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The role of dipeptidyl peptidase-IV and homologous proteases in migration and invasion.
Fejfarová, Edita ; Bušek, Petr (advisor) ; Rösel, Daniel (referee)
Migration and invasion are processes which naturally occur in organism during embryogenesis, immune reactions or wound healing. These processes are very important factors in some serious diseases like rheumatoid arthritis and carcinogenesis. There is no doubt about contribution of proteases in these processes-many of them degrade extracellular matrix and thereby facilitate the movement of cells. While dipeptidylpeptidase-IV cleaves solely two amino acids from N-terminus so it is not considerably involved in ECM degradation. DPPIV and its homologues recognize peptides with proline on penultimate position, which causes resistance to ordinary types of proteases. Substrates of DPPIV and its activity homologues include chemokines implicated in signalling of migration - their cleavage and thus inactivation present DPPIV and DPPIV-like molecules as modulators of cell migration signalling in choriocacinoma, neuroblastoma, on Sézary cells or epithelial cells migrating in response to injury. Another activity of some DPPIV-like proteases is binding to the extracellular matrix proteins, when are helping in the attachment of cells and thus affect the migratory ability of the cells like ovarian cancer cells, prostate cancer cells, melanoma cells or kidney cells. Effects on migration and invasion have also...
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Analysis of Src dynamics in cellular structures
Pelantová, Markéta ; Rösel, Daniel (advisor) ; Rozbeský, Daniel (referee)
Src kinase is a key element in many signaling pathways affecting cellular processes such as differentiation, proliferation, motility, or migration. Deregulation of its activity is associated with the promotion of cancer. Therefore, understanding its cellular function is vital. Src activity directly correlates with its structure; when Src is active, it adopts opened conformation, when inactive, it is in closed conformation stabilized by intramolecular interactions. Detection of the conformation can be used to analyze Src activity. In this thesis, conformation-sensitive FRET-based Src biosensor was improved using mNeonGreen as a new acceptor fluorophore in the existing design and the properties of the new biosensor were compared with the original Src biosensor. The new biosensor is able to detect changes in Src conformation and can be stably expressed in cells. Src activity in focal adhesion was analyzed and higher Src activity in these structures was confirmed. Although the new biosensor did not exhibit significantly better sensitivity to Src conformational changes, it still proved to be a useful tool to study Src activity, and mNeonGreens higher brightness makes it more suitable for microscopic experiments. Key words: Src, FRET, biosensor, live-cell imaging, mNeonGreen
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Construction and evaluation of a novel protein mechanosensor
Kolomazníková, Veronika ; Rösel, Daniel (advisor) ; Novotný, Ivan (referee)
The protein p130Cas (human ortholog BCAR1) is a major substrate for phosphorylation by the Src family kinase and plays a central role in oncogenic transformation. Increased level of BCAR1 correlates with primary tumour growth and cancer progression. Localized to focal adhesion, p130Cas serves as a mechanosensor and mediates key interactions with the extracellular environment. The structure of p130Cas is crucial for its function, mainly the anchoring domains SH3 and CCH, together with the substrate domain which is extended when under tension. This Master's thesis presents a newly developer FRET mechanosensor based on the structure of p130Cas. The sensor utilizes the anchoring domains of p130Cas for proper localization to focal adhesions, where it can detect tension in living cells. Key words: p130CAS, FRET, focal adhesions, mechanosensing
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Identification of novel substrates of PKN3 kinase and characterization of the role of phosphorylation in the regulation of Rho GAP activity
Dibus, Michal ; Rösel, Daniel (advisor) ; Vomastek, Tomáš (referee) ; Petrák, Jiří (referee)
Protein phosphorylation represents one of the most important posttranslational modifications in signal transduction and plays a crucial role in regulation of most of the cellular processes including cell cycle, communication with extracellular environment, cell migration or apoptosis. Phosphorylation is mediated by protein kinases, deregulation of which often negatively affects development and overall homeostasis and leads to development of several diseases, including cancer. In the first part of this work we focused on identification of new substrates of PKN3 kinase, which is a known player in regulation of cytoskeletal organization and pro-malignant tumor growth. Using an analog-sensitive mutant of PKN3 we performed a phosphoproteomic screen and identified 281 proteins that could potentially be phosphorylated by PKN3. Among these, we selected ARHGAP18, a protein from Rho GAP family, for further study. We confirmed PKN3 is able to phosphorylate ARHGAP18 on Thr154, Ser156 and Thr158 and that the two proteins are able to interact with one another in an ARHGAP18 isoform-specific manner. We further showed that substitution of the three candidate sites for phosphomimicking aspartate led to the activation of ARHGAP18 GAP domain which resulted in decreased levels of active RhoA, suggesting the existence...
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The role of PKN family kinases in cancer
Novotná, Petra ; Rösel, Daniel (advisor) ; Ramaniuk, Volha (referee)
This bachelor thesis is focused on the PKN family of Ser/Thr kinases. This family includes three isoforms PKN1, PKN2 and PKN3. Especially it deals with the kinase PKN3 in more detail. These are kinases related to protein kinase C, belonging to the AGC superfamily. PKN kinases are activated via small G proteins of the Rho GTPase family or unsaturated fatty acids. PKN kinases are involved in many cellular processes, such as the regulation of cytoskeletal rearrangements, affect cell adhesion, cell movement, embryonic development and the cell cycle. Expression of PKN3 is particularly increased in cancer cells but is only present in small amounts in normal body cells. Therefore, PKN3 appears to be a very interesting therapeutic target for the treatment of cancer. Studies have shown that PKN3 has a significant effect on the motility of cancer cells, thus contributing to their migration and ability to form metastases.
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Mechanosignaling and mechanosensory proteins in adherens junctions
Hejnarová, Marie ; Rösel, Daniel (advisor) ; Rozbeský, Daniel (referee)
Adherens junctions are part of intercellular contacts, where they help to connect actin cytoskeleton of two or more neighboring cells. Within the tissue, they enable the sensing of me- chanical forces acting on cells and subsequently develop adequate response. These functions are provided by proteins, assembled into the mechanosensitive complexes of adherens junctions. In their base form-in the initial intercellular contacts, they are composed of cadherins, β-catenins, and α-catenins. Extrinsic forces acting on adherens junctions, propagate to the conformatio- nal changes of these proteins, mostly in α-catenin, allowing the creation of new interactions with additional binding partners. Such interactors include -for example- EPLIN, vinculin, or α-actinin. They allow the interconnection of the cadherin-catenin complex with actin filaments, which mediates the tension-dependent change of cell morphology. Furthermore, these proteins can transform the mechanical force to a regulatory signal, which activates important pathways, such as cell proliferation, differentiation, or migration. Thus mechanosensitive activity of ad- herens junctions plays an important role in the processes that maintain tissue integrity. Any knowledge how the mechanosensing in adherens junctions affects tissue physiology contribu-...
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The Role of Lck Kinase in T-cell Antigen Receptor Signaling
Němec, Dušan ; Štěpánek, Ondřej (advisor) ; Rösel, Daniel (referee)
LCK activity is crucial for the triggering of the entire T cell activation process. The primary function of LCK is to convert the signal of TCR:pMHC ligation into the intracellular environment. The outcome of the LCK-triggered pathway is T cell activation, cytokine production, differentiation, and clonal expansion. This thesis provides a summary of recent knowledge about the unique position of LCK in the T cell signaling machinery as well as an overview of molecules and interacting partners that regulate LCK activity. It describes the importance of the LCK-coreceptor association for optimal TCR signaling and physiological thymocyte development and mentions discussed adaptor role of LCK in the T cells. Keywords: LCK, T-cell, antigen, kinase, enzyme
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Construction and evaluation of a novel protein mechanosensor
Kolomazníková, Veronika ; Rösel, Daniel (advisor) ; Novotný, Ivan (referee)
The protein p130Cas (human ortholog BCAR1) is a major substrate for phosphorylation by the Src family kinase and plays a central role in oncogenic transformation. Increased level of BCAR1 correlates with primary tumour growth and cancer progression. Localized to focal adhesion, p130Cas serves as a mechanosensor and mediates key interactions with the extracellular environment. The structure of p130Cas is crucial for its function, mainly the anchoring domains SH3 and CCH, together with the substrate domain which is extended when under tension. This Master's thesis presents a newly developer FRET mechanosensor based on the structure of p130Cas. The sensor utilizes the anchoring domains of p130Cas for proper localization to focal adhesions, where it can detect tension in living cells. Key words: p130CAS, FRET, focal adhesions, mechanosensing
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Genetically encoded biosensors of cellular tension and their use in cellular biology
Pelantová, Markéta ; Rösel, Daniel (advisor) ; Lánský, Zdeněk (referee)
1 Abstract and key words Mechanical forces have great impact on the life of cells. They influence cell proliferation, migration or differentiation and defects in cellular mechanosensing were reported to be the cause of various diseases, such as deafness, atherosclerosis or cancer. However, mechanisms of mechanical sensing are not thoroughly examined and not many tools for doing such research are available. Genetically encoded FRET-based biosensors are one of the existing methods for studying transfer of mechanical signal in cells. It is a non-invasive method allowing to observe changes in mechanical tension across proteins in living cells. In this thesis, different types of existing genetically encoded FRET-based tension biosensors are introduced together with the process of their development and knowledge gained by their use in research. Key words: mechanical force, mechanosensing, FRET, tension sensor, biosensor development
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