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Study of differential potential of germ stem cells in vitro in higher vertebrates
Smolík, Ondřej ; Krylov, Vladimír (advisor) ; Krulová, Magdaléna (referee)
Spermatogonial stem cells are unipotent male germ cells which provide spermatogenesis during the whole life. In 2004, an important experiment was conducted. During in vitro cultivation mouse spermatogonial stem cells gained the characteristics of embryonic stem cells, pluripotency by all means. Those cells had the feature to spontaneously differentiate into all three germ layers, endoderm, ectoderm and mesoderm. They also could pass its genetic information to the next generation and they could give rise to teratomas. By this event, experiments started on other vertebrates including rodents, domestic animals and also human. Differentiation of these cells can be directed in vitro to generate specific cell types. On base of these facts, spermatogonial stem cells are alternative source of pluripotent cells which possess many applications in life sciences. The purpose of this thesis is to summarize actual knowledge about differentiation potential in vitro of spermatogonial stem cells in higher vertebrates and try to identify tendencies which they prefer during differentiation, if they exist. Powered by TCPDF (www.tcpdf.org)
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Etiology and mechanisms of pathogenesis of congenital neural crest disorders
Poštulková, Adéla ; Fábik, Jaroslav (advisor) ; Krylov, Vladimír (referee)
The neural crest is an extensively migrating population of cells found only in vertebrates. It is formed in the dorsal part of the neural tube during the early embryonic development by a process called neurulation, in which the neural plate forms and the neuroepithelium subsequently sinks into the embryo. Neural crest cells detach during development and then migrate through the body to their final destinations, where they differentiate into various mesenchymal and non-mesenchymal cell types (derivatives) under the influence of signals. These derivatives include tissues of the peripheral nervous system, tissues of the endocrine system, pigment cells, facial bones, and other connective tissues. Defective migration of neural crest cells and disruption of their differentiation leads to maldevelopment of the neural crest cells derivatives, and to development a group of syndromes known as neurocristopathies, which include a wide range of malformations. The aim of this thesis is to summarize the current knowledge about individual neural crest diseases and syndromes such as Hirschsprung's disease, Treacher-Collins syndrome, piebaldism and neurofibromatosis, their causes, mechanisms of pathogenesis and possible treatment options. Key words: Neural crest, migration, differentiation, neurocristopathies,...
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Mechanisms determining the identity of intestinal epithelial cells
Berková, Linda ; Valenta, Tomáš (advisor) ; Krylov, Vladimír (referee) ; Klíma, Jiří (referee)
(ENG) The intestinal epithelium has been heavily studied for decades. It is a traditional model to study the natural renewal of tissue during homeostasis and upon the damage, carcinoma establishment and progression and regulation of stem cells by Wnt signalling. Since the main interest in the tissue is focused on epithelial stem cells, it has been described quite recently that also all differentiated epithelial cells from the small intestine including enterocytes undergo drastic changes in properties during their short life. This phenomenon came to be known as villus zonation. In parallel with that observation, the heterogeneity of intestinal mesenchymal cells and their impact on intestinal epithelium have been gradually revealed. Distinct populations of mesenchymal cells secrete specific signalling molecules, which determine the destiny of epithelial cells. Those signalling molecules regulate stem cell maintenance as well as villus zonation. Our work combines those novel findings of intestinal enterocytes and mesenchymal cells. We studied the effect of distinct Bmp ligands produced by mesenchymal cells on the differentiation of enterocytes. We treated small intestinal epithelial cells with recombinant Bmp proteins in vitro and analysed changes in their gene expression by RNA sequencing. We...
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Immunomodulatory potential of Sertoli cell progenitors during heart regeneration after injury in Xenopus
Žabková, Světlana ; Krylov, Vladimír (advisor) ; Procházka, Jan (referee)
The high percentage of mortality associated with cardiovascular diseases shows the importance of studying cardiac muscle regeneration. An interesting approach for heart regeneration may be the use of a cell culture of Sertoli progenitor cells and peritubular myoid cells derived from juvenile testes of Xenopus tropicalis that was named XtiSC. These cells were found to share a number of characteristics which are typical for mesenchymal stem cells. Mesenchymal stems cells are well-known for their immunomodulatory and anti-inflammatory abilities that promote heart regeneration. The goal of this master thesis was to test the immunomodulatory potential of Sertoli progenitor cells during heart regeneration after heart apex amputation in Xenopus tropicalis without macrophages. The reason for macrophages depletion was the assumption that XtiSC behave like macrophages in many aspects. Clodronate encapsulated in liposomes was chosen as a tool for macrophage depletion. It was confirmed that XtiSC can positively influence heart regeneration in a classic model of heart regeneration, but also in a model with depletion of macrophages. It was revealed that Xenopus tropicalis adults without macrophages injected with XtiCS into the hindlimb skeletal muscle 3 days before apical resection showed reduced fibronectin...
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The role of cumulus cells during the maturation of mammalian oocytes
Meniuková, Kateřina ; Krylov, Vladimír (advisor) ; Drutovič, David (referee)
Cumulus cells in the ovarian follicles emerge by differentiation from somatic granulosa cells. They are located around the oocytes in stratified clusters and their innermost layer is called the corona radiata. Cytoplasmic microvilli of cumulus cells pass through zona pellucida to the proximity of the oolema, where formation of gap junctions enables signalling and metabolic codependency. Oocytes produce factors affecting processes in cumulus cells, including their metabolism and viability. By modulating the abundance of cyclic nucleotides in oocytes, cumulus cells allow the maintenance of meiotic arrest, providing time for oocytes to finish their growth and gain competence for maturation. Because oocytes have a very limited ability to process glucose, metabolic cooperation with cumulus cells enables them to gain the energy needed for processes associated with growth and maturation. Several metabolic pathways of glucose processing create energy substrates for the formation of ATP, which they then transport to the oocytes. They also regulate the amount of lipid droplets in oocytes and protect them from cellular toxicity. An increase in the concentration of luteinizing hormone in the follicle just before ovulation induces the transmission of the signal for cumulus expansion. Interruption of the...
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Identification of basolateral localization motifs of the urate transporter GLUT9 using functional studies.
Pácalová, Eliška ; Krylov, Vladimír (advisor) ; Pavelcová, Kateřina (referee)
The GLUT9 transporter, coded by the SLC2A9 gene, is one of the key proteins enabling the trasnfer of uric acid across the membrane in epithelial cells of the proximal tubule. In humans, this protein is naturally expressed in two variants: long (GLUT9L) and short (GLUT9S), which differ from one another by their N-terminus sequence. Each of these isoforms is localized on a different pole of the epithelial cell. The signal sequence/motif responsible for this difference is presumed to be located in the aforementioned N-terminus domain. Numerous allelic variants influencing the transport properties of the protein have also been described. The first aim of this thesis is to verify the influence of a newly discovered variant, characterized by substituting of valine for leucine in the 114th position (V114L, in the short form its corresponding variant V85L) on the ability to transport uric acid. Second aim is to verify the influence of mutations in selected motifs, which could be responsible for the localization of the protein, thus also changing its transport properties. Two dileucine motifs 12LGL14 and 33LL34 and one tyrosine motif 84YIKA87 were tested. Functional studies using 14 C radiolabeled urate demonstrated significant decrease of transport ability for the V114L/V85L allelic variant in both...
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Evolution of the gene regulatory network underlying the formation of the gastrula organizer
Macháčová, Simona ; Kozmikova, Iryna (advisor) ; Krylov, Vladimír (referee) ; Buchtová, Marcela (referee)
During gastrulation, the vertebrate embryo is organized from the clump of cells into a bilaterally symmetric body. This organization process is driven by the gastrula organizer. Its establishment is induced by maternal Wnt/β-catenin signaling and Nodal/Activin signaling localized in the presumptive dorsal region of the embryo. The regulative environment then triggers the expression of the organizer-specific genes which create morphogen gradients in the embryonic body and therefore give each cell positional information. However, the evolution of vertebrate organizer establishment remains vague. Here we aim to test the compatibility of the invertebrate cis-regulatory modules with the vertebrate gene regulatory network (GRN). We introduced fluorescent reporter genes under the control of the invertebrate regulatory sequence of organizer-specific genes into a vertebrate model to observe their behavior in the context of the vertebrate GRN. We found and functionally verified a 500 bp-long amphioxus sequence (an enhancer) that is necessary and sufficient to drive a correct Chordin gene expression in the gastrula organizer in zebrafish. Chordin is a prominent organizer-specific gene antagonizing Bone Morphogenetic Protein (BMP) signaling. We tested also other invertebrate genes for their compatibility with...
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Immunomodulatory potential of Sertoli cell progenitors in Xenopus tadpoles during the healing of amputated tail
Mertová, Irem ; Krylov, Vladimír (advisor) ; Procházka, Jan (referee)
A cell culture of common Sertoli and peritubular myoid cells progenitors derived from the testes of male X. tropicalis, called XtiSC, was established in the Laboratory of Developmental Biology. XtiSCs exhibit similar properties as more well-known mesenchymal stem cells that are used in cell therapy for their immunomodulatory and proregenerative properties. Microinjection of XtiSC into the dorsal vein of the tail of the tadpoles has increased macrophage numbers 7 days after tail amputation (dpa), both in controls and in tadpoles after depletion of macrophages by the application of clodrosomes. Macrophage depletion also reduces the migratory ability of XtiSC to the site of tail amputation. Macrophage depletion also led to a reduction in the number of satellite cells 1 dpa. On the seventh day after tail amputation and XtiSC injection, there was a significant increase in their number compared to the control group without XtiSC injection. Using DAF-2DA probe, nitric oxide production was confirmed by injected XtiSC and at the injury site. For future research of the proliferation, differentiation and migration of satellite cells in vivo, a vector expressing EGFP under endogenous Pax7 promoter and Katushka-RFP under gama-crystalline promoter was created and integrated into the X. tropicalis genome using...
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Left-right organizer of body asymmetries in ray-finned fishes
Kupková, Anežka ; Soukup, Vladimír (advisor) ; Krylov, Vladimír (referee)
Left-right asymmetry of the body occurs across a number of organisms from invertebrates to vertebrates, and is mostly exhibited by the asymmetry of internal organs. These asymmetries are established at early stages of embryonic development due to the action of temporary structures called organizers of left-right asymmetry. In ray-finned fishes, the most-studied organizer is the so-called Kupffer's vesicle present in the teleosts. It is a hollow structure composed of monociliary cells. The cilia of these cells rotate and generate a leftward flow of extracellular fluid. The flow subsequently triggers the induction of the Nodal signalling cascade, which is responsible for left-right organ orientation and is considered evolutionarily conserved in vertebrates. The main participants in this pathway are the Nodal, Pitx2 and Lefty factors. In contrast to teleosts, the left-right organiser of non-teleost ray-finned fishes resembles the gastrocoel roof plate present in amphibians, which is apparently ancestral for ray-finned fishes. This bachelor thesis evaluates the origin and function of Kupffer's vesicle, describes the Nodal signalling cascade triggered by this organizer, and compares Kupffer's vesicle with the organizer of non-teleost ray-finned fishes.
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