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Danio rerio as a model of serious human diseases
Hason, Martina ; Bartůněk, Petr (advisor) ; Živný, Jan (referee) ; Divoký, Vladimír (referee)
(ENGLISH) Over the last five decades, zebrafish (Danio rerio) has become a useful vertebrate model organism for the field of developmental biology and disease control. Using zebrafish in xenotransplantation studies is becoming more popular and progressed towards drug screening of anti-cancer drugs. Zebrafish are particularly suitable for high-throughput pre-clinical drug screening, due to the small size of embryos and the striking evolutionary conservation of cancer- related pathways between human and zebrafish. The fast, large-scale evaluation of the cancer- drug response in vivo could facilitate progress in personalized cancer therapy. Nevertheless, there is still a lack of methods which would allow for rapid and sensitive evaluation of tumor cell growth to facilitate high-throughput screening of drugs in vivo. In our bioluminescent zebrafish transplantation model, we proposed and validated a new screening platform for pre-clinical drug discovery in zebrafish embryos. In our experiments we used the NanoLuc luciferase, which enabled us to rapidly screen inhibitors of cancer growth in a sensitive and quantitative way with very low background compared to the conventional fluorescence signal. In our screen we evaluated the in vivo drug response of 180 kinase inhibitors in zebrafish embryos...
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Development of instrumentation and high-throughput screening methods for peptide ligand discovery and validation
Kryštůfek, Robin ; Konvalinka, Jan (advisor) ; Jiráček, Jiří (referee)
Peptides are used as synthetically available and easily derivatizable scaffold upon which it is possible to develop ligands targeting broad spectrum of biological targets. A time-tested approach to peptide binder identification is the preparation and screening of combinatorial libraries. Bypassing of this complicated procedure is possible by using biological systems for presentation, identification and selection of peptides based on the principle of in vitro evolution - i.e. display techniques. There are two complementary automated solutions for peptide binder identification described in this work. First is the SPENSER parallel peptide synthesizer, developed as a part of this diploma project, which can be used for peptide ligand discovery and optimization as well as validation of ligands identified using display techniques. Several libraries consisting of a total of 1 052 peptides have been prepared and then used to describe its potential applications. A sample of 154 preparations, representing 14.6 % analytical coverage of the prepared libraries, showed an average purity of 67 ± 19 % according to LC-MS. The libraries presented illustrate that SPENSER is a suitable tool for the parallel synthesis of linear and disulfide-cyclized peptides with limited variability, or libraries consisting of short...
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Development of high-throughput screening assay for the identification of inhibitors targeting influenza A polymerase
Karlukova, Elena ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Influenza virus A circulates in birds and mammals and causes severe infectious disease that affects from 3 to 5 million people each year. There are two classes of anti-influenza drugs currently available: neuraminidase and M2 channel inhibitors. However, increasing resistance against these two types of inhibitors along with the potential emergence of new viral strains and unpredictability of pandemic outbreaks emphasize an unmet need for new types of inhibitors. RNA-dependent influenza polymerase serves as a novel promising target for the development of anti-influenza medications. The aim of this master thesis is to develop in vitro high-throughput assays for screening of compounds targeting influenza RNA polymerase, particularly, its cap binding and endonuclease domains. For cap-binding domain the screening is based on DIANA (DNA-linked Inhibitor ANtibody Assay) method that was recently developed in our laboratory; for endonuclease domain, the method is based on AlphaScreen technology. For the purposes of the methods development, recombinant cap binding domain of PB2 subunit and N-terminal endonuclease domain of PA subunit of influenza polymerase were expressed with appropriate fusion tags and purified using affinity and gel permeation chromatography. The probes for the screening assays were...
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Srovnání indukce a regulace autofagocytózy v proliferujících a senescentních nádorových buňkách
Pešina, František ; Anděra, Ladislav (advisor) ; Rudolf, Emil (referee)
Autophagy, senescence and apoptosis are tightly linked processes which together determine the fate of cells in response to various stresses. There is ample evidence supporting the notion that senescent cells are highly dependent on autophagy and this process is here much more intensive than in nonsenescent cells. Autophagy may to some extent compensate increased energetic and metabolic demands of senescent cells and also helps with removal of toxic products such as oxidized proteins, protein aggregates and damaged organelles resulting from an overloaded metabolism of some senescent cells. In addition, some studies reported the need of autophagy for the adoption of senescent phenotype. However, there are also studies with seemingly contradictory results claiming that increased autophagy prevents or delays cellular senescence. Relationship of autophagy to apoptosis is similarly ambivalent. Whereas intact autophagy is necessary for the cell, while slightly increased autophagy still has a rather positive impact, excessive autophagy may lead to degradation of critical components necessary for cell function and survival and can trigger one of the modes of programmed cell death. In the first part of this work, we focused on the analysis of autophagic response in senescent and proliferating pancreatic...
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