Uloga endocitoze posredovane klatrinom u ekspresiji upalnih citokina nakon infekcije adenovirusom tipa 26

master's thesis

• Title (english): Role of clathrin mediated endocytosis in adenovirus type 26 induced expression of inflammatory cytokines
• Author: Lea Vrbančić
• Mentor: Dragomira Majhen (mentor)
• Mentor: Silvija Černi (sumentor)
• Committee member: Silvija Černi (predsjednik povjerenstva)
• Committee member: Julija Erhardt (član povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Committee member: Duje Lisičić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-11-16, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Adenovirusni vektori se već dugi niz godina koriste u genskoj terapiji te za razvoj cjepiva protiv zaraznih bolesti. Najbolje proučeni adenovirusi tipa 2 i 5 imaju visoku seroprevalenciju u ljudskoj populaciji što ograničava njihovo korištenje kao vektora. Vektori temeljeni na niskoseroprevalentnom adenovirusu tipa 26 ističu se kao dobri kandidati za razvoj cjepiva jer potiču snažan i dugotrajan imunosni odgovor. Cilj ovog istraživanja bilo je ispitati ulogu endocitoze posredovane klatrinom u imunosnom odgovoru epitelnih stanica na infekciju adenovirusom tipa 26. Epitelnim stanicama adenokarcinoma pluća inhibirana je endocitoza posredovana klatrinom nakon čega su inficirane adenovirusom tipa 26. Stanicama je potom metodom qPCR određena ekspresija gena za pro-upalne citokine IL-6, IL-8 i IFN-α te metodom ELISA izmjerena količina izlučenih citokina IL-6 i IL-8 24 sata nakon infekcije. Kako bi se istražio utjecaj endocitoze posredovane klatrinom na aktivaciju signalnih puteva uslijed infekcije, westernskom analizom određena je aktivacija MAP kinaza ERK i p38 te transkripcijskog faktora NFkB. Rezultati pokazuju da inhibicija endocitoze posredovane klatrinom povećava ekspresiju gena za citokine te njihovo izlučivanje iz stanica uslijed infekcije adenovirusom tipa 26. Pokazano je da ovaj imunosni odgovor stanica nastaje aktivacijom signalnih puteva p38, ali ne i signalnih puteva ERK, neovisno o transkripcijskom faktoru NFkB.

Abstract (english)

Adenovirus have long been used as vectors in gene therapy and for development of vaccines against infectious diseases. The most investigated adenovirus type 2 and 5 have a high seroprevalence in human population which limits their use as vectors. Vectors based on adenovirus type 26, which has low seroprevalence, are recognized as good candidates for vaccination because they elicit a strong and long-lasting immune response. The aim of this study was to investigate the role of clathrin mediated endocytosis in adenovirus type 26 induced immune response in epithelial cells. Clathrin mediated endocytosis was inhibited in human lung adenocarcinoma cells, after which they were infected. After treatment and infection, the level of gene expression for pro-inflammatory cytokines IL-6, IL-8 and IFN-α was determined in cells by qPCR and the level of cytokines IL-6 and IL-8 secreted 24 hours after infection was determined by ELISA. In order to investigate the role of clathrin mediated endocytosis in activation of signaling pathways induced by infection, activation of MAP kinases ERK and p38, as well as transcription factor NFkB was determined by Western blot analysis. Results show that inhibition of clathrin mediated endocytosis increases adenovirus type 26 induced gene expression of cytokines as well as their secretion. Observed immune response was generated via activation of p38, but not ERK signaling pathways, independently of NFkB.

• Keywords: urođeni imunosni odgovor; adenovirusni vektori
• Keywords (english): innate immune response; adenoviral vectors
• Language: croatian
• URN:NBN: urn:nbn:hr:217:013682
• Study programme: Title: Experimental Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra eksperimentalne biologije (magistar/magistra eksperimentalne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2023-11-16
• Created on: 2021-11-23 07:38:38