B A Margulis

Russian Academy of Sciences, Moskva, Moscow, Russia

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Publications (86)214.64 Total impact

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    ABSTRACT: The Hsp70 chaperone is known to elicit cytoprotective activity and this protection has a negative impact in anti-tumor therapy. In cancer cells subjected to oxidative stress Hsp70 may bind damaged polypeptides and proteins involved in apoptosis signaling. Since one of the important targets of oxidative stress is glyceraldehyde-3-phospate dehydrogenase (GAPDH) we suggested that Hsp70 might elicit its protective effect by binding GAPDH. Microscopy data show that in C6 rat glioma cells subjected to hydrogen peroxide treatment a considerable proportion of the GAPDH molecules are denatured and according to dot ultrafiltration data they form SDS-insoluble aggregates. Using two newly developed assays we show that Hsp70 can bind oxidized GAPDH in an ATP-dependent manner. Pharmacological up- or down-regulation of Hsp70 with the aid of U133 echinochrome or triptolide, respectively, reduced or increased the number of C6 glioma cells containing GAPDH aggregates and dying due to treatment with hydrogen peroxide. Using immunoprecipitation we found that Hsp70 is able to sequester aggregation-prone GAPDH and this may explain the anti-oxidative power of the chaperone. The results of this study led us to conclude that in cancer cells constantly exposed to conditions of oxidative stress, the protective power of Hsp70 should be abolished by specific inhibitors of Hsp70 expression.
    Full-text · Article · Dec 2015 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the most abundant targets of the oxidative stress. Oxidation of the enzyme causes its inactivation and the formation of intermolecular disulfide bonds, and leads to the accumulation of GAPDH aggregates and ultimately to cell death. The aim of this work was to reveal the ability of chemicals to break the described above pathologic linkage by inhibiting GAPDH aggregation. Using the model of oxidative stress based on SK-N-SH human neuroblastoma cells treated with hydrogen peroxide, we found that lentivirus-mediated down- or up-regulation of GAPDH content caused inhibition or enhancement of the protein aggregation and respectively reduced or increased the level of cell death. To reveal substances that are able to inhibit GAPDH aggregation, we developed a special assay based on dot ultrafiltration using the collection of small molecules of plant origin. In the first round of screening, five compounds were found to possess anti-aggregation activity as established by ultrafiltration and dynamic light scattering; some of the substances efficiently inhibited GAPDH aggregation in nanomolar concentrations. The ability of the compounds to bind GAPDH molecules was proved by the drug affinity responsive target stability assay, molecular docking and differential scanning calorimetry. Results of experiments with SK-N-SH human neuroblastoma treated with hydrogen peroxide show that two substances, RX409 and RX426, lowered the degree of GAPDH aggregation and reduced cell death by 30%. Oxidative injury was emulated in vivo by injecting of malonic acid into the rat brain, and we showed that the treatment with RX409 or RX426 inhibited GAPDH-mediated aggregation in the brain, reduced areas of the injury as proved by magnetic resonance imaging, and augmented the behavioral status of the rats as established by the "beam walking" test. In conclusion, the data show that two GAPDH binders could be therapeutically relevant in the treatment of injuries stemming from hard oxidative stress.
    Full-text · Article · Dec 2015 · Free Radical Biology and Medicine
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    ABSTRACT: The Hsp70 chaperone is one of the major components of tumor microenvironment displaying multiple not yet established functions. It was shown to stimulate innate and adaptive anti-tumor immunity in a variety of cancer models. These effects were due to active release of endogenous Hsp70 to an extracellular matrix and therefore the extracellular chaperone can be a trigger of multiple events in the whole tumor. Factors inducing Hsp70 release are heat stress, inhibitors of important signaling proteins, anticancer drugs and X-ray treatment. Recently we have shown that delivery of Hsp70 can be efficient pusher of its intracellular analogue to extracellular milieu. In this study we show that intracellular cycling of exo- and endogenous Hsp70s increases the sensitivity of cancer cells to cytotoxic lymphocytes. Moreover, the results of in vivo studies employing B16 mouse melanoma and C6 rat glioblastoma as targets proved the therapeutic relevance of exogenous Hsp70 in intra-tumoral application.
    Preview · Article · Nov 2015 · EJC Supplements
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    D. Meshalkina · M. Shevtsov · B. Margulis · I. Guzhova
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    ABSTRACT: Glioblastoma is one of the most malignant cancer types. Its median survival is 15months with combined radio- and chemotherapy and only 4months without therapy. Molecular chaperones play a very multifaced role in tumor development. Depletion of Hdj1 in cancer cells accelerates tumor growth. Decrease of Hdj2 level correlates with the increase of tumor aggressiveness, but also attenuates tumor protection against radiotherapy. Hsp70 provides considerable survival advantages to the cancer cells, but at the same time can act as a “chaperokine”, activating antitumoral immunity. For assessment of chaperone’s role in glioma progression, invasiveness and metastasis formation we chose rat model of intracranial injection of 105 C6 cells. We developed three C6-based cell lines with protein knock-down by RNA-interference: C6 shHsp70 (on 83%), C6 shHdj1 (on 96%) and C6 shHdj2 (on 52%). The last differed in roundish and easily detachable morphology. Following intracranial injection of the modified tumor cells, the animals’ survival was estimated. As compared to the groups of control C6 (25.4±3.9days) and C6 shHdj1 (25.5±3.8) we observed a nearly 1.5-fold decrease in survival in C6 shHdj2 (16.8±3.5days) (P<0.05). On the contrary, in C6 shHsp70 group the survival increased up to 42.5±12.0days (the increase is completely explainable by the slower growth rate of the culture). Subsequent MR imaging and histological analysis of tumors demonstrated elevated invasiveness and metastatic activity in C6 shHdj2 group in comparison to C6 shHsp70, C6 shHdj1 and control C6. High migration activity and the ability of floating C6 shHdj2 cells to adhere and settle on the substrate was proved in wound-healing assay, spot-healing assay, colony forming assay and transwell migration assay. Adhesion assay showed decreased adhesion ability of C6 shHdj2 cells and increased – of C6 shHsp70 cells on all types of tested extracellular matrixes. Immunofluorescence analyses showed loss of membrane- expressed N-cadherin and loss of intercellular contacts mediated by N-cadherin in C6 shHdj2 cells in comparison to other considered cell lines (although its level in western blot was elevated). Actin staining with rhodamine-falloidin revealed highly abundant leading edges in C6 shHdj2 culture. Matrix metalloprotease zymography proved an increased activity in gelatinases (mmp2 and mmp9) as well as in caseinases (mmp1 and mmp8) in C6 shHdj2 culture supernatant. Assay of stemness marker CD133 expression showed its 11.8 times increase in C6 shHdj2.
    Preview · Article · Nov 2015 · EJC Supplements
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    ABSTRACT: Brain tumor targeting efficiency and biodistribution of the superparamagnetic nanoparticles conjugated with heat shock protein Hsp70 (SPION–Hsp70) were evaluated in experimental glioma model. Synthesized conjugates were characterized using the method of longitudinal nonlinear response of magnetic nanoparticles to a weak ac magnetic field with measurements of second harmonic of magnetization (NLR-M2). Cellular interaction of magnetic conjugates was analyzed in 9L glioma cell culture. The biodistribution of the nanoparticles and their accumulation in tumors was assessed by the latter approach as well. The efficacy of Hsp70-conjugates for contrast enhancement in the orthotopic model of 9L glioma was assessed by MR imaging (11 T). Magnetic nanoparticles conjugated with Hsp70 had the relaxivity properties of the MR-negative contrast agents. Morphological observation and cell viability test demonstrated good biocompatibility of Hsp70-conjugates. Analysis of the T2-weighted MR scans in tumor-bearing rats demonstrated the high efficacy of Hsp70-conjugates in contrast enhancement of the glioma in comparison to non-conjugated nanoparticles. High contrast enhancement of the glioma was provided by the accumulation of the SPION–Hsp70 particles in the glioma tissue (as shown by the histological assay). Biodistribution analysis by NLR-M2 measurements evidenced the many-fold increase (~40) in the tumor-to-normal brain uptake ratio in the Hsp70-conjugates treated animals. Biodistribution pattern of Hsp70-decorated nanoparticles differed from that of non-conjugated SPIONs. Coating of the magnetic nanoparticles with Hsp70 protein enhances the tumor-targeting ability of the conjugates that could be applied in the MR imaging of the malignant brain tumors.
    Full-text · Article · Aug 2015 · Journal of Magnetism and Magnetic Materials

  • No preview · Article · Aug 2015 · Cancer Research
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    ABSTRACT: Proteins with long polyglutamine repeats form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which enhances aggregation and cytotoxicity in models of Huntington disease. The aim of this study was to develop a novel assay for the screening of anti-aggregation compounds with a focus on the aggregation-promoting capacity of GAPDH. The assay includes a pure Q58 polyglutamine fragment, GAPDH, and a transglutaminase that links the two proteins. The feasibility of the new assay was verified using two GAPDH binders, hydroxynonenal and -(-)deprenyl, and the benzothiazole derivative PGL-135 which exhibits anti-aggregation effect. All three substances were shown to reduce aggregation and cytotoxicity in the cell and in the fly model of Spinocerebellar ataxia. Copyright © 2015. Published by Elsevier B.V.
    No preview · Article · Jan 2015 · FEBS Letters
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    ABSTRACT: Chaperone Hsp70 can cross the plasma membrane of living cells using mechanisms that so far have not received much research attention. Searching the part of the molecule that is responsible for transport ability of Hsp70, we found a cationic sequence composed of 20 amino acid residues on its surface, KST peptide, which was used in further experiments. We showed that KST peptide enters living cells of various origins with the same efficiency as the full-length chaperone. KST peptide is capable of carrying cargo with a molecular weight 30 times greater than its own into cells. When we compared the membrane-crossing activity of KST peptide in complex with Avidin (KST-Av complex) with that of similarly linked canonical TAT peptide, we found that TAT peptide penetrated SK-N-SH human neuroblastoma cells at a similar rate and efficiency as the KST peptide. Furthermore, KST peptide can carry protein complexes consisting of a specific antibody coupled to the peptide through the Avidin bridge. An antibody to Hsp70 delivered to SK-N-SH cells with high expression level of Hsp70 reduced the protective power of the chaperone and sensitized the cells to the pro-apoptotic effect of staurosporine. We studied the mechanisms of penetration of KST-Av and full-length Hsp70 inside human neuroblastoma SK-N-SH and human erythroleukemia K-562 cells and found that both used an active intracellular transport mechanism that included vesicular structures and negatively charged lipid membrane domains. Competition analysis of intracellular transport showed that the chaperone reduced intracellular penetration of KST peptide and conversely KST peptide prevented Hsp70 transport in a dose-dependent manner.
    Full-text · Article · Nov 2014 · Cell Stress and Chaperones
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    ABSTRACT: Chaperone Hsp70 can activate adaptive immunity suggesting its possible application as an anti-tumor vaccine. To assess the therapeutic capacity of Hsp70 we administered purified chaperone into a C6 glioblastoma brain tumor and explored the viability and tumor size as well as IFNγ production and cytotoxicity of lymphocytes in the treated animals. Targeted intratumoral injection of Hsp70 resulted in its distribution within the area of glioblastoma, and caused significant inhibition of tumor progression as confirmed by MRI. The delay in tumor growth corresponded to the prolonged survival of tumor-bearing animals of up to 31 days versus 20 days in control. Continuous administration of Hsp70 with an osmotic pump increased survival even further (39 days). Therapeutic efficacy was associated with infiltration to glioblastoma of NK cells (Ly-6c+) and T lymphocytes (CD3+, CD4+, and CD8+) as well as with an increase in the activity of NK cells (granzyme B production) and CD8+ T lymphocytes as shown by IFNγ ELISPOT assay. Furthermore, we found that Hsp70 treatment caused concomitantly, with a 10-fold elevated IFNγ production, an increase in anti-C6 tumor cytotoxicity of lymphocytes. In conclusion, continuous intratumoral delivery of Hsp70 demonstrates high therapeutic potential and therefore could be applied in the treatment of glioblastoma. © 2014 Wiley Periodicals, Inc.
    No preview · Article · Nov 2014 · International Journal of Cancer

  • No preview · Article · Oct 2014 · International Journal of Stroke
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    ABSTRACT: Superparamagnetic nanoparticles (SPIONs) due to their unique properties could be applied for theranostic approach in neuro-oncology. Accumulation and retention of the nanoparticles in the tumour can be directly imaged by MRI proving its application as contrast agents. Due to high heating capacity SPIONs could also be applied for thermotherapy of brain tumours. To further increase the biocompatibility and targeted delivery of SPIONs we have functionalized the surface of particles with recombinant heat shock protein Hsp70. Nuclear magnetic resonance (NMR) spectra and magnetic relaxation times of the synthesized conjugates T1, T2, T2* were measured with the help of an NMR spectrometer (CXP-300, Bruker) in a magnetic field of 7.1 T. Hsp70-SPIONs relaxivity corresponded to the properties of negative contrast agents with a hypointensive change of resonance signal in MR imaging. In series of in vitro experiments we observed enhanced internalization of the SPION-Hsp70 conjugates by C6 glioma cells in comparison to control SPIONs. The contrast-enhancing ability of SPION-Hsp70 conjugates was analyzed in the model of intracranial C6 glioma in rodents. The rat images were acquired with the help of a Bruker Avance II NMR spectrometer equipped with a microtomographic accessory at a magnetic field of 11 T. In vivo analysis of the conjugates distribution in the model of intracranial glioma revealed the retention of the functionilzed particles inside the tumour site. Negative contrast tumor enhancement in the T2-weighted MR images was higher in the case of Hsp70-SPIONs in comparison to non-modified SPIONs. Histological analysis of the brain sections confirmed the retention of the Hsp70-SPIONs in the glioma tumor but not in the adjacent normal brain tissues. Immunofluoresence studies confirmed that retention in the glioma of nanoparticles was due to the receptor-mediated endocytosis of the SPION-Hsp70 conjugates via the CD40 receptors. The uptake of SPION-Hsp70 by tumour cells permitted MRI contrast enhancement after systemic delivery while sparring surrounding normal brain tissues.
    Full-text · Article · Sep 2014 · Neuro-Oncology
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    ABSTRACT: Intratumoral injections of recombinant heat shock protein (Hsp)70 were explored for feasibility in patients with brain tumors. Patients aged 4.5-14 years with untreated newly diagnosed tumors (n=12) were enrolled. After tumor resection, five injections of recombinant Hsp70 (total 2.5 mg) were administered into the resection cavity through a catheter. Before administration of Hsp70 and after the last injection, specific immune responses to the autologous tumor lysate were evaluated using the delayed-type hypersensitivity test. Further, peripheral blood was monitored to identify possible changes in lymphocyte subpopulations, cytokine levels, and the cytolytic activity of natural killer cells. The follow-up period in this trial was 12 months. Intratumoral injections of Hsp70 were well tolerated by patients. One patient had a complete clinical response documented by radiologic findings and one patient had a partial response. A positive delayed-type hypersensitivity test was observed in three patients. In peripheral blood, there was a shift from cytokines provided by Th2 cells toward cytokines of a Th1-cell-mediated response. These data corresponded to changes in lymphocyte subpopulations. Immunosuppressive T-regulatory cell levels were also reduced after injection of Hsp70, as well as production of interleukin-10. The cytolytic activity of natural killer cells was unchanged. The present study demonstrates the feasibility of intratumoral delivery of recombinant Hsp70 in patients with cancer. Further randomized clinical trials are recommended to assess the optimum dose of the chaperone, the treatment schedule, and clinical efficacy.
    Full-text · Article · Jun 2014 · OncoTargets and Therapy
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    ABSTRACT: Recombinant 70 kDa heat shock protein (Hsp70) is an antiapoptotic protein that has a cell protective activity in stress stimuli and thus could be a useful therapeutic agent in the management of patients with acute ischemic stroke. The neuroprotective and neurotherapeutic activity of recombinant Hsp70 was explored in a model of experimental stroke in rats. Ischemia was produced by the occlusion of the middle cerebral artery for 45 minutes. To assess its neuroprotective capacity, Hsp70, at various concentrations, was intravenously injected 20 minutes prior to ischemia. Forty-eight hours after ischemia, rats were sacrificed and brain tissue sections were stained with 2% triphenyl tetrazolium chloride. Preliminary treatment with Hsp70 significantly reduced the ischemic zone (optimal response at 2.5 mg/kg). To assess Hsp70's neurotherapeutic activity, we intravenously administered Hsp70 via the tail vein 2 hours after reperfusion (2 hours and 45 minutes after ischemia). Rats were then kept alive for 72 hours. The ischemic region was analyzed using a high-field 11 T MRI scanner. Administration of the Hsp70 decreased the infarction zone in a dose-dependent manner with an optimal (threefold) therapeutic response at 5 mg/kg. Long-term treatment of the ischemic rats with Hsp70 formulated in alginate granules with retarded release of protein further reduced the infarct volume in the brain as well as apoptotic area (annexin V staining). Due to its high neurotherapeutic potential, prolonged delivery of Hsp70 could be useful in the management of acute ischemic stroke.
    Full-text · Article · May 2014 · Drug Design, Development and Therapy
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    Full-text · Article · May 2014 · European Journal of Cancer
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    ABSTRACT: Hsp70 chaperone is known to stimulate anti-tumour immunity in a variety of cancer models. Here we demonstrated that the addition of purified recombinant Hsp70 to the culture medium facilitated cancer cell cytolysis by lymphocytes. Importantly, exogenous Hsp70 triggered secretion of the intracellular Hsp70 to a cell surface and extracellular milieu, which played a role in cytolysis because down-regulation of the endogenous Hsp70 reduced both its presence at the cell surface and the lymphocyte-mediated cytolysis. Inhibitors that target both the ATPase and the peptide-binding domains of Hsp70 molecule potently decreased its anti-tumor effect. Using a variety of cell transport markers and inhibitors, we showed that the exchange of exogenous and intracellular Hsp70 is supported by classical and non-classical transport pathways, with a particular role of lipid rafts in the chaperone's intracellular transport. In conclusion, exogenous Hsp70 can eject endogenous Hsp70, thus exerting anticancer activity.
    Full-text · Article · Mar 2014 · Oncotarget
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    Full-text · Article · Mar 2014
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    ABSTRACT: Background Superparamagnetic iron oxide nanoparticles (SPIONs), due to their unique magnetic properties, have the ability to function both as magnetic resonance (MR) contrast agents, and can be used for thermotherapy. SPIONs conjugated to the heat shock protein Hsp70 that selectively binds to the CD40 receptor present on glioma cells, could be used for MR contrast enhancement of experimental C6 glioma.Methods The magnetic properties of the Hsp70-SPIONs were measured by NMR relaxometry method. The uptake of nanoparticles was assessed on the C6 glioma cells by confocal and electron microscopes. The tumor selectivity of Hsp70-SPIONs being intravenously administered was analyzed in the experimental model of C6 glioma in the MRI scanner.ResultsHsp70-SPIONs relaxivity corresponded to the properties of negative contrast agents with a hypointensive change of resonance signal in MR imaging. A significant accumulation of the Hsp70-SPIONs but not the non-conjugated nanoparticles was observed by confocal microscopy within C6 cells. Negative contrast tumor enhancement in the T2-weighted MR images was higher in the case of Hsp70-SPIONs in comparison to non-modified SPIONs. Histological analysis of the brain sections confirmed the retention of the Hsp70-SPIONs in the glioma tumor but not in the adjacent normal brain tissues.Conclusion The study demonstrated that Hsp70-SPION conjugate intravenously administered in C6 glioma model accumulated in the tumors and enhanced the contrast of their MR images.
    Full-text · Article · Dec 2013 · Neuro-Oncology
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    ABSTRACT: Abstract Purpose: Heat shock protein 70 (HSPA family) is a multi-functional protein which protects individual cells from proteotoxic shock and the whole organism from microbial, viral and oncogenic pathogens. These diverse functions may depend upon 'chaperone' activity that allows Hsp70 to regulate the mechanism of damaged protein recovery or utilisation inside a cell and to be a potent adjuvant, stimulating immune activity against a variety of viral or tumour antigens. The aim of this review is to present recent data on specific roles of intracellular and extracellular Hsp70 in cancerous tissue. Conclusion: The data presented in this paper show that endogenous Hsp70 protects cancer cells of different origins from a variety of cytotoxic threats including cancer cell therapeutics. In contrast, however, Hsp70 released from stressed cancer cells can serve as a danger signal or may recruit cells responsible for the generation of innate and adaptive immune responses against tumour cells.
    No preview · Article · Jul 2013 · International Journal of Hyperthermia
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    ABSTRACT: Polyglutamine diseases are a group of pathologies affecting different parts of the brain and causing dysfunction and atrophy of certain neural cell populations. These diseases stem from mutations in various cellular genes that result in the synthesis of proteins with extended polyglutamine tracts. In particular, this concerns huntingtin, ataxins, and androgen receptor. These mutant proteins can form oligomers, aggregates, and, finally, aggresomes with distinct functions and different degrees of cytotoxicity. In this review, we analyze the effects of different forms of polyQ proteins on other proteins and their functions, which are considered as targets for therapeutic intervention.
    Full-text · Article · May 2013 · FEBS letters
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    ABSTRACT: Most neurodegenerative pathologies stem from the formation of aggregates of mutant proteins, causing dysfunction and ultimately neuronal death. This study was aimed at elucidating the role of the protein factors that promote aggregate formation or prevent the process, respectively, glyceraldehyde-3-dehydrogenase (GAPDH) and tissue transglutaminase (tTG) and Hsp70 molecular chaperone. The siRNA technology was used to show that the inhibition of GAPDH expression leads to a 45-50% reduction in the aggregation of mutant huntingtin, with a repeat of 103 glutamine residues in a model of Huntington's disease (HD). Similarly, the blockage of GAPDH synthesis was found for the first time to reduce the degree of aggregation of mutant superoxide dismutase 1 (G93A) in a model of amyotrophic lateral sclerosis (ALS). The treatment of cells that imitate HD and ALS with a pharmacological GAPDH inhibitor, hydroxynonenal, was also shown to reduce the amount of the aggregating material in both disease models. Tissue transglutaminase is another factor that promotes the aggregation of mutant proteins; the inhibition of its activity with cystamine was found to prevent aggregate formation of mutant huntingtin and SOD1. In order to explore the protective function of Hsp70 in the control of the aggregation of mutant huntingtin, a cell model with inducible expression of the chaperone was used. The amount and size of polyglutamine aggregates were reduced by increasing the intracellular content of Hsp70. Thus, pharmacological regulation of the function of three proteins, GAPDH, tTG, and Hsp70, can affect the pathogenesis of two significant neurodegenerative diseases.
    Full-text · Article · Apr 2013 · Acta Naturae

Publication Stats

1k Citations
214.64 Total Impact Points

Institutions

  • 1988-2015
    • Russian Academy of Sciences
      • • Institute of Cytology
      • • Pavlov Institute of Physiology
      Moskva, Moscow, Russia
  • 2006
    • Institute of Cytology and Genetics
      Novo-Nikolaevsk, Novosibirsk, Russia
  • 2004
    • Saint Petersburg Medical Academy
      Sankt-Peterburg, St.-Petersburg, Russia
  • 1999
    • Sergei Kirov Military Medical Academy
      Sankt-Peterburg, St.-Petersburg, Russia
  • 1991-1998
    • Uppsala University
      • Department of Medical Cell Biology
      Uppsala, Uppsala, Sweden
  • 1997
    • Centro Universitário Fluminense
      São Salvador dos Campos, Rio de Janeiro, Brazil
    • Universidade Estadual do Norte Fluminense
      • Center of Biosciences and Biotechnology – CBB
      Rio de Janeiro, Rio de Janeiro, Brazil