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Cell growth on biomaterials for skin replacements and wound dressings
Kudláčková, Radmila ; Bačáková, Lucie (advisor) ; Rösel, Daniel (referee) ; Eckhardt, Adam (referee)
Tissue engineering is an emerging interdisciplinary field developing new ways of treatment of patient's tissue defects using artificial substitutes. Skin tissue engineering is developing skin substitutes and wound dressings that would replace current treatment using autologous, allogeneic or xenogenic substitutes. There are high demands on materials which should serve as a scaffolds for dermal fibroblasts and keratinocytes. They must be non-cytotoxic and biodegradable with a rate proportional to formation of a new tissue. The materials should support adhesion and proliferation of the cells and even they could release growth factors and antimicrobial substance to enhance healing and new tissue formation. In this master thesis, the cell adhesion and proliferation were evaluated on sodium carboxymethyl cellulose (Hcel® NaT), poly-ε-caprolactone (PCL), poly-L-lactide-co-ε-caprolactone (PLA/PCL) and cellulose acetate (AC) nanofiber membranes. Primary human dermal fibroblasts and HaCaT cell line keratinocytes were selected for evaluation. The cell adhesion was observed by fluorescent microscopy, the proliferation was determined by metabolic assay (WST-1) and the material cytotoxicity was evaluated in xCELLigence® system. Materials did not show cytotoxic effects on the cells. However, the materials did...
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The role of 53BP1 in the cellular response to double-strand DNA breaks
Liďák, Tomáš ; Macůrek, Libor (advisor) ; Rösel, Daniel (referee)
DNA damage may result in various pathological conditions and contributes to aging and development of cancer. Evolutionarily conserved DNA damage response prevents the acumulation of mutations and protects against genomic instability. Tumor suppressor p53-binding protein 1 (53BP1) is an important regulator of the cellular response to DNA double-strand breaks (DSB) and is a canonical component of ionizing radiation-induced foci which are formed at DNA DSB following radiation exposure. Recently, new insights have been gained into its functions in the DNA damage response. Apart from its subtle role in the DNA damage checkpoints signaling, 53BP1 is a well established player in the DNA DSB repair pathway choice. The outcome of DNA repair is influenced by 53BP1 in several contexts. 53BP1 controls 5' end resection at DNA ends, improves DSB repair in heterochromatin, promotes the mobility of uncapped telomeres and mediates synapsis of DNA ends during V(D)J and class switch recombination. 53BP1 contributes to repair defect in BRCA1 (breast cancer type 1 susceptibility protein)-deficient cells, which may have an impact on the treatment of some types of breast cancer. The aim of this bachelor's thesis is to summarize new findings about the role of 53BP1 in the cellular response to DNA DSB. Powered by TCPDF (www.tcpdf.org)
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The role of Ser/Thr phosphorylation of p130CAS in signaling
Dibus, Michal ; Rösel, Daniel (advisor) ; Macůrková, Marie (referee)
p130Cas is an important adaptor protein that plays an essential role in many intracellular signaling processes. Given the fact that p130Cas is a well-known substrate for wide spectrum of kinases, its function is regulated mostly by phosphorylation on tyrosine, threonine and serine residues. This work is focused on Serine/Threonine phosphorylation and its role in regulation of p130Cas signaling. Although it is known that Serine/Threonine phosphorylation of p130Cas is regulated by cell cycle, integrin-mediated cell adhesion and association with BCAR3, the mechanisms leading to the phosphorylation are still not well understood and the kinases involved in these processes are unknown. Being p130Cas an important regulator of cell migration and tumor invasiveness, understanding of these mechanisms should provide a useful tool in developing new strategies in aiming of anti-cancer drugs.
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The effect of Crk SH3domain inhibition in invasiveness of cells
Tomášová, Lea ; Rösel, Daniel (advisor) ; Vomastek, Tomáš (referee)
Protooncogene Crk was found to be upregulated in tumours with aggressive and invasive potential. The adaptor protein Crk has an important role in cell signaling: it integrates signals from activated integrins and growth factors receptors via its SH2 domain and transmits the signal to its SH3 domain binding partners that activate the small GTPases Rac1, Rap1 and Ras. This leads to regulation of cell migration, proliferation and survival. The aim of this thesis project was to inhibit the Crk dependent signaling by a competitive inhibition of the Crk SH3 domain, using a high affinity CrkSH3 binding peptoid. Binding of the inhibitor to the Crk SH3 domain prevents binding of cellular Crk SH3 interaction partners and the corresponding signal transmission is impaired. In this thesis project the effect of the Crk SH3 inhibition on the invasiveness of cancer cells was analyzed. The observed inhibitory effect on cell invasion as well as on anchorage independent growth provides a proof of therapeutical relevance of targeting CrkSH3N domain by peptoide-based inhibitors. Powered by TCPDF (www.tcpdf.org)
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Microtubule-active drugs: mechanism of action and resistance
Dostál, Vojtěch ; Libusová, Lenka (advisor) ; Rösel, Daniel (referee)
Microtubular cytoskeleton represents a target for a myriad of diverse chemical compounds, referred to as microtubule-active drugs. Produced by certain plants, animals or microbes, the substances often effectively elicit cell death - especially in animals and also in plants to a certain extent, but never in species which produce them to defend against their predators. Nowadays, several microtubule-active substances constitute hallmarks of anti-cancer treatment and agricultural weed control. There is an enormous sum of knowledge about the action of paclitaxel (taxol), vinca alkaloids and colchicine, three best-known microtubule active compounds used in medicine, and new research often challenges the previously accepted theories. This work investigates the mechanism of action of microtubule-active drugs from the angle of biochemistry and cell biology, as well as from the physiological standpoint. Effects on microtubule levels and dynamics and the path towards the cell death are reviewed. In the last chapter, attention is given to drug activity in both animal and plant bodies and, finally, to drug-producing plant species which often show substantial resistance.
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Odpověď metastatických buněčných linií karcinomu prostaty na genotoxický stres
Imrichová, Terezie ; Hodný, Zdeněk (advisor) ; Rösel, Daniel (referee)
Prostate cancer is the fourth most frequent cause of cancer-related deaths in men worldwide. One of current successful approaches to treat prostate cancer is radical prostatectomy followed by radiotherapy. However, this treatment is not 100% successful, as 53% patients develop secondary tumors. Our hypothesis is, that ionizing radiation itself contributes to the development of metastases by inducing changes in cell phenotype, particularly in terms of epithelial-to-mesenchymal transition and stemness. To test this hypothesis, we irradiated the cells of metastatic prostate cancer cell line DU145 by fractionated radiation 2 x 10 Gy and we compared the expression of selected epithelial, mesenchymal and stem-cell markers prior to and after irradiation. Besides we focused on a subpopulation of so called floating cells which arise during irradiation. These cells can survive the radiation treatment and after some time they are able to reattach and give rise to readherent population. We wanted to asses what is the cell cycle profile of these cells and whether and how fast they proliferate. In this thesis we have shown that radiation causes only minor changes in epithelial/mesenchymal and stem-like character of adherent fraction of the DU145 cell line. However, we have also described that small population of...
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The role of proto-oncogene crk in invasiveness
Tomášová, Lea ; Rösel, Daniel (advisor) ; Ševčík, Jan (referee)
Proto-oncogene Crk was identified as an oncogenic product of an avian retrovirus in 1988. It is an adaptor protein containing SH2 and SH3 binding domains. Thanks to these domains Crk facilitates protein-protein interactions and therefore plays a crucial role in signal transduction. Crk forms signal complexes with several proteins and hence impacts many cellular processes, among them cell migration, tumorigenezis and invasion of the surrounding tissues. The increased invasiveness allows the tumour cells to detach from the primary tumour and form metastasis which is very problematic feature of cancer. Overexpression of Crk was observed in several tumour tissues, it correlates with an aggressive and metastatic phenotype of the tumours. The subject of this thesis is to describe the mechanisms of how Crk can regulate cellular motility and invasiveness.
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The role of NG2 glycoprotein in the regulation of Rho/ROCK signaling.
Kratochvílová, Magdalena ; Rösel, Daniel (advisor) ; Libusová, Lenka (referee)
NG2 is a transmembrane glycoprotein mainly expressed in developing tissue, and often re-expressed in tumor cells. NG2 glycoprotein is an important regulator of cell migration and adhesion. Increased expression of NG2 enhances the metastatic potential of cancer cells. However, the molecular mechanisms of these processes are still not fully understood. An increasing number of evidences, in recent years, have shown that NG2 can be responsible for Rho/ROCK activation, which is essential for effective amoeboid invasiveness. In this thesis, we analysed the role of NG2 glycoprotein, especially the role of its PDZ- binding motif on amoeboid phenotype induction, and activation of Rho/ROCK signaling. Our results demonstrate the importance of the NG2 PDZ-binding motif on mesenchymal- amoeboid transition of cells in a 3D environment. Surprisingly, they show that the expression of both the NG2 cytoplasmatic domain and the truncated version, lacking the PDZ-binding motif, do not change the amount of Rho-GTP or the activation of the Rho/ROCK signaling pathway in 2D.
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Design and construction of Src biosensor
Pavlík, Vojtěch ; Rösel, Daniel (advisor) ; Brdička, Tomáš (referee)
Src kinase is a well-known proto-oncogene that contributes to cell migration and proliferation and is often found deregulated in tumors. Yet, biology of Src is not fully understood and great effort is made to find new tools for broadening knowledge about the kinase. In this thesis is described design and construction of a novel Src biosensor that exploits genetically encodable fluorophores derived from green fluorescent protein and Förster/fluorescence resonance energy transfer (FRET). The fluorophores are inserted directly into the structure of full-length c-Src in the way that should not impair the inner regulatory mechanisms of Src. The created biosensor proved to be sensitive to various stimuli, which also activate c-Src, by increase of activating autophosphorylation on Tyr4162 and decrease in FRET. Preliminary experiments indicate that the Src biosensor can be used to reflect Src activation in fixed cell as well.
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The search for novel interaction partners of SH3 domain of an adaptor protein p130Cas
Gemperle, Jakub ; Rösel, Daniel (advisor) ; Forstová, Jitka (referee)
Protein p130Cas is the major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. P130Cas plays an important role in invasiveness and metastasis of Src-transformed cells. In breast cancer patients, high p130Cas levels are associated with higher recurrence of disease, poor response to tamoxifen treatment and lower overall survival. In non-transformed cells, after the stimulation of integrins, protein p130Cas is phosphorylated in substrate domain affecting cell migration and cytoskeletal dynamics. For this signalling is the SH3 domain of p130Cas indispensable. In this thesis, was for the first time using the Phage display method analysed and subsequently characterized the binding motif of SH3 domain of p130Cas. Based on this high-affinity motif [AP]-P-[APMS]-K-P-[LPST]-[LR]- [LPST], we predicted new interaction partners of protein p130Cas and subsequently confirmed the interaction with the Ser/Thr kinase PKN3. This kinase colocalizes with p130Cas in the nucleus and perinuclear region and could phosphorylate p130Cas. In this thesis, we also analysed the effect of phosphomimicking mutation of tyrosine from sequence ALYD, which is conserved in the sequence of SH3 domains, on ability of these domains to bind ligands. This mutation reduced binding by about 3 orders of...
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