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Preparation of human Ca2+/calmodulin-dependent protein kinase kinase 2 phosphorylated at Ser100 and Ser511
Koupilová, Nicola ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
5 Abstract Ca2+ /calmodulin-dependent protein kinase kinases (CaMKK) are serine/threonine kinases involved in the calcium signaling pathway. Two CaMKK isoforms were described in mammals: CaMKK1 and CaMKK2. The increase in calcium concentrations induces Ca2+ /CaM binding to the C-terminal segment of CaMKK, thus relieving autoinhibition by disrupting the interaction between the autoinhibitory segment and the kinase domain. Active CaMKK then phosphorylate and activate their downstream kinases CaMK1 and CaMK4, and in the case of CaMKK2 also AMPK. The activity of CaMKK is also regulated by phosphorylation mediated by cAMP-dependent protein kinase A (PKA). This phosphorylation creates two binding motifs recognized by the regulatory 14-3-3 proteins. Previous studies have suggested that the 14-3-3 protein keeps phos- phorylated CaMKK1 in the inhibited state by blocking the dephosphorylation of the inhibitory phosphorylation site and it has been speculated that CaMKK2 is regulated in a similar manner. However, the role of 14-3-3 protein in the regulation of CaMKK2 is unclear. In order to study this protein complex, it is necessary to prepare recombinant CaMKK2 fully phosphorylated at both 14-3-3 binding motifs. The main aim of this bachelor thesis was to optimize the protocol for the phosphorylation of human CaMKK2...
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Mitochondrial permeability transition pore
Sereghy, Ursula ; Hansíková, Hana (advisor) ; Čapková, Markéta (referee)
The mitochondrial permeability transition pore (mPTP) is highly evolutionarily conserved channel found in the inner membrane of mitochondria. This pore is non-selectively permeable for molecules below 1,5 kD. Consequences of the pore opening due to an increase of Ca2+ or reactive oxygen species (ROS) and following depolarization of the membrane involve a disruption of the proton gradient, decrease in the production of ATP and prevalently a cell death. Death of a cell as a result of the mPTP opening is a physiopathological mechanism which follows ischemic diseases and neurodegenerative disorders such as Alzheimers and Huntingtons disease. Study of a structure and function of mPTP is essential for the research of mechanisms and progression of diseases, and it is crucial for the development of responding drugs and an overall decrease in the morbidity of the patients. This work compiles the course of the research into structure and function of the channel under physiological and pathological conditions and briefly puts down some of the experimental methods.
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The effect of the mast cell activation on the microtubule organisation
Hájková, Zuzana ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee)
The activation of bone marrow-derived mast cells (BMMCs) induces a number of cell processes such as degranulation, proliferation and cytoskeleton rearrangements. Although microtubules are important in these processes, molecular mechanisms that control changes in microtubule organisation during cell activation are unknown. Activation of BMMCs can be achieved in several ways. Under physiological conditions, the aggregation of IgE receptors (FcRI) on the surface of BMMCs leads to the initiation of specific signaling pathways. Cells can be also activated nonspecifically by a tyrosine phosphatase inhibitor pervanadate, or by thapsigargin that inhibits Ca2+ ATPase pumps located on the endoplasmic reticulum. In this diploma thesis it was found out that rapid morphological changes can be monitored when BMMC are immobilised on the fibronectin before their activation. It was proved that specific and nonspecific activation events lead to microtubule reorganization, as well as to generation of a large number of microtubule-dependent protrusions. In the course of FcRI aggregation, generation of microtubule protrusions depends on the activity of Src family protein tyrosine kinases and on the intracellular Ca2+ concentration. STIM1, an endoplasmic reticulum Ca2+ sensor, which participates in the activation of...
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