U T Brunk

University of Michigan, Ann Arbor, MI, United States

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Publications (291)923.92 Total impact

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    ABSTRACT: Endothelial cells (ECs) of thin-walled blood vessels form a barrier between blood and tissue. As a response to inflammation, the EC junctions widen and gaps form, resulting in compromised barrier functions. Although the mechanisms behind the establishment of these changes are still incompletely understood, one known reason is actomyosin-dependent actin rearrangement. Here, by using atomic force microscopy and a combination of confocal microscopy methods, we are the first to report that thermal injury induces general venular hyperpermeability and that serum from burned rats induces EC actin rearrangement, contraction, as well as tight-junction damage. Inhibition of the p38 mitogen-activated protein kinase (p38MAPK) largely ameliorates resulting vascular dysfunction by significantly reducing EC stress-fiber formation, contraction, volume changes and tight-junction damage, thereby greatly reducing the appearance of EC gaps. The findings may be of importance for the design of future pharmacotherapies aiming to ease the severe general vascular dysfunction that follows extensive burns.
    Apmis 01/2014; · 2.07 Impact Factor
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    ABSTRACT: The objective of this study was to elucidate possible reasons for the remarkable resistance of human retinal pigment epithelial (RPE) cells to oxidative stress. Much oxidative damage is due to hydrogen peroxide meeting redox-active iron in the acidic and reducing lysosomal environment, resulting in the production of toxic hydroxyl radicals that may oxidize intralysosomal content, leading to lipofuscin (LF) formation or, if more extensive, to permeabilization of lysosomal membranes. Formation of LF is a risk factor for age-related macular degeneration (AMD) and known to jeopardize normal autophagic rejuvenation of vital cellular biomolecules. Lysosomal membrane permeabilization causes release of lysosomal content (redox-active iron, lytic enzymes), which may then cause cell death. Total cellular and lysosomal low-mass iron of cultured, immortalized human RPE (ARPE-19) cells was compared to that of another professional scavenger cell line, J774, using atomic absorption spectroscopy and the cytochemical sulfide-silver method (SSM). It was found that both cell lines contained comparable levels of total as well as intralysosomal iron, suggesting that the latter is mainly kept in a non-redox-active state in ARPE-19 cells. Basal levels and capacity for upregulation of the iron-binding proteins ferritin, metallothionein and heat shock protein 70 were tested in both cell lines using immunoblotting. Compared to J774 cells, ARPE-19 cells were found to contain very high basal levels of all these proteins, which could be even further upregulated following appropriate stimulation. These findings suggest that a high basal expression of iron-binding stress proteins, which during their normal autophagic turnover in lysosomes may temporarily bind iron prior to their degradation, could contribute to the unusual oxidative stress-resistance of ARPE-19 cells. A high steady state influx of such proteins into lysosomes would keep the level of lysosomal redox-active iron permanently low. This, in turn, should delay intralysosomal accumulation of LF in RPE cells, which is known to reduce autophagic turnover as well as uptake and degradation of worn out photoreceptor tips. This may explain why severe LF accumulation and AMD normally do not develop until fairly late in life, in spite of RPE cells being continuously exposed to high levels of oxygen and light, as well as large amounts of lipid-rich material.
    Experimental Eye Research 11/2013; 116:359-65. · 3.03 Impact Factor
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    ABSTRACT: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
    Autophagy 04/2012; 8(4):445. · 12.04 Impact Factor
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    Autophagy 04/2012; 8(4):1-100. · 12.04 Impact Factor
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    ABSTRACT: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process);5,6 thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
    Autophagy 04/2012; 8(4). · 12.04 Impact Factor
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    [Show abstract] [Hide abstract]
    ABSTRACT: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
    Autophagy 04/2012; 8(4):445-544. · 12.04 Impact Factor
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    Autophagy 04/2012; 4454(8):445-544. · 12.04 Impact Factor
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    ABSTRACT: Normally, cell proliferation and death are carefully balanced in higher eukaryotes, but one of the most important regulatory mechanisms, apoptosis, is upset in many malignancies, including hepatocellular-derived ones. Therefore, reinforcing cell death often is mandatory in anticancer therapy. We previously reported that a combination of tumor necrosis factor-α (TNF) and cycloheximide (CHX) efficiently kill HTC cells, a rat hepatoma line, in an apoptosis-like mode. Death is actively mediated by the lysosomal compartment, although lysosomal ceramide was previously shown not to be directly implicated in this process. In the present study, we show that TNF/CHX increase lysosomal ceramide that is subsequently converted into sphingosine. Although ceramide accumulation does not significantly alter the acidic compartment, the sphingosine therein generated causes lysosomal membrane permeabilization (LMP) followed by relocation of lysosomal cathepsins to the cytoplasm. TNF/CHX-induced LMP is effectively abrogated by siRNAs targeting acid sphingomyelinase or acid ceramidase, which prevent both LMP and death induced by TNF/CHX. Taken together, our results demonstrate that lysosomal accumulation of ceramide is not detrimental per se, whereas its degradation product sphingosine, which has the capacity to induce LMP, appears responsible for the observed apoptotic-like death.
    The Journal of Lipid Research 03/2012; 53(6):1134-43. · 4.39 Impact Factor
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    ABSTRACT: In a Wistar rat model, prolonged supplementation of mustard seed (MS) to the diet significantly ameliorates the induction of colorectal carcinomas by 1,2-dimethylhydrazine (DMH). The expression of the splenocyte major histocompatibility complex class I (MHCI) was found significantly enhanced, whereas that of the major histocompatibility complex class II (MHCII) was significantly decreased. Compared to that of control animals, the proportion of spleenic B- and dendritic cells (DC) was amplified in the MS group. The expressions of MHCI, as well as that of MHCII, were increased in DC cells; whereas in B cells, MHCI expression was augmented but that of MHCII moderately decreased. The percentages of CD8+CD28+ and CD4+CD28+ cells were increased in the MS group, while the CD4+CD25+Foxp3+ subset was depressed. Plasma analysis showed that DMH-exposure induced amplified amounts of interleukin (IL)-4, IL-5, IL-10, and transforming growth factor-beta, whereas MS feeding counteracted this effect but enhanced IL-2, IL12p70, IL21, TNF-alpha, and interferon-gamma. In the SW480 colon adenocarcinoma cell-line, the cytotoxicity of spleenic T-cells from MS-fed animals was significantly increased. In the DMH-exposed rats, the expression of perforin in the spleenic T-cells was dramatically decreased, whereas MS abolished this depression. In summary, dietary MS suppresses DMH-induced immuno-imbalance as well as colon carcinogenesis in rats.
    Nutrition and Cancer 03/2012; 64(3):464-72. · 2.70 Impact Factor
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    ABSTRACT: The main objective of this study was to investigate the activity of polydatin on mitochondrial dysfunction and lysosomal stability of arteriolar smooth muscle cells (ASMCs) in severe shock. The experimental animals (rats) were divided into five groups: control, hemorrhagic shock, shock + CsA, shock + Res, and shock + PD (exposed to cyclosporin A, resveratrol, or polydatin following induction of hemorrhagic shock, respectively). The calcein-Co(2+) technique revealed opening of ASMC mitochondrial permeability transition pores (mPTP) after shock with resulting mitochondrial swelling, decreased mitochondrial membrane potential (ΔΨm), and reduced intracellular ATP levels. These alterations were all inhibited by exposure to PD, which was significantly more effective than CsA and Res. PD also preserved lysosomal stability, suppressed activation of K(ATP) channels, ASMC hyperpolarization, and reduced vasoresponsiveness to norepinephrine that normally follows severe shock. The results demonstrate that exposure to PD after initiation of severe shock effectively preserves ASMC mitochondrial integrity and has a significant therapeutic effect in severe shock. The effects may partially result from lysosomal stabilization against shock-induced oxidative stress and depressed relocation of hydrolytic enzymes and redox-active lysosomal iron that, in turn, may induce mPTP opening.
    AJP Regulatory Integrative and Comparative Physiology 01/2012; 302(7):R805-14. · 3.28 Impact Factor
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    Autophagy 01/2012; 4454(8):445-544. · 12.04 Impact Factor
  • Hua Zhao, Ulf T Brunk, Brett Garner
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    ABSTRACT: Vitamin-B(12) is a generic term for corrinoid compounds that exhibit the biological activity of cyanocobalamin and are collectively referred to as cobalamins. Methylcobalamin and 5-deoxyadenosylcobalamin are the active cobalamins in human metabolism. Cobalamin plays a crucial role in the maintenance of homocysteine and methylmalonyl-CoA homeostasis and is required for erythrocyte formation and DNA synthesis. Data from human and animal studies indicate that cobalamin deficiency impairs neuronal function; a process that is thought to contribute to age-related cognitive decline and dementia. Cobalamin deficiency also results in dysfunction of the peripheral nervous system; among other disorders. Although there is a detailed understanding of the biochemical pathways that are perturbed in cobalamin deficiency, the mechanisms underlying age-related dyshomeostasis in such pathways remain to be addressed. Because cobalamin utilization is dependent on its efficient transit through lysosomes, and mounting evidence indicates that lysosomal function deteriorates in aging long-lived post-mitotic cells such as neurons, in the present article we review published data that supports the proposition that impaired lysosomal processing of cobalamin may play a significant role in age-related (neuro) degenerative diseases.
    Cellular and Molecular Life Sciences CMLS 12/2011; 68(24):3963-9. · 5.62 Impact Factor
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    ABSTRACT: Different procedures were tested for the purpose of circumventing the technical obstacles encountered in SEM of the scorpion hepatopancreas and stomach gland. These problems are caused by the relatively high osmotic pressure of the body fluid of this animal, the irregular surface contour, and the very high content of fat in the organs under investigation. The best results were obtained following glutaraldehyde fixation at an effective osmotic pressure approaching that of scorpion body fluid, ensuring stabilization of fat by post-fixation in osmium tetroxide, and overcoming the charge effects via the OTOTO (Osmium-Thiocarbohydrozide) method in combination with metal coating. Dehydration by CPD or FD both gave rise to inevitable artefacts (shrinkage or cracking). Used together, however, CPD and FD were complementary.The surfaces of hepatopancreatic and stomach gland tubules showed a distinct similarity. Tubules of both organs were lined mostly with cells equipped with parallel arrays of microvilli on the apical surface and containing large amounts of fat.
    Scanning 10/2011; 3(4):279 - 282. · 1.29 Impact Factor
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    Tino Kurz, John W Eaton, Ulf T Brunk
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    ABSTRACT: Iron is the most abundant transition metal in the earth's crust. It cycles easily between ferric (oxidized; Fe(III)) and ferrous (reduced; Fe(II)) and readily forms complexes with oxygen, making this metal a central player in respiration and related redox processes. However, 'loose' iron, not within heme or iron-sulfur cluster proteins, can be destructively redox-active, causing damage to almost all cellular components, killing both cells and organisms. This may explain why iron is so carefully handled by aerobic organisms. Iron uptake from the environment is carefully limited and carried out by specialized iron transport mechanisms. One reason that iron uptake is tightly controlled is that most organisms and cells cannot efficiently excrete excess iron. When even small amounts of intracellular free iron occur, most of it is safely stored in a non-redox-active form in ferritins. Within nucleated cells, iron is constantly being recycled from aged iron-rich organelles such as mitochondria and used for construction of new organelles. Much of this recycling occurs within the lysosome, an acidic digestive organelle. Because of this, most lysosomes contain relatively large amounts of redox-active iron and are therefore unusually susceptible to oxidant-mediated destabilization or rupture. In many cell types, iron transit through the lysosomal compartment can be remarkably brisk. However, conditions adversely affecting lysosomal iron handling (or oxidant stress) can contribute to a variety of acute and chronic diseases. These considerations make normal and abnormal lysosomal handling of iron central to the understanding and, perhaps, therapy of a wide range of diseases.
    The international journal of biochemistry & cell biology 09/2011; 43(12):1686-97. · 4.89 Impact Factor
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    ABSTRACT: Motexafin gadolinium (MGd) sensitizes malignant cells to ionizing radiation, although the underlying mechanisms for uptake and sensitization are both unclear. Here we show that MGd is endocytosed by the clathrin-dependent pathway with ensuing lysosomal membrane permeabilization, most likely via formation of reactive oxygen species involving redox-active metabolites, such as ascorbate. We propose that subsequent apoptosis is a synergistic effect of irradiation and high MGd concentrations in malignant cells due to their pronounced endocytic activity. The results provide novel insights into the mode of action of this promising anti-cancer drug, which is currently under clinical trials.
    Cancer letters 08/2011; 307(2):119-23. · 5.02 Impact Factor
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    ABSTRACT: To test the consequences of lysosomal degradation of differently iron-loaded ferritin molecules and to mimic ferritin autophagy under iron-overload and normal conditions, J774 cells were allowed to endocytose heavily iron loaded ferritin, probably with some adventitious iron (Fe-Ft), or iron-free apo-ferritin (apo-Ft). When cells subsequently were exposed to a bolus dose of hydrogen peroxide, apo-Ft prevented lysosomal membrane permeabilization (LMP), whereas Fe-Ft enhanced LMP. A 4-h pulse of Fe-Ft initially increased oxidative stress-mediated LMP that was reversed after another 3h under standard culture conditions, suggesting that lysosomal iron is rapidly exported from lysosomes, with resulting upregulation of apo-ferritin that supposedly is autophagocytosed, thereby preventing LMP by binding intralysosomal redox-active iron. The obtained data suggest that upregulation of the stress protein ferritin is a rapid adaptive mechanism that counteracts LMP and ensuing apoptosis during oxidative stress. In addition, prolonged iron starvation was found to induce apoptotic cell death that, interestingly, was preceded by LMP, suggesting that LMP is a more general phenomenon in apoptosis than so far recognized. The findings provide new insights into aging and neurodegenerative diseases that are associated with enhanced amounts of cellular iron and show that lysosomal iron loading sensitizes to oxidative stress.
    Free Radical Biology and Medicine 03/2011; 50(11):1647-58. · 5.27 Impact Factor
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    ABSTRACT: Cellular deposits of oxidized and aggregated proteins are hallmarks of a variety of age-related disorders, but whether such proteins contribute to pathology is not well understood. We previously reported that oxidized proteins form lipofuscin/ceroid-like bodies with a lysosomal-type distribution and up-regulate the transcription and translation of proteolytic lysosomal enzymes in cultured J774 mouse macrophages. Given the recently identified role of lysosomes in the induction of apoptosis, we have extended our studies to explore a role for oxidized proteins in apoptosis. Oxidized proteins were biosynthetically generated in situ by substituting oxidized analogues for parent amino acids. Apoptosis was measured with Annexin-V/PI (propidium iodide), TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling), MMP (mitochondrial membrane permeabilization), caspase activation and cytochrome c release, and related to lysosomal membrane permeabilization. Synthesized proteins containing the tyrosine oxidation product L-DOPA (L-3,4-dihydroxyphenylalanine) were more potent inducers of apoptosis than proteins containing the phenylalanine oxidation product o-tyrosine. Apoptosis was dependent upon incorporation of oxidized residues, as indicated by complete abrogation in cultures incubated with the non-incorporation control D-DOPA (D-3,4-dihydroxyphenylalanine) or when incorporation was competed out by parent amino acids. The findings of the present study suggest that certain oxidized proteins could play an active role in the progression of age-related disorders by contributing to LMP (lysosomal membrane permeabilization)-initiated apoptosis and may have important implications for the long-term use of L-DOPA as a therapeutic agent in Parkinson's disease.
    Biochemical Journal 01/2011; 435(1):207-16. · 4.65 Impact Factor

Publication Stats

11k Citations
923.92 Total Impact Points

Institutions

  • 2012
    • University of Michigan
      • Life Sciences Institute
      Ann Arbor, MI, United States
  • 1982–2012
    • Linköping University
      • Faculty of Health Sciences
      Linköping, Östergötland, Sweden
  • 2011
    • Umeå University
      Umeå, Västerbotten, Sweden
    • Nanfang Hospital
      Shengcheng, Guangdong, China
  • 2009–2011
    • Southern Medical University
      • Department of Pathophysiology
      Guangzhou, Guangdong Sheng, China
    • University of Sydney
      Sydney, New South Wales, Australia
    • Karolinska University Hospital
      • Department of Clinical Pathology and Cytology
      Stockholm, Stockholm, Sweden
  • 1997–2011
    • University of Wollongong
      • • Illawarra Health and Medical Research Institute
      • • School of Chemistry
      • • Department of Biomedical Science
      Wollongong, New South Wales, Australia
  • 1971–2011
    • Karolinska Institutet
      • • Institutionen för medicinsk biokemi och biofysik
      • • Clinical Research Center
      Solna, Stockholm, Sweden
  • 1972–2009
    • Uppsala University
      • Division of Analytical Pharmaceutical Chemistry
      Uppsala, Uppsala, Sweden
    • Lund University
      • Department of Obstetrics and Gynecology
      Lund, Skåne, Sweden
  • 2004–2008
    • University of New South Wales
      • • Prince of Wales Medical Research Institute
      • • School of Medical Sciences
      Kensington, New South Wales, Australia
    • Newcastle University
      Newcastle-on-Tyne, England, United Kingdom
  • 1992–2006
    • University Hospital Linköping
      • Department of Ophthalmology
      Linköping, Östergötland, Sweden
  • 2005
    • Albert Einstein College of Medicine
      • Department of Anatomy and Structural Biology
      New York City, NY, United States
  • 2002
    • Aarhus University
      • Institute of Anatomy
      Aars, Region North Jutland, Denmark
  • 2001
    • University of Southern California
      • School of Pharmacy
      Los Angeles, CA, United States
  • 1999–2001
    • Ludwig-Maximilians-University of Munich
      München, Bavaria, Germany
  • 1984–1997
    • Stockholm University
      • Department of Biochemistry and Biophysics
      Stockholm, Stockholm, Sweden
  • 1995
    • Humboldt-Universität zu Berlin
      Berlín, Berlin, Germany
  • 1993
    • Charité Universitätsmedizin Berlin
      • Institute of Pathology
      Berlin, Land Berlin, Germany
  • 1989–1992
    • Southern Methodist University
      • Department of Biological Sciences
      Dallas, TX, United States
  • 1988
    • Kenya Medical Research Institute
      Nairoba, Nairobi Area, Kenya