magistrsko delo
Andreja Kamnik (Author), Nataša Poklar Ulrih (Mentor)

Abstract

Namen naloge je bil iz hipertermofilne arheje Aeropyrum pernix K1 izolirati majhne organske molekule (termolite) ter preučiti njihov vpliv na agregacijo/fibrilacijo inzulina. Začeli smo z izolacijo termolitov z metodo HPLC ter nadaljevali s preučevanjem agregacije inzulinskih verig pri različnih pogojih. Iz arheje smo izolirali dve majhni organski molekuli, vendar ju nismo uspeli identificirati. Agregacijo inzulinskih verig smo spremljali z emisijsko fluorescenčno spektrometrijo z barvilom tioflavinom T (ThT). Veriga B je hitreje fibrilirala v nevtralnem kot v kislem območju, veriga A je tvorila fibrile samo pri pH 2,0. Fibrilacijo verige B smo potrdili s cirkularnim dikroizmom z določitvijo za fibrile značilne β-strukture. Proces fibrilacije sta najbolj zavirala manoza in N-acetilglukozamin, slabše je učinkovala glukoza, glicerol je preprečil nastanek fibril pri visoki koncentraciji, mio-inozitol ni imel vpliva. Z določitvijo sekundarne strukture polipeptidov ob dodatku molekul smo potrdili oziroma ovrgli nastanek fibril. Z nativno poliakrilamidno gelsko elektroforezo smo ugotovili, da nekatere molekule inzulinskih verig ostanejo po fibrilaciji v monomerni obliki in se ne vgradijo v fibrile. Glede na molekulsko sidranje se nobena izmed testiranih majhnih organskih molekul ne veže močno na verigo B. V nalogi smo uspešno postavili metodo za izolacijo termolitov, ugotovili katere molekule vplivajo na fibrilacijo inzulinskih verig, vendar nismo uspeli potrditi/ovreči delovne hipoteze glede vpliva termolitov na agregacijo inzulina.

Keywords

Aeropyrum pernix;majhne organske molekule;agregacija;inzulin;emisijska fluorescenčna spektrometrija;cirkularni dikroizem;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [A. Kamnik]
UDC: 602.3:582.233:612.349.8:604.2:547.1(043.2)
COBISS: 81422851 Link will open in a new window
Views: 232
Downloads: 48
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Other data

Secondary language: English
Secondary title: Effect of small organic compounds from hyperthermophilic archaeon Aeropyrum pernix K1 on processes of insulin aggregation
Secondary abstract: The purpose of this study was to isolate small organic compounds (thermolytes) from hyperthermophilic archaeon Aeropyrum pernix K1 and research their effect on insulin aggregation/fibrillation. We started our research by isolating thermolytes with HPLC and continued to study the aggregation of insulin chains at different conditions. We isolated two small organic compounds from the archaeon, but we were unable to identify them. Aggregation of insulin chains was firstly studied using emission fluorescent spectroscopy with the dye thioflavin T (ThT). Chain B formed more fibrils at neutral than at acidic pH, while chain A fibrillated solely at pH 2.0. Measurements of circular dichroism after the fibrillation showed the formation of β-structure by chain B, which is typical for fibrils. Mannose and N-acetylglucosamine were the best inhibitors of fibrillation, glucose had small effect, glycerol only affected fibrillation at high concentration, myo-inositol did not inhibit fibrillation at all. By determining the secondary structure of polypeptides with added compounds, the formation of fibrils was confirmed or refuted. Native polyacrylamide gel electrophoresis indicated that during fibrillation some molecules of insulin chains remained in monomeric form and were not incorporated into fibrils. The molecular docking data exhibited that tested small organic compounds bind weakly to chain B. In this thesis we successfully set a method for thermolyte isolation, found out which molecules affect insulin chains fibrillation, but unfortunately, we were not able to confirm or refute the hypothesis about the effect of thermolytes on insulin aggregation.
Secondary keywords: small organic compounds;aggregation;insulin;emission;fluorescence spectroscopy;circular dichroism;
Type (COBISS): Master's thesis/paper
Study programme: 0
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Pages: XI, 55 f.
ID: 13700558