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The role of p130Cas substrate domain mediated signaling in cancer cell migration, invasiveness and metastasis of cancer cells
Zemanová, Kateřina ; Brábek, Jan (advisor) ; Čáslavský, Josef (referee)
p130Cas (Crk-associated substrate) was first described over 30 years ago as a protein that associates with the v-src and v-crk oncoproteins and undergoes tyrosine phosphorylation. Proteins of the CAS family are an important part of cellular biological processes in normal and pathological situations. The existence of 15 YXXP repetitive motifs is characteristics for substrate domain. p130Cas is an adapter protein that allows interactions between proteins that lead to assembly of multiprotein complexes. The p130Cas protein regulates these multiprotein complexes, which further drive chemotaxis, apoptosis, differentiation and migration. Overproduction of CAS proteins was found in connection with a poor prognosis and an increased incidence of metastases. Also, the elevated expression of proteins of the CAS family is related to resistance to some types of chemotherapeutics.
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Analysis of PKCα Influence on Cancer Cell Invasion.
Szabadosová, Emília ; Brábek, Jan (advisor) ; Nováková, Olga (referee)
7 Abstract Protein kinase C alpha (PKCα) is a serine/threonine protein kinase. PKCα is an important protein regulating cell polarity, protein secretion, apoptosis, cell proliferation and differentiation and tumorogenesis. Previous research has shown a role of PKCα also in a cancer cell migration and cancer cell invasion. The aim of this study was to investigate the role of protein kinase C alpha (PKCα) played in amoeboid mode of cancer cell invasion. We showed that higher expression of PKCα resulted in mesenchymal-amoeboid transition of K2 and MDA mesenchymal cancer cell lines, which was accompanied with decreased cancer cell invasive capability in 3D collage matrix. PKCα overexpression had no effect on the cell morphology of A375m2, however, the results showed a trend in increased invasive potential of A375m2 cells. Conversely, the expression of dominant-negative PKCα resulted in amoeboid-mesenchymal transition of A375m2 cells, and it was associated with decreased invasive potential of K2 and MDA cell lines. Furthermore, a linkage between PKCα and phosphatidylinositol 3-kinase (PI3K) was tested. The results revealed that increased activity of PKCα was accompanied with decreased level of active Akt in K2 cell line. To summarize, our results suggest a probable role of PKCα in regulation of amoeboid...
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Mebrane adaptor proteins in hematopoiesis and immune response
Pavliuchenko, Nataliia ; Brdička, Tomáš (advisor) ; Brábek, Jan (referee) ; Smrž, Daniel (referee)
Membrane adaptor proteins are proteins associated with cellular membranes that do not themselves serve as receptors. Instead, they propagate or modify the signals of these receptors by recruiting other signaling and regulatory proteins and arranging them into supramolecular complexes. In this thesis, I sought to describe selected membrane adaptor proteins and their roles in inflammation and regulation of hematopoiesis in mouse models using a reverse genetics approach. The main part of the work focused on the role of the membrane adaptor protein PSTPIP2 in suppressing inflammation. In mice, missense mutations in the Pstpip2 gene causing loss of PSTPIP2 protein lead to the development of autoinflammatory disease chronic multifocal osteomyelitis (CMO) characterized by sterile inflammatory lesions in the bones and adjacent soft tissue. These mice represent a model of the human autoinflammatory disease, chronic recurrent multifocal osteomyelitis. At the molecular level, neutrophils in the absence of PSTPIP2 exhibit pathological hyperactivity of pathways regulating IL-1β and reactive oxygen species (ROS) production, which are both implicated in the etiology of the disease. PSTPIP2 interacts with several signaling regulators, including PEST family protein tyrosine phosphatases (PEST-PTPs) and inositol...
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The influence of the RACK1 scaffold protein and the ERK signalling pathway on cell polarity
Klímová, Zuzana ; Vomastek, Tomáš (advisor) ; Brábek, Jan (referee) ; Varga, Vladimír (referee)
The establishment of cell polarity is an essential step in cell migration, as it provides cells with information about the direction of migration. It is a highly dynamic process that leads to an asymmetric distribution of cytoskeletal networks, cell organelles, protein complexes and signalling pathways, which is reflected in the typical polarised cell shape. The cell shape is determined by the interplay between the dynamics of the actin cytoskeleton, cell adhesions and the cell membrane towards the extracellular surface. Cell adhesion and spreading on the extracellular matrix is a morphogenetic process in which cells initially spread isotropically from the point of first contact and then spontaneously break their radial symmetry and develop a migratory polarity with spatially separated protruding cell front and non-protruding cell rear. It is unclear how these symmetry break events, both complex and stochastic, are organised and regulated. In this study, I show that symmetry breaking in isotropically spreading fibroblasts begins with the establishment of a non-protruding cell rear delineated by large but sparse focal adhesions. Development of the non-protruding regions requires the scaffold protein RACK1, which promotes adhesion-mediated activation of ERK2. ERK2 and RACK1 inhibit p190RhoGAP...
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Role of NAV3 in glioblastoma cells invasiveness
Legátová, Anna ; Brábek, Jan (advisor) ; Libusová, Lenka (referee)
The invasion of tumor cells from the primary lesion and the formation of metastases are the main reasons for the severe impact of cancer diseases. An option for dealing with this poor impact is the development of drugs (so-called migrastatics) that would target these processes and thus limit the spread of tumor cells from the site of the primary tumor. However, to develop such drugs, it is essential to clarify the molecular mechanisms that control or promote cell migration. One of the possible strategies for migrastatics development is the targeting of cytoskeletal structures, which play an indispensable role in cell migration. This work is focused on Neuron navigator 3 (NAV3), a protein that binds to + ends of microtubules (MTs), participates in their stabilization, and is able to mediate crosstalk between MTs and the actin network. The function of NAV3 is important for directing MTs into growing axons and proper neurite outgrowth, which is necessary for brain development. The results of this thesis suggest that NAV3 could act as a pro-tumor factor, which localizes not only to the + ends of MTs, but also to the cell protrusions, and whose presence supports the cell expansion and increases the invasive potential of glioblastoma cell lines. Key words: neuron navigator 3, microtubules, cancer,...
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High grade serous ovarian carcinoma: molecular background and platinum-based chemotherapy challenges
Ivančinová, Jana ; Heneberg, Petr (advisor) ; Brábek, Jan (referee)
Ovarian carcinoma (O.C.) represent a group of various disease entities derived from ovaries. The most common malignant gynaecological cancer is high-grade serous ovarian carcinoma (HGSOC). HGSOC is associated with a high mortality rate due to its aggressive behaviour and insufficient early-stage detection. The survival rate has not been significantly improved since 1970s. The most effective treatment of HGSOC patients is by cytoreductive surgery (for early stages I/II) and followed by platinum-based chemotherapy (HGSOC presented in advanced stage III/IV) combined with taxane or potentially with PARP inhibitors (for BRCA1/2 mutation carriers). Multiple factors affect the patient's outcome and prognosis. Chemoresistance, molecular mutational patterns, stage at presentation of HGSOC are one of the clinical challenges contributing to common relapses even though patients often initially respond well to the HGSOC chemotherapy. This thesis overviews the fundamental biology of HGSOC, the major obstacles in clinical management and its improvements by implementing of multitherapy approaches. Key words: CA-125; platinum−based chemotherapy treatment; homologous recombination deficiency; ovarian carcinoma; resistance; Tp53; mortality; survival rate
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Drug repurposing for the inhibition of invasiveness and metastasis of cancer cells
Turchyna, Kateryna ; Brábek, Jan (advisor) ; Jakubek, Milan (referee)
Metastasis is the primary cause of death from cancer. Current lack of medicines for metastasis makes it a topical issue and encourages the global search for molecules that may potentially inhibit invasiveness of cancer cells, and thus prevent metastatic spread. Drug repurposing is a strategy of identifying new uses for approved drugs that are outside the scope of the original medical indication. The aim of this thesis is to summarize knowledge on the repurposing potential of selected groups of drugs with emphasis on molecular mechanisms of their action. In particular, the work is focused on selected antipsychotics, antidepressants, beta- blockers and statins, whose abilities to inhibit cancer invasiveness have been indicated.
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Molecular basis of fluoropyrimidine toxic effect etiology with focus on palmar-plantar erythrodysesthesia and potential antidote use
Hartinger, Jan ; Veselý, Pavel (advisor) ; Květina, Jaroslav (referee) ; Brábek, Jan (referee)
(thesis): Palmar-plantar erythrodysesthesia (PPE) frequently accompanies the therapy with a continuous 5-FU infusion or peroral capecitabine (5-FU prodrug). In the most severe cases this adverse effect leads to discontinuation of a needful therapy. Local 10 % uridine ointment is used to prevent and treat the said adverse event. Nevertheless, this method is not generally accepted as an effective one because it has never been proved in a randomized controlled clinical trial. Most probably, a direct effect of a cytostatic compound on the skin of hands and foots causes PPE. The toxicity of 5-FU is mediated primarily by its incorporation into RNA and by thymidylate synthase (TS) inhibition and subsequent DNA synthesis disruption. The importance of particular 5-FU toxicity mechanisms varies in different cell types. For choosing the best PPE local antidote it is necessary to find out which molecular mechanism applies in keratinocytes. We have chosen pyrimidine nucleosides as the most suitable compounds for the local PPE therapy because the uridine ointment is already being used in several oncology centers in the Central Europe. In order to find out the 5-FU toxicity mechanism, we further tested the effect of calciumfolinate (CF) which strengthens the TS inhibition by 5-FU. We studied also uracil and...
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The role of protein kinase C alpha in individual cancer cell invasiveness
Szabadosová, Emília ; Brábek, Jan (advisor) ; Hlaváčková, Markéta (referee)
Protein kinase C alpha was one of the first identified isoenzyme of PKC family. Since then PKC has been shown to be important in various signal cascades. One of the best known role is in tumor invasiveness and in development of metastases. A role of protein kinase C alpha was pointed out in regulation of Rho/ROCK pathway in amoeboid invasiveness and also Raf/MAPK signaling cascade of mesenchymal movement. ERM proteins, which act in cancer invasiveness, are regulated by protein kinase C too.
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