Ts19 and its fragments (I-III) are also discussed here. A mechanism of post-translational processing (post-splitting) is suggested to explain Ts19 fragments production. In addition to the discovery of this new toxin, this report provides further evidence for the existence of several compounds in the scorpion venom contributing to the diversity of the venom arsenal. Copyright © 2015. Published by Elsevier Inc.
    Peptides 06/2015; DOI:10.1016/j.peptides.2015.06.004 · 2.61 Impact Factor
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    ABSTRACT: Kbot55 is a 39 amino acid peptide isolated from the venom of the Tunisian scorpion Buthus occitanus tunetanus. This peptide is cross-linked by 3 disulfide bridges and has a molecular mass of 4128.65Da. Kbot55 is very low represented in the venom and thus represents a challenge for biochemical characterization. In this study, Kbot55 has been subjected to a screening on ion channels expressed in Xenopus laevis oocytes. It was found that Kbot55 targets voltage-gated potassium channels with high affinity. Kbot55 shows very low amino acid identity with other scorpion potassium toxins and therefore was considered a bona fide novel type of scorpion toxin. Sequence alignment analysis indicated that Kbot55 is the first representative of the new α-Ktx31 subfamily and therefore was classified as α-Ktx31.1. Copyright © 2015. Published by Elsevier Inc.
    Peptides 06/2015; DOI:10.1016/j.peptides.2015.05.015 · 2.61 Impact Factor
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    ABSTRACT: Sponges of the genus Agelas produce compounds that modulate the activity of voltage-gated sodium ion channels and contribute novel scaffolds for the development of compounds with activity against a plethora of biological targets. In particular, clathrodin and dibromosceptrin were reported to decrease the average maximum amplitude of inward sodium currents in isolated chick embryo sympathetic ganglia cells; we envisaged these compounds as a starting point to design novel Nav channel modulators. This endeavor was part of our long-term goal of designing a comprehensive library of Agelas alkaloid analogs that would cover a broader chemical space and allow us to examine the activity of such compounds on Nav channels. Our series of compounds was designed by maintaining the terminal structural features found in clathrodin while rigidizing the central part of the molecule and replacing the 3-aminopropene linker with a 4-methylenepiperazine moiety. Synthesised compounds were screened for inhibitory action against the human voltage-gated sodium channel isoforms Nav 1.3, Nav 1.4, cardiac Nav 1.5, and Nav 1.7 using an automated patch clamp electrophysiology technique. The results demonstrate that we have obtained a series of compounds with a modest but selective inhibitory activity against the Nav 1.3 channel isoform. The most potent compound showed selective activity against the Nav 1.3 channel isoform with an IC50 of 19 μM and is a suitable starting point for further development of selective Nav 1.3 channel modulators. Such compounds could prove to be beneficial as a pharmacological tool towards the development of novel therapeutically useful compounds in the treatment of pain.
    Medicinal Chemistry Research 06/2015; 24(6):2366-2380. DOI:10.1007/s00044-014-1300-x · 1.61 Impact Factor
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    ABSTRACT: As a cornerstone of quality management in the laboratory, External Quality Assessment (EQA) schemes are used to assess laboratory and analytical method performance. The characteristic function is used to describe the relation between the target concentration and the EQA standard deviation, which is an essential part of the evaluation process. The characteristic function is also used to compare the variability of different analytical methods. We fitted the characteristic function to data from the Belgian External Quality Assessment program for serum ethanol. Data included results from headspace gas chromatography and the enzymatic methods of Abbott, Roche, Siemens, and Ortho-Clinical Diagnostics. We estimated the characteristic function with weighted nonlinear regression. By introducing dummy variables, we rewrote the original formula of the characteristic function to assess statistical inference for comparing the variability of the different analytical methods. The characteristic function fitted the data precisely. Comparison between methods showed that there was little difference between the estimated variability for low concentrations, and that the increase in SD with increasing target concentration was slower for Abbott and Roche than for the other methods. The characteristic function can successfully be introduced in clinical schemes, although its applicability to fit the data should always be assessed. Because of its easy parameterization, it can be used to assess differences in performance between analytical methods and to assess laboratory performance. The characteristic function also offers an alternative framework for coefficients of variation to describe variability of analytical methods. © 2015 American Association for Clinical Chemistry.
    Clinical Chemistry 05/2015; DOI:10.1373/clinchem.2015.240176 · 7.77 Impact Factor
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    ABSTRACT: Tityus serrulatus (Ts) venom is composed of mainly neurotoxins specific for voltage-gated K(+) and Na(+) channels, which are expressed in many cells such as macrophages. Macrophages are the first line of defense invasion and they participate in the inflammatory response of Ts envenoming. However, little is known about the effect of Ts toxins on macrophage activation. This study investigated the effect of Ts5 toxin on different sodium channels as well as its role on the macrophage immunomodulation. The electrophysiological assays showed that Ts5 inhibits the rapid inactivation of the mammalian sodium channels Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6 and Nav1.7. Interestingly, Ts5 also inhibits the inactivation of the insect Drosophila melanogaster sodium channel (DmNav1), and is therefore classified as the first Ts α-like toxin. The immunological experiments on macrophages reveal that Ts5 is a pro-inflammatory toxin inducing the cytokine production of tumor necrosis factor (TNF)-α and interleukin (IL)-6. On the basis of recent literature, our study also stresses a possible mechanism responsible for venom-associated molecular patterns (VAMPs) internalization and macrophage activation and moreover we suggest two main pathways of VAMPs signaling: direct and indirect. This work provides useful insights for a better understanding of the involvement of VAMPs in macrophage modulation. Copyright © 2015. Published by Elsevier B.V.
    Biochimie 04/2015; 115. DOI:10.1016/j.biochi.2015.04.010 · 3.12 Impact Factor
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    ABSTRACT: Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) for the analysis of intact hair is a powerful tool for the detection of drugs of abuse in toxicology and forensic applications. Here we present a quick, easy, and reproducible method of preparing longitudinal sections of single hairs. This method improves the accessibility of chemicals embedded in the hair matrix for molecular imaging with mass spectrometry. The images obtained from a single, sectioned hair sample show molecular distributions in the exposed medulla, cortex, and a portion of the cuticle observed as a narrow layer surrounding the cortex. Using MALDI-MS/MS imaging, the distribution of cocaine was observed throughout five longitudinally sectioned drug-user hair samples. The images showed the distribution of the product ion at m/z 182, derived from the precursor ion of cocaine at m/z 304. MetA-SIMS images of longitudinally sectioned hair samples showed a more detailed distribution of cocaine at m/z 304, benzoylecgonine the major metabolite of cocaine at m/z 290 and other drugs such as methadone which was observed at m/z 310. Chronological information of drug intake can be obtained more sensitively. The chronological detail is in hours rather than months, which is of great interest in clinical as well as forensic applications. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.
    Drug Testing and Analysis 04/2015; DOI:10.1002/dta.1812 · 2.82 Impact Factor
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    ABSTRACT: In the last decades, Ts1 has not only been the subject of many studies, it has also been considered as a very useful tool to investigate NaV channels and to explore the exact role of NaV channels in channelopathies. Ts1 is believed to modulate the activation process of NaV upon interaction at the neurotoxin binding site 4. Our aim was to carry out an in depth functional characterization of Ts1 on a wide array of Nav channels, in order to investigate its mechanism of action and to verify if Ts1 can indeed be considered as a prototype site 4 selective toxin, valid for all the Nav isoforms we know currently. Ts1 has been subjected to an in-depth functional investigation on 9 NaV isoforms expressed in Xenopus laevis oocytes. Ts1 does not only interfere with the activation process but also modulates the inactivation in a bell-shaped voltage-dependent matter. Furthermore, Ts1 altered the ion selectivity through insect NaV. without influencing the tetrodotoxin selectivity of the channels. Finally, Ts1 was also found to inhibit the sodium current through the cardiac Nav1.5 isoform. On the basis of the totally unexpected plethora of Nav modulations as induced by Ts1, we demonstrate that caution is required in interpretation the in vivo experiments when using Ts1. The electrophysiological characterization of Ts1 indeed shows that the general accepted contours of NaV binding sites are much more obscure than believed and that interpretation of NaV pharmacology upon toxin binding is more complex than believed thus far. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Neuropharmacology 04/2015; 95. DOI:10.1016/j.neuropharm.2015.03.027 · 4.82 Impact Factor
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    ABSTRACT: Scorpion venom consists of a complex mixture of molecules including biologically active compounds. Because of their high potency and selectivity, toxins have medical applicability. In the last decades, scorpion toxins have thus gained considerable interest among scientist in the fields of pharmacology, biophysics and neurobiology. Identification of scorpion venom peptides and toxins can be achieved based on transcriptome approaches. We constructed the first cDNA library and Expressed Sequence Tag (EST) study to explore the transcriptomic composition of the telson from the southern African scorpion Hottentotta conspersus, belonging to the family Buthidae. We obtained 21 new venom-related sequences (8 contigs and 16 singlets) from a total of 98 ESTs analyzed, including putative neurotoxins (chloride, potassium, sodium and calcium channel toxins), bradykinin-potentiating peptides and other venom peptides without established function. These novel toxin-related sequences might serve as basis for further research both of pharmaceutical and phylogenetic nature. Copyright © 2015. Published by Elsevier Ltd.
    Toxicon 03/2015; 99. DOI:10.1016/j.toxicon.2015.03.015 · 2.58 Impact Factor
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    ABSTRACT: The lesser Asian scorpion Mesobuthus eupeus (Buthidae) is one of the most widely spread and dispersed species of the Mesobuthus genus and its venom is actively studied. Nevertheless, a considerable amount of active compounds is still under-investigated due to the high complexity of this venom. Here we report a comprehensive analysis of putative potassium channel toxins (KTxs) from the cDNA library of M. eupeus venom glands and compare the deduced KTx structures with peptides purified from the venom. For the transcriptome analysis we used conventional tools as well as a search for structural motifs characteristic of scorpion venom components in the form of regular expressions. We found 59 candidate KTxs distributed in 30 subfamilies and presenting the cysteine-stabilized α/β (CSα/β) and inhibitor cystine knot (ICK) types of fold. M. eupeus venom was then separated to individual components by multi-stage chromatography. A facile fluorescent system based on the expression of the KcsA-Kv1.1 hybrid channels in Escherichia coli and utilization of a labeled scorpion toxin was elaborated and applied to follow Kv1.1 pore-binding activity during venom separation. As a result, eight high-affinity Kv1.1 channel blockers were identified including five novel peptides, which extend the panel of potential pharmacologically important Kv1 ligands. Activity of the new peptides against rat Kv1.1 channel was confirmed (IC50 in the range of 1-750 nM) by the two-electrode voltage-clamp technique using a standard Xenopus oocyte system. Our integrated approach is of general utility and efficiency to mine natural venoms for KTxs. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 03/2015; 290(19). DOI:10.1074/jbc.M115.637611 · 4.60 Impact Factor
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    ABSTRACT: Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the Kv1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.
    PLoS ONE 01/2015; 10(1):e0116369. DOI:10.1371/journal.pone.0116369 · 3.53 Impact Factor
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    ABSTRACT: Conotoxins are venom peptides from cone snails with multiple disulfide bridges that provide a rigid structural scaffold. Typically acting on ion channels implicated in neurotransmission, conotoxins are of interest both as tools for pharmacological studies and as potential new medicines. δ-Conotoxins act by inhibiting inactivation of voltage-gated sodium channels (Nav). Their pharmacology has not been extensively studied because their highly hydrophobic character makes them difficult targets for chemical synthesis. Here we adopted an acid-cleavable solubility tag strategy that facilitated synthesis, purification, and directed disulfide bridge formation. Using this approach we readily produced three native δ-conotoxins from Conus consors plus two rationally designed hybrid peptides. We observed striking differences in Nav subtype selectivity across this group of compounds, which differ in primary structure at only three positions: 12, 23, and 25. Our results provide new insights into the structure-activity relationships underlying the Nav subtype selectivity of δ-conotoxins. Use of the acid-cleavable solubility tag strategy should facilitate synthesis of other hydrophobic peptides with complex disulfide bridge patterns.
    Journal of Biological Chemistry 12/2014; 289(51):35341-35350. DOI:10.1074/jbc.M114.610436 · 4.60 Impact Factor
  • Eva Cuypers, Elien Rosier, Jan Tytgat
    TIAFT 2014; 11/2014
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    ABSTRACT: We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called ″inhibitor cystine knot″ (ICK) or ″knottin″ fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μM Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and feature a ″membrane-access″ mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of ICK toxins from Araneomorphae and Mygalomorphae suborders.
    Journal of Biological Chemistry 10/2014; 290(1). DOI:10.1074/jbc.M114.595678 · 4.60 Impact Factor
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    ABSTRACT: Cone snails (Conus sp.) are poisonous animals that can be found in all oceans where they developed a venomous strategy to prey or to defend. The venom of these species contains an undeniable source of unique and potent pharmacologically active compounds. Their peptide compounds, called conotoxins, are not only interesting for the development of new pharmaceutical ligands, but they are also useful for studying their broad spectrum of targets. One conotoxin family in particular, the α-conotoxins, acts on nicotinic acetylcholine receptors (nAChRs) which dysfunctions play important roles in pathologies such as epilepsy, myasthenic syndromes, schizophrenia, Parkinson's disease and Alzheimer's disease. Here we define a new subclass of the α-conotoxin family. We purified the venom of a yet unexplored cone snail species, i.e. Conus australis, and we isolated a 16-amino acid peptide named α-conotoxin AusIA. The peptide has the typical α-conotoxin CC-Xm-C-Xn-C framework, but both loops (m/n) contain 5 amino acids, which has never been described before. Using conventional electrophysiology we investigated the response of synthetically made globular (I-III, II-IV) and ribbon (I-IV, II-III) AusIA to different nicotinic acetylcholine receptors. The α7 nAChR was the only receptor found to be blocked with a similar potency by both peptide-configurations. This suggests that both α5/5 conotoxin isomers might be present in the venom gland of C. australis. NMR spectroscopy showed that no secondary structures define the peptides' three-dimensional topology. Moreover, the ribbon configuration, which is generally considered to be non-native, is more stable than the globular isoform. Accordingly, our findings show relevancy concerning the α-conotoxin classification which might be helpful in the design of novel therapeutic compounds.
    Toxicon 09/2014; DOI:10.1016/j.toxicon.2014.08.074 · 2.58 Impact Factor
  • Alternative Sampling Strategies in Toxicology and Therapeutic Drug Monitoring, Ghent, Belgium; 09/2014
  • Alternative Sampling Strategies in Toxicology and Therapeutic Drug Monitoring; 09/2014
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    ABSTRACT: Today, forensic hair analysis is considered to be a standard method for identifying chronic drug users since information about drug use stored and located in hair can cover several months to even years. When interpreting these results, one should be aware of all kind of pitfalls. External factors such as bleaching might influence the analytical result. Although the effect of hydrogen peroxide on cocaine in a solution was described before, it was never investigated whether the described reaction products (ecgonine methylester, benzoylecgonine, hydroxynorcocaine and dihydroxycocaine) are indeed found on contaminated or user hair. Since it is of great importance in forensic hair analysis to know whether cocaine and/or reaction products are detectable in hair after bleaching, matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was used to study the effect of hydrogen peroxide treatment on incorporated cocaine in hairs. Cocaine oxidation products were identified in a solution based on MS/MS spectra and spatial distribution of these products in hair was explored using MALDI TOF-MS. All images were accomplished by spraying α-Cyano-4-hydroxycinnamic acid (CHCA) as a MALDI-matrix. Images revealed a loss of detectability of cocaine and its reaction products in hairs already after a short bleaching period. Since all compounds of interest are found in the hydrogen peroxide and wash solution, these findings indicate that all evidence of cocaine use might be lost after a hair bleaching treatment. Therefore, forensic toxicologists should take into consideration whether hair samples were bleached before making any conclusions from hair analysis results.
    Forensic Science International 07/2014; 242C:103-110. DOI:10.1016/j.forsciint.2014.06.035 · 2.12 Impact Factor

Publication Stats

5k Citations
898.04 Total Impact Points


  • 1996–2015
    • Universitair Psychiatrisch Centrum KU Leuven
      Cortenberg, Flemish, Belgium
  • 1994–2015
    • University of Leuven
      • • Laboratory for Toxicology and Food Chemistry
      • • Faculty of Pharmaceutical Sciences
      Louvain, Flemish, Belgium
  • 2014
    • The Catholic University of America
      Washington, Washington, D.C., United States
  • 2006–2012
    • Catholic University of Louvain
      Лувен-ла-Нев, Wallonia, Belgium
  • 2011
    • Nanyang Technological University
      Tumasik, Singapore
    • University of Wisconsin–Madison
      Madison, Wisconsin, United States
  • 2010
    • University of Chicago
      Chicago, Illinois, United States
  • 2005
    • Hunan University
      • College of Chemistry and Chemical Engineering
      Ch’ang-sha-shih, Hunan, China
  • 2004
    • Universität Ulm
      Ulm, Baden-Württemberg, Germany
    • Shanghai Jiao Tong University
      Shanghai, Shanghai Shi, China
  • 2003
    • Architecture et Fonction des Macromolécules Biologiques
      Marsiglia, Provence-Alpes-Côte d'Azur, France
    • University of Debrecen
      • Department of Biophysics and Cell Biology
      Debrecen, Hajdu-Bihar, Hungary
  • 1992
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 1990
    • Martin Luther University Halle-Wittenberg
      • Julius Bernstein Institute for Physiology
      Halle-on-the-Saale, Saxony-Anhalt, Germany