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Development and optimalization of sectioning technique for the study of migration and differentiation potential of testicular stem cells in X. tropicalis tadpoles
Bláhová, Monika ; Krylov, Vladimír (advisor) ; Pšenička, Martin (referee)
Thanks to their ability to differentiate into variable cell types and migrate to the site of an injury mesenchymal stem cells (MSC) are broadly used in regenerative medicine. Their relative easy availability together with the property to control the immune system determines them as a cure of autoimmune diseases or a recovery of wounded tissues. Similar features posses Sertoli cells which take place in the seminiferous tubule of testis. Cell culture of testicular stem cells from juvenile male testes of X. tropicalis (XtTSC) was established in supervisor's laboratory. This cell culture showing both MSC's and SeC's properties was transformed to carry red fluorescent protein RFP. The aim of this diploma thesis was to investigate an behavior of transformed XtTSC in living organism, therefore cells were transplanted into the X. tropicalis tadpoles in stage 41. Subsequently, their migration potential was explored. To study of XtTSC's differentiation potential it was necessary to introduce a reliable sectioning techniques for the subsequent immunohistochemical analysis. Based on our experiments, we found that the XtTSC's cell culture contains precursors of SeC and peri-tubular myoid cells, however in vivo these cells turned into the dedifferentiated MSC-like state allowing a strong migration through the...
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Gene expression in chicken embryo: micromanipulation and visualization methods
Bendová, Michaela ; Dvořák, Michal (advisor) ; Hirsch, Ivan (referee) ; Krylov, Vladimír (referee)
The aim of this work was to obtain better insight into the principles of cell structures and organs in the chicken embryo development. To reach this goal special methods of micromanipulations and visualization in vitro, ex vivo, ex ovo and in ovo were implemented and adjusted. These methods were used to study gene expression in neural crest development and eye development. In the course of long term research in our laboratory we observed that oncoprotein v-Myb influences the development of the neural crest and has the capacity to change natural cell fate. We performed a series of experiments to investigate v-Myb protein influence on neural crest cells differentiation, especially melanocyte lineage development, and its influence on gene expression in the neural crest. Therefore we focused on Gremlin 2 (PRDC), the gene upregulated by v-Myb in the neural crest. The established procedure of electroporation in ovo was adjusted to transfect cells of the developing eye and used to study gene expression during lens induction. The results obtained from chicken embryo experiments endorsed the study performed on mouse embryos. Futhermore, the electroporation technique was slightly modified for manipulations of the neural retina in the developing eye in ovo. Thereafter, the retinas were processed ex vivo and...
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Effect of ABCG2 allelic variants on the transport of uric acid
Vávra, Jiří ; Krylov, Vladimír (advisor) ; Ježek, Petr (referee)
Uric acid is a main metabolite of purine degradation in humans and in higher primates. Its increased plasmatic level is called hyperuricemia and may be the cause of gout and many other similar diseases. Uricemia is controlled by many transporters, which are located in proximal tubule of human kidney. When some transporter have abnormal function, the physiological plasmatic level of uric acid may be impaired. In genome wide association study (GWAS) it was discovered that some hyperuricemia or gout patients have ABCG2 protein damaged. This protein carries out uric acid from epithelial cell to the urine. The goal of this diploma thesis is the determination of transport capacity of ABCG2 allelic variants found via GWAS (Institute of Rheumatology of 1st medical faculty UK in Prague) in vitro with Xenopus laevis oocyte expression system. Uric acid secretion was compared with wild type variant. Keywords: Uric acid, GWAS study, Xenopus laevis, membrane transport protein, ABCG2
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Genetics and molecular basis of spina bifida
Kim, Alice ; Krylov, Vladimír (advisor) ; Hovořáková, Mária (referee)
Spina bifida is a neural tube defect that evolves during neurulation when the neural folds fail to fuse and result in an open neural tube. New studies have shown that our understanding of neural tube closure might be wrong and suggested a single site neural tube closure in humans. Various factors like nutrition, genetics, and environment lead to the formation of a neural tube defect. Folic acid and Vitamin B12 have been shown as effective supplements when it comes to lowering the risk of developing NTD and. Genetic mutations of MTHFR, CUBN, CHKA, SARDH, MTRR, Grhl-3, which are all involved in methylations are considered important risk factors for NTD's. Wrong methylation or hypomethylation of Hox and Vangl genes have shown to be also playing a role in NTD's. Par1/Par2 mutations in mice have shown to cause Spina Bifida. Keywords: Spina Bifida, NTD, Neural tube defects, Myelomeningocele, Neurulation
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Cellular and molecular basis of a skin regeneration in amphibians and mammals
Hybešová, Michaela ; Krylov, Vladimír (advisor) ; Jendelová, Pavla (referee)
Wound healing of skin in mammals and its regeneration in Amphibians are crucial biomedicine topics in the last few decades. The most important aspect in humans is the scrarring proccess and the effort to substitute it with the regeneration producing functional and differentiated tissues. To modulate the formation of scar it is neccessary to compare both proccesses. The core animal model is axolotl (Caudata) where regeneration takes place during whole lifespam. On the other hand, in frogs (Anurans) this phenomenon is restricted up to metamorphosis. After metamorphosis, the immune system of Anurans is similar if compared to mammals. Similarly, the transition from early embryonic development in mammals, where the fetus was able to completely regenerate damaged tissue to an adult type of healing, goes hand-in-hand with the development of the immune system and structural differentiation of damaged tissue. Thus, the inflammatory cells and their regulation, the formation of ECM, which includes fibroblast proliferation, and the production of appropriate cytokines are key factors that distinguish the process of healing with and without scarring.
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Sperm cell penetration and membrane fusion with oocyte during fertilization in mammals
Klimková, Veronika ; Krylov, Vladimír (advisor) ; Frolíková, Michaela (referee)
Fertilization is a process involving multiple steps, which are in continuity and complement each other. Spermatozoa become competent to fertilize after capacitation, hyperactivation and acrosome reaction. Oocytes are surrounded by a layer of cumulus cells and give a signals for spermatozoa to activation and orientation. This study focuses on the latest knowledges about a roles of cumulus cells, production of chemoattractants and the possibility that cumulus cells induce the acrosome reaction after binding a novel sperm protein NYD-SP8 to the cumulus. It seems that progesterone induce the acrosome reaction and also is the best chemoattractant secreted by cumulus cells. Next I am focus on gamete fusion including the role of surface proteins such as Juno and IZUMO1. Key words: membrane fusion, cumulus cells, acrosome reaction, chemotaxis sperm, oocyte, fertilization
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The study of Xenopus tropicalis testis-derived stem cells
Nguyen, Thi Minh Xuan ; Krylov, Vladimír (advisor) ; Bartůněk, Petr (referee) ; Hovořáková, Mária (referee)
The study of Xenopus tropicalis testis-derived stem cells Nguyen Thi Minh Xuan Abstract The substances secreted by Sertoli cells (SCs) are crucial to determine male sex characteristics in embryos and regulate spermatogenesis in adulthood. The failure in SC maturation can cause sterility in men. Before puberty, SCs keep the ability to proliferate and have been considered as immature cells. They differ remarkably from mature cells in connection with their morphology and biochemical activity and thus they probably play a part in maintaining spermatogonia stem cells in an undifferentiated stage. The transient presence of cytokeratin in immature SCs has been reported in many species, but not in Xenopus yet. We investigated which molecules are expressing only in immature Sertoli cells of X. tropicalis testes. The regulation of cytokeratin and β-catenin was revealed by fluorescent immunostaining. Cytokeratin and membrane β-catenin co- expressed in X.tropicalis juvenile testes and in cultured SC progenitors, called XtiSCs, but they were absent in adulthood. There was no signal of cytokeratin in migrating SCs (pre-SCs) located outside the seminiferous tubules. The suppression of cytokeratin along with the breakdown of β-catenin-based cell contacts have been observed in XtiSCs after the treatment with a small...
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Effect of sperm ubiquitination in early embryonic development of porcine embryos
Petelák, Aleš ; Krylov, Vladimír (advisor) ; Šušor, Andrej (referee) ; Procházka, Radek (referee)
The PhD thesis is focused on the effect of porcine sperm cell extracellular ubiquitination on early embryonic development up to the blastocyst stage after ICSI. In addition, it also presents a potential improvement of the technique of in vitro fertilization using oocyte incubation with ion channels regulators. To address these aims, we established an entirely novel methodology for sperm cell sorting using flow cytometry and subsequent cryopreservation. We determined the conditions for successful sperm cell sorting based on extracellular ubiquitination rate providing highly specific selection as well as sufficient numbers of viable sperms for fertilization using the ICSI method. Concerning the following cryopreservation, established methods were optimized to enable freezing of a minimal sperm cell suspension volume with low cell numbers. The performed experiments showed a direct relationship between the rate of extracellular ubiquitination and the capability of sperms to give rise to a properly developing embryo. Highly ubiquitinated sperm cells were less successful regarding the embryonic development to the blastocyst stage if compared with the lowly ubiquitinated group (6,2 % vs. 16,7 %, P<0,001). Interestingly, the rate of extracellular ubiquitination showed no effect on the pronuclear formation...
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Transdifferentiation of somatic cells into hepatocytes and clinical relevant edition of the Tight junction protein 2 gene
Fryntová, Lucie ; Janečková, Lucie (advisor) ; Krylov, Vladimír (referee)
Transdifferentiation induces chromatin reconstructions and epigenetic changes that affect gene expression spectum and cause cell remodeling in general. Direct conversion of mature somatic cell line into another mature cell type occures during the transdifferentiation thereby differences betweeen individual germ layers are eliminated. The aim of the master thesis is transdifferentation of mesenchymal cells - mouse embryonic fibroblast into endodermal cells - hepatocytes in vitro, using combination of transcripion factors Hnf4α and Foxa1. Detection of fibroblasts transformation has been initiated immediately after retroviral transduction and final generation of induced hepatocyte culture was confirmed by morphological and function analysis. The population of mouse induced hepatocytes served as a possible model for human liver disease in case of a pacient whose liver proteins could not be detected immunohistochemically. Genome editing of induced hepatocytes was realized by CRISPR/Cas9 technology which is based on cooperation of guideRNA and Cas9 nuclease followed in addition to generation of DNA-specific double strand breaks. These specific breaks in the Tight junction protein 2 gene were repaired via homologous recombination that induced a missense mutation with amino acid changes in the target...
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Analyses of Monocercomonoides genome sizes, ploidies and karyotypes
Kornalíková, Martina ; Hampl, Vladimír (advisor) ; Krylov, Vladimír (referee)
Oxymonads are a group of flagellate protists living in low oxygen environments - mainly the guts of insects and vertebrates. In this study, we focus on the analysis of ploidy and karyotype of various species of oxymonads using Fluorescence In Situ Hybridization (FISH) with probes against single copy genes and telomeric repeats as well as estimating the DNA content in the nuclei of these oxymonads using flow cytometry. Using specific FISH probes against SufDSU gene, which is present in a single copy in the haploid genome, we showed that all studied strains are probably haploid. From the genome of Monocercomonoides exilis strain PA203 we know that oxymonads have the ancestral type of telomeric repeat (TTAGGG). Using a probe against these repeats we tried to label chromosome ends and estimate the number of chromosomes for seven strains (five species) of Monocercomonoides. With a single exception, the average number of signals per nucleus was below 20 indicating number of chromosomes below 10. In the strains of M. mercovicensis, we observed much higher number of signals suggesting that the cells have much higher number of chromosomes. Finally, we established the DNA content for several strains using flow cytometry. We used as a standard M. exilis strain PA203 knowing that the haploid genome size is...
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