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Functional analysis of posttranscriptional gene regulation by TENT5A in biomineralization and metabolism
Aranaz Novaliches, Goretti ; Sedláček, Radislav (advisor) ; Hovořáková, Mária (referee) ; Tencerová, Michaela (referee)
Non-canonical poly-A polymerases, such as TENT5A, belong to the Terminal nucleotidyl transferases (TENTs) family and are crucial for mRNA protection, stability, and translation. A Tent5a knock-out (KO) mouse model was generated in our laboratory, which exhibited a phenotype in teeth, skeleton structure, and metabolism. In my PhD project, I aimed to characterize the molecular mechanism underlying these phenotypes and explore their potential connection to rare human diseases. I focused on the biological function of Tent5a gene in enamel development (amelogenesis) and mRNA stabilization. Micro-computed tomography and scanning electron microscopy revealed that Tent5a KO mice displayed thin, hypomineralized enamel with disrupted microstructure, a condition known as Amelogenesis imperfecta. Direct mRNA sequencing demonstrated that TENT5A is responsible for polyadenylation of amelogenin (AmelX) and other secreted proteins, leading to a shortened poly-A tail in Tent5a KO ameloblasts. Moreover, Tent5a KO mice disclosed impaired self- assembly of enamel matrix proteins (EMPs) such as AMELX and ameloblastin (AMBN), leading to compromised hydroxyapatite deposition and enamel formation. In addition to its role in teeth, I investigated the physiological functions of EMPs in other tissues, considering that EMP...
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Influence of the epicardium on cardiac regeneration after cryoinjury during embryonic development - analysis of morphological and physiological changes
Neffeová, Kristýna ; Kolesová, Hana (advisor) ; Hovořáková, Mária (referee)
Millions of people die from myocardial infarction every year. In the heart, damaged cardiomyocytes do not regenerate and fibrotic scar forms. Surface layer of the heart - epicardium, improve heart regeneration processes by producing signalling factors. Epicardial cells undergo epithelial-mesenchymal transition and give rise to various cells involved in heart regeneration (coronary vessel formation and possibly new cardiomyocytes). To study the effect of epicardium on cardiac regeneration, I introduced a novel method of myocardial cryoinjury in avian embryos. In embryonic stages, where the heart is already covered by a layer of epicardium. Probe cooled in liquid nitrogen, injured the left ventricle of the heart and simulate myocardial infarction. Cryoinjury is an appropriate alternative to induce myocardial infarction as the coronary ligation is not feasible in early embryos. After cryoinjury, were observed changes in the epicardium. Epicardium is activated (expression of WT1) and detached from myocardium and form protrusions. In subepicardium we observed a large number of migrating cells. These cells undergo epithelial-mesenchymal transition were detected by cytokeratin and vimentin antibodies. We observed α-SMA positive cells and undifferentiated cardiomyocytes (N2.261 antibody) in the...
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Pathogenesis of limbs in Sprouty2/Sprouty4 mice
Cimlerová, Markéta ; Hovořáková, Mária (advisor) ; Buchtová, Marcela (referee)
Sprouty proteins play the role of negative regulators of receptor tyrosine kinases (RTKs), including the fibroblast growth factor (FGF) signaling pathway. During embryonic development, this pathway is essential for the regulation of cell proliferation, migration, differentiation and apoptosis, and its activity is crucial for normal limb development. An integral part of this pathway are also FGF receptors (FGFR), of which, for example, FGFR3 significantly affects chondrocytes in the growth plate and its mutations lead to the development of chondrodysplastic syndromes. Sprouty proteins play important roles during the formation of body tissues in the process of organogenesis. It has been shown that Sprouty2 and Sprouty4 are involved in the formation of many organs, such as the brain, lungs, teeth, but also in the formation of limbs. Sprouty2 is a regulator of enchondral ossification, modulates signaling in osteoblasts and chondrocytes and is essential for normal chondrocyte proliferation and differentiation. Sprouty4 is expressed in the progress zone of the limb bud. Disruption of Sprouty2 and Sprouty4 gene expression leads to limb defects, which has been demonstrated, for example, in mouse or chicken embryos. In the presented work we deal with the development of the limbs of transgenic mice with...
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Bone regeneration and its analysis
Tesařová, Adéla ; Hovořáková, Mária (advisor) ; Zahradníček, Oldřich (referee)
Bone regeneration can take place throughout life as a natural process (remodeling) or because of trauma when fractures heal in which the damaged or missing part is replaced by new tissue. The aim of my bachelor thesis was to create a literature search on the issue of bone regeneration. The introductory part deals with the bone as such - its structure and the process of new formation (ossification) and remodeling. The following section describes some of the methods used to support bone regeneration in therapy - selected types of carriers and molecules that play an important role in regeneration and are part of the carriers or therapies used. At the end of the thesis, the basic methods used to analyze bone regeneration are described and provide us with an insight into the success of the tested therapeutic approaches, how well the bone heals and how effective the methods used are. The bachelor thesis was processed in the form of a literary search.
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Mapping of the cell population expressing Sonic hedgehog during the embryonic development of heart
Břežná, Veronika ; Hovořáková, Mária (advisor) ; Šaňková, Barbora (referee)
Sonic hedgehog is one of three members of the Hedgehog family, whose signaling pathway plays a crucial role in controlling the development of vertebrates. Sonic hedgehog has an important role in organizing the developmental processes of majority of organ systems. Interestingly, its direct expression or a contribution of cell lineages expressing this signaling molecule in the myocardium has never been described. The aim of this work was to try to detect the cells that expressed Sonic hedgehog directly in the heart tissue with a focus on the myocardium. We monitored these cells from embryonic day 10.5 to 16.5 and then postnatally. We also evaluated current Sonic hedgehog expression in cardiac tissue from embryonic day 12.5 to embryonic day 15.5. We used the CreLoxP system, X-gal staining, fluorescence and confocal microscopy to detect cell lines expressing Sonic hedgehog. We also assessed the presence of cells that expressed Sonic hedgehog in the developping heart in the past using immunohistochemistry. According to our results, the presence of a descendant cell lineage expressing Sonic hedgehog in the past was demonstrated prenatally and postnatally in the mouse myocardium. The performed analysis shows that these cells can be detected in cardiac tissue from embryonic day 10.5 till the postnatal...
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Morphological variability of the incisor in mutant mice
Lochovská, Kateřina ; Hovořáková, Mária (advisor) ; Churavá, Svatava (referee)
Myš je nejfrekventovaněji používaným experimentálním modelovým organismem pro studium vývoje zubů. Myší funkční dentice obsahuje jeden řezák oddělený od tří molárů dlouhou bezzubou diastemou v každém čelistním kvadrantu. Cílem této práce bylo shrnout poznatky o myší dentici a jejím vývoji se zaměřením na myší řezáky a jejich patologie. Myší řezák je díky svým vlastnostem jedinečným zubem. Charakteristickou vlastností hlodavců jsou právě kontinuálně rostoucí řezáky. Tyto řezáky jsou pokryté sklovinou pouze na labiální straně. Lingvální povrch je tvořen pouze dentinem. Toto je spojeno s asymetrickou abrazí. Nicméně je také častým cílem mutací, ať už v podobě delece nebo jiné modifikace genů. Tyto mutace dentálních signálních drah jsou studovány na mutantních myších jako jsou například Tabby myši, Sprouty nebo Small eye (Sey) mutantní myši a mnoho dalších. Některé mutace jsou homologní k lidským onemocněním. Například X-vázaný tabby (Ta) syndrom u myší je považován za homolog hypohidrotické ektodermální dysplásie (HED) u lidí. Tato mutace napadá velikost řezáku, jeho tvar a pozici stejně jako cytodiferenciaci. Dále se může objevit hypodoncie, anodoncie nebo nějaké morfologické změny napadající ostatní existující zuby. Abnormality v počtu, velikosti a tvaru zubu byly dobře zdokumentovány v lidské...
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Eda gene in the development of teeth
Dalecká, Linda ; Hovořáková, Mária (advisor) ; Buchtová, Marcela (referee)
Ectodysplasin A (EDA) is a transmembrane protein of the TNF family, which plays an important role in the development of ectodermal derivates, such as teeth, hair or glands. Mutation in Eda gene causes the Hypohidrotic ectodermal dysplasia (HED) in humans. Sonic hedgehog (Shh) as a downstream of Eda signalling pathway is an important signalling molecule involved in the initiation of tooth development. In frame of the present study, we aimed to evaluate the involvement of Eda gene during the development of mice teeth and its relation to Shh signalling, since Shh is an important marker of the normal tooth development. We focused on Shh expression in Eda mutant mice teeth with spontaneous mutation in Eda gene, since these mice represent a natural model for X-linked HED. First, we compared the Shh expression pattern during the tooth development in Eda mutant mice with CD1 control using the dissociation of dental epithelia and fluorescent microscopy. Consequently, we focused on the cell line expressing Shh observation using Cre-loxP system. We also visualized SHH expression in Eda mutants using imunohistochemistry and qantified SHH protein in the embryonic jaws using western blot. According to our results, the development of teeth in Eda mutants seems to be approximately one day delayed compared to...
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Analysis of embryotoxic effect of hydrocortisone using chick embryotoxicity screening test (CHEST).
Janíková, Michaela ; Peterka, Miroslav (advisor) ; Hovořáková, Mária (referee)
Cleft lip is one of the most common human birth deffects. Its etiopathogenesis is multifactorial and many aspects of its occurrence remain unknown in the fields of both genetics and teratology. One of the set of known negative external factors causing cleft lip is chemical hydrocortisone. Its effect on cell proliferation is highly heterogeneous and depends on attributes of a specific cell population. In this work we studied the cleft beak origin after the hydrocortisone treatment on the basis of Chick Embryotoxicity Screening Test (CHEST). Our main aim was to detect cell cycle changes in the chick frontonasal process after hydrocortisone injection via flow cytometry analysis. Hydrocortisone caused S phase arrest within a minor subpopulation of highly granular cells with specific cell cycle. This sensitive subpopulation was localized in the areas of previously defined proliferative centers within the frontonasal process using immunohistochemistry of frozen sections. Quantitative analysis of cells in these areas revealed significant decrease of M phase portion in the hydrocortisone treated samples in comparison with the control samples. The TUNEL staining of histological sections was used to determine the apoptotic rate in the frontonasal process. The comparison between the control and the...
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Developmental and structural analysis of similarities among calcified and keratinized tooth structures in mouth of vertebrates
Karpecká, Zuzana ; Černý, Robert (advisor) ; Hovořáková, Mária (referee)
Calcified teeth comprise the hardest vertebrate tissues (enamel and dentine) and they undoubtedly represent a key evolutionary advantage for their users. Nevertheless, teeth have been lost many times and they were often replaced by keratinised structures, such as rhamphoteca of birds or of turtle beaks, or labial tooth structures and scratching laminae of many aquatic vertebrates. This thesis is intended to analyse levels of similarities among keratinised teeth and structures of three phylogenetically distinct lineages of amniotes and to compare them to morphogenesis of true teeth. Detailed developmental and structural characteristics of oral apparatus possessing keratinous teeth of lampreys, Loricarid cathfishes and frog tadpoles were identified and distinguished. Keratinous structures of cathfishes include only horny projections from single cell surface (unculi), whereas in frog tadpoles both horny jaw sheet and unicellular labial teeth composed by cell columns were found; the lamprey oral apparatus consists of multicellular teeth and laminae instead. Disparate phenotypes of these keratinous structures can, however, developmentally derive from a general process of keratinization: morphological disparities arise by distinct gradual accretion of keratinous layers and collumns during development. My...
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The signs of odontogenesis externally to the prospective functional dentition in the experimental model
Fábik, Jaroslav ; Hovořáková, Mária (advisor) ; Buchtová, Marcela (referee)
6 Abstract The development of the mouse tooth primordium is an important model for studying odontogenesis, as well as general organogenesis. The development of the mouse lower incisor is of remarkable interest. The epithelial anlage of the mouse lower incisor is interconnected with the vestibular anlage via the epithelial bridges. According to some authors, the epithelial bridges represent an area, where the transient rudimentary incisor germ appears. From a morphological point of view, the first sign of ongoing epithelial-mesenchymal interactions during early odontogenesis is the thickening of oral epithelium. From a molecular point of view, it is the expression of the Shh, Eda, Edar, Pitx2, Bmp2, Bmp4 and Dlx2 genes. Except for the transcription domain, representing the proper signalling centre of a developing prospective incisor, a transient transcription domain, localized anteriorly and superficially, appears in the odontogenic zone of the epithelium during early development. The anterior transcription domain originates in the area of epithelial bridges, and according to some authors, from an evolutionary point of view, it corresponds with the signalling centre of the rudimentary incisor germ. The aim of this diploma thesis was to compare the temporospatial dynamics of SHH and EDA protein expression in...
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