diplomsko delo
Gašper Struna (Avtor), Miha Pavšič (Mentor)

Povzetek

Wnt proteini so glikoproteini, ki imajo vezan palmitoleat in so pomembni pri proliferaciji celic in raku. Pri človeku sodelujejo pri več signalnih poteh, ki se delijo na kanonične in nekanonične signalne poti. Pri kanonični signalni poti je pomemben transkripcijski regulator β-katenin, na katerega se med drugim lahko veže tudi znotrajcelični del tumorskega označevalca EpCAM. Zunajcelični del EpCAM (EpEX) vsebuje hidrofobni žep, v katerega se lahko veže palmitat. Ker se proteini EpCAM in proteini Wnt signalne poti v literaturi omenjajo pogosto v istem kontekstu, smo želeli nekaj izbranih proteinov izraziti v celicah HEK293T in preveriti, ali jih lahko veže EpEX preko svojega hidrofobnega žepa. Celice HEK293T smo pri različnih pogojih transfecirali s plazmidi, ki so vsebovali zapise za Wnt proteine z oznako V5. Po transfekciji smo izvedli test »pull down« z Wnt proteini in nemutirano oziroma mutirano (zaprt žep) obliko EpEX. Obe obliki smo predhodno pripravili v insektnih celicah in očistili z nikljevo afinitetno kromatografijo. Rezultati po transfekcijah so pokazali, da se Wnt proteini v celicah izražajo v majhnih količinah. Na membrani po testu »pull down« in sledečem western prenosu nismo zaznali nobene lise. Vzrok neprisotnosti lis bi bila lahko majhna količina izraženih Wnt proteinov zaradi česar signala ne moremo zaznati. Druga možnost pa je, da interakcije med Wnt in EpEX ni ali pa je ta šibka. V nadaljnjih študijah bi bilo torej potrebno optimizirati pogoje transfekcije in ponovno preveriti potencialno interakcijo med EpEX in Wnt.

Ključne besede

Wnt;EpCAM;zunajcelična domena EpCAM;EpEX;transfekcija;interakcije;diplomska dela;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.11 - Diplomsko delo
Organizacija: UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik: [G. Struna]
UDK: 577.112(043.2)
COBISS: 169094403 Povezava se bo odprla v novem oknu
Št. ogledov: 41
Št. prenosov: 3
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: Preparation of Wnt proteins and test of Wnt:EpCAM interaction
Sekundarni povzetek: Wnt proteins are glycoproteins that have palmitoleate attached and are important in cell proliferation and cancer. In humans, they participate in several signaling pathways, which are divided into canonical and non-canonical signaling pathways. In the canonical signaling pathway, the transcriptional regulator β-catenin is important, to which, the intracellular part of the tumor marker EpCAM can bind. The extracellular part of EpCAM (EpEX) contains a hydrophobic pocket into which palmitate can bind. As EpCAM proteins and Wnt signaling pathway proteins are often mentioned in the literature in the same context, we wanted to express some selected proteins in HEK293T cells and check whether EpEX can bind them via its hydrophobic pocket. HEK293T cells were transfected under different conditions with plasmids containing transcripts for V5-tagged Wnt proteins. After transfection, we performed a pull-down assay with Wnt proteins and the unmutated or mutated (closed pocket) form of EpEX. Both forms were previously prepared in insect cells and purified by nickel affinity chromatography. The results after the transfections showed that Wnt proteins are expressed in small amounts in the cells. We did not detect any band on the membrane after the pull-down assay and the following Western blot. The reason for the absence of band could be a small amount of expressed Wnt proteins, due to which the signal cannot be detected. Another possibility is that the interaction between Wnt and EpEX is absent or weak. In further studies, it would therefore be necessary to optimize the transfection conditions and recheck the potential interaction between EpEX and Wnt.
Sekundarne ključne besede: Wnt;transfection;EpEX;interaction;Beljakovine;Univerzitetna in visokošolska dela;
Vrsta dela (COBISS): Diplomsko delo/naloga
Študijski program: 1000371
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Strani: 30 str.
ID: 19904869