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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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Structural characterization of interaction between transcription factors and DNA
Filandrová, Růžena ; Novák, Petr (advisor) ; Vondrášek, Jiří (referee) ; Wimmerová, Michaela (referee)
Structural characterization of interaction between transcription factors and DNA Mgr. Růžena Filandrová Abstract Transcription factors are proteins that mediate gene expression regulation through interactions with DNA and other factors. They allow a cell to respond to various stimuli and play a crucial role in many biological processes such as control of cell cycle progression, differentiation of cells during development or immune response. To understand these processes, the knowledge of the transcription factors 3D structure together with the mechanism of their interaction with DNA is essential. However, some of the typical features of transcription factors, such as is for example the presence of intrinsically unstructured regions, make the 3D structure determination by the commonly used high resolution methods challenging. Therefore, utilization of complementary methods like structural mass spectrometry (MS), which was used in this thesis, might prove to be beneficial to explore the structural basis of the transcription factor-DNA interaction. In first part of this work, a set of structural mass spectrometry methods with the main focus on hydrogen/deuterium exchange mass spectrometry (HDX-MS) was optimized and tested on two transcription factor-DNA complexes and their DNA binding motifs and proved to be...
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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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Preparation of the transcription factor FOXK1 DNA binding domain
Procházková, Valérie ; Novák, Petr (advisor) ; Košek, Dalibor (referee)
Transcription factors are proteins that regulate gene expression in different cell types. They play an important role in many cellular processes including regulation of cell cycle and cell differentiation. They possess DNA binding domains to recognize and bind specific DNA sequences. One type of DNA binding domain is the forkhead domain, which contains a region of 100-110 amino acid residues. This sequence is referred to DBD FOX and its spatial arrangement resembles a "winged helix". Proteins of the FOX family interact with double- stranded DNA via the α-helix H3, which represents highly conserved region within the proteins of this family. Other regions of the DBD further contribute in DNA binding, but as not significantly conserved, and their different properties are responsible for variable affinities of individual FOX proteins against binding motifs. Differences in three-dimensional structure may also alter biological functions of FOX proteins in the organism. FOX proteins are divided into 19 subfamilies, including the FOXK subfamily, consisting of two members, FOXK1 and FOXK2. FOXK proteins regulate aerobic glycolysis, cell proliferation and carcinogenesis. Their increased expression has been reported in cancer cells of skeletal tissue, stomach, colon, breast, lung, ovary, etc. However, the...
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Comparison of apo- and holoforms of the transcription factor "Bach1"
Vávra, Jakub
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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Transcriptional regulation in the development of the cardiac sympathetic system
Matějková, Kateřina ; Pavlínková, Gabriela (advisor) ; Holzerová, Kristýna (referee)
To improve modern therapeutic and diagnostic methods, it is crucial to understand the development of the cardiac sympathetic system and to identify the genes involved in its regulation. Neural crest cells give rise to the sympathetic precursors that migrate towards the dorsal aorta. This migration is regulated by the NRP1/SEMA3A and neuregulin/ERBB signaling. The differentiation towards the sympathetic phenotype is regulated by transcriptional factor networks, including ASCL1, PHOX2A/B, GATA3, HAND2, HIF1A and ISL1. Next, neurons migrate to the final paravertebral position, which is regulated by the BDNF/TRKB signaling. The final step in the development of cardiac sympathetic neurons is the axon growth and guidance towards the heart. This is regulated by the NGF/TRKA and NRP1/SEMA3A signaling. This thesis aims to map current knowledge of different regulation pathways involved in the cardiac sympathetic development (especially in the mouse model) with emphasis on transcriptional factors. This type of information should help us better understand the pathophysiology of some cardiovascular diseases associated with the dysfunctional sympathetic system, such as arrhythmias, congestive heart failure or myocardial infarction, which remain to be main causes of death worldwide.
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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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