I am a PhD student at the Center for Laser Microscopy, situated at the Faculty of Biology, University of Belgrade. Our group, led by Prof. Dr. Pavle Andjus, has long been devoted to studies of extracellular matrix glycoprotein Tenascin C (TnC), up-regulated during embryogenesis, in wound healing, and in tumor tissues. Our published studies concerned the contribution of TnC to promotion of neuronal structural and functional plasticity during murine developmental stages. Our current research aims at investigating its role in wound healing after CNS trauma. Glial scar, formed by astrocytes around the injury has been regarded as barrier to full neuronal regeneration since the mid-twentieth century, but lately as potentially necessary for axon regeneration, being the source of growth factors and other permissive molecules. TnC is strongly upregulated and secreted mainly by astrocytes into the extracellular matrix after CNS injury. Its diverse effects on cell proliferation, and activation are attributed to alternatively spliced isoforms, with varying number of domains. Hence, we investigate the role of individual recombinant fragments (alternatively spliced domains: Fn A, FnC, FnD) of TnC in glial scar formation. In addition to TnC experiments, our studies concerning disease mechanism in Amyotrophic lateral sclerosis (ALS) might also interest WG4. Early studies have shown that in ALS a significant feature of the immune-inflammatory process is a production of anti-neuronal IgG that affect motoneurons. Our previous experiments indicated further non-cell autonomous characteristics with effects recorded also from astroglia. I am conducting a multifactorial analysis of calcium signaling induced by ALS IgG in different brain cell types, and trying to find potential membrane receptor(s) specific for ALS IgG and further signaling pathways. We believe that the results will correlate IgG to specific score and type of disease, thus allowing better patient stratification.
I am experienced in preparation and maintenance of primary rodent neuroglial cell cultures: cortical astrocytes and microglia, as well as hippocampal, cortical and granular cerebellar neurons.
The technique I employ the most in my research is calcium imaging in cell cultures. Our laboratory, the Center for Laser Microscopy, situated at the Faculty of Biology, University of Belgrade has inverted epifluorescent microscope (AxioObserver A1, Carl Zeiss, Germany) equipped with water, glycerine and oil immersion objective LD LCI Plan-Apochromat 25x/0.8 (Carl Zeiss, Germany) and combined with VisiFluor Calcium Ratio Imaging System. The excitation light source is Xenon Short Arc lamp (Ushio, Japan) combined with high speed polychromator system (VisiChrome, Visitron Systems, Germany). Time-lapse images are obtained by “evolve”-EM 512 Digital Camera System (Photometrics, USA). We assess intracellular calcium changes using the cell-permeate acetoxymethyl (AM) ester of Fluo-4 (Fluo-4 AM, Molecular Probes). We also have customized delivery system, via glass pipettes (0.8 mm inner diameter, positioned ~350 µm away and ~1 mm above the cells, at the angle of 45º) connected to High Speed Solution Exchange System (ALA Scientific Instruments, USA) with pinch valves and VC3 electronic valve controller. The delivery system enables us to control with high precision application of up to 3 desired solutions during live imaging. In addition to calcium imaging, I use the system for dynamic video microscopy for monitoring free radical production and cell organelles motility.
I am also skilled at Scratch wound assay as a model of in vitro CNS injury, as well as in spinal cord lesions in vivo.
Furthermore, I am skilled at immunocytochemistry, western blot and RT- PCR.
I am interested in completing training in super resolution microscopy such as STED, as well as in using such technique for obtaining better resolution for my ongoing experiments.
I would like to collaborate with experts in the field of extracellular matrix in order to understand better potential mechanisms of cell signalling that might occur upon binding of immune-inflammatory components to the extracellular matrix.
Additionally, I am especially interested in the work of IIBB/CSIC group and their investigation of the influence of mitochondrial fusion/fission on neuronal synapse loss in neurodegeneration.
Milošević M, Milićević K, Božić I, Lavrnja I, Stevanović I, Bijelić D, Dubaić M, Živković I, Stević Z, Giniatullin R, Andjus P. (2017) Immunoglobulins G from Sera of Amyotrophic Lateral Sclerosis Patients Induce Oxidative Stress and Upregulation of Antioxidative System in BV-2 Microglial Cell Line. Front Immunol. 8:1619.
doi: 10.3389/fimmu.2017.01619. eCollection 2017.
Kapadia M, Bijelić D, Zhao H, Ma D, Stojanovich L, Milošević M, Andjus P, Šakić B. (2017) Effects of sustained i.c.v. infusion of lupus CSF and autoantibodies on behavioral phenotype and neuronal calcium signaling. Acta Neuropathol Commun. 5:70.
Milošević M, Bataveljić D, Nikolić L, Bijelić D, Andjus P. (2016) The effect of amyotrophic lateral sclerosis-linked exogenous SOD1-G93A on electrophysiological properties and intracellular calcium in cultured rat astrocytes. Amyotroph Lateral Scler Frontotemporal Degener. 17:443-51.