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Role of tumour suppressor PML in nucleolar functions
Kučerová, Alena ; Hodný, Zdeněk (advisor) ; Stejskalová, Eva (referee)
The cell nucleus is a complex structure composed of different parts, the nucleolus and PML nuclear bodies are important compartments of the nucleus. In the nucleolus, transcription of ribosomal DNA and biogenesis of ribosomes take place. The nucleolus may regulate the expression of proteins and thus the subsequent cell growth through regulating the amount of ribosomes. The nucleolus is also a sensor of stress. PML nuclear bodies play an important role in many cellular process - response to stress, virus infection or DNA damage. PML nuclear bodies consist of many proteins, the major protein is PML protein (Promyelotic leukemia protein). PML protein is coded by PML gene, it is spliced postrancriptionally and it has several isoforms. PML protein is an important cellular regulator and also a tumor suppressor. The nucleolus and PML protein cooperate together and have a functional relationship, which is not entirely clear. It was shown that PML protein changes its localization after exposure to stress and it goes near the nucleolus or into the nucleolus and this happens mainly in primary cells (the reason can be that the level of PML protein downregulates in tumour cells). The relationship between the nucleolus and PML nuclear bodies is important for cell response to stress. Keywords: nucleolus,...
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Changes in oxidative phosphorylation during development of cellular senescence
Zima, Michal ; Hodný, Zdeněk (advisor) ; Kašparová, Dita (referee)
Cellular senescence represents a state of permanent cell cycle arrest. It is considered to be an active response of the cell to various extrinsic and intrinsic types of stress, which are damaged and/or uncapped telomeres, activation of certain oncogenes, DNA damage and effects of several cytokines. This thesis describes current mechanisms which may result in establishment of senescence phenotype, putting those facts in association with changes in oxidative phosphorylation. In thesis are also mentioned features of senescence cells and their impact on the neighborhood. Special attention is focused on the role of reactive oxygen species in promotion of cellular senescence, mechanisms of their elevation, the role of NADPH oxidases and the inhibition of mitochondrial oxidative phosphorylation complexes by activity of cytokine signaling pathways STAT3 and TGFbeta. Key words: cellular senescence, reactive oxygen species, cytokines, mitochondria, oxidative phosphorylation chain, NADPH oxidases, Signal Transducer and Activator of Transcription 3 (STAT3), TGF-β, DNA damage response (DDR)
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The role of cellular senescence in carcinogenesis and aging of the brain
Paroubková, Michaela ; Hodný, Zdeněk (advisor) ; Horníková, Daniela (referee)
The risk of developing many pathological conditions and ageing-releated diseases increases persistently throughout a lifetime. A dramatic increase in the number of people suffering from one of these diseases, such as atherosclerosis, Alzheimer's disease or Parkinson's disease, is caused by constant elevation of human life's length due to advancements in modern medicine and changes in life style. Several recent studies have demonstrated that senescent cells accumulate in aged and ill tissues. Senescent cells are metabolically active, but unable of proliferation and unlike the terminally differented cells, they secrete many factors that contribute to the transformation of the tissue microenviroment. The role of senescence as anticancer barier is known for a long time, but its importance in physiological processes and aging is mainly a matter of a recent time. While there is also a lot of studies focusing on cellular senescence in peripheral tissues, their involvement in or contribution to cognitive decline with aging of the central nervous system (CNS) remains relatively unknown. Recent data of many laboratories suggest that senescence-associated secretory phenotype of the non-neuronal senescent cells in brain can cause chronic level of inflammation and thus accompany aging and ageing-related...
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The role of sumoylation in cellular senescence
Kopová, Ivana ; Hodný, Zdeněk (advisor) ; Švadlenka, Jan (referee)
Organisms with renewable tissues require mechanisms to prevent the development of cancer. One such mechanism is cellular senescence, which irreversibly arrests the growth of cells at risk for neoplastic transformation. In this study, we show that 100 μM 5-bromo-2-deoxyuridine, 0.5 μM camptothecin, 0.5 μM aphidicolin and 2.5 mM thymidine cause chemically-induced premature senescence in different human cancer cell lines and they induce an increasing conjugation of SUMO-2/3 isoforms. Chemically- induced premature senescence also induces formation of SUMO-1 and SUMO-2/3 foci, which are colocalized with PML nuclear bodies. In addition, we describe that aphidicolin induces premature senescence in stable HeLa cell line expressing His6-tagged SUMO-1. HeLa-His6-SUMO-1 cell line has decreased number of PML bodies, which do not colocalize with SUMO-2/3 foci. Moreover, the number of PML bodies in HeLa cell line with ectopic expression of His6-SUMO-1 is not increasing during aphidicolin-induced senescence. This demonstrates that increasing number of PML nuclear bodies is not essential for aphidicolin-induced senescence. Absence of SUMO-2/3 foci and increased number of PML nuclear bodies support the theory that SUMO-1 acts as a SUMO-2/3 polymeric chain terminator. On the other hand, in stable HeLa cell lines expressing...
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Cytokine expression in chemically-induced senescence
Nováková, Zora ; Hodný, Zdeněk (advisor) ; Hampl, Aleš (referee) ; Dvořák, Michal (referee)
AbstractJanderova-Rossmeislova L., Novakova Z., Vlasakova J., Philimonenko V., Hozak P., Hodny Z., 2007. PML protein association with specific nucleolar structures differs in normal, tumor and senescent human cells. J. Struct. Biol. 159, 56-70. Cellular senescence, widely recognized as a potent suppressor of tumorigenesis, represents response to cellular stress and DNA damage which results in irreversible cell growth arrest. Growing evidence signalizes crucial role of cytokine production in senescence phenomenon. Recently, many research groups have efforted to define exact character of senescence-associated secretory phenotype and its function in senescence development and maintentance. Factors secreted by senescent cells, mainly of proiflammatory character, were found to have pronounced effects on their environment as well as on its own producer. These observations were obtained preferentially on models of replicative senescence and oncogene-induced senescence. Vlasakova J, Novakova Z, Rossmeislova L, Kahle M, Hozak P, Hodny Z. 2007. Histone deacetylase inhibitors suppress IFNalpha-induced up-regulation of promyelocytic leukemia protein. Blood 109: 1373-80 Novakova Z, Man P, Novak P, Hozak P, Hodny Z. 2006. Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis....
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Mechanisms of phenotypic plasticity induced by genotoxic stress
Přibyl, Miroslav ; Hodný, Zdeněk (advisor) ; Remešová, Hana (referee) ; Vomastek, Tomáš (referee)
Therapy resistance of malignant cells represents the main reason responsible for the failure of cancer therapy. The growth of malignant cells at primary tumour sites but most importantly the dissemination of tumour cells and their growth at secondary sites, are the main reasons why patients eventually succumb to the disease. Even novel immune-based therapies find their limitation in most tumour types. The therapy resistance is mediated by the tumour cells but also by other cellular components of the tumour microenvironment. Understanding the tumour cells mechanisms and the tumour microenvironment features responsible for therapy resistance enables the development of novel therapeutic strategies. Here, we show that ionizing irradiation, 5-azacytidine, and IFNγ treatments induced expression of suprabasin (SBSN) and therapy-resistant low-adherent phenotype in cancer cells. Knockdown of SBSN resulted in suppression of the phenotype. Next, we identified aberrantly elevated SBSN in the bone marrow of a subgroup of myelodysplastic syndromes (MDS) patients. SBSN was expressed by myeloid-derived suppressor cells (MDSCs) and showed significant anti-correlation with T cell abundance and CCL2 levels, hence promises a prognostic value in clinical use. We compiled the most of the relevant knowledge of SBSN...
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Real-time monitoring of cellular processes - current approaches
Švecová, Iva ; Hodný, Zdeněk (advisor) ; Groušl, Tomáš (referee)
This thesis aims to provide an overview of real-time live-cell imaging methods with a focus on the signalling pathways. The first, most thorough section is about fluorescence methods and is followed by sections about bioluminescence and label-free methods. In the fluorescence section, we will at first introduce the types of fluorophores and respective labelling approaches. Subsequently, we will go through the individual techniques, starting with single-fluorophore and FRET biosensors, continuing with kinetic modelling approaches, a FLIM method used to detect changes in the cellular environment, and ending with two methods used to improve the resolution. With each technique, we will shortly explain the working principle and look at the examples at which this method was used. Finally, we will look at the example of live-cell imaging of one signalling cascade.
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Mechanisms of phenotypic plasticity induced by genotoxic stress
Přibyl, Miroslav ; Hodný, Zdeněk (advisor) ; Remešová, Hana (referee) ; Vomastek, Tomáš (referee)
Therapy resistance of malignant cells represents the main reason responsible for the failure of cancer therapy. The growth of malignant cells at primary tumour sites but most importantly the dissemination of tumour cells and their growth at secondary sites, are the main reasons why patients eventually succumb to the disease. Even novel immune-based therapies find their limitation in most tumour types. The therapy resistance is mediated by the tumour cells but also by other cellular components of the tumour microenvironment. Understanding the tumour cells mechanisms and the tumour microenvironment features responsible for therapy resistance enables the development of novel therapeutic strategies. Here, we show that ionizing irradiation, 5-azacytidine, and IFNγ treatments induced expression of suprabasin (SBSN) and therapy-resistant low-adherent phenotype in cancer cells. Knockdown of SBSN resulted in suppression of the phenotype. Next, we identified aberrantly elevated SBSN in the bone marrow of a subgroup of myelodysplastic syndromes (MDS) patients. SBSN was expressed by myeloid-derived suppressor cells (MDSCs) and showed significant anti-correlation with T cell abundance and CCL2 levels, hence promises a prognostic value in clinical use. We compiled the most of the relevant knowledge of SBSN...
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Cell response to genotoxic stress-based anti-cancer therapies
Imrichová, Terezie ; Hodný, Zdeněk (advisor) ; Rossmeislová, Lenka (referee) ; Rotrekl, Vladimír (referee)
The dissertation deals with a cell response to genotoxic stress, specifically to anti-cancer treatments with a genotoxic mechanism of action. In principle, cells can respond to these perturbing stimuli in several ways: in case of severe DNA damage, they usually undergo apoptosis or enter senescence. In case of minor DNA damage, or upon defective checkpoint mechanisms, they may continue the cell cycle, either with successfully repaired DNA or with mutations of various kind. Thanks to selection pressure, the mutations that provide cells with a certain growth advantage under conditions of continuing genotoxic stress, gradually accumulate and render the tumor treatment-resistant. In my thesis, I focus on several aspects of this whole process. First, I participated in a characterization of a radioresistant and anoikis-resistant population of prostate cancer cells. This population was generated by irradiating cells 35 times by 2 Gy, a regime used in clinics. After this treatment, a population of low-adherent cells emerged that demonstrated increased expression of EMT- and stem cell markers. The low-adherent state of these cells was maintained by Snail signaling and their anoikis resistance by ERK1/2 signaling. Interestingly, after a protracted period of time, these cells were able to re-adhere and...
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