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Study of regenerative potential of Sertoli cell progenitors in Xenopus tropicalis tadpoles after tail amputation.
Wróblová, Aneta ; Krylov, Vladimír (advisor) ; Procházka, Jan (referee)
African clawed frogs (Xenopus) represent an ideal model organism for study of regeneration mechanisms. In frogs, complete regeneration occurs in the tadpole stage. In later stages the regeneration capacity is lost. The Laboratory of Developmental biology was successful in establishment of cell culture called Xenopus tropicalis immature Sertoli cells (XtiSCs) derived from X. tropicalis testes. These cells are common progenitors of Sertoli cells and peritubular myoid cells. XtiSCs show similar characteristics as mesenchymal stem cells. MSCs hold interest of scientists for their immunomodulatory properties and multipotent differential and regeneration potential. In this thesis, we studied regeneration and migration potential of XtiSCs after X. tropicalis tadpole's tail amputation in developmental stage 47 - 50. Transgenic XtiSCs culture expressing RFP was prepared to facilitate transplantation experiments. Transplantation experiments showed preferential migration of XtiSCs into the site of injury. XtiSCs transplantations in X. laevis tadpoles with downregulated NO synthases eNOS and nNOS revealed their migratory dependence on nitric oxide signalization. Imunocytochemical staining of XtiSCs in vitro showed positive iNOS, nNOS and Pax7 expression. Imunohistochemical staining of tadpole's tail vibratome...
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Role of transcription factors Meis during embryogenesis Danio rerio
Brežinová, Veronika ; Machoň, Ondřej (advisor) ; Krylov, Vladimír (referee)
Meis transcription factors belong to the group of TALE (three amino acids loop extension) homeodomain proteins. Meis2 proteins have a potential role in regulation of neural crest cells development and in differentiation of their derivates. Zebrafish genome has two paralogues of meis2 gene, meis2a and meis2b. CRISPR/Cas9 technology was used to prepare mutant lines of both paralogues, meis2a and meis2b, for the purpose of study of function of Meis2 transcription factors. Specific morpholinos that reduce the expression of meis2a and meis2b were used as controls. Craniofacial and cardiac development in mutant fish was analyzed in the meis2a line by RNA in situ hybridization, histological cartilage staining, and computed tomography. While we observed impaired craniofacial and cardiac development after injection of specific Morpholinos, we did not detect similar changes in the meis2a KO line. Our genetic approach has not clearly shown that the meis2a paralogue itself plays an important role in craniofacial development and cardiac development. For more detailed analysis, further experiments on fish lines with combined meis2a and meis2b knock-outs are needed. Key words Mutagenesis CRISPR, Danio rerio, neural crest cells, Meis2, transcription factor
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The effect of urate transporter polymorphisms on uric acid excretion
Mančíková, Andrea ; Krylov, Vladimír (advisor) ; Novotný, Jiří (referee) ; Ježek, Petr (referee)
Uric acid excretion disorders are the most common cause of primary dysuricemia. The kidneys eliminate two-thirds of uric acid production and the other third is eliminated in the gastrointestinal tract. Renal reabsorption and secretion occur through the polarised epithelial cells in the proximal tubules. Uric acid transporters are expressed on these cell membranes. Reabsorption deficiency leads to hypouricemia and elevated fraction excretion associated with urolithiasis, nephrolithiasis or acute renal injury. Decreased uric acid secretion in the kidneys and small intestine leads to hyperuricemia, which develops into gout in 10% of individuals. Genome wide association studies detected a strong effect of SLC22A12 (URAT1), SLC2A9 (GLUT9) reabsorbing transporters and ABCG2 (ABCG2) secreting transporter on uric acid serum concentration variability. This thesis aimed to map out urate transporter allelic variants in a cohort of primary dysuricemia patients and identification of the variants causing defective uric acid excretion. Six non-synonymous variants were described in SLC22A12 (URAT1) and SLC2A9 (GLUT9) genes in hypouricemic individuals, which had not been identified previously in any population studies. Significant decreases in uric acid transport have been demonstrated experimentally in vitro,...
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Regenerative potential of Sertoli cell progenitors regarding heart injury in Xenopus tropicalis
Onhajzer, Jakub ; Krylov, Vladimír (advisor) ; Procházka, Jan (referee)
Cardiac failure is one of the leading cause of deaths worldwide. Potential therapeutic approach, which overcome invasive organ transplantation and delivery of immunosuppressive drugs, is lacking nowadays. However, research of mesenchymal stem cells (MSCs) therapy displays immunomodulation potential, which can further promote variety of organ regeneration without need of drug treatment. Xenopus tropicalis immature Sertoli cells (XtiSCs) culture was established in our laboratory from juvenile Xenopus tropicalis male. XtiSCs possess immunomodulatory capacity and differentiation to cardiomyocytes after the treatment with the inhibitor of glycogen synthase kinase-3 (GSK-3) CHIR99021. To test the survival rate of transplanted XtiSCs we firstly microinjected treated cells directly inside tadpole's heart. XtiSCs proliferated there for the whole tested time period (30 days). However, after direct heart XtiSCs injection and subsequent cardiac injury in adult frog, no cells were localized in wound area. Thus, we focused on remote control of cardiac regeneration using XtiSCs without CHIR99021 treatment. We injected cells inside skeletal muscle bed and confirmed their survival and proliferation. Moreover, if cells were transplanted 3 days before heart injury, it resulted in significant reduction of fibronectin...
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Bisphenol S influence on selected markers of meiotic maturation of porcine oocytes
Černíková, Terezie ; Petr, Jaroslav (advisor) ; Krylov, Vladimír (referee)
Bisphenol A is a widely used chemical in the manufacture of plastics. The presence of BPA in the environment adversely affects human health due to contamination of air, drinking water and food. Growing concerns about the effects of BPA have led to its regulation in production and development of alternative chemicals to BPA, such as bisphenol S (BPS). However, the effects of BPS were not properly tested before its introduction to production and the effects on human reproduction are still unknown. For this reason, it is desirable to test the effect of BPS on mammalian oocyte development. This study hypothesizes that BPS exposure causes inhibition of meiotic maturation of porcine oocytes in vitro. This study aims to investigate the potency of BPS at low concentrations corresponding to normal human exposures to selected porcine oocyte proteins. The results of this study demonstrate the negative effect of BPS on the progression of meiotic maturation and reaching the mature oocyte stage. In addition, the results show an increase in the formation of defective meiotic spindles and a disruption of mitochondrial integrity after exposure to BPS concentrations. However, the effect of BPS on double-strand breaks was not demonstrated in this study, in contrast to the case of BPA. Taken together, the results show...
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Mechanism and risk factors for the development of monozygotic twins in mammals
Žabková, Světlana ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
Monozygotic twins result from the splitting of one embryo in early embryonic development. The developmental stage, in which the splitting occurs, is the main factor determining the degree of sharing fetal sacs. The etiology and mechanism of monozygotic twinning are still unknown. The human and two species of armadillo, Dasypus novemcinctus and Dasypus hybridus are the only mammals that regularly produce monozygotic multiple pregnancies. The spontaneous occurrence of monozygotic twinning is 0,45 % of all births. The monozygotic twin pregnancies have been reported to occur at a higher rate following the increasing interest of assisted reproduction technologies. In certain consideration, we understand them as a side effect of infertility treatment because monozygotic twins result in a higher rate of prenatal mortality, premature birth and congenital anomalies than singleton pregnancies. This bachelor thesis aims to summarize knowledge about the formation of monozygotic twins in mammals. Also, it tries to discuss potential mechanisms and risk factors which could influence their formation. Monozygotic twins are an important model for explaining the genetic predisposition of some diseases. The thesis also introduces the ways of experimental production of monozygotic twins. Keywords: monozygotic twins,...
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Genetic and molecular basis of Meckel-Gruber syndrome
Václavková, Vendula ; Krylov, Vladimír (advisor) ; Niederlová, Veronika (referee)
Meckel-Gruber syndrome (MKS) is rare multisystemic, autosomal recessive hereditary disorder, which appears in different places around the world. MKS is classified as a ciliopathy. These disorders are caused by defects of primary cilium, cell's signaling organelle, during embryogenesis. Meckel-Gruber syndrome represents the most severe form of ciliopathy in human population. MKS is caused by mutation in several genes, involved in correct formation of primary cilium. Until this day, 13 genes have been confirmed. As a result we distinguish 13 types of MKS. More genes are also included in MKS origin, but they do not define solo type of MKS. They are called MKS-related genes. The syndrome was recognized mainly on the basis of clinical cases. A big amount of cases was described in consanguineous families. The MKS is characterized by occipital encephaloce, polycystic kidney disease and polydactyly of hands and feet. These symptoms are common with all 13 types. Syndrome also goes with disorder of nervous and renal system, face defects or undefined genitals. Key words: Meckel-Gruber syndrome, ciliopathy, primary cilium, autosomal recesive, hereditary disease, consanguineous origin, occipital encephaloce, polycystic kidney disease, polydactyly
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Shared mechanisms of development of vertebrate body appendages
Vintr, Jan ; Soukup, Vladimír (advisor) ; Krylov, Vladimír (referee)
Vertebrate body appendages include a number of structures such as limbs, paired and median fins, the tail bud, the cloacal region, barbels or pharyngeal arch derivatives. Despite differences in form and function, these appendages share a number of developmental genetic mechanisms. Development of these structures is chiefly regulated by Fgf, Shh, Wnt, Tgf-β and retinoic acid signalling pathways, activity of transcription factors such as Hox or Tbx along with cis-regulatory elements controlling the genes in which these factors are encoded. A recurring feature shared by some of the herein discussed anatomical structures is a transient epithelial ridge whose activity influences production and proliferation of the adjacent mesenchyme. Some are also instructed by a signalling centre, which confers asymmetry to the structure. In this text, the various appendages are compared on the basis of their development in relation to tetrapod limbs and theories evaluating the emergence of vertebrate paired appendages. The fin fold theory and archipterygial theory are discussed in the light of recent data provided by evolutionary developmental biology. All together, this thesis evaluates shared and derived characteristics of vertebrate body appendages with the aim to provide a basis for the upcoming work on the role...
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The role of nitric oxide during embryonic wound healing and regeneration
Abaffy, Pavel ; Šindelka, Radek (advisor) ; Brábek, Jan (referee) ; Krylov, Vladimír (referee)
The study of the mechanisms that control wound healing is an attention-drawing area within the fields of biology and medicine. Wound healing can be usually defined as two basic types. The first type is adult wound healing, which is slow and results in the scar formation. The second type is referred to as embryonic wound healing, which is in contrast fast and scarless. Wound healing is a complicated process that includes many steps, which are regulated by various types of molecules. One of these important molecules is nitric oxide (NO). Its function is usually connected with the regulation of inflammation and angiogenesis during adult wound healing. However, there is currently no information on its role during embryonic wound healing, where the immune and vascular systems are not yet developed. In this work, we explore and describe the role of the NO during the healing of the early embryos. The highest concentration of the NO post wounding is produced during the first 30 minutes after injury. This applies to all developmental stages, from the blastula stage all the way to the swimming tadpole stage. The main role of the NO during embryonic wound healing is the regulation of the gene expression that is connected with the stress response and the regulation of cellular metabolism. Additionally, we...
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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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