Utjecaj broja odmrzavanja i zamrzavanja te temperature pohrane krvne plazme na N-glikane ukupnih plazmatskih proteina i imunoglobulina G

master's thesis

• Title (english): Impact of the number of freeze-thaw cycles and storage temperature of blood plasma on N-glycans of plasma proteins and immunoglobulin G
• Author: Vedran Bogdanić
• Mentor: Olga Gornik (mentor)
• Committee member: Olga Gornik
• Committee member: Sandra Šupraha Goreta
• Committee member: Lidija Bach-Rojecky
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2015-06-11, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Glikozilacija je visokospecifičan i reguliran posttranslacijski proces kovalentnog vezanja složenih glikanskih struktura na proteine i lipide, a glikoproteini su uključeni u gotovo sve biološke procese na intracelularnoj i intercelularnoj razini. Predmet istraživanja ovog rada su N-vezani plazmatski glikoproteini s posebnim fokusom na imunoglobulin G kao iznimno važan i istraživan glikoprotein. Posljednjih godina provode se velike populacijske te cijelogenomske asocijacijske studije i kohortna istraživanja s ciljem identifikacije genske podloge glikozilacije, pronalaženja novih dijagnostičkih biljega te općenitog razumijevanja uloge glikozilacije. Ova istraživanja podrazumijevaju upotrebu velikog broja uzoraka iz različitih zemalja te njihov transport, pohranu i višestruke analize. S obzirom na navedeno, od velike je važnosti istražiti kako opetovano zamrzavanje i varijabilni uvjeti tijekom transporta uzoraka, koji često bivaju analizirani i više puta, mogu utjecati na stabilnost glikanskih struktura i rezultate same analize N-glikozilacije. S ukupnih proteina različito zamrzavane plazme, ali i iz takve plazme izoliranog imunoglubulina G, odvojeni su N-glikani koji su zatim fluorescentno obilježeni te analizirani tekućinskom kromatografijom vrlo visoke djelotvornosti koja se bazira na hidrofilnim interakcijama (UPLC – HILIC). Na temelju rezultata se može zaključiti kako ponavljano i dugotrajno odmrzavanje može utjecati na rezultate analize N-glikanskih struktura imunoglobulina G i ukupnih plazmatskih proteina. Ovaj utjecaj nije velik, ali se povećava drastičnijom manipulacijom uzorcima. Za ispitivanje stvarne statističke značajnosti istraživanog predanalitičkog uvjeta trebalo bi provesti mnogo veće istraživanje.

Abstract (english)

Glycosylation is a highly specific and delicately regulated posttranslational process of covalent bonding of complex glycan structures and proteins or lipids. Glycoproteins are invovlved in most intercellular and intracellular biological processes. The topic of this master thesis are N-linked plasma glycoproteins with a focus on immunoglobulin G as an important and widely investigated glycoprotein. In the last few years huge population studies, genome wide association studies and cohort studies have been conducted with the goal of identifying genetic foundation of glycosylation, discovering new diagnostic glycobiomarkers and deepening the understanding of glycosylation in general.All these above mentioned studies encompass the repeated analysis of a large number of samples collected from multiple countries, and variable transport and storage conditions. It is of great importance to determine how repeated freeze-thaw cycles and variable transport conditions as well as the reuse of samples affect the stability of plasma glycan structures and the results of N-glycosylation analysis. In this experiment, N-glycans have been removed from the plasma proteins and the previously isolated IgG both of which have undergone different freeze-thaw cycles. Purified N-glycans have been labeled with a flourescent marker and separated with Hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC). The result of the experiment points to the conclusion that the repeated freeze-thaw cycles and a longer thawing period may have an impact on the glycan structure analysis of immunoglubulin G and other plasma proteins. The effect was not very large so a much larger study should be conducted in order to determine its real statistical significance.

• Keywords: glikozilacija; protein plazme; glikoprotein; imunoglobulin G
• Keywords (english): glycosylation; plasma protein; glycoprotein; immunoglobulin G
• Language: croatian
• URN:NBN: urn:nbn:hr:163:796742
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2016-01-18 14:49:10