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Molecular mechanisms of amoeboid invasion of cancer cells
Paňková, Daniela ; Brábek, Jan (advisor) ; Dvořák, Michal (referee) ; Vomastek, Tomáš (referee)
Tumour cell invasion is one of the most critical steps in malignant progression. It includes a broad spectrum of mechanisms, including both individual and collective cell migration, which enables them to spread towards adjacent tissue, and form new metastases. Understanding the mechanisms of cell spreading, and invasion, is crucial for effective anticancer therapy. Two modes of individual migration of tumour cells have been established in a three-dimensional environment. Mesenchymally migrating cells use proteases to cleave collagen bundles, and thus overcome the ECM barriers. Recently described protease-independent amoeboid mode of invasion has been discovered in studies of cancer cells with protease inhibitors. During my PhD study, I have focused on determining the molecular mechanisms involved in amoeboid invasion of tumour cells. We have examined invasive abilities in non-metastatic K2 and highly metastatic A3 rat sarcoma cell lines. We have shown that even though highly metastatic A3 rat sarcoma cells are of mesenchymal origin, they have upregulated Rho/ROCK signalling pathway. Moreover, A3 cells generate actomyosin-based mechanical forces at their leading edges to physically squeeze through the collagen fibrils by adopting an amoeboid phenotype. Amoeboid invasiveness is also less dependent on...
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Regulation of transcription by proteins of the Early growth response and Myb families
Čermák, Vladimír ; Dvořák, Michal (advisor) ; Vomastek, Tomáš (referee) ; Elleder, Daniel (referee)
The regulation of transcription of tens of thousands of genes in a vertebrate organism is an enormously complex phenomenon which entails the participation of thousands of various regulatory proteins. The largest functional category of these regulators is accounted for by sequence-specific DNA-binding proteins known as transcription factors. Proteins of the EGR and Myb families of transcription factors are long-studied regulators of a variety of physiological processes including cellular proliferation and differentiation. The structural and physical aspects of their function have been well characterized. Their cell-type specific participation in complex gene-regulatory networks, on the other hand, is still incompletely understood and represents a major challenge in the respective research areas. Preliminary analysis of gene expression data from metastasizing PR9692 and non- metastasizing PR9692-E9 chicken sarcoma cell lines revealed that the transcription factor EGR1 is expressed at a higher level in metastasizing cells and can thus take part in the regulatory processes that underlie the differences between the two cell lines. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from PR9692...
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Mechanizmy regulace signální transdukce povrchovými proteiny leukocytu*.
Štěpánek, Ondřej ; Brdička, Tomáš (advisor) ; Brábek, Jan (referee) ; Vomastek, Tomáš (referee)
The core of the doctoral thesis "Regulation of signal transduction by leukocyte surface proteins" consists of three publications in international peer-reviewed journals dealing with leukocyte signaling both at the level of individual signaling pathways and in the context of a multicellular organism. Most attention is paid to signaling via the T cell receptor (TCR), which plays a central role in the development and function of T cells and represents a key signaling pathway for proper function of the adaptive component of the immune system. Transmembrane protein tyrosine phosphatase CD148 was considered a negative regulator of TCR signaling through dephosphorylation of LAT and PLCγ1 proteins. This study brings evidence that CD148 is able to modulate signaling also at the level of Lck, both positively and negatively. The net effect of CD148 activity on the TCR signaling is determined by the intracellular biochemical context, notably, the presence of another tyrosine phosphatase CD45. The second project dealt with the characterization of a transmembrane adaptor protein PRR7. This adapter inhibits TCR signaling via down-regulation of the intracellular Lck and cell surface TCR levels. The research concerning the signaling in the environment of a multicellular organism is represented by the analysis of...
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The regulation of the ERK signalling pathway by scaffold protein RACK1
Bráborec, Vojtěch ; Vomastek, Tomáš (advisor) ; Filipp, Dominik (referee)
The ERK signalling cascade comprised of protein kinases Raf, MEK and ERK is an evolutionarily conserved member of MAPK family that is activated in response to wide range of extracellular stimuli. The ERK pathway controls fundamental cellular functions including cell proliferation, differentiation, apoptosis or cell motility. To control such a diverse cellular responses by a single pathway cells have evolved regulatory mechanisms that channel the extracellular signals towards the specific biological response. Crucial to this control are non- enzymatic proteins termed scaffolds that associate with and enhance functional interaction of the components of MAPK pathways and can regulate amplitude, timing, specificity and location of signals. Scaffold protein RACK1 associates with several components of cell migration machinery including integrins, FAK, Src and the ERK pathway core protein kinases. RACK1 regulates distinct steps of cell migration such as establishment of cell polarity and focal adhesion turnover, however, the molecular mechanism by which RACK1 regulates these processes remains largely unknown. The main aim of this study was to investigate the functional role of RACK1 in cell motility, in particular to identify new effector proteins utilized by the ERK pathway and RACK1 in the regulation of...
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The regulation of cofilin by the ERK signaling cascade
Rasl, Jan ; Vomastek, Tomáš (advisor) ; Gahura, Ondřej (referee)
Cofilin is small ubiquitous actin binding protein, which is required for polymerization and depolymerization of actin fibers. Cofilin is involved in numerous cellular processes where the remodeling of actin cytoskeleton is required, such as cell division and cell migration. In order to precisely and dynamically regulate the cofilin activity, cells utilize large network of interconnected signaling pathways. One of these signaling pathways is the MAP-kinase cascade ERK (extracellular signal-regulated kinase), although the molecular mechanisms by which ERK regulates cofilin activity are not fully understood. Much evidence suggests that ERK controls the cofilin activity mainly through the regulation of Rho family of small GTPases. The ERK signaling cascade can modulates the Rho GTPase pathway signaling components, such as GAPs (GTPase activating proteins), GEFs (guanine nucleotide exchange factors) or Rho GTPases effectors. The ERK signaling cascade utilizes two different mechanisms for the regulation of Rho GTPases signaling pathways. The first mechanism is on transcriptional and translational level, where ERK regulates the transcription and subsequently translation of key regulatory proteins. Second mechanism, which is far more dynamic, occurs at the level of posttranslational modification,...
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In-vitro analysis of amoeboid-mesenchymal transition of A375m2 melanoma cells
Kasalová, Lenka ; Rösel, Daniel (advisor) ; Vomastek, Tomáš (referee)
The invasion of cancer cells is an important aspect of cancer progression. Single tumor cells exhibit at least two types of invasion in 3D environment, mesenchymal and amoeboid invasion. Tumor cells can switch between these two modes of movement depending on cellular status and surrounding environment. Amoeboid-mesenchymal transition (AMT) is less explored then mesenchymal-amoeboid transition (MAT). We performed a proteomic analysis of amoeboid-mesenchymal transition of human melanoma cell line A375M2. We have induced amoeboid-mesenchymal transition by treatment with a ROCK inhibitor Y27632 in 3D matrigel matrices and in 2D environment. Induction of the amoeboid-mesenchymal transition has changed a level of expression of 92 proteins and a level of phosphorylation of 15 proteins. Expression of only 17 proteins and phosphorylation of 8 proteins was identically changed in both of these environments. We found that PKCα regulates amoeboid migration and that treatment of cells with a PKCα inhibitor Gö6976 induces amoeboid-mesenchymal transition. Analysis of the proteomics data have further shown that induction of AMT by the ROCK inhibitor Y27632 leads to activation of antiapoptotic signals and activation of signaling pathways involved in regulation of actin cytoskeleton especially regulation of focal...
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Impact of NKR-P1 polymorphism on Ly49 receptors expression in hybrid mouse strains (C57BL/6 x Balb/c, F10-12)
Holubová, Martina ; Fišerová, Anna (advisor) ; Vomastek, Tomáš (referee)
Impact of NKR-P1 polymorphism on Ly49 receptors expression in hybrid mouse strains (C57BL/6 x Balb/c, F10-12) Abstract Natural killer (NK) cells constitute the subpopulation of large granular lymphocytes which mediate spontaneous immune response against infected, transformed or allogeneic cells and thus represent an important component of the innate immunity. NK cells express a wide repertoir of surface receptors which can be either activating or inhibitory and which mediate NK cell recognition and regulation of cytolytic activity. NKR-P1 and Ly49 receptor families belong to the most important murine NK receptors. Both NKR-P1 and Ly49 families are members of C-type lectin-like superfamily of receptors encoded by natural killer gene complex (NKC) on chromosome 6 and include both activating and inhibitory members. The aim of this diploma thesis was to elucidate the impact of Nkr-p1c gene divergence on Ly49 receptors expression and to find out whether the Ly49 and Nkr-p1 gene clusters (which are localized on opposite ends of NKC) are inherited independently or whether the NKC domain is inherited as a complex. The second research interest was to illustrate the influence of the above mentioned divergence on cytotoxic activity of NK cells and tumor growth. In this study, inbred mouse strains C57BL/6 and Balb/c...
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Molecular mechanisms of signal transduction by the ERK signaling cascade.
Bráborec, Vojtěch ; Rösel, Daniel (referee) ; Vomastek, Tomáš (advisor)
The MAPK (mitogen-activated protein kinase) cascade represents an evolutionary conserved mechanism by which cells sense extracellular signals and convert them into variety of context-dependent responses. The best studied member of the MAPK protein family is protein kinase ERK (extracellular signal-regulated kinase). Together with protein kinases Raf and MEK (MAPK/ERK kinase) comprise a prototypical signaling pathway which regulates broad-spectrum of biological processes such as cellular proliferation, differentiation, cellular migration, adhesion or apoptosis. To modulate such a multitude of distinct responses by a single pathway, cells utilize mechanisms such as signal strength and duration, distinct protein localization, communication with other signaling pathways, differential substrate selection and the selection of interactive partners. All presented means of regulation are influenced by proteins with non-enzymatic functions - scaffold proteins, protein inhibitors and anchoring proteins. These protein modulators channel the signals leading to particular cellular response, and thus represent the key element of signal transduction. Despite increasing importance of protein modulators in cellular signaling, their biological roles remain mostly unknown. The physiological importance of protein modulators is...
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