Utjecaj komponenata citoskeleta na diobu i diferencijaciju modelnog tkiva

master's thesis

• Title (english): Influence of cytoskeletal components on division and differentiation of model tissue
• Author: Ana Rastija
• Mentor: Marijeta Kralj (mentor)
• Mentor: Maja Matulić (mentor)
• Committee member: Maja Matulić (predsjednik povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Committee member: Duje Lisičić (član povjerenstva)
• Committee member: Inga Marijanović (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2017-12-14, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Poznavanje mehanizama odgovornih za diferencijaciju tkiva i razvoj organizma veliki je izazov razvojne biologije, ali i molekularne onkologije. Koordinirano ponašanje i kretanje stanica nužni su za morfogenezu, zacijeljivanje rana i tumorigenezu. Karakteristika stanica koje se gibaju ili diferenciraju jest promjena oblika ovisna o citoskeletu. U ovom radu istražili smo važnost citoskeleta u rastu i diferencijaciji stanica kružne kolonije MDCK II (Madin-Darby canine kidney epithelial cell) i razlike između dva tipa stanica koje nastaju treći dan rasta. Zbijene središnje stanice pojačano fosforiliraju eIF2α što upućuje na povećani stanični stres. eIF2α i fosforilirani eIF2α detektirali smo metodom Western blot. Istom metodom smo pratili razinu citoskeletnih (aktin, β-tubulin, vimentin, vinkulin) i adhezijskih proteina (β-katenin, E kadherina). Pokazali smo da se dva tipa stanica značajno razlikuju u ekspresiji vimentina, β katenina i E kadherina. Narušavanjem aktina i mikrotubula kemijskim spojevima te utišavanjem ekspresije vimentina molekulama siRNA, stanice kružne kolonije su izgubile diferencirani izgled. Metodom PCR istražili smo razinu ekspresije nesprina - proteina koji povezuju stanični i jezgrin skelet - na razini glasničke RNA. Narušavanje aktina i vimentina dovodi do promjene ekspresije nesprina 1 i nesprina 3. Rezultati ovog rada ukazuju na razlike između dva tipa stanica kružne kolonije te važnost integriteta citoskeleta u procesu rasta i diferencijacije modelnog tkiva.

Abstract (english)

Knowing the mechanisms responsible for tissue differentiation and development of the organism is a major challenge both to developmental biology and molecular oncology. Coordinated behavior and cell movement are necessary for morphogenesis, wound healing and tumorigenesis. Change of the shape that is dependent on the cytoskeleton is the characteristic of the cells that move or differentiate. Here we investigated importance of the cytoskeleton in the growth and differentiation of MDCKII (Madin-Darby canine kidney epithelial cell) circular colonies and the differences between the two types of cells that emerged on the third day of growth. Compacted central cells increase phosphorylation of eIF2α which indicates increased cellular stress. eIF2α and phosphorylated eIF2α were detected by the Western blot analysis. By the same method we analyzed the level of cytoskeletal (actin, β-tubulin, vimentin, vinkulin) and adhesion proteins (β-catenin, E cadherin). We have shown that two types of cells differ significantly in the expression of vimentin, β catenin and E cadherin. Circular colony cells lost a differentiated appearance because of disruption of actin and microtubules by chemical compounds and suppresion of the vimentin expression by siRNA. Expression of nesprin – protein linking the cellular and nuclear skeleton – was analyzed via PCR method at the level of the messenger RNA. Disruption of actin and vimentin leads to a change in expression of nesprin 1 and nesprin 3. The results of this research point out the differences between the two types of cells inside circular colonies and the importance of cytoskeletal integrity in the growth and differentiation of the model tissue.

• Keywords: nesprini; kružna kolonija; latrunkulin B; kolhicin; siRNA
• Keywords (english): nesprins; circular colonies; latrunculin B; colchicine; siRNA
• Language: croatian
• URN:NBN: urn:nbn:hr:217:411463
• 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: Open access; Embargo expiration date: 2019-12-14
• Created on: 2018-01-31 10:04:52