doktorska disertacija
Tjaša Plaper (Avtor), Mojca Benčina (Mentor)

Povzetek

V zadnjem stoletju so cepiva najučinkovitejši način preprečevanja smrti zaradi nalezljivih bolezni. Napredek v znanosti je ključno gonilo razvoja cepiv, zato so raziskave na področju cepiv zelo pomembne. Raziskovanje različnih pristopov načrtovanja novih cepiv je pomembno orodje za izboljšanje njihovih lastnosti. Cepiva ločimo na živa in neživa. Razlika med prvo in drugo skupino je v tem, da prva vsebuje oslabljene patogene, ki se lahko v telesu razmnožujejo, druga pa vsebuje mrtve patogene ali njihove komponente. V doktorskem delu smo se osredotočili na peptide, ki tvorijo obvite vijačnice (ang. coiled coils, CC-peptide) kot nosilce, s katerimi bi združili imunološko aktivne komponente v učinkovito ne-živo cepivo. Zaradi svoje modularne narave, so nam CC-peptidi služili kot dobri gradniki za načrtovanje nanocepiva, ki omogoča hkratno dostavo antigena in adjuvansa v tarčno celico, kar je za učinkovitost cepiva zelo pomembno. Načrtovali smo več parov stabilnih in med seboj ortogonalnih CC-peptidov. Pri tem smo upoštevali dobro definirana pravila umeščanja posamezne aminokisline znotraj CC-peptida. Z energijskega vidika je najpomembnejši položaj tisti, ki tvori hidrofobno sredico, elektrostatske interakcije med nasprotno nabitimi aminokislinskimi ostanki pa so drugi pomemben doprinos k stabilizaciji obvite vijačnice ter ključne za selektivnost parjenja. Sintetične peptide smo, po določitvi njihovih lastnosti in vitro, smo jih uporabili kot dimerizacijske domene za sestavljanje cepljenih proteinov. Na ta način smo se prepričali, ali načrtovani peptidi ohranijo svoje lastnosti v fuziji z večjimi domenami (proteini), kar je bilo ključno za nadaljnje eksperimente. Peptidna para z najustreznejšimi lastnostmi smo uporabili kot sestavna dela nanoogrodja, kjer smo načrtovane peptide izrazili v fuziji s fluorescenčnimi proteini ter s celičnimi dostavnimi domenami. Fluorescenčni protein nam je omogočal spremljanje celične dostave s pomočjo konfokalne mikroskopije ali pretočne citometrije. Ker je za cepivo poleg izbire ustreznega antigena in adjuvansa pomembna tudi tarčna dostava v celice, smo preizkusili več dostavnih domen. Po izbiri najučinkovitejše dostavne domene, smo nanoogrodje uporabili kot nanocepivo s katerim smo tarčno dostavili antigen ter adjuvans (kratek flagelin) v modelno antigen predstavitveno celico. Dokazali smo, da s CC-interakcijami posredovana dostava imunsko aktivnih molekul izboljša vnetni odziv.

Ključne besede

peptidi;obvite vijačnice;cepiva;nosilci;imunski odziv;imunologija;disertacije;Molekularna biologija;Disertacije;DNA;Imunologija;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.08 - Doktorska disertacija
Organizacija: UL MF - Medicinska fakulteta
Založnik: [T. Plaper]
UDK: 577.27:615.371(043.3)
COBISS: 128807427 Povezava se bo odprla v novem oknu
Št. ogledov: 366
Št. prenosov: 75
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: Modular assembley of immunologically active molecules based on coiled coils
Sekundarni povzetek: In the last century, vaccines have been the most effective way to prevent deaths caused by infectious diseases. Advances in science are a key driver of vaccine development, so research in the field of vaccines is very important. Exploring different approaches to designing new vaccines is important for improvement of vaccine efficacy. Vaccines are separated in two types: live-attenuated and inactivated. The difference between the two is that the first contains attenuated pathogens that can reproduce in our body, while non-living ones contain dead pathogens or their components. In this doctoral dissertation, we focused on peptides that form coiled coils (CC-peptides) as carriers with which to combine immunologically active components into an effective inactivated vaccine. Due to their modular nature, CC-peptides have served as good building blocks for the design of a nanovaccine that allows simultaneous delivery of antigen and adjuvant to the target cell, which is important for vaccine effectiveness. We designed several sets of stable and orthogonal CC-peptides. Well-defined rules for individual position within a CC peptide were followed. Most important positions are those that form the hydrophobic core. Electrostatic interactions between oppositely charged amino acid residues are another important contribution to the stabilization of the coiled helix and crucial for selectivity. After determining the properties of synthetic peptides in vitro, they were used as dimerization domains for the assembly of split proteins. Using split proteins method, we made sure the designed peptides retained their properties in fusion with larger domains (proteins), which was crucial for further experiments. The peptide pairs with most suitable properties were used as components of the nanoscaffold, where the designed peptides were expressed in fusion with fluorescent proteins and cell delivery domains. The fluorescent protein added to the nanostructure allowed us to monitor cell delivery by confocal microscopy or flow cytometry. In addition to selecting the appropriate antigen and adjuvant, target delivery into cells is also important for vaccine efficacy. We thus focused on several delivery domains. After selecting the most efficient delivery domain, the nano framework was used as a nanovaccine to target the antigen and adjuvant (short flagellin) to the model antigen-presenting cell. We have shown that CC-mediated delivery of immunologically active molecules improves the inflammatory response.
Sekundarne ključne besede: Molekularna biologija;Imunologija;Imunski odzivi;Biomolekule;Cepiva;Univerzitetna in visokošolska dela;
Vrsta dela (COBISS): Doktorsko delo/naloga
Študijski program: 0
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Medicinska fak.
Strani: XI, 77 str.
ID: 14554072