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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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The applicability of criminal law statues
Sýkora, Michal ; Jelínek, Jiří (advisor) ; Musil, Jan (referee) ; Fryšták, Marek (referee)
The applicability of criminal law statues (abstract) The submitted thesis is focused on the applicability of criminal law statutes and aims to summarize the current legislation regarding the applicability of criminal law statutes, assess its current state, draw attention to any legislative gaps and propose possible de lege ferenda changes. The usual focus of university textbooks might suggest the applicability of criminal legislation is a matter more or less brief, which does not require a special level of attention. However, the opposite is true. The applicability of criminal legislation is a rather broad and diverse topic, its legal framework can be found both in substantive and procedural law, as well as in regular law, constitutional law and law of international treaties. A detailed theoretical research and the solution of individual cases uncovers a number of subquestions, not only in criminal law, but also in a number of other areas of law. There are four basic aspects of applicability: temporal, territorial, personal and subject matter. Every single one of those has a rich history in theoretical, statutory and case law development. Despite such history, it remains an ongoing issue, both from the perspective of professional and general public alike. The matter is also a part of the regular occupation...
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Aronia as a raw material for cosmetic production
Palkovská, Kateřina ; Sýkora, Michal (referee) ; Zemanová, Jana (advisor)
This bachelor thesis deals with extraction of polyphenols from fruits and leaves of aronia and consequent use of obtained extract in cosmetic production. In the theoretical part are information about the origin of the aronia, the botanical characteristics of Aronia Melanocarpa and about its utilization. Furthermore, the chemical composition of the plant material of the aronia is described, which is also common for fruits. At the end of the theoretical part are described extraction and separation techniques, methods of determination of total polyphenolic compounds and brief characteristics of the material safety data sheet. The experimental part describes the optimization of the prepared extract so that the extract contains as many polyphenolic compounds as possible. 50% ethanol at room temperature was shown to be optimal for leaf and fruit extraction, and the maceration time was found to have a significant effect on the polyphenolic compounds obtained. For the obtained optimized macerate, some chemical and physical properties such as pH, density, refractive index were determined for the purpose of producing a material safety data sheet and product specification. At the end of the experimental part were prepared cosmetic products with the addition of macerate from crushed aronia fruits.
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Replication strategies of newly discovered giant viruses of amoebas
Blaško, Michal ; Forstová, Jitka (advisor) ; Sýkora, Michal (referee)
Giant viruses are a group of viruses with genome composed of double-stranded DNA molecule. They are characterized by the creation of giant viral particles, the size of which varies between 150-1500 nm. Also, their genomes are huge reaching sizes of up to 2,5 Mbp. The viruses replicate either in the cytoplasm or they exploit for their replication both nucleus and cytoplasm. Therefore, they are called, nucleocytoplasmic large DNA viruses (NCLDVs). During their replication cycle, the giant viruses induce the creation of viral factories, which provide morphogenesis of new virions. The aim of this thesis is to summarise current knowledge of selected representatives of the giant viruses, and to describe their replication strategies. Furthemore, this work aims to discuss discoveries made in relation to this particular group if viruses. Thanks to the discovery of giant viruses, another group of small viruses was identified - the so-called virophages (viruses of viruses). Virophages have the ability to take advantage of the giant virus infecting an amoeba to realize their own replication strategy. In some members of the Mimiviridae family there was described a presence of an genome element providing a unique way of immunoprotection of giant viruses from being infected by the Zamilon virophage. Key words:...
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Assessment of fatty acids in processed cheese
Mitra, Michal ; Sýkora, Michal (referee) ; Vítová, Eva (advisor)
This bachelor thesis deals with determination of fatty acids in samples of processed cheeses made at Tomas Bata University in Zlín. In the theoretical part, lipids and fatty acids are characterized. Subsequently, the issue of production, distribution and composition of natural and processed cheeses is discussed. The conclusion of the theoretical part is focused on the assessment of fatty acids, i.e. their extraction and the determination by gas chromatography. In the experimental part, the fatty acid content of the seven samples of processed cheese was compared on the basis of different temperatures and the time of their production. The ČSN EN ISO 1735 method was used for extraction of fat. The method with methanolic potassium hydroxide solution was used for esterification. The determination of fatty acid methyl esters alone was accomplished by gas chromatography with a flame ionization detector. Based on the comparison with the standards, 17 fatty acids were identified in the samples.
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