IZOLACIJA I KARAKTERIZACIJA DIVLJEGA TIPA STANICA MDCK S NISKOM IZRAŽENOŠĆU GENA mdr1 I NJIHOVA PRIMJENA U ISTRAŽIVANJU MEMBRANSKE PROPUSNOSTI

doctoral thesis

• Title (english): ISOLATION AND CHARACTERISATION OF MDCK CELLS WITH LOW EXPRESSION OF mdr1 GENE AND THEIR USE IN MEMBRANE PERMEABILITY SCREENING
• Author: Ana Bokulić
• Mentor: Astrid Milić (mentor)
• Committee member: Domagoj Đikić (predsjednik povjerenstva)
• Committee member: Donatella Verbanac (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2022-03-01, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology
• Universal decimal classification (UDC): 577 - Biochemistry. Molecular biology. Biophysics

Abstract

U ovoj disertaciji je opisana izolacija klona divljega tipa stanica MDCK s niskom
izraženošću gena mdr1 koji kodira P-glikoprotein. Klonovi su izolirani metodom velikog
razrjeđivanja, te uspoređeni mjerenjem dvosmjernog transporta prema razini efluksa
amprenavira. Klon 79, koji je pokazao najmanji omjer efluksa amprenavira, kao i tri puta
manju izraženost mRNA za mdr1 u odnosu na divlji tip (MDCK-WT), odabran je kao klon
MDCK s niskim efluksom za daljnje istraživanje. Na standardnom setu spojeva klon 79
pokazao je znatno manji efluks od MDCK-WT, iako je kod nekih spojeva i dalje
detektirana ostatna aktivnost P-glikoproteina.
Transport 12 makrocikličkih spojeva ispitan je na klonu 79 i nekoliko dodatnih staničnih
linija, Caco-2, MDCK-MDR1, MDCK-WT, te MDCK bez ekspresije P-glikoproteina. Nije
uočeno značajno poboljšanje permeabilnosti makrolida u staničnim modelima s niskom ili
bez izraženosti P-glikoproteina u odnosu na MDCK-MDR1 i MDCK-WT stanični model.
Proučen je utjecaj vremenske komponente na permeabilnost makrocikličkih spojeva u
staničnom modelu dvosmjernog transporta. Dobiveni rezultati analizirani su
multivarijantnim statističkim metodama, te su uspješno razvijeni modeli permeabilnosti i
bioraspoloživosti.

Abstract (english)

In this thesis, a clone of wild type MDCK cells with low expression of the mdr1 gene that
encodes the efflux pump P-glycoprotein, was isolated using the limiting dilution method.
The clones were ranked based on amprenavir efflux level in a bidirectional transport assay.
Clone 79 was selected as a low efflux MDCK clone for further investigation since it had
the lowest efflux ratio of amprenavir and three fold lower mdr1 mRNA expression in
comparison to MDCK wilde type. When profiled using standard compounds, clone 79 had
substantially reduced efflux compared to the wild type, although residual P-glycoprotein
activity was still observed with some compounds.
The transport of 12 macrocyles was tested using clone 79 and several additional cell lines,
including Caco-2, MDCK-MDR1, MDCK-WT, and MDCK with no P-glycoprotein
expression. No significant improvement in macrolide permeability was observed in cell
models with low or no P-glycoprotein expression in comparison to the MDCK-MDR1 and
MDCK-WT cells. The influence of the time component on the permeability of macrocyclic
compounds in the cellular transport model was further investigated. The obtained results
were analyzed by multivariate statistical methods and models of permeability and
bioavailability were successfully developed.

• Keywords: permeabilnost; MDCK; makrolid; izolacija klona; P-glikoprotein
• Keywords (english): permeability; MDCK; macrolide; clone isolation; P-glycoprotein
• Language: croatian
• URN:NBN: urn:nbn:hr:217:213679
• Promotion: 2022
• Study programme: Title: Biology; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija (doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-11-22 11:41:39