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Quantitative aspects of mitochondrial genome in cancer and diabetes
Alán, Lukáš ; Ježek, Petr (advisor) ; Teisinger, Jan (referee) ; Modrianský, Martin (referee)
Mitochondria are cellular powerhouses and physiological regulators with many features resembling their prokaryotic ancestors. They maintain their own mitochondrial DNA (mtDNA) encoding 13 inner mitochondrial membrane proteins of oxidative phosphorylation machinery, 22 transfer RNAs and two ribosomal RNAs. Healthy mitochondria in normal cells form a dynamic network consisting of highly interconnected tubules. The mitochondrial disorders are very heterogeneous and difficult to diagnose and cure. It is due to combining products of two genomes, the complexity of mitochondrial structure and due to insufficiency of current state of knowledge. To understand the mitochondrial nucleic acid species distribution, we have developed and established new techniques to visualize mitochondrial network, nucleoids and different RNA species together with qPCR techniques for monitoring the mitochondrial intactness. We determined nucleoid distribution and mtDNA amount following rotenone mediated respiratory inhibition or following degeneration of the electrical component of the protonmotive force by valinomycin treatment. Native mitochondria were mostly tubular with average nucleoid spacing 1.1 +/- 0.2µm which we termed as a nucleoid code. Subsequently induced fission resulted in mitochondrial network fragmentation and each...
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Preparation of the 14-3-3 Protein Binding Partners for Structural Studies.
Kopecká, Miroslava ; Obšil, Tomáš (advisor) ; Teisinger, Jan (referee)
Tyrosine hydroxylase belongs to the group of hydroxylases of aromatic acids and catalyzes a key step in the biosynthesis of catecholamine neurotransmitters. The tyrosine hydroxylase possesses the homotetrameric structure and contains three structural domains: the N-terminal regulatory domain, the catalytic domain and the C-terminal tetramerization domain. The activity of tyrosine hydroxylase is regulated by phosphorylation and through the regulation of its expression. Phosphorylation at Ser-19 induces binding of the 14-3-3 protein, which affects the structure of the regulatory domain and protects it against both dephosphorylation and degradation. Since the structure of the regulatory domain is still unknown, we decided to perform its structural characterization using NMR techniques. First, the expression and purification protocol of the regulatory domain of tyrosine hydroxylase was optimized. The protein was expressed as a His-tag fusion protein and its purification is composed from two steps: the chelating chromatography and the size-exclusion chromatography. The dynamic light scattering and the 1 H nuclear magnetic resonance were used to verify its monodispersity, and hence its suitability for further experiments.
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Localization and characterization of binding sites for Ca2+ binding proteins and phosphatidylinositol phosphates on intracellular termini of TRP channels
Boušová, Kristýna ; Teisinger, Jan (advisor) ; Žáčková, Markéta (referee) ; Krůšek, Jan (referee)
This dissertation concerns with characterization of binding sites for calcium binding protein S100A1 and phosphatidylinositol phosphates on intracellular regions of transient receptor potential channels (TRPs), particular from canonical (TRPC), vaniloid (TRPV) and melastatin (TRPM) families. TRPs represent superfamily of important mediators that play critical roles in sensory physiology: contributions to taste, olfaction, vision, hearing, touch and thermo- and osmo- sensation. They serve as non-selective and nociceptive membrane receptors responsible for the modulation of driving force for cations entry into the cell. TRPs are composed from six transmembrane domains and N- and C- termini intracellular regions. Overall four monomer units form a characteristic assembly of functional channel. It was demonstrated that most of this almost thirty-member family transporters are activated by a variety of different stimuli and function as signal integrators. The most examined intracellular TRPs modulators are cytosolic calcium binding proteins and membrane anchored phosphatidylinositol phosphates (PIPs). These signal integrators bind specific domains in intracellular termini of TRPs, thereby change their structure and activate or inhibit the transportation function of receptor. To identify a novel ligand...
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Study of the structure of 14-3-3:phosducin complex
Kacířová, Miroslava ; Obšil, Tomáš (advisor) ; Teisinger, Jan (referee)
The aim of this diploma thesis is a biophysical characterization of the protein complex that consists of two regulatory proteins, the phosducin (Pd) and the 14-3-3 protein. These proteins are involved in the regulation of a signal cascade in vertebrate eye's retina. Pd is a 33kDa protein located in photoreceptor cells in retina, but it has been found in other tissues as well. In retina, phosducin affects transfer of light signaling from eye to brain, by binding Gtβγ subunit of transducin that is the main part of G-protein signaling. In light-adapted retina, unphosphorylated phosducin down-regulates the light response by binding to Gtβγ. This process is important for protecting retina in eyes in response to very intense light. It has also been found that phosducin affects hypertension. Phosducin reduces blood pressure of human and mice, especially during sleep. The function of phosducin is regulated in dark-adapted retina by 14-3-3. 14-3-3 is a 28kDa protein that has been found in many eukaryotic tissues, e.g. brain, and is involved in many processes, e.g. apoptosis. The 14-3-3 protein binds phosphorylated Pd and keeps him in a rod inner segment. For 14-3-3 to Pd binding, two sites on Pd must be phosphorylated, Ser54 and Ser73. This interaction, hinders Pd binding to Gtβγ, and hence enables the...
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Effect of cholesterol depletion on signalling cascade initiated with receptors coupled to G protein class Gq/G11
Ostašov, Pavel ; Svoboda, Petr (advisor) ; Teisinger, Jan (referee) ; Hof, Martin (referee)
Membrane domains are an important structure in plasamatic membrane. They concentrate various signaling molecules. Their main structural component is cholesterol and by its removal the membrane domains are disrupted. The aim of our work was to examine the effect of cholesterol depeletion on signaling initiated thyreothropin releasing hormone (TRH). Although its signaling cascade is located within membrane domains the receptor itself is not. We showed that cholesterol depletion by -cyclodextrin caused release of Gq/11 proteins and caveolin 2 from membrane domains. We also discovered that cholesterol depletion decreases potency of TRH to activate G proteins as well as induction of release of intracellular Ca2+ In the last part we investigated the effect of disruption of the cell membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility and internalization in HEK293 cells stably expressing TRH-R-eGFP fusion protein. CLSM studies indicated that the internalization of receptor molecules initiated by TRH stimulation was significantly attenuated. The detailed analysis of recovery of TRH-R-eGFP fluorescence in bleached spots of different sizes indicated that cholesterol depletion results in an increase of overall receptor mobility. We suggest that migration of...
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