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The role of a specific miRNAs in the regulation of apoptosis during physiological and pathophysiological processes in the CNS
Kaslová, Tereza ; Romanyuk, Natalyia (advisor) ; Klassen, Ruslan (referee)
MicroRNAs are small non-coding RNAs of 20 to 24 nucleotides in size that are able to post- transcriptionally regulate gene expression by binding to mRNA. This paper focuses on how these microRNAs are generated and how they are able to regulate at the level of proteins involved in programmed cell death - apoptosis. By what mechanisms apoptosis occurs, what proteins are involved and what changes the cell undergoes are further discussed in this thesis. The precise influence of this post-transcriptional regulation is presented by using selected microRNAs that influence apoptosis during the development of the central nervous system, as well as during and as a consequence of the neurodegenerative diseases and damage that can affect it. Finally, it will also introduce the use of microRNAs as potential biomarkers, due to changes in their levels associated with various diseases, and as direct therapeutic targets. Keywords Apoptosis, microRNA, cell death, central nervous system, neurodegenerative diseases, gene expression regulation
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Characterization of the effect of human mutated huntingtin on the neuronal stem cell differentiation.
Budková, Kateřina ; Vodičková, Kateřina (advisor) ; Romanyuk, Natalyia (referee)
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expansion of the CAG codon repeat in the huntingtin gene (HTT). This expansion causes a change in the biochemical properties of the huntingtin protein (HTT), its aggregation and cellular toxicity, which leads to the degeneration of brain neurons, especially in the striatum. Induced pluripotent cells (iPSC) derived directly from HD patient cells can serve as a model system for in vitro modeling of this disease. Because neuronal dysfunctions occur in HD patients years before the first clinical symptoms manifest, this model system may help elucidate the mechanisms that precede the onset of the disease. The aim of this thesis was to differentiate iPSCs (derived from fibroblasts of HD patients and healthy controls) into neural stem cells (NSCs) and subsequently into neuronal cell populations and to monitor molecular changes in their differentiation associated with the effect of mutated HTT. The differentiation process was monitored based on selected markers using immunofluorescence, western blot and qRT-PCR. We were able to generate stable NSC lines derived from 3 control and 3 HD iPSC lines. All 6 NSC lines were able to further differentiate into neural populations. At the transcriptional level, we found a higher...
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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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The role of miRNA in injury and regeneration of spinal cord tissue
Šprincl, Vojtěch ; Romanyuk, Natalyia (advisor) ; Mrózková, Petra (referee)
MicroRNAs are small non-coding RNA molecules of a length about 20-24 nucleotides, that regulate gene expression post-transcriptionally. They interfere mRNA molecules via base- pairing with complementary sequences. Recently it was shown that they play an important role in injury and regeneration of nervous tissue. The aim of this bachelor thesis is to describe possible role of miRNAs in central nervous system injury with focus on spinal cord injury.
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The effect of the canonical Wnt singalling pathway on the differentiation of polydendrocytes after ischemic brain injury
Knotek, Tomáš ; Anděrová, Miroslava (advisor) ; Romanyuk, Natalyia (referee)
Polydendrocytes, or NG2 glia, are fourth type of glial cells in mammal central nervous system. In the adult brain, NG2 glia represent important cell type with respect to their role in gliogenesis and nervous tissue regeneration following injury. Ligands from the Wingless/Int (Wnt) family play key role in proliferation and differentiation of NG2 glia and they can also influence regeneration of nervous tissue after ischemia. The aim of this thesis was to elucidate the role of NG2 glia in neurogenesis and gliogenesis following ischemic brain injury and investigate the impact of Wnt signalling on the reaction of NG2 glia to this type of injury. To fulfil these aims, transgenic mouse strains with tamoxifen-inducible recombination, that enabled simultaneous expression of red fluorescent dye and either activation or inhibition of the Wnt signalling pathway in NG2 glia, were employed. To induce ischemic injury, middle cerebral artery occlusion model was used. Changes in differentiation and electrophysiological properties of NG2 glia were analysed using patch-clamp technique. Activation of the Wnt signalling pathway under physiological conditions and 7 days after ischemic injury led to increased differentiation of NG2 glia toward astrocytes, while 3 days after ischemic injury activation of this signalling...
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The role of miRNA in injury and regeneration of spinal cord tissue
Šprincl, Vojtěch ; Romanyuk, Natalyia (advisor) ; Mrózková, Petra (referee)
MicroRNAs are small non-coding RNA molecules of a length about 20-24 nucleotides, that regulate gene expression post-transcriptionally. They interfere mRNA molecules via base- pairing with complementary sequences. Recently it was shown that they play an important role in injury and regeneration of nervous tissue. The aim of this bachelor thesis is to describe possible role of miRNAs in central nervous system injury with focus on spinal cord injury.
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The effect of the canonical Wnt singalling pathway on the differentiation of polydendrocytes after ischemic brain injury
Knotek, Tomáš ; Anděrová, Miroslava (advisor) ; Romanyuk, Natalyia (referee)
Polydendrocytes, or NG2 glia, are fourth type of glial cells in mammal central nervous system. In the adult brain, NG2 glia represent important cell type with respect to their role in gliogenesis and nervous tissue regeneration following injury. Ligands from the Wingless/Int (Wnt) family play key role in proliferation and differentiation of NG2 glia and they can also influence regeneration of nervous tissue after ischemia. The aim of this thesis was to elucidate the role of NG2 glia in neurogenesis and gliogenesis following ischemic brain injury and investigate the impact of Wnt signalling on the reaction of NG2 glia to this type of injury. To fulfil these aims, transgenic mouse strains with tamoxifen-inducible recombination, that enabled simultaneous expression of red fluorescent dye and either activation or inhibition of the Wnt signalling pathway in NG2 glia, were employed. To induce ischemic injury, middle cerebral artery occlusion model was used. Changes in differentiation and electrophysiological properties of NG2 glia were analysed using patch-clamp technique. Activation of the Wnt signalling pathway under physiological conditions and 7 days after ischemic injury led to increased differentiation of NG2 glia toward astrocytes, while 3 days after ischemic injury activation of this signalling...
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Treating spinal cord injury with a combination of human fetal neural stem cells and hydrogel modified with serotonin agonists
Růžička, Jiří ; Romanyuk, Natalyia (advisor) ; Vargová, Lýdia (referee)
Spinal cord injury (SCI) results in the loss of nervous tissue and consequently the loss of motor and sensory function. The transplantation of neural stem cells (NSCs) and a porous hydrogel material may support spinal cord repair. In my Master of Science thesis we evaluate the biocompatibility of the human fetal neural stem cell (hfNSC) line SPC-01_GFP3 in combination with hydroxy ethyl methacrylate hydrogel modified with a serotonin agonist (P2544-1). Moreover, we evaluate the effect of a combination of SPC-01-derived progenitors and P2544-1 hydrogel on functional improvement and tissue reconstruction. As a model of SCI, a spinal cord lateral hemisection at the Th8-9 level in adult Wistar rats was used. A P2544-1 hydrogel seeded with SPC-01 cells was applied immediately after the hemisection surgery (n=11) in the treated animals, while the control group was only hemisected (n=20). Locomotor (BBB) and sensitivity (plantar test) evaluations were performed weekly for three months. An immunohistochemical analysis (IHC) of the cells and hydrogel was made in vitro before the surgery and also at the conclusion of the experiment. IHC and the behavioural tests showed that this combination of NSCs and hydrogel material is highly biocompatible in vitro, but that after transplantation it was unable to quickly...
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