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Neuronal cell culture in vitro
Kohoutová, Šárka ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Neuronal cell cultures are in vitro cultures of dissociated neurons that have become an essential part of many neurobiological experiments in the last century. Cultured neurons not only allow to answer questions about their physiology under complex in vivo conditions, but also can serve as a model of neurodegenerative diseases. Neuronal cells can either be isolated directly from the nervous tissue of animals at the prenatal or adult stage of development, or they can be obtained through targeted manipulations of stem cells and secondary cell lines that lead to their neuronal differentiation. Primary neurons are considered the gold standard of neurobiological research not only because of their long tradition of cultivation, but also because primary neurons retain typical neuronal properties under in vivo conditions. There are several disadvantages associated with primary neurons, including the fact that fully differentiated neurons do not proliferate and are relatively demanding in terms of culture conditions For this reason, their role is often replaced by mitotically active secondary cell lines or stem cells. This bachelor thesis summarizes the knowledge about cell cultures used to study the functions of neuronal cells and highlights the advantages and disadvantages of their use. Key words Primary...
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Small extracellular vesicles as microRNA carriers and their role in neural cell regeneration
Šprincl, Vojtěch ; Romanyuk, Natalyia (advisor) ; Kriška, Ján (referee)
Acute spinal cord injury is a serious type of injury, the treatment of which still represents a challenge for contemporary medicine. Neural stem cells (NSCs) transplantation is one of many promising ways to contribute to the regeneration of damaged tissue. NSCs communicate with the rest of the tissue by means of small extracellular vesicles (sEVs), in which their regenerative potential is thus assumed. The aim of this thesis is to verify the antiapoptotic effect of sEVs isolated from the culture medium of two types of NSCs in an rat in vitro model of spinal cord injury. To meet this goal, different methods of isolating sEVs from the culture medium were tested. Subsequently, sEVs were characterized according to their size and the presence of surface markers. This thesis includes an PCR analysis of the cargo of sEVs, which showed an abundance of neuroprotective and antiapoptotic miRNAs. Fluorescent staining of sEVs proved that sEVs penetrate into the cytoplasm of stem cells. Finally, sEVs were applied to a rat in vitro model of spinal cord injury and their neuroprotective effect was demonstrated using the immunoblotting method. Keywords Small extracellular vesicles, exosomes, acute spinal cord injury, regeneration of nervous tissue, miRNA, microRNA, neural stem cells
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Methods of isolation of individual subpopulations of neural stem cells
Zelený, Martin ; Zíková, Martina (advisor) ; Vargová, Ingrid (referee)
Stem cells have huge medical potential, but they are not explored enough for the use in medicine yet. Because of that, their further research is important. One group of the stem cells are neural stem cells, which are differentiating into the neural and glial cells in the brain. Neural stem cells are present at the two areas in the adult brain, subventricular zone and subgranular zone of hippocampus. Neural stem cells are not one homogenous population of cells, but rather various subpopulations with different cell expression. In my bachelor thesis I described the methods, which are used in the research of neural stem cell subpopulations. Described methods focused mainly on the isolation and gene expression. Keywords: neural stem cells, neurogenic zones, molecular markers
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Cell therapies designed to restore radiation-induced brain injury
Nuriakhmetova, Elina ; Zíková, Martina (advisor) ; Weissová, Romana (referee)
Neural stem cells are undifferentiated cells that are defined by their replication potential and their ability to differentiate into different types of neuronal and glial cells. The adult brain contains two neurogenic zones located in the subventricular zone of the lateral ventricles and in the dentate gyrus of the hippocampus. In both of these zones, neural stem cells produce new neurons. The subject of the bachelor thesis is a summary of current knowledge about neural stem cells and molecular mechanisms underlying changes in neurogenic zones after radiotherapy. At the same time, cell therapies used to minimize damage associated with radiation are discussed.
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Human in vitro models for epilepsy research
Hebenstreitová, Kristýna ; Danačíková, Šárka (advisor) ; Valeš, Karel (referee)
Epilepsy is the most common chronic neurological disease that affects around 1 % of the world population. It is a multifactorial disease, which is characterised by recurrent seizures. Present pharmacological treatment is symptomatic and approximately one third of the patients develop pharmacorezistant epilepsy. This bachelor thesis presents an overview of current knowledge about the modelling of epilepsy, while focusing on human in vitro models. Cell lines appear to be very promising in vitro models for modelling genetic epilepsies. For example, cell lines derived from human induced pluripotent stem cells or human neural stem cells, which both allow for introduction of potentially pathological mutations and further differentiation into many different cell types. Human in vitro models of epilepsy play important role in understanding the process of epileptogenesis, ictogenesis, mechanism of antiepileptic drugs effects and are used in the search for new active substances. Epilepsy comes with great burden of disease for the patients, that is why it is very important to research not only the molecular mechanisms of epileptogenesis, but also advancements of personalised therapy, which could give hope to many (even pharmacorezistant) patients.
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The role of morphogenes in stem cell neurogenic differentiation in mammals
Slováková, Lucie ; Tlapáková, Tereza (advisor) ; Kyclerová, Hana (referee)
Stem cells are non-differentiated self-renewing cell population that can derive different kinds of cell types according to their differential potential. Neurogenic differentiation is the process of generating of all three types of nervous systems from the neural stem cells. This process is common for embryonic development, however neurogenesis appears to be present also in adult mammalian brain. It continues to generate new neurons within its microenvironments called niches and we can find two major areas of neurogenesis. One is the subventricular zone of the forebrain, the other is the subgranular zone within the hippocampal dental gyrus. In these niches we can find specific signaling molecules called morphogens. Morphogens function in regulating neural stem cell activity. They play a part in proliferation, differentiation and cell migration, thus determining the fate of neural cells. In addition, morphogens play an important role in many diseases and cancers.
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The role of the Wnt signalling pathway in proliferation and differentiation of neural stem cells in the neonatal and adult mouse brain
Koleničová, Denisa ; Anděrová, Miroslava (advisor) ; Janečková, Lucie (referee)
The canonical Wnt/β-catenin signalling pathway plays an important role in proliferation and differentiation of neural progenitors during embryogenesis as well as postnatally. In the present study, the effect of the Wnt signalling pathway on the differentiation potential of neonatal and adult neural stem cells (NS/PCs) isolated from subventricular zone (SVZ) of lateral ventricles and their membrane properties were studied eight days after the onset of in vitro differentiation. To manipulate Wnt signalling at different cellular levels, three transgenic mouse strains were used, which enabled inhibition or activation of the pathway using the Cre- loxP system. We showed that the activation of the Wnt signalling pathway leads to higher expression of β-catenin in both postnatal as well as adult NS/PCs, while Wnt signalling inhibition results in the opposite effect. To follow the fate of NS/PCs, the patch-clamp technique, immunocytochemistry, and Western blot were employed. After eight days of NS/PCs differentiation we identified three electrophysiologically and immunocytochemically distinct cell types of which incidence was significantly affected by the canonical Wnt signalling pathway, only in differentiated neonatal NS/PCs. Activation of this pathway suppressed gliogenesis, and promoted neurogenesis,...
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