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The role of NG2 glycoprotein in cancer cell invasiveness
Obr, Adam ; Tlapáková, Tereza (referee) ; Brábek, Jan (advisor)
NG2 proteglycan is a novel membrane - spanning proteoglycan, expressed in general in developing tissue whose cells are characteristic for its increased level of proliferation and motility. NG2 proteoglycan is considered to be an anchor for cell adhesion capabilities on different substrata as well as a signaling transmembrane structure which is capable of affecting actin cytoskeleton and causing increased cell migration. This bibliographic search shows the considered effect of NG2 proteoglycan to the migration abilities of cancer cells via different molecular mechanisms, such as NG2 - mediated, integrin - independent cell interactions with collagens and other ECM substrata, effect of phosphorylation with two different kinases leading to diverse signaling and different behavior in response to phosphorylation and finally the interaction with scaffolding protein MUPP1 and possible connection with signaling pathway to RhoA GTPase, which is involved in cytoskeleton regulation.
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The role of the exocyst in development and maintaining of cell migration structures
Vaškovičová, Katarína ; Žárský, Viktor (referee) ; Brábek, Jan (advisor)
The exocyst is a hetero-octameric protein complex which mediates tethering secretory vesicles to specific sites of plasma membrane for polarized exocytosis. The exocyst was long known to contribute to processes such as yeast budding, cytokinesis, epithelia polarization and neurite outgrowth. Recently, the role of the exocyst in regulation of actin cytoskeleton and cell migration was discovered. It was shown, that the exocyst is important for formation of cell migration structures such as lamellipodia and filopodia in motile cells and invadopodia in invasive cancer cells. These structures are all actin-based membrane protrusions and the exocyst can through its Exo70 subunit interact with the Arp2/3 complex, the activator of actin nucleation. By binding and activating the Arp2/3 complex, the exocyst mediates actin polymerization resulting in formation of these membrane protrusions. Furthermore, the exocyst probably targets the Arp2/3 complex to specific sites of plasma membrane that are intended to become membrane protrusions. In addition, the exocyst mediates secretion of matrix metalloproteinases (MMPs) in invadopodia. MMPs are important for degradation of the extracellular matrix, an essential process in cancer cell invasion. The exocyst seems to be part of the cascade downstream of cytokines...
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Enhancement of the efficacy of DNA vaccines against the cervical cancer with helper epitopes
Peřinová, Lucie ; Brábek, Jan (referee) ; Šmahel, Michal (advisor)
The human papillomaviruses (HPV) are the etiological agent of cervical cancer. Their oncoproteins E6 and E7 are involved in the transformation of an infected cell into a neoplastic cell, thereby they are the target antigenes for the development of DNA vaccines. Helper epitopes activating CD4+ T cells are under study because they enhance the efficacy of DNA vaccines through increasing the number of cytotoxic T lymphocytes and thereby removal of the tumor. There are already being used epitopes derived directly from oncoproteins, synthetic epitopes or bacterial epitopes for the general enhancement of the immune response. Sufficient number of comparative studies which would establish the exactly most efficient helper epitopes has not been made. The research aims at combining more peptide types using immunostimulatory molecules.
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The role of NADPH oxidase and ros in invadopodia formation
Hanušová, Kristýna ; Vrbacký, Marek (referee) ; Brábek, Jan (advisor)
Invadopodia as specific organelles enabling tumour cells movement, spreading over the organism and ultimately formation of metastasis are possible and promising targets of tumour therapy. Recently, many interesting facts about assembly and mechanism of function of invadopodia were discovered. Invadopodia are centres of ECM degradation by extra-cellular proteases facilitating an invasion of tumour cells. For creation of invadopodia a precisely localized increased production of ROS is necessary. ROS work as crucial signalling molecules and participate in many processes resulting in invadopodia formation. ROS in tumour cells are produced by specific extra-mitochondrial NADPH oxidases (Nox). Several regulatory molecules participating in activation and localization of Nox to invadopodia have been discovered recently (Tks organizer proteins). Furthermore, a regulatory role of Src kinase in ROS production and subsequent invadopodia formation was confirmed. Key words: ECM degradation, invadopodia, invasion, proteases, Nox, ROS, Src kinase, Tks proteins
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Clonning and characterization of the membrane microdomain targeted componets
Falteisek, Lukáš ; Černý, Jan (advisor) ; Brábek, Jan (referee)
3 Abstract Lipid rafts are defined as islets in the membranes of eucaryotic cells with different composition from the rest of the membrane. They appear spontaneously due to phase separation of different membrane components and are proposed to serve as platforms for concentration of selected signaling proteins. However, evidence for their existence is still indirect, despite more than decade of intense research. Some new approaches show that the fluctuations of membrane composition are more diverse and are caused more likely by presence of proteins than by lipid phase separation. We performed bioinformatical search looking for new signaling proteins targeted to putative rafts. We have identified several proteins and out of them phosphodiesterase 8a (PDE 8a) was salected for further research. To prepare the mouse monoclonal antibody we expressed and purified fragment of PDE 8a fused with GST. After immunisation we obtained one clone producing antibodies tentatively positive on western blot and by indirect immunofluorescence. We predicted that PDE 8a is targeted to putative rafts by N-terminal myristoylation and palmitoylation. To clarify, whether these modifications are present in the strusture of PDE 8a we prepared mutants of the PDE 8a N-terminal region lacking myristoylation, palmitoylation or both. These...
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Molecular mechanisms of checkpoint signalling and termination
Benada, Jan ; Macůrek, Libor (advisor) ; Brábek, Jan (referee) ; Truksa, Jaroslav (referee)
Cells employ an extensive signalling network to protect their genome integrity, termed DNA damage response (DDR). The DDR can trigger cell cycle checkpoints which prevent cell cycle progression and allow repair of DNA damage. The failures in these safeguarding mechanism are represented by serious human malignancies, most predominantly by cancer development. This work aims to contribute to the understanding of how do the cells negatively regulate DDR and cell cycle checkpoint signalling. We focused mainly on Wip1 (PPM1D) phosphatase, which is a major negative regulator of DDR and is indispensable for checkpoint recovery. Firstly, we have shown that Wip1 is degraded during mitosis in APC-Cdc20 dependent manner. Moreover, Wip1 is phosphorylated at multiple residues during mitosis, resulting in inhibition of its enzymatic activity. We suggest that the abrogation of Wip1 activity enables cells to react adequately even to low levels of DNA damage encountered during unperturbed mitosis. In the following publication, we have investigated why the mitotic cells trigger only early events of DDR and do not proceed to the recruitment of DNA repair factors such as 53BP1. We showed that 53BP1 is phosphorylated within its ubiquitination-dependent recruitment domain by CDK1 and Plk1. These phosphorylations prevents...
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