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Intracellular dinucleoside polyphosphates and methods of their detection
Říha, Jan ; Macíčková Cahová, Hana (advisor) ; Šanderová, Hana (referee)
(EN) Dinucleoside polyphosphates are already known for more than 50 years, but their role inside a cell is still unclear. Some theories discuss their possible role as alarmones during stress conditions, others connect them to DNA damage or proliferation. One new theory is that dinucleoside polyphosphates act as 5' RNA caps. To elucidate their role in organisms, it is important to know their concentration in normal and stress conditions. This work will try to determine basal concentration in both bacterial and eukaryotic cells, and the changes of their concentration under stress conditions, from already known data. Measurement of concentration of any compound inside a cell depends on the used method. I also present basic overview of methods for detecting dinucleoside polyphosphates, from older luciferase- based techniques to new precise mass spectrometry-based techniques. Keywords: dinucleoside polyphosphates, Ap4A, RNA caps, cellular stress, LC-MS detection and quantification
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Ap4A-RNA in IgE activated mast cells
Potužník, Jiří František ; Macíčková Cahová, Hana (advisor) ; Černý, Jan (referee)
Mast cells are tissue resident members of the immune system. They have a wide range of functions and receptors including the FcεRI receptor, which gets activated by binding to IgE bound to an antigen. When the cells are activated in this manner, a process termed the LysRS- Ap4A-MITF signalling pathway occurs, resulting in the translocation of the Lys tRNA synthetase into the nucleus and an activation of its moonlighting activity - the production of diadenosine tetraphosphate (Ap4A). Ap4A is a dinucleoside polyphosphate, a type of ubiquitous molecule present in all domains of life. They are made up of two nucleosides joined together by a 5' to 5' phosphodiester bridge of variable lengths. Recently, these molecules have been shown to serve as non-canonical initiating nucleotides during bacterial transcription, where they function as 5' RNA caps, similar to the well-known 7- methylguanosine eukaryotic mRNA cap. In this thesis, I present proof of existence of Ap 4A capped RNA in mast cells, a previously unknown 5' RNA structure in eukaryotic cells, and I attempt to pinpoint its role in the activation of these cells and in the wider context of mast cell mediated immune response. Keywords: mast cells, RNA caps, Dinucleoside polyphosphates, Ap 4A, RNA modification, IgE, FcεRI receptor, Lysine tRNA synthetase
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