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The role of inflammatory signaling in cancer cell invasiveness
Šůchová, Anna-Marie ; Brábek, Jan (advisor) ; Brdička, Tomáš (referee)
Metastasizing is responsible for 90% of death in cancer patients. Metastatic tumour cells have several strategies that they use to invade surrounding tissues - they can migrate together or individually. When individual cells migrate, tumour cells adopt two different morphologies. They are either elongated and migrate using the proteolytically active mesenchymal mode, or they are rounded and migrate in the amoeboid mode. Metastatic tumour cells can switch between these modes, which complicates the development of effective migrastatics. In this work, we focused on the effect of inflammatory signalling on metastatic cell migration. We worked with cell lines of malignant human melanoma, which adopt a mixed morphology and show both amoeboid and mesenchymal phenotype during migration. Upon stimulation of melanoma human cells with interferon beta, a mesenchymal to amoeboid transition occurs. Interferon beta appears to induce amoeboid morphology by maintaining high levels of the ISGF3 complex, which is composed of the heterodimer of STAT 1 and STAT 2 proteins and the IRF9 protein. Upon blocking of Jak / Stat signalling pathway by negative regulators, human melanoma cells return to mesenchymal morphology. Key words - invasiveness, mesenchymal-ameboid transition, interferons, inflammation, migration, metastases
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Structural and regulatory aspects of Src kinase activation
Koudelková, Lenka ; Brábek, Jan (advisor) ; Brdička, Tomáš (referee) ; Hejnar, Jiří (referee)
Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that...
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The role of galectins in cancer cell invasiveness
Remišová, Michaela ; Brábek, Jan (advisor) ; Kovář, Marek (referee)
Galectins are family of β-galactosidase binding proteins that serve many functions in all kind of mammalian cells. In the past years galectins, namely galectin-1 and galectin-3, have been revealed to play a major role in various cancer processes including cancer cell invasiveness, a process indispensable for the formation of metastasis. Both extracellular and intracellular forms of galectins modify the process of invasiveness in various ways, through interacting with different components of the cell or of the cell signalling pathways. The aim of this bachelor's thesis is to summarize mechanisms by which galectins promote cancer cell invasiveness. Keywords: galectins, galectin-1, galectin-3, invasiveness, cancer, metastasis
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The plasticity of melanoma cell invasiveness
Gandalovičová, Aneta ; Brábek, Jan (advisor) ; Truksa, Jaroslav (referee)
and keywords: During metastasis, cancer cells can invade the extracellular matrix using various strategies. When invading individually, they employ either the amoeboid invasion mode, during which the cell body dynamically deforms by enhanced contractility to squeeze through pores within the matrix, or protease dependent mesenchymal migration that takes advantage of the possibility to digest the surrounding matrix. Cells migrating in one mode can actively switch to the other by mesenchymal-amoeboid (MAT) or amoeboid-mesenchymal transitions (AMT). This enables escape mechanisms and considerably complicates anti-metastatic treatment. It is well known that Rho GTPases are master regulators of cytoskeleton re-arrangements and thus, unsurprisingly, play a major role in both invasion modes and can directly drive the transitions. However, upstream activation of these pathways is still largely unclear. This thesis aimed to optimize 3D conditions suitable for studying plasticity of cell invasion in vitro, establish AMT and MAT in melanoma cells based on manipulation of Rho GTPases and verify novel candidates regulating cell invasion plasticity based on previous RNA sequencing of cells before and after MAT. Last, by synthesis of published data, results from sequencing and new findings presented in this...
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The role of cell polarity signaling in the plasticity of cancer cell invasiveness
Gandalovičová, Aneta ; Brábek, Jan (advisor) ; Cvrčková, Fatima (referee)
Throughout the last few years cancer research has focused on studying the origin of secondary tumors, i.e. metastases, which are a direct outcome of the ability of cancer cells to disseminate from the primary tumor and invade the adjacent tissue. Generally, cancer cells migrate by two distinct mechanisms- amoeboid or mesenchymal. Whereas the mesenchymal migration mode can be described as "path generating", the amoeboid mode resembles a "path finding" way of migration. Both types of invasion are regulated by divergent signaling pathways that are closely related to cell polarity and cytoskeleton reorganization. Responsible for cell polarization are not only the polarity complexes Par, Scribble and Crumbs, but also phosphoinositides and Rho GTPases Rac, Rho and Cdc42, which, additionally, regulate the dynamics of the cytoskeleton. By a mutual interplay they regulate cell motility. It cannot come as a surprise that their deregulation commonly results in tumorigenesis. A more thorough comprehension of the signaling pathways leading to cancer cell invasiveness is a necessary step towards understanding the complex problem of metastasis. Key words: invasiveness, amoeboid, mesenchymal, cell polarity, motility, Rho GTPases, polarity complexes
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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 use of CAM assay for characterization and study of cancer cell invasive properties
Vágnerová, Lenka ; Dvořák, Michal (advisor) ; Geryk, Josef (referee)
The chorioallantoic membrane (CAM) of chicken embryos belongs to the in vivo model systems frequently used for the study of angiogenesis and cell invasiveness. Using CAM assay we have tested selected chicken sarcoma cell lines characterized by different angiogenic properties and different ability to form metastasis. In addition to CAM assay, several other methods have been used to characterize the phenotype of these cell lines. We have selected a few proteins which could significantly influence the angiogenic and metastatic properties of investigated cell lines. We have established cell lines stably overexpressing these genes and compared their phenotypes with parental cell lines. We have shown that genes encoding ISL1, ARNT2, PROM1, HOXA11 proteins participate, in our experimental model, in activation of programes controlling angiogenesis and cell invasion.
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Effect of polyploidization on species invasive success
Líblová, Zuzana ; Münzbergová, Zuzana (advisor) ; Rooks, Frederick (referee)
Polyploid variants of many species of plants are strikingly frequently found among alien plants on all continents. They also very often have a much larger distribution range of its occurrence, compared to diploid plants in the place of their origin. In many cases, the polyploid cytotype also has increased tolerance to various stress factors or a physiological and morphological characteristics that allow them to survive the conditions in which the diploid plants would have little chance to survive. All this suggests that polyploidy is likely to bring plants an evolutionary advantage over their diploid ancestors, and polyploids therefore can successfully colonize new territories. This thesis summarizes the findings about the possible consequences of polyploidy at different levels in relation to their effects on the properties supporting plant invasive ability. It presents also known hypotheses dealing with possibilities of why plants become invasive after introduction. This is followed by sections devoted to flow cytometry, an important modern method for determining genome size and ploidy level. In conclusion it briefly describes the model species bird vetch (Vicia cracca) and the results of measurements of the degree of ploidy of seeds of this plant from Alaska and Japan.
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The biological importance of CAS SH3 domain tyrosine phosphorylation
Janoštiak, Radoslav ; Brábek, Jan (advisor) ; Dvořák, Michal (referee)
Protein CAS is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes. It is a multidomain adaptor protein, which serves as a scaffold for assembly of signalling complexes which are important for migration and invasiveness of Src-transformed cells. A novel phosphorylation site in N-terminal SH3 domain was identified - tyrosine 12 located on binding surface of CAS SH3 domain. To study biological importance of tyrosine 12 phosphorylation, non-phosphorylable (Y12F) and phosphomimicking ( Y12E) mutant of CAS were prepared. We found that phosphomimicking mutation Y12E leads to decreased interaction of CAS SH domain with kinase FAK a phosphatase PTP-PEST and also reduce tyrosine phosphorylation of FAK. Using GFP-tagged CAS protein, we show that Y12E mutation caused delocalization of CAS from focal adhesion but has no effect on localization of CAS to podosome-type adhesion. Non-phosphorylable mutation Y12F cause hyperphosphorylation of CAS substrate domain and decrease turnover of focal adhesion and associated cell migration of mouse embryonal fibroblasts (MEFs) independent to integrin singalling. Analogically to migration, CAS Y12F decrease invasiveness of Src-transformed MEF. The results of this diploma thesis show that phosphorylation of Tyr12 in CAS SH3 domain is...
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Unix Tools for Application and System Profiling
Dressler, David ; Chalupníček, Kamil (referee) ; Kašpárek, Tomáš (advisor)
The main goal of this thesis was to demonstrate usage of tools for application and system profiling. Initially, these tools was found and studied. They was also divided into categories according to their purpose. After that, these tools was compared according to their complexity of use and invasiveness. As the result of this comparison, these tools was divided into three groups, that express measure of complexity and invasiveness. As technology, used for creating models, was chosen Apache server and NFS server. Virtualization by hyper-v technology was used for putting these models into operation. There was created four virtual machines. Fist one for Apache server, another one for NFS server. Third was for mirroring content of Apache server and the last one for load generation. The last part of this thesis was to demonstrate usage of found tools on the created models.
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