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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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In vitro differentiation of Xenopus tropicalis and Mus musculus testicular somatic cells.
Hlaviznová, Michaela ; Tlapáková, Tereza (advisor) ; Hovořáková, Mária (referee)
Sertoli cells (SCs) are somatic cells of testicular tissue that are involved in spermatogenesis and maturation of germ cells. They are currently being extensively studied for their immunomodulatory abilities, and recent studies have shown that they share some properties with mesenchymal stromal cells (MSCs). Detailed characterization of SCs and clarification of their role in testicular tissue is crucial for potential use of SCs as a therapeutic tool in regenerative medicine. Cell culture of Xenopus tropicalis immature Sertoli cells (XtiSCs) and Mus musculus (mSCs) Sertoli cells were established in the Laboratories of Developmental Biology and Immunoregulations, Faculty of Science, Charles University. Previous research has characterized XtiSCs and demonstrated their multipotent potential by in vitro differentiation into a mesodermal line. Following this research, one of the goals of the diploma project was the induction of in vitro differentiation of XtiSC into other cell types, which would verify the differentiation potential of XtiSCs. The mSC expression profile confirmed the somatic origin of this culture as well as the transcription of Sertoli cell gene markers. Differentiation of mSCs along the mesodermal line into osteoblasts, chondrocytes and adipocytes has been successfully induced in vitro....
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Differentiation of Sertoli cell progenitors and preparation of testicular 3D cultures of Xenopus tropicalis.
Slováková, Lucie ; Tlapáková, Tereza (advisor) ; Hovořáková, Mária (referee)
Sertoli cells represent the only somatic cell type within the seminiferous tubules with direct contact to germ cells. Sertoli cells significantly contribute to the development of the testicular niche in a male embryo. Their role during postnatal life is in the regulation and nutrition of germ cells and the formation of the blood-testis barrier to protect these cells. In our laboratory, we have been successful in establishing a cell line of X. tropicalis immature Sertoli cells (XtiSCs) derived from juvenile testes of X. tropicalis. The objective of this thesis was to induce the differentiation process of XtiSCs into mature Sertoli cells. In vitro experiments using several factors or primary culture from adult male X. tropicalis did not show any mature markers in differentiated XtiSCs. Another experiment using cell culture derived from pubertal mice was partially successful in the induction of the differentiation process. These results indicate that XtiSCs do have some differentiation potential into mature Sertoli cells. Part of this work was to test the ability of testicular cells isolated from juvenile males of X. tropicalis to form de novo organoids. In vitro experiments were successful when these cells were cultured in a three-layer matrigel.
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Maternal and fetal microchimerism.
Hynková, Marie ; Tlapáková, Tereza (advisor) ; Pačes, Jan (referee)
Microchimerism is defined as the presence of small amount of genetically distinct cells in one individual. It most often arises during pregnancy and breastfeeding. It can also arise from blood transfusion or organ transplantation. During pregnancy there occurs a bidirectional migration of cells through the placenta between the mother and the fetus. Cells which are transferred during pregnancy and breastfeeding can persist in the offspring until adulthood. During breastfeeding, a big number of immune cells is transferred to the offspring via mother's milk. These cells protect the offspring against pathogens and are involved in modulation of its immune system. Fetal cells persist in mother organism even decades after giving birth and can have long-lasting effect on mother's health condition. These fetal cells can help to regenerate mother's damaged tissues, but they can also contribute to the development of serious autoimmune diseases.
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Folate and its role in early embryonic development of higher vertebrates.
Zahradníková, Hana ; Tlapáková, Tereza (advisor) ; Procházková, Michaela (referee)
Folate or vitamin B9 is an essential water-soluble nutrient that takes part in important cellular processes. These include amino acid metabolism, methylation, and nucleotide synthesis, the last two of which play a key role in early embryonic development. It was approximately 30 years ago when folic acid supplementation was confirmed to help prevent embryonic neural tube defects and since then an intake of 400 µg of folate a day has been recommended to all women of childbearing age. Despite this, folate deficiency is a relatively common phenomenon and during pregnancy can lead to congenital malformations and pregnancy complications. This bachelor thesis summarizes the latest findings on the association of neural tube defects and miscarriages with single-nucleotide polymorphisms in genes encoding folate metabolism enzymes in humans, focusing mainly on the gene encoding the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR). Furthermore, this thesis deals with potential mechanisms of the folate's protective effect which could participate in the prevention of neural tube defects and are studied mainly on model organisms. Key words: folate, embryo, neural tube defects, MTHFR, homocysteine, miscarriage, single-nucleotide polymorphisms
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Cell senescence with a focus on telomerase theory
Laštůvková, Viktorie ; Tlapáková, Tereza (advisor) ; Hanus, Robert (referee)
Cell senescence is an irreversible state in which the cell cycle ends. The cell remains metabolically active, but its physiological functions are limited. Cell cycle arrest is a response to the action of a number of factors, most of which affect the secondary structure of DNA in various ways and cause damage to it. Other initiators of cell entry into the senescent state are oncogenes and chemotherapeutics. One of the best studied factors, which is also in a way the "fairest", is the shortening of the telomere. Telomeres are non-coding regions of DNA at the chromosome ends that shorten slightly with each division of the cell. If the cell does not have mechanisms to compensate for this loss, the Hayflick limit is reached after a certain time. In general, senescence can be considered as a protective mechanism to prevent DNA damage pathologies. However, the presence of senescent cells in tissues is often ambivalent, accumulates with age, and can cause many degenerative diseases. The senescent phenotype includes a wide range of signs such as changes in cell size and shape, increased granularity, or altered range of expressed genes. A significant phenomenon of senescent cells is the secretion of biologically active factors contributing to physiological or pathological changes in the organism. Key words:...
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The effect of maternal age on the level of DNA damage in oocytes
Koubovská, Markéta ; Šolc, Petr (advisor) ; Tlapáková, Tereza (referee)
Female germ cells are oocytes, whose number defines the length of the reproductive period. Besides quantity the quality of oocytes is also an important factor. The quality is not the same throughout whole life, but it decreases with increasing age. The loss of quality is mainly due to the accumulation of DNA damage. The most serious damage is considered to be double-strand breaks (DSBs) because of both DNA strands break and subsequent translocation or deletion. There are also products caused by reactive oxygen species, UV or γ radiation, modified DNA section or mismatch sequence. All these lesions are repaired by oocyte repair mechanisms. However, the older a woman is, the more gene expression of DNA repair genes is decreased. That is why the repair is not efficient and DNA damage is not removed. Higher age is associated with unsuccessful attempts to conceive, a higher risk of miscarriage or an ectopic pregnancy. To avoid the risks that pregnancy in old age represents, the reproductive ability has been lost. In humans, it ends around the age of 50 with so- called menopause.
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The role of integrins in gametes prior to fertilization and during their interaction
Foldynová, Veronika ; Komrsková, Kateřina (advisor) ; Tlapáková, Tereza (referee)
Integrins are heterodimeric transmembrane glycoproteins that represent a large group of cell adhesion receptors involved in cell-cell, cell-extracellular matrix and cell-pathogen interacti- ons. Up to now, 24 different integrin heterodimers have been detected in mammals. They are involved in a wide range of processes such as immune response, lymphocyte homing, platelet aggregation, also in wound healing, cell differentiation, migration, proliferation and even in cell survival. Integrins have also been detected on germ cells and are now known to play an important role in reproductive processes such as fertilization, embryo implan- tation, and embryonic development. The main aim of this thesis is to introduce integrins from the perspective of reproduction, integrin physiology, occurrence and localization of individual subunits in male and female germ cells. A significant part of the work is devoted to a discussion of the role of integrins in gametes, both during maturation processes (egg maturation in the ovary, capacitation and the acrosomal reaction of sperm), in sperm mi- gration, oviductal reservoir formation and their direct and indirect involvement in adhesion and fusion of the gametes during fertilization. Keywords: integrins, sperm, egg, fusion, fertilization
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