Utjecaj UV zračenja na proteine Bcl-2 i Bax u kulturi mišjih embrionalnih fibroblasta

master's thesis

• Title (english): The effect of UV irradiation on Bcl-2 and Bax proteins in mouse embryonic fibroblast culture
• Author: Tamara Džinić
• Mentor: Inga Marijanović (mentor)
• Committee member: Inga Marijanović (predsjednik povjerenstva)
• Committee member: Dubravka Hranilović (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: 2012-09-14, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

UV zračenje je potencijalni karcinogen koji potiče apoptozu preko membranskih receptora smrti ili aktivacijom unutarnjeg puta apoptoze. Bcl-2 djeluje kao antiapoptotski, a Bax kao proapoptotski protein u unutarnjem putu apoptoze. Protein FADD (Fasu pridružen protein s domenom smrti) kao pozitivni regulator apoptoze i negativni regulator nekroptoze sudjeluje u prijenosu signala sa staničnog receptora smrti na kaspazu 8, odnosno kinazu RIP1. Western imunodetekcijom proteina Bcl-2 i Bax ispitan je utjecaj UVB zračenja na mišje embrionalne fibroblaste divljeg tipa i knock-out mutante za FADD i uključenost unutarnjeg puta apoptoze u UVB induciranu apoptozu. Metodom TUNEL ispitivana je apoptoza na staničnoj razini korištenjem fluorescencijske mikroskopije za adherentne stanice te je kvantitativno određen postotak stanica u apoptozi korištenjem protočne citometrije. Pokusi su uključivali tretman stanica UVB zračenjem od 600 J/m2 i nekrostatinom-1, inhibitorom kinaze RIP1. Istraživanja su pokazala da mišji fibroblasti divljeg tipa nakon izlaganja UVB zračenju umiru procesima apoptoze i nekroptoze. U FADD-/- stanicama, bez obzira na zračenje i inhibiciju kinaze RIP1, nije detektirana fragmentacija DNA. UVB zračenje uzrokuje smanjenje količine antiapoptotskog proteina Bcl-2 u svim ozračenim stanicama bez obzira na odsutnost FADD-a i inhibiciju kinaze RIP1. UVB zračenje uzrokuje povećanje količine proapoptotskog proteina Bax kod fibroblasta divljeg tipa. Količina Bax proteina ovisi o prisutnosti FADD-a i aktivnosti RIP1 kinaze. Nedostatak jedne komponente FADD ili RIP1 dovodi do degradacije Bax- nakon zračenja, dok nedostatak obje komponente dovodi do povećanja količine Bax nakon zračenja.

Abstract (english)

UV irradiation is a potential carcinogen which induces apoptosis trough membrane death receptors or by activation of intrinsic apoptotic pathway. Bcl-2 acts as anti-apoptotic and Bax acts as pro-apoptotic protein in intrinsic apoptotic pathway. FADD (Fas- associated death domain) protein as a positive regulator of apoptosis and negative regulator of necroptosis acts in signal conduction from cell death receptors to caspase 8, respectively to RIP1 kinase. The effect of UVB irradiation on wild type and knock-out mouse embryonic fibroblasts for FADD and involvement of intrinsic apoptotic pathway in UVB induced apoptosis by Western blotting of Bcl-2 and Bax proteins were investigated. Experiments included exposure of cells to 600 J/m2 of UVB irradiation and treatment with necrostatin-1, inhibitor of RIP1 kinase. Apoptosis on cellular level was investigating using TUNEL method for fluorescence microscopy in adherent cells and the percentage of cells in apoptosis was quantitatively determined using TUNEL method for flow citometry. Researches showed that wild type of mouse embryonic fibroblasts die by processes of apoptosis and necroptosis after exposure to UVB irradiation. In FADD-/- fibroblasts, no matter of irradiation and inhibition of RIP1 kinase, no DNA fragmentation was detected. UVB irradiation causes decrease of anti-apoptotic Bcl-2 protein level in irradiated cells no matter to the absence of FADD or inhibition of RIP1 kinase. UVB irradiation causes increase of Bax protein level in wild type of fibroblasts. The amount of Bax protein depends on the presence of FADD and the activity of RIP1 kinase. Defect of only one component, FADD or RIP1, leads to degradation of Bax after irradiation, whereas defect of both components leads to increase of Bax level after irradiation.

• Keywords: UVB; apoptoza; nekroptoza; Bcl-2; Bax
• Keywords (english): UVB; apoptosis; necroptosis; Bcl-2; Bax
• Language: croatian
• URN:NBN: urn:nbn:hr:217:235326
• 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: 2017-04-28 12:59:14