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Large Extracellular Vesicles in Cell Culture and Blood: Role in Prion Transmission and Detection by Flow Cytometry
Soukup, Jakub
Prions (PrP) are the main cause of neurodegenerative diseases such as Scrapie in sheep, bovine spongiform encephalopathy, chronic wasting disease in deer, and Creutzfeldt-Jakob disease in humans. Although the cellular PrP (PrPC ) is involved in many cellular processes, its precise function still needs to be discovered. The disease is caused by the accumulation of a pathological form of PrP (PrPTSE ), which is caused by direct contact of PrPTSE and PrPC . PrP is anchored in the membrane by GPI and can be transmitted by cell-to-cell contact, tunnelling nanotubes, or extracellular vesicles (EVs). EV factions are divided by different biogenesis into exosomes, microvesicles, and apoptotic bodies. PrPTSE was found in exosomes and microvesicles, but these fractions were never compared to each other. The first aim of the doctoral thesis is a comparison of PrP content, prion-converting activity and infectivity in these fractions on CAD5 and N2a-PK1 cellular models of infection. We isolated a fraction of large EVs (20,000× g) and small EVs (110,000× g) by centrifugation from a conditioned medium. We characterised EVs by cryo-electron microscopy and western blot with Alix, TSG-101, CD63, CD9, and HSP70 markers. The contamination from other cellular compartments was checked by calnexin. EV fractions differed...
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Large Extracellular Vesicles in Cell Culture and Blood: Role in Prion Transmission and Detection by Flow Cytometry
Soukup, Jakub ; Holada, Karel (advisor) ; Šebestová Janoušková, Olga (referee) ; Živný, Jan (referee)
Prions (PrP) are the main cause of neurodegenerative diseases such as Scrapie in sheep, bovine spongiform encephalopathy, chronic wasting disease in deer, and Creutzfeldt-Jakob disease in humans. Although the cellular PrP (PrPC ) is involved in many cellular processes, its precise function still needs to be discovered. The disease is caused by the accumulation of a pathological form of PrP (PrPTSE ), which is caused by direct contact of PrPTSE and PrPC . PrP is anchored in the membrane by GPI and can be transmitted by cell-to-cell contact, tunnelling nanotubes, or extracellular vesicles (EVs). EV factions are divided by different biogenesis into exosomes, microvesicles, and apoptotic bodies. PrPTSE was found in exosomes and microvesicles, but these fractions were never compared to each other. The first aim of the doctoral thesis is a comparison of PrP content, prion-converting activity and infectivity in these fractions on CAD5 and N2a-PK1 cellular models of infection. We isolated a fraction of large EVs (20,000× g) and small EVs (110,000× g) by centrifugation from a conditioned medium. We characterised EVs by cryo-electron microscopy and western blot with Alix, TSG-101, CD63, CD9, and HSP70 markers. The contamination from other cellular compartments was checked by calnexin. EV fractions differed...
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Consumer credit
Soukup, Jakub ; Elischer, David (advisor) ; Dvořák, Jan (referee)
Consumer credit Abstract This diploma thesis deals with the topic of consumer credit, focusing on a systematic and comprehensive analysis of the development of consumer credit legislation not only in the Czech Republic, but also with a focus on essential legislation of the European Community and the European Union. The thesis is also focused on answering the theses set out in the introduction, which focus mainly on the issue of assessing the creditworthiness of consumers and the issue of regulation of providers and intermediaries and their impact on the activities of these entities in practice. For this purpose, the thesis is structured into seven parts, in which the legal regulation of consumer credit is systematically described, and certain essential or problematic passages are analysed in more detail. The first part of the thesis defines the basic concepts that are used in all aspects of consumer credit legislation and simultaneously in this thesis. The second part follows, which deals with the historical development of consumer credit from its very beginnings, through the development of consumer credit legislation in Community law, to the development of consumer credit legislation in our legal system. In the third part of this thesis, I analyse the general legal regulation of consumer credit and related...
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Optimization of methods for analysis of early steps of mouse polymavirus life cycle
Soukup, Jakub ; Španielová, Hana (advisor) ; Němečková, Šárka (referee)
Mouse polyomavirus is a type species of Polyomaviridae family and serves as model for studying viral infection of human pathogenic polyomaviruses. Minor proteins of viral capsid have been found to be necessary for effective initiation of infection. In order to study their role in the early steps of infection we utilized the novel Cre-LoxP system for production of the viral mutant lacking both minor proteins. Virus produced this way was compared with virus produced by standard method and we found that both systems facilitate production of mutant virus with the comparable quality and quantity. The mutant virus contained reduced amount of viral DNA and formed virions with impaired stability. For further studies of intracellular virion trafficking we prepared virions with genomes modified by thymidine analogues 5- bromo-2'-deoxyuridine (BrdU) and 5-Ethynyl-2'-deoxyuridine (EdU) and optimized the methods for analogue detection. The viral genome become accessible for detection 4 hours post infection. For ultramicroscopic analysis of translocation of virus to the nucleus we used freeze substitution. All this methods will be utilized for detailed study of distinct steps in viral infection. Key words: Mouse polyomavirus, minor proteins,...
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Retrocyclins
Soukup, Jakub ; Španielová, Hana (advisor) ; Beranová, Jana (referee)
Defensins are peptides with antimicrobial, antifungal and antiviral activity. Defensins are likely to occur in all vertebrate species. There are three subfamilies of defensins - α, β and θ-defensins. The α-defensins exists only in mammals and θ-defensins were described only in macaques and baboons but their genes occur in genomes of all old world monkeys except Homininae subfamily. In humans no θ-defensins are produces due to premature stop codon in their coding sequence. However, they were produced synthetically in vitro and named retrocyclins. Retrocyclins not only maintain antimicrobial activity - but also exhibit unusual antiviral activity. Retrocyclins successfully protect cells from HIV, HSV, influenza and dengue fever virus infections with no detectable cytotoxicity to host cells. Retrocyclin RC- 101 is close to the clinical testing as microbicide to prevent heterosexual transmission of HIV-1.
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