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The development and maintenance of neurons in the inner ear
Wirthová, Natália ; Pavlínková, Gabriela (advisor) ; Pysanenko, Kateryna (referee)
Hearing loss is among the most frequent disabilities. Neurosensory hearing loss is permanent and results from the death of neurons or sensory cells, which have little ability to regenerate in the inner ear (cochlea). Identifying the genes that are associated with generating differentiated and functional sensory cells, neurons, and with hearing loss could provide tools for neurosensory regeneration therapy and preventive measures. Recent data suggest that the prevention of neuronal loss and enhancement of long- term maintenance of neurons are the most important targets for the immediate future. This work is focused on transcription factors and signaling pathway networks that regulate the development and maintenance of neurons in the inner ear.
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Differentiation of pancreatic stem cells into insulin producing β-cells.
Leontovyč, Ivan ; Saudek, František (advisor) ; Štechová, Kateřina (referee) ; Holáň, Vladimír (referee)
Diabetes mellitus (DM) is a severe and frequent disease with increasing prevalence. It is not possible to achieve long term cure without late complications. Recent advances in cell fate modifications open a pathway to alternative cell therapies for DM cure. My doctoral thesis "Differentiation of pancreatic stem cells into insulin producing β- cells" is focused on the development of a new source of insulin secreting cells for transplantation. Combinatorial testing of numerous potential transcription factors and epigenetic modifiers resulted in a final protocol for the reprogramming pancreatic of exocrine cells into insulin secreting cells. The key transcriptional factors TF (Pdx1, Ngn3 a MafA) were applied in the form of synthetic mRNA. In four independent experiments we applied transcriptional factors in a specific sequence, thus obtaining 14.3 ± 1.9 % insulin positive cells. When challenged in vitro by the glucose levels of 2.5 and 20 mmol/l glucose, respectively, these cells exhibited glucose-sensitivity of insulin secretion (842 ± 72 and 1 157 ± 58 pg insulin/µg DNA/ml, n=5). They also demonstrated a sensitivity of insulin secretion (863 ± 78 and 1 025 ± 66 pg insulin/µg DNA/ml, n=5) to the concentration of depolarization agent KCl applied at 0 and 30 mmol/l, respectively together with 2.5...
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Comparison of apo- and holoforms of the transcription factor "Bach1"
Vávra, Jakub ; Martínková, Markéta (advisor) ; Brynychová, Veronika (referee)
Hemoproteins represent very important components of many living organisms. Participation in the processes of oxygen transport and storage, electron transport or enzymatic catalysis of reactions involving oxygen or hydrogen peroxide are commonly known functions of hemoproteins. Recently, there has been discovered a new group of hemoproteins. The main feature of this new group of proteins is their ability to detect changes in heme concentration (heme-responsive proteins) or changes in diatomic gas concentration (gas-responsive heme-containing sensor proteins) in their vicinity. Detection of these concentration changes generates signals that induce structural changes of the respective sensor proteins. Finally, the structural changes of the respective sensor proteins affect their functions or activities. The subject of this diploma thesis is the preparation and characterization of the eukaryotic heme sensor Bach1. We especially focused on the ability of Bach1 to bind heme molecules and on the comparison of various Bach1 properties in its apoform and holoform. Determination of the exact amount of heme molecules that specifically interact with heme sensor Bach1 represents very important part of this thesis. We also studied the effect of different redox states of heme iron and the presence of interaction...
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Chromatin immunoprecipitation of selected transcription factors
Smetanová, Jitka ; Vališ, Karel (advisor) ; Převorovský, Martin (referee)
The TEAD family of transcription factors regulates expression of genes affecting cell proliferation, differentiation and apoptosis. The activity of a particular transcription factor called TEAD1 is regulated by the Hippo signalling pathway. The Hippo pathway has been implicated to play a role in cancer suppression, however its precise mechanism remains unclear. MYC and GLUT1, genes which are coding two key regulators of glycolysis, were recently described as potential targets of the Hippo signalling pathway in human leukemia cells. In this diploma thesis, I tried to confirm the proposed interaction of the transcription factor TEAD1 with regulatory sequences of MYC and GLUT1 genes using chromatin immunoprecipitation (ChIP) analysis in human leukemic cells. However, I failed to successfully isolate TEAD1 complexes using ChIP. So, I discuss in my diploma thesis also possible reasons for this outcome, including biological and methodological issues. (In Czech) Key words: Transcriptional regulation, TEAD transcription factors, chromatin immunoprecipitation, leukemia
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The transcription factor C/EBPƴ as a novel regulator in mast cell development and function
Jedlička, Marek ; Alberich Jorda, Meritxell (advisor) ; Černý, Jan (referee)
Mast cells contribute to the activities of innate and adaptive branches of the immune system. They participate in pro-inflammatory responses to a wide range of pathogens, such as parasites, bacteria, and other foreign agents. These beneficial properties are in contrast to the contribution of mast cells to certain pathologies, such as asthma, allergy, autoimmune disorders, anaphylaxis, and systemic mastocytosis. Thorough knowledge of mast cell biology in health and disease is critical for the development of new therapeutic approaches. However, molecular mechanisms that control mast cell development and function are still incompletely defined. Our preliminary data indicate that the transcription factor C/EBP is a key player in mast cell biology. Here, using in vitro and in vivo models, we determine how C/EBP regulates the commitment of hematopoietic progenitors towards mast cells, and modulates mast cells function. These efforts provide novel insights to the role of C/EBP in hematopoiesis, and contribute to a better understanding of the mechanisms governing mast cell biology. Key words Mast cells, C/EBP, transcription factors, bone marrow-derived mast cell cultures, mast cell development, Cebpg conditional knockout mice
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The regulation of primary response genes by the ERK signaling pathway
Chvalová, Věra ; Vomastek, Tomáš (advisor) ; Doubravská, Lenka (referee)
The ERK signaling pathway represents an evolutionary conserved mechanism that enables cells to perceive various extracellular signals and convert them to a diverse array of biological outcomes such as proliferation, differentiation, cell cycle control, apoptosis or cell migration. Key components of this pathway are protein kinases Raf, MEK and the effector protein kinase ERK. In addition to its physiological role, continuous activation of the ERK pathway caused by somatic mutations of some of its components or upstream regulators appears to be significant cause of many human tumor diseases. That is why this pathway plays an important role also from the biomedical viewpoint. The multistep changes in gene expression are primarily responsible for these physiological and pathological events. Changes in genes expression are induced by activated kinase ERK that after translocation into the nucleus phosphorylates transcription factors (TFs) whose activation, in turn, leads to transcription of so-called immediate early genes (IEGs), many of which also code for other TFs (e.g. c-Fos, c-Jun or c-Myc). The latter TFs then regulate expression of further genes for structural and signaling proteins. This causes global changes in gene expression and leads to functional reprogramming of the cells. This thesis...
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Potential of genetic engineering for breeding plants tolerant to abiotic stresses: cold resistance in rice
Lotová, Gabriela ; Lipavská, Helena (advisor) ; Janská, Anna (referee)
With increasing population and climate change, there has been an increase in efforts to breed more efficient crops. Genetic engineering has opened unprecedented breeding possibilities in developing plants with desired traits. Transgenic crops with better qualities, including resistance to adverse environmental conditions, can contribute to solving problems of hunger and malnutrition in developing countries. Although society perceives genetically modified crops rather negatively, these crops are widely used as feed for livestock and outside Europe also for human nutrition. Because of the complexity of resistance to abiotic stress, the utility of genetic manipulations for the breeding of resistant plants was previously not anticipated. However, it turned out that modification of the stress signalling cascade or transcription factors can lead to success. This thesis summarizes the possibilities of genetic modification of crops, which may result in better tolerance to cold, and is mainly focused on rice. Part of the work deals with transduction of cold signal, whose modification can also result in increased tolerance to cold. Another part deals with transcription factors that activate expression of low temperature- resistant genes. The greatest attention is paid to CBF/DREB transcription factors that...
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Chromatin immunoprecipitation of selected transcription factors
Smetanová, Jitka ; Vališ, Karel (advisor) ; Převorovský, Martin (referee)
The family of transcription factors TEAD regulates the expression of genes that affect cell proliferation, differentiation and apoptosis. Activity of TEAD1 is regulated via the Hippo signaling pathway. General mechanism of tumor cell suppression by the Hippo signaling pathway remains unclear. C-MYC and GLUT1, the two key regulators of glycolysis, were recently described as targets of the Hippo signaling pathway in human leukemia cells. In this diploma thesis, the interaction of TEAD1 with M-CAT binding motifs was experimentally confirmed in the first exon of C-MYC gene. In addition, a new interaction of TEAD1 with M-CAT binding motifs has been found in the enhancer of C-MYC promoter and enhancer of GLUT1 promoter by ChIP analysis. Regulation of glucose metabolism by the Hippo signaling pathway may represent a new mechanism of tumor cell suppression. Key words: Gene regulation, transcription factors, chromatin immunoprecipitation, bioinformatics
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Visual system development in Platynereis dumerilii: insight from genetic engineering approach
Dobiášovská, Ivana ; Kozmik, Zbyněk (advisor) ; Vopálenský, Václav (referee)
Gene regulatory networks, underlying the molecular regulation of eye development are conserved across many animal phyla. Genes from the Pax family of transcription factors are one of the most conserved members through the evolution, regulating the development of crucial parts of eye, including the photoreceptor cells. Pax transcription factors are considered to be regulators of opsins, molecules providing the conversion of the light stimulus into the electrochemical signalisation in the photoreceptors cells. In this thesis, pax6 and pax2/5/8 transcription factors are investigated as potential regulators of eye development in Platynereis dumerilii. pax6 and pax2/5/8 transcription factors are tested as potential regulators of the r-opsin in Platynereis, based on the observed early expression onsets of these genes. Wild-type expression analysis of pax6 and pax2/5/8 using the whole mount RNA in-situ hybridization is provided, accompanied by the initial analysis of the Platynereis pax6 knockout line. pax6 heterozygote mutants are shown to be viable and able to reproduce, however, homozygote mutation of pax6 in Platynereis is lethal. Our data suggest that transcription factors pax2/5/8, otx and six3 are not regulated by the pax6 in Platynereis. Concerning the r-opsin present in the Platynereis eyes, pax6...
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Prediction of p53 Protein Binding Sites
Radakovič, Jozef ; Vogel, Ivan (referee) ; Martínek, Tomáš (advisor)
Protein p53 which is encoded by gene TP53 plays crucial role in cell cycle as a regulator of transcription of genes in cases when cell is under stress. Therefore p53 acts like tumor suppressor. Understanding the pathway of p53 regulation as well as predicting its binding sites on p53 regulated genes is one of the major concerns of modern research in genetics and bioinformatics. In first part of this project we aim to introduce basics from molecular biology to better understand the p53 protein pathway in gene transcription and introduction to analysis of prediction of p53 binding sites. Second part is about implementation and testing of tool which would be able to predict transcription factor binding sites for protein p53.
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