Important research infrastructure

The infrastructure of the Institute was obtained mostly thanks to 3 projects supported by Structural Funds of EU. It is intensively used not only by IEPhT, but also by other institutes of SAS and universities.

 

Department of Cellular Pharmacology and Developmental Toxicology

Cell Culture Unit. The research staff of the laboratory has the appropriate qualification and expertise in a range of techniques for the study of pathological processes in cells associated with oxidative stress, inflammation and ageing. The Laboratory utilises a spectrum of standard cellular and molecular biology methods such as fluorescence-based methods for assessment of oxidative stress and cytotoxicity assisted by the fluorescence microscopy and flow cytometry, colorimetric cytotoxicity tests, ELISA and western blot analysis. The laboratory disposes modern standard equipment necessary for the work with cell cultures. Supplementary equipment, such as spectrophotometer TECAN and system for electrophoresis and western blot, serve for the biochemical processing of cultured cells. Based on cooperation with Institute of Chemistry SAS within the Centre of Excellence for Glycomics ITMS 26240120031 and other projects the Laboratory can utilise also Droplet Digital PCR (BioRad) and flow cytometer Beckman Coulter FC500.

Cardiovascular Unit. The research staff of the department has the appropriate qualification and expertise in a range of techniques for the study of pathological processes in the heart, brain and vessels associated with metabolic syndrome, oxidative stress, degeneration and ageing. The department utilises a spectrum of specified methods to determine function deficits of organs and tissues, such as perfusion of isolated heart under Langendorff method, measurement of smooth muscle tension of vessels under isometric conditions, the non-invasive method of blood pressure measurement, extracellular electrophysiological measurement of electrically evoked potentials in hippocampal slices, as well as a spectrum of standard biochemical laboratory methods for determination of biochemical variables playing role in metabolic syndrome, oxidative stress, aging, etc. The department disposes equipment for this techniques such as the McIllwain tissue chopper, the Grass stimulator, the DigiData 1332A (Molecular Devices), the Tektronix osciloscope, the AxoScope software, cold centrifuge, plate reader etc. Based on cooperation with Medical Faculty of Comenius University, there is possibility to analyse morphological changes in stained tissue preparates from brain/hippocampus, liver, abdominal aorta, heart etc. Based on the cooperation with Pharmaceutical Faculty CU, there is possibility to determine changes in expression of pro-inflammatory markers in tissue due to RT PCR method and Western blot analysis. Based on cooperation with Faculty of Chemical and Food Technology, Slovak University of Technology we can determine proton nuclear magnetic resonance (1H-NMR) spectra of brain metabolites measured on the 600 MHz Varian VNMRS spectrometer in nano-probe of hippocampal extracts in vitro. Further, based on the co-operation with the above mentioned faculty there is possibility to determine volume changes of hippocampus, ventricle and whole brain connected with trimethyltin-induced neurodegeneration measured by non-invasive method of magnetic resonance imaging (MRI) as well as determine proton magnetic resonance spectra (MRS) from dorsal hippocampus in vivo.

Cellular Pharmacology Unit. The research team of the department is experienced in pharmacological studies concerning the effects of drugs and biologically active substances on functions of immune and inflammatory cells. Studies are concentrated on human phagocytes (control and from patients with rheumatoid arthritis) and on phagocytes of rats with adjuvant arthritis. Modern molecular-biologic approaches are applied to the identification of cellular and molecular mechanisms participating in the effects studied: chemiluminescence methods (formation of reactive oxygen species in blood, neutrophils and in tissues e.g. joint, spleen, blood vessels), SDS-PAGE, western blotting and immunodetection with phosphospecific antibodies (phosphorylation of PKC isoforms and NADPH oxidase subunits), flow cytometry (oxidative burst, phagocytic activity, apoptosis, calcium mobilisation), ELISA (caspase-3 and PKC activity, concentration of cytokines), fluorescence microscopy (formation of neutrophil extracellular traps). The department possesses its own devices (luminometers, spectrophotometer, equipment for gel electrophoresis and western blotting, lumiaggregometer, laminar box, CO2 incubator, haematological analyser) and can share the utilisation of another devices needed (flow cytometer, fluorimeter, fluorescent and confocal microscope).

Developmental Toxicology Unit. The broad area of research interest in our department involves studies to elucidate mechanisms of hypoxia during sensitive stages of development and involvement of drug during organogenesis and “fine tuning” of central nervous system. Using a variety of animal models of human diseases in conjunction with biochemical, histology and behavioral methods of analysis, we seek to determine the extent to which these early influences can change the quality of later life. Screening for new prospective psychoactive drugs is also one of the research focuses. The Department utilises a spectrum of standard toxicological and behavioral methods such as screening for developmental anomalies, neurodevelopmental analysis, imunohistochemical-based assessment, anxiety- and depression-like behaviour observations, cognition and social behaviour evaluation. The department disposes modern equipment necessary for teratological and behavioural studies (ActiTrack and ANYmaze systems for recording of animal movement).

Bioorganic Chemistry Unit. The research staff is focused on studying oxidative degradation of a high-molar-mass hyaluronan induced by cupric ions and ascorbate. We assess protective effects of various drugs and antioxidants to prevent hyaluronan degradation. As a method we use rotational viscometry, which allows monitoring time-dependent changes in dynamic viscosity of hyaluronan solutions. Radical scavenging capacity of examined antioxidants/drugs is assessed by ABTS assay, DPPH assay, site- and non-site specific hydroxyl radical-mediated 2-deoxy-d-ribose degradation. The laboratory is equipped with two rotational viscometers LVDV Brookfield Pro-II and spectrophotometer UV-Vis 1800.

 

Department of Biochemical Pharmacology

The research of our department focuses on the etiology and possible prevention of diabetic complications as well as on the maintenance of calcium homeostasis from the point of view of sarcoplasmic reticulum Ca2+-ATPase function (SERCA). Our staff has the appropriate qualification and expertise in a range of techniques for the study of interactions of drugs/xenobiotics with proteins. The department utilizes a spectrum of standard enzymology methods at the level of isolated enzymes. The complex questions of biological activity/bioavailability and toxicity of drugs under study are solved by applying standard pharmacological approaches both at cellular and isolated organ levels as well as in intact animals. In silico methods comprise the molecular modeling from the quantum-chemical DFT level through docking to full optimization of protein-ligand complexes by means of molecular dynamics and simulated annealing. These methods are used for interpreting of experimental results and drug design, together with ADMET prediction and advanced QSAR techniques. Equipment used with methods: 3 HP PCs with the remote access to the HPC Aurel at the Computing Centre of SAS. Available software: Spartan08 (quantum-chemical and conformational calculations), Yasara (docking and ligand-complex optimization, molecular dynamics), Dragon (molecular descriptors, activity prediction), Pymol (visualization), Statistica (advanced statistical methods), NMAD (large scale molecular dynamics). Spectrophotometer Specord 40 (measurement of aldose reductase activity and inhibition; determination of SERCA activity and its enzyme kinetics); FluoroMax-4 spectrofluorometer (evaluation of conformational changes of SERCA). In the laboratory of cell cultures we utilise a spectrum of fluorescence and colorimetric methods and flow cytometry (Beckman Coulter FC500) for determination of cytotoxicity of pancreatic ß-cells induced by oxidative stress or methylglyoxal. To the standard methods of the laboratory belongs also the electrophoresis, ELISA, western blot used for evaluation of impaired SERCA and pancreatic ß-cells as well as standard antioxidant tests (DPPH, AAPH, DOPC lipid peroxidation). For in vivo experiments we used animal rat models of diabetes and colitis.

 

Department of Pharmacology of Inflammation

The pharmacological research is focusing on new therapeutic combinations of the classical immunosuppressive treatment (represented by methotrexate – MTX) with immunomodulators and compounds affecting redox homeostasis for treatment of rheumatoid arthritis (RA). In particular, natural substances capable to improve standard therapy of RA by MTX and minimize the administration of biological therapy are studied in the animal model of adjuvant arthritis. Moreover, elucidation of the mechanism of action of these substances in treatment of chronic inflammatory diseases could bring them into the evidence-based medicine. For this purpose, the studies are focused on the connection between systemic and local oxidative stress and inflammatory processes in the organism. The laboratory is equipped for measurement of markers of oxidative stress (malondialdehyde, 4-hydroxynonenal, protein carbonyls, heme oxygenase-1 and F2-isoprostanes) and inflammation (CRP, TNF-alpha, IL-1beta, MCP-1, IL-17, MMP-9, IL-6, oxidative burst and phagocytosis of neutrophils and monocytes) in plasma and different tissues using ELISA, Western blot, flow cytometry and spectrophotometry methods.