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Preparation of Xenopus tropicalis transgenic testicular stem cell culture.
Vegrichtová, Markéta ; Tlapáková, Tereza (advisor) ; Drobná Krejčí, Eliška (referee)
Testicular stem cells (TSCs) are relatively accessible potential source of pluripotent cells, which are particularly important for their application in regenerative medicine. Xenopus tropicalis is a useful model organism to study the migration and differentiation potential of stem cells. This amphibian is characteristic by outer fecundation and embryonic development of a great amount of embryos after fertilization. Oocytes and embryos are large enough (about 1 mm) to be suitable for micromanipulation micromanipulations. Laboratory of Developmental Biology, Faculty of Science, Charles University in Prague succeeded in the establishment of a mixed cell culture of TSCs growing on feeder layer of pre- Sertoli cells. This culture was derived from the testes of juvenile Xenopus tropicalis male. In the study of their differentiation potential it was found, that leukemia inhibitory factor (LIF) is the decisive factor allowing rapid proliferation of stem cells and their forming into characteristic colonies. This protein is produced by both types of cells which are present in the culture. The mouse LIF has the same positive effect on the proliferative potential of stem cells, which points at the evolutionary conservation of metabolic pathways associated with the maintenance of the stemness. RT-PCR analysis...
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Changes in embryonal programing induced by diabetes mellitus
Landsmann, Lukáš ; Pavlínková, Gabriela (advisor) ; Tlapáková, Tereza (referee)
Embryonic development is sensitive to environmental changes. These changes may lead to changes in the embryonic programming. Changes in programming embryos can occur due to inadequate nutrition, stress, treatment with chemicals and also due to diabetes. Epigenome reacts sensitively to environmental factors regulating gene transcriptional activity. Changes in the epigenome lead to a changes in gene expression, which can have a negative impact on the physiology and metabolism of organism. Maternal diabetes may alter embryonic and fetal development and may result in diabetic embryopathy. Furthermore, maternal diabetic enviromental plays an important role in the predisposition of offspring to a number of chronic diseases later in life. The offspring of diabetic pregnancies demonstrate differences in metabolic, cardiovascular, and inflammatory variables, compared to the offspring of nondiabetic mothers. This thesis summarizes the genetic and epigenetic factors involved in the development of diabetic embryopathy and in the embryonic programming. Key words: Diabetes mellitus, diabetic embryopathy, transcriptional regulation, genetic and epigenetic factors , embryonic programming, genome
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Induction of testicular stem cell differentiation in mammals.
Strnadová, Karolína ; Tlapáková, Tereza (advisor) ; Krulová, Magdaléna (referee)
Stem cells represent a unique cell source with potential usage in regenerative medicine and organ transplantation. As is known, spermatogonial stem cells are unipotent giving rise to a single cell type, which is sperm. Pluripotency was achieved by isolation and cultivation of these testicular stem cells in a number of researches. Testicular pluripotent stem cells differentiated in conditions in vitro to derivatives of all three germ layers identically as embryonic stem cells. The aim of this thesis is to characterize stem cells and summarize the findings of testicular stem cell research. The main focus of this thesis is on studies of cultivated pluripotent stem cells derived from mouse and human testes and their ability to differentiate under determinate conditions into the cells of ectoderm, mesoderm and endoderm.
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The Role of Bmp4 and Bmp8b proteins in genesis of mouse primordial germ cells
Vegrichtová, Markéta ; Tlapáková, Tereza (advisor) ; Petelák, Aleš (referee)
Primordial germ cells are precursors of sexual breeding organisms. For induction of their formation are necessary growth factors from extraembryonic ectoderm. There are key proteins BMP4 a BMP8b among these factors. These proteins form both homodimers and heterodimers and through receptor complexes and SMAD proteins are indicating the competent cells of proximal epiblast, which give rise to the primordial germ cells and other structures of embryonic mezoderm. Although BMP4 and BMP8b proteins signal similarly, they are mutually indispensable and though for induction of primordial germ cells are both necessary. Up to now, it was proposed several models of their interaction, but not yet completely accurate.
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Formation of blastema during limb regeneration in Amphibia
Paušlyová, Lucia ; Tlapáková, Tereza (advisor) ; Paňková, Daniela (referee)
Total limb regeneration among vertebrates is basically restricted to some amphibians. Urodeles have the ability to regenerate amputated limbs through their life span. Anurans have the ability of complete regeneration of amputated limbs only in their larval stage. The key process of the limb regeneration is the formation of undifferentiated cell group which is called blastema. There are many cell types that contribute to formation of the blastema while the most important part in this process belongs to the skeleton muscle tissue and dermal fibroblasts. Another critical factor in formation of the blastema and its growth are the nerves in the area of wound and neurotrophic factors produced by them. In the last 20 years it has been great improvement in using different markers for tracking the fate of blastema cells.
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Characterization of the porcine induced pluripotent stem cells
Svobodová, Eliška ; Vodička, Petr (advisor) ; Tlapáková, Tereza (referee)
Induced pluripotent stem cells (iPSCs) are the cells established by introducing several transcription factors into the somatic cells and culturing them in embryonal stem cell (ESCs) culture conditions. Factors used for the establishment of the first iPSCs are OCT3/4, SOX2, KLF4 and c-MYC. IPSCs created by these means resemble closely to the ESCs. IPS technology may be used to derive iPS cells of individual pacients and apply these cells for their treatment in the cases where the use of ESCs represents an ethical and imunological problem. Therefore, it is important to establish an appropriate animal model for the longtime safety testing of iPSCs before acceding to their medicinal aplication.
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The role of NG2 glycoprotein in cancer cell invasiveness
Obr, Adam ; Tlapáková, Tereza (referee) ; Brábek, Jan (advisor)
NG2 proteglycan is a novel membrane - spanning proteoglycan, expressed in general in developing tissue whose cells are characteristic for its increased level of proliferation and motility. NG2 proteoglycan is considered to be an anchor for cell adhesion capabilities on different substrata as well as a signaling transmembrane structure which is capable of affecting actin cytoskeleton and causing increased cell migration. This bibliographic search shows the considered effect of NG2 proteoglycan to the migration abilities of cancer cells via different molecular mechanisms, such as NG2 - mediated, integrin - independent cell interactions with collagens and other ECM substrata, effect of phosphorylation with two different kinases leading to diverse signaling and different behavior in response to phosphorylation and finally the interaction with scaffolding protein MUPP1 and possible connection with signaling pathway to RhoA GTPase, which is involved in cytoskeleton regulation.
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Transfection of stem cells and other testicular cells of Xenopus tropicalis
Šídová, Monika ; Tlapáková, Tereza (advisor) ; Petr, Jaroslav (referee)
Xenopus tropicalis represents one of the most important model organism used in developmental and cellular biology. Laboratory of Developmental Biology at the Faculty of Science, Charles University in Prague has successfully established a long-term culture of X. tropicalis juvenile testicular cells. Based on expression profiling analysis of selected specific markers (Sox9, WT1, etc) it was proven that the major cell type in this culture is pre-Sertoli cells. Furthermore these pre-Sertoli cells allow a longterm cultivation of the germinal stem cells. By performing a histochemical test for the presence of alkaline phosphatase in the colony of these cells were proven the features of stem cells. In this diploma thesis we focused on optimization of work with the mixed cell culture. In particular we define conditions of dissociation and subsequent separation of a feeder-layer formed by the pre-Sertoli cells. We also attempted to develop suitable conditions for transfection of the germinal cells. With these techniques we will to investigate the functional properties of the germinal stem cells. Moreover, it provides us a powerful tool for performing another experiments focused on transgenesis and/or different gene inactivation. Powered by TCPDF (www.tcpdf.org)
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Description of Xenopus tropicalis pre-Sertoli cells in tissue culture
Ševčíková Jonáková, Jana ; Tlapáková, Tereza (advisor) ; Drda Morávková, Alena (referee)
5 Abstract Xenopus tropicalis is an important model organism used in developmental and cell biology. Long term primary mixed cell culture, derived from juvenile testis of Xenopus tropicalis, is the first tissue culture of this specie. Characterization of this tissue culture is important for its further handling and subsequent experiments. The culture is composed of Sertoli cell precursors. Feeder layer of this cells enables long term germinal stem cell cultivation. Influence of condicioned medium plays important role during the cell culture cultivation. Result of this task approved effect of condicioned medium for cell culture growth. Cell culture and testis gene expression analyse revealed, that in process of Sertoli cell differentiation many growth and differentiation factors are involved. Presence of marker genes has been approved by RT-PCR and in situ hybridization. This in vitro system can contribute to clarification of different roles of pre-Sertoli and Sertoli cells in testis differentiation, and therefore it establishes a new spermatogenesis model offering the possibility to distinguish parts of the pre-Sertoli and Sertoli cells differentiation, which is difficult in mammal models. Keywords: Xenopus tropicalis, tissue culture, pre-Sertoli cells, Sertoli cells, testicular differentiation, gene expression
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Formation of spatio-temporal molecular gradients in early embryonic development of Xenopus laevis.
Šídová, Monika ; Tlapáková, Tereza (advisor) ; Pěknicová, Jana (referee) ; Petr, Jaroslav (referee)
Clarifying the underlying spatio-temporal mechanisms that determine body pattern is important for detailed understanding of embryonic development. A crucial question of vertebrate embryogenesis remains: when and how are single blastomeres determined for differentiation that subsequently leads to body axes specification and the formation of different tissues and organs? The answer to this question will be beneficial for primary research as well as in the field of applied medicine. The main aim of the presented thesis was to study spatio-temporal molecular gradients of cell fate determinants during early embryonic development. The African clawed frog Xenopus laevis was used as a model organism because of their large size of oocytes and external embryonic development. Due to late activation of embryonic transcription, a crucial mechanism of early blastomeres determination is dependent on asymmetric localization of maternal factors within oocyte and their uneven distribution into single blastomeres during early cell division. Two main localization patterns were identified along the animal-vegetal axis of the mature Xenopus oocyte using qPCR tomography. The localization gradient with preference in either animal or vegetal hemisphere was found for maternal mRNA as well as miRNAs. Moreover, two vegetal...
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