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Development of Biophysical Interpretation of Quantitative Phase Image Data
Křížová, Aneta ; Jákl, Petr (referee) ; Vomastek, Tomáš (referee) ; Chmelík, Radim (advisor)
This doctoral thesis deals with biophysical interpretation of quantitative phase imaging (QPI) gained with coherence-controlled holographic microscope (CCHM). In the first part methods evaluating information from QPI such as analysis of shape and dynamical characteristics of segmented objects as well as evaluation of the phase information itself are described. In addition, a method of dynamic phase differences (DPD) is designed to allow more detailed monitoring of cell mass translocations. All of these methods are used in biological applications. In an extensive study of various types of cell death, QPI information is compared with flow cytometry data, and preferably a combination of QPI and fluorescence microscopy is used. The DPD method is used to study mass translocations inside the cell during osmotic events. The simplified DPD method is applied to investigate the mechanism of tumor cell movement in collagen gels.
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Biophysical interpretation of quantitative phase imaging of live cells generated by coherence-controlled holographic microscopy
Šuráňová, Markéta ; Rösel,, Daniel (referee) ; Vomastek, Tomáš (referee) ; Veselý, Pavel (advisor)
The dissertation thesis deals with the biophysical interpretation of quantitative phase imaging (QPI – Quantitative Phase Imaging) obtained using coherence-controlled holographic microscopy (CCHM – Coherence-Controlled Holographic Microscopy) in the Q-PHASE microscope, Telight, Brno). The theoretical part of this thesis deals with the characteristics of quantitative phase imaging, which provides non-invasive information on the activity of living cells in vitro. The main part of the work consists in elaborating a concept and verifying it of a new methodology (PAMP – Primary Assessment of Migrastatic Potential) for the first critical evaluation of drugs for expected anti-migratory/metastatic potential. The result of this method is considered the first sorting evaluation when considering specific migrastatic agents for future complex oncological treatment. PAMP evaluates the speed of cell migration, the growth of tumor cells and controls the risk of appearance of invasive phenotypes. Furthermore, the correlation microscopy method between the Q-PHASE microscope and the laser scanning confocal microscope (LSCM) is proposed to evaluate cell behavior and the occurrence of focal adhesions after drug application. The quantitative phase image obtained using the Q-PHASE microscope is compared with the quantitative phase image from the HoloMonitor (PHI AB, Sweden), on which the PAMP method has been positively verified.
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Functional analysis of the ERK signaling pathway in epithelial cells
Galvánková, Kristína ; Vomastek, Tomáš (advisor) ; Rösel, Daniel (referee)
The MAPK/ERK pathway, which is evolutionarily conserved in eukaryotes is one of the most intensively studied signaling pathways and consists of a three-tier cascade of Raf- MEK-ERK protein kinases. A variety of extracellular signals are transduced from receptors to hundreds of substrates by a series of sequential phosphorylations leading from Raf to MEK to ERK. The ERK pathway regulates a plethora of cell- and extracellular signal- specific responses such as gene expression, proliferation, differentiation, migration, and apoptosis. The proper execution of these physiological processes requires a precise temporal and spatial regulation of the pathway and disruption of the regulatory mechanisms leads to pathological consequence such as tumor transformation. Specificity and regulation of signal transduction are provided in part by the presence of isoforms at each level of the ERK signaling pathway. The functional differences between the effector protein kinases ERK1 and ERK2 have been controversial for a long time, but it is still unclear how important they are in achieving an appropriate cellular response. In this work, we focused on the functional characterization of ERK1 and ERK2 isoforms in MDCK epithelial cells. Specifically, we examined the effects of ERK2 inactivation on cell morphology and...
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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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Regulation of epithelial plasticity by ERK1 and ERK2 isoforms
Rasl, Jan ; Vomastek, Tomáš (advisor) ; Rösel, Daniel (referee) ; Libusová, Lenka (referee)
The ERK pathway is an evolutionarily conserved three-tier signaling cascade comprised of protein kinases Raf, MEK, and ERK. These core kinases are arranged in a hierarchical order and the signal is transduced from Raf to MEK to ERK. The ERK pathway is activated by diverse extracellular signals and in response regulates many cellular processes including cell proliferation, differentiation, apoptosis, migration or epithelial plasticity. Given the role of the ERK pathway in regulating such fundamental cellular processes, the ERK pathway signaling is tightly controlled and its dysregulation has pathological consequences such as cancer development and progression. Although much is known about mechanisms underlying the signal transduction by the ERK signaling pathway, much less is known about how two highly homologous ERK1 and ERK2 isoforms contribute to the signaling by this pathway. In this thesis, I studied isoform-specific functions of ERK1 and ERK2 using epithelial Madin- Darby Canine Kidney (MDCK) cells overexpressing either ERK1 or ERK2. Obtained data show that overexpression of ERK2, but not ERK1, had significant effects on the morphology and functional phenotype of MDCK cells. Both ERK1 and ERK2 expressing cells were able to form cohesive clusters, but the only ERK2 overexpression affected...
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Tracing intestinal tumorigenesis driven by BRAF V600E oncogene
Herrmannová, Terezie ; Hrčkulák, Dušan (advisor) ; Vomastek, Tomáš (referee)
Colorectal carcinoma is one of the most commonly diagnosed tumor diseases worldwide and is the cause of more than nine percent of deaths due to neoplasia. Colorectal cancer develops through different ways and one of them is the so-called serrated pathway, which is characterized by the presence of the BRAF V600E oncogenic mutation. Tumors arising through serrated pathway do not respond to classical therapy, and therefore are currently being studied at the molecular level. The oncogenic variant of the BRAF kinase activates MAPK signaling and is considered to be the main cause of serrated intestinal tumor formation. However, the mere presence of this oncogene is not sufficient for tumor development that requires further changes within the genome of the cell. In this thesis, we try to clarify what effect the BRAF V600E mutation has on the cells of the intestinal epithelium. In addition, we try to identify a possible cooperation between BRAF gene mutation and disruption of p53 and Wnt signaling, whose components are also frequently mutated in colorectal cancer. As a model for studying the processes associated with BRAF V600E activation, we use a mouse strain with conditional expression of a mutant variant of the Braf gene. We isolate intestinal organoids from these mice and subsequently perform in vitro...
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Characterization of perinuclear actin fibers and their role in cell migration
Hlaváčková, Tereza ; Vomastek, Tomáš (advisor) ; Binarová, Pavla (referee)
Cell migration is crucial for such physiological and pathological processes as wound healing, emryonal development, immune response, and methastasizing of the cancer cells. It is tightly coupled with cell polarization, nuclear traslocation, and turnover of actin cytoskeleton. Substantial, but so far poorely explored, part of actin cytoskeleton is perinuclear actin cap - dome-like structure above the nucleus costructed from perinuclear actin fibers. At the apical side of the nucleus perinuclear actin fibers are associated with LINC complex through nesprin proteins; at the edges of the cell they are anchored to focal adhesions. In the literature there were assumptions that this type of actin fibers can generate traction forces for nuclear reorientation during cell migration. The aim of this thesis is to elucidate the mechanism involved in the attachment of perinuclear actin to the LINC complex and the nucleus, thereby regulating the formation of the perinuclear actin cap. In addition, we aimed to establish a semi- automatic tool for perinuclear actin fibers quantification. Rat2 fibroblasts were used as the model cell line because they contain well-developed perinuclear actin cap. We focused on the inactivation of LINC complex components, namely Giant nesprin proteins (nesprin 1 and nesprin 2) and...
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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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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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