National Repository of Grey Literature 26 records found  1 - 10nextend  jump to record: Search took 0.00 seconds. 
Development and implementation of new approaches for proteomic characterization of bone tissues in oral surgery
Michalus, Iva ; Hynek, Radovan (advisor) ; Šebela, Marek (referee) ; Hrabal, Richard (referee)
1 Abstract Proteomics is a booming field with application in many areas of medicine, including dentistry. Nevertheless, proteomic characterization of bone tissues in oral surgery is not still commonly used. The main reason is involvement of demanding analytical approaches due to insoluble chatacter of bone tissues. The goal of this work was to develop and apply straightforward methodology that could lead to the routine use of proteomics in this area as well. Using porcine jawbones as a model samples, a technique was developed allowing identifying about hundreds of proteins thanks to their trypsin digestion directly in bone tissues ("in-bone digestion") followed by analysis using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This technique was subsequently applied to the analysis of human maxillary and mandibular bone tissues obtained during surgical procedures. In both maxillary and mandibular bone samples, it was possible to identify a considerable amount of proteins using the "in-bone digestion" technique. Additionaly, the mathematical analysis of the obtained data was able to distinguish between the inflammatory and healthy tissues. The approach based on direct cleavage was subsequently successfully extended to the analysis of in vitro models of human bone tissues. Direct...
Development of new methodology for accurate determination of molecular geometry
Socha, Ondřej ; Dračínský, Martin (advisor) ; Munzarová, Markéta (referee) ; Hrabal, Richard (referee)
This thesis employs the combined strengths of Nuclear Magnetic Resonance (NMR) and computational methodologies to investigate chemical phenomena that influence molec- ular structure across the solid, liquid, and gas states. In the solid phase, the research focuses on chlorine quadrupole couplings, and their use in structure analysis, highlight- ing the advantages of NMR in conjunction with Density Functional Theory for data interpretation. In the liquid state, the tautomeric conversion of isocytosine is examined, underlining the role of NMR in analyzing intricate chemical exchanges. The gas phase study explores the dimerization of acetic acid using NMR, addressing previous inconsis- tencies and evaluating calculations of molecular structure and properties. The combined use of NMR and computational techniques has been instrumental in resolving existing discrepancies and providing a deeper understanding of these chemical systems. The find- ings underscore the importance of careful data interpretation and emphasise the strengths and constraints of the combined methodologies. 1
Structural Interpretation of NMR Spectroscopic Parameters in Nucleic Acids
Fukal, Jiří ; Sychrovský, Vladimír (advisor) ; Hrabal, Richard (referee) ; Munzarová, Markéta (referee)
Title: Structural Interpretation of NMR Spectroscopic Parameters in Nucleic Acids Author: Mgr. Jiří Fukal Supervisor: Dr. Vladimír Sychrovský Institutions: Charles University, Faculty of Mathematics and Physics, and Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry Abstract: Nuclear Magnetic Resonance (NMR) is versatile spectroscopy technique applicable to determination of both chemical and 3D structure of molecules. Elementary characterization of peptides, proteins and nucleic acids employs the 1 H and 13 C NMR measurements. Probing of 15 N and 17 O atoms usually requires advanced chemical preparation of molecules (labelling with relevant isotopes). 31 P NMR is in that regard exceptional due to 100% natural abundance of the isotope. Hence, NMR structural studies of a nucleic acid's backbone are aimed at the phosphate group. Pertinent spectral parameters include δ31P NMR shift and J-couplings involving the phosphorus atom (e.g. 3 JP,H3'). Structural interpretation of NMR spectra requires usage of adequate quantum chemistry method (QM method) and inclusion of effects due to molecular dynamic (MD). That is why plausible interpretation of the spectral parameters in nucleic acids may become computationally demanding. The benchmark of QM methodology for 31 P NMR calculation made possible...
Characterization of protein-protein interactions between Forkhead box O (FOXO) and p53 transcription factors
Mandal, Raju ; Obšil, Tomáš (advisor) ; Hrabal, Richard (referee) ; Pavlíček, Jiří (referee)
The transcription factor p53 plays a key role in cell cycle arrest, DNA repair, apoptosis, tumor suppression, and maintaining cellular homeostasis. Under cellular stress, p53 directly interacts with the Forkhead box O (FOXO) 4 transcription factor, thereby upregulating the expression of the p21 gene, resulting in the induction of cellular senescence. However, the detailed molecular mechanism behind FOXO4-p53 interaction remains unclear due to the unavailability of structural data. Therefore, main goal of this doctoral thesis was the characterization of the interaction between FOXO4 and p53 using several biophysical techniques including sedimentation velocity analytical ultracentrifugation (SV AUC), nuclear magnetic resonance (NMR) spectroscopy and chemical cross-linking coupled to mass spectrometry. Furthermore, we also investigated the DNA binding properties of both proteins with their respective consensus DNA sequences in the presence or absence of their binding partners by fluorescence anisotropy measurements along with the comparison of p53-binding surfaces of the Forkhead domain of three different FOXO proteins by NMR spectroscopy. In addition, we also optimized small molecule compounds for the inhibition of FOXO3-DNA interaction. Our results revealed that the p53 interacts with FOXO4 through...
Determination of structure and dynamics of biomolecules by theoretical calculations of NMR spectroscopic parameters
Benda, Ladislav ; Sychrovský, Vladimír (advisor) ; Munzarová, Markéta (referee) ; Hrabal, Richard (referee)
iv Abstract Subject: Determination of structure and dynamics of biomolecules by theoretical calcu- lations of NMR spectroscopic parameters Author: Ladislav Benda, ladislav.benda@gmail.com Department/Institute: Institute of Organic Chemistry and Biochemistry, AS CR Supervisor: Dr. Vladim'ır Sychrovsk'y, Institute of Organic Chemistry and Biochemistry, AS CR, vladimir.sychrovsky@uochb.cas.cz Abstract: This doctoral work was focused on theoretical modeling of nuclear magnetic resonance (NMR) parameters in peptides and nucleic acids. Dependences of NMR para- meters on molecular structure and solvation were primarily modeled. Great emphasis was put on the comparison of the calculated data with the NMR experiment. The molecular models studied included the l-alanyl-l-alanine di-peptide (AA) and the phosphate group of nucleic acid backbone. Conformations of all three charged forms of AA in solution were determined and the respective pH-induced changes of experimental NMR chemical shifts and nuclear spin-spin coupling constants were explained. Dependences of NMR cross-correlated relaxation rates on the AA backbone geometry were calibrated. The 31 P NMR parameters in nucleic acid phosphate were systematically calculated in dependence on the backbone conformation and the phosphate solvation pattern. Qualitative rules...
Měření C-13 NMR relaxací karbonylových skupin v proteinech
Grocký, Marián ; Hrabal, Richard (referee)
Title: Measurement of 13 C NMR relaxation of Carbonyl Groups in Proteins Author: Marián Grocký Department: Department of Low Temperature Physics Supervisor: RNDr. Jan Lang, PhD. Supervisor's email address: Jan.Lang@mff.cuni.cz Abstract: I have investigated measurement methods of 13 C NMR longitudial and transversal relaxation constants in proteins labeled by 13 C, 15 N. The Mason-Pfizer Matrix protein (M-PMV) has been studied in it's wild form (WT) and mutated form (R55F) by these methods in order to reveal its dynamics. It has been known that point mutation of arginine for phenylalanine at the 55th position of matrix protein changes virus's life cycle. Reproduction of M-PMV than resembles HIV virus reproduction. Nitrogen T1, T2 and NOE relaxation experiments have also been carried out. All relaxation data were used for Lipari-Szabó model-free approach in order to get and compare carbonyl and amide group motion parameters. There has been found a high similarity between carbonyl and nitrogen motion parameters within WT protein. Nitrogen data of R55F protein approved higher mobility of particular sequences in that protein, which is assumed to be responsible for changes in mutant's life cycle. Both carbonyl and nitrogen data significantly enlarged our current knowledge of dynamics of both WT and R55F...
Synthesis and characterization of the new insulin analogues with the aim to clarify the interaction of insulin with its receptor
Kletvíková, Emília ; Jiráček, Jiří (advisor) ; Obšil, Tomáš (referee) ; Hrabal, Richard (referee)
The objective of this thesis is to characterize insulin analogues modified at the C-terminus of the B-chain with the aim to observe the impact of the inserted modifications on the insulin-insulin receptor (IR) interaction and the ability of the analogues to dimerize. Therefore, a series of analogues with modifications at B24-B26 positions was prepared. Using the synthetic and semisynthetic methods we inserted coded and non-coded amino acids to this part of B-chain. We studied full-length analogues and analogues truncated by three to four amino acids. Binding affinity of all analogues to the insulin receptor was determined by competition of analogue with radioactive (125I) human insulin. Dissociation constant in the dimer dissociation process of selected analogues (especially of those with N- methylation of B23-B24, B24-B25 and B25-B26 peptide bonds) was determined by isothermal titration microcalorimetry. The crystal structures of several analogues were resolved by X-ray crystallography and nuclear magnetic resonance. The structural results showed the consequences of inserted modifications to the insulin molecule. We characterized analogues with higher, equipotent and lower binding affinity to the IR. The results...
Structure and interactions of selected forkhead transcription factors
Kohoutová, Klára ; Obšil, Tomáš (advisor) ; Hrabal, Richard (referee)
This diploma thesis is a part of a project aiming to develop and study specific inhibitors of FOXO3 transcriptional activity. FOXO3 belongs together with FOXO1, FOXO4 and FOXO6 to FOXO subfamily of forkhead family transcription factors. FOXO transcription factors are evolutionary conserved proteins playing important roles in numerous cellular processes, such as apoptosis, cell cycle regulation and metabolism. Due to their ability to induce apoptosis and to block the cell cycle they are considered tumor suppresors. However, it has been shown that increased activity of FOXO proteins is connected with many kinds of cancer. In such cases FOXO proteins function to maintain cell homeostasis. They promote tumor resistance against chemotherapy as well as they speed up its growth. The aim of this project is to develop specific inhibitors able to bind to FOXO3 DNA-binding domain (DBD, residues 156-269) and to block its interaction with target DNA. Development of specific inhibitors of FOXO3 transcriptional programme requires knowledge of solution structure of all FOXO DBDs and detailed insight into their interaction with target DNA. So far crystal structures of complexes of FOXO1, FOXO3 and FOXO4 with target DNA and solution NMR structures of apo DBDs of FOXO3 and FOXO4 have been solved. One of the goals of...
Structural study of S6A mutant of matrix protein from Mason-Pfizer monkey virus
Kamal, Rašíd ; Hrabal, Richard (advisor) ; Ulbrich, Pavel (referee)
Mason-Pfizer monkey virus (M-PMV) belongs to the morphogenetic type D retroviruses which produce viral particles in the cytoplasm. This process is temporally and spatially separated from the cytoplasmic membrane budding and therefore M-PMV is a suitable model organism for studying the life cycle of retroviruses. Matrix protein, which is N-terminal portion of Gag polyprotein precursor, plays a central role in this life cycle. One of the key areas of the protein is its myristoylated N-terminus which is particularly important for binding to the cytoplasmic membrane during budding from host cells. A hydrogen bond exists between serine 6 and glutamate 9 of the wild-type protein. We have studied its influence on the structure and molecular mechanics of the corresponding area of the protein by means of disruption of this interaction by replacing serine 6 with alanine. This thesis describes the preparation of recombinant mutant of the M-PMV matrix protein, resonance assignment of its backbone atoms with nuclear magnetic resonance spectroscopy and compares observed secondary structure with that of other mutants and the wild type.
Interactions in solutions and gels of stimuli-responsive polymer systems investigated by NMR spectroscopy
Konefał, Rafał ; Spěváček, Jiří (advisor) ; Hrabal, Richard (referee) ; Štěpánek, Miroslav (referee)
Stimuli-responsive (stimuli-sensitive, intelligent, or smart) polymers are polymer materials which, after small external stimuli, evidently change their physical or chemical properties. Smart polymers can be classified according stimuli they respond to such as: temperature changes, mechanical stress, light irradiation, ultrasonic treatment, application of external magnetic as well as electric field, changes of pH, ionic strength, addition of the chemical agents and presence of biomolecules and bioactive molecules. Stimuli-responsive synthetic polymer systems has attracted considerable attention due to wide range of applications, i.e. controlled drug delivery and release systems, diagnostics, tissue engineering and 'smart' optical systems, as well as biosensors, microelectromechanical systems, coatings, and textiles. Among the types of stimuli for this dissertation temperature, pH and reactive oxygen species (ROS) responsive polymer systems were studied. In case of thermoresponsive polymers, when polymer chains are molecularly dissolved in a good solvent, changes (increasing or decreasing) of temperature result in insolubility (globular nanoparticles formation) of polymer chains, called temperature induced phase-separation. pH responsive polymers change properties such as: solubility, volume (gels),...

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