Aktivirani signalni putevi stanične smrti kod mišjih embrionalnih fibroblasta nakon UVB zračenja

master's thesis

• Title (english): Activated cell death signaling pathways of mouse embrionic fibroblasts after UVB irradiation
• Author: Josipa Skelin
• Mentor: Inga Marijanović (mentor)
• Committee member: Inga Marijanović (predsjednik povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Committee member: Mirjana Kalafatić (član povjerenstva)
• Committee member: Maja Matulić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2014-01-22, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

UVB zračenje može inducirati staničnu smrt putem izazivanja oštećenja DNA, direktno aktivirajući površinske receptore na stanici te putem formacije reaktivnih kisikovih vrsta. Fasu pridruženi protein s domenom smrti ključni je adapterski protein koji sudjeluje u nizu signalnih procesa u stanici, uključujući i vanjski signalni put apoptoze i signalni put nekroptoze. U ovom radu ispitan je utjecaj FADD proteina na odabir signalnog puta stanične smrti koja nastupa uslijed UVB zračenja s 300J/m2. Stanice divljeg tipa te stanice kojima nedostaje FADD protein su 24 sata prije zračenja tretirane pankaspaznim inhibitorom zVAD-fmk, koji inhibira proces apoptoze, inhibitorom kinaze RIP1 necrostatinom-1, koji inhibira proces nekroptoze, te s oba tretmana istovremeno. Protočnom citometrijom utvrđene su populacije živih stanica, stanica u ranoj apoptozi te stanica u kasnoj apoptozi/nekrozi. Elektoforezom DNA detektirana je apoptoza. Aktivnosti kaspaza-3, -8 i -9 detektirane su korištenjem kolorimetrijskih supstrata.Western imunodetekcijom proteina detektirani su proteini PARP-1, PARP-2 i protein p53 fosforiliran na serinu15. Lančanom reakcijom polimeraze u stvarnom vremenu utvrđena je aktivnost gena bax i bcl-2. Rezultati pokazuju kako mišji embrionalni fibroblasti divljeg tipa uslijed zračenja umiru vanjskim i unutarnjim putem apoptoze i nekroptozom. Moguća je i prisutnost alternativnog puta stanične smrti koji nastupa prilikom inhibicije nekroptoze. FADD-/- mišji embrionalni fibroblasti pokazuju smanjeno preživljenje uslijed zračenja. Rezultati dobiveni za ovaj tip stanica ukazuju na prisutnost oba tipa apoptoze i nekroptoze, no ne kao dominantnih tipova stanične smrti.

Abstract (english)

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.

• Keywords: FADD; UV; stanična smrt; apoptoza; nekroptoza
• Keywords (english): FADD; UV; cell death; apoptosis; necroptosis
• Language: croatian
• URN:NBN: urn:nbn:hr:217:739935
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-04-26 12:21:48