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The role of transcription factors in mouse eye development
Sunny, Sweetu Susan ; Kozmik, Zbyněk (advisor) ; Pavlínková, Gabriela (referee) ; Fafílek, Bohumil (referee)
Vision is a complex process that begins with the transmission and refraction of light through a highly specialised transparent tissue called the cornea. The cornea acts as a protective barrier and contributes to the focusing power of the eye. The development of mammalian cornea is a multiphase process involving the formation of the corneal epithelium (CE), stroma and endothelium (CEn) during embryogenesis, followed by the postnatal stratification of epithelium and constant renewal of desquamated outermost cells. Paired box protein (Pax) 6 is an evolutionarily conserved transcription factor important for the proper development of the eye. To provide further insights into the role of Pax6 in corneal development, we took the advantage of Cre-loxP system for selectively inactivating Pax6 in two ocular domains, specifically, the postnatal CE and the ocular surface epithelium (OSE) (cornea, limbus, and conjunctiva). We generated a novel postnatal CE-specific Cre-expressing transgenic mouse line, Aldh3-Cre. Inactivation of Pax6 in the postnatal CE using Aldh3-Cre resulted in the abnormal thin cornea with defective cell-cell adhesion, thus providing direct evidence for the function of Pax6 in postnatal corneal development. Subsequently, the OSE-specific depletion of Pax6 using K14-Cre, resulted in the...
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Regulation of gene expression by thyroid hormone receptors
Oltová, Jana ; Bartůněk, Petr (advisor) ; Fafílek, Bohumil (referee)
Hormones coordinate various processes in living organisms and their action is mediated either by cell membrane receptors or nuclear receptors. Thyroid hormones (THs) have prominent effects on the growth, development, and many aspects of metabolism, embryogenesis and early life. Their receptors (TRs) belong to the nuclear receptor superfamily, whose members function as ligand-activated transcription factors, and depending on the context, they can act both as activators or as inhibitors of transcription. Thyroid hormone receptors are encoded by two genes, TRα and TRβ, and by alternative splicing multiple isoforms are generated. The major form of TR binds to T3-response element as a heterodimer with retinoid X receptor (RXR). TRs are able to bind T3-response elements (TREs) independently of ligand occupancy. TREs contain two or more subsequent half-site sequences of AGGTCA, which are usually arranged as direct repeats with four nucleotide spacing (DR4). TH-responsive target genes are involved in a wide range of cellular pathways. Example of such gene is Disp3, which might link TH and cholesterol metabolism in certain cell types.
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Epigenetics in gene regulation and chromatin structure.
Lađinović, Dijana ; Vacík, Tomáš (advisor) ; Fafílek, Bohumil (referee) ; Stixová, Lenka (referee)
2. Abstract Histone methylation plays an important role in almost all cellular processes and its homeostasis is maintained by histone methyltransferases and histone demethylases. Misregulation of histone methylation levels is associated with gene expression misregulation and consequently also with various developmental defects and diseases. In this thesis we focus on the lysine demethylases KDM2A and KDM2B and on their demethylation deficient isoforms KDM2A-SF and KDM2B-SF. The lysine specific demethylases KDM2A and KDM2B have been predominantly studied for their demethylation function on CpG island-rich gene promoters. However, KDM2A-SF and KDM2B-SF have not been studied in detail. Therefore, the main goal of this thesis was to characterize KDM2A-SF more in detail and to focus on the role that KDM2A/B-SF might potentially play in canonical Wnt signaling pathway. We found that the KDM2A-SF mRNA arises through the action of an alternative intronic promoter and not by alternative splicing. We showed that the KDM2A-SF start codon is located in the exon that corresponds to KDM2A exon 14 and we thus determined the exact amino acid sequence of the KDM2A-SF protein. Furthermore, using an isoform specific knockdown assay we showed that KDM2A-SF, unlike KDM2A-LF, forms distinct nuclear foci on pericentromeric...
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Epigenetics in gene regulation and chromatin structure.
Lađinović, Dijana ; Vacík, Tomáš (advisor) ; Fafílek, Bohumil (referee) ; Stixová, Lenka (referee)
2. Abstract Histone methylation plays an important role in almost all cellular processes and its homeostasis is maintained by histone methyltransferases and histone demethylases. Misregulation of histone methylation levels is associated with gene expression misregulation and consequently also with various developmental defects and diseases. In this thesis we focus on the lysine demethylases KDM2A and KDM2B and on their demethylation deficient isoforms KDM2A-SF and KDM2B-SF. The lysine specific demethylases KDM2A and KDM2B have been predominantly studied for their demethylation function on CpG island-rich gene promoters. However, KDM2A-SF and KDM2B-SF have not been studied in detail. Therefore, the main goal of this thesis was to characterize KDM2A-SF more in detail and to focus on the role that KDM2A/B-SF might potentially play in canonical Wnt signaling pathway. We found that the KDM2A-SF mRNA arises through the action of an alternative intronic promoter and not by alternative splicing. We showed that the KDM2A-SF start codon is located in the exon that corresponds to KDM2A exon 14 and we thus determined the exact amino acid sequence of the KDM2A-SF protein. Furthermore, using an isoform specific knockdown assay we showed that KDM2A-SF, unlike KDM2A-LF, forms distinct nuclear foci on pericentromeric...
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Study of the role of selected endocytosis regulators in the Wnt signalling pathway
Knop, Filip ; Macůrková, Marie (advisor) ; Fafílek, Bohumil (referee)
Wnt signalling pathway is indispensable for a proper development and homeostasis in most of the multicellular organisms. Its evolutionary conservation and wide spectrum of diseases caused by its improper regulation only underscores its importance. Wnt signalling pathway can be conveniently divided into two halves. The first one covers processes that take place in Wnt ligand producing cells and the second one includes Wnt signal transduction in Wnt receiving cells. One of the vital steps in both types of cells is the process of endocytosis. Via regulation of this process, outcome of Wnt signalling itself is regulated. In this work, nematode Caenorhabditis elegans was used as a model organism to characterize new regulator of the Wnt signalling pathway which acts at the level of endocytosis. This new regulator described here is SEL-5 protein kinase. SEL-5 protein kinase belongs to a NAK kinase family and its function seems to be conserved throughout evolution. Its role in Wnt signalling of C. elegans was estimated by mining database information about its human homolog AAK1 and based on these information experiments were designed that would allow detailed investigation of its involvement in this pathway. Genetic interaction of sel-5 with components of the retromer complex was uncovered and it was...
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Rodina proteinů obsahujících sterol-sensing doménu
Ditrychová, Karolína ; Zíková, Martina (advisor) ; Fafílek, Bohumil (referee)
1. Abstract Sterol sensing domain (SSD) is a putative transmembrane region consisting of 5 helices and in few cases it was shown that it interacts with cholesterol. Proteins containing this domain play a role in many cellular pathways connected to cholesterol. It was first described in a study of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). It is an enzyme playing role in cholesterol biosynthesis and its transcription is regulated by sterol regulatory element binding protein cleavage activating protein (SCAP). HMG-CoA reductase and SCAP were the first proteins where SSD was recognized. After that, other proteins were described; one more protein functioning in cholesterol biosynthesis was described as containing SSD - 7-dehydrocholesterol reductase. Also two proteins playing role in cholesterol endocytosis - Niemann-Pick type C 1 liked 1 - and in its efflux from lysosomes - Niemann-Pick type C 1 - were recognized. Furthermore, proteins playing role in Hedgehog signalling Patched, Patched-related and Dispatched were found as SSD containing structures. In the end, tumour-suppressor gene TRC8 was also recognized as member of SSD family. In this text, structure and function of these proteins is summarized. Key words: sterol sensing domain, Hedgehog signalling pathway, sterol binding protein,...
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Adjusting Wnt signaling, new regulatory mechanisms of the Wnt pathway
Fafílek, Bohumil ; Kořínek, Vladimír (advisor) ; Stopka, Tomáš (referee) ; Machoň, Ondřej (referee)
4 Abstract The Wnt pathway is one of the major signaling cascades contributing to multiple cellular processes during embryogenesis, and adult tissue homeostasis and regeneration. Moreover, aberrant activation of the Wnt signaling pathway is connected with development of neoplasia, notably colorectal cancer. The aim of the thesis was to identify new ways of the Wnt pathway regulation to understand better physiological as well as non-physiological mechanisms of Wnt signaling. The results are summarized in four publications. The first article deals with TROY, a member of tumor necrosis factor receptor family. We identified TROY as a Wnt target gene during our search for Wnt responsive genes in colorectal cancer cell lines. Additionally, we detected expression of Troy in tumors of two mouse models of intestinal cancer. In the healthy gut, Troy is produced in fast cycling intestinal stem cells where negatively regulates the Wnt pathway. The second study focuses on processing and posttranslational modification of murine Wnt1 and Wnt3a. Wnts are glycosylated and double acetylated by lipid adducts and our results revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. Moreover, acylation of Wnts is connected with their signaling activity which is related to Wnt1 and...
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Mechanismy regulace aktivity proteinu MTM-6 na endosomech.
Horázná, Monika ; Macůrková, Marie (advisor) ; Fafílek, Bohumil (referee)
Wnt signalling belongs to conserved pathways and mediates cell fate decision, development, regeneration and adult tissue homeostasis. Disruption or misregulation of Wnt signalling pathway often leads to disease. Wnt proteins are hydrophobic glycoproteins which need a special receptor for transport from Golgi Apparatus to cell surface, which is called MIG-14 in Caenorhabditis elegans and Wntless (Wls) in mammals. In this study, I focus on understanding mechanisms that regulate MTM-6 protein activity. MTM-6, a lipid phosphatase associated with endosomal membrane, has been recently identified as a regulator of MIG-14/Wls trafficking in Caenorhabditis elegans. Silencing of mtm-6 leads to misregulation of some Wnt-directed processes, such as migration of Q neuroblasts progeny. This study reports identification of novel mtm-6 genetic interactors that have been found to influence migration of Q neuroblasts progeny through Wnt signalling. New knowledge about mtm-6 genetic interactions bring us near to understanding of Wnt signalling regulation. Keywords: Caenorhabditis elegans, MTM-6, SEL-5, Wntless, Wnt, endosomes, phosphoinositides, retromer
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Generation of transgenic mouse model to study biological role of KLK5 in epidermis
Kašpárek, Petr ; Sedláček, Radislav (advisor) ; Fafílek, Bohumil (referee)
A number of studies provide strong evidence that KLK5 is one of the most important serine proteases in the epidermis and is involved in processes such as desquamation, processing of antimicrobial peptides or induction of inflammatory reaction. The role of KLK5 has been deduced from in vitro experiments and thus its functions should be verified in vivo. This work aimed to develop a specific tissue targeting strategy to study the role of murine kallikreins in the epidermal compartment in vivo and to generate a transgenic model overexpressing mKlk5 in the mouse epidermis. Using the modified promoter of human involucrin, transgenic mice expressing the fluorescent marker, tdTomato, were generated in the first step. This transgenic reporter mouse showed specific targeting pattern of the reporter in the upper epidermal layers and, thus, the modified involucrin promoter could be employed for targeting further gene of interest in the differentiated epidermal compartment. In the second step transgenic mouse lines expressing murine kallikrein 5 were successfully generated. Among them two lines exhibited approximately 6-9 fold overexpression at the mRNA and protein levels, however it appeared that the immature protease was not activated under normal healthy conditions. Therefore two models of epidermal...
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