Uloga endocitoze posredovane klatrinom u in vitro aktivaciji inflamasoma nakon infekcije adenovirusom tipa 26

master's thesis

• Title (english): Role of clathrin mediated endocytosis in adenovirus type 26 induced inflammasome activation in vitro
• Author: Ana Runtić
• Mentor: Dragomira Majhen (mentor)
• Mentor: Silvija Černi (komentor)
• Committee member: Silvija Černi (predsjednik povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Committee member: Dijana Škorić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2023-09-21, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Vektorske vakcine na bazi adenovirusa čovjeka tipa 26 (HAdV-D26) intenzivno se istražuju
unazad nekoliko godina. Unatoč tome mehanizmi aktivacije urođenog imunosnog sustava ovim
virusom još nisu u potpunosti istraženi. Inflamasom je signalni kompleks koji preko kaspaze-1
aktivira proupalne interleukine važne za upalni odgovor i zaštitu od patogena. Cilj ovog rada
bio je istražiti kako inhibicija endocitoze posredovane klatrinom utječe na aktivaciju
inflamasoma u epitelnim stanicama čovjeka inficiranima virusom HAdV-D26. Metodom
reverzne trenskripcije i lančanom reakcijom polimerazom u stvarnom vremenu utvrđeno je
smanjenje ekspresije gena za proupalne interleukine IL-1β i IL-18 i kaspazu-1 nakon infekcije,
a inhibicijom endocitoze posredovane klatrinom ona se povećala. Aktivacija inflamasoma je
bila analizirana i na razini proteina westernskom metodom otiska i metodom
imunofluorescencije. Pritom nisu uočene značajne promjene u ekspresiji proteina NLRP3,
AIM2 i ASC koji sudjeluju u nastanku inflamasoma, kao ni u njihovoj unutarstaničnoj
lokalizaciji, neovisno o inhibiciji endocitoze posredovane klatrinom. Također nisu bili
detektirani ni aktivni oblici kaspaze-1 i proupalnih interleukina IL-1β i IL-18. Na temelju toga
se može zaključiti da infekcija virusom HAdV-D26 ne aktivira inflamasom u epitelnim
stanicama čovjeka. No s druge su strane infekcija virusom HAdV-D26 i inhibicija endocitoze
posredovane klatrinom potaknule oslobađanje katepsina B iz lizosoma što može biti signal za
sklapanje i aktivaciju inflamasoma.

Abstract (english)

Vector vaccines based on human adenovirus type 26 (HAdV-D26) have been intensively
researched for several years. Regardless, the mechanisms of activation of the innate immune
system by this virus have not yet been fully investigated. The inflammasome is a signaling
complex that via caspase-1 activates pro-inflammatory interleukins important for the
inflammatory response and protection against pathogens. The aim of this study was to
investigate how inhibition of clathrin-mediated endocytosis affects inflammasome activation in
human epithelial cells infected with HAdV-D26. The quantitative polymerase chain reaction
revealed a decrease in gene expression for pro-inflammatory interleukins IL-1β i IL-18 and
caspase-1 after infection, while inhibition of clathrin-mediated endocytosis increased it.
Inflammasome activation was also analyzed at the protein level by western blot and
immunofluorescence. No significant changes were observed in the expression of proteins
NLRP3, AIM2 and ASC which are involved in the formation of inflammasome, nor in their
intracellular localization, regardless of the inhibition of clathrin-mediated endocytosis. Active
forms of caspase-1 and pro-inflammatory interleukins IL-1β i IL-18 were also not detected.
Based on this, it can be concluded that infection with HAdV-D26 does not activate
inflammasome in human epithelial cells. However, infection with HAdV-D26 and inhibition of
clathrin-mediated endocytosis stimulated the release of cathepsin B from lysosomes, which
could be the signal for inflammasome assembly and activation.

• Keywords: HAdV-D26; epitelne stanice čovjeka; urođeni imunosni odgovor
• Keywords (english): HAdV-D26; human epithelial cells; innate immune response
• Language: croatian
• URN:NBN: urn:nbn:hr:217:918756
• 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: Embargoed access; Embargo expiration date: 2025-09-21
• Created on: 2023-10-16 10:16:51