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COST Action CA15203 (2016-2021): MitoEAGLE
Evolution-Age-Gender-Lifestyle-Environment: mitochondrial fitness mapping

Cizmarova Beata

MitoPedia topics: MitoPedia topic::EAGLE 

COST: Has COST::Member COST WG1: Has COST WG1::WG1

COST WG4: Has COST WG4::WG4

Name	Has lastname::Velika Has firstname::Beata, Has title::PhD, RNDr.
Institution	[[Has institution:: Department of Medical and Clinical Biochemistry Faculty of Medicine Pavol Jozef Šafárik University in Kosice]]
Address	Has address::Trieda SNP 1, Has area code::040 11
City	In city::Košice
State/Province	In state::
Country	In country::Slovakia
Email	Has mailaddress::[email protected]
Weblink	Weblink::
O2k-Network Lab	{{#ask:has member::Cizmarova Beata}}

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MITOEAGLE Short-Term Scientific Mission

• • • • STSM Grant Period 1

Motivation letter

My name is Beáta Veliká and I work as Research specialist at the Pavol Jozef Šafárik University in Kosice, Department of Medical and Clinical Biochemistry, Faculty of Medicine, Slovakia. I graduated at the Faculty of Natural Science, at the University of Pavol Jozef Šafárik in Košice, Slovakia. The field of my study was biology and chemistry. After Master’s studies I continued in PhD study, at the Faculty of Medicine, at the University of Pavol Jozef Šafárik in Košice, Slovakia. The major field of my studies was Clinical Biochemistry. The topic of my dissertation work was Antioxidant properties of natural and synthetic compounds.

During my study and work I specialized to fluorescence spectroscopy method, I have experiences with measuring of SFF, EEM, synchronous spectra of different biological fluids, and I have experiences with different isolation methods, including mitochondria isolation, and measuring of their native fluorescence and respiration by Clark electrode.

Overview of the proposed activities will be performed:

The short–term project is focused on the optimalization of protocols for cryopreservation of human cells which are used as the study model of mitochondria respiration and H2O2 production. The usage of human blood cells will provide a significant stimulus for the entire field of mitochondrial medicine, because the mitochondrial function of blood cells is linked with numerous diseases and metabolic conditions in the human body, and the protocols developed during the project will lead to the standardization of the procedures, and should be used for studying mitochondria physiological and pathological mechanisms. The research project should show optimized conditions of cryopreservation for valid subsequent respirometric measurements of stored cells. The establishment of adequate conditions is very important, because reports by numerous groups that have previously evaluated the use of cryopreserved cells and tissue samples for later use in respirometry were both positive as well as negative.

We will work on the standardization of the procedures how to separate or isolate blood cells. We will test the different conditions of freezing and optimize the freezing protocols by testing appropriate media for cryopreservation (10, 20, 30% DMSO in FCS), we will also test the proper cell density at which cells should be stored (concentrated, diluted) and the time, for how long the cells can be maintained in a cryopreserved state. We will also optimize the thawing procedure of the cells, the effect of quick and slow freezing with freeze container enabling freezing with 1°C/min, and their viability and recovery after freezing. After that we will optimize protocols with defined substrate combinations, uncoupler and inhibitor titrations (SUIT - substrate-uncoupler-inhibitor titration protocol), which will be applied for the measurement of respiration in combination with fluorescence methods. We will evaluate the respiration and H2O2 production of permeabilized blood cells. We will apply high-resolution respirometry with using the Oxygraph 2k and the O2k-Fluo LED2-Module, in collaboration with experts in the OROBOROS laboratory, and we will apply fluorometry with Amplex Red reagent (AmR), to detect hydrogen peroxide (H2O2). The most appropriate conditions for the respirometric study of the different cell types will be determined, anticipating that cells with different functions may require different respiration media and/or substrate combinations for the adequate respirometric characterization. These measurements will be performed with Oxygraph 2k and the O2k-Fluo LED2-Module, in collaboration with experts in the OROBOROS laboratory.

I would like to carry out the project in cooperation with the research group at the Mitochondrial Research Laboratory of Dr. Erich Gnaiger in Innsbruck, Austria. The laboratory of Dr. Erich Gnaiger contains very high standard laboratory equipment, with a number of specialists in the field of mitochondrial physiology and instrument development working in the lab. The project will be realized at the Mitochondrial Research Laboratory of Prof. Dr. Erich Gnaiger (Medical University of Innsbruck; K-Regio project MitoFit) in collaboration with the scientific company OROBOROS INSTRUMENTS Corp.

STSM request: download pdf