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Influence of freezing and thawing process on cryopreserved cells nuclei and surfaces. Functions and physico-chemical properties of cryoprotectants.
Golan, Martin ; Kratochvílová, Irena (advisor) ; Raška, Milan (referee) ; Schneider, Bohdan (referee)
1 Abstract: Cryopreservation of cells is a complex process with many useful applications in basic biological research, medicine and agriculture. In this work we deepened the current understanding of the cryopreservation process both at physical and biological level. Results include characteristics of selected cryoprotectants (primarily DMSO, trehalose, antifreeze protein ApAFP752) in liquid phase, during phase transition and in solid phase, as well as their impact on cryopreserved cells states. Specifically, the level of cell viability, state of cell membrane and condition of cell nucleus (nuclear membrane, chromatin condensation, DNA strand breaks) are monitored over several time points after thawing. It is shown that S-phase cells (NHDF and MCF7 lines) suffer massive collapse of replication forks during cryopreservation which makes them much less suitable for cryopreservation than cells in other phases of the cell cycle. Several methods (most importantly Atomic Force Microscopy, Confocal Fluorescence Microscopy and Flow Cytometry) were used to examine the post-thaw state of cryopreserved cells. The acquired insights into cryodamage of cells can lead to optimization of current cryopreservation protocols and to more thorough evaluation of efficacy of future novel cryoprotectants.
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Study of regulatory mechanisms of selected protein kinases
Petrvalská, Olívia ; Obšil, Tomáš (advisor) ; Jiráček, Jiří (referee) ; Schneider, Bohdan (referee)
Through binding interactions with more than 300 binding partners, 14-3-3 proteins regulate large amount of biologically relevant processes, such as apoptosis, cell cycle progression, signal transduction or metabolic pathways. The research discussed in this dissertation thesis was focussed on investigating the role of 14-3-3 proteins in the regulation of two selected protein kinases ASK1 and CaMKK2. The main goal was to elucidate the mechanisms by which phosphorylation and 14-3-3 binding regulate functions of these protein kinases using various biochemical and biophysical methods, such as site-directed mutagenesis, enzyme activity measurements, analytical ultracentrifugation, small-angle X-ray scattering, chemical crosslinking, nuclear magnetic resonance and fluorescence spectroscopy. A structural model of the complex between the catalytic domain of protein kinase ASK1 with 14-3-3ζ, which was calculated using the small-angle X-ray scattering and chemical crosslinking data, suggested that this complex is conformationally heterogeneous in solution. This structural model together with data from time-resolved fluorescence and nuclear magnetic resonance suggested that the 14-3-3ζ protein interacts with the catalytic domain of ASK1 in the close vicinity of its active site, thus indicating that the complex...
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Bioinformatic methods of detection of protein coevolution
Pařízková, Hana ; Schneider, Bohdan (advisor) ; Hampl, Vladimír (referee)
The term coevolution describes the situation when two or more species or biomole- cules reciprocally affect each others' evolution. On the protein level, it is thought to be the main mechanism ensuring correct folding, interactions and function of a protein, and it can be observed both on the level of interacting protein families and individual amino acid residues. Coevolution studies have been proved to be a powerful tool for prediction of protein structure, function, interaction partners, etc. In this thesis, different algorithms used for detection of protein coevolution are described, as well as their applications and limitations. Keywords: coevolution, protein family, protein structure prediction, interac- tion partners, correlated mutations, mirrortree, mutual information, direct cou- pling analysis
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Molecular mechanisms and functions of 14-3-3 proteins
Šilhán, Jan ; Obšil, Tomáš (advisor) ; Krůšek, Jan (referee) ; Schneider, Bohdan (referee)
Závěr Hlavním cílem této doktorské práce bylo objasnění molekulárních mechanismů funkce 14-3-3 proteinů a vlivu na proteiny FOXO4 a tyrosinhydroxylasu. V první časti této práce (publikace I) byla potvrzena předložená hypotéza polohy Cterminálního konce molekuly 14-3-3. Bylo ukázáno, že v nepřítomnosti ligandu se Cterminální konec nachází ve vazebném místě a brání tak vstupu ligandů. Po vazbě fosforylovaných ligandů, dochází k velmi silné vazbě a vytěsnění C-terminálního konce 14-3- 3 proteinu z vazebného místa. Tyto výsledky jsou v souladu s původními pracemi, které navrhly důležitost tohoto segmentu jako inhibitoru nepatřičných ligandů. Druhá část této doktorské práce poskytuje rozsáhlý popis vlivu 14-3-3 proteinů na transkripční faktory FOXO4. S použitím stacionární a časově-rozlišené fluorescence byla studována interakce 14-3-3 proteinu s fosforylovaným transkripčním faktorem FOXO4. Navázání 14-3-3 proteinu způsobuje rozpad komplexu FOXO4:DNA. Tato část práce charakterizuje interakci 14-3-3 proteinu s DNA-vazebnou doménou FOXO4. Výsledky neprokázaly výrazné konformační změny v rámci DNA-vazebné domény. Spíše dochází ke sterickému bránění vazby DNA (publikace II). Ve třetí části se práce zabývá studiem interakcí 14-3-3 s fosforylovaným ligandem odvozeným od C-konce enzymu serotonin N-acetyltransferasa...
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Molecular mechanisms of regulation of forkhead transcription factor FOXO4
Bouřa, Evžen ; Obšil, Tomáš (advisor) ; Konvalinka, Jan (referee) ; Schneider, Bohdan (referee)
I 7. Abstract The main goal of this PhD thesis was to investigate the structure of FOXO4-DBD/DNA complex and the molecular mechanism of FOXO4 DNA binding properties. Especially, the role of protein kinase B phosphorylation and the regulatory role of 14-3-3 proteins. This work has been published in four original papers (for full citation see page no. 3). Paper l: The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation- dependent manner. 14-3-3 proteins are thought to play a direct role in the regulation of subcellular localization of FoxO forkhead transcription factors. It has been suggested that the interaction with the 14-3-3 protein affects FoxO binding to the target DNA and interferes with the function of nuclear localization sequence (NLS). Masking or obscuring of NLS could inhibit interaction between FoxO factors and nuclear importing machinery and thus shift the equilibrium of Foxo localization toward the cýoplasm. According to our best knowledge, there is no experimental evidence showing a direct interaction between the 14-3-3 protein and NLS of FoxO. Therefore, the main goal of this work was to investigate whether the phosphorylation by protein kinase B, the 14-3-3 protein, and DNA binding affect the structure of FoxO4 NLS. We have...
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Coevolution of cytokines from the interleukin 10 family and their receptors
Harazim, Markéta ; Schneider, Bohdan (advisor) ; Novotný, Marian (referee)
Interleukin 10 family (FIL-10) is an important family of cytokines triggering immune response of different outcome, from antiinflammatory factor interleukin (IL) 10 through epithelia related subfamily of IL-19, IL-20 and IL-24, to IL-22 and IL-26 with role in infection immunity. The family is closely related to interferons (IFNs), several of which (Interferon λs) are commonly placed into FIL-10 for its functional and structural similarities with FIL-10 proteins. FIL-10 interleukins share several receptors, which in different combinations of receptors and interleukin result in different immune response. As the family proteins are expressed in as evolutionary old taxa as cartilaginous fish, we presumed a coevolution in the protein family and the corresponding receptors would be detectable in the sequences of genes and subsequently proteins of FIL-10. Using statistical methods, evolutionary relations within the group of FIL-10 and group of their receptors were resolved. Coevolution of the cytokine-receptor combination in FIL-10 was detected in some of the expected cases. IL10RB seems to be consistently in coevolution with IL-10. Evolution of IL20RB receptor, which is shared within the group by IL-19, IL-20 and IL-24, is directed by IL-20 evolution, coevolution with IL-19 and IL-24 is in most cases...
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Coevolution of cytokines from the interleukin 10 family and their receptors
Harazim, Markéta ; Schneider, Bohdan (advisor) ; Novotný, Marian (referee)
Interleukin 10 family (FIL-10) is an important family of cytokines triggering immune response of different outcome, from antiinflammatory factor interleukin (IL) 10 through epithelia related subfamily of IL-19, IL-20 and IL-24, to IL-22 and IL-26 with role in infection immunity. The family is closely related to interferons (IFNs), several of which (IFN s, and in this study alsoλ IFN ) are commonly placed into FIL-10 for its functional and structural similarities with FIL-10γ proteins. FIL-10 interleukins share several receptor subunits, which in different combinations of receptors and interleukin bound result in different immune response. As the family proteins are expressed in as evolutionary old taxa as cartilaginous fish, we presumed a coevolution in the protein family and the corresponding receptors would be detectable in the sequences of genes and subsequently proteins of FIL-10. Using statistical and structure biological methods, evolutionary relations within the group of FIL-10 and group of their receptors were resolved, with notable division of IFN into independent groups of fish and the later vertebrates. Coevolutionγ of the ligand receptor combination in FIL-10 was detected in most cases, with exception of some interactions of IL10RB, the most widely used receptor subunit in the family,...
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Bioinformatic analysis of protein/DNA interactions
Božíková, Paulína ; Schneider, Bohdan (advisor) ; Hašek, Jindřich (referee)
In this thesis, we focused on local structural features of the DNA backbone in protein-complexed DNA and non-complexed (naked) DNA, and its dependence on types of a base pairing in DNA, and on the base sequence. To reach this goal we analyzed about 1,400 crystal structures of DNA in complexes with proteins and more than 400 crystal structures of naked DNA. DNA local conformations were structurally classified into 38 dinucleotide conformers ntCs, which were described previously (Svozil et al. Nucleic Acids Res. 2008). The ntC were further clustered into 16 structural alphabet classes ntA to reduce the number of analyzed variables. We assembled base-paired dinucleotides from double helical DNA structures accord- ing to their assigned structural alphabet classes into so called Association matrices. Three basic Association matrices were analyzed; two compare ntA/ntA associations between dinucleotides forming only Watson-Crick base pairs in protein/DNA com- plexes and in naked DNA, respectively; the third one ntA/ntA associations between dinucleotides base-paired also by non-Watson-Crick pairs. We also analyzed As- sociation matrices of dinucleotides as a function of their sequences. The analyzes revealed differences in structural behavior of various ntA and their dependence on dinucleotide sequences.
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Modelling mechanical properties of RNA and DNA
Dršata, Tomáš ; Lankaš, Filip (advisor) ; Banáš, Pavel (referee) ; Schneider, Bohdan (referee)
Structural and mechanical properties of nucleic acids play a key role in a wide range of biological processes, as well as in the field of nucleic acid nanotechnology. The thesis presents results of several studies focused on modelling these properties. Extensive unrestrained atomic-resolution molecular dynamics (MD) simulations are used to investigate structural dynamics of nucleic acids, and to parametrize their mechanical models. The deformation energy is assumed to be a general quadratic function of suitably chosen internal coordinates. Two types of models are employed which differ in the level of coarse- graining. The first one is based on the description of conformation at the level of individual bases and the second, coarser one is used to study global bending and twisting flexibility. The models are applied to explain mechanical properties of A-tracts in the context of DNA looping and nucleosome positioning, to characterize twist-stretch cou- pled deformations in DNA and RNA, and to predict changes in the properties of damaged DNA that are likely to be relevant for damage recognition and repair. Besides that, we propose a general model of DNA allostery, applied to study the effect of minor groove binding of small ligands and the allosteric coupling between proteins mediated by the DNA. A careful...
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Increasing affinity of Interferon gamma receptor 1 to Interferon gamma by combining molecular modeling and experimental methods
Mikulecký, Pavel ; Schneider, Bohdan (advisor) ; Šulc, Miroslav (referee) ; Vaněk, Ondřej (referee)
Protein-protein interactions play an important role in nearly all processes of the living cells and the function of many proteins is dependent on their specific interactions with other biomolecules. A reliable tool to modulate these interactions would be invaluable for the development of molecules suitable for diagnostics, medicine, and biotechnology. In this work, we aimed to study the specificity of interactions in the model system of Interferon gamma receptor 1 (IFNgR1) and its natural ligand Interferon gamma (IFNg), important in innate immunity. We searched for mutations within the interferon receptor molecule IFNgR1 to modulate (increase as well as decrease) its affinity to IFNg by in silico analysis of the existing crystal structures of the complex between IFNgR1 and IFNg. We modeled amino acid substitutions and gauged how they influenced the interaction using empirical force field implemented in software FoldX. All selected promising IFNgR1 variants were expressed in Escherichia coli, purified to homogeneity, characterized, and kinetics of their interactions with IFNg was measured by Surface Plasmon Resonance (SPR). The first set of IFNgR1 variants included mutations on the interface of the IFNg/IFNgR1 complex. According to our SPR measurements, the affinity of most of these receptor...
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