1) The role of melanin and melanocytes in mechanisms of the drugs’ side-effects directed to pigmented tissues:

—Purpose: to examine the effect of the tested drugs on melanogenesis and antioxidant defense system in cultured normal human melanocytes light (HEMn-LP) and dark pigmented (HEMn-DP).

—Analyzed drugs: Aminoglycoside antibiotics, Tetracyclines, Fluoroquinolones, Phenothiazine neuroleptics, Nicotine and Cotinine.

—Methods and applied techniques:

—Cell viability - the WST-1 colorimetric assay, Melanin content, Tyrosinase activity, Hydrogen peroxide (H2O2) content and Antioxidant enzmymes (SOD, CAT, GPx) activity assays – spectrophotometric methods using a microplate reader UVM 340.

—Reactive oxygen species (ROS) detection assay – DCFDA fluorimetric assay using a multimode microplate reader TECAN INFINITY,

—Changes of genes expression involved in melanogenesis (MITF, TYR, TRP2, CREB) and antioxidant defense system (SOD1, SOD2, CAT and GPx) using real-time polymerase chain reaction (RT-PCR) technique with LIGHT CYCLER 96 SYSTEM (Roche).

—Changes of protein levels involved in melanogenesis (MITF, TYR, TRP2, CREB) and antioxidant defense system (SOD, CAT and GPx) using western blot technique and G:BOX SYSTEM (Syngene) for chemiluminescent and multicolor fluorescent imaging system.

—Analysis of the morphology of cells using a microscope Nikon Eclipse Ti-E with confocal equipment A1R -SI and superresolution N-SIM.

—The studies are also performed in the presence of UVA radiation (filtered lamp BVL-8.LM - Vilber Lourmat).

2) Searching for the potential anticancer drugs:

—Purpose: to examine the effect of the tested drugs on cell viability and apoptosis of different cancer cell lines.

—Analyzed drugs and cancer cells: Fluoroquinolone antibiotics, Phenothiazine neuroleptics, Nonsteroidal anti-inflammatory drugs (Sulindac) – melanotic (COLO-829) and amelanotic (C32) melanoma, BH3 mimetics (MIM-1) – glioblastoma astrocytoma (U-87 MG), melanotic (COLO-829) and amelanotic (C32) melanoma.

—Methods and applied techniques:

—Cell cycle analysis, DNA fragmentation, Mitochondrial potential, Caspase 3/7, 8, 9 and Annexin V assay - using the NUCLEOCOUNTER NC-3000 SYSTEM (Chemometec).

—Changes of genes expression involved in apoptosis (BAX, BCL-2, P-53) using real-time polymerase chain reaction (RT-PCR) technique with LIGHT CYCLER 96 SYSTEM (Roche)

—Changes of protein levels involved in apoptosis (BAX, BCL-2, P-53) using western blot technique and G:BOX SYSTEM (Syngene) for chemiluminescent and multicolor fluorescent imaging system.

—Analysis of the apoptosis: determination of apoptotic bodies using a microscope Nikon Eclipse Ti-E with confocal equipment A1R -SI and superresolution N-SIM.

3) Research on the neuroprotective properties of nicotine and its main metabolites:

—Purpose: to examine the effect of the tested drugs on apoptosis, antioxidant status and morphological changes of different normal human cell lines. The obtained results may be helpful to assess the potential of the tested drugs as neuroprotective agents.

—Analyzed drugs: Nicotine and its metabolites.

—Methods and applied techniques:

—Analysis of the apoptosis: determination of apoptotic bodies using a microscope Nikon Eclipse Ti-E with confocal equipment A1R -SI and superresolution N-SIM.

—Imaging of the oxidative stress in normal human cells under the influence of nicotine and its metabolites using a microscope Nikon Eclipse Ti-E with the confocal equipment A1R-SI and superresolution N-SIM.

Mitochondrial potential assay - using the NUCLEOCOUNTER NC-3000 SYSTEM (Chemometec).