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Research of epigenetic aspects of hematopoietic and spermatogenesis stem cells.
Hybešová, Michaela ; Pimková, Kristýna (advisor) ; Děd, Lukáš (referee)
Stem cell differentiation is controlled by coordinated regulation of gene transcription. One of the regulatory factors is the loosening of chromatin and the accessibility of DNA to transcription factors. Chromatin remodeling is mediated by remodeling complexes. The ISWI chromatin remodeling ATPase Smarca5 (S5) is an important factor of remodeling complexes. It is a highly conserved chromatin-remodeling factor forming a catalytic subunit that can be found in several oligosubunit complexes. In these complexes, it actively regulates nucleosome structure and remodeling during DNA replication, repair and transcription. S5 has been identified as a key protein in embryonic development. Its deficiency leads to defects in hematopoiesis and male genital development. In the presented study, we focused on the role of S5 in hematopoiesis and spermatogenesis. Using a mouse model with transgenic expression of S5, co-immunoprecipitation and mass spectrometry, we identified S5 complexes in hematopoietic and testicular cells. We also studied the phenotypic consequences of S5 deficiency in mouse testes and found that it leads to impaired sperm development and male sterility. Using transcriptomic and proteomic analysis, we identified several molecular programs that could lead to reproductive disorders. Our work...
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Stimulation of mesenchymal stem cells osteogenic differentiation using perfusion bioreactor
Šljivnjak, Erna ; Rampichová, Michala (advisor) ; Rösel, Daniel (referee)
Bone cells in vivo reside in a dynamic environment exposed to constant chemical and mechanical stimuli caused by the interstitial fluid flow. It is hypothesized that perfusion of the medium through the scaffold increases the mass transport and creates at the same time shear stress, thereby in vitro simulating the interstitial fluid effects and bone tissue formation conditions. This work examined the effects of perfusion flow rates on cell viability, proliferation, migration and osteogenic differentiation of human mesenchymal stem cells within cell-seeded 3D poly-ε-caprolactone scaffolds. Scaffolds were perfused for 21 days at flow rates 1, 3 and 5 mL/min and were compared to the scaffolds from static culture. Cells were most viable, had upregulated expression of osteogenic markers collagen type I and highest alkaline phosphatase activity under flow rate 1 mL/min when compared to their static counterparts. Cells proliferated the most under flow rate 3 mL/min when compared to their static counterparts. Flow rate 5 mL/min did not significantly differ from the static culture in any of the examined parameters. Cell migration into the scaffold was not improved upon exposure to perfusion. This data confirms that medium perfusion may benefit cell proliferation and osteogenic differentiation by enhancing...
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Significance of the major histocompatibility complex for hemopoietic stem cell transplantation
Graman, Vojtěch ; Slavčev, Antonij (advisor) ; Dobeš, Jan (referee)
The genes of the major histocompatibility complex are located on the short arm of chromosome 6 and encode surface glycoproteins (HLA glycoproteins), which ensure the presentation of self and foreign peptides on the cell surface. These glycoproteins are subsequently recognized by T-lymphocytes and by other cells of the immune system. When the HLA-peptide complex is recognized as foreign, T-lymphocytes and other components of the immune system are activated, and the foreign cell is destroyed. Therefore, in hematopoietic stem cell transplantation (HSCT), HLA incompatibility between donor and recipient causes a strong immune response against the transplanted cells, and is therefore a major criterion in selecting suitable stem cell donors. This work briefly summarizes the current knowledge about the structure and function of HLA class I and class II antigens. The work focuses on HLA typing techniques to help understand the HLA system, which include serological typing methods, as well as modern molecular typing methods based on PCR and next-generation sequencing, and their relevance for HSCT. We also focus on HSCT processes and preparatory therapy, but the main emphasis is on the importance of HLA incompatibilities between stem cell recipients and donors and their effect on HSCT outcome.
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Proteomic analysis of posttranslation modifications in breast cancer cell line profiles
Predná, Nikola ; Laštovičková,, Markéta (referee) ; Strouhalová,, Dana (advisor)
Estrogenové a progesteronové receptory, stejně jako HER2 protein, jsou v současnosti klinicky nejužitečnějšími metabolickými markery u karcinomu prsu. Tyto markery umožňují určit typ nádoru a nejlepší možnosti jeho léčby. Jeden z nejagresivnějších typů tohoto onemocnění, triple-negative breast cancer (TNBC), však tyto klinicky stanovené biomarkery postrádá. To znamená, že hormonální terapie nebo cílené léky nepřicházejí v úvahu, takže je na výběr méně možností léčby. Aby bylo možné vyvinout nové léky na míru, je zásadní pochopení molekulárního základu onemocnění. V poslední době se mnoho studií zaměřuje na hledání biomarkerů na úrovni proteinů pomocí proteomiky. Proteiny, zejména jejich post-translační modifikace (PTM), jsou jádrem mnoha buněčných událostí a jejich odhalení může pomoci při pochopení mechanismů rakoviny prsu. Pro objevení molekulárních rysů TNBC, je cílem této studie porovnat proteomická data neléčených rakovinných buněčných linií s buňkami, které podstoupily retinoidní terapii. Důraz bude kladen na PTM, zejména glykosylaci a fosforylaci, Vimentinu a CD44, které byly navrženy jako potenciální biomarkery TNBC v předchozích studiích. Proteinová separace bude provedena pomocí 1D a 2D gelové elektroforézy nebo pomocí SEC-HPLC. Vzorky budou také podrobeny enzymatickému štěpení před identifikací pomocí MALDI-TOF hmotnostní spektrometrie. V případě fosfoproteinového selektivního záchytu bude obohacení provedeno afinitní chromatografií s použitím hrotů pro obohacení fosfopeptidu TiO2 (TopTip). Glykosylované proteiny budou obohaceny pomocí WGA lektinové afinitní chromatografie. Proteiny s významnými rozdíly v PTM mezi ošetřenými a neošetřenými buňkami budou blíže hodnoceny pomocí proteinových databází (MASCOT, STRING a další). Data získaná ze studie budou případně použita k navržení potenciálních biomarkerů pro TNBC.
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The effect of carbon nanostructures on human cell behavior and the role of fetal bovine serum in cell adhesion
Verdánová, Martina ; Hubálek Kalbáčová, Marie (advisor) ; Brábek, Jan (referee) ; Smetana, Karel (referee)
Graphene (G) and nanocrystalline diamond (NCD) are carbon allotropes and promising nanomaterials with an excellent combination of their properties, such as high mechanical strength, electrical and thermal conductivity, possibility of functionalization and very high surface area to volume ratio. For these reasons, G and NCD are employed next to electronics in biomedical applications, including implant coating, drug and gene delivery and biosensing. For a fundamental characterization of cell behavior on G and NCD, we studied osteoblast adhesion and proliferation on differently treated G and NCD. Generally, both G and NCD exhibited better properties for osteoblast cultivation than control tissue culture polystyrene. Better cell adhesion but lower cell proliferation were observed on NCD compared to G. The most surprising finding was that hydrophobic G with nanowrinkled topography enhanced cell proliferation extensively, in comparison to hydrophilic and flat G and both NCDs (hydrophobic and hydrophilic) with slightly higher roughness. Promoted cell proliferation enables faster cell colonization of G and NCD substrates, meaning faster new tissue formation which is beneficial in biomedical applications. Furthermore, it was shown that osteoblast adhesion was promoted in the initial absence of fetal bovine...
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Mesenchymal stem cells for treatment of spinal cord injury
Dubišová, Jana ; Kubinová, Šárka (advisor) ; Hock, Miroslav (referee)
Poranenie miechy a jeho terapia pomocou mezenchymálnych kmeňových buniek Abstract Patients with serious spinal cord injury (SCI) have a little chance of a full return to their original life. This kind of injuries leaves lifelong consequences as on the patients themselves, as well as their surroundings and family. Considerable financial resources are, worldwide, granted for reintegratation of these patients into society. This work focuses on the description of spinal cord injury, its complexity and the possibility of treatment by new therapeutic methods of mesenchymal cells. The first section discusses the severity of spinal cord injury and its pathophysiology with the performance of subsequent clinical phases. The second part is dedicated to new treatment methods and approaches, with an emphasis on cell therapy. The third part presents treatment method using mesenchymal stem cells (MSCs), which immunomodulatory and anti-inflammatory properties, such as their ability to migration to the injured area and the possibility of genetic modification, made them a great candidates for the treatment of serious injuries. Keywords Spinal cord injury, stem cells, mesenchymal stem cells, cell therapy, regenerative medicine
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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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Experimental models of a transfer of stem cells for therapeutic purposes
Faltýsková, Helena ; Krulová, Magdaléna (advisor) ; Indrová, Marie (referee)
Experimental models of a transfer of stem cells for therapeutic purposes Abstract Stem cell therapy currently represents a standard procedure of treating a wide variety of hereditary diseases and serious injuries. Development of the most suitable way of transfer of stem cells into the patient body remains very important question concerning this type of therapy. In our experiments we used nanofiber scaffolds for stem cell cultivation and their subsequent transfer. These nanofibers were prepared by the original needleless electrospun NanospiderTM technology. Allogeneic cornea or skin graft were transplanted from B6 mice to BALB/c mice. The grafts were covered by a nanofibrous scaffold with cultivated stem cells. Stem cells were stained by an imunofluorescent dye to enable us to monitore their migration from nanofibers into tissues and consequent distribution in the body and characterize changes of this distribution in the time. The methods of ELISA and PCR were used to confirm that mesenchymal stem cells support the production of antiinflammatory cytokines IL-4 and IL-10 and contribute to inhibition of production of proinflammatory cytokines IL-1, IFNγ and inducible nitric oxide synthase. We confirmed an important beneficial role of nanofiber scaffolds in transplantation of mesenchymal stem cells. Nanofiber...
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Potential and significance of extended embryo culture
Uher, Petr ; Rokyta, Zdeněk (advisor) ; Slípka, Jaroslav (referee) ; Pilka, Ladislav (referee)
The aim of this dissertation thesis is to emphasize the sense of extended cultivation of embryos to the stadium of blastocyst and its influence on success of assisted reproduction and facilitation of pre implantation diagnosis, analysis of cultivation media and derivation of human embryonic stem cells. Author summarizes current literary findings in assisted reproduction and examines the currently used methods. Author also submits his own published experimental works, in which he compares his own results of infertility treatment with usage of extended cultivation to blastocyst with results of other techniques. Furthermore author submits his own published experimental works which are using extended cultivation for pre implantation diagnosis and its improvement. Another experimental works includes possibility of stem cells derivation. Usage of extended cultivation to blastocyst convincingly leads, according to author's own experiments and simultaneously to available literary findings, to higher success of infertility treatment. This is especially significant by middle-aged mothers. Sufficient term of cultivation enables not just selection, but also biopsy and its generic treatment. Long-term cultivation also enables analysis of cultivation media - but these didn't met the expectations for increase of...
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