Kloniranje urokinaznog plazminogenskog aktivatora, njegovog inhibitora i receptora te njihova ekspresija u ljudskim stanicama u kulturi

master's thesis

• Title (english): Cloning of urokinase plasminogen activator, its inhibitor and receptor and their expression in human cells in culture
• Author: Tihana Balent
• Mentor: Maja Matulić (mentor)
• Committee member: Maja Matulić (predsjednik povjerenstva)
• Committee member: Željka Soldin (član povjerenstva)
• Committee member: Ines Radanović (član povjerenstva)
• Committee member: Draginja Mrvoš-Sermek (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2022-02-25, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Urokinazni plazminogenski aktivator (uPA) ili urokinaza zaslužan je za aktivaciju
plazminogena i njegovu pretvorbu u plazmin, proteolitički enzim koji razgrađuje
ekstracelularni matriks. Urokinaza je dio sustava koji obuhvaća i receptor uPA (uPAR) koji je
veže na staničnu membranu te njene inhibitore, PAI-1 i PAI-2. Cilj ovog rada bio je klonirati
sekvence koje kodiraju za urokinazu, njen receptor i inhibitor PAI-1 te ih eksprimirati u
različitim staničnim linijama. Analiza ekstracelularne enzimske aktivnosti i ekspresije
pojedinih elemenata sustava u različitim tipovima stanica ukazala bi na mehanizme regulacije
urokinazne aktivnosti. Konstruirani su plazmidi sa sekvencama gena za uPA, PAI-1 i uPAR te
transficirane stanične linije A1235, HeLa i HEK293. Stanice HEK293 i A1235 transficirane
plazmidom sa sekvencom za uPA imale su bazalnu aktivnost urokinaze. Sojevi stanica A1235
i HEK293 transficirani plazmidom sa sekvencom za PAI-1, pokazali su smanjenu aktivnost
urokinaze. Stanice HeLa nisu pokazale urokinaznu aktivnost. Unos plazmida sa sekvencom
uPAR nije utjecao na urokinaznu aktivnost stanica. Analiza ekspresije proteina pokazala je da
plazmidi sa sekvencom urokinaze nisu promijenili razinu ekspresije ovog proteina u staničnim
lizatima. Kod stanica A1235 i HEK293 detektirano je povećanje ekspresije uPAR i PAI-1
nakon transfekcije plazmidima s njihovim sekvencama. Uzgojene stanične linije s različitom
urokinaznom aktivnosti mogu poslužiti kao model za istraživanje utjecaja urokinaznog sustava
na staničnu proliferaciju, migraciju i preživljenje.

Abstract (english)

Urokinase plasminogen activator (uPA) or urokinase is responsible for the activation of
plasminogen and its conversion into plasmin, a proteolytic enzyme that degrades the
extracellular matrix. Urokinase is a part of a system that includes the uPA receptor (uPAR) that
binds urokinase to the cell membrane and its inhibitors, PAI-1 and PAI-2. The aim of this study
was to clone sequences encoding urokinase, its receptor and PAI-1, and to express them in
different cell lines. Analysis of extracellular enzyme activity and expression of individual
urokinase system elements in different cell types, could indicate the regulatory mechanisms of
urokinase activity. Plasmids with gene sequences for uPA, PAI-1 and uPAR were constructed
and A1235, HeLa and HEK293 cell lines were transfected with plasmids. HEK293 and A1235
cells transfected with the uPA sequence plasmid had basal urokinase activity. A1235 and
HEK293 cell strains transfected with plasmid containing PAI-1 sequence showed reduced
urokinase activity. HeLa cells did not have urokinase activity. Expression of plasmids with the
uPAR sequence did not affect urokinase activity in the transfected cell lines. Analysis of protein
expression showed that plasmids with the urokinase sequence did not change the expression
levels of this protein in the cell lysates. In A1235 and HEK293 cells, an increase in uPAR and
PAI-1 expression was detected after transfection with plasmids with corresponding sequences.
Cell strains with different urokinase activity can serve as a model for investigating the impact
of the urokinase system on cell proliferation, migration and survival.

• Keywords: urokinaza; PAI-1; uPAR; tumorske stanice; ekspresija; aktivnost
• Keywords (english): urokinase; PAI-1; uPAR; tumor cells; expression; activity
• Language: croatian
• URN:NBN: urn:nbn:hr:217:049696
• Study programme: Title: Integrated undergraduate and graduate university study of Biology and Chemistry Education; specializations in: Integrated undergraduate and graduate university study of Biology and Chemistry Education; Course: Integrated undergraduate and graduate university study of Biology and Chemistry Education; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra edukacije biologije i kemije (magistar/magistra edukacije biologije i kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2024-02-25
• Created on: 2022-03-23 13:23:49