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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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Study of Ectonucleotidases and Adenosine Deaminases in Drosophila
PREUER, Kristina
Extracellular adenosine triphosphate and extracellular adenosine are important regulatory molecules in the human immune system. The concentrations of these molecules are in turn regulated by ectonucleotidases and adenosine deaminases. In this thesis I attempt to test the gene silencing efficiency of RNA interference for three different genes coding for such enzymes in the model organism Drosophila melanogaster.
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Transcription factor PU.1 is a target of 5-azacitidine during differentiation therapy of myelodysplastic syndrome
Čuřík, Nikola ; Stopka, Tomáš (advisor) ; Kleibl, Zdeněk (referee) ; Trka, Jan (referee)
PU.1 is a key hematopoietic transcription factor. Knock-out of PU.1 in mouse is embryonic lethal due to complete depletion or several disruption of differentiation of multiple blood cell lineages. Low level of PU.1 and the disruption of its regulation are associated in vivo with acute myeloid leukemia and other hematologic malignancies. Myelodysplastic syndrome (MDS) is hematopoietic stem cell disorder with extremely heterogeneous features and outcome. It is characterized by improper differentiation of blood cells resulting in loss of function, dysplasia and blasts accumulation in bone marrow. About one third of MDS cases transforms into AML. MDS is also characterized by silencing of gene expression caused by aberrant DNA hypermethylation. Using DNA Methyltransferase inhibitors (DNMTi) such as 5-azacitidine (AZA) has good clinical results for the MDS patients with higher risk of disease. Indeed, AZA became standard therapy of high risk MDS in recent years. Nonetheless, our understanding of molecular mechanisms of AZA remains incomplete. This PhD thesis reports about the role of transcription factor PU.1 in MDS. We found that significant subset of high risk MDS patients express low level of PU.1 due to DNA hypermethylation of PU.1 upstream regulatory element (URE). We also found significant...
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Yeast gene silencing.
Tarabová, Eva ; Kuthan, Martin (advisor) ; Schierová, Michaela (referee)
Each cell contains a complete copy of the entire genetic equipment of the organism. However not all genes are expresed, cells are differentiated in higher eukaryots and only certain proteins are transcribed in each cell. This is possible thanks to a gene silencing, that is stable throughout the whole cell cycle and epigeneticaly inherited from one generation to another. Gene silencing serves also in the maintainance of the chromosomal integrity, it is connected with the right progression of the cell division. It even enables mating type switching and ensures right cells' identity in yeasts. The basis is compact and a higher-ordered structure of chromatin called heterochromatin. The mechanism is common to many various organisms, although the proteins, which ensure silencing, are different.
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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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Transcriptomics of bovine preimplantation embryo genome activation in vivo and in in vitro culture conditions
Vodičková Kepková, Kateřina ; Kaňka, Jiří (advisor) ; Petr, Jaroslav (referee) ; Krylov, Vladimír (referee)
The goal of the thesis was to characterize transcriptional profiles of in vivo and in vitro derived embryos during bovine minor and major embryonic genome activation and to identify mRNA transcripts newly synthesized during these stages. In our first work we have concentrated on the study of minor genome activation at the 4-cell stage of embryo. Using SSH, we have identified 31 amplicons homologous with already identified genes. We have selected 5 of these for detailed study of their expression during the whole period of preimplantation development: centromere protein, 350/400 kDa (CENPF, mitosin), splicing factor arginine/serine-rich 3 (SRFS3), high mobility group nucleosomal binding domain 2 (HMGN2) protein and eukaryotic translation initiation factors EIF4A2 a EIF4E. All these genes play an important role in the early embryo development. SRFS3 is the first described gene with an important function in preimplantation development, which is expressed already during bovine minor genome activation, and its transcription is α-amanitin sensitive during this period. We have selected CENPF gene for a more thorough study. By silencing its expression by the injection of CENPF dsRNA into the zygote, we have studied its function throughout the whole preimplantation development of bovine embryo....
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Methylation profile in malignancy
Stojčeva, Nina ; Vodička, Pavel (advisor) ; Vondrejs, Vladimír (referee)
Epigenetic changes represent chemical modifications of the DNA molecule and histone proteins by which gene expression is altered. Among them, DNA methylation is a known mechanism of silencing of tumor-suppressor and DNA repair genes, with an important role in carcinogenesis. Many studies have been done in order to identify the methylation signatures of these genes in different types of cancer. In our study, we investigated the methylation status of promoter regions of eight mismatch repair genes (MLH1, MSH2, MSH3, MLH3, PMS1, PMS2, MSH6 and EXO1) in 45 sporadic colorectal cancer cases and 12 head and neck cancer patients. Two out of eight genes, MLH1 and MLH3, exhibited promoter methylation. The results from both groups of patients were concordant. We summarize that the methylation profiles of MLH1 and MLH3 promoters could be potential candidates for epigenetic biomarkers in colorectal cancer, and eventually in head and neck cancer. Further investigations, which would confirm this theory, should be carried out.
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The occurrence of Coccidiosis in the intestine of sucking pigs before and after weaning
KOTILOVÁ, Jiřina
In two years of observation, (spring 2006, autumn 2006, spring 2007 and autumn 2007) were being screened for parasites in total 495 faecal samples coming out of three farms from Ceske Budejovice (285 samples of sucking pigs not older then 28 days and 174 samples of piglets not older then 8 weeks). The method used to examine those samples was a flotation-concentrating method (Sheather{\crq}s carbohydrate fusion) and in the year of 2007 was also used a specific aniline-carbol-methyl violet staining method to detect the Cryptosporidium spp. followed by positive molecular characterized (direct sequencing of partial SSU rRNA partial genes and PCR-RFLP at the SSU rRNA). In screened samples were mainly detected parasites named Cryptosporidium spp., found in 4,1% of faecal samples in 2006 and in 2007 in 32,8% faecal samples, out of which 14,4% was found in pre-weaned piglets samples and 26,4% in post-weaned piglets. Based on genotyping provided on positive samples out of the year 2007, using method of sequensing analysis SSU rRNA, was the occurence of C. suis, Cryptosporidium pig genotype II aC. Muris described. High prevalence of Isosporou suis 13,9 % (64/459) was also detected with its appearance, in particular, in pre-weaned piglets 21,4 % (61/285). Further on, some of other identified group was Eimerie spp. 5,7 % (26/459) infecting, in the main, post-weaned piglets 10,9% (19/174) and Giardia intestinalis 2,4 % (10/459). Most of the samples mentioned occured in conditioned faeces and there is no seasonal relationship to the parasital occurance.
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Side-effects of siRNA - induced gene silencing
Feřtek, Marina ; Štaud, František (advisor) ; Nachtigal, Petr (referee)
Vedlejší účinky siRNA byly testovány na buněčných liniích po "umlčení" genu kódujícího cyklooxygenázu. Teoretickým základem tohoto experimentu byl poznatek, že siRNA mohou vyvolat imunitní odpověď. Z tohoto důvodu byla testována exprese IFN-indukovaných genů v intaktní Hep2 buněčné linii a ve třech liniích odvozených od Hep2 buněk, kde byla různými metodami vnesena siRNA. Buněčné linie byly odvozeny z Hep2 pomocí různých metod transfekce siRNA (tj. nuzkleofekce, effectene technologie a klonování). RNA byla izolována z těchto buněčných linií a cDNA byla získaná reverzní transkripcí. Genová exprese byla měřena pomocí QRT PCR a kvantifikována jako počet kopií/mikrogram RNA. Stupeň exprese byl porovnáván s intaktní linií Hep2. Jako houskeeping gen byl použit NUP. Výsledky byly vyhodnoceny pomocí softwarů REST verze 2 a rotoru Gene verze 6. Mělo být testováno devět IFN-indukovaných genů, RNL, PKR, IRF1, IRF3, IFITM, IFIT1, OAS1, OAS2 a OAS3. Z nich OAS1 a OAS2 nebylo možné izolovat a tedy měřit. Exprese PKR byla up-regulovaná; exprese všech ostatních testovaných genů byly down-regulovány. Další rozsáhlé studie jsou nezbytné pro vysvětlení těchto výsledků.
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