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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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3D models of brain tumors
Fišer, Ondřej ; Novák, Josef (advisor) ; Bohačiaková, Dáša (referee)
Despite intensive research, glioblastoma multiforme remains one of the tumours of the central nervous system with the worst prognosis. The ability of glioblastoma cells to infiltrate brain tissue by forming invasive microtubular structures is stimulated by contact with adjacent non- tumor cells. Intercellular communication and the influence of the extracellular matrix create a specific microenvironment that affects cell signaling, proliferation, differentiation and response to pharmaceuticals. The recurrent form of glioblastoma often displays a much faster progression than the initial disease, which is attributed to the development of resistance to therapeutics and the preservation of the proliferative capacity of some tumour cells. The discovery of the stem- cells ability to self-aggregate in suspension has led to the creation of 3D in vitro models - brain organoids. They are much more complex that the established 2D models and their heterogeneity provides an environment simulating the in vivo state. This thesis aims to describe their use in brain tumour research and techniques for culturing 3D aggregates of neural lineage formed from induced or embryonic human stem cells with respect to their gradually increasing complexity. It also presents methods of addressing issues of hypoxia, organoid...
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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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Pathobiochemistry of lysosomal storage disorders: Study of Fabry disease and generation of cellular models of X-linked disorders.
Rybová, Jitka
Human autopsy or biopsy tissue samples, mouse models and cell cultures of various types represent the most common materials in the investigation of cell pathogenesis of inherited diseases. This dissertation is devoted to all these approaches in the study of two X-linked lysosomal storage diseases, Fabry disease (FD,α-galactosidase A (AGAL) deficiency) and mucopolysaccharidosis type II (MPSII, idunorate-2- sulfatase (IDS) deficiency). The primary goal of the work was analysis of lipid blood group B antigens with terminal α-galactose (B-GSL) in the pancreas of FD patients with blood group B (FD-B).,In addition to the main glycosphingolipid (GSL) substrate, globotriaosylceramide (Gb3Cer), B-GSLs represent another minor substrate of AGAL. The deposition of undegraded B-GSL has been demonstrated in FD-B pancreas where it was significantly higher than in other organs such as the kidneys and lungs which accumulate mainly Gb3Cer. High concentration of lipid and non-lipid B-antigens was primarily confirmed in exocrine acinar epithelial cells of FD-B, accompanied by massive accumulation of ceroid (secondary sign of lysosomal storage). Unlike acini, the endocrine portion of the pancreas remained unaffected by accumulation of AGAL substrates. This interesting phenomenon of cell biology shows how a specific...
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Preparation and characterization of cell models of lysosomal hereditary diseases - Mucopolysaccharidoses
Presová, Gabriela ; Dobrovolný, Robert (advisor) ; Dvořáková, Lenka (referee)
Mucopolysaccharidoses are a group of diseases that belong to lysosomal storage disorders. A common sign of these monogenic multisystem diseases is a gene mutation leading to a deficiency of the lysosomal enzyme participating in glycosaminoglycan degradation. It results to their accumulation in the tissues and organs, where they cause a progressive damage. There is no efficient treatment available for most mucopolysaccharidoses. Moreover, the research is complicated because of the low prevalence and type of affected tissues. Animal models of these human diseases are used for an evaluation of newly developed therapeutic approaches. However, they also have many limitations due to the different pathogenesis and catabolic pathways of the accumulated substrates between humans and animals. Therefore, animal models are replaced by human cell models. In this thesis, the development of four mucopolysaccharidoses human cell models is reported (MPS IIID, MPS IVA, MPS IVB, MPS VI). Corresponding genes (GNS, GALNS, GLB1, ARSB) were inactivated using CRISPR/Cas9 technology, where plasmids containing specific inserts are delivered to the target human induced pluripotent stem cells (iPSC), using electroporation. Isolated clones, which represent iPSC disease models, were characterized by Sanger sequencing, enzyme...
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Pathobiochemistry of lysosomal storage disorders: Study of Fabry disease and generation of cellular models of X-linked disorders.
Rybová, Jitka
Human autopsy or biopsy tissue samples, mouse models and cell cultures of various types represent the most common materials in the investigation of cell pathogenesis of inherited diseases. This dissertation is devoted to all these approaches in the study of two X-linked lysosomal storage diseases, Fabry disease (FD,α-galactosidase A (AGAL) deficiency) and mucopolysaccharidosis type II (MPSII, idunorate-2- sulfatase (IDS) deficiency). The primary goal of the work was analysis of lipid blood group B antigens with terminal α-galactose (B-GSL) in the pancreas of FD patients with blood group B (FD-B).,In addition to the main glycosphingolipid (GSL) substrate, globotriaosylceramide (Gb3Cer), B-GSLs represent another minor substrate of AGAL. The deposition of undegraded B-GSL has been demonstrated in FD-B pancreas where it was significantly higher than in other organs such as the kidneys and lungs which accumulate mainly Gb3Cer. High concentration of lipid and non-lipid B-antigens was primarily confirmed in exocrine acinar epithelial cells of FD-B, accompanied by massive accumulation of ceroid (secondary sign of lysosomal storage). Unlike acini, the endocrine portion of the pancreas remained unaffected by accumulation of AGAL substrates. This interesting phenomenon of cell biology shows how a specific...
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Pathobiochemistry of lysosomal storage disorders: Study of Fabry disease and generation of cellular models of X-linked disorders.
Rybová, Jitka ; Ledvinová, Jana (advisor) ; Entlicher, Gustav (referee) ; Živný, Jan (referee)
Human autopsy or biopsy tissue samples, mouse models and cell cultures of various types represent the most common materials in the investigation of cell pathogenesis of inherited diseases. This dissertation is devoted to all these approaches in the study of two X-linked lysosomal storage diseases, Fabry disease (FD,α-galactosidase A (AGAL) deficiency) and mucopolysaccharidosis type II (MPSII, idunorate-2- sulfatase (IDS) deficiency). The primary goal of the work was analysis of lipid blood group B antigens with terminal α-galactose (B-GSL) in the pancreas of FD patients with blood group B (FD-B).,In addition to the main glycosphingolipid (GSL) substrate, globotriaosylceramide (Gb3Cer), B-GSLs represent another minor substrate of AGAL. The deposition of undegraded B-GSL has been demonstrated in FD-B pancreas where it was significantly higher than in other organs such as the kidneys and lungs which accumulate mainly Gb3Cer. High concentration of lipid and non-lipid B-antigens was primarily confirmed in exocrine acinar epithelial cells of FD-B, accompanied by massive accumulation of ceroid (secondary sign of lysosomal storage). Unlike acini, the endocrine portion of the pancreas remained unaffected by accumulation of AGAL substrates. This interesting phenomenon of cell biology shows how a specific...
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Treatment of spinal cord injury by transplantation different types of stem cells
Dubišová, Jana ; Kubinová, Šárka (advisor) ; Cizkova, Dasa (referee)
Spinal cord injury (SCI) is complicated injury with serious socioeconomic consequences for the patient and his whole family. Big difficulty cause also extremely high living expenses for the patient with this type of injury. That's why there is a need for therapeutic methods which would help patients after SCI to recover the lost functions and be able at least partially to return to their normal life. Different therapeutic methods are being used for SCI treatment. In this study we used four various types of stem cells: human bone marrow stem cells (hBM-MSCs), human umbilical cord mesenchymal stem cells (hUC-MSCs), neural precursors derived from induced pluripotent stem cells (iPS-NPs) and neural stem cell line derived from human fetal spinal cord tissue (SPC-01). These cells have been transplanted intrathecally or intraspinally 7 days after induction of the experimental model of SCI in the rat. We studied expressions of genes related to neurogenesis, growth factors and inflammation 10 and 28 days after SCI. Our analysis showed significant changes in gene expression 10 days after SCI. Significant up-regulation in expression of vascular endothelial growth factor (Vegf), ciliary neurotrophic factor (Cntf) and interferon regulatory factor 5 (Irf5) were found after transplantation of hBM-MSCs and hUC-...
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