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Automated bioreactor for the cultivation of living cells
Ukropcová, Iveta ; Tolde, Ondřej (referee) ; Dostál, Zbyněk (advisor)
Control of cultivation conditions in the~live cell imaging extends the possibilities of biological experiments and makes the experimental results more reliable. In order to change the~cultivation conditions in a controlled manner and increase the reproducibility of the experiments, it is necessary to reduce the amount of manual operations and replace them with automated procedures. Therefore, the concept of a new automated culture device (bioreactor) was created. This device controls the exchange of medium in the observation chamber, ensures the circulation and exchange of the atmosphere and controls its composition. The bioreactor is intended for use in the Laboratory of Experimental Biophotonics. This laboratory is equipped with coherence-controlled holographic microscope (CCHM), which uses quantitative phase imaging (QPI) method. Thus, the bioreactor is adapted to the current requirements of this laboratory and optical elements of the bioreactor meet the requirements of the QPI method. This text specifies the cultivation conditions of the living cells and summarizes, how the conditions could be controlled in the live cell microscopy. Next some commercially available culture devices are described and assessed, whether they are convenient for the~use in Laboratory of Experimental Biophotonics. The crucial part of the thesis is the~design, construction and testing of the new bioreactor.
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Cytoskeleton-membrane protein interaction network in sperm
Adamová, Zuzana ; Komrsková, Kateřina (advisor) ; Tolde, Ondřej (referee)
In order to fertilize the egg, sperm cell undergoes several subsequent maturation processes. The final one called acrosome reaction is an exocytosis of acrosome vesicle, which is filled with lytic enzymes. Acrosome reaction is crucial for penetration of the sperm cell through the egg surroundings, especially zona pellucida, as well as for reorganization of a membrane protein composition on its surface. This rearrangement leads to the exposure of proteins essential for fertilization, mainly for gamete recognition, binding and fusion in specific compartments of the sperm head. One of such protein is CD46, which is located in the acrosomal membrane of an intact sperm and after acosomal exocytosis it relocates to the equatorial segment of a sperm head, which is known to be the initial site of interaction of sperm with the egg plasma membrane. The relocation of CD46 is disrupted by inhibition of actin, which reorganization within sperm head is known to play a role in onset of acrosome reaction, however, the precise mechanism of CD46 interaction with actin in sperm is unknown. In this thesis, ezrin - a crosslinker of membrane proteins and actin - has been studied in context of CD46 and its relocation across the sperm head. Analysis of the immunofluorescent detection of ezrin revealed its mutual...
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Regulation of actin cytoskeleton and epithelial cells morphology by the ERK signaling pathway
Rasl, Jan ; Vomastek, Tomáš (advisor) ; Tolde, Ondřej (referee)
The ERK signalling cascade belongs to a familly of the signalling pathways conserved in eukaryotic cells, which responds to the wide spectrum of extracellular stimuli and convert these stimuli to appropriate response. In epithelial cells the ERK signalling cascade induces disintegration of epithelial architecture and induces morphological changes leading to the gain of the autonomy of the epithelial cells. During morphological changes of the epithelial structure, the ERK signalling cascade participates in the remodelling of the actin cytoskeleton, which leads to the disassembly of cell-cell adhesions and the loss of the epithelial polarity. Subsequently ERK activates the migration programme, which enables epithelial cells to use individual mesenchymal-like mode of migration. The so called peripheral actin is one of the least explored actin structures that forms at the periphery of the epithelial cells and surrounds the colony of epithelial cells. Peripheral actin is located at the basal side of the cell and it probably takes part in the integrity of epithelial tissue. Nevertheless, up to date it is not know if and how ERK signalling cascade regulatesthe peripheral actin and if remodeling of peripheral actin takes part in the cell migration. In this thesis we show, that ERK signalling cascade is...
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Mechanisms of invasiveness and transcription regulation in cancer cells
Tolde, Ondřej ; Folk, Petr (advisor) ; Kovář, Jan (referee) ; Brdička, Tomáš (referee)
The mechanisms of invazivity and regulation of transcription of cancer cells Cancer originates in cells that overcome the control mechanisms of the organism. Cancer cells can be eventually released from the site of origin and spread through tissues. Cancer cells can acquire certain mechanisms that enable them to more effectively invade surrounding tissue or layers of other cells. The research on the migration of cancer cells is important for the understanding of the origin and spreading of metastases and consequently for anticancer therapy. In my Ph.D. work, I participated in the research of the properties of invasive metastatic cells. We compared non-invasive rat sarcoma cell line with a higly metastatic cell line derived from it. We showed that cells of the invasive cell line use amoeboid mode of migration, have upregulated Rho/ROCK signaling, and have accumulated actin and myosin at the leading edge. It is at the leading edge where the cells generate their traction forces. Cells of non-invasive cell line use mesenchymal mode of migration and generate forces mainly at their retracting end. We also compared two breast cancer cell lines derived from a single carcinoma. We showed that the more invasive cell line, derived from its parental line by neoplastic transformation, displayed elevated cytoskeletal...
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Physiological and pathophysiological aspects of hydrogen sulfide metabolism in humans
Neminářová, Kristýna ; Kožich, Viktor (advisor) ; Tolde, Ondřej (referee)
Hydrogen sulfide (H2S), known as a toxic gas for a long time, was recently shown to be an important signaling molecule. Hydrogen sulfide is produced in small concentration in organism and exhibits a physiological role in many tissues (brain, blood vessels, lungs). Hydrogen sulfide is mostly formed enzymatically from L-cysteine by two enzymes - cystathionin β-synthase (CBS, EC 4.2.1.22, L- serine hydro-lyase) occurring especially in the brain and cystathionine γ-lyase (CSE, EC 4.4.1.1, L- cystathionine cysteine-lyase) generating hydrogen sulfide mainly in the small intestine, portal vein and thoracic aorta. In vessels hydrogen sulfide acts as a vasorelaxant factor and reduces blood pressure while in the brain it is involved in neuronal transmission. In addition to these effects, hydrogen sulfide plays a role in inflammatory processes as well as in the transmission of pain. Hydrogen sulfide acts through activation of KATP channel (in blood vessels, digestive tract, and in inflammation), activation of NMDA receptors (brain), reduces molecules causing oxidative stress (lungs, brain) and affects influx of Ca2+ ions into the cells (retina). Based on these findings it is apparent that the modulations of metabolism of hydrogen sulfide may have a therapeutic potential, e.g. in vascular disease or in...
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Mechanisms of invasiveness and transcription regulation in cancer cells
Tolde, Ondřej
The mechanisms of invazivity and regulation of transcription of cancer cells Cancer originates in cells that overcome the control mechanisms of the organism. Cancer cells can be eventually released from the site of origin and spread through tissues. Cancer cells can acquire certain mechanisms that enable them to more effectively invade surrounding tissue or layers of other cells. The research on the migration of cancer cells is important for the understanding of the origin and spreading of metastases and consequently for anticancer therapy. In my Ph.D. work, I participated in the research of the properties of invasive metastatic cells. We compared non-invasive rat sarcoma cell line with a higly metastatic cell line derived from it. We showed that cells of the invasive cell line use amoeboid mode of migration, have upregulated Rho/ROCK signaling, and have accumulated actin and myosin at the leading edge. It is at the leading edge where the cells generate their traction forces. Cells of non-invasive cell line use mesenchymal mode of migration and generate forces mainly at their retracting end. We also compared two breast cancer cell lines derived from a single carcinoma. We showed that the more invasive cell line, derived from its parental line by neoplastic transformation, displayed elevated cytoskeletal...
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Automated bioreactor for the cultivation of living cells
Ukropcová, Iveta ; Tolde, Ondřej (referee) ; Dostál, Zbyněk (advisor)
Control of cultivation conditions in the~live cell imaging extends the possibilities of biological experiments and makes the experimental results more reliable. In order to change the~cultivation conditions in a controlled manner and increase the reproducibility of the experiments, it is necessary to reduce the amount of manual operations and replace them with automated procedures. Therefore, the concept of a new automated culture device (bioreactor) was created. This device controls the exchange of medium in the observation chamber, ensures the circulation and exchange of the atmosphere and controls its composition. The bioreactor is intended for use in the Laboratory of Experimental Biophotonics. This laboratory is equipped with coherence-controlled holographic microscope (CCHM), which uses quantitative phase imaging (QPI) method. Thus, the bioreactor is adapted to the current requirements of this laboratory and optical elements of the bioreactor meet the requirements of the QPI method. This text specifies the cultivation conditions of the living cells and summarizes, how the conditions could be controlled in the live cell microscopy. Next some commercially available culture devices are described and assessed, whether they are convenient for the~use in Laboratory of Experimental Biophotonics. The crucial part of the thesis is the~design, construction and testing of the new bioreactor.
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Basement membrane and the role of matrix metalloproteinases during embryonic wound healing
Kadlčíková, Dominika ; Šindelka, Radek (advisor) ; Tolde, Ondřej (referee)
The healing process is an attractive topic in biology and medicine. There are two types of wound healing - in embryos and in adults. In the case of embryonic wound healing the whole process is simplified and accelerated. An inflammatory reaction typical for adult healing is eliminated and the wound is healed without scars. Better understanding of embryonic wound healing could lead to more effective treatment of injuries, burns and chronic wounds in human population. The studying of molecular mechanisms in the healing process is also meaningful in the context of understanding the cancer regulation. The wound healing could be compared with embryonic development in many ways. The body's axes are essential for a spatial activation of genes involved in development. Our hypotesis was that the axis are also important for wound healing. It has been suggested that the cell's polarity and motility depends whether the wound is vertical or horizontal. There are also connections between wound healing and cancer development. One of them is the degradation of the basement membrane (BM) and extracellular matrix (ECM). The process of degradation is catalyzed by the coordinated action of several classes of enzymes. Some of them - matrix metalloproteinases (MMP) and their role in embryonic wound healing have been...
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Cytoskeleton-membrane protein interaction network in sperm
Adamová, Zuzana ; Komrsková, Kateřina (advisor) ; Tolde, Ondřej (referee)
In order to fertilize the egg, sperm cell undergoes several subsequent maturation processes. The final one called acrosome reaction is an exocytosis of acrosome vesicle, which is filled with lytic enzymes. Acrosome reaction is crucial for penetration of the sperm cell through the egg surroundings, especially zona pellucida, as well as for reorganization of a membrane protein composition on its surface. This rearrangement leads to the exposure of proteins essential for fertilization, mainly for gamete recognition, binding and fusion in specific compartments of the sperm head. One of such protein is CD46, which is located in the acrosomal membrane of an intact sperm and after acosomal exocytosis it relocates to the equatorial segment of a sperm head, which is known to be the initial site of interaction of sperm with the egg plasma membrane. The relocation of CD46 is disrupted by inhibition of actin, which reorganization within sperm head is known to play a role in onset of acrosome reaction, however, the precise mechanism of CD46 interaction with actin in sperm is unknown. In this thesis, ezrin - a crosslinker of membrane proteins and actin - has been studied in context of CD46 and its relocation across the sperm head. Analysis of the immunofluorescent detection of ezrin revealed its mutual...
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