Activated cell death signaling pathways of mouse embrionic fibroblasts after UVB irradiation

Skelin, Josipa (2014) Activated cell death signaling pathways of mouse embrionic fibroblasts after UVB irradiation. Diploma thesis, Faculty of Science > Department of Biology.

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Fas-associated death domain protein is a key adaptor protein which takes part in a series of signaling pathways within a cell, including the extrinsic and intrinsic apoptosis pathways and the pathway of necroptosis. UVB irradiation can induce cell death through DNA damage, by directly activating cell surface receptors and through formation of reactive oxigen species. This paper examined the effect of FADD protein in the cell death responce after 300 J/m2 UVB irradiation. Wild type cells and cells lacking the FADD protein were treated with a pancaspase inhibitor zVAD-fmk, which inhibits apoptosis, a RIP1 kinase inhibitor necrostatin-1, which inhibits necroptosis and with both of the treatments simultaneously. Populations of living cells, of early apoptotic cells and of cells in late aapoptosis/necrosis were detected with flow cytometry. Apoptosis was detected with DNA electrophoresis. The activity of caspases-3, -8 and -9 was examined using colorimetric supstrates. Western blot immunodetection was used to detect PARP-1, PARP-2 and serine 15 phosphorilated p53 proteins. Polymerase chain reaction in real time was used to examine the activity of BAX and BCL-2 genes. The results show that mouse embryonic fibroblasts die after irradiation through the extrinsic and intrinsic pathways of apoptosis and through the pathway of necroptosis. There is a possibility that an alternative cell death pathway is activated after the inhibition of necroptosis. FADD-/- knock-out mouse embryonic fibroblasts show decreased survival after irradiation. The results for this type of cells show the presence of apoptosis and necroptosis, but not as dominant cell death pathways.

Item Type: Thesis (Diploma thesis)
Keywords: FADD, UV, cell death, apoptosis, necroptosis
Supervisor: Marijanović, Inga
Co-supervisor: Antunović, Maja
Date: 2014
Number of Pages: 55
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Grozdana Sirotic
Date Deposited: 02 Apr 2014 09:54
Last Modified: 02 Apr 2014 09:54

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