magistrsko delo
Sara Friškovec (Avtor), Darja Žgur-Bertok (Recenzent), Matej Butala (Mentor)

Povzetek

Bakterija Aggregatibacter actinomycetemcomitans je glavni povzročitelj lokaliziranega agresivnega parodontitisa (LAP). Pri tej bolezni pride do hitrega uničenja obzobnega tkiva in se pogosto pojavi že pri najstnikih. Interakcije med proteini in molekulami RNA imajo ključno vlogo pri sintezi proteinov, kar vodi v spremembo delovanja celic. Bioinformatska analiza je predhodno nakazala, da bakterija A. actinomycetemcomitans sintetizira proteine, ki bi lahko interagirali z gostiteljevimi molekulami RNA. Naš cilj je bil analizirati interakcijo proteinov DnaK, HU, YjgF in Pnp te bakterije z molekulami evkariontske RNA. Ti proteini so bili prepoznani v zunanjih veziklih, ki se sproščajo iz bakterije in predvidoma vežejo molekule RNA. Izolirali smo rekombinantne proteine in celokupno RNA iz humane celične linije. Interakcijo protein-RNA smo poskušali analizirati s predhodno vezavo posameznega proteina na stacionarno fazo, Ni-NTA agarozo, nato smo dodali celokupno RNA izolirano iz evkariontske celične linije in vezano RNA eluirali z elucijskim pufrom za izolacijo RNA. V nadaljevanju smo molekule mRNA prevedli v molekule cDNA. Tako izvedena afinitetna izolacija tarčnih molekul RNA se je izkazala za zahtevno in s to metodo nismo dokazali vezave izbranih proteinov na molekule RNA. V nadaljevanju študije smo se posvetili analizi vezave proteina DnaK z molekulami RNA. Meritve triptofanskega spektra proteina DnaK, v prisotnosti ali odsotnosti molekul RNA nam nakazujejo, da pride do vezave. Predvidevamo, da RNA z vezavo na protein DnaK spremeni konformacijo proteina tako, da se aminokislinski ostanek triptofan proteina DnaK izpostavi na površini.

Ključne besede

Aggregatibacter actinomycetemcomitans;parodontitis;RNA vezavni proteini;molekularne interakcije;protein DnaK;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UL BF - Biotehniška fakulteta
Založnik: [S. Friškovec]
UDK: 579.873:577.2.088:575.112
COBISS: 5060216 Povezava se bo odprla v novem oknu
Št. ogledov: 719
Št. prenosov: 202
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: Identification of Aggregatibacter acinomycetemcomitans RNA binding proteins
Sekundarni povzetek: Bacterium Aggregatibacter actinomycetemcomitans is the major cause of localized aggressive periodontitis (LAP). This disease causes rapid destruction of the dental tissue and occurs early in life. The RNA-protein interaction plays a key role in the proteins synthesis, which directs processes in cells. The bioinformatics analysis previously indicated that certain A. actinomycetemcomitans synthesized proteins may interact with RNA molecules of the eukaryotic host. Our goal was to analyze the interaction of the proteins DnaK, HU, YjgF and Pnp with the eukaryotic RNA molecules. These proteins were identified in the outer membrane vesicles released from the bacterium and are putative RNA binding proteins. The recombinant proteins and the RNA molecules were isolated from the human cell lines. The proteins were then immobilized onto the Ni-NTA agarose. Immobilized proteins were afterwards exposed to the isolated RNA and the RNAs bound to proteins were later eluted with elution buffer. The mRNA molecules were converted into cDNA molecules that we tried to identify. The performed affinity isolation of the target RNA molecules proved to be difficult and this methoddid not prove the binding of the selected proteins to the RNA molecules. In the continuation of the study, the binding of the DnaK protein with RNA molecules was analyzed. Measurements of the tryptophan spectrum of DnaK protein, in the presence or in the absence of RNA molecules, indicate that DnaK protein interacts with RNA molecules. According to the data obtained, we assume that binding the RNA molecules to the DnaK protein changes the conformation of the protein in such a way that the triptofan residue is brought to the surface.
Sekundarne ključne besede: Aggregatibacter actinomycetemcomitans;periodontitis;RNA binding proteins;molecular interactions;DnaK protein;
Vrsta dela (COBISS): Magistrsko delo/naloga
Študijski program: 0
Komentar na gradivo: Univ. v Ljubljani, Biotehniška fak., Študij mikrobiologije
Strani: XII, 48 f., [5] f. pril.
ID: 11155054