Optimiziranje uvjeta mjerenja superoksidnog radikala fluorescentnom mikroskopijom

master's thesis

• Title (english): Optimization of superoxide radical measurement by fluorescence microscopy
• Author: Luka Režan
• Mentor: Ana-Marija Domijan (mentor)
• Committee member: Ana-Marija Domijan (predsjednik povjerenstva)
• Committee member: Petra Turčić (član povjerenstva)
• Committee member: Kristina Radić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmaceutical botany); Zagreb
• Defense date and country: 2024-09-16, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Superoksidni radikal (O2∙−) primarni je kisikov radikal, koji u glavnini nastaje tijekom procesa
oksidativne fosforilacije u mitohondrijima i pokreće kaskadu stvaranja drugih reaktivnih kisikovih
vrsta (ROS) u stanici. Neravnoteža između stvaranja ROS-ova i mogućnosti biološkog sustava da ih
detoksificira i da popravlja štetu koja je u nastanku, dovodi do oksidacijskog stresa (OS), koji je
povezan s razvojem mnogih bolesti. Zbog pretpostavke da je O2∙− potencijalno koristan u procjeni
razine OS-a i činjenice da bi mjerenje O2∙− moglo imati dijagnostičku svrhu, cilj ovog rada bio je
optimizirati metodu za mjerenje O2∙− u stanicama na fluorescentnom mikroskopu. Za detekciju O2∙−
korištena je fluorescentna proba dihidroetidij (DHE), a pokus je proveden na bukalnim stanicama.
U prvom je koraku istraživanja optimizirana metoda mjerenja O2∙− u stanicama, u smislu
koncentracije DHE i vremena inkubacije stanica s DHE. Rezultati su pokazali da su optimalni uvjeti
za mjerenje O2∙− u stanicama 10-minutna inkubacija stanica na sobnoj temperaturi (25ºC) s otopinom
DHE u koncentraciji od 20 μM zbog najoptimalnijeg obojenja citoplazme. Nakon toga je
optimizirana metoda primijenjena za mjerenje O2∙− u stanicama koje su tretirane s H2O2 da bi se
inducirao OS. Potvrđeno je da je H2O2 uspješan induktor OS-a, odnosno da uspješno potencira
stvaranje O2∙− u stanicama. Do indukcije povišene razine O2∙− u bukalnim stanicama došlo je nakon
10-minutne inkubacije stanica s 30 mM otopinom H2O2. Razvijenu je metodu moguće primijeniti za
praćenje razine O2∙−, a time i OS-a, u stanicama.

Abstract (english)

The superoxide radical (O2∙−) is the primary oxygen radical, predominantly formed during the process of
oxidative phosphorylation in mitochondria, and it initiates a cascade of other reactive oxygen species
(ROS) production in the cell. An imbalance between ROS production and the biological system's ability
to detoxify them and repair the resulting damage leads to oxidative stress (OS), which is associated with
the development of many diseases. Due to the assumption that O2∙− could be potentially useful in
assessing OS levels and the fact that measuring O2∙− could have diagnostic purposes, the aim of this
study was to optimize a method for measuring O2∙− in cells using a fluorescent microscope. The
fluorescent probe dihydroethidium (DHE) was used for detecting O2∙−, and as experimental model,
buccal cells were used. In the first step of the research, the method for measuring O2∙− in cells was
optimized in terms of DHE concentration and cell incubation time with DHE. Results indicate that the
optimal conditions for measuring O2∙− in cells are 10 minutes incubation of cells at room temperature
(25ºC) with a DHE solution at a concentration of 20 μM for the most optimal cytoplasmic staining.
Subsequently, the optimized method was applied to measure O2∙− in cells treated with H2O2 to induce
OS. It was confirmed that H2O2 is a successful inducer of OS, meaning that it effectively enhances the
production of O2∙− in cells. Elevated levels of O2∙− in buccal cells were induced after 10 minutes
incubation of the cells with a 30 mM H2O2 solution. The developed method can be applied to monitor
O2∙− levels and, consequently, OS in cells.

• Keywords: oksidacijski stres; superoksidni radikal; reaktivne kisikove vrste; fluorescentni mikroskop; DHE
• Keywords (english): oxidative stress; superoxide radicals; reactive oxygen species; fluorescence microscopy; DHE
• Language: croatian
• URN:NBN: urn:nbn:hr:163:435264
• Study programme: Title: Medical biochemistry - integrated undergraduate and graduate - university study; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: sveučilišni magistar medicinske biokemije i laboratorijske medicine (sveučilišni magistar medicinske biokemije i laboratorijske medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-10-08 11:41:46