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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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The localization of maternal RNAs during early development
Kováčiková, Petra ; Šindelka, Radek (advisor) ; Děd, Lukáš (referee)
The maternal determination of a developmental plan has been described in many animal species. The most well-known examples are found in the embryogenesis of C. elegans and D. melanogaster. In vertebrates, these studies are often limited by the size of oocytes to just a small subset of model organisms. The most comprehensive research can be found dealing with the early development in the frog, Xenopus laevis. Its oocytes, displaying differently pigmented animal and vegetal hemispheres, are patterned by an asymmetric distribution of maternal factors. These proteins and RNAs of maternal origin are stored in the oocyte and provide the only source of cell differentiation until the zygotic genome activation. Here, we address the role of RNA localization in this process. We assessed the localization patterns of both, maternal mRNAs and lncRNAs and found that, in addition to protein coding transcripts, lncRNAs also form concentration gradients along the animal-vegetal axis. Furthermore, using RT-qPCR tomography, we have now been able to demonstrate a difference between actively localized extremely animal group and spontaneously localized animal group of transcripts, a distinction emerging from the transcriptome characterization performed in our laboratory. Additionally, we found that the extremely animal...
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Gene expression in early development of Xenopus laevis
Šindelka, Radek ; Jonák, Jiří (advisor) ; Beneš, Vladimír (referee) ; Svoboda, Petr (referee)
Everyhigherorganismconsistsof severalhundredsof differentcell types.The cell differentiationis a processwhich requireshighlypreciseregulationof mRNA productionand subsequentlyproteinsynthesis.Duringdevelopmentof an organism eachcellgainsa uniquepaletteof mRNAand proteinmolecules,whichreflectsthe cell'sfate.To understandthe basicfunction and regulationof genesthat are importantduringdevelopment,mRNA expressionprofilingis an irreplaceabletool. However, most techniquesto determine the mRNA content, such as Northern blot, microarraysand in situ hybridizationhave some limitationsin their specificity,dynamicrangeand/or sensitivity.Quantitativereal-timePCR analysis(qPCR)for nucleicacids quantificationwas introduced in the last decade and it has overcome the above mentioned drawbacks.qPCR has rapidly becomethe goldenstandardin basicresearchas well as in manyaspectsof appliedresearchsuchas moleculardiagnostic,food pathogendetectionand geneticallymodifiedorganism(GMO)analysis. We decidedto applyqPCRin studieswhere it helpsus to understandbasicbiologicalprocessesthattake placein the developingorganism.One of the focusareasof the Laboratoryof gene expressionat IMG AS CR,where Idid my PhDthesis,was the role of Src tyrosine kinasesin the early developmentof vertebratesstudied on the African clawed frog Xenopus...
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Microsatellite region in the murine oncogene src - its primary structure and variability during evolution
Fučík, Vladimír ; Šindelka, Radek ; Beran, Jaroslav ; Ferjentsik, Zoltán ; Jonák, Jiří
In the fifth intron of the Mus musculus oncogene src a complex microsatellite was discovered that consists of a CA run and 31 bp apart followed by track of TG dinucleotides interrupted by extra Gs in specific intervals. As the 31 bp interlink can assume a hairpin structure, the whole region can be viewed as an imperfect inverted repeat. The microsatellite was sequenced in imbred lines of M. m. musculus and M. m. domesticus, as well as in mice of both subspecies trapped near the hybrid zone. Differences in the length of repeat regions, insertion/deletions and simple base changes characterize the strains. Hybrids between the two species could be identified by sequencing of cloned PCR products. The structure of the microsatellite in more distant species was found to be conspicuously different. Instead of frequent (CA)18-25, we can see only (CA)8 in M. spretus, CATGCGCCCCCCA in Mus pahari and CACCCCCC in Mus caroli. No similar structure is present in the corresponding human src intron.
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