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Preparation and characterization of modified viral particles derived from mouse polyomavirus for the transport of genes to increase the efficiency of transduction
Škvára, Petr ; Španielová, Hana (advisor) ; Sýkora, Michal (referee)
Viral particles derived from mouse polyomavirus can be potentially used as a delivery system for therapeutic genes and drugs into target cells. This thesis focuses on preparation and characterization of polyomaviral particles that are modified with cell-penetrating peptides in order to increase efficiency of transduction of reporter genes into human cells. Viral particles that are composed of major capsid protein VP1 in combination with minor capsid protein VP2 and minor capsid protein VP3 that is modified with octaarginine, LAH4 peptide or with transduction domain of adenoviral protein VI are analysed in transduction assays. The thesis also provides information about the effect of the modification on encapsidation of heterologous DNA. The results of transduction assays performed with modified particles containing encapsidated luciferase gene revealed that efficiency of transduction did not increase but decreased in comparison with unmodified particles. These findings help to elucidate the role of polyomaviral minor capsid proteins in gene transfer mediated by viral particles and contribute to the design of new strategies for modifications of viral particles derived from mouse polyomavirus for their successful application in nanomedicine. Key words: mouse polyomavirus, pseudovirions, virus-like...
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Genome editing using programmable endonucleases
Hanečková, Radmila ; Sedláček, Radislav (advisor) ; Sýkora, Michal (referee)
Programmable endonucleases are engineered proteins that recognize specific nucleotide sequences and that are capable of introducing double-strand breaks within these sequences. Zinc-finger nucleases have been used extensively as a tool in genome editing, the practice of introducing changes into genomes of cell lines or whole organisms as a way to study gene function. Recently, new types of programmable endonucleases have emerged in the form of transcription activator like effector (TALE) nucleases and the CRISPR/Cas system. The types differ in respect to their mechanism of function, accessibility, selectivity, frequency of off-target cleavage and cytotoxic effects. Here, we compare zinc-finger nucleases, TALENs and the CRISPR/Cas system and explore their current and possible future applications in a broad spectrum of research ranging from developing genetically modified organisms to gene therapy. Powered by TCPDF (www.tcpdf.org)
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Temporal applicability of criminal legislation and certain selected issues occurring in practice
Sýkora, Michal ; Šámal, Pavel (advisor) ; Císařová, Dagmar (referee)
1 Abstract The essay is taking through the current problems with the time applicability of the criminal (penal) law in the Czech republic, especially with the view of new Criminal Code no. 40/2009, which has taken place on 1st January 2010 (but the new Criminal Code was in progress for several years). The theoretical part of the essay is about the basic questions of the criminal (penal) law's time applicability, the sources of the law (both national and international) from the past and present, also the basic theoretical insights into the time applicability and finally its current adaptation in the Czech law system and the changes brought by the new Criminal Code. The most significant and serious change in the Criminal Code regarding to the time applicability is to be able to establish the exact time of the criminal act. The previous absence of this adjustment in the Criminal Code no. 140/1960 have caused disagreements between the theory and the judicature, whether the actual time of the criminal act is once the crime has been accomplished or when the criminal act hase been committed. The new Criminal Code have uniquely determined this to be once the criminal act has been committed which means no future disagreements with that issues. The practical part of the essay is describing and dealing with problems...
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Artificial phospholipid membranes - method of prepatation, properties and their usage
Hryzáková, Klára ; Fišer, Radovan (advisor) ; Sýkora, Michal (referee)
Heterogenita biologických membrán vedla ke vzniku širokého spektra zjednodušených modelových systémů, jejichž uspořádání, velikost a tvar se dají přizpůsobovat různým aktuálním požadavkům. Existují dva rozdílné přístupy k vytváření umělých fosfolipidových membrán. První z nich je založen na vzniku membrán ve vodném prostředí. Do této skupiny patří černé lipidické membrány, dvojvrstvy na pevném podkladu, dvojvrstvy vzniklé z monovrstev na rozhraní vody a vzduchu a liposomy. Ve druhém případě vznikají dvojvrstevné membrány v množství organické fáze metodou dvojvrstev na kapičkovém rozhraní. Každý typ umělých membrán má své experimentální výhody a nevýhody, což se používá ke studiu různých problémů sahajících od chování jednotlivých fosfolipidů a proteinů až po fúze membrán. Umělé fosfolipidové membrány jsou vhodným nástrojem pro elektrickou charakterizaci dvojvrstev a nebo membránových proteinů. Tato práce je ucelený přehled nejpoužívanějších metod vhodných pro vznik umělých fosfolipidových membrán. Klíčová slova: membrána, lipid, fosfolipidová dvojvrstva, liposom, černá lipidová membrána, dvojvrstvy na pevném podkladu, dvojvrstva na kapičkovém rozhraní Abstract The heterogeneity of biological membranes has led to development of a wide spectrum of simplified model systems whose composition, size and...
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Catalytic function of DNA-dependent RNA polymerases
Sýkora, Michal ; Vopálenský, Václav (advisor) ; Lichá, Irena (referee)
DNA-dependent RNA polymerase is a highly organised protein complex that is responsible for gene expression and its regulation. Multisubunit RNA polymerase with its several catalytic activities is responsible for transcription of genes to RNA copies in all cellular organisms. During transcription RNA polymerase undergoes substantial conformational changes depending on the conditions in a particular cell. RNA polymerase in a state designated as an elongation complex passes through repetitive cycles of adding a nucleotide to the growing RNA chain. The active center contains two magnesium ions which coordinate the reactive groups of substrates. Furthermore, the active center contains structural elements that participate in binding of substrate, propper orientation of substrate towards the template strand and translocation of the RNA polymerase. The most important of these mobile structural elements are the bridge helix and the trigger loop whose conformational changes accompanies nucleotide addition cycle. Advances in the structural and biochemical characterization of RNA polymerase open new possibilities in the understanding of the transcription mechanism, its fidelity and control.
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Phenomena affecting the behavior of Czech nobility in the Hussite revolution. Example Hanus of Kolovraty
Sýkora, Michal ; Foltýn, Dušan (advisor) ; Charvátová, Kateřina (referee)
This thesis focuses on the factors affecting the behavior of Czech nobility (especially Czech lords) during the Hussite revolution. Phenomena such as family, economic and regional relations, state affiliation,and the contemporary military situation will be confronted with the life of Hanus of Kolovraty. Hanus of Kolovraty was chosen for this purpose for several reasons. As a member of the Pilsen landfried he was an ally of Sigismund of Luxemburg for a long time. For us it will be interesting to see how Hanus of Kolovraty contributed to the secularization of church property, which strategically built his estate and how the Hussite pressure forced him into radical change in the opposite doctrinal camp. The research will be completed by the year 1448, when George of Podebrady took Prague and Hanus of Kolovraty was defeated and withdrew from the political scene. Various behavioral theories of the Czech nobility will be confronted not only with the life of Hanus of Kolovraty, but also with other examples of prominent leaders of the Hussite revolution. This thesis will attempt to answer the question whether it is possible to find some sort of unifying theory of a behavior of the Czech nobility in the first half of the 15th century, or whether this theory is definitely shattered by the detailed...
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The project of Boris Akunin - an example of literary strategy at the turn of XX-XXI centuries
Volkova, Natalia ; Glanc, Tomáš (advisor) ; Janáček, Pavel (referee) ; Sýkora, Michal (referee)
This work deals with the literaturary project "B. Akunin", which was defined by it's creator G. Chkhartishvili as a "quality literature for the mass reader". When they wish to characterise the project, critics emphasize the use of an adventure plot combined with interesting style and intertextual play. We see a tendency to place the project in the "middle level", which in their hierarchy is somewhere between the literature created for the "happy few" and consumer fiction. However convergence of mass and elite culture is too general a trend in postmodernist literary strategy to be sufficiently characteristic of a concrete author. Our research covers all the texts published to the present day under the names of both G. Chkhartishvili and B. Akunin. It was the part of the extratextual strategy of the author to construct an image of himself as a writer of serious, almost scientific prose, who began to write detective stories for his own and his readers' enjoyment. The author generally uses mass clichés whenever he describes his heroes and action scenes in the story. He uses a great many simple references to the other literaturary works in his texts which should be understandable by every reader. At the same time we find other, more complicated references requiring knowledge of a wider literary "encyclopedia"....
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Characterization of non-canonical RNA polymerase encoded by the yeast linear plasmids
Sýkora, Michal
Transcription is the control point of gene expression. This process relies on protein complex of multisubunit RNA polymerases, which are extremely conserved among all cellular organisms. Transciption of extrachromosomal hereditary elements such as organelles, viruses and plasmids is dependent on host cellular RNA polymerases or intrinsic RNA polymerase is contained within these elements. Putative non-canonical two-subunit RNA polymerase is also encoded by linear cytoplasmic plasmids of the yeast Kluyveromyces lactis and most likely transcribes genes of these plasmids. Besides the two subunits of RNA polymerase encoded by linear plasmids of Kluyveromyces lactis there are another two estimated components of the transcription apparatus, namely capping enzyme that adds the cap to 5' mRNA ends and putative DExD/H box helicase. Characterization of the unique and underexplored transcription machinery of Kluyveromyces lactis plasmids was the principal objective of this work. The main goal was to: 1) clarify evolutionary origin of the linear plasmid transcription apparatus; 2) describe architecture of the linear plasmid transcription complex in vivo focused on putative RNA polymerase binding partners; 3) reveal mechanisms of transcription initiation and termination of the yeast linear plasmids. The main...
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Changes of sensory active substances during ripening of selected type of natural cheese
Sýkora, Michal ; Buňková, Leona (referee) ; Gregor,, Tomáš (referee) ; Buňka, František (advisor)
The presented dissertation is a study focused on the characterization of sensory quality, especially the flavour of model samples of natural cheeses. Attention is aimed to the composition and content of volatile (sensory active) substances as important markers of flavour and free/bound fatty acids as their precursors. The headspace solid phase microextraction in conjunction with gas chromatography with mass spectrometry was used to determine volatiles. For the determination of fatty acids, the lipids were isolated from the sample with a solvent mixture of diethyl ether and petroleum ether, followed by esterification using a methanolic solution of boron trifluoride as a catalyst and final analysis by gas chromatography with flame ionization detection. The experimental part was divided into two experiments; in experiment I, Moravský bochník type cheeses (45 % fat in dry matter., 60 % dry matter) were analysed, in experiment II, Edam type cheeses (45 % fat in dry matter, 50 % dry matter). Model samples of cheeses were made at Tomas Bata University in Zlín. For all samples, a classical mesophilic culture containing Lactococcus spp. and Leuconostoc spp. was used, this was always supplemented with a suitable monoculture of thermophilic bacteria; in the case of Moravský bochník cultures of Streptococcus thermophilus and Lactobacillus helveticus and in the case of Edam cheeses Lactobacillus casei and Lactobacillus plantarum. The essence of both experiments was to assess the influence of the used microbial cultures on the above-mentioned parameters, at the same time their changes during the ripening of the cheeses were monitored. The results showed that the addition of thermophilic cultures, as well as the maturation process did not affect the number of fatty acids or volatiles, but significant differences were found in their content. The content of bound fatty acids decreased during cheese ripening due to ongoing lipolytic changes while increasing the content of free fatty acids and their subsequent degradation to volatile substances, which ultimately contribute to the taste and aroma of the cheese. Ketones and acids were the most quantitatively present in all samples. During maturation, contrary to expectations, the content of identified volatiles also decreased. The addition of the thermophilic cultures didn’t have a clear effect and varied depending on the maturation phase. In the first phase, although they showed more pronounced lipolytic activity, the subsequent metabolic processes - the breakdown of free fatty acids into volatile substances, compensated for these differences. These results confirm a significant share of the basic mesophilic culture in the formation of volatile substances, in particular the flavour of cheeses.
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