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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...
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Development and applications of affinity carrier for isolation of human carbonyl-reducing enzymes
Andrýs, Rudolf ; Wsól, Vladimír (advisor) ; Šebela, Marek (referee) ; Šatínský, Dalibor (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Rudolf Andrýs Supervisor: Prof. Ing. Vladimír Wsól, Ph.D Title of dissertation thesis: DEVELOPMENT AND APPLICATION OF AFFINITY CARRIER FOR ISOLATION OF HUMAN CARBONYL-REDUCING ENZYMES For several millennia the human medicine is based on application of small bioactive molecules that are administered in the form of plant extracts or synthetic compounds. However, their use in modern medicine is not possible without a detailed understanding of their biochemical effects and identification of their molecular targets. Chemical proteomics based on the specific recognition between the bioactive molecule and the target molecule is currently the most widely used techniques for identification of molecular targets of small molecules. Compared to conventional biochemical methods (e.g. 2D electrophoresis), chemical proteomics represents particularly sensitive and very selective technique that enable successful identification of biomolecules from complex biological samples that are naturally presented in very small concentrations. Carbonyl- reducing enzymes, which play an important role in physiology due to their involvement in metabolism of various endogenous (e.g. prostaglandins, steroid...
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Molecular mechanisms of Diamond-Blackfan anemia
Handrková, Helena ; Petrák, Jiří (advisor) ; Šebela, Marek (referee) ; Trka, Jan (referee)
Diamond-Blackfan anemia (DBA) is a rare congenital syndrome that presents with ane- mia and selective deficiency of erythroid precursors, while other blood lineages are usu- ally unaffected. Approximately half of the patients display additional somatic anoma- lies and growth retardation. The therapy is mostly symptomatic and is dominated by corticosteroids, other modalities include regular blood transfusions or hematopoietic stem cell transplantation. At the beginning of this work, only two DBA causal genes were known, RPS19 and RPS24, being mutated in approximately 1/4 of all DBA patients. The goals of this work were to study the consequences of the known DBA causal mutations on cellular level and to find novel DBA causal genes. To date, over a half of DBA patients have been reported to carry a mutation in one of nine known DBA causal genes, including RPS17, RPL11 and RPL5, that are reported in this dissertation. All confirmed DBA causal genes encode for ribosomal proteins (RPs) that were essential for ribosome assembly. We further hypothesized a non- ribosomal protein participating in this process might be involved in DBA pathogenesis, too. In one DBA patient, we identified a rare sequence variant in one such candidate, a protein arginine methyltransferase 3 (PRMT3). We reported that the patient PRMT3...
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Structural characterization of biotechnologically and medicinally important proteins.
Filandr, František ; Man, Petr (advisor) ; Šebela, Marek (referee) ; Škultéty, Ľudovít (referee)
A large number of biological processes depends on dynamics of protein structure and specific protein-protein and protein-ligand interactions occurring under specific native conditions in or outside of cells. Standard methods for protein structure analysis like x-ray crystallography, nuclear magnetic resonance or cryo-EM are able to obtain important atomic or near- atomic resolution protein structures, however these are usually a static snapshot of protein locked in a specific conformation and mostly in non-native conditions. Structural mass spectrometry on the other hand, allows to describe protein structure dynamics, protein-protein and protein-ligand interactions and obtain inter- and intraprotein distance constraints between amino acid residues, all while working with proteins in their native conditions and needing only a fraction of sample. In this work, hydrogen/deuterium exchange mass spectrometry (HDX-MS) and classical proteomic approaches were used together with other methods to analyse biotechnologically important proteins of fungal cellulolytic system lytic polysaccharide monooxygenase (LPMO) and cellobiose dehydrogenase (CDH) as well as plant-derived photosensitizer protein LOV2 with potential use in biologically targeted photodynamic therapy. These methods allowed us to follow...
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Structure and dynamics of mouse C-type lectin-like receptors.
Wallenfels, Lucie ; Novák, Petr (advisor) ; Obšilová, Veronika (referee) ; Šebela, Marek (referee)
Natural killer (NK) cells represent indispensable part of the innate immunity as they are capable of promptly identifying virally infected or tumor cells and participating in the regulation of adaptive immune responses. These functions are ensured by the interplay between NK receptors, creating a complex regulatory system. Solving the receptors' structure may contribute to an overall understanding of NK cell biology. Presented thesis describes an elucidation of the structure of the inhibitory C-type lectin-like receptor (CTLR) Nkrp1b with an emphasis toward structural features (stalk, loop and oligomerization state) which might affect conformation or interactions of this receptor. The interaction of Nkrp1b with its ligand, Clr-b protein, is immunologically significant as it regulates NK cells' activity independently and monitors changes that are not visible to cytotoxic T lymphocytes. To study individual structural aspects of Nkrp1b, two protein variants were recombinantly prepared in bacterial expression system: entire ectodomain and ligand-binding domain lacking the stalk. Using a range of mass spectrometric techniques in combination with homology modeling and molecular dynamics, we proposed the Nkrp1b structure including its monomeric and dimeric arrangements. In addition, the oligomerization...
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Study of polyamine metabolism in cell division and their role in physiological plant processes
Gemperlová, Lenka ; Cvikrová, Milena (advisor) ; Šebela, Marek (referee) ; Havel, Ladislav (referee)
3. AIMS To study the role of PA biosynthesis,oxidation and conjugation in maintenanceof free intracellular PA levelsduring the growth and cell cycle of tobacco BY-2 cell culture (Nicotiana tabacum L.cv. Bright Yellow-2). Partial aims: o To describe alterations in the levels of free PA and PA conjugated with HCA during the growth and cell cycle oftobaccoBY-2 cell culture. o To charaďerjzechangesin the activities of PA bioqynthetic en4/mes(ADC, ODC and SAMDC) during the growth and cell cycle oftobacco BY-2 cell culture. o To detenrrinatechangesin the activiý of PA degradation en4fme (DAO) duringthegrowthandcell cycle of tobacco BY-2 cell culture. Following (next)aims: o To evaluatealterationsin the levels of intracellular free and conjugatedPA in tobacco BY-2 cell culture under Cd2*- inducedoxidativestress. o To studythemechanismsof PA homeostasis(biosynthesis, degradation and conjugation) during the diurnal cycle oftobacco plant(Nicotiana tabacumL. cv. Wisconsin 38). o To evaluate the role of excreted PA in PA homeostasis dwing the growth cycle of tobacco BY-2 and alfalfa (MedicagosativaL.) cell suspensionculture. o To comparechangesin the PA metabolismduring somatic and zygotic embryodevelopmentof Norway spruce(Picea abiesL. (Kars|). 7
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Study of conformations and conformational changes of proteins using mass spectrometric methods.
Kádek, Alan ; Man, Petr (advisor) ; Šebela, Marek (referee) ; Hnízda, Aleš (referee)
Mass spectrometry (MS) techniques have, over the last twenty years, found their stable place in the structural biology toolkit. They are not only employed to provide information on the protein primary sequence, but are increasingly used to probe higher orders of protein structure as well. They may not boast the atomic resolution and the ability to directly provide structural coordinates, but on the other hand suffer from very few experimental limitations as they are able to work under native conditions in solution, provide data fast, with low sample consumption and for proteins and complexes of vastly differing sizes. Perhaps most importantly, they may often be employed to study conformational dynamics of proteins and can thus complement other methods with higher spatial resolution in integrative structural biology approaches. The main focus of this Ph.D. thesis was hydrogen / deuterium exchange coupled to MS (HXMS), which is one of the most widespread structural MS methods. Recombinantly produced aspartic protease nepenthesin-1 from Nepenthes pitcher plants was characterized, immobilized and extensively tested with the intention to expand the portfolio of aspartic proteases in HXMS workflow and to improve the spatial resolution of the technique. Following successful implementation of nepenthesin-1...
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Development and applications of affinity carrier for isolation of human carbonyl-reducing enzymes
Andrýs, Rudolf ; Wsól, Vladimír (advisor) ; Šebela, Marek (referee) ; Šatínský, Dalibor (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Rudolf Andrýs Supervisor: Prof. Ing. Vladimír Wsól, Ph.D Title of dissertation thesis: DEVELOPMENT AND APPLICATION OF AFFINITY CARRIER FOR ISOLATION OF HUMAN CARBONYL-REDUCING ENZYMES For several millennia the human medicine is based on application of small bioactive molecules that are administered in the form of plant extracts or synthetic compounds. However, their use in modern medicine is not possible without a detailed understanding of their biochemical effects and identification of their molecular targets. Chemical proteomics based on the specific recognition between the bioactive molecule and the target molecule is currently the most widely used techniques for identification of molecular targets of small molecules. Compared to conventional biochemical methods (e.g. 2D electrophoresis), chemical proteomics represents particularly sensitive and very selective technique that enable successful identification of biomolecules from complex biological samples that are naturally presented in very small concentrations. Carbonyl- reducing enzymes, which play an important role in physiology due to their involvement in metabolism of various endogenous (e.g. prostaglandins, steroid...
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Cytochrome P-450: Study of structure and interactions using chemical modification, photo-initiated cross-linking and mass spectrometry
Ječmen, Tomáš ; Šulc, Miroslav (advisor) ; Petrák, Jiří (referee) ; Šebela, Marek (referee)
ABSTRACT Mixed function oxygenase system participates in biosynthesis of endogenous and metabolism of exogenous substances (e.g. drugs or chemical procarcinogens) in an organism. Substrates are biotransformed by terminal oxygenases - cytochromes P450 (P450). Catalytic properties of certain P450s (e.g. studied isoform 2B4) are altered in the presence of a redox partner - cytochrome b5 (cyb5). Both cytochromes are anchored by hydrophobic domains in a lipid membrane of endoplasmic reticulum whereas their catalytic domains are exposed to cytosol. Two zero-length cross-linking approaches were employed to extend present knowledge of P450 2B4 and cyb5 protein structure and protein-protein interactions: (1) interlinking of carboxylate and primary amine groups of amino acids by water soluble 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and (2) photo-initiated cross-linking by photo-labile methionine analog (pMet), which links to any amino acid after activation by UV-irradiation, either in hydrophilic or hydrophobic environment. pMet was incorporated to methionine site(s) of cyb5 during recombinant expression in E. coli, which was carried out in limit medium supplemented with amino acid analog. Optimization of experimental conditions led to ~20-30% substitution of the natural amino acid. Covalent...
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Human Membrane-bound Carbonyl Reductases
Štambergová, Hana ; Wsól, Vladimír (advisor) ; Wimmerová, Michaela (referee) ; Šebela, Marek (referee)
ABSTRACT Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Hana ŠTAMBERGOVÁ Supervisor prof. Ing. Vladimír WSÓL, Ph.D. Title of Doctoral Thesis Human membrane-bound carbonyl reductases Human membrane bound enzymes involved in the metabolism of carbonyl containing compounds consitute a highly interesting group of enzymes. Despite the great effort of scientists over the world most of them still remain uncharacterized, mainly those from the large short-chain dehydrogenases/reductases superfamily (SDR). At present, 75 SDR members have been indentified in the human genome whereas only 20 % of them is considered to be well characterized. On the other hand 30 % SDRs remain completly uncharacterized. SDR enzymes are involved in the metabolism of steroids, saccharides, retinoids or prostaglandins and therefore play a crucial role in a number of physiological pathways as well as several serious diseases (e.g. hormon dependent cancer, metabolic syndrome, diabetes mellitus). Moreover, SDR proteins contribute to the biotransformation of some xenobiotic compounds. Several SDR enzymes have been identified to be involved in the phase I metabolism of drugs such as doxorubicin, dolasetron, benfluron, metyrapone or ketoprofen, but so far only one...
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