Karakterizacija i validacija sojeva stanične linije melanoma dobivenih ugradnjom sekvence SHH u genom

master's thesis

• Title (english): Characterization and validation of SHH knock-in melanoma cell lines
• Author: Ana Brtan
• Mentor: Maja Sabol (mentor)
• Mentor: Sofia Ana Blažević (sumentor)
• Committee member: Sofia Ana Blažević (predsjednik povjerenstva)
• Committee member: Jasna Lajtner (član povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Committee member: Jasna Hrenović (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-02-26, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

U okviru ovog diplomskog rada identificirana je stanična linija dobivena ugradnjom sintetske sekvence koja kodira za protein sonic hedgehog. Aktivni oblik proteina sonic hedgehog, SHH-N, odgovoran je za autokrinu i parakrinu aktivaciju signalnog puta vezanjem za transmembranski receptor Patched 1. Provođenjem signala u stanicu, aktivacijski oblici transkripcijskih faktora glioma associated oncogenes ulaze u jezgru i aktiviraju transkripciju gena koji sudjeluju u ovom signalnom putu. Glavna uloga ovog signalnog puta je u organizaciji polarnosti organizma tijekom embriogeneze, dok je u odraslom organizmu većinom inaktivan, osim tijekom proliferacije i diferencijacije stanica. Zbog činjenice da regulira rast i razvoj, promjene u ovom signalnom putu mogu dovesti do nastanka tumora, poticanja njihovog rasta i invazije. Iz pojedinačnih transfeciranih stanica uspostavljene su klonske stanične linije kojima je pomoću PCR-a testirana prisutnost inserta SHH u genomu, ekspresija proteina SHH i SHH-N. Na temelju tih rezultata izabrane su četiri linije kojima su westernskom metodom otiska i imunofluorescencijom analizirane količine proteina HH/GLI signalnog puta (PTCH1, GLI1, GLI2, GLI3) i markera EMT (vimentin, E-kadherin). Identifikacijom prikladne stanične linije koja stabilno eksprimira SHH-N bit će moguće daljnje istraživanje ovog signalnog puta, osobito u stanicama tumora.

Abstract (english)

This master thesis contains the identification of one knock-in cell line for the protein SHH. Its active form, SHH-N is responsible for autocrine and paracrine activation of the HH/GLI signaling pathway by binding to its transmembrane receptor PTCH1. Transduction of the HH/GLI signal into the cell leads to the localization of activator forms of transcription factors GLI in the nucleus where they induce transcription of GLI target genes. This signaling pathway is mostly active during embryonic development, during which it regulates the polarity of the organism. In adults, under normal conditions, the pathway is inactivated or very weakly activated, except during cell proliferation and differentiation. Because of its role in regulating growth and development, changes in this signaling pathway can lead to tumor development, stimulating their growth and invasion. Cell lines developed from individual transfected cells were tested by PCR to detect the SHH insert, and by Western blot to identify the lines with increased SHH and SHH-N protein expression. Four candidate cell lines were selected based on PCR and Western blot results and additionally tested by Western blot and immunofluorescence to analyze the levels of HH/GLI components (PTCH1, GLI1, GLI2, GLI3) and EMT markers (vimentin, E-cadherin). By finding the appropriate cell line with a stable expression of SHH-N, it becomes possible to further study this signalling pathway, especially in tumor cells.

• Keywords: Hedgehog-GLI; CHL1; melanom; transfekcija
• Keywords (english): Hedghehog-GLI; CHL1; melanoma; transfection
• Language: croatian
• URN:NBN: urn:nbn:hr:217:353167
• 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: 2023-02-26
• Created on: 2021-03-09 07:56:19