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The role of cytoskeleton in cell chirality
Jandjuková, Anna ; Libusová, Lenka (advisor) ; Soukup, Vladimír (referee)
The cytoskeleton is one of the key structures inside the cell. The cytoskeleton includes microfilaments, microtubules, intermediate filaments, and more recently, septins. Cytoskeletal proteins play a role in intracellular transport, cell movement, cell division, and other structural functions. A chiral object is one that is not identical to its mirror image. The concept of chirality is most commonly used to describe chemical molecules. Cells of living organisms are composed of molecules, most of which are chiral. In recent years, cellular chirality has been observed, likely involving the cytoskeleton. Cellular chirality refers to the cell shape, organelle positioning, stable cell movement and directionally biased growth of cell culture. The latest findings suggest a probable influence of cell chirality on the development of right-left asymmetry during the embryonic development of animals. This work summarizes current knowledge regarding the role of the cytoskeleton in establishing cellular chirality and its likely connection to the development of right-left asymmetry during embryonic development. Key words Cell chirality, cytoskeleton, embryonal development
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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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Cytoskeletal orchestration of early mammalian development
Novotný, Jan ; Libusová, Lenka (advisor) ; Mašek, Jan (referee)
Structures of the cytoskeleton play a critical role in early mammalian development. Microtubules, intermediate filaments and actin microfilaments provide structural and mechanical basis for major developmental transitions of preimplantation development. Microvilli supported by bundles of actin filaments become restricted to one pole of the blastomere during the process of polarisation. All components of the cytoskeleton network undergo apparent reorganisation in embryo compaction. Cells flatten and adhere to each other thanks to the formation of cytoskeleton-linked intercellular junctions and oscillatory contractions generated by actomyosin complexes. Filopodia, actin based protrusions, are debated for their potential role in blastomere apposition and cell signalling. The first process of cell line specification in mammalian development is that which defines outer cells of morula as presumptive trophectoderm and inner cells as presumptive inner cell mass. Proteins and structures of cytoskeleton are asymmetrically distributed between these two populations helping to determine the fate of each blastomere. The aim of the thesis is to summarise the roles of cytoskeleton in blastomere polarisation, embryo compaction and specification of inner and outer cell populations in mammalian preimplantation...
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The role of vent genes family in early development and brain development
Fabian, Peter ; Kozmík, Zbyněk (advisor) ; Němec, Pavel (referee) ; Balaštík, Martin (referee)
6 III ABSTRACT (ENGLISH) In chordates, the central nervous system (CNS) is derived from the dorsal part of gastrula. Induced dorsal part of the embryo - the neural plate - gives rise to the neural tube or primordial brain. The developing dorsal part of the embryo is shaped by BMP/Smad signaling from the ventral part. Using the basal chordate amphioxus, we show here the conserved evolutionary role BMP/Smad signaling in axial cell fate determination. Pharmalogical inhibition of BMP/Smad signaling induces dorsalization of Branchiostoma floridae (amphioxus) and Oryzias latipes (medaka) embryos and expansion of neural plate markers. We provide evidence for the presence of the positive regulatory loop within the BMP/Smad signaling network of amphioxus. Thus, our data suggest that early emergence of a positive feedback loop within the BMP/Smad signaling network may represent a crucial molecular event in the evolutionary history of the chordate cell fate determination. The dorso-ventral body axis formation is mediated by genes of the vent family, which are the direct targets of BMP/Smad signaling. The function of vent gene family in early development is relatively well known, however, its role in developing CNS is not yet clear. Therefore, we decided to manipulate vox transcription factor, a vent family member....
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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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Functional role of Islet1 in pancreatic development
Malfatti, Jessica ; Pavlínková, Gabriela (advisor) ; Krausová, Michaela (referee)
1 Abstract Diabetes mellitus is characterized by the dysfunction and reduction of insulin-producing cells, resulting in hyperglycemia, which in long term harms the organism. For future therapy, it is crucial to understand the function of various factors participating in the differentiation and maturation of endocrine pancreatic cells. The aim of this study was to unravel the functional role of ISL1 during the development of the pancreas. ISL1 is expressed in all endocrine cells of the islets of Langerhansbut its function remains unclear, especially during early pancreatogenesis. As the global deletion of this gene is embryonically lethal, we used the tissue specific deletion of Isl1 in Neurod1 possitive cells using the Cre-loxP system. In this work we studied the effect of this deletion on the structure of islets of Langerhans, the formation of endocrine cell types and relative expression of genes during early pancreatic development. A defective achitecture of islets together with postnatal absence of α-cells was found in the Isl1 deletion mutant. Also, the expression of genes important for the specification of α-cell lineage and their subsequent function was decreased. The secondary outcome was the optimalization of a protocol for effective sorting of endocrine cells using fluorescent flow cytometry, which...
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