H Bujard

Central Institute of Mental Health, Mannheim, Baden-Württemberg, Germany

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Publications (153)951.83 Total impact

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    ABSTRACT: Plasmodium falciparum is the causative agent of the most severe form of malaria in humans. The merozoite, an extracellular stage of the parasite lifecycle, invades erythrocytes in which they develop. The most abundant protein on the surface of merozoites is Merozoite Surface Protein 1 (MSP1), which consists of four processed fragments. Studies have indicated that MSP1 interacts with other peripheral merozoite surface proteins to form a large complex. Successful invasion of merozoites into host erythrocytes is dependent on this protein complex; however, the identity of all components and its function remain largely unknown. We have shown that the peripheral merozoite surface proteins, MSPDBL1 and MSPDBL2 are part of the large MSP1 complex. Using Surface Plasmon Resonance, we determined the binding affinities of MSPDBL1 and MSPDBL2 to MSP1 to be 171.6 nM and 445.4 nM respectively. Both proteins bind to 3 of the 4 proteolytically cleaved fragments of MSP1 (P42, P38 and P83). In addition, MSPDBL1 and MSPDBL2, but not MSP1, bind directly to human erythrocytes. This demonstrates that the MSP1 complex acts as a platform for display of MSPDBL1 and MSPDBL2 on the merozoite surface for binding to receptors on the erythrocyte and invasion.
    The Journal of biological chemistry. 07/2014;
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    ABSTRACT: The adaptive immune system is involved in tumor establishment and aggressiveness. Tumors of the ovaries, an immune-privileged organ, spread via transceolomic routes and rarely to distant organs. This is contrary to tumors of non-immune privileged organs, which often disseminate hematogenously to distant organs. Epigenetics-based immune cell quantification allows direct comparison of the immune status in benign and malignant tissues and in blood. Here, we introduce the "cellular ratio of immune tolerance" (immunoCRIT) as defined by the ratio of regulatory T cells to total T lymphocytes. The immunoCRIT was analyzed on 273 benign tissue samples of colorectal, bronchial, renal and ovarian origin as well as in 808 samples from primary colorectal, bronchial, mammary and ovarian cancers. ImmunoCRIT is strongly increased in all cancerous tissues and gradually augmented strictly dependent on tumor aggressiveness. In peripheral blood of ovarian cancer patients, immunoCRIT incrementally increases from primary diagnosis to disease recurrence, at which distant metastases frequently occur. We postulate that non-pathological immunoCRIT values observed in peripheral blood of immune privileged ovarian tumor patients are sufficient to prevent hematogenous spread at primary diagnosis. Contrarily, non-immune privileged tumors establish high immunoCRIT in an immunological environment equivalent to the bloodstream and thus spread hematogenously to distant organs. In summary, our data suggest that the immunoCRIT is a powerful marker for tumor aggressiveness and disease dissemination.
    Epigenetics: official journal of the DNA Methylation Society 09/2013; 8(11). · 4.58 Impact Factor
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    ABSTRACT: BACKGROUND: Turning gene expression on and off at will is one of the most powerful tools for the study of gene function in vivo. While several conditional systems were successful in invertebrates, in mice the Cre/loxP recombination system and the Tet-controlled transcription activation system are predominant. Both expression systems allow for spatial and temporal control of gene activities, in the case of Tet regulation, even for the reversible activation/inactivation of gene expression. Although the rat is the principal experimental model in biomedical research, in particular in studies of complex diseases and in neuroscience, conditional rat transgenic systems are exceptionally rare in this species. RESULTS: We addressed this lack of technology and established and thoroughly characterized CreERT2 and tTA transgenic rats with forebrain specific transgene expression, controlled by the CaMKII alpha promoter. In addition, we developed new universal rat reporter lines for both transcription control systems and established inducible and efficient reporter gene expression in forebrain neurons. CONCLUSIONS: We demonstrate that functional conditional genetic manipulations in the rat brain are both feasible and practicable and outline advantages and limitations of both systems.
    BMC Biology 09/2012; 10(1):77. · 7.43 Impact Factor
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    ABSTRACT: The merozoite surface protein (MSP)-1 of Plasmodium falciparum, the causative agent of malaria tropica, is considered to be a promising vaccine candidate. Although its stable cloning and expression has been difficult in the past, adenoviral vectors expressing the complex protein are described in the present study. Codon-optimized msp-1 was used to construct a set of first generation (ΔE1Ad) and high-capacity adenovirus (HC-Ad) vectors, and cellular and humoral immune responses induced by the vectors were characterized in detail in mice. Generation of stable ΔE1Ad and HC-Ad vectors expressing full-length MSP-1 and their production to high vector titers was found to be feasible. Epitope identification and analysis of frequencies of specific CD8 T-cells revealed that MSP-1 expressing HC-Ad vectors induced higher frequencies of interferon-γ + CD8 T-cells than ΔE1 vectors. Irrespective of the vector format, higher titers of MSP-1 specific antibodies were generated by Ad vectors expressing MSP-1 from a chicken β-actin (CAG) promoter comprising the cytomegalovirus early enhancer element and the chicken β-actin promoter. The findings of the present study suggest that Ad vectors expressing full-length codon-optimized MSP-1 are promising candidate vaccines against P. falciparum infections. Use of the HC-Ad vector type for delivery, as well as the CAG promoter to control MSP-1 expression, may further increase the efficacy of this vaccine candidate.
    The Journal of Gene Medicine 11/2011; 13(12):670-9. · 2.16 Impact Factor
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    ABSTRACT: The establishment of functional transgenic mouse lines is often limited by problems caused by integration site effects on the expression construct. Similarly, tetracycline (Tet) controlled transcription units most commonly used for conditional transgene expression in mice are strongly influenced by their genomic surrounding. Using bacterial artificial chromosome (BAC) technology in constitutive expression systems, it has been shown that integration site effects resulting in unwanted expression patterns can be largely eliminated. Here we describe a strategy to minimize unfavourable integration effects on conditional expression constructs based on a 75 kb genomic BAC fragment. This fragment was derived from a transgenic mouse line, termed LC-1, which carries the Tet-inducible genes luciferase and cre (Schönig et al. 2002). Animals of this mouse line have previously been shown to exhibit optimal expression properties in terms of tightness in the off state and the absolute level of induction, when mated to appropriate transactivator expressing mice. Here we report the cloning and identification of the transgenic LC-1 integration site which was subsequently inserted into a bacterial artificial chromosome. We demonstrate that this vector facilitates the efficient generation of transgenic mouse and rat lines, where the Tet-controlled expression unit is shielded from perturbations caused by the integration site.
    Transgenic Research 06/2011; 20(3):709-20. · 2.61 Impact Factor
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    ABSTRACT: Conditional regulation of gene expression by the combined use of a lung-specific promoter and the tetracycline-regulated system provides a powerful tool for studying gene function in lung biology and disease pathogenesis in a development-independent fashion. However, the original version of the reverse tetracycline-dependent transactivator (rtTA) exhibited limited doxycycline sensitivity and residual affinity to its promoter (P(tet)), producing leaky transgene expression in the absence of doxycycline. These limitations impeded the use of this system in studying gene dosage effects in pulmonary pathogenesis and repair mechanisms in the diseased lung. Therefore, we used a new-generation rtTA, rtTA2(s)-M2, with no basal activity and increased doxycycline sensitivity, and the rat Clara cell secretory protein (CCSP) promoter to target its expression to pulmonary epithelia in mice. Novel CCSP-rtTA2(s)-M2 founder lines were crossed, with bi-transgenic reporter mice expressing luciferase and Cre recombinase. Background activity, doxycycline sensitivity, tissue and cell-type specificity, inducibility, and reversibility of doxycycline-dependent gene expression were determined by luciferase activity, immunohistochemistry, morphometry, and bioluminescence measurements in neonatal and adult lungs. We generated two distinct novel CCSP-rtTA2(s)-M2 activator mouse lines that confer tight and doxycycline dose-dependent regulation of transgene expression, with high inducibility, complete reversibility, and no background activity, in airway and alveolar epithelia. We conclude that rtTA2(s)-M2 enables quantitative control of conditional gene expression in respiratory epithelia of the murine lung, and that the new CCSP-rtTA2(s)-M2 activator mouse lines will be useful in the further elucidation of the pathogenesis of complex lung diseases and in studies of lung repair.
    American Journal of Respiratory Cell and Molecular Biology 02/2011; 44(2):244-54. · 4.15 Impact Factor
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    Journal of Cystic Fibrosis - J CYST FIBROS. 01/2011; 10.
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    ABSTRACT: The malaria parasite Plasmodium falciparum invades erythrocytes where it replicates to produce invasive merozoites, which eventually egress to repeat the cycle. Merozoite surface protein-1 (MSP1), a prime malaria vaccine candidate and one of the most abundant components of the merozoite surface, is implicated in the ligand-receptor interactions leading to invasion. MSP1 is extensively proteolytically modified, first just before egress and then during invasion. These primary and secondary processing events are mediated respectively, by two parasite subtilisin-like proteases, PfSUB1 and PfSUB2, but the function and biological importance of the processing is unknown. Here, we examine the regulation and significance of MSP1 processing. We show that primary processing is ordered, with the primary processing site closest to the C-terminal end of MSP1 being cleaved last, irrespective of polymorphisms throughout the rest of the molecule. Replacement of the secondary processing site, normally refractory to PfSUB1, with a PfSUB1-sensitive site, is deleterious to parasite growth. Our findings show that correct spatiotemporal regulation of MSP1 maturation is crucial for the function of the protein and for maintenance of the parasite asexual blood-stage life cycle.
    Molecular Microbiology 10/2010; 78(1):187-202. · 4.96 Impact Factor
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    ABSTRACT: Plasmodium falciparum merozoites expose at their surface a large protein complex, which is composed of fragments of merozoite surface protein 1 (MSP-1; called MSP-183, MSP-130, MSP-138, and MSP-142) plus associated processing products of MSP-6 and MSP-7. During erythrocyte invasion this complex, as well as an integral membrane protein called apical membrane antigen-1 (AMA-1), is shed from the parasite surface following specific proteolysis. Components of the MSP-1/6/7 complex and AMA-1 are presently under development as malaria vaccines. The specificities and effects of antibodies directed against MSP-1, MSP-6, MSP-7 on the growth of blood stage parasites were studied using ELISA and the pLDH-assay. To understand the mode of action of these antibodies, their effects on processing of MSP-1 and AMA-1 on the surface of merozoites were investigated. Antibodies targeting epitopes located throughout the MSP-1/6/7 complex interfere with shedding of MSP-1, and as a consequence prevent erythrocyte invasion. Antibodies targeting the MSP-1/6/7 complex have no effect on the processing and shedding of AMA-1 and, similarly, antibodies blocking the shedding of AMA-1 do not affect cleavage of MSP-1, suggesting completely independent functions of these proteins during invasion. Furthermore, some epitopes, although eliciting highly inhibitory antibodies, are only poorly recognized by the immune system when presented in the structural context of the intact antigen. The findings reported provide further support for the development of vaccines based on MSP-1/6/7 and AMA-1, which would possibly include a combination of these antigens.
    Malaria Journal 03/2010; 9:77. · 3.49 Impact Factor
  • Kai Schönig, Hermann Bujard, Manfred Gossen
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    ABSTRACT: Tetracycline-controlled transcriptional activation systems are widely used to control gene expression in transgenic animals in a truly conditional manner. By this we refer to the capability of these expression systems to control gene activities not only in a tissue specific and temporal defined but also reversible manner. This versatility has made the Tet regulatory systems to a preeminent tool in reverse mouse genetics. The development of the technology in the past 15 years will be reviewed and guidelines will be given for its implementation in creating transgenic rodents. Finally, we highlight some recent exciting applications of the Tet technology as well as its foreseeable combination with other emerging technologies in mouse transgenesis.
    Methods in enzymology 01/2010; 477:429-53. · 1.90 Impact Factor
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    ABSTRACT: The performance of the tetracycline controlled transcriptional activation system (Tet system) depends critically on the choice of minimal promoters. They are indispensable to warrant low expression levels with the system turned "off". On the other hand, they must support high level of gene expression in the "on"-state. In this study, we systematically modified the widely used Cytomegalovirus (CMV) minimal promoter to further minimize background expression, resulting in an improved dynamic expression range. Using both plasmid-based and retroviral gene delivery, our analysis revealed that especially background expression levels could be significantly reduced when compared to previously established "standard" promoter designs. Our results also demonstrate the possibility to fine-tune expression levels in non-clonal cell populations. They also imply differences regarding the requirements for tight regulation and high level induction between transient and stable gene transfer systems. Until now, our understanding of mammalian transcriptional regulation including promoter architecture is limited. Nevertheless, the partly empirical modification of cis-elements as shown in this study can lead to the specific improvement of the performance of minimal promoters. The novel composite Ptet promoters introduced here will further expand the utility of the Tet system.
    BMC Biotechnology 01/2010; 10:81. · 2.17 Impact Factor
  • Kai Schönig, Hermann Bujard, Manfred Gossen
    [Show abstract] [Hide abstract]
    ABSTRACT: Tetracycline-controlled transcriptional activation systems are widely used to control gene expression in transgenic animals in a truly conditional manner. By this we refer to the capability of these expression systems to control gene activities not only in a tissue specific and temporal defined but also reversible manner. This versatility has made the Tet regulatory systems to a preeminent tool in reverse mouse genetics. The development of the technology in the past 15 years will be reviewed and guidelines will be given for its implementation in creating transgenic rodents. Finally, we highlight some recent exciting applications of the Tet technology as well as its foreseeable combination with other emerging technologies in mouse transgenesis.
    Methods in Enzymology - METH ENZYMOLOGY. 01/2010; 477:429-453.
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    ABSTRACT: The generation of cell type specific inducible Cre transgenic mice is the most challenging and limiting part in the development of spatio-temporally controlled knockout mouse models. Here we report the generation and characterization of a B lymphocyte-specific tamoxifen-inducible Cre transgenic mouse strain, LC-1-hCD19-CreER(T2). We utilized the human CD19 promoter for expression of the tamoxifen-inducible Cre recombinase (CreER(T2)) gene, embedded in genomic sequences previously reported to give minimal position effects after transgenesis. Cre recombinase activity was evaluated by cross-breeding the LC-1-hCD19-CreER(T2) strain with a strain containing a floxed gene widely expressed in the hematopoietic system. Cre activity was only detected in the presence of tamoxifen and was restricted to B lymphocytes. The efficacy of recombination ranged from 27 to 61% in the hemizygous and homozygous mice, respectively. In conclusion, the LC-1-hCD19-CreER(T2) strain is a powerful tool to study gene function specifically in B lymphocytes at any chosen time point in the lifecycle of the mouse.
    genesis 08/2009; 47(11):729-35. · 2.58 Impact Factor
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    ABSTRACT: CD226 is known to be expressed on many types of peripheral lymphoid cells and involved in T cell differentiation, activation, and cytotoxicity. In this study, we report that CD226 is also expressed on mouse thymocytes at varying developmental stages, and its expression is associated with resistance of thymocytes to apoptosis. The levels of CD226 expression appeared to be closely coupled with thymocyte development, in that it was preferentially expressed on CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes at all stages during mouse development, and was markedly increased on the cells in neonatal mice. Of the CD4(+)CD8(+) population, CD226 was predominantly expressed by the cells also positive for CD69, suggesting that CD226 expression may be induced in thymocyte-positive selection. Inhibition of CD226 by short hairpin RNA in a fetal thymus organ culture model led to reduced thymus cellularity, which was associated with enhanced apoptotic cell death. In contrast, CD226-transgenic mice displayed enlarged thymus lobes resulting from increased thymus cellularity. CD226 on thymocytes seemed to play a role in regulating the expression of survivin, as inhibition of CD226 down-regulated survivin, but overexpression of CD226 rescued thymocytes from apoptosis through up-regulation of survivin. In addition, overexpression of CD226 reduced sensitivity of EL-4 thymoma cells to apoptosis by up-regulating the expression of survivin. Taken together, these results indicate that CD226 is an antiapoptotic molecule and may play an important role in murine thymocyte development.
    The Journal of Immunology 06/2009; 182(9):5453-60. · 5.52 Impact Factor
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    ABSTRACT: Conditional gene expression systems have developed into essential tools for the study of gene functions. However, their utility is often limited by the difficulty of identifying clonal cell lines, in which transgene control can be realized to its full potential. Here, we describe HeLa cell lines, in which we have identified-by functional analysis-genomic loci, from which the expression of transgenes can be tightly controlled via tetracycline-regulated expression. These loci can be re-targeted by recombinase-mediated cassette exchange. Upon exchange of the gene of interest, the resulting cell line exhibits the qualitative and quantitative properties of controlled transgene expression characteristic for the parent cell line. Moreover, by using an appropriate promoter, these cell lines express the tetracycline controlled transcription activator rtTA2-M2 uniformly throughout the entire cell population. The potential of this approach for functional genomics is highlighted by utilizing one of our master cell lines for the efficient microRNA-mediated knockdown of the endogenous human lamin A/C gene.
    Nucleic Acids Research 04/2009; 37(7):e50. · 8.81 Impact Factor
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    ABSTRACT: The malaria parasite Plasmodium falciparum replicates within an intraerythrocytic parasitophorous vacuole (PV). Rupture of the host cell allows release (egress) of daughter merozoites, which invade fresh erythrocytes. We previously showed that a subtilisin-like protease called PfSUB1 regulates egress by being discharged into the PV in the final stages of merozoite development to proteolytically modify the SERA family of papain-like proteins. Here, we report that PfSUB1 has a further role in 'priming' the merozoite prior to invasion. The major protein complex on the merozoite surface comprises three proteins called merozoite surface protein 1 (MSP1), MSP6 and MSP7. We show that just before egress, all undergo proteolytic maturation by PfSUB1. Inhibition of PfSUB1 activity results in the accumulation of unprocessed MSPs on the merozoite surface, and erythrocyte invasion is significantly reduced. We propose that PfSUB1 is a multifunctional processing protease with an essential role in both egress of the malaria merozoite and remodelling of its surface in preparation for erythrocyte invasion.
    The EMBO Journal 03/2009; 28(6):725-35. · 9.82 Impact Factor
  • Nucleic Acids Research, v.37 (2009). 01/2009;
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    ABSTRACT: We describe a transgenic mouse line, Pax8-rtTA, which, under control of the mouse Pax8 promoter, directs high levels of expression of the reverse tetracycline-dependent transactivator (rtTA) to all proximal and distal tubules and the entire collecting duct system of both embryonic and adult kidneys. Using crosses of Pax8-rtTA mice with tetracycline-responsive c-MYC mice, we established a new, inducible model of polycystic kidney disease that can mimic adult onset and that shows progression to renal malignant disease. When targeting the expression of transforming growth factor beta-1 to the kidney, we avoided early lethality by discontinuous treatment and successfully established an inducible model of renal fibrosis. Finally, a conditional knockout of the gene encoding tuberous sclerosis complex-1 was achieved, which resulted in the early outgrowth of giant polycystic kidneys reminiscent of autosomal recessive polycystic kidney disease. These experiments establish Pax8-rtTA mice as a powerful tool for modeling renal diseases in transgenic mice.
    Nature medicine 09/2008; 14(9):979-84. · 27.14 Impact Factor
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    ABSTRACT: Plasmodium falciparum infection causes cerebral malaria (CM) in a subset of patients with anti-malarial treatment protecting only about 70% to 80% of patients. Why a subset of malaria patients develops CM complications, including neurological sequelae or death, is still not well understood. It is believed that host immune factors may modulate CM outcomes and there is substantial evidence that cellular immune factors, such as cytokines, play an important role in this process. In this study, the potential relationship between the antibody responses to the merozoite surface protein (MSP)-1 complex (which consists of four fragments namely: MSP-1(83), MSP-1(30), MSP-1(38) and MSP-1(42)), MSP-6(36) and MSP-7(22) and CM was investigated. Peripheral blood antibody responses to recombinant antigens of the two major allelic forms of MSP-1 complex, MSP-6(36) and MSP-7(22) were compared between healthy subjects, mild malaria patients (MM) and CM patients residing in a malaria endemic region of central India. Total IgG and IgG subclass antibody responses were determined using ELISA method. The prevalence and levels of IgG and its subclasses in the plasma varied for each antigen. In general, the prevalence of total IgG, IgG1 and IgG3 was higher in the MM patients and lower in CM patients compared to healthy controls. Significantly lower levels of total IgG antibodies to the MSP-1(f38), IgG1 levels to MSP-1(d83), MSP-1(19) and MSP-6(36) and IgG3 levels to MSP-1(f42) and MSP-7(22) were observed in CM patients as compared to MM patients. These results suggest that there may be some dysregulation in the generation of antibody responses to some MSP antigens in CM patients and it is worth investigating further whether perturbations of antibody responses in CM patients contribute to pathogenesis.
    Malaria Journal 08/2008; 7:121. · 3.49 Impact Factor
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    ABSTRACT: We describe two new transgenic mouse lines for studying pathological changes of Tau protein related to Alzheimer's disease. They are based on the regulatable expression of the four-repeat domain of human Tau carrying the FTDP17 (frontotemporal dementia and parkinsonism linked to chromosome 17) mutation deltaK280 (Tau(RD)/deltaK280), or the deltaK280 plus two proline mutations in the hexapeptide motifs (Tau(RD)/deltaK280/I277P/I308P). The deltaK280 mutation accelerates aggregation ("proaggregation mutant"), whereas the proline mutations inhibit Tau aggregation in vitro and in cell models ("antiaggregation mutant"). The inducible transgene expression was driven by the forebrain-specific CaMKIIalpha (calcium/calmodulin-dependent protein kinase IIalpha) promoter. The proaggregation mutant leads to Tau aggregates and tangles as early as 2-3 months after gene expression, even at low expression (70% of endogenous mouse Tau). The antiaggregation mutant does not aggregate even after 22 months of gene expression. Both mutants show missorting of Tau in the somatodendritic compartment and hyperphosphorylation in the repeat domain [KXGS motifs, targets of the kinase MARK (microtubule affinity regulating kinase)]. This indicates that these changes are related to Tau expression rather than aggregation. The proaggregation mutant causes astrogliosis, loss of synapses and neurons from 5 months of gene expression onward, arguing that Tau toxicity is related to aggregation. Remarkably, the human proaggregation mutant Tau(RD) coaggregates with mouse Tau, coupled with missorting and hyperphosphorylation at multiple sites. When expression of proaggregation Tau(RD) is switched off, soluble and aggregated exogenous Tau(RD) disappears within 1.5 months. However, tangles of mouse Tau, hyperphosphorylation, and missorting remain, suggesting an extended lifetime of aggregated wild-type Tau once a pathological conformation and aggregation is induced by a proaggregation Tau species.
    Journal of Neuroscience 02/2008; 28(3):737-48. · 6.91 Impact Factor

Publication Stats

14k Citations
951.83 Total Impact Points

Institutions

  • 2011
    • Central Institute of Mental Health
      Mannheim, Baden-Württemberg, Germany
  • 1975–2010
    • Universität Heidelberg
      • Center for Molecular Biology (ZMBH)
      Heidelberg, Baden-Wuerttemberg, Germany
  • 2007–2008
    • Max Planck Research Unit for Structural Molecular Biology at DESY
      Hamburg, Hamburg, Germany
  • 2006
    • National Institutes of Health
      Maryland, United States
  • 2002
    • Max-Delbrück-Centrum für Molekulare Medizin
      Berlín, Berlin, Germany
  • 2000
    • ETH Zurich
      • Institute of Cell Biology
      Zürich, ZH, Switzerland
  • 1999
    • Philipps University of Marburg
      Marburg, Hesse, Germany
  • 1976
    • Heidelberg University
      Tiffin, Ohio, United States