prikaz prve stranice dokumenta Epigenetička manipulacija gena MGAT4A i MGAT5 upotrebom molekularnog alata VPR-dCas9 u modelnoj staničnoj liniji gušterače 1.1B4
  Download
PDF 19.27 MB
master's thesis
Epigenetička manipulacija gena MGAT4A i MGAT5 upotrebom molekularnog alata VPR-dCas9 u modelnoj staničnoj liniji gušterače 1.1B4
Zagreb: University of Zagreb, Faculty of Science, 2022. urn:nbn:hr:217:058743
Lalić, Dora
University of Zagreb
Faculty of Science
Department of Biology

Institutional repository: Repository of the Faculty of Science

Cite this document

Lalić, D. (2022). Epigenetička manipulacija gena MGAT4A i MGAT5 upotrebom molekularnog alata VPR-dCas9 u modelnoj staničnoj liniji gušterače 1.1B4 (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:058743

Lalić, Dora. "Epigenetička manipulacija gena MGAT4A i MGAT5 upotrebom molekularnog alata VPR-dCas9 u modelnoj staničnoj liniji gušterače 1.1B4." Master's thesis, University of Zagreb, Faculty of Science, 2022. https://urn.nsk.hr/urn:nbn:hr:217:058743

Lalić, Dora. "Epigenetička manipulacija gena MGAT4A i MGAT5 upotrebom molekularnog alata VPR-dCas9 u modelnoj staničnoj liniji gušterače 1.1B4." Master's thesis, University of Zagreb, Faculty of Science, 2022. https://urn.nsk.hr/urn:nbn:hr:217:058743

Lalić, D. (2022). 'Epigenetička manipulacija gena MGAT4A i MGAT5 upotrebom molekularnog alata VPR-dCas9 u modelnoj staničnoj liniji gušterače 1.1B4', Master's thesis, University of Zagreb, Faculty of Science, accessed 23 December 2024, https://urn.nsk.hr/urn:nbn:hr:217:058743

Lalić D. Epigenetička manipulacija gena MGAT4A i MGAT5 upotrebom molekularnog alata VPR-dCas9 u modelnoj staničnoj liniji gušterače 1.1B4 [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2022 [cited 2024 December 23] Available at: https://urn.nsk.hr/urn:nbn:hr:217:058743

D. Lalić, "Epigenetička manipulacija gena MGAT4A i MGAT5 upotrebom molekularnog alata VPR-dCas9 u modelnoj staničnoj liniji gušterače 1.1B4", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2022. Available at: https://urn.nsk.hr/urn:nbn:hr:217:058743

Metadata
TitleEpigenetička manipulacija gena MGAT4A i MGAT5 upotrebom molekularnog alata VPR-dCas9 u modelnoj staničnoj liniji gušterače 1.1B4
Title (english)Epigenetic manipulation of the MGAT4A and MGAT5 genes in pancreatic model cell line 1.1B4 using VPR-dCas9 molecular tool
AuthorDora Lalić
MentorVlatka Zoldoš (mentor)
Committee memberVlatka Zoldoš (predsjednik povjerenstva)
Committee memberTomislav Ivanković (član povjerenstva)
Committee memberJasna Lajtner (član povjerenstva)
Committee memberŽeljka Vidaković-Cifrek (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Biology)
Zagreb
Defense date and country2022-10-27, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Biology
Abstract
Transporteri GLUT (engl. Glucose Transporter) nalaze se na membranama beta-stanica gušterače te su odgovorni za unos glukoze iz krvotoka, što u konačnici dovodi do ispuštanja inzulina koji regulira razinu glukoze u krvi. U miševa je dokazano da nedostatak glikoziltransferaza GnT-4a i GnT-V, kodiranih genima MGAT4A i MGAT5, uzrokuje nepravilnu glikozilaciju transportera GLUT i smanjenje njihove količine u membranama, što posljedično dovodi do razvitka dijabetesa tipa 2. Čini se da isti mehanizam postoji i u ljudima, ali mreža molekularnih događaja uključenih u nepravilnu regulaciju glikoproteina GLUT nije još razjašnjena. U ovom je istraživanju ciljano povećana ekspresija gena MGAT4A i MGAT5 pomoću molekularnog sustava CRISPR/dCas9 koji na sebe ima fuzioniranu aktivacijsku domenu VPR kako bi se ispitao učinak na količinu i lokalizaciju transportera GLUT1 u stanicama 1.1B4, koje su model za ljudske beta-stanice gušterače. Ekspresija gena uspješno je povećana na razini transkripcije te je potvrđena reverznom transkripcijom i lančanom reakcijom polimeraze u stvarnom vremenu (qPCR). Metodom Western blot analizirana je količina proteina GnT-4a i GnTV te je uočen porast količine koji nije statistički značajan. Imunofluorescencijskim bojanjem analizirana je količina i lokalizacija transportera GLUT1 te je uočen gotovo dupli porast u količini transportera između tretiranih i kontrolnih stanica. Kako bi se istražilo na koji je način povećana količina enzima utjecala na povećanje količine transportera GLUT1, trebalo bi analizirati glikanski profil transportera.
Abstract (english)
GLUT transporters (Glucose Transporters), localized in membranes of pancreatic beta cells, are responsible for uptake of glucose from the bloodstream. That leads to release of insulin which regulates blood sugar level. It has been proven in mice that the absence of glycosytransferases GnT-4a and GnT-V, encoded by genes MGAT4A and MGAT5, leads to improper glycosylation of GLUT transporters and a decrease in their abundance, which leads to development of type 2 diabetes. It appears the same mechanism exists in people, however the network of exact molecular events that lead to impaired glycosylation of GLUT transporters has not yet been resolved. In this study, expression of genes MGAT4A and MGAT5 was upregulated using a CRISPR/dCas9 system to which an activating VPR domain was fused. Cell line 1.1B4 was used, which is a model cell line for human pancreatic beta cells, so the effect on GLUT transporters could be investigated. Gene upregulation was confirmed on a transcription level by reverse transcription and quantitative polymerase chain reaction (qPCR). Protein analysis was done by Western blot and an increase in protein quantity was observed, but it was statistically unsignificant. Imunofluorescent staining was used to analyse the quantity and localization of GLUTs. Treated cells had almost double the quantity of GLUTs compared to the control group. To investigate the mechanism by which elevated levels of GnT-4a and GnT-V affected on GLUT quantity, glycan profile of transporters would have to be analysed.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:058743
Study programmeTitle: Molecular Biology
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2022-11-17 11:31:11