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Bioinformatics analysis of sequences required for localization of RNA during development
Naraine, Ravindra ; Šindelka, Radek (advisor) ; Fulková, Helena (referee) ; Tichý, Boris (referee)
The development of a complex organism from the fusion of two cells (oocyte and sperm) has been a fascinating aspect of developmental biology. It is now known that certain spatially and temporally regulated molecules tightly regulate embryogenesis. The asymmetrical gradient of these molecules within a given cell or within groups of cells helps to guide the differentiation of certain parts of the developing embryo. In fishes and frogs, the establishment of the maternal animal-vegetal transcript gradient within the egg produces the first developmental axis and subsequent formation of the ectoderm, mesoderm, and endoderm regions. Despite this important process, most of our knowledge on this initial animal-vegetal distribution in vertebrates has been limited primarily to the Xenopus laevis model, involved the analysis of only few transcripts and also analyzed only polar regions of the oocyte. This thesis aims to address this deficit in knowledge by leveraging high throughput analysis (RNA sequencing) to characterize and compare the maternal transcriptome and its sub-compartmentalization within the egg of four distantly related models. Additionally, we analyzed different stages of oocyte maturation to determine where the observed localization occurs. The models used were the African clawed frog (Xenopus...
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Studying the evolution of chromosome rearrangements in pipid frogs using in situ hybridization
Bergelová, Barbora ; Knytl, Martin (advisor) ; Johnson Pokorná, Martina (referee)
In African pipid frogs we can find at least eight polyploidization events in which a lot of levels of ploidy arose. This group includes diploid, tetraploid, octoploid, and/or dodecaploid species. Due to this great variability in ploidy levels, representatives of the genus Xenopus are excellent model organisms for studying the evolution of whole-genome duplications and chromosomal rearrangements. Currently, little information is known about chromosomal rearrangements within the Xenopus species, specifically, there are only two known cases of large-scale rearrangements. The first is a fusion of chromosomes 9 and 10 in the subgenus Xenopus and the other rearrangement is a non-reciprocal translocation occurring between chromosomes 2 and 9 in the subgenus Silurana. In this diploma thesis, we attempted to expand the knowledge about chromosomal rearrangements using the fluorescence in situ hybridization method. We confirmed the fusion of chromosomes 9 and 10 in other species of the Xenopus genus, such as X. pygmaeus, and proposed a hypothesis about the possibility that this fusion occurred in the common diploid ancestors of this subgenus. We also gained further insights into the origin of non-reciprocal translocation within the Silurana subgenus. This translocation was not detected in the species X....
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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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The role of nitric oxide (NO) during Xenopus laevis embryonic epidermis development
Tománková, Silvie ; Šindelka, Radek (advisor) ; Krylov, Vladimír (referee) ; Soukup, Vladimír (referee)
Nitric oxide (NO) is an interesting molecule, which is involved in many important biological processes such as vasodilatation, neurotransmission, immune response and cell proliferation. This work presents the crucial role of NO during the Xenopus laevis embryonic epidermis development. The outer layer of the embryonic epidermis is composed of 4 cell types (small secretory cells, multi-ciliated cells, ionocytes and goblet cells). Embryonic epidermis composition reflects specialized epithelia such as a respiratory epithelium of mammals. Therefore, Xenopus embryonic epidermis has become a suitable model for the study of human mucosal and mucociliary epithelium and their defects. I found that NO is mainly produced in ionocytes and multi-ciliated cells by using molecular and immunohistochemical approaches. The study of molecular and cellular phenotype changes in embryos with inhibited NO production revealed the necessity of this molecule for correct formation and function of the mucociliary epithelium. NO inhibition caused reduction of specialized epidermal cell types (small secretory cells, ionocytes and multi-ciliated cells) and structural changes in multi-ciliated cells. I also showed that NO affects the development of the embryonic epidermis through the sGC-cGMP-PKG signaling pathway, probably by...
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Basement membrane and the role of matrix metalloproteinases during embryonic wound healing
Kadlčíková, Dominika ; Šindelka, Radek (advisor) ; Tolde, Ondřej (referee)
The healing process is an attractive topic in biology and medicine. There are two types of wound healing - in embryos and in adults. In the case of embryonic wound healing the whole process is simplified and accelerated. An inflammatory reaction typical for adult healing is eliminated and the wound is healed without scars. Better understanding of embryonic wound healing could lead to more effective treatment of injuries, burns and chronic wounds in human population. The studying of molecular mechanisms in the healing process is also meaningful in the context of understanding the cancer regulation. The wound healing could be compared with embryonic development in many ways. The body's axes are essential for a spatial activation of genes involved in development. Our hypotesis was that the axis are also important for wound healing. It has been suggested that the cell's polarity and motility depends whether the wound is vertical or horizontal. There are also connections between wound healing and cancer development. One of them is the degradation of the basement membrane (BM) and extracellular matrix (ECM). The process of degradation is catalyzed by the coordinated action of several classes of enzymes. Some of them - matrix metalloproteinases (MMP) and their role in embryonic wound healing have been...
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The role of nitric oxide (NO) during Xenopus laevis embryonic epidermis development
Tománková, Silvie ; Šindelka, Radek (advisor) ; Krylov, Vladimír (referee) ; Soukup, Vladimír (referee)
Nitric oxide (NO) is an interesting molecule, which is involved in many important biological processes such as vasodilatation, neurotransmission, immune response and cell proliferation. This work presents the crucial role of NO during the Xenopus laevis embryonic epidermis development. The outer layer of the embryonic epidermis is composed of 4 cell types (small secretory cells, multi-ciliated cells, ionocytes and goblet cells). Embryonic epidermis composition reflects specialized epithelia such as a respiratory epithelium of mammals. Therefore, Xenopus embryonic epidermis has become a suitable model for the study of human mucosal and mucociliary epithelium and their defects. I found that NO is mainly produced in ionocytes and multi-ciliated cells by using molecular and immunohistochemical approaches. The study of molecular and cellular phenotype changes in embryos with inhibited NO production revealed the necessity of this molecule for correct formation and function of the mucociliary epithelium. NO inhibition caused reduction of specialized epidermal cell types (small secretory cells, ionocytes and multi-ciliated cells) and structural changes in multi-ciliated cells. I also showed that NO affects the development of the embryonic epidermis through the sGC-cGMP-PKG signaling pathway, probably by...
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