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Epigenetic factors CTCF a SMARCA5 control expression of hematopoietic transcription factor SPI1 in cells of acute myeloid leukemia and myelodysplastic syndrome.
Dluhošová, Martina ; Stopka, Tomáš (advisor) ; Machová Poláková, Kateřina (referee) ; Kozák, Tomáš (referee)
CCCTC-binding factor (CTCF) can both activate as well as inhibit transcription by forming chromatin loops between regulatory regions and promoters. In this regard, Ctcf binding on the non-methylated DNA and its interaction with the Cohesin complex results in differential regulation of the H19/Igf2 locus. Similarly, a role for CTCF has been established in normal hematopoietic development; however its involvement, despite mutations in CTCF and Cohesin complex were identified in leukemia, remains elusive. CTCF regulates transcription dependently on DNA methylation status and can if bound block interactions of enhancers and promoters. Here, we show that in hematopietic cells CTCF binds to the imprinting control region of H19/Igf2 and found that chromatin remodeller Smarca5, which also associates with the Cohesin complex, facilitates Ctcf binding and regulatory effects. Furthermore, Smarca5 supports CTCF functionally and is needed for enhancer-blocking effect at imprinting control region. We identified new CTCF-recognized locus near hematopoietic regulator SPI1 (PU.1) in normally differentiating myeloid cells together with members of the Cohesin complex. Due to DNA methylation, CTCF binding to the SPI1 gene is reduced in AML blasts and this effect was reversible by DNA methylation inhibitor 5-azacitidine.
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Production and secretion of virulence factors in Bordetella pertussis
Držmíšek, Jakub ; Večerek, Branislav (advisor) ; Petráčková, Denisa (referee)
Bordetella pertussis is a strictly human pathogen and causative agent of infectious respiratory disease called whooping cough. In order to establish successful infection and colonization of the host, B. pertussis uses a broad spectrum of virulence factors such as adhesins (filamentous hemagglutinin, pertactin, and fimbriae) and toxins (adenylate cyclase and pertussis toxins). In addition, the type 3 secretion system (T3SS) was also found in the genus Bordetella. In connection to our previous characterisation of B. pertussis strain lacking the gene encoding RNA chaperone Hfq (Δhfq), which proved that Hfq is required for T3SS functionality, the recombinant T3SS proteins BopB, BopD, BopC and BopN were purified to homogeneity. Next, the specific antibodies were obtained using purified recombinant proteins in order to study the production of the T3SS components in B. pertussis. Using refined anti- BopC antibodies it was for the first time shown that laboratory-adapted B. pertussis strain secretes BopC protein into medium. The recombinant translocators BopB and BopD were also used to examine their pore-forming activity using planar black lipid membranes. Based on the characterisation of hfq deletion mutant, having impaired production of membrane proteins when compared to the wild type, mass spectrometry...
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BacSeqer: Read simulator for bacterial RNA-Seq
Fialová, Adéla ; Sedlář, Karel
The RNA-Seq method has become a key tool in transcriptomics, providing deep insight into the genetic functioning of organisms. It is essential for understanding how genes are expressed, for discovering new gene variants and for understanding pathologies at the genetic level. RNA-Seq simulators are great for testing and validating bioinformatics tools, they offer a way to compare different computational algorithms and allow examining different sequencing protocols, parameter settings and sample sizes to ensure the most efficient use of resources. This study introduces BacSeqer, an RNA-Seq simulator specifically designed for bacterial transcriptomes with the aim of producing data that faithfully resembles those obtained in real experiments. It allows to take into account the specificities of the bacterial genome, especially an existence of only exons and their organization in operons that are not normally present in eukaryotes.
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Lizard growth and the ontogeny of sexual size dimorphism
Meter, Brandon ; Starostová, Zuzana (advisor) ; Kupfer, Alexander (referee) ; Vrtílek, Milan (referee)
Růst byl u plazů historicky považovaný za neukončený. Tento obecně sdílený předpoklad byl v posledních letech zpochybněn a narůstaly důkazy o ukončeném růstu u ještěrů. Tento obrat způsobila především analýza růstových plotének kostí, která ukázala jejich uzavření a tím nemožnost dalšího prodlužování kostí, což způsobuje dosažení konečné velikosti těla. Paralelně s touto změnou v nahlížení na ukončený růst byly zpochybněny i dlouhodobé předpoklady o zásadní přímé roli energetického rozpočtu a s ním spojených trade-offs v ovlivňování růstu. Tento posun v perspektivě ovlivňuje nejen naše chápání individuálního růstu, ale také se odráží ve studiu souvisejícího znaku - pohlavního dimorfismu ve velikosti (SSD) - rozdílu ve velikosti mezi pohlavími v rámci druhu. Existují tři hlavní hypotézy o příčinách SSD u plazů: pohlavně-specifické náklady na reprodukci, ovlivnění růstu samčími gonadálními androgeny a ovariálními hormony. Tato práce přináší další důkazy o ukončeném růstu u ještěrů a diskutuje jeho důsledky pro evoluci SSD. Review o růstu zaměřené na gekona Paroedura picta, kde jsou větší samci než samice, zdůrazňuje značnou kanalizovanost růstu a současně poskytuje základ o úvahách týkajících se proximátních příčin SSD. V této práci dále předkládám podporu pro pravděpodobnou roli ovariálních hormonů...
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Long term sublethal stress in colonies of Saccharomyces cerevisiae deleted in WHI3
Krampotová, Ester ; Schierová, Michaela (advisor) ; Dušková, Michala (referee)
The yeast species Saccharomyces cerevisiae is a suitable model for studying many biological processes, including adaptation to stress conditions. Over the past 40 years, multiple signaling pathways have been uncovered to play an important role in the response to acute stress induced by high temperature, osmotic stress, or high concentrations of metals in S. cerevisiae. The effect of sublethal dose of stressor is rather neglected. The advantage of the WHI3 deletion strain used in this work is its sensitivity to the presence of stressor in the solid medium not only by reducing the growth rate but also by changing the colony morphology. The Whi3 protein stimulates not only flocculin Flo11 production but also the activity of the general transcription factors Msn2 and Msn4. The diploma project aimed to determine whether changes in the expression of genes coding for stress proteins induced by sublethal doses of the stressor were dependent on the genotype WHI3, YAP6, and MPT5. Transcription factor Yap6 and RNA binding protein Mpt5 are both involved in stress response and cause repression of FLO11. Their levels depend on Whi3. To induce mild stress, low concentrations of NaCl and CdSO4 have been applied to colonies for 4 and 6 days. Their genotype varied in the WHI3, YAP6, and MPT5 genes. Our experiments...
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Read simulator for bacterial RNA-Seq
Fialová, Adéla ; Jurečková, Kateřina (referee) ; Sedlář, Karel (advisor)
This thesis is dedicated to the analysis of the bacterial genome, measurement of its expression mainly by RNA-Seq technology and simulation of RNA-Seq data. The first part of the thesis provides a theoretical background on the structure of the bacterial genome, its expression and methods to study it, including modern sequencing techniques. Several selected simulators of RNA-Seq data are also mentioned. In the second part, the actual simulator implementation is presented, designed to take into account the characteristics of the bacterial genome, in particular the presence of operons.
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Identification and analysis of regulon’s structures in Arabidopsis thaliana
Janigová, Patrícia ; Bartoň, Vojtěch (referee) ; Schwarzerová, Jana (advisor)
This bachelor thesis focuses on the identification and analysis of regulon structures in a model organism from the plant kingdom Arabidopsis thaliana. The first part of the thesis deals with the regulation of gene expression in eukaryotes, techniques for inferring transcriptional units and tools for the analysis of these units. Subsequently, the laboratory data from Arabidopsis thaliana, including their preparation and preprocessing, are described in detail. The next section describes the process of deriving regulons using specific methods such as correlation coefficient and mutual information. This is followed by a detailed description of a custom algorithm for inferring potential regulon structures in Arabidopsis thaliana, which is implemented in Python. In the conclusion of the bachelor thesis, the obtained results are visualized, evaluated and compared with the available literature. The identification of potential MBF1 and ABI3 regulons is also discussed in detail, thus confirming the correctness and efficiency of the proposed approach.
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Use of molecular techniques for study of thermophilles
Dvořáková, Dominika ; Kouřilová, Xenie (referee) ; Buchtíková, Iva (advisor)
Polyhydroxyalkanoates are microbial storage polymers that represent a green alternative to petrochemical plastics. However, their high production cost limits wider industrial utilization. Use of waste materials as a substrate and/or utilization of extremophilic organisms is an option to reduce production costs. The strain of thermophilic bacteria Aneurinibacillus appears to be a promising producer because of its ability to synthesize wide range of non traditional copolymers. In addition, PHA synthesis is independent of nutrient limitation, which was the subject of this study. Experiments were focused on the expression of genes involved in PHA synthesis in the bacterium Aneurinibacillus sp. AFn2 on different types of production media. The presence of selected genes was verified using classical PCR. After that the cultivation experiments were performed on mineral and complex media to simulate environments with different nutrient availability. Biomass concentration was determined gravimetrically from the samples collected in time during the growth curve measurement. PHA content was measured simultaneously using gas chromatography. Finally, the expression of the studied genes was analysed by RTqPCR. Significant differences were observed in the transcription of individual genes depending on the media type and time. The results confirmed the ability of Aneurinibacillussp.AFn2 to produce PHA independently on nutrient limitation, however, the production rate and cell filling depended on the media type. This fact may be related to the different course of expressed genes involved in PHA synthesis. A higher percentual yield of P(3HB) in cells were observed in complex media along with an increasing trend in the overall gene expression. On the other hand, in mineral medium, the transcription of individual genes only decreased after initial strong expression, and the cell filling of P(3HB) was also lower. The findings lead to understanding not only the expression of selected genes but also the overall synthesis of PHA. At the same time, it offers the way to optimize production with the aim of reducing cost and expanding their market applications.
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A tool for prediction of small RNA in RNA-Seq data
Pomykalová, Barbora ; Čejková, Darina (referee) ; Jurečková, Kateřina (advisor)
This diploma thesis focuses on the detection of small RNA (sRNA) in the bacterial genome. sRNAs are short non-coding transcripts that play a key role in gene expression. To date, there are several algorithms focusing on the detection of sRNAs from RNA-Sequencing (RNA-Seq) data that can be obtained by some of the sequencing platforms. The most frequently used platforms are Illumina and Ion Torrent belonging to the next generation sequencing and PacBio with Oxford Nanopore belonging to the third generation of sequencing. In this work, the workflow of sRNA detection using freely available tools was described and then an own unique tool for sRNA detection – the SEARCHsRNA tool – was designed. Two open-source software tools – Rockhopper and DETR'PROK, together with newly created tool, were tested on RNA-Seq data for bacteria Vibrio atlanticus LGP32.
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Studying the dynamics of gene expression and the role of NAC complex in male gametophyte development
Klodová, Božena ; Fíla, Jan (advisor) ; Fajkus, Jiří (referee) ; Hudzieczek, Vojtěch (referee)
This Ph.D. thesis presents a comprehensive study of gene expression regulation within the male gametophyte development focusing on transcriptional, translational, and post- translational levels. The research introduces an online tool, GOLEM, designed for the visualization and analysis of motif distribution within the DNA of various plant species. Utilizing RNA-seq data, GOLEM enables the study of gene expression across different tissues and developmental stages, as well as a comparative analysis across the evolution of plant lineages. Through an integrated multi-omics approach, combining transcriptomic and proteomic data, the thesis enriches the understanding of gene expression dynamics in male gametophyte development, identifying significant trends and categorizing gene families based on their expression patterns. This multifaceted dataset provides a valuable resource for future functional genomics studies. Additionally, prospects and challenges of studying gene expression regulation in male gametophyte are discussed. Furthermore, the thesis explores the regulatory potential of the nascent polypetide- associated complex (NAC) protein family in male gametophyte development, particularly their role in translation during pollen tube growth. Experiment results indicate that NACβ subunit knock-down...
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