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Molecular basis of endothelial sysfunction: endothelial nitric oxide synthase and heme oxygenase 1 genetic variations
Král, Aleš ; Martásek, Pavel (advisor) ; Baxová, Alice (referee) ; Schneider, Bohdan (referee)
Endothelial dysfunction is a pathologic state characterized by an altered equilibrium among vasodilatory and antithrombotic mediators and vasoconstrictive and prothrombotic mediators produced by the vascular endothelium. Multiple factors induce impaired production or increased consumption nitric oxide (NO), the key mediator of vascular homeostasis, produced by the nitric oxide synthase enzymes (NOS). Endothelial dysfunction represents one of the initial steps in the development of atherosclerosis, a chronic inflammatory disease of the vascular wall. The inducible enzyme heme oxygenase 1 (HO-1) represents one of the main cellular defense mechanisms against increased oxidative stress and decreased NO bioavailability accompanying endothelial dysfunction and atherosclerosis. We studied the genetic determinants of endothelial dysfunction and atherosclerosis by evaluating the association of the G894T endothelial NOS (eNOS) polymorphism and the HO-1 (GT)n promoter polymorphism with coronary artery atherosclerosis severity and risk profile and their evolution during hypolipidaemic treatment. In addition, we searched for genetic variations in exons 25 and 26 of eNOS gene, encoding the C-terminal part of the protein, deemed crucial for proper enzyme function and the 3'- untranslated region crucial for eNOS...
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Molecular pathology of selected inherited hyperbilirubinemias
Šlachtová, Lenka ; Martásek, Pavel (advisor) ; Schneider, Bohdan (referee) ; Králová, Jarmila (referee)
Inherited hyperbilirubinemias are a group of metabolic disorders, characterized by increased levels of total serum bilirubin or its conjugated fraction. Most of these hyperbilirubinemias are inherited autosomal recessively and are manifested in young age. Increased bilirubin reflects the genetic disturbances in one of the enzymes of heme degradation pathway, the defect of bilirubin conjugation (UGT1A1 gene) or its transport (ABCC2, OATP1B1, OATP1B3). All of these proteins are involved not only in elimination of bilirubin, but various substrates; therefore the performed studies have a great pharmacogenomics impact. We have studied the molecular pathology of hereditary hyperbilirubinemias in Caucasian and Roma population and to compare the clinical and biochemical results with the molecular genetic data. We described the impact of compound defect of c.-3279T>G and g.175492_175493insTA on total serum bilirubin and calculated the linkage disequlibrium of these two variants in promoter region of UGT1A1 gene. We also verified, that the population distribution of both variants is in concordance with the literature. In our second study, we have described the rare conjugated hyperbilirubinemia Dubin-Johnson type among 7 Roma families. We have found a novel variant NG_011798.1:c.[1013_1014delTG] together with...
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Study of structural features of single stranded DNA by biophysical techniques and crystallography
Svoboda, Jakub ; Schneider, Bohdan (advisor) ; Pavlíček, Jiří (referee)
DNA is the fundamental molecule in all domains of life, its role in heredity is well established. Although the famous double helical complementary form is indispensable for replication mechanism DNA can occupy wide range of conformations. In the past studies performed in the laboratory, DNA oligomers related to single stranded bacterial Repetitive Extragenic Palindromic (REP) showed spectral behavior suggesting complex equilibria including double helical, hairpin, and tetraplex conformations. The studies presented in this thesis extended the scope of analyzed sequences and employed circular dichroism spectroscopy and X-ray crystallography. We report spectral data and X-ray structures of three successfully crystalized oligonucleotides. All three structures acquire double helical architecture with two consecutive T- T mismatches in the center. To improve the convergence of the refinement process of the crystal structures we used novel dinucleotide conformational classes, NtC classes. The NtC class classification was also used to analyze geometries of selected non-canonical base pairs in all DNA crystal structures in the Protein Data Bank. We measured the fit between geometries of the dinucleotides involved in the non-canonical base pairing and the NtC classes and correlated this fit to the electron...
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Modulation of interactions between interleukins and their receptors
Nepokojová, Tereza ; Schneider, Bohdan (advisor) ; Obšilová, Veronika (referee)
Scaffolds are proteins with high conformational stability, allowing us to implement multiple mutations into specific parts of the protein. Even with these mutations, the structural integrity of the protein is maintained as well as its physical-chemical properties. These mutations give the specific scaffold new properties. In most cases it is the binding specificity towards previously chosen target. The biggest advantages of scaffolds are their small size, stability, low-cost manufacturing, and easiness of preparation. Scaffold utilized in this thesis is unique for having two binging surfaces designed on which it can be mutated. Each of those two surfaces can be separately mutated to develop a binging site for two different proteins. In our case these mutations led to binding two nonidentical receptors of a human cytokine. Mutations are made with a use of yeast display, one of the methods of directed evolution. The main focus of this thesis is changing an expression system of the binding proteins from the yeast system to a bacterial one, their production and purification followed by characterization of those binding proteins using biophysical methods. These methods were used to evaluate structural and thermal stability, and binding affinity to both receptors of the beforementioned binding proteins....
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Study of DNA hydration by analysis of structural data from databases
Šrůtková, Alžběta ; Schneider, Bohdan (advisor) ; Hoksza, David (referee)
The deoxyribonucleic acids play an essential role in the living organisms by storing the genetic information. The process of the genetic information transfer, translation and transcription is carried out by other molecules, mostly proteins, which cause DNA bend and locally deform. These protein-DNA interactions, together with the nucleotide sequence, result in different DNA conformations. The description and classification of the DNA local structural diversity at a level of dinucleotides is provided by so-called dinucleotide conformer classes, NtC. The NtC classification system developed for description of the local conformations of nucleic acids consists of 97 NtC classes. In this work we use 44 classes important for the description of DNA conformations. All biomolecules, including DNA are heavily hydrated. The first hydration layer around DNA is largely localized. The structure of this hydration layer depends on the sequence and conformation of the analyzed biomolecule. The DNA structural classification by the NtC classes allows specific study of the first hydration layer with a sufficient sequence and structural granularity. Based on the data available in the structural databases, we studied hydration of double helical tetranucleotide fragments in the three biologically most important DNA conformational...
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Study of single stranded DNA by biophysical techniques
Svoboda, Jakub ; Schneider, Bohdan (advisor) ; Novák, Petr (referee)
DNA is the fundamental functional molecule of all domains of life. One of its characteristics is the ability to self-associate to form a double stranded helix. Nevertheless, even single stranded DNA can form non-canonical structures such as hairpins, triplexes or tetraplexes. One of the interesting single stranded sequences are REP elements. These sequences form a part of bacterial transposable elements, so-called insertion sequences, which can be found in a great number of copies in wide range of bacterial species. This thesis studies structure and properties of selected REP sequences. It presents the results of spectral measurements by technique of circular dichroism, melting curves from differential scanning calorimetry, and monocrystal diffraction data. Key words Single-stranded DNA, crystallography, circular dichroism, calorimetry, transpozone, REP elements, insertion sequences.
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Targeted modifications of the protein-protein interactions: Ternary complex of interferon-γ as a model system
Zahradník, Jiří ; Schneider, Bohdan (advisor) ; Obšilová, Veronika (referee) ; Vaněk, Ondřej (referee)
A key prerequisite for a deeper understanding of biological processes at molecular level is a detailed description of the three-dimensional structure of interaction partners and their complexes. We adopted the IFN-γ complex as our model system. Even though IFN-γ is one of the key modulators of the immunity response, which has been studied intensively for more than 60 years, the structure of the accessory receptor chain and the understanding of the IFN-γ complex is still lacking. In this work we firstly discussed the binary system between IFN-γ and its high affinity receptor R1 which is structurally known. Using a new innovative methodology we focused on the modulation of the affinity between IFN-γ and its receptor R1. Our approach was based on the modulation of protein - protein stability by mutating cavities in the proteins' structure and increasing the affinity about seven-fold. Secondly, we crystallized and solved the structure of the IFN-γ receptor 2, the accessory receptor molecule. Our analysis of variable residues on the surface of the structures of type II family receptors, to which receptor 2 belongs, revealed the putative binding site for IFN-γ. In the third part of our work, we crystallized IFN-γ from olive flounder Paralichthys olivaceus and solved its structure at 2.3 Å resolution (PDB...
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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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