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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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Functional analysis of the internally disordered structure of the substrate domain in p130Cas protein biology
Hejnarová, Marie ; Rösel, Daniel (advisor) ; Gemperle, Jakub (referee)
The p130Cas protein is an important mechanosensor in cell adhesive structures such as focal adhesions and podosomes. There, the protein is subjected to mechanical tension that underlie its participation in integrin signaling. At the level of p130Cas, the mechanical force is transduced into a chemical signal. Although p130Cas does not exhibit enzymatic activity, its high binding potential turns it into an important signaling junction that ensures signal distribution to different cellular pathways. As a result, p130Cas has a profound effect on fun- damental cellular processes such as cell proliferation, differentiation and motility. In addition to its irreplaceable role in embryonic development, its involvement in the origin of patholo- gies has also been reported. As a major substrate for Src kinase, p130Cas can participate in signaling leading to tumor transformation and further malignant development. Its upregulated expression is ofen observed in aggressive types of metastasizing tumors, such as breast, prostate, and melanoma cancer. Therefore, in recent years, the possible use of the p130Cas protein as a potential target for migrastatics under development has been discussed. Within this thesis, we focus on the functional analysis of the p130Cas substrate domain. This domain is respon- sible for...
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Analysis of functional interaction between PKN3 kinase and CARMIL1 protein
Novotná, Petra ; Rösel, Daniel (advisor) ; Groušl, Tomáš (referee)
Cancer cell motility and cytoskeletal rearrangements are crucial for metastasis formation. These complex changes involve multiple cellular processes affected by many different proteins. One such protein is the Ser/Thr kinase PKN3. This kinase has been shown to be essential for metastasis formation in some aggressive types of breast and prostate cancer. Interestingly, the PKN3 kinase is not only important in malignant cancers but also in normal tissues. In endothelial cells, the PKN3 kinase can alter their adhesion, or in osteoclasts it helps to promote bone resorption. The effects of the PKN3 kinase on cancer malignancy and cell motility are well documented, but the mechanism behind these effects is still unclear. Therefore, our laboratory seeks to identify novel substrates and interaction partners of the PKN3 kinase. This work focuses on a novel potential substrate of the PKN3 kinase, CARMIL1. This protein is involved in actin cytoskeleton rearrangements by regulating actin polymerisation and thus cell motility. Here we provide evidence that the PKN3 kinase interacts with CARMIL1. Key words: PKN3, CARMIL1, actin cytoskeleton, cancer, invasion
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The role of the WASH complex in endolysosomal homeostasis
Beránková, Pavla ; Libusová, Lenka (advisor) ; Rösel, Daniel (referee)
The WASH (WASP and SCAR homologue) complex is an actin nucleation promoting factor essential for endosomal cargo sorting. Upon WASH complex depletion, endosomal cargoes are mislocalized and the endolysosomal system collapses. Here, we employed high-speed vesicle tracking and real-time rescue experiments to test the effect of WASH complex depletion on endolysosomal homeostasis. We found that large lysosome-like vacuoles emerge in knockout cell lines of individual WASH complex subunits, although the overall dynamics of the lysosomal network does not substantially change. Follow-up experiments revealed that the WASH complex does not act directly on the vacuoles during their rescue. Overall, the data indicate that the emergence of vacuoles in WASH complex knockouts is a secondary process that depends on the WASH complex indirectly.
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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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The detection of hypoxic markers in head and neck tumours
Šťovíčková, Eliška ; Šmahelová, Jana (advisor) ; Rösel, Daniel (referee)
Head and neck cancers (HNSCC) are heterogeneous group of tumours. Risk factors are mainly smoking and alcohol consumption. Some of these tumours are associated with human papillomavirus (HPV) infection, which is a significant positive prognostic factor. These tumours differ from HPV-negative tumours in clinicopathological characteristics, tumour microenvironment and response to treatment. Hypoxia is commonly found in tumors including HNSCC and is a significant prognostic and predictive factor. Elucidating the degree of hypoxia in relation to HPV infection could partly explain the differences in prognosis of these patients and allow more appropriate choice of therapy. Aspartate-β-hydroxylase is also a significant negative prognostic factor in a number of tumours, but its role in HNSCC has not yet been investigated. I focused on the detection of hypoxic markers and aspartate-β-hydroxylase expression in samples from HNSCC patients at the mRNA level by quantitative PCR and at the protein level by multispectral immunohistochemistry. The expression levels of these markers were compared in tumors stratified according to viral etiology and other characteristics such as smoking, localization or tumor stage. HPV infection had the highest impact on the expression of these markers, but the results cannot point to a...
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The effect of phosphorylation in the regulation of Rho-GTPases
Novotná, Michaela ; Rösel, Daniel (advisor) ; Hála, Michal (referee)
This bachelor thesis is focused on regulating of the activity of the Rho GTPases. Rho GTPases are important signalling proteins that are involved in regulation of many cellular processes, for example dynamic cytoskeleton changes (especially actin fibres) associated with cell adhesion and migration, vesicle trafficking or cell cycle. The activity of the Rho GTPases is primarily regulated by other proteins affecting the presence GTP/GDP or by posttranslational modifications. This thesis is focused on a narrow section of these modifications, namely phosphorylations. Phosphorylation of the Rho GTPases affects for example their activity, localization, or degradation. Understanding the mechanism of regulation of Rho GTPase activity is important for comprehension of cellular processes. Rho GTPases are involved, for example, in tumour cell invasiveness. By studying Rho GTPases and their regulation, it is possible to better target potential treatment or even prevent the occurrence of the metastases.
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Epigenetic regulation of gene expression in cancer progression and HIV-1 latency
Kovářová, Denisa ; Hejnar, Jiří (advisor) ; Černá, Marie (referee) ; Rösel, Daniel (referee)
Epigenetic mechanisms of transcriptional regulation and modulation of gene expression using RNA interference have become a powerfull tool for studying various cellular mechanisms, including tumor progression and viral latency. Advances in expression profiling have provided technology to detect candidate genes implicated in this complex process and bring other possibilities in the diagnosis and treatment of tumor diseases. In this work, I compare the gene expression profiles of v-src-transformed metastatic and nonmetastatic cells using microarray chip technology. Transcription factor homeodomain only protein X (HOPX) was identified as one of the differentially expressed genes. Activity of HOPX gene in several cancer types is usually controlled by promoter methylation. The role HOPX in metastatic formation was assessed by inoculation of cells with modulated expression of HOPX in a syngeneic chicken model system. After HOPX knockdown using shRNA, originally metastatic line showed decreased in vivo metastatic capacity. Further genomic analyses identified a cadre of genes affected by HOPX knockdown. These data demonstrate that HOPX is a metastasis-associated gene and that its knockdown decreases the metastatic activity of v-src-transformed cells through altered gene expression patterns. In the second...
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Stimulation of mesenchymal stem cells osteogenic differentiation using perfusion bioreactor
Šljivnjak, Erna ; Rampichová, Michala (advisor) ; Rösel, Daniel (referee)
Bone cells in vivo reside in a dynamic environment exposed to constant chemical and mechanical stimuli caused by the interstitial fluid flow. It is hypothesized that perfusion of the medium through the scaffold increases the mass transport and creates at the same time shear stress, thereby in vitro simulating the interstitial fluid effects and bone tissue formation conditions. This work examined the effects of perfusion flow rates on cell viability, proliferation, migration and osteogenic differentiation of human mesenchymal stem cells within cell-seeded 3D poly-ε-caprolactone scaffolds. Scaffolds were perfused for 21 days at flow rates 1, 3 and 5 mL/min and were compared to the scaffolds from static culture. Cells were most viable, had upregulated expression of osteogenic markers collagen type I and highest alkaline phosphatase activity under flow rate 1 mL/min when compared to their static counterparts. Cells proliferated the most under flow rate 3 mL/min when compared to their static counterparts. Flow rate 5 mL/min did not significantly differ from the static culture in any of the examined parameters. Cell migration into the scaffold was not improved upon exposure to perfusion. This data confirms that medium perfusion may benefit cell proliferation and osteogenic differentiation by enhancing...
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