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Evolution of protein-RNA and protein-cofactor interactions
Sanchez Rocha, Alma Carolina ; Hlouchová, Klára (advisor) ; Lepšík, Martin (referee) ; Berka, Karel (referee)
Genetický kód je překládán do bílkovin s využitím abecedy 20 proteinogenních aminokyselin. Přes současnou dominanci kanonicke abecedy je pravděpodobné, že primordiální proteiny byly složeny pouze z části tohoto aminokyselinového repertoáru. V této práci jsme prozkoumali soubor peptidů a proteinů s cílem objasnit, jak vyvíjející se repertoár aminokyselin ovlivnil jejich fyzikálně-chemické a biologické vlastnosti a sklon k interakci s evolučně konzervovanými molekulami, jako jsou kofaktory a RNA. V první části jsme prozkoumali soubor nejkonzervovanejších protoribozomálních peptidů s cilem odhalit jejich strukturní a funkční charakteristiky. Pomocí experimentálních a bioinformatických analýz jsme potvrdili převážně nestrukturovanou povahu těchto peptidů a odhalili jejich úlohu při stabilizaci peptidyltransferázového centra a ochraně RNA před degradací. Tyto procesy odráží nejstarší pochody koevoluce RNA a peptidů jež předcházely zrodu posledního univerzálního společného předka (LUCA z angl. Last Universal Common Ancestor). Dále jsme se zaměřili na pre-ribozomálni peptidy s potenciálním prebiotickým významem. Pomocí bioinformatických metod jsme prozkoumali strukturní charakteristiky kompletních knihoven peptidů zahrnujících kanonické a prebioticky přípustné nestandardní aminokyseliny. Predikce...
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Capturing chromatin-associated proteins in nucleosomal context
Koutná, Eliška ; Veverka, Václav (advisor) ; Hlouchová, Klára (referee) ; Bařinka, Cyril (referee)
| 9 ABSTRACT Eukaryotic DNA is stored in the nucleus wrapped around histone octamers in the form of nucleosomes. These basic chromatin units can further associate with DNA-binding factors through DNA and the N-terminal histone tails that are subjected to various covalent posttranslational modifications that, in combination with DNA modifications, define the epigenetic code. Eukaryotic transcription is dependent on these specific histone modifications - their recognition by chromatin reader proteins triggers complex processes relying on the coordinated association of transcription regulatory factors. Although various modification states of a particular histone residue often lead to differential outcomes, it is not entirely clear how they are discriminated at a molecular level. Moreover, the contribution of intrinsically disordered regions outside of the specialized reader domains to nucleosome binding remains unexplored. In this thesis, the main focus is put on the transcription coactivator LEDGF/p75, capable of reading the H3K36me2/3 histone mark, and the pioneer transcription factor Sox2. Using structural biological and biophysical techniques, the interaction of LEDGF and Sox2 with nucleosomes is dissected, describing the peculiarities of intrinsically disordered DNA interacting motifs. Two...
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Strategies and results in the development of insulin derivatives for the treatment of diabetes
Pokorný, Štěpán ; Jiráček, Jiří (advisor) ; Hlouchová, Klára (referee)
The aim of this bachelor's thesis is to present insulin analogs and to analyse the direction of development of these organic compounds. The text contains basic information on this subject (e.g. the history of Diabetes Mellitus, the first treatment, the discovery of insulin (1921) and the experiments that preceded this discovery...). The thesis also explains the basic differences between type 1 and type 2 Diabetes Mellitus and the resulting differences in treatment. It mainly summarizes, the specific types of analogs, their chemical composition and the nature of their action. The main contribution of this work lies in the analysis of insulin analogs and the overview of the course in which the development of these analogues is proceeding or what innovations we can expect in diabetes therapy in the upcoming years. The thesis draws primarily from peer-reviewed scientific papers, articles and books (older as well as more recent ones) and from the expertise of the IOCB laboratory. All sources are well cited at the end of the work.
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Whole genome synthesis: methodology and applications
Kučera, Vítězslav ; Hlouchová, Klára (advisor) ; Nedvědová, Jana (referee)
Genomes contain the information needed to support life and viruses. Understanding this information is no small task but an essential one for the understanding of life and its evolution. Genome sequencing can provide useful information about the meaning of genetic information, although it is now clear that sequencing alone cannot provide all the answers. For deeper comprehension of life's intricacies, synthesis of genomic DNA will extend our current knowledge and offer a platform for further applications. The immense growth in the methodology of DNA synthesis in the last decades facilitated the construction of the first cellular genome in 2008. Continuous advances in genome synthesis and engineering are the driving forces of synthetic biology. The history, current state and future applications of the whole genome synthesis are presented in this thesis. Key words: DNA assembly, synthetic biology, minimal cell, genome editing, genome transplantation, DNA synthesis, top-down approach, bottom-up approach
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Inhibitors of rhomboid proteases as tools for cell biology
Kuzmík, Ján ; Stříšovský, Kvido (advisor) ; Hlouchová, Klára (referee)
Rhomboid intramembrane serine proteases cleave polypeptide chains within lipid bilayer. Rhomboid proteases were originally discovered in Drosophila melanogaster where they regulate ontogenesis of the fly, but they are present in all domains of life. Nowadays, various diseases, such as malaria, amoebiasis, Parkinson's disease, various tumour malignancies, and diabetes, have been linked with rhomboid proteases. However, natural substrates and function of most rhomboids remain elusive. Cell biology tools are needed for unravelling functions of rhomboids, as well as for potential pharmacological applications, and this together fuels the effort to develop specific rhomboid inhibitors. The inhibitors known to date always bear an electrophilic warhead attacking the nucleophilic serine of the atypical serine-histidine catalytic dyad of rhomboid. From the various developed inhibitors, peptidyl -ketoamides substituted at the ketoamide nitrogen by hydrophobic groups, discovered in our laboratory, hold the biggest potential. They are potent, reversible, selective, tunable, and are built around a pharmacophore already approved for medical use. Here, I set out to improve peptidyl -ketoamides by exploring the chemical space in the active site of rhomboid and testing substituents of the ketoamide nitrogen of increasing...
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Biochemical tools for targeting immunomodulatory receptor CD73
Poukarová, Magdalena ; Ormsby, Tereza (advisor) ; Hlouchová, Klára (referee)
New approaches to cancer treatment are investigated because cancer remains one of the most abundant causes of death. One of the promising fields of oncology is immunotherapy, which is based on the activation of immunity to eliminate malignant cells. Several immunotherapy drugs have already been approved and are being used; however, only a part of patients responds with the desired efficiency. In recent years, ecto-5'-nucleotidase CD73 emerged as a potential new target of immunotherapy. The expression of this GPI-anchored enzyme at the extracellular membrane is significantly up-regulated tumour cells and in immune cells of the tumour microenvironment. CD73 catalyses the hydrolysis of AMP to immunosuppressive adenosine, therefore contributing to the immunosuppressive environment in the tumour and participating in resistance to immunotherapy. CD73 appears to be a promising target for a combination treatment to improve the effect of other immunotherapy drugs. So far, several clinical trials of CD73 antibodies and small-molecule inhibitors have been conducted. This diploma thesis describes the development of new biochemical tools for CD73. DNA- linked Inhibitor ANtibody Assay (DIANA), a sensitive method that enables the measurement of the binding affinity of a small-molecule inhibitor to a protein, was...
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Functional screening of de novo proteins
Melikov, Aleksandr ; Hlouchová, Klára (advisor) ; Míšek, Jiří (referee)
Synthetic biology relies upon working with two main types of biological macromolecules - nucleic acids and proteins. Natural proteins represent only a small percentage of the whole amino-acid sequence space. Most of it may conceal an enormous potential (unexplored by nature as well as scientific endeavor), which has started to be carefully explored only in the recent decades. Characterization of non-native proteins includes several key aspects: structure and its stability, function, patterns of interaction with other molecules (of different chemical nature) and in vivo tolerance. This work focuses on the functional testing of de novo polypeptide molecules, either appearing as novelties of genome non-coding regions or as products of artificial bioengineering design. Key words: de novo proteins, function screening, protein libraries, protein design, sequence space
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LacZ-alpha complementation peptide as a tool for molecular evolution studies
Ptáčková, Barbora ; Hlouchová, Klára (advisor) ; Vaněk, Ondřej (referee)
Proteins are the key structural and functional molecules of living organisms. Although the last decades have brought a lot of knowledge about their structural and functional characteristics, science still lacks very basic answers about how these properties evolved. Current predominant opinions suggest that early genetic code contained only a subset of today's canonical amino acids. Both exogenous and endogenous sources of prebiotic amino acids imply that even though the prebiotic amino acid repertoire was very broad, only about half of the proteinogenic amino acids were present. It follows that the ''evolutionary new'' amino acids were added to the genetic coding system only after the evolution of their biosynthetic pathways. From the current scientific knowledge it is unclear whether proteins composed of "evolutionary old" amino acids could serve basic metabolic functions and if today's proteins could be "reversely-evolved" to be composed of only such a subset of amino acids while maintaining their structural and functional integrity. These questions lie at the core of this study. This thesis aims to test a starting methodology that would randomize "new" amino acid positions by "old" amino acids in the sequence of LacZ-alpha peptide. This peptide was selected as a target model protein because it...
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Preparation of a library of methionine sulfoxide reductase for applications in synthesis of chiral sulfoxides
Havelka, Václav ; Míšek, Jiří (advisor) ; Hlouchová, Klára (referee)
Chiral sulfoxides are important compounds in the pharmaceutical and chemical industries, however, their enantioselective synthesis providing only one desired enantiomer is not fully mastered. Some natural enzymes can be used for the biocatalytic preparation of chiral sulfoxides. One of such enzymes is methionine sulphoxide reductase. Methionine sulphoxide reductase is an enzyme limiting the effects of reactive oxygen radicals in the organism resulting from oxygen metabolism. Its function is the reduction of methionine sulfoxide in proteins to methionine. There are two types of methionine sulphoxide reductase, methionine sulphoxide reductase A reducing only (S)-methionine sulphoxide and methionine sulphoxide reductase B reducing (R)-methionine sulphoxide. Methionesulfoxide reductase B is suitable for the preparation of (S)-sulfoxides, however its catalytic activity is not sufficient for practical use. Using the recombinant DNA and mutagenic PCR techniques, a methionine sulphoxide reductase B mutant library was prepared, and the extent and nature of mutation introduced was determined. This library will serve as a starting point for the controlled evolution of the enzyme to obtain clones with increased activity and reduced substrate specificity.
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Vascular Endothelial Growth Factor Expression and its Application in Vascular Tissue Engineering
Mikulová, Barbora ; Konvalinka, Jan (advisor) ; Hlouchová, Klára (referee)
This paper deals with the expression of vascular endothelial growth factor (vascular endothelial growth factor, VEGF) and its use in tissue engineering of vascular wall. During the work interaction of endothelial cells with the modified fibrin-based biomaterial into which vascular endothelial growth factor (VEGF-A121) has been incorporated was monitored. This modification supported the adhesion and growth of endothelial cells. Vascular endothelial growth factor VEGF-A121 is signal glycoprotein that activates transmembrane receptors on endothelial cells. VEGF-A121 is a key regulator in vasculogenesis, angiogenesis, proliferation, migration and survival of endothelial cells. In this work, this protein was heterologously expressed at a thioredoxin fusion partner in an expression system of E. coli Origami B (DE3). Recombinant VEGF-A121 was additionally coexpressed with bacterial chaperones GroEL/GroES for potential increase of its solubility and biological activity. In the next part of this work thin fibrin network was prepared by catalytic action of thrombin on the polystyrene-bound monolayer of fibrinogen. This network has been further enriched by vascular endothelial growth factor (VEGF-A121), which was covalently incorporated in it by enzyme activity of transglutaminase (factor XIIIa). The last...
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