| Grant number: | 21/02906-3 |
| Support Opportunities: | Scholarships in Brazil - Post-Doctoral |
| Start date: | May 01, 2021 |
| End date: | August 07, 2022 |
| Field of knowledge: | Biological Sciences - Physiology - Compared Physiology |
| Principal Investigator: | Marisa Narciso Fernandes |
| Grantee: | Mariana Morozesk |
| Host Institution: | Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil |
| Associated research grant: | 19/08491-0 - Atmospheric particulate material and environmental contamination. Impact assessment in the aquatic biota in an integrated ecophysiotoxicological approach, AP.TEM |
Abstract The action of atmospheric particulate matter (MPA) from the steel industry will be evaluated in: (1) Nile Tilapia (Oreochromis niloticus), a euryhaline species of commercial interest, whose physiological and biochemical parameters are well known; (2) a top-of-the-chain species, Sea Bass (Centropomus parallelus), which is an indicator of the real effects on the estuary environment. Specimens of each fish species will be exposed to, at least, 2 concentrations of MPA (MPA groups) to be determined and respective control groups (groups C), kept in water without MPA addition. After exposure tissues with high concentration of ionocytes (gills and kidneys) will be removed and processed according to the technique to be used for the determination of ionic transport proteins (Na+ /K+ -ATPase, H-ATPase, cotransporter Na+ /K+ /2Cl- , chloride channels) by immunohistochemistry (Dang et al., 2000; McCormick et al., 2003) and the distribution of these cells in each of the organs analyzed under fluorescence and confocal microscope will be evaluated. The plasticity and dynamics of the different types of ionocytes will be evaluated in relation to the environment and level of contamination. In relation to cardiomyocytes (heart), the action of MPA will be evaluated in relation to myocardial contractility through the analysis of the expression of proteins involved in excitation-contraction coupling and intracellular management of Ca2+. The expression of Ca2+ proteins -APTase of the sarcoplasmic reticulum (SERCA), phospholamban (PLB), Na+ /Ca2+ exchanger (NCX) and Ca2+ channel type L (LTCC) will be analyzed by Western blot. After electrophoresis of protein samples in SDS-polyacrylamide gel followed by electrical transfer to a polyvidylene fluoride membrane (PVDF) and protein block the membranes will be incubated overnight at 4 oC with the primary antibodies: anti-SERCA rabbit polyclonal antibody (Abcam Plc, Cambridge, UK); anti-PLB mouse monoclonal antibody (Millipore Corporation, Billerica, MA); anti-NCX rabbit monoclonal antibody (Abcam Plc, Cambridge, UK); Monoclonal anti-LTCC mouse antibody (Abcam Plc, Cambridge, UK) diluted in a 5% BSA solution in a TBS-T buffer. Subsequently, the membranes will be incubated with secondary antibodies conjugated with alkaline phosphatase: anti-rabbit IgG (Santa Cruz Biotechnology Inc., CA, USA) and anti-mouse IgG (Millipore Corporation, Billerica, MA). The membranes will be digitized, the bands will be analyzed by densitometry in the ImageJ software and the results will be expressed in arbitrary units (U.A.) of optical density normalized by the amount of GADPH (Santa Cruz Biotechnology Inc., CA, USA) detected in the respective samples. (AU) | |
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