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Bioinformatics analysis of sequences required for localization of RNA during development
Naraine, Ravindra ; Šindelka, Radek (advisor) ; Fulková, Helena (referee) ; Tichý, Boris (referee)
The development of a complex organism from the fusion of two cells (oocyte and sperm) has been a fascinating aspect of developmental biology. It is now known that certain spatially and temporally regulated molecules tightly regulate embryogenesis. The asymmetrical gradient of these molecules within a given cell or within groups of cells helps to guide the differentiation of certain parts of the developing embryo. In fishes and frogs, the establishment of the maternal animal-vegetal transcript gradient within the egg produces the first developmental axis and subsequent formation of the ectoderm, mesoderm, and endoderm regions. Despite this important process, most of our knowledge on this initial animal-vegetal distribution in vertebrates has been limited primarily to the Xenopus laevis model, involved the analysis of only few transcripts and also analyzed only polar regions of the oocyte. This thesis aims to address this deficit in knowledge by leveraging high throughput analysis (RNA sequencing) to characterize and compare the maternal transcriptome and its sub-compartmentalization within the egg of four distantly related models. Additionally, we analyzed different stages of oocyte maturation to determine where the observed localization occurs. The models used were the African clawed frog (Xenopus...
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Regeneration initiation of Xenopus laevis tail-functional study
Netušil, Jiří ; Šindelka, Radek (advisor) ; Petr, Jaroslav (referee)
The recent introduction of high-throughput sequencing techniques rapidly changes our perception of vertebrate regeneration. One of the model organisms intensely studied for its regenerative potential is the embryonic tadpole stage of African clawed frog (Xenopus laevis). This thesis aims to review and functionally validate some of the latest findings gained by RNA sequencing of regenerating Xenopus laevis tail. We specifically focus on gene expression changes during the early phases of regeneration and how their absence affects the progression and phenotypic outcome of this process. Our analysis confirms that Regeneration initiating cells (RICs) identified by single-cell and spatial RNA sequencing represent a vital element in successful tail regeneration. The RIC marker genes mmp9 and pmepa1 affect the processes linked to extracellular matrix remodelling and the migration pattern of previously described Regeneration organizing cells (ROCs) while not interfering with the myeloid cell lineage. We further demonstrate a novel use of Vivo Morpholino oligonucleotides in a transient knockdown assay and offer an assessment of its benefits and limitations. This thesis highlights the importance of spatio-temporal regulation happening on both cellular and molecular levels during the initial 24 hours of...
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Detection and quantification of maternal RNA localization during early development
Šimková, Kateřina ; Šindelka, Radek (advisor) ; Frolíková, Michaela (referee)
The asymmetric localization of maternal RNAs and proteins is a crucial mechanism for the body plan development in many animal species. These maternal factors are expressed during the oogenesis and they are used for the regulation of early developoment. In this diploma thesis, I addressed the role of asymmetrically localized RNAs along animal- vegetal axis in the early development of Ambystoma mexicanum. The second part of my thesis is focused on RNA localization in dorso-ventral and left-right axes. I identified the localization patterns of many known RNAs along the animal-vegetal axis using RT-qPCR and I also detected several genes, which can be involved in the dorso-ventral or lef-right patterning. Furthermore, we performed transcriptome wide analysis, which revealed changes classified into the following categories: RNAs relocalization, de novo synthesis before the onset of MBT and RNAs degradation during early development. Many vegetally localized genes in Xenopus laevis with the important role in the development of primordial germ cells have been previously described. Surprisingly, my results show that many of these genes are degraded during the early development of A. mexicnum. I believe that this degradation may be due to a different mechanism of PGC development in A. mexicanum and X. laevis....
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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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Gene expression differences during regeneration in model organisms
Netušil, Jiří ; Šindelka, Radek (advisor) ; Hason, Martina (referee)
With new progressive methods allowing us to study natural regeneration in model organisms, we have an opportunity to gain important insights into the very essence of this process. These insights might help us radically improve the current state of therapeutic approaches based on tissue replacement. Many different animal models display an incredible ability to restore various body parts, allowing them to escape predators and avoid premature death. While invertebrate models give us a chance to investigate the fundamental elements of regeneration, vertebrates represent systems often more resembling human biology. This thesis outlines the variability of regeneration in frequently studied model organisms with a special emphasis on the impact of gene expression. Keywords: regeneration, model organism, gene expression, injury
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Role of matrix metalloproteinases in regulation of late embryonic healing process
Kikinderová, Paulína ; Šindelka, Radek (advisor) ; Procházka, Jan (referee)
Cutaneous wound healing could be distinguished into two main types: embryonic and adult. Embryonic healing in contrast to adult is faster, scar-less and consists of early, middle, and late phases. Actin ring is formed during the early phase and its cables pull the edges of the wound towards apposite sites during the following middle phase. De novo expression of healing specific genes is initiated also during middle phase. However, process of the wound healing continues under the closed wound in the late phase which has been poorly described. Adult wound healing is more complex, longer, and is divided into 4 phases: haemostasis, inflammation, proliferation, and remodelling phase. Adult wound healing might end with the scar. Pivotal role in the wound healing is given to matrix metalloproteinases (MMPs). These remodelling enzymes are important for releasing cytokines, inducing apoptosis, and degradation of extracellular matrix. Our laboratory performed temporal RNA-sequencing of the healing tissue using tailbud stage and swimming tadpole embryos. Results showed predominant expression of four mmps: mmp1.L, mmp7.S, mmp8.S, and mmp9.L. Injury or amputation caused the upregulation and their expression level peaked at 3-6 hours post injury which corresponds with late phase of healing. Thus, I focused on...
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The analysis of plasticity of cancer cell invasiveness
Merta, Ladislav ; Brábek, Jan (advisor) ; Šindelka, Radek (referee) ; Staněk, David (referee)
The ability of cancer cells to adopt various invasive modes (the plasticity of cancer cell invasiveness) represents a significant obstacle in the treatment of cancer metastasis. Cancer invasiveness involves various modes of migration. Cells can move together (with the preserved intercellular junctions; collective invasiveness) or individually. Within individual invasiveness, we distinguish two principal invasive modes - mesenchymal and amoeboid. The mesenchymal mode of migration is characterized by an elongated shape, proteolytic degradation of the fibres of the extracellular matrix, and the formation of strong contacts with the extracellular matrix. The amoeboid mode of migration is not dependent on proteolytic activity, the cells are characterized by a round shape and increased contractility, which they use to squeeze themselves through the pores of the extracellular matrix. This thesis deals with the analysis of the plasticity of cancer cell invasiveness, specifically the transitions between individual amoeboid and mesenchymal migration modes, in the 3D environment of the collagen gel as a model of extracellular matrix. The work presents models of mesenchymal-to-amoeboid transition (MAT), which include BLM, HT1080 and MDA-MB-231 cell lines, in which MAT is induced by the expression of...
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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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Primary mouth formation in basal fishes
Psutková, Viktorie ; Černý, Robert (advisor) ; Šindelka, Radek (referee)
The mouth is a crucial structure for every organism since it forms an important connection between internal and external environments. The primary mouth develops from the ectodermal-endodermal region at the anteriormost part of the forming head during early embryogenesis. In the majority of vertebrates the primary mouth develops through the stomodeal invagination, establishment of the oral membrane, and by its rupture the mouth gets opened. However, the mouth development significantly differs in several groups of vertebrates and the three main modes of the mouth development were recognized according to ectoderm-endoderm interactions. The most common is "stomodeal invagination", the "stomodeal collar" is in salamanders and lungfishes, and "stomodeal wedge" in teleosts. Here, I analyse the development of the oral region in embryos of basal actinopterygian fishes, bichir (Polypterus senegalus), sturgeon (Acipenser ruthenus) and gar (Atractosteus tropicus), which could give us better understanding of the evolution and morphogenesis of the primary mouth in vertebrates. In bichir embryos, the mouth development generally resembles development of the stomodeal wedge in teleost fishes, with some morphogenetic differences. In sturgeons, the mouth forms via modified stomodeal collar, whereas in gars the mouth...
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