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Gene expression in chicken embryo: micromanipulation and visualization methods
Bendová, Michaela
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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Molecular logic of the Notch ligands in development and disease
Trampotová, Eliška ; Mašek, Jan (advisor) ; Rozbeský, Daniel (referee)
The Notch signalling pathway is one of the key signal transduction pathways in the embryonic development of all metazoa. In mammals, the "canonical" signalling occurs on four Notch receptors, and it is triggered by five ligands varying in both their structures and functions. The importance of the pathway for embryonic development is reflected by the fact that mutations of either receptors or ligands result in various congenital disorders. A prime example is the Alagille syndrome - a rare multisystemic condition caused by mutations of the Notch ligand Jagged1 in 94 % of cases. Much of the research effort so far was concentrated into elucidating how the Notch receptors operate, however one could argue the importance of ligand activity regulation is even higher. This thesis thus aims to provide a comprehensive comparison of the five mammalian Notch ligands' structures and roles in developmental processes. The main focus of the thesis is the variability of ligand-receptor interactions and biophysical mechanisms of signalling, highlighting the importance of the Notch ligands' intracellular domains' interactions.
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Asymetric segregation of cell determinants in embryos with regulatory development
Chramostová, Kamila ; Krylov, Vladimír (advisor) ; Černý, Robert (referee)
Asymmetric segregation of cell determinants specific the cell fates in early embryonic development. In C. Elegans somatic and germ cells are separeted in early development. On the basis of the first five asymmetric divisions AB, MS, C, E, D, P4 are found. In this specifications, par genes and genes responsible for the specification of blastomers are involved. P granules are segragated to P4 cells. Xenopus as a representative of amphibians, belonging to a group with regulative embryogenesis , exhibit features of mosaic development like C. Elegans. Maternal RNAs in germ plasm determine germ cells already in oocyte. These RNAs are transported through two distinct pathways. METRO pathways is used for a transportation of mitochondrial cloud RNA. It is specific for Xcat-2, Xpat, XIsirt, Xdazl. Vg1 and VegT RNAs are involved in the second pathway called Vg1 . This RNA contribute to the specification of mesoderm and endoderm. Although mammals reveal regulative development they also exhibit some features of the mosaic embryos. As an example, in the mouse embryo, overexpressing cadherin leads to compaction at the 8 -cell stage. Then the cells of embryo are polarized. Polarized internal cells give rise to ICM. Conversely, the outer nonpolarized cells give rise to TE. Specific factor for TE is Cdx2, for ICM is...
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Physiological role of SIGIRR in early embryonic development.
Hanusová, Zdeňka ; Filipp, Dominik (advisor) ; Brdička, Tomáš (referee)
IL-1 receptor/Toll-like receptor (IL-1R/TLR) supefamily represents a group of proteins that share highly conserved TIR domain in their cytoplasmic region. Signal transduction mediated by TIR-containing proteins involves the activation of NF-κB transcription factor and thus the members of this superfamily play a key role in many physiological responses related to innate immune defense and inflammation. SIGIRR (single immunoglobulin IL1R-related molecule) is a recently discovered member of the IL-1R family, however it differs from the other group members by its unique structural features. SIGIRRhas been so far considered to be an 'orphan' receptor as no SIGIRR ligand has been identified yet. Moreover, SIGIRR itself is not capable to induce the NF-κB activation. Instead, SIGIRR is supposed to act as a negative regulator for IL- 1Rs/TLRs mediated inflammation. Its inhibitory function has been implemented in several signalling pathways in various cell types and tissues including the kidney, the digestive tract and the lung. Recent reports also suggest that SIGIRR could play a role in early embryonic development. The main aim of this thesis is to characterize the mechanism how SIGIRR negative regulatory function in IL-1R/TLR signalling pathway is delivered. Here we describe the establishment of...
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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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Molecular mechanisms of coronary vasculature development
Neffeová, Kristýna ; Kolesová, Hana (advisor) ; Neckář, Jan (referee)
The cardiovascular system is the first functional system that develops in vertebrates during embryonic development. Its irreplaceable function is the transport of nutrients and the removal of waste products. During the development the heart not only grows, but also acts as a pump that drives the blood circulation of the embryo. With advancing development, it is necessary to ensure an adequate supply of oxygen to the heart, for that reason coronary arteries are formed. Each cardiomyocyte is surrounded by at least one capillary, therefore the interaction between cardiomyocytes and endothelial cells plays an indispensable role in the proper functioning of the heart. Understanding, how cardiomyocytes and endothelial cells communicate, is essential for medical research in cardiac tissue regeneration. A number of factors involved in coronary development are described in the literature. However, these factors are described as separate signaling pathways, not as a system of mutually interacting mechanisms. The main goal of my bachelor thesis is to connect individual signaling cascades important in cardiomyocyte-endothelial cell communication and describe their interactions. The main factors overviewed are VEGF, Notch, PDGF, Angiopoietin and others. Factors function and signalization is reviewed in details....
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Formation of spatio-temporal molecular gradients in early embryonic development of Xenopus laevis.
Flachsová, Monika
Clarifying the underlying spatio-temporal mechanisms that determine body pattern is important for detailed understanding of embryonic development. A crucial question of vertebrate embryogenesis remains: when and how are single blastomeres determined for differentiation that subsequently leads to body axes specification and the formation of different tissues and organs? The answer to this question will be beneficial for primary research as well as in the field of applied medicine. The main aim of the presented thesis was to study spatio-temporal molecular gradients of cell fate determinants during early embryonic development. The African clawed frog Xenopus laevis was used as a model organism because of their large size of oocytes and external embryonic development. Due to late activation of embryonic transcription, a crucial mechanism of early blastomeres determination is dependent on asymmetric localization of maternal factors within oocyte and their uneven distribution into single blastomeres during early cell division. Two main localization patterns were identified along the animal-vegetal axis of the mature Xenopus oocyte using qPCR tomography. The localization gradient with preference in either animal or vegetal hemisphere was found for maternal mRNA as well as miRNAs. Moreover, two vegetal...
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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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Asymetric segregation of cell determinants in embryos with regulatory development
Chramostová, Kamila ; Krylov, Vladimír (advisor) ; Černý, Robert (referee)
Asymmetric segregation of cell determinants specific the cell fates in early embryonic development. In C. Elegans somatic and germ cells are separeted in early development. On the basis of the first five asymmetric divisions AB, MS, C, E, D, P4 are found. In this specifications, par genes and genes responsible for the specification of blastomers are involved. P granules are segragated to P4 cells. Xenopus as a representative of amphibians, belonging to a group with regulative embryogenesis , exhibit features of mosaic development like C. Elegans. Maternal RNAs in germ plasm determine germ cells already in oocyte. These RNAs are transported through two distinct pathways. METRO pathways is used for a transportation of mitochondrial cloud RNA. It is specific for Xcat-2, Xpat, XIsirt, Xdazl. Vg1 and VegT RNAs are involved in the second pathway called Vg1 . This RNA contribute to the specification of mesoderm and endoderm. Although mammals reveal regulative development they also exhibit some features of the mosaic embryos. As an example, in the mouse embryo, overexpressing cadherin leads to compaction at the 8 -cell stage. Then the cells of embryo are polarized. Polarized internal cells give rise to ICM. Conversely, the outer nonpolarized cells give rise to TE. Specific factor for TE is Cdx2, for ICM is...
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Formation of spatio-temporal molecular gradients in early embryonic development of Xenopus laevis.
Flachsová, Monika
Clarifying the underlying spatio-temporal mechanisms that determine body pattern is important for detailed understanding of embryonic development. A crucial question of vertebrate embryogenesis remains: when and how are single blastomeres determined for differentiation that subsequently leads to body axes specification and the formation of different tissues and organs? The answer to this question will be beneficial for primary research as well as in the field of applied medicine. The main aim of the presented thesis was to study spatio-temporal molecular gradients of cell fate determinants during early embryonic development. The African clawed frog Xenopus laevis was used as a model organism because of their large size of oocytes and external embryonic development. Due to late activation of embryonic transcription, a crucial mechanism of early blastomeres determination is dependent on asymmetric localization of maternal factors within oocyte and their uneven distribution into single blastomeres during early cell division. Two main localization patterns were identified along the animal-vegetal axis of the mature Xenopus oocyte using qPCR tomography. The localization gradient with preference in either animal or vegetal hemisphere was found for maternal mRNA as well as miRNAs. Moreover, two vegetal...
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