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Specificita interakcí protein-protein a jejich modulace
Pham, Phuong Ngoc ; Schneider, Bohdan (advisor) ; Damborský, Jiří (referee) ; Vaněk, Ondřej (referee)
(EN) Protein-protein interactions (PPI) have essential roles in life processes, and abnormal PPI are associated with many human diseases. Given their importance, PPI have received increasing attention and became drug targets. However, the design of specific PPI and their modulation is challenging. Cytokine-receptor interactions are especially important in the regulation of the immune system. Interleukin-10 (IL-10) over-production results in excessive immunosuppressive effects, tumor growth and infection. The interaction between interferon gamma receptor 2 (IFN- γR2) and interferon gamma (IFN-γ) leads to activation of downstream signaling pathways but the mechanism of such interaction is elusive. Interleukin-24 (IL-24) is another cytokine that signals through receptors sharing the interleukin-20 receptor two (IL-20R2) subunit and has important roles in autoimmunity and cancer. The aims of this Ph.D. thesis are to study PPI from several aspects emphasizing their specificity. The first goal is to develop a novel protein scaffold and subsequently evolve it into a high-affinity binder specific for human IL-10. The second goal is to understand the structural basis for receptor specificity of human IFN-γ. The third goal is to modulate the binding affinity between human IL- 24 and its receptor IL-20R2 by...
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Directed evolution of enzymes on microfluidic chip
Kohúteková, Táňa ; Chmelíková, Larisa (referee) ; Prokop,, Zbyněk (advisor)
Tato práce se zaměřuje na řízenou evoluci enzymů pomocí fluorescenčně řízeného kapénkového třídení (ang. zkratka FADS) na mikrofluidním čipu. Byl sestaven nový třídicí systém, který byl validován pro vysoce výkonnou optickou detekci mikrofluidních kapének spojenou s dielektrickým tříděním. Systém byl testován pomocí dvou různých (DhaA31, ancHLD-RLuc) populací haloalkan dehalogenas (HLD) exprimovaných v buňkách Escherichia coli a jedné populace prázdného vektoru pIDR9 v poměru 5:45:50, což imituje proteinovou knihovnu. Naměřená a analyzovaná data z experimentu ukazují, že intenzity kapek ve validačním experimentu byly zaznamenány v stejném poměru jako teoretický poměr. Poté byla knihovna HLD mutantů s inzercemi a delecemi tříděna pomocí FADS, za účelem získaní 1 % nejaktivnějších variant. Program pro analýzu dat pro FADS byl programován v prostředí LabVIEW s využítím jazyka Python, s funkcemi pro počáteční analýzu, predikci prahového napětí, validaci a grafickou interpretaci dat a konečnou zprávu o experimentu.
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Engineering and selection of protein binders recognising medically important cytokines
Huličiak, Maroš
Protein engineering attracts more attention as a powerful tool of biotechnology and medicine. Small, engineered proteins derived from protein molecules of stable fold, the so called scaffolds, are potential replacements of supplements of more widely used antibodies. In this thesis, I introduce utilization of two scaffold molecules designed in our laboratory for development of stable and specific protein binders of high affinity. This thesis discusses the development of binders interacting with medically important human cytokines and their cellular receptors, interleukin-10, interleukin-28 receptor, and interleukin-9 receptor alpha. Recombinant cytokine and receptor proteins were expressed in eukaryotic cells in high yields and quality and served as molecular targets for selections using various display methods of directed evolution. We demonstrated that application of ribosome and yeast display methods or their unconventional combination in a newly developed integrated pipeline leads to successful generation of high affinity and specificity binders based on newly designed protein scaffolds called 57aBi and 57bBi.
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Engineering and selection of protein binders recognising medically important cytokines
Huličiak, Maroš ; Schneider, Bohdan (advisor) ; Pichová, Iva (referee) ; Kukačka, Zdeněk (referee)
Protein engineering attracts more attention as a powerful tool of biotechnology and medicine. Small, engineered proteins derived from protein molecules of stable fold, the so called scaffolds, are potential replacements of supplements of more widely used antibodies. In this thesis, I introduce utilization of two scaffold molecules designed in our laboratory for development of stable and specific protein binders of high affinity. This thesis discusses the development of binders interacting with medically important human cytokines and their cellular receptors, interleukin-10, interleukin-28 receptor, and interleukin-9 receptor alpha. Recombinant cytokine and receptor proteins were expressed in eukaryotic cells in high yields and quality and served as molecular targets for selections using various display methods of directed evolution. We demonstrated that application of ribosome and yeast display methods or their unconventional combination in a newly developed integrated pipeline leads to successful generation of high affinity and specificity binders based on newly designed protein scaffolds called 57aBi and 57bBi.
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Modulation of interactions of cytokines and their receptors
Kolářová, Lucie ; Schneider, Bohdan (advisor) ; Rozbeský, Daniel (referee) ; Osička, Radim (referee)
Protein-protein interactions and interactions with other molecules including DNA and RNA, play an important role in a range of biological activities and processes in all living cells. Understanding of protein-protein interactions, new approaches, and tools for their modulations are valuable for medicine, biotechnology, and drug development. We used the interleukin-10 family of cytokines as a model system for our research of biological interactions and modulation of their functions. A key prerequisite to study biological processes and a detailed understanding of biomolecular interactions is a recombinant protein that is stable under a broad range of conditions. Recombinant protein expression in sufficient yield and quality is often a challenging task. Therefore, we aimed at developing new approaches in protein design and production. In the first part of our study, we modified IL-24, a member of the IL-10 family to increase its expression and stability. We demonstrated that protein engineering is a powerful tool in research of difficult protein targets. In the second part of our study, we adopted new approaches in designing new protein scaffolds suitable for use in the ribosome and yeast display techniques. Protein scaffolds have become promising alternatives to antibodies in protein drug...
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Konstrukce mutantních enzymů pro degradaci antropogenních polutantů
Kratochvíl, Jakub
Enzymes are naturally occurring catalysts with a broad spectrum of catalysed chemical reactions, and they therefore represent environmentally-friendly and economically costeffective tools in many industrial processes, bioremediation, biomedical and pharmacological applications. Among such biocatalysts belong haloalkane dehalogenases (HLDs), which catalyse the cleavage of the carbon-halogen bond of halogenated compounds, resulting in corresponding products – alcohols, halide ions and protons. Howe-ver, catalytic properties of naturally occurring HLDs very often do not fulfill reqirements of industrial applications which limits their practical potential. Methods of directed evolution and ratoional design are therefore often used to generate genetic diversity and cosntruct novel protein varaits with deired function. In this work, a protein engineering strategy was applied to improve catalytic parameters of haloalkane dehalogenase DhaA31 towards anthropogenic pollutant 1,2,3-trichloropropane.Formula clause:Please postpone publication of materials, methods, results and conclusions of this thesis for a period of 3 years. These results are part of a project that has not yet been published.
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