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Molecular characterization of selected PHA producers
Kubáčková, Eliška ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.
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Study on gene expression in Cupriavidus necator and other selected polyhydroxyalkanoates producers
Centnerová, Radmila ; Šedrlová, Zuzana (referee) ; Pernicová, Iva (advisor)
The aim of this bachelor thesis was study on gene expression in bacterium Cupriavidus necator H16 that is known as a model bacterium for the metabolism of polyhydroxyalkanoates. In the first part of this thesis, the optimalization of RT-qPCR method was performed. The optimized method was implemented on the study on gene expression. Furthermore, there were tested several commercial isolation kits for the genomic DNA isolation, the RNA isolation and the reverse transcription of the RNA and synthesis of the complementary DNA. These kits were compared in order to choose the one that would have provided the most relevant results and also the kit handling would have been simple and safe. There were different results accomplished for all kits. This means the kit used for the isolation had unneglectable impact on the quality of the isolated nucleic acid and therefore also on the success of the whole measurement. Isolated genomic DNA was used for optimalization and calibration. Isolated RNA and complementary DNA were used in the second part of the thesis. In this part, the studied bacterium was cultivated under various conditions and carbon sources. Subsequently, the optimized RT-qPCR method was performed and used for study on gene expression of chosen genes involved in the biosynthesis of polyhydroxyalkanoates. There were more significant differences in gene expression observed for fructose as a carbon source, compared to -butyrolacton as a carbon source. The greatest increase of the gene expression for fructose as a carbon source was measured for gene encoding 4-hydroxyphenylacetate-3-hydroxylase. There were more considerable differences in gene expression observed for -butyrolacton as a carbon source only for gene encoding 4-hydroxybutyrate dehydrogenase. Therefore, the choice of the carbon source impacts fundamentally the gene expression.
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Potenciální vliv tkáňově specifických transkripčních faktorů na expresi metalothioneinů
Michálková, Nikola
Metallothioneins represent a group of intracellular proteins capable of binding metal ions, and their function includes the regulation of cellular levels of these metal ions. Transcription factors are central regulators of gene expression and play an important role in regulating the expression of metallothionein genes, which represents the basic mechanism for the cellular response to changes in metal ion levels. Although transcription factors that bind to the promoter regions of metallothioneins are known, their role in inducing the expression of metallothionein genes is far from clear. The subject of this thesis was the induction of gene expression of metallothioneins by zinc ions in HUH7 (hepatocellular carcinoma) and T47D (breast cancer) cell lines, and the subsequent analysis of gene expression of selected transcription factors. The selection of transcription factors was based on TF ChIP-seq analysis of ENCODE database. For the experimental part, the transcription factors FOXA1, FOXA2, HNF4A, and HNF4G were chosen, whose high levels are typical for liver tissue. Furthermore, gene expression of the ubiquitinated transcription factors JUND, NR3C1, RXRA, STAT3, SP1, and MTF1 was monitored. Statistically significant changes in gene expression (RT-qPCR) after short-term exposure (4-6 h) to 100 μM ZnSO4 were observed for FOXA1, JUND, and MTF1. In the case of FOXA1, a decrease in gene expression was observed in both tumor lines compared to the control, whereas an increase in expression was observed in JUND and MTF1 after ZnSO4 treatment. In the case of MTF1, a significant increase was observed only in the HUH7 tumor line. The results show that, in addition to the validated zinc-sensitive transcription factor MTF1, the transcription factors FOXA1 and JUND could also be involved in the regulation of metallothionein gene expression. FOXA1 could represent a potential repressor and inducer of JUND metallothionein expression in response to increased exposure to zinc ions.
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Post-transcriptional regulation of TbIF1 in life cycle of Trypanosoma brucei
GRATZL, Sascha
TbIF1, a protein Inhibitor of F1-ATPase in Trypanosoma brucei, is expressed exclusively in the insect stage of the parasite. In the bloodstream form, TbIF1 is switched off, because its activity interferes with the essential role of the ATP synthase in the maintenance of the mitochondrial membrane potential. Here, we employ a series of reporter genes to study the impact of 3'UTR of TbIF1 on mRNA stability and translatability to get insight into the tight post-transcriptional control of TbIF1. We provide evidence that developmentally regulated RNA binding protein Rbp10 is critical for downregulation of TbIF1 on translation level in bloodstream-form trypanosomes.
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Study on gene expression in Cupriavidus necator and other selected polyhydroxyalkanoates producers
Centnerová, Radmila ; Šedrlová, Zuzana (referee) ; Pernicová, Iva (advisor)
The aim of this bachelor thesis was study on gene expression in bacterium Cupriavidus necator H16 that is known as a model bacterium for the metabolism of polyhydroxyalkanoates. In the first part of this thesis, the optimalization of RT-qPCR method was performed. The optimized method was implemented on the study on gene expression. Furthermore, there were tested several commercial isolation kits for the genomic DNA isolation, the RNA isolation and the reverse transcription of the RNA and synthesis of the complementary DNA. These kits were compared in order to choose the one that would have provided the most relevant results and also the kit handling would have been simple and safe. There were different results accomplished for all kits. This means the kit used for the isolation had unneglectable impact on the quality of the isolated nucleic acid and therefore also on the success of the whole measurement. Isolated genomic DNA was used for optimalization and calibration. Isolated RNA and complementary DNA were used in the second part of the thesis. In this part, the studied bacterium was cultivated under various conditions and carbon sources. Subsequently, the optimized RT-qPCR method was performed and used for study on gene expression of chosen genes involved in the biosynthesis of polyhydroxyalkanoates. There were more significant differences in gene expression observed for fructose as a carbon source, compared to -butyrolacton as a carbon source. The greatest increase of the gene expression for fructose as a carbon source was measured for gene encoding 4-hydroxyphenylacetate-3-hydroxylase. There were more considerable differences in gene expression observed for -butyrolacton as a carbon source only for gene encoding 4-hydroxybutyrate dehydrogenase. Therefore, the choice of the carbon source impacts fundamentally the gene expression.
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Genetické aspekty domestikačního znaku pukavosti lusku u hrachu
Čevelová, Lucie
The aim of this thesis is the study of the genetic substance of the important domestication sign pod dehiscence. Two types of pea were analyzed, with indehiscence pods JI92 (Pisum sativum subsp. sativum) and wild field pea with dehiscent pods JI64. (Pisum sativum subsp. elatius). By reciprocal crossbreeding of these two lines, were created recombinant inbred lines (RILs), of a total of 134 RILs lines were selected with 9 contrast lines. We utilized the massive parallel sequencing of the 3'ends of the cDNA, obtained by reverse transcription of mRNA isolated from the seam. Thanks to this method, 3 candidate genes were generated. Subsequently, we determined the expression of these three candidate genes for the using quantitative Real-Time PCR (RT-qPCR). Amplification curves and Ct values generated from the RT-qPCR were subsequently used to generate graphs to show the degree of expression of the candidate genes. The most suitable candidate was the Ps15 gene, which is present in LGIII in the Dpo1 region, and therefore could be responsible for pod dehiscence.
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Vliv mírného teplotního stresu na expresi genů metabolismu cytokininů a kinetiku růstu klíčních rostlin Arabidopsis thaliana
Truong, Thanh Huong
Climate change and thermal stress poses a problem for sustainable agriculture. Research in the adaptation and acclimation of plants to the elevated temperature is therefore one of the current scientific issues. Plants are exposed to different ambient temperatures during their life. The response to adverse temperatures includes a number of signalling pathways affecting development and growth processes in plants. Coordination of develomental processes under elevated temperature and other external factors is primarily controlled by plant hormones. Here effect of cytokinins on plant morphology, growth kinetics, gene expression and quanification in combination with increased temperature was observed. By determination of the hypocotyl growth during increased temperature, cytokinins were found to play important role in this process. Cytokinins were found to inhibit temperature induced hypocotyl growth and inversly seedlings treated with higher temperatutre showed decreased level of cytokinins which was confirmed on the level of marker gene expression and determination of levels of cytokinins.
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Role cytokininů při teplotou indukovaném přechodu rostlin z vegetativní do generativní fáze
Malých, Veronika
Recent numerous studies have been focused on the impact of the climate changes on the crop production. The response of plants to increased temperature include hypocotyl and petiole elongation, leaf hyponasty, alternations in timing of the flowering and even the inhibition of the flower bud development which may lead to reduced crop yields. Acclimation and stress responses are co-regulated by levels of plant phytohormones such as auxins, gibberellins, brassinosteroids and cytokinins. Although cytokinins play important role in physiological and developmental processes the effect of the interplay of temperature and cytokinin in growth and developmental processes has not been studied till now. This thesis is focused on the impact of cytokinins on thermomorphology and time of flowering of Arabidopsis thaliana in combination with increased temperature under long day conditions. It was observed that modulation of cytokinin levels reduce biomass and area of leaf rosette and this effect is dependent on temperature. As next, it was confirmed that interactions of cytokinins and temperature play important role in transition to generative developmental stage of plant Arabidopsis.
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Molecular characterization of selected PHA producers
Kubáčková, Eliška ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.
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Comparison of methods of isolating double-stranded RNA from plant tissue with a focus on viral fraction yield.
MATYÁŠOVÁ, Alena
High-throughput sequencing is one of methods used for diagnostics of plant pathogens and has advantage of unspecific unbiased detection of all nucleic acids present in a sample. Input material for HTS can be prepared using by different approaches that reflect purpose of the planned task. During viral infections of plants, viral double stranded RNAs are generated as replication intermediates or transcription products. Thus, they are often used for HTS. Nevertheless, such preparations contain large amount of plant RNAs. The work aimed for comparison of two methods of double stranded RNA enrichment - widely used cellulose chromatography and differential centrifugation with lithium chloride. Both qualitative and quantitative profiles of viral nucleic acids were estimated. An isolate HZ2 of red clover (Trifolium pratense L.) was used for the study. Previously, there were detected eight different RNA viruses with HTS-aimed analyses in the plant. To compare qualitative profile, RNA was extracted by each method, transcribed into cDNA, and specific viral fragments were amplified using PCR, followed by agarose gel electrophoresis. Quantitative profiles were analyzed using three selected viruses with single- and double-stranded RNA genomes. Their relative quantification was estimated using RT-qPCR approach. Further, normalized (to 26S rRNA as a reference) expressions were calculated using Bio-rad CFX Manager 3.1 software. All eight viruses were successfully detected using RNA material obtained by each method. After evaluating the quantification data and performing statistical tests (F-test, T-test; with a significance level of = 0.05), no significant difference was found between the compared methods.
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