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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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RNAi of the a subunit of human translation initiation factor 3 (eIF3).
Peclinovská, Lucie ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Translation initiation is the first step of protein synthesis that captures the flow of gene expression pathway in all living organisms. The advantage of regulation of gene expression at the level of translation initiation is that it allows for more rapid changes in the proteome and serves as the rate limiting step under certain conditions such as stress. This process is masterminded by many initiation factors. One of them, a multisubunit eukaryotic initiation factor 3 (eIF3), is a very efficient player in this field taking a part in the most of the initiation steps. The largest subunit of the eIF3 complex is called eIF3a p170 and TIF32 in mammals and yeast, respectively, and at least in yeast, it was shown to represent an essential constituent of the translational machinery. This work is based on all that has been learned about the eIF3a roles in translation initiation in the model organism of yeast Saccharomyces cerevisiae in effort to examine the degree of the functional conservation with its human ortholog. This is achieved by the RNAi-mediated knock-down of eIF3a in HeLa and HEK cell lines followed by variety of well established assays to monitor translational status of eIF3a depleted cells. In the first part, I describe optimization of the RNA interference protocol with respect to the choice...
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Substrate cleavage by mammalian Dicer isoforms
Kubíková, Jana ; Svoboda, Petr (advisor) ; Pospíšek, Martin (referee)
Host organisms evolved antiviral responses, which can recognize the viral infection and deal with it. One of the frequent signs of viral infection in a cell is appearance of double-stranded RNA (dsRNA). One of the pathways responding to dsRNA is RNA interference (RNAi), which functions as the key antiviral defence system in invertebrates and plants. Mammals, however, utilize for antiviral defence a different dsRNA-sensing pathway called the interferon response. RNAi functions only in mammalian oocytes and early embryonal stages although its enzymatic machinery is present in all somatic cells, where it is employed in the microRNA pathway. A previous study indicated that the functionality of RNAi in mouse oocytes functions due to an oocyte-specific isoform of protein Dicer (DicerO ), which is truncated at the N-terminus. In my thesis, I aimed to assess whether DicerO processes RNAi substrates more efficiently in vitro than the full-length Dicer (DicerS ), which is found in somatic cells. Therefore, I developed Dicer purification protocol for obtaining both recombinant mouse Dicer isoforms of high purity. I examined their activity in a non-radioactive cleavage assay using RNA substrates with structural features characteristic of RNAi substrates. My results suggest that recombinant DicerO and DicerS do not...
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Viannia development in the vector
Hlaváčová, Jana ; Volf, Petr (advisor) ; Dostálová, Anna (referee)
Leishmania of the subgenus Viannia are protozoan parasites transmitted by phlebotomine sandflies (Diptera: Phlebotominae). They occur in tropical and subtropical areas in South America, where they cause cutaneous and mucocutaneous leishmaniasis. In this thesis, we studied developmental pattern of Viannia group and factors affecting its development within the sand fly gut. First, we investigated Leishmania braziliensis development within the Lutzomyia longipalpis digestive tract. Using GFP-labeled strain we demonstrated peripylar development: promastigotes escaped from the endoperitrophic space, colonized the hindgut and then migrated anteriorly. Four morphological forms were found within the Lu. longipalpis digestive tract: elongated nectomonads, short nectomonads, metacyclic promastigotes and paramastigotes. Furthermore, using the histological methods we demonstrated parasite attachment in pylorus region, while there were only free promastigotes in the midgut; neither form was found attached to the midgut epithelium. The next part was devoted to the effect of temperature on Viannia in Lu. longipalpis. We compared development of two closely related species L. peruviana and L. braziliensis at 20 řC and 26 řC. Leishmania braziliensis developed well in both temperatures tested, L. peruviana developed...
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The influence of RDR6 activity and mode of RNAi induction on dynamics and mechanism of silencing of the reporter GFP gene in tobacco cell line BY-2
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Kovařík, Aleš (referee)
RNA interference (RNAi) is a process mediated by small RNAs (sRNA), which is significantly involved in the regulation of gene expression in plants. Diverse RNAi pathways can be divided into two basic mechanisms, which are post-transcriptional and transcriptional gene silencing (PTGS and TGS). Production of sRNAs is dependent on the presence of a double-stranded RNA molecule (dsRNA), which is cleaved by one of DCL proteins to produce sRNAs usually of 21-24 nt in length. One strand of the sRNA is subsequently loaded onto AGO protein. During PTGS, the AGO-sRNA complex interacts with the target RNA based on its sequence complementarity to the sRNA and cleaves it or blocks its translation. In the case of TGS, AGO interacts with plant-specific RNA Pol V and its transcripts, which are again complementary to the sRNA. This interaction allows assembling of a protein complex facilitating DNA and histone methylation inhibiting RNA Pol II transcription. There are numerous ways the dsRNA can arise. A significant part of dsRNA cell production is dependent on synthesising the complementary strand of the dsRNA by RDR6 (RNA-dependent RNA polymerase 6). RDR6 is also involved in the process of the secondary sRNA formation. The significance of RDR6 during PTGS was examined using a GFP reporter gene either during...
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Dynamics and variability of induced transgene silencing in tobacco cell line BY-2
Čermák, Vojtěch ; Fischer, Lukáš (advisor) ; Pečinka, Aleš (referee) ; Lafon Placette, Clément (referee)
RNA interference (RNAi) is an important mechanism regulating gene expression. In plants, RNAi is triggered by double-stranded RNA (dsRNA) which is processed into small RNAs (sRNAs), usually 21-24 nt long. The sRNAs are loaded into Argonaut (AGO) protein and recognize the target based on sequence complementarity. When the target is mRNA, they can slice it or block translation leading to posttranscriptional gene silencing (PTGS). When the target is DNA, they can induce DNA methylation and chromatin changes, which when present in the promoter can lead to transcriptional gene silencing (TGS). The individual components of RNAi are well described, but less is known about the impact of different types of dsRNA precursors on the dynamics of RNAi. To study these aspects of RNAi, we used tobacco BY-2 cell line expressing GFP reporter and inducible silencers. The silencers used different ways of triggering the dsRNA formation by transcripts from antisense (AS), unterminated sense (UT) and inverted repeat (IR) GFP sequence to initiate PTGS. Additionally, one IR silencer based on the CaMV 35S promoter initiated TGS. This allowed us to study RNAi from the beginning throughout the steady state level and till the recovery phase, all in the highly homogeneous system. Using this system, we described several features...
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MicroRNAs encoded by polyomaviruses.
Zachovalová, Veronika ; Bruštíková, Kateřina (advisor) ; Malík, Radek (referee)
MicroRNAs are small regulating molecules of RNA that are encoded by orgamism's genome. Biogenesis of microRNA takes place partly in the nucleus and partly in the cytoplasm. Result of this biogenesis is a 22 nt long microRNA molecule. They are able to silence the genes thanks to sequence- specific degradation of a target mRNA or thanks to the repression of translation of target, complementary mRNA. In mammalian cells the mechanism of translational repression is more common. During this mechanism the microRNA molecule is not entirely complementary to 3'UTR of its target mRNA. Polyomaviruses are small, non-enveloped dsDNA viruses with a circular genome and icosahedral capsid composed of VP1 protein pentamers. These viruses belong in a group called onkoviruses, which can transform infected cells and contribute to development of serious illnesses such as Merkell cell carcinoma. Their genome encodes regulating proteins called T antigens, structural capsid proteins and also microRNAs. My main focus in this thesis will be SV40, MPyV, MCPyV, BKPyV and JCPyV encoded microRNA molecules. Key words: polyomaviruses, small interfering RNA, microRNA, siRNA, RNA interference, mouse polyomavirus, BK virus, JC virus, SV40
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Study of RNAi mechanisms in tobacco BY-2 cell line and potato plants
Tyč, Dimitrij ; Fischer, Lukáš (advisor) ; Kovařík, Aleš (referee) ; Moravec, Tomáš (referee)
Knowledge of the processes of RNA interference, the regulation of gene expression by small RNAs (sRNAs), has grown at an unprecedented rate over the last 30 years. Some of the findings were literally revolutionary, as they revealed events that overturned many long-held notions. Many phenomena have been shown to be highly conserved and common to organisms of different species, but others are specific to certain lineages or have not yet been fully explored. There is also a lack of knowledge about the interconnection of numerous pathways - for example between silencing at the transcriptional (TGS, leading to the promoter methylation) and post-transcriptional levels (PTGS, affecting mRNA stability or translation). The present work summarizes the findings of two published and two unpublished works and attempts to describe some of the less known sites of RNA interference using various plant model organisms. Research on Solanum tuberosum transgenic lines has revealed the ability of 5-azacytidine to restore the expression of transcriptionally silenced transgenes at the whole plant level. De novo regeneration from leaves of such plants can lead to re-silencing of reactivated transgenes and thus serves as a selection method to exclude lines prone to spontaneous silencing. The nature of changes in the...
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Biocompatible protein cages for encapsulation and internatization of small interfering RNA
Mokrý, Michal ; Balvan, Jan (referee) ; Heger, Zbyněk (advisor)
This thesis is focused on creation of apoferritin nanocarrier with encapsulated small interfering RNA marked with fluorescent dye. Main objectives are optimization of pH and amount of siRNA encapsulated into apoferritin cavity and physicochemical characteristics of created nanocarrier. First part deals with theoretical knowledge necessary for understanding concept of this thesis. Second part describes used methods and evaluated results. Created apoferritin nanocarriers were optimal in size with great hemocompatibility, but long-term stability didn’t meet our expectations.
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