magistrsko delo
Sanja Terzić (Avtor), Sabina Berne (Recenzent), Roman Jerala (Mentor)

Povzetek

Protein β-katenin, ki ga kodira gen CTNNB1, sodeluje v signalni poti Wnt in ima pomembno vlogo v zgodnjem nevrološkem razvoju. Pomanjkanje ß-katenina povzroča redek sindrom CTNNB1, za katerega so značilne motnje v kognitivnem in motoričnem razvoju. V magistrski nalogi smo želeli povečati količino citosolnega ß-katenina z zaviranjem delovanja aksina, ki je vpleten v razgradnjo β-katenina s pomočjo destrukcijskega kompleksa. V ta namen smo uporabili protismiselne oligonukleotide (angl. antisense oligonucleotides, ASOs), ki ciljajo vezavna mesta spajalno-izrezovalnega kompleksa na RNA za aksin. Pripravili smo delecijske konstrukte aksina brez izbranih eksonov ter s prenosom western, konfokalno mikroskopijo in pretočno citometrijo preverili njihovo izražanje, ko-lokalizacijo z ß-kateninom ter vpliv na aktivnost ß-katenina v celicah HEK293T. Na podlagi rezultatov smo načrtovali kratke protismiselne oligonukleotide ter testirali, ali lahko sprožijo preskakovanje eksonov na nivoju RNA ter kakšen je njihov vpliv na raven endogenega aksina in ß-katenina v celicah HEK293T. Protismiselni oligonukleotidi so povzročili preskakovanje tarčnih eksonov, znižali raven endogenega aksina ter povišali raven β-katenina v celicah. Po uporabi ASO:eks5 smo potrdili prisotnost dveh različic endogenega aksina. Dobljeni rezultati kažejo na možnost razvoja terapije za zdravljenje sindroma CTNNB1 na osnovi protismiselne tehnologije.

Ključne besede

sindrom CTNNB1;beta-katenin;destrukcijski kompleks;aksin-1;protismiselni oligonukleotidi;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: KI - Kemijski inštitut
Založnik: [S. Terzić]
UDK: 601.4:577.21(043.2)
COBISS: 193608707 Povezava se bo odprla v novem oknu
Št. ogledov: 74
Št. prenosov: 191
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Inhibition of endogenous ß-catenin inhibitors with designed short antisense oligonucleotides
Sekundarni povzetek: The protein β-catenin, which is encoded by the CTNNB1 gene, is involved in the Wnt signalling pathway and plays an important role in early neurological development. Deficiency of ß-catenin causes a rare CTNNB1 syndrome, which is characterized by impaired cognitive and motor development. Our aim was to increase the amount of cytosolic ß-catenin by inhibiting the activity of Axin, which is involved in the degradation of ß-catenin by the destruction complex. For this purpose, we used antisense oligonucleotides (ASOs) targeting the binding sites of the splicing complex on Axin RNA. Deletion constructs of Axin without selected exons were prepared and their expression, co-localisation with ß-catenin and their influence on ß-catenin activity in HEK293T cells were investigated by western blot, confocal microscopy, and flow cytometry, respectively. We designed short ASOs and tested their ability to induce exon skipping at the RNA level and their effect on endogenous levels of Axin and ß-catenin in HEK293T cells. We demonstrated that exon skipping was successfully induced by ASOs at the RNA level. Treatment with ASOs led to a decrease in endogenous Axin levels and an increase in endogenous β-catenin levels. Two variants of endogenous Axin were detected after treatment with ASO:eks5. These results suggest the possibility of developing a therapy for the treatment of CTNNB1 syndrome based on antisense technology.
Sekundarne ključne besede: CTNNB1 syndrome;beta-catenin;destruction complex;Axin-1;antisense oligonucleotides;
Vrsta dela (COBISS): Magistrsko delo/naloga
Študijski program: 0
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Biotehniška fak., Oddelek za agronomijo
Strani: 1 spletni vir (1 datoteka PDF (XII, 54 str.))
ID: 23306936