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Production and characterisation of therapeutical antibody Farletuzumab - Thiomab E154C S378C
Mochánová, Michaela ; Brynda, Jiří (advisor) ; Dračínská, Helena (referee)
5 Abstract The human folate receptor α (FRα) is a receptor that binds folic acid, which as one of the vitamins is required for basic cellular processes, cellular growth, and differentiation. FRα's expression is strictly regulated in healthy tissue but is highly overexpressed in some tumor types. A humanized monoclonal antibody against folate receptor α called Farletuzumab has been developed. Binding of Farletuzumab to FRα leads to the activation of the immune system via antibody-dependent cytotoxicity and complement-dependent cytotoxicity. To enhance the antitumor effect, selected amino acids in the Farletuzumab antibody molecule were mutated to cysteines, allowing the subsequent use of THIOMAB technology. These cysteines are used to specifically conjugate the antibody to an artificial effector molecule that triggers the cGAS-STING (cyclic GMP-AMP synthase - stimulator of interferon genes) signaling pathway. The combination of the antibody's own anti-tumor effect and the triggering of the immune response to the cGAS-STING signaling pathway maximizes the immune response against cancer cells. Therefore, the aim of my work was to express the antibody in a suitable eukaryotic cell system and then to do a basic characterization of the protein. The Farletuzumab Thiomab antibody was prepared by heterologous...
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Cloning, expression and characterization of human serine racemase mutants
Nováková, Ilona ; Konvalinka, Jan (advisor) ; Brynda, Jiří (referee)
AAbbssttrraacctt Human serine racemase (hSR) is a cytosolic pyridoxal-5'-phosphate dependent enzyme localized in the central nervous system. It synthesizes D-serine, which is an endogenous coagonist for the N-methyl-D-aspartate (NMDA) receptors and plays a key role in excitatory neurotransmission in the brain. Thus, human serine racemase is a promising target for the treatment of neurodegenerative diseases connected with NMDA receptors. However, few specific inhibitors have been identified to date and the crystal structure of hSR has become available only very recently. We decided to perform a random mutagenesis to determine the amino acid residues critical for the enzyme activity. Ser 84 was reported as a catalytic residue along with Lys 56. After analysis of a double mutant S84G/P111L which retained its capability to convert L-serine to pyruvate, we prepared and characterized the single mutant S84G in order to exclude potential effect of the P111L mutation.on the activity of the analyzed enzyme. KKeeyy wwoorrddss:: D-serine; Serine racemase; PLP-dependent enzymes; Random mutagenesis; Racemases
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Novel binding proteins derived from small protein domains targeting diagnostically important molecules
Vaňková, Lucie ; Malý, Petr (advisor) ; Brynda, Jiří (referee) ; Trefil, Pavel (referee)
The rapid development of the gene engineering techniques, especially methods for in vitro directed evolution and combinatorial mutagenesis, has triggered the generation of new binding agents to almost any antigen of interest as an alternative to broadly used antibodies. These so-called non-Ig scaffolds are often derived from proteins with useful biophysical properties. While the therapeutic market is still dominated by monoclonal antibodies, the easy option of desired customization of non-Ig binders by conventional methods of gene engineering predestine them largely for the use in the diagnostic area. The ABD scaffold, derived from a three-helix bundle of albumin-binding domain of streptococcal protein G, represents one of the small non-Ig scaffolds. In our laboratory, we have established a highly complex combinatorial library developed on the ABD scaffold. This ABD scaffold-derived library was used to generate unique binders of human prostate cancer (PCa) biomarkers PSP94, KLK2, KLK11 for the more precise diagnosis of PCa. The second part of the thesis describes the generation of ABD-derived binders selectively recognizing different phenotypes of circulating tumor cells as a binding component of the cell capture zone of microfluidic chip for lung adenocarcinoma diagnosis. Beside this already...
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Glutamate Carboxypeptidase II - Structural and Biochemical Characterization and Structure-Assisted Drug Design
Ptáček, Jakub ; Bařinka, Cyril (advisor) ; Obšil, Tomáš (referee) ; Brynda, Jiří (referee)
Glutamate carboxypeptidase II (GCPII) is a human membrane-bound metallopeptidase discovered more than 30 years ago. It has attracted attention of biomedical scientists thanks to its diverse tissue expression profile and different biological functions. GCPII is detected on the surface of astrocytes in both central and peripheral nervous systems where it is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG), the most abundant mammalian peptidic neurotransmitter. Glutamate, one of the hydrolytic products, is a potent excitatory neurotransmitter and its overproduction has been shown to be responsible for cell death in various neurological disorders by a so-called glutamate excitotoxicity mechanism. Together with the fact that NAAG acts neuroprotectively it has been postulated (and later confirmed) that GCPII inhibition has a therapeutic potential in such disorders. Prostate cancer (PCa) is the second most prevalent cancer in men and despite its slow progression it is prone to metastasize thus posing a life threat. GCPII has been found to be overexpressed in prostate tumor cells compared to the healthy tissue (therefore it is also termed prostate-specific membrane antigen - PSMA) thus representing an excellent biomarker of PCa validated by many publications and clinical studies....
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Production of mouse recombinant prion protein and analysis of its properties
Krejčiříková, Anna ; Konvalinka, Jan (advisor) ; Brynda, Jiří (referee)
Title: Production of mouse recombinant prion protein and analysis of its properties Author: Anna Krejčiříková Department: Department of Biochemistry Supervisor: doc. RNDr. Jan Konvalinka, CSc., Supervisor's e-mail: konval@uochb.cas.cz Consultant: dr. Ing. Karel Holada, IIM 1stFM CU Abstract: Although prion diseases represent only a small fraction of all known neurodegenra- tive illnesses, they deserve our attention mainly due to the fact that they are lethal, incurable by today, and having a potential to cause a serious epidemic. Even though this topic has been studied for many years, there are still many unanswered questions concerning both the mechanism, and the spread of prion diseases. The most promising current theory is the protein-only hypothesis, which explains the fundamental of the ill- ness by the conversion of cellular prion protein (PrPC ) into pathological prion protein (PrPSc ). The important tool in proving this theory is also a recombinant prion pro- tein. The presented thesis summarizes existing successes in the proving of protein-only hypothesis. In the experimental part, we prepared the mouse recombinant prion pro- tein (mrPrP) in E. coli bacteria. The structure of the purified and renaturated protein was verified by CD spectroscopy. Next, we focus on the effects of the...
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Structural studies of 14-3-3 protein complexes and their stabilization by small molecule compounds
Lentini Santo, Domenico ; Obšil, Tomáš (advisor) ; Brynda, Jiří (referee) ; Pavlíček, Jiří (referee)
Protein-protein interactions (PPIs) play a crucial role in almost all biological processes. Many proteins require a number of dynamic interactions with other proteins and/or biomolecules to function. Proteomic studies have suggested that human protein-protein interactome consists of several hundred thousands of protein complexes. A detailed insight into these PPIs is essential for a complete understanding of the processes mediated by these protein complexes. Because many PPIs are involved in disease-related signaling pathways, such PPIs are important targets for pharmaceutical interventions, especially in situations where a more conventional target (e.g. the active site of an enzyme, the binding site of a receptor) cannot be used. This doctoral thesis focuses on 14-3-3 proteins, a family of eukaryotic adaptor and scaffolding proteins involved in the regulation of many signaling pathways. The 14-3-3 proteins function as interaction hubs and critical regulators of many enzymes, receptors and structural proteins. The main aim was to structurally characterize selected 14-3-3 protein complexes and investigate their stabilization by small molecule compounds. Using combination of protein crystallography, differential scanning fluorimetry, fluorescence polarization and analytical ultracentrifugation, the...
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Use of Sodium Bicarbonate for Flue Gas Treatment in Small Scale.
Zach, Boleslav ; Pohořelý, Michael ; Šyc, Michal ; Svoboda, Karel ; Václavková, Šárka ; Moško, Jaroslav ; Brynda, Jiří ; Punčochář, Miroslav
The trend of reduction of landfilling can lead, especially in areas with low population density, to the need to build new waste-to-energy capacities in the form of small units. However, flue gas treatment in small scale has to be sufficiently simple to decrease capital costs and allow the construction of such facilities. For that reason, the possibility of one-step dry flue gas treatment at compromise conditions was investigated as well as the limitation of flue gas composition.\n\n
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Novel binding proteins derived from small protein domains targeting diagnostically important molecules
Vaňková, Lucie ; Malý, Petr (advisor) ; Brynda, Jiří (referee) ; Trefil, Pavel (referee)
The rapid development of the gene engineering techniques, especially methods for in vitro directed evolution and combinatorial mutagenesis, has triggered the generation of new binding agents to almost any antigen of interest as an alternative to broadly used antibodies. These so-called non-Ig scaffolds are often derived from proteins with useful biophysical properties. While the therapeutic market is still dominated by monoclonal antibodies, the easy option of desired customization of non-Ig binders by conventional methods of gene engineering predestine them largely for the use in the diagnostic area. The ABD scaffold, derived from a three-helix bundle of albumin-binding domain of streptococcal protein G, represents one of the small non-Ig scaffolds. In our laboratory, we have established a highly complex combinatorial library developed on the ABD scaffold. This ABD scaffold-derived library was used to generate unique binders of human prostate cancer (PCa) biomarkers PSP94, KLK2, KLK11 for the more precise diagnosis of PCa. The second part of the thesis describes the generation of ABD-derived binders selectively recognizing different phenotypes of circulating tumor cells as a binding component of the cell capture zone of microfluidic chip for lung adenocarcinoma diagnosis. Beside this already...
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Glutamate Carboxypeptidase II - Structural and Biochemical Characterization and Structure-Assisted Drug Design
Ptáček, Jakub ; Bařinka, Cyril (advisor) ; Obšil, Tomáš (referee) ; Brynda, Jiří (referee)
Glutamate carboxypeptidase II (GCPII) is a human membrane-bound metallopeptidase discovered more than 30 years ago. It has attracted attention of biomedical scientists thanks to its diverse tissue expression profile and different biological functions. GCPII is detected on the surface of astrocytes in both central and peripheral nervous systems where it is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG), the most abundant mammalian peptidic neurotransmitter. Glutamate, one of the hydrolytic products, is a potent excitatory neurotransmitter and its overproduction has been shown to be responsible for cell death in various neurological disorders by a so-called glutamate excitotoxicity mechanism. Together with the fact that NAAG acts neuroprotectively it has been postulated (and later confirmed) that GCPII inhibition has a therapeutic potential in such disorders. Prostate cancer (PCa) is the second most prevalent cancer in men and despite its slow progression it is prone to metastasize thus posing a life threat. GCPII has been found to be overexpressed in prostate tumor cells compared to the healthy tissue (therefore it is also termed prostate-specific membrane antigen - PSMA) thus representing an excellent biomarker of PCa validated by many publications and clinical studies....
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Properties of Biochar Produced by Slow Pyrolysis of Stabilized Sewage Sludge.
Moško, Jaroslav ; Pohořelý, Michael ; Skoblia, S. ; Beňo, Z. ; Brynda, Jiří ; Zach, Boleslav ; Šyc, Michal ; Václavková, Šárka ; Jeremiáš, Michal ; Svoboda, Karel
Direct application of stabilized sewage sludge on agricultural soil is currently a big concern because of its significant content of detectable organic micropollutants. Proper sludge treatment should suppress its negative features and produce material suitable for soil amendment. Slow pyrolysis is one of such treatments. We studied the influence of pyrolysis temperature (400-800 °C) on material and energy balances and on elementary composition of the pyrolysis solid residue (biochar). Pyrolysis at higher temperatures resulted in lesser biochar yield and promoted gas yield. The macronutrient content of biochar increased with an increase in pyrolysis temperature (except for nitrogen).
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