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Analysis of cell signaling mediated by the adapter protein Daxx
Švadlenka, Jan ; Anděra, Ladislav (advisor) ; Forstová, Jitka (referee) ; Stopka, Tomáš (referee)
2 Abstract Multifunctional adapter protein and histone chaperone Daxx has been described in nu- merous cellular processes, including the regulation of apoptotic and stress signalling, antiviral response and processes connected to chromatin (e. g. transcription). Its influ- ence on chromatin-related processes is mainly carried out by several associated en- zymes, such as DNA-methyltransferase-1, histone deacetylases and chromatin- remodelling ATPase ATRX. In the complex with ATRX Daxx functions as a chaperone of histone-3.3, maintaining the constitutive heterochromatin e. g. at centromeric and telomeric regions. The main aim of this Thesis was a better understanding of the Daxx cellular functions through identification and functional characterization of its novel interacting proteins. Using the yeast two-hybrid screen, several such new Daxx-interacting proteins were identified. These proteins were mainly nuclear, connected to the regulation of chroma- tin-related processes. More detailed analysis focused on the interaction of Daxx with chromatin-remodelling ATPase Brg1. This interaction was confirmed both in vitro and in the cells, where Daxx and Brg1 associated mainly in high molecular weight pro- tein complexes. These likely chromatin-remodelling complexes contain, in addition to Brg1, several...
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The analysis of cancer invasion plasticity in a 3D environment
Škarková, Aneta ; Brábek, Jan (advisor) ; Anděra, Ladislav (referee) ; Bryja, Vítězslav (referee)
iii Abstract Cells have evolved multiple mechanisms of cellular motility ranging from the migration of large cell cohorts to specialized migration of individual cells. The wide range of invasion modes has been exploited by cancer cells to their advantage, which has rendered the metastatic process so difficult to defeat. To allow for a better understanding of cancer invasion plasticity, we have employed studies on cancer cells that adopt the proteolytically active, adhesion-dependent, elongated mesenchymal invasion mode, the protease-independent, low adhesion, rounded amoeboid invasion mode, or combination of both. To study invasion plasticity directly, we have established two model systems of the mesenchymal- amoeboid transition (MAT) that allow for regulated induction of MAT in 3D in vitro environments. Using these systems, MAT was induced in HT1080 fibrosarcoma cells and the acquisition of a motile, invasive amoeboid phenotype was confirmed. We then observed the mesenchymal and amoeboid invasion strategies within 3D collagen in more detail using a digital holographic microscope. Further, HT1080 cells before and after MAT were subject to high throughput proteomic and transcriptomic studies. Comparison of gene expression and protein levels of mesenchymal and amoeboid cells disclosed an inflammatory-like...
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Sensing of MPyV infection by innate immunity sensors
Rjabčenko, Boris ; Forstová, Jitka (advisor) ; Anděra, Ladislav (referee) ; Mělková, Zora (referee)
Host sensors that recognize pathogen associated molecular patterns and the mechanisms of innate immune response to mouse polyomavirus (MPyV) infection were the main topics of current work. We found that MPyV did not induce interferon (IFN) production during early events of infection, but induced interleukin-6 (IL-6) and other cytokine production without inhibiting virus multiplication. Cytokine microenvironment changed the phenotype of adjacent non infected fibroblasts toward the cancer-associated fibroblast (CAF)-like phenotype. We identified Toll-like receptor 4, a sensor of the innate immunity system, to be responsible for infection dependent IL-6 production. In an effort to determine whether and where virions are released from endosomal compartments into the cytosol, we found that the hydrophobic domains of minor capsid proteins, exposed on the surface of virions after their partial disassembly in the ER, play an important role in effective escape of virions from the lumen part of endoplasmic reticulum into the cytosol, Although naked, partially disassembled virions appear before translocation to the nucleus in the cytosol, viral DNA is not recognized by cytosolic sensors at this phase of infection Sensing of MPyV resulting in IFN production occurs first during viral replication. Mutant virus,...
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Characterization of a role of selected antiapoptotic Bcl-2 family proteins in mitochondrial metabolism.
Antoš, Šimon ; Anděra, Ladislav (advisor) ; Brábek, Jan (referee)
Proteins from the Bcl-2 family are now for over 30 years widely studied mainly for their key role in apoptosis, a principal mode of regulated cell death. In the last ten years Bcl-2 proteins were also linked to the regulation of cellular signaling, mainly cellular metabolism and respiration. In this study we aimed to analyze non-apoptotic function of Bcl-2 proteins by their genetic elimination using the CRISPR-Cas12a approach and by the subsequent analysis of mitochondrial respiration, glycolysis and metabolic profiling. Our results confirmed that Bcl-2 proteins can modulate the level of mitochondrial respiration. The elimination of anti-apoptotic proteins Bcl-2, Bcl-XL and Mcl-1 decreased high respiration of cells lacking pro-apoptotic proteins Bax and Bak to the levels observed in parental U87-MG glioblastoma cells. Therefore, the loss of anti-apoptotic Bcl-2 proteins has greatly impacted mitochondrial respiration and it points to their role in a regulation of oxidative phosphorylation.
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Molecular mechanisms of apoptosis regulation by fatty acids in pancreatic β-cells
Němcová, Vlasta ; Kovář, Jan (advisor) ; Anděra, Ladislav (referee) ; Mělková, Zora (referee)
The incidence of type 2 diabetes is growing rapidly and represents a big threat for the human health care and economy system as well in the 21st century. The association of type 2 diabetes with obesity is apparent and dysfunction and apoptosis of pancreatic β-cells caused by elevated levels of fatty acids in circulation are considered as an important factor contributing to the development of this disease. However, molecular mechanisms that underlie these detrimental effects of fatty acids are only partially understood. The aim of this research project was to contribute to elucidation of mechanisms by which saturated and unsaturated fatty acids regulate viability and apoptosis induction in human pancreatic β-cells in vitro. Employing human pancreatic β-cell line NES2Y, we showed that increased levels of relevant dietary saturated fatty acids (palmitic and stearic acid) induce apoptosis of pancreatic β-cells, in contrast to relevant dietary unsaturated fatty acids (e.g. palmitoleic and oleic acid). We found that stearic acid-induced apoptosis is accompanied by significant activation of caspase-2, -6, -7, -8 and -9, but not by significant activation of caspase-3. Nevertheless, it was not associated with significant cytochrome c release, alteration in PIDD, Fas receptor and Fas ligand expression and...
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Activation and regulation of cell death in senescent cancer cells.
Holíček, Peter ; Anděra, Ladislav (advisor) ; Drbal, Karel (referee)
Cellular senescence is a distinct cell state, characteristic by cessation of cell proliferation and it is accompanied by specific morphological and biochemical alterations. Increasing and persisting incidence of senescence cells has been shown to have detrimental effect on an organism largely contributing to its ageing. Senescent cells also positively support tumour growth and can even stimulate carcinogenic transformation of surrounding cells. Moreover, senescence can be induced even in tumour cells spontaneously or by chemotherapy. Regardless of an initial stimuli and type of cells, there are two main senescence inducing pathways p16/pRb and p53/p21. Both senescent cells as well as senescent cancer cells seems to have modified apoptotic signalling at the level of mitochondria and Bcl-2 family proteins. In this study, we aimed to analyse effect of senescent state as well as pre-senescent (growth arrested state) induced by p16/pRb and p53/p21 signalling pathways on the response of H28 mesothelioma cancer cells-derived clonal cultures to various cell death-inducing stimuli. By inducible expression of p16 and p21 proteins in doxycycline-dependent manner, we forced cells to acquire senescent-like phenotype, which we detailly characterised. Our results showed that senescent-like phenotype, manifests...
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Molecular biomarkers related to DNA damage and repair: their role in carcinogenesis, patients' treatment and monitoring
Vodenková, Soňa ; Vodička, Pavel (advisor) ; Anděra, Ladislav (referee) ; Černá, Marie (referee)
Genome instability represents one of the leading forces driving the onset and development of cancer. It arises as a consequence of the combined effect of DNA damage and errors made by the DNA repair system. In many cancers, DNA damage tolerance and DNA repair pathways are disrupted or deregulated, thereby promoting cancer progression. DNA repair also appears to play a substantial role in cancer therapy response. This Dissertation Thesis was performed in response to several unclear and unresolved issues of the role of DNA damage and DNA repair in cancer pathogenesis. The aim of the Thesis was to search for potential novel biomarkers and confirmation of the validity of already existing biomarkers related to DNA damage and DNA repair, which may be associated with cancer susceptibility and patient's clinical outcome. We also explored the biological basis of different biomarkers and their associations. The major outcomes of this Thesis are: 1) The elevated chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBLs) may serve as a biomarker of cancer susceptibility and partially affects patients' clinical outcome. While telomere shortening contributes to the formation of CAs in PBLs only in healthy individuals, less efficient DNA double- strand break repair in PBLs is associated with telomere...
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Novel protein binders targeting marker of epithelial cells
Huličiak, Maroš ; Malý, Petr (advisor) ; Anděra, Ladislav (referee)
Fast and precise quantification of circulating tumour cells (CTC) in lung adenocarcinoma is a pivotal step in acceleration of diagnosis, selection of early therapy and estimation of treatment prognosis. Development of a new type of microfluidic device based on detection and quantification of epithelial- and mesenchymal-type CTC by high-affinity and cell-type specific protein binders anchored to a microfluidic chip surface represents a highly innovative approach. In this work, we used EpCAM membrane glycoprotein as a target for generation of epithelial cell- specific protein binders by a directed evolution of proteins selected from highly complex combinatorial libraries derived from albumin-binding domain scaffold (ABD) or human muscle protein domain-derived "Myomedin" scaffold. Collections of EpCAM-binding candidates from the both used libraries were generated and particular binding variants were further characterized by DNA sequencing, biochemically and by functional cell-surface binding assays. The best candidates might serve as robust anchor proteins of a microfludic chip. Key words: epithelial cell, EpCAM, protein binder, ribosome display, combinatorial library, protein scaffold
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Novel protein binders targeting marker of epithelial cells
Huličiak, Maroš ; Malý, Petr (advisor) ; Anděra, Ladislav (referee)
Fast and precise quantification of circulating tumour cells (CTC) in lung adenocarcinoma is a pivotal step in acceleration of diagnosis, selection of early therapy and estimation of treatment prognosis. Development of a new type of microfluidic device based on detection and quantification of epithelial- and mesenchymal-type CTC by high-affinity and cell-type specific protein binders anchored to a microfluidic chip surface represents a highly innovative approach. In this work, we used EpCAM membrane glycoprotein as a target for generation of epithelial cell-specific protein binders by a directed evolution of proteins selected from highly complex combinatorial libraries derived from albumin-binding domain scaffold (ABD) or human muscle protein domain-derived "Myomedin" scaffold. Collections of EpCAM-binding candidates from the both used libraries were generated and particular binding variants were further characterized by DNA sequencing, biochemically and by functional cell-surface binding assays. The best candidates might serve as robust anchor proteins of a microfludic chip. Key words: epithelial cell, EpCAM, protein binder, ribosome display, combinatorial library, protein scaffold
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Pro-apoptotic and anti-apoptotic properties of mesenchymal stem cells
Benešová, Iva ; Kössl, Jan (advisor) ; Anděra, Ladislav (referee)
Mesenchymal stem cells (MSC) display different ways of influencing not only surroundig cells but also the function of the whole organism. Besides well known immunomodulatory properties, MSC are capable of inducing and inhibiting of apoptosis in various conditions, effecting mostly cancer and immune cells. They are able to protect other cell types against apoptosis. On the contrary, anti-apoptotic and apoptotic effect of MSC is not always beneficial. Deeper and more precise understanding of influence of MSC on the apoptosis is needed to allow the usage of MSC in the biological therapy and to facilitate the treatment of many diseases. MSC are exhibited to oxidative stress, hypoxia and many pro-apoptotic factors, which cause their senescence and apoptosis. However, without this microenvironment they will not be activated and will not gain all their properties. Nowadays is an intensive research in prolongation of MSC's lifespan done, because it would improve potential biological therapy. Key words: mesenchymal stem cells, apoptosis, biological therapy
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