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Physiological interactions and stabilities of inner mitochondrial membrane complexes.
Kafková, Anežka ; Trnka, Jan (advisor) ; Mráček, Tomáš (referee) ; Vaňhara, Petr (referee)
Inner mitochondrial membrane (IMM) is composed of a phospholipid bilayer with a typically high ratio of protein complexes forming different complexes which execute different functions being reflected into the physiology of the whole organism. To maintain all these functions, as well as to have an ability to respond to various stress stimuli, the individual complexes of the respiratory chain interact, most likely to enhance the electron flow between these complexes, allowing the respiration steps from electron transfer from NADH to molecular oxygen to occur more efficiently. Other interactions between the complexes of the respiratory chain and other IMM complexes have been suggested. However, the underlying molecular details and functional relevance of such interactions are far from being elucidated. One of these many uncharacterised interactions involves the cytochrome c oxidase (COX), the terminal oxidase complex of the respiratory chain, and mitochondrial calcium uniporter complex (MCUC), the main gate to calcium entry into the mitochondrial matrix. Perhaps surprisingly, preliminary data from a systematic unbiased BioID analysis of MCUC subunits has consistently revealed an interaction between MCUC and COX subunits and assembly factors. In addition, SILAC-based quantitative proteomics also showed...
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The effect of psychotropic drugs on the mitochondrial functions.
Cikánková, Tereza ; Rečková Hroudová, Jana (advisor) ; Herink, Josef (referee) ; Vaňhara, Petr (referee)
Psychopharmaca are a large group of drugs widely used not only in psychiatry. Their systemic administration affects both the main diagnosis and the organism as a whole. The subject of our experiments is the effect of psychopharmaca on the changes in mitochondrial functions, which is beneficial for understanding of molecular mechanisms of therapeutic and adverse effects of drugs. The aim of this thesis was to study the in vitro effects of selected drugs on the cell energy metabolism. Selected antipsychotics (chlorpromazine, levomepromazine, haloperidol, risperidone, ziprasidone, zotepine, aripiprazole, clozapine, olanzapine, and quetiapine), antidepressants (bupropion, fluoxetine, amitriptyline, imipramine) and mood stabilizers (lithium, valproate, valpromide, lamotrigine, carbamazepine) were tested. In vitro effects of selected psychopharmaca were measured on isolated pig brain mitochondria. The activities of citrate synthase (CS) and electron transport chain (ETC) complexes (I, II+III, IV) were measured spectrophotometrically. Drug-induced changes of mitochondrial respiration rates linked to complex I (supported by malate and pyruvate) and complex II (supported by succinate) were evaluated by high resolution respirometry. Complex I was significantly inhibited by lithium, carbamazepine, fluoxetine,...
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The role of glycolytic enzymes in the development of cancer and metabolic disorders
Šimčíková, Daniela ; Heneberg, Petr (advisor) ; Šimíček, Michal (referee) ; Vaňhara, Petr (referee)
In this Ph.D. thesis, we aimed to focus on molecular mechanisms that underlie the roles of hexokinases in health and disease. First, we focused on the molecular basis of GCK-MODY and possibilities how to predict effects of variations in genes causing Mendelian disorders in general. We performed in vitro experiments on GCK and its variants carrying activating, neutral or inactivating variations. Subsequently, we compared these experimental results with outcomes from the state-of-the-art prediction algorithms with distinct backgrounds. As a result of analyses, we realized that the prediction algorithms commonly suffered from low specificity. Therefore, we suggested a method how to tailor numerical outcomes of these prediction algorithms in order to increase specificity. Furthermore, we determined pH optimum of human GCK and HK2 and investigated the influence of ATP concentrations on buffering capacity of commonly used buffers in hexokinase assays. In the part concerning the role of HKs in tumorigenesis, we studied in vitro somatic cancer-associated variations in GCK, which did not give meaningful evidence for a role of GCK in tumorigenesis, although a subset of somatic cancer-associated variations were activating, thus potentially advantageous for tumors. Therefore, we rather moved to the study of...
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Detection and clonogenic assay of cancer stem-like cells using flow cytometry
Fedr, Radek ; Souček, Karel (advisor) ; Vaňhara, Petr (referee)
The Diploma Thesis deals with an implementation of the new method for an assessment of a cloning efficiency of the cancer stem cells separated by a high speed cell sorter. The cell-sowing on the microtitration plates was performed by the flow cytometry method in a combination with the high speed cell sorter. In the first part of the Diploma Thesis the new method was introduced and tested on the selected cell lines. The obtained results were compared with the results of the limiting dilution assay within four cell lines. As for the second part of my Diploma Thesis, the method was practically applied to analysis of the cloning capacity of two subpopulations of cE2 cells based on the expressions of characteristic markers of stem and cancer stem cells - CD44 and CD 133. Based on the findings, the new method can be introduced as an approved proceeding for the cloning capacity assessment of cancer stem cells in other workplaces that possess analogical device equipment.
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