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master's thesis
Konstrukcija i validacija vektora za ekspresiju represorskog oblika proteina GLI3 u stanicama čovjeka
Zagreb: University of Zagreb, Faculty of Science, 2023. urn:nbn:hr:217:079669
Brtan, Lucija
University of Zagreb
Faculty of Science
Department of Chemistry

Institutional repository: Repository of the Faculty of Science

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Brtan, L. (2023). Konstrukcija i validacija vektora za ekspresiju represorskog oblika proteina GLI3 u stanicama čovjeka (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:079669

Brtan, Lucija. "Konstrukcija i validacija vektora za ekspresiju represorskog oblika proteina GLI3 u stanicama čovjeka." Master's thesis, University of Zagreb, Faculty of Science, 2023. https://urn.nsk.hr/urn:nbn:hr:217:079669

Brtan, Lucija. "Konstrukcija i validacija vektora za ekspresiju represorskog oblika proteina GLI3 u stanicama čovjeka." Master's thesis, University of Zagreb, Faculty of Science, 2023. https://urn.nsk.hr/urn:nbn:hr:217:079669

Brtan, L. (2023). 'Konstrukcija i validacija vektora za ekspresiju represorskog oblika proteina GLI3 u stanicama čovjeka', Master's thesis, University of Zagreb, Faculty of Science, accessed 26 May 2024, https://urn.nsk.hr/urn:nbn:hr:217:079669

Brtan L. Konstrukcija i validacija vektora za ekspresiju represorskog oblika proteina GLI3 u stanicama čovjeka [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2023 [cited 2024 May 26] Available at: https://urn.nsk.hr/urn:nbn:hr:217:079669

L. Brtan, "Konstrukcija i validacija vektora za ekspresiju represorskog oblika proteina GLI3 u stanicama čovjeka", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2023. Available at: https://urn.nsk.hr/urn:nbn:hr:217:079669

Metadata
TitleKonstrukcija i validacija vektora za ekspresiju represorskog oblika proteina GLI3 u stanicama čovjeka
Title (english)Construction and validation of the expression vector for the repressor form of the GLI3 protein in human cells
AuthorLucija Brtan
MentorMaja Sabol (mentor)
Committee memberMorana Dulić (predsjednik povjerenstva)
Committee memberŽeljka Soldin (član povjerenstva)
Committee memberIva Juranović Cindrić (član povjerenstva)
Committee memberMarko Močibob (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Chemistry)
Zagreb
Defense date and country2023-02-16, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Chemistry
Abstract
Proteini GLI važni su sudionici signalnog puta Hedgehog-GLI čiji se razni poremećaji u signalizaciji povezuju se s razvojnim deformacijama te onkološkim oboljenjima. U fokusu ovog rada nalazi se protein GLI3, jedan od tri izoforme proteina GLI. Protein GLI3 u svom punom obliku (GLI3FL) ima ulogu transkripcijskog aktivatora, dok njegov C-terminalni skraćeni oblik ima ulogu represora (GLI3R). Cilj ovog istraživanja je razvitak in vitro alata za ispitivanje različitih funkcija GLI3FL i GLI3R u tumorskim stanicama. Konstruiran je vektor za ekspresiju GLI3R, a njegova validacija izvršena je na staničnim linijama s različitom proteinskom ekspresijom proteina GLI3. Uvjeti transfekcije su optimizirani na staničnoj liniji fibroblasta prostate WPMY-1 s niskom razinom ekspresije, a efekt transfekcije ispitan je na staničnoj liniji metastatskog melanoma A375 s visokom razinom ekspresije, te uspoređen s djelovanjem spojeva koji mijenjaju omjere GLI3FL/GLI3R. Nakon transfekcije staničnih linija, analizom po Westernu dokazana je povećana ekspresija proteina GLI3R, a RT-qPCR metodom je potvrđena povećana ekspresija gena GLI3. Imunofluorescencijskom metodom proteini GLI3 su lokalizirani u citoplazmi te u jezgri stanica.
Abstract (english)
GLI proteins are essential members of the Hedgehog-GLI signaling pathway, and aberrations in which are associated with developmental deformities and oncological diseases. This thesis focuses on the GLI3 protein, one of the three isoforms of GLI proteins. The GLI3 protein in its full form (GLI3FL) has the role of a transcriptional activator, while its C-terminal shortened form has the repressor effect (GLI3R). This research aims to develop an in vitro tool for testing the different functions of GLI3FL and GLI3R in tumor cells. A GLI3R expression vector was constructed and validated on cell lines with different basal expression of the GLI3 protein. The transfection conditions were optimized on the low-expressing WPMY-1 cell line, and the transfection effect was tested on the high-expressing A375 cell line, and compared with the effect of compounds which affect the GLI3FL/GLI3R ratio. After the transfection of the cell lines, increased protein expression of GLI3R was demonstrated by the Western blot, and increased gene expression of GLI3 was confirmed by the RT-qPCR method. GLI3 proteins were localized in the cytoplasm and the cell nucleus using immunofluorescent staining.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:079669
ProjectNumber: IP-2018-01-4889
Title: Regulacija GLI koda u tumorima ovisnim o BRAF/NRAS mutacijama
Acronym: GLIcode
Leader: Maja Sabol
Jurisdiction: Croatia
Funder: HRZZ
Funding stream: IP
Study programmeTitle: Graduate university studies in chemistry; specializations in: Research programme
Course: Research programme
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2023-03-22 13:34:08