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Monitoring of cell signaling pathways via interaction of fluorescently tagged proteins

Final Report Summary - MONINTERFLUOPROT (Monitoring of cell signaling pathways via interaction of fluorescently tagged proteins)

At present, fundamental questions in biomedical translational research require the use of non invasive nanometer resolution techniques that facilitate the study of cell structure and function in vivo. The present project was highly interdisciplinary: it encompassed advanced research techniques in biophysics, cell physiology, and molecular biology. The long term aim of the project was to establish a cutting-edge multidisciplinary program in biomedical and translational research at the Department of Biophysics at the P.J. Safarik University in Kosice, Slovakia.

The foundation of this program was laid out within EU structural fund projects CEVA I and II and SEPO I and II. The reintegrated scientist's, Dr. Stroffekova's, advanced knowledge of cell physiology and biophysics was well within scope of our goal, and therefore she was employed to plan and design the infrastructure for above mentioned EU projects, and to transfer her research at the Department of Biophysics. The reintegrated scientist's advanced experience in in vivo techniques of cell physiology, molecular biology, and fluorescent microscopy including FRET greatly enhanced multidisciplinary approaches and scope to study cell physiological and patho-physiological processes at the Department, and at the same time complements and enhanced development of a newly emerging field of Nanobiophotonics in Slovakia.

The specific objectives of the present project were to introduce new research methodologies that will be used within infrastructure and to expand the scientific potential at the Department of Biophysics. Specifically, the project was designed to introduce and establish electrophysiological measurements by whole cell patch clamp, site-directed mutagenesis and creation of fluorescent fusion proteins (FFPs) for application in confocal microscopy and fluorescence resonance energy transfer (FRET) measurements, and immunocytochemistry. All these methodologies were proposed to expand research capabilities for a molecule targeted photodynamic therapy of cancer. Further, the project implementation was designed to enable targeted researcher to establish a continuation and transfer of her research from USA to Slovakia, to introduce proposed methodologies in the Department of Biophysics, and to firmly anchor her long term career in Slovakia.

The knowledge and experience of reintegrated scientist have been successfully transferred to the Department. Fully functioning laboratories of cell physiology (whole patch clamp) (see online), molecular biology (fusion fluorescent proteins, Western blot analysis; (see online)) and confocal fluorescent microscopy (FRET) are the best proofs. The reintegrated scientist, Dr. Stroffekova, established within Department a group which studies regulation mechanisms of programmed cell death (apoptosis). This research is interdisciplinary as includes all the approaches she brought into Department such as fusion fluorescent proteins, immunocytochemistry, whole cell patch clamp and measurements metabolic flux rates in live cells. Her group comprises her and two PhD students (2nd and 3rd year, respectively) and two students at bachelor level. Her research very well complements long term goals of the Department's main research focus, which is to increase the sensitivity of cancer detection and the efficacy of cancer treatment by interdisciplinary approaches.

The reintegrated scientist's knowledge and research within the Department added to the Department strength, and therefore enabled the Department to receive one national research project, as well as EU project Fostering Excellence in Multiscale Cell Imaging - CELIM. Celim is designed to energize the research potential of the University of P. J. Safarik in Kosice, Slovakia, by the enhancement of the existing network of collaborators with the selected excellent European Partners, centered on the theme of cell imaging and by the acquisition of a new technical infrastructure.

Besides participating in the enhancement of the technological, methodological and experimental abilities of the Biophysics Department, the reintegrated scientist also actively participated in the Department's PhD program. She is a project manager of EU structural fund project 'DOKTORAND' regarding improvement and creating new PhD programs as well as dissipation of knowledge within EU by creating double diploma PhD studies. Besides that she taught, trained and advised students. At the present she is advisor of two PhD students. As a result, her work and activities significantly contribute to the socio-economic development of Slovakia. Overall, this project leads to the improvement of the quality of teaching activities and enhancement of higher interest of the young generation in biomedical sciences and multidisciplinary approaches.

Dissemination activities:

The reintegrated scientist research and transfer of her knowledge and experiences resulted in the national two research projects, which were funded and within which she is co-PI. She also submitted her own national independent project, which is now under review. Other results of her re-integrations are the publications with co-authors from our University (Faculty of Sciences and Medicine) and active participation on international and national meetings:

Peer-reviewed journal articles
1.Stroffekova K. (2011) The IQ motif is crucial for Cav1.1 function. J. Biomedicine and Biotechnology, Volume 2011, Article ID 504649, 9 pages, doi:10.1155/2011/
2.Skyba P.,Ukropec J.,Pobeha P., Ukropcova B.,Joppa P.,Kurdiova T., Stroffekova K., Brusik M., Klimes I., Tkac I., Gasperikova D.,and Tkacova R. (2010) Metabolic Phenotype and Adipose Tissue Inflammation in Patients with Chronic Obstructive Pulmonary Disease. Mediators of Inflammation Volume 2010, Article ID 173498, doi:10.1155/2010/173498
3.Balogova L., Maslanakova M., Dzurova L., Miskovsky P. and K. Stroffekova (2013) Bcl-2 proapoptotic proteins distribution in U-87 MG glioma cells before and after Hypericin photodynamic action. Gen. Physiol. Biophys. 32: 179-187
4.Dzurova L., Huntosova V., Petrovajova D., Nad'ova Z. , Miskovsky P. and K. Stroffekova (2013) Role of anti-apoptotic protein kinase Ca in response to hypericin photodynamic action in U-87 MG cells. Photochem. Photobiol. Under review.

Published Abstracts:
1.Stroffekova K., Balogova L., Maslanakova M., Dzurova L., Miskovsky P. (2013) BCL-2 Proapoptotic Proteins Distribution in U-87 MG Glioma Cells before and after Hypericin Photodynamic Action. Biophys. J. 104(2) Suppl.1: 656A-656A; 57th Annual Meeting of the Biophysical-Society. Philadelphia, PA (FEB 02-06, 2013)
2.D. Petrovajova, L. Dzurova, K. Stroffekova, Z. Nadova and P. Miskovsky. (2012) Interaction of hypericin with Protein Kinases Cα and Cδ and its Influence on U-87 MG Cells Death. Regional Biophysics Conference, 03.-07.09.2012 Kladovo (Serbia)
3.L. Balogova, M. Maslanakova, P. Miskovsky, K. Stroffekova. (2012) Proapoptotic proteins distribution in U-87 MG glioma cells before and after photodynamic action. 5th Slovak Biophysical Symposium, Bratislava, Book of abstracts pp 50.
4.L. Dzurova, D. Petrovajova, K. Stroffekova, Z. Nad'ova, P. Miskovsky. (2012) Activity of PKC isoforms in U-87MG glioma cells before and after PDT treatment- Western blot study. 5th Slovak Biophysical Symposium, Bratislava, Book of abstracts pp 52.
5.A. Strejckova, G. Bano, J. Stanicova, K. Stroffekova, G. Fabriciova, P. Miskovsky. (2012) Hypericin fluorescence in bilayer lipid membranes. 5th Slovak Biophysical Symposium, Bratislava, Book of abstracts pp. 39
6.Skyba P.,Ukropec J.,Pobeha P., Ukropcova B.,Joppa P.,Kurdiova T., Stroffekova K., Brusik M., Klimes I., Tkac I., Gasperikova D.,and Tkacova R. (2011) Metabolic Phenotype and Adipose Tissue Inflammation in Patients with Chronic Obstructive Pulmonary Disease. Am. J. Respir. Crit. Care Med 183: A4108. Annual American Thoracic Society Meeting, Denver, Colorado, USA, May 2011
7.Strejckova A.; Stanicova J.; Bano G.; et al. The incorporation of Hypericin and Emodin into artificial lipid membranes Conference: 8th EBSA European Biophysics Congress Budapest, HUNGARY Date: AUG 23-27, 2011; EUROP. BIOPHYSICS J. w BIOPHYSICS LETT. 40(1): 60-60
8.Dancakova A., Pacakova L., Stanicova J., Bano G., Stroffekova K., Fabriciova G., and Miskovsky P. (2010) The kinetics of Hypericin incorporation into bilayer lipid membranes. SPEC 2010, 26 June - 1 July 2010, Manchester, Book of abstracts PS50.