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Variability and silencing of transgene expression in potato plants and in tobacco cell line BY-2
Nocarová, Eva ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee) ; Kovařík, Aleš (referee)
Conclusions In suspension cultures of tobacco BY-2 cell line derived from calli after transformation about 90 % of lines contained cells with various GFP fluorescence level after transformation. Newly introduced cloning method allowed obtaining nearly 50 % of clones with homogeneous GFP expression from primarily heterogeneous BY-2 lines. Heterogeneity of GFP expression in transgenic BY-2 lines had two causes - genetic (primary lines contained cells with different T-DNA insertions) and epigenetic one. Epigenetic heterogeneity of BY-2 lines was connected with transgene silencing, formation of stable epigenetic states early after transformation, and "permanent heterogeneity" with fluctuating levels in GFP expression. Reduction or silencing of transgene expression in potato was predominantly observed in lines with higher number of T-DNA insertions and with higher initial GFP expression. Silencing in potato always gradually affected both introduced genes. Silencing of GFP expression preceded (in months) loss of resistance to kanamycin (silencing of NPTII gene) in all monitored cases that indicates interconnections between silencing of both transgenes. The same sequence of silencing of both transgenes in potato was also observed in silenced lines after reactivation of transgene expression by 5-azacytidine, which...
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Silencing of Transposable Elements in Plants and Implications for Rationalised Transgene Design
Tobiáš, Andrej ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
(English) Transposable elements (TEs) are integral parts of all plant genomes. Their ability to change their position (transpose) or copy to a new position and induce mutations within these genomes has driven evolution for billions of years. Despite its importance, unrestrained transposition can lead to irreversible genomic damage. Therefore, plants have evolved special silencing mechanisms to regulate transposition and "silence" TEs. This allows plants to harness the mutagenic potential of TEs, tame their new insertions, and thus potentially acquire genomic novelties. Silencing mechanisms operate mostly at the epigenetic/transcriptional level, where they induce methylation on target DNA or histones. This accounts for RNA-dependent DNA methylation pathways (RdDMs). Additionally, they operate at the post-transcriptional level, where they degrade target mRNA through a pathway known as post-transcriptional gene silencing (PTGS). These silencing mechanisms have also become proficient in recognizing both natural and artificial transgenes. Recognition and subsequent silencing of transgenes diminishes the efficiency of transgenesis and, thus, successful production of genetically modified organisms (GMOs). Extensive research into these silencing pathways and TEs can lead to a better understanding of what...
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Petrol Direct Injection
Moravec, Tomáš ; Janoušek, Michal (referee) ; Dundálek, Radim (advisor)
This bachelor thesis is about the system of direct injection petrol engines within cars. The first chapter is designated to the history of the direct injection petrol system and the development of different concepts. The thesis then concentrates on the mixing of air with fuel and describes the injection system in detail. Within the content of my work is a list of specific engines, using the direct injection system. The last chapter is devoted to the future development of direct injection petrol system and those with the compression-ignition system.
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Design and optimization of injector bracket
Moravec, Tomáš ; Prokop, Aleš (referee) ; Řehák, Kamil (advisor)
The present Master thesis entitled "Engineering Design and Optimization of Injector Holder" deals with the design of clamping holder, whose function is to ensure the exact position of injector system Common Rail. The shape of the injector holder is designed in the modelling program, both with respect to the installation space of internal combustion engine and also the method of manufacture. The proposed geometry is then subjected to stress analysis in the program using FEM. The final design phase is to optimize the geometry of the injector holder using the same method with the aim to reduce the weight of the component.
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AGO-hook domains in RNA-directed DNA methylation in Arabidopsis thaliana
Teznerová, Kateřina ; Čermák, Vojtěch (advisor) ; Moravec, Tomáš (referee)
RNA-directed DNA methylation (RdDM) is an important pathway that regulates gene expression by inducing DNA methylation and is involved in regulation of gene expression and defence against invading DNA elements (especially transposons). Argonaut (AGO) proteins with small RNAs (sRNAs) that have sequence complementarity to the target DNA play a key role in the RdDM pathway. Domains called AGO-hooks are able to interact with Argonaut proteins. In plants, two proteins with AGO-hook domains are involved in the RdDM pathway: NRPE1 and SPT5L. Recently, a third protein, SPT6L, has been discovered at the investigator's site to be part of the Pol V complex (as well as the two proteins mentioned above). The role of SPT6L has not yet been described, but we hypothesize that it also plays a role in the RdDM pathway. This work focuses on the study of all three AGO-hook domains in Pol V complex and their involve in the RdDM pathway in Arabidopsis thaliana, from the preparation of mutants lacking different combinations of these AGO-hook domains to the study of their role and substitution in DNA methylation at different loci. Key words AGO-hook, Arabidopsis thaliana, NRPE1, SPT5L, SPT6L, siRNA, epigenetic chromatin labelling, Argonaut protein
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RNA directed DNA methylation in Arabidopsis thaliana
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The differential transcriptional activity of the genome is provided by epigenetic modifications, which include DNA methylation, alteration of histone N-terminal amino acids and changes in histone variants. RNA interference is a regulatory process, in which transcriptional or post-transcriptional silencing of exogenous or endogenous sequences is mediated by the action of small RNAs derived from these sequences. The 24-nucleotide siRNAs, forming a fraction of small RNAs, direct de novo DNA methylation and participate in the maintenance of DNA methylation (RNA-directed DNA methylation; RdDM), which facilitates transcriptional silencing of heterochromatin and transposable elements representing a large part of plant genomes. The presence of two RNA polymerases involved in this pathway is characteristic for flowering plants, which were discovered for the first time in the genome of Arabidopsis thaliana, which has also become the main plant model for the study of RdDM. Polymerase IV transcribes siRNA precursors; siRNAs are subsequently associated with AGO4 proteins and guide methylation enzymes to the target sequences via complementarity with polymerase V transcripts.
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Study of the mechanism of posttranscriptional and transcriptional transgene silencing in tobacco BY-2 cell line
Čermák, Vojtěch ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The RNA interference is a mechanism, which allows cells to regulate their genes functions, to establish and maintain heterochromatin and to defend them against invasive nucleic acids. In plants, RNA interference is initiated by double-stranded RNA, which is processed by Dicer into small RNAs, usually 20-24nt long. These small RNAs form a complex with Argonaut protein that participates in different processes based on sequence complementarity. This complex can guide mRNA cleavage, translation blocking and chromatin modifications, resulting either into posttranscriptional silencing (by preventing translation of already existing mRNA, PTGS) or transcriptional silencing (by preventing transcription of mRNA, TGS). The first step of this thesis was to establish different ways of triggering PTGS and to evaluate their functionality and efficiency. The next step was a preparation of a system which would allow to study the transition from posttrancriptional to transcriptional silencing. These so called "indicator lines" should allow to observe the timing and dynamics of this process by utilizing fluorescent proteins. This system is also going to enable to evaluate, how different factors are involved in this process - one of the factors is RNA-dependent RNA polymerase 6 (RDR6) which plays an essential role in...
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Insolvency regulation 1346/2000/ES - COMI
Moravec, Tomáš ; Brodec, Jan (advisor) ; Dobiáš, Petr (referee)
The thesis deals with the issue of COMI and the term of COMI in European insolvency regulation. It concerns on set of COMI for both natural and legal person and fact on that the fiction of COMI could be rebutted. In the thesis is analysed also judicature of European court of Justice and national court which concern to the COMI. Also is dealt with draft of European insolvency regulation. Further the thesis focus on the problem of forum shopping and the possibility of COMI shift to another member state. Also thesis concerns with the set the COMI by company groups.
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In planta production of TMV (Tobacco mosaic virus) nanoparticles of specific length
Dlabalová, Lucie ; Moravec, Tomáš (advisor) ; Šafářová, Dana (referee)
Tobacco mosaic virus (TMV) is one of the most investigated viruses and its attributes and structure are therefore well-known. In this work, we have chosen TMV as a biotemplate for the adjustable-length particles production in plants. The viral RNA and coat protein of TMV self-assemble into particles under physiological conditions. The particle length depends on the length of packaged RNA. The encapsidation signal that is necessary for preferential viral RNA packaging by coat protein disks is known and characterized since the 1980's. In this work, we have proposed a two-component system based on a Nicotiana bentamiana plants infection with packaging competent defective RNA (dRNA) and a helper virus RNA which provides all the components necessary for dRNA replication and packaging. The encapsidation signal in the helper virus sequence was removed to avoid formation of particles of incorrect length. Some of our helper viruses contained a coat protein with modified region of the particle's inner channel. This modification should allow specific binding of metal atoms within the core of the rod shaped particle. Several variants of dRNA and helper viruses were prepared to identify individual areas important for the replication, encapsidation and nanoparticle stability. We focused on the particle formation...
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