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Differentiation of yeast colonies: The role of selected transcription factors and metabolic proteins
Plocek, Vítězslav
5 Abstract Although yeasts are unicellular microorganisms, they form complex multicellular formations such as biofilms and colonies under natural conditions. Within these structures, processes such as cell differentiation, specialization by particular cell populations and cell signalling, which are typical of multicellular organisms, take place. The literature introduction to this thesis summarizes current knowledge regarding the development of biofilms and colonies, in particular those of the model organism, Saccharomyces cerevisiae, and some selected regulations that are important for the formation of multicellular structures. In the results section, I focus on two lines of research. The first is directed towards mechanisms, involved in the formation of multicellular structures. In studying the formation of SLI biofilms (biofilms at the solid/liquid interface), we have documented the antagonistic role of the regulators CYC8 and TUP1 in their formation and have also described the effect of the presence of glucose on the development and stability of SLI biofilms of strain BR-F. During this study we[D1] have developed an imaging method that allows us to prepare and observe the internal structure (vertical cross-section) of SLI biofilms, as well as the growth of unattached cells, under physiological...
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Immune response in mammalian species against progenitor cell types including induced pluripotent stem cells (iPSCs)
Kovandová, Barbora ; Drbal, Karel (advisor) ; Krulová, Magdaléna (referee)
Stem cells may be very useful tool for regenerative medicine. They are able to repair any tissue in a human body and cure any damage caused by injury, sickness or aging. But at first, we have to deal with problems, which are connected with their usage - especially their immunogenicity. This bachelor thesis is focused on immunogenicity of embryonal (ESC), induced pluripotent (iPSC) and adult stem cells (ASC). Tissues derived from ESC are in vivo described as strongly immunogenic, although they seem to be immunosuppressive in vitro. Another danger of their usage is their tumorigenic potential. There also exist ethical issues connected with their usage. iPSC were supposed to be a good replacement for ESC, because no immunological nor ethical problems were expected. Surprisingly, they were described as immunogenic, too, even in autologous environment. These cells were also described as tumorigenic; this is the main reason for now why they cannot be used for the replacement therapy. Immunogenicity, so as tumorigenicity of iPSC may be a consequence of their dedifferentiation from somatic back to stem cells. ASC are the only stem cells, which are already used for the replacement therapy (transplantation of bone marrow). Some of them are described as immunosuppressive or tumor-suppressive, other are...
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Differentiation of yeast colonies: The role of selected transcription factors and metabolic proteins
Plocek, Vítězslav ; Palková, Zdena (advisor) ; Heidingsfeld, Olga (referee) ; Sychrová, Hana (referee)
5 Abstract Although yeasts are unicellular microorganisms, they form complex multicellular formations such as biofilms and colonies under natural conditions. Within these structures, processes such as cell differentiation, specialization by particular cell populations and cell signalling, which are typical of multicellular organisms, take place. The literature introduction to this thesis summarizes current knowledge regarding the development of biofilms and colonies, in particular those of the model organism, Saccharomyces cerevisiae, and some selected regulations that are important for the formation of multicellular structures. In the results section, I focus on two lines of research. The first is directed towards mechanisms, involved in the formation of multicellular structures. In studying the formation of SLI biofilms (biofilms at the solid/liquid interface), we have documented the antagonistic role of the regulators CYC8 and TUP1 in their formation and have also described the effect of the presence of glucose on the development and stability of SLI biofilms of strain BR-F. During this study we[D1] have developed an imaging method that allows us to prepare and observe the internal structure (vertical cross-section) of SLI biofilms, as well as the growth of unattached cells, under physiological...
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Evaluation of influence of mechanical loading on differentiation of stem cells into smooth muscle cells
Pražák, Šimon ; Filová, Elena (advisor) ; Maxová, Hana (referee)
Cultivation of cells in bioreactors with mechanical load simulates the physiological conditions to which cells in the body are exposed. This technology has been used to induce the differentiation of stem cells from adipose tissue towards the phenotype of vascular smooth muscle cells, which can further serve to form vascular replacements. At present, there is no established strategy for cultivating stem cells while being exposed to mechanical stress. The main aim of this work was therefore to optimize the cultivation strategy and determine the ideal load parameters. Differentiation was analyzed by immunofluorescence of specific smooth muscle cell markers, α-actin and h1-calponin, which were quantified by Western blot. Extracellular matrix production was also detected by immunofluorescence staining. The outcome of this work is the establishment of ideal conditions of cell culture in a bioreactor with mechanical load, during which they differentiate into smooth muscle cells. Three types of scaffolds were used for cultivation; plasma treated glass, fibrin-coated glass and decelularized pericardium. Preliminary results show that smooth muscle differentiation was succesfully induced in human and porcine adipose tissue stem cells. Cells were analyzed after 3 and 7 days of culture. Developing a stem cell...
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Androgenesis
Kočová, Helena ; Honys, David (advisor) ; Kocábek, Tomáš (referee)
(anglicky) Androgenesis in the plant kingdom is an interesting phenomenon, in which a new individual is regenerated from male gametophyte. Having gametophytic, i.e. haploid number of chromosomes, these plants are potentially useful in research as well as for the generation of new genotypes. Duplication of their genetic information then results in fully homozygous plants, that can be used for breeding. At the same time, microspores represent a unique system for studying totipotency, cell proliferation, differentiation and embryogenesis. However, in many important crops as well as in some model species, such technology has not yet been efficiently managed. The aim of this thesis is to summarize the knowledge about androgenesis, from the historical context to the latest discoveries, including methods, development, complications and at the end also the possible use of obtained doubled haploid plants. Keywords: androgenesis, male gametophyte, microspore embryogenesis, pollen, totipotency, cell differentiation, stress, organogenesis, haploid
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The role of Disp3 gene in cell proliferation
Ditrychová, Karolína ; Zíková, Martina (advisor) ; Pospíchalová, Vendula (referee)
Dispatched 3 (DISP3), sterol - sensing domain (SSD) - containing protein, is a key focus of our laboratory. It was described as a gene regulated by thyroid hormone and its expression is mainly localized within neural tissue. Our preliminary data suggested increased DISP3 expression in medulloblastoma, a highly common pediatric cerebellar tumour, therefore we wanted to examine DISP3 role in human cancer cells. The aim of this thesis is to perform DISP3 overexpression and downregulation in human medulloblastoma cell lines and in mouse neural progenitors and analyse its effect on cell proliferation and differentiation. For this purpose, we chose DAOY and D341, human medulloblastoma cell lines with low and high expression of DISP3 and mouse multipotent neural progenitor cell line, C 17.2, with low DISP3 expression. We showed, that DISP3 ectopic expression leads to increase in cell proliferation in both DAOY and C 17.2 cells. Next, we examined the ability of C 17.2 cells to differentiate into neurons and astrocytes and observed, that cells overexpressing DISP3 reveal delay in differentiation, what we proved by analysis of cell specific markers. Using CRISPR-Cas9 targeting system, we reduced DISP3 expression within D341 cells and observed decrease in their proliferation. Finally, we analysed cell cycle...
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Immune response in mammalian species against progenitor cell types including induced pluripotent stem cells (iPSCs)
Kovandová, Barbora ; Drbal, Karel (advisor) ; Krulová, Magdaléna (referee)
Stem cells may be very useful tool for regenerative medicine. They are able to repair any tissue in a human body and cure any damage caused by injury, sickness or aging. But at first, we have to deal with problems, which are connected with their usage - especially their immunogenicity. This bachelor thesis is focused on immunogenicity of embryonal (ESC), induced pluripotent (iPSC) and adult stem cells (ASC). Tissues derived from ESC are in vivo described as strongly immunogenic, although they seem to be immunosuppressive in vitro. Another danger of their usage is their tumorigenic potential. There also exist ethical issues connected with their usage. iPSC were supposed to be a good replacement for ESC, because no immunological nor ethical problems were expected. Surprisingly, they were described as immunogenic, too, even in autologous environment. These cells were also described as tumorigenic; this is the main reason for now why they cannot be used for the replacement therapy. Immunogenicity, so as tumorigenicity of iPSC may be a consequence of their dedifferentiation from somatic back to stem cells. ASC are the only stem cells, which are already used for the replacement therapy (transplantation of bone marrow). Some of them are described as immunosuppressive or tumor-suppressive, other are...
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Regulation of alternative splicing via chromatin modifications
Hozeifi, Samira ; Staněk, David (advisor) ; Krásný, Libor (referee) ; Lanctôt, Christian (referee)
Alternative splicing (AS) is involved in expansion of transcriptome and proteome during cell growth, cell death, pluripotency, cell differentiation and development. There is increasing evidence to suggest that splicing decisions are made when the nascent RNA is still associated with chromatin. Here, I studied regulation of AS via chromatin modification with main focus on histone acetylation. First, we demonstrate that activity of histone deacetylases (HDACs) influences splice site selection in 700 genes. We provided evidence that HDAC inhibition induces histone H4 acetylation and increases RNA Polymerase II (RNA Pol II) processivity along an alternatively spliced element. In addition, HDAC inhibition reduces co-transcriptional association of the splicing regulator SRp40 with the target fibronectin exon. Further we showed that histone acetylation reader, Brd2 protein, affect transcription of 1450 genes. Besides, almost 290 genes change their AS pattern upon Brd2 depletion. We study distribution of Brd2 along the target and control genes and find that Brd2 is specifically localized at promoters of target genes only. Surprisingly, Brd2 interaction with chromatin cannot be explained solely by histone acetylation, which suggests that other protein-domains (in addition to bromodomains) are important for...
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Differentiation within yeast populations of Saccharomyces cerevisiae: influence of cultivation conditions
Šimek, Jan ; Palková, Zdena (advisor) ; Dostál, Jiří (referee)
Long-time research of chronologically aging yeast populations of Saccharomyces cerevisiae laboratory strains revealed that yeasts are able to differentiate into specialized cell types. Differentiation of liquid cultures growing in glucose rich medium and differentiation of colonies growing on solid glycerol medium has been previously studied. These populations create two fractions of cells with diverse morphology which adapt their metabolism and physiology to enable a long-term survival of the yeast population in environment with limited nutritional potential. In this study, yeast subpopulations isolated from colonies growing on solid glucose medium and liquid cultures cultivated in glycerol medium were characterized. Newly isolated cell types were compared with already known cell types isolated from colonies and liquid cultures. Selected metabolic processes and stress resistance were analysed in studied populations. Based on previous studies of yeast differentiation, a spectrum of GFP-labelled marker proteins was choosen and production and localization of these marker proteins was monitored within yeast populations. Results of the analyses showed that type of medium and cultivation influence development and metabolism of each yeast cell type. Key words: Saccharomyces cerevisiae, BY4742, cell...
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