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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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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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Characterization of nut oils
Coufalová, Dominika ; Sýkora, Michal (referee) ; Vítová, Eva (advisor)
This bachelor thesis deals with the characterization of selected nut oils, almond and pistachio virgin oils. The teoretical part concentrates on the description of nut fruits, the characterization of aroma-active compounds, fatty acids and their content in the nut oils. After that, the fatty characteristics, which determines the oil quality, and instrumental methods selected for individual assignment are described. In the experimental part there were 42 volatile compounds identified in the nut oils by the headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry, 35 were found in almond oil and 24 were found in pistachio oil. Free and bound fatty acids were converted into methyl esters using acid-catalysed esterification with boron trifluoride-methanol solution as a catalyst and then they were determined by gas chromatography with flame ionization detection. There were 12 fatty acids identified in almond oil and 14 fatty acids in pistachio oil. Oleic acid and linoleic acid predominated in both samples. The quality and properties of nut oils were valued by determining the fatty characteristics (acid value, saponification value, ester value, iodine value by Hanus method and peroxid value). The increased value of peroxide value was found in both oils which correlates with the trait and storage time of samples.
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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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Characterization of non-canonical RNA polymerase encoded by the yeast linear plasmids
Sýkora, Michal ; Vopálenský, Václav (advisor) ; Macíčková Cahová, Hana (referee) ; Valášek, Leoš (referee)
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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