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Structural characterization of influenza A polymerase PA subunit domains in complex with novel inhibitors
Radilová, Kateřina ; Kožíšek, Milan (advisor) ; Rumlová, Michaela (referee) ; Obšil, Tomáš (referee)
Influenza RNA-dependent RNA polymerase is a heterotrimeric complex and has an essential role in the life cycle of the virus. It is responsible for viral replication and transcription. One of its subunits, the polymerase acidic protein, interacts with the PB1 subunit via a crucial protein- protein interaction at its C-terminal domain. This 310 helix-mediated intersubunit interaction is required for the whole heterotrimer assembly. The N-terminal domain carries the endonuclease active site with two manganese ions. Both domains are considered promising drug targets. Current strategies to fight the influenza virus are limited to seasonal vaccines, and there are only a few anti-influenza drugs targeting mostly other viral proteins. Many used antivirals are susceptible to rapid resistance mutations development or cause severe side effects. This thesis provides structural insights into the two domains of the PA subunit. The first part is devoted to the characterization and optimization of a PB1-derived minimal peptide interacting with the C-terminal domain. Results from this part may be considered as a starting point for the rational design of first-in-class anti-influenza inhibitors of the PA-PB1 protein-protein interaction. In the other half, we have explored the inhibitory potency of flavonoids and...
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Entropically driven cascade hybridization reactions for detection of microRNA
Runová, Alžbeta ; Cígler, Petr (advisor) ; Kožíšek, Milan (referee)
The emerging potential of miRNA molecules as diagnostic biomarkers calls for the development of a new quantification method. Current approaches usually require time-consuming and costly miRNA isolation for proper sample analysis. In this thesis, a new, isolation-free, oligonucleotide- modified gold nanoparticle (AuNP/DNA) system is proposed and designed for miRNA detection and quantification in living cells. This cascade, entropy-driven, and enzyme-free amplification system provides fluorescence signal upon selective interaction with the target miRNA. For this purpose, citrate-stabilized gold nanoparticles were synthesized, and their diameters were determined by dynamic light scattering and transmission electron microscopy. The AuNP/DNA conjugates were prepared following a recently published "freezing method". Their reaction kinetics with the target miRNA and selectivity to various miRNAs were compared with those of an analogous DNA system without AuNPs in a series of fluorescence measurements. Furthermore, stability experiments in glutathione environment were conducted, as well as DNA electrophoresis, demonstrating the mechanistic aspects of the reaction. The reaction yields and selectivity to target miRNA of 42.31 ± 2.91 nm AuNP/DNA constructs, containing approximately 25 DNA complexes per AuNP,...
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Study of DNA-compatible Reactions and Their Utilisation for DNA Encoded Libraries
Havelka, Václav ; Míšek, Jiří (advisor) ; Kožíšek, Milan (referee)
DNA-encoded peptide libraries are the basis for in vitro selection methods that use various biological systems (phage display; yeast display; mRNA display). Despite the great success of these selection methods, their obvious disadvantage is the limited number of building blocks, which consist of only twenty proteinogenic amino acids. The involvement of other non- proteinogenic amino acids and other building blocks could significantly expand the range of possible applications of these selection methods. For example, the introduction of chemical modifications in amino acid side chains in such libraries would allow the effective study of post-translational modifications (phosphorylation, acylation, glycosylation, methylation, etc.) in living organisms. The aim of this work was to develop a method for preparation of a fully synthetic DNA encoded library of peptides. The basic steps for the preparation were the chemical synthesis of the peptide and associated enzymatic synthesis of encoding DNA. Compatibility of chemical reactions with DNA is essential for the synthesis of DNA-encoded peptide libraries. Because the final acidic deprotection of the side chains in the peptide is not compatible with DNA, two approaches have been tested to overcome this problem. The first was an attempt to develop finer...
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The influence of S159A mutation on the oligomeric state of human NK cell receptor NKR-P1A
Hausleitner, Filip ; Vaněk, Ondřej (advisor) ; Kožíšek, Milan (referee)
Natural killer cells (NK cells) are lymphocytes that possess cytotoxic activity against tumour or virally infected cells independent of preceding antigen sensitisation. To kill such cells, they utilise their activating and inhibitory surface receptors that interact with target cell surface molecules. The immune response carried by NK cells depends on the balance of both activating and inhibitory signals. Human NK cell surface receptor NKR-P1A belongs to the structural family of C-type lectin-like receptors. This receptor interacts with its ligand LLT1, which belongs to the same protein family, with low affinity and high specificity. The NKR-P1A:LLT1 complex formed between NK cell and its target cell inhibits NK cell cytotoxicity, and hence is a part of the regulation of immune response. This thesis studied the effect of S159A mutation on the stoichiometric state of soluble human NKR-P1A ectodomain in solution. Therefore, a mutant form of NKR-P1A G90-S225 S159A ectodomain was successfully produced in stably transfected human embryonic kidney cells 293 (HEK293S GnTI" ). This construct was purified by affinity and size-exclusion chromatography, and analysed by SDS-PAGE and analytical ultracentrifugation. Our results show that the preclusion of N-linked glycosylation in the position 157 promotes the...
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Overcoming drug resistance: The discovery, design and characterization of new nonpeptidic inhibitors of HIV - 1 protease
Kožíšek, Milan ; Konvalinka, Jan (advisor) ; Majer, Pavel (referee) ; Obšil, Tomáš (referee)
Ph.D. thesis abstract Overcoming drug resistance : The discovery, design and characterization of new nonpeptidic inhibitors of HIV-1 protease Milan Kožíšek, M.Sc. Supervisor : Jan Konvalinka, Ph.D. PPrraagguuee 22001100 Department of Biochemistry Faculty of Science Charles University, Prague, Czech Republic Institute of Organic Chemistry and Biochemistry Gilead Sciences & IOCB Research Centre Academy of Sciences of the Czech Republic 3 Abstract HIV-1 protease is an aspartic protease which plays an essential role in the life cycle of HIV virus. It is responsible for the cleavage of the viral polyproteins into the structural and functional proteins during viral maturation. The efficient inhibition of the protease thus leads to the formation of immature and non-infectious viral particles. The introduction of protease inhibitors dramatically changed the treatment of retroviral infection. The viral replication was reduced to undetectable level and the rate of disease progression was significantly lowered. However, resistance to the inhibitors was observed. The first inhibitors had limited bioavailability, caused severe side effects and easily developed resistance. To combat these negative factors, second-generation inhibitors have been developed. Understanding the mechanisms of resistance toward inhibitors is...
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