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Cell signalling and molecular complexes of the TRH receptor
Drastichová, Zdeňka ; Novotný, Jiří (advisor) ; Hodný, Zdeněk (referee) ; Říčný, Jan (referee)
1 Summary The first part of this thesis is preoccupied with the identification of protein alterations in the membrane fraction of HEK293-E2M11 cells after prolonged TRH treatment. The isolated membrane fraction enriched in plasma membranes contained markedly increased the amount of Na,K-ATPase, TRH receptor and G-proteins compared to the postnuclear supernatant. By using 2D electrophoresis and mass spectrometry, the levels of 42 proteins were identified to be altered in samples of PM- enriched fractions from TRH-treated (16 h; 10 μM) cells. Out of these proteins only ezrin and stomatin-like 2 are known to be localized in the plasma membrane. Five proteins (mitofilin, MTHSP75, prohibitin, stomatin like-2, peroxiredoxin III) whose levels were increased after the prolonged TRH treatment represent proteins localized in mitochondria. All of them are important for proper structure and function of mitochondria. The ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax was markedly higher in cells treated with TRH than in control untreated cells. Hence, it can be concluded that prolonged TRH treatment may significantly affect mitochondrial membrane and function of mitochondria. The second part of this thesis deals with the identification of molecular protein complexes of TRH-R and/or Gq/11 protein. The presumed...
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Role of STAT3 signalling in oncogenesis and cancer therapy
Machalová, Veronika ; Hodný, Zdeněk (advisor) ; Brábek, Jan (referee)
STAT3 (Signal Transducer and Activator of Transcription 3) is considered to be one of the possible targets of cancer treatment. The ability of STAT3 constitutive activation to form tumors is a foundation of such theories. Additionally, constitutively activated STAT3 is present in many types of cancer with high occurrence, such as breast and prostate carcinoma. This protein is required in normal body cells as well. STAT3 is a transcription factor targeting many genes that are essential for the cell. STAT3 is activated by phosphorylation of its tyrosine residue and homodimerization. Proteins transcribed with help of STAT3 function in cell cycle progression, cell growth, replication, negative regulation of apoptosis, and other roles, typical for cancer. Moreover, STAT3 is modulating mitochondrial function and maintaining ROS production in mitochondria, but in form of transcriptionally inactive monomers. The purpose of this Thesis is to review known data about STAT3 in oncogenesis and by that, to show STAT3 has great potential to become the target of cancer treatment. This Thesis contains a short overview of known STAT3 inhibitors as well. Key words: Signal Transducer and Activator of Transcription 3 (STAT3), JAK/STAT3 pathway, constitutive activation, cancer, tumor, inhibitor, mitochondria, apoptosis
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Postranslational modifications in DNA damage response
Moudrý, Pavel ; Hodný, Zdeněk (advisor) ; Malcová, Ivana (referee) ; Mistrík, Martin (referee)
Our genetic material is continually challenged by varieties of factors and processes that represent enormous threat to our ability to transmit genetic information to our offspring and to our survival as well. Cells possess many mechanisms capable of detecting and repairing damaged DNA, collectively known as DNA damage response (DDR). DDR activities must be tightly regulated in a spatiotemporal and DNA lesion-specific manner to optimize repair and prevent alterations in DNA during normal cellular processes. Defects in DNA repair drive genomic instability and may ultimately lead to cancer and neurodegenerative diseases. This work is a compilation of 4 projects focused on molecular mechanisms of DNA damage response and assembly of multiprotein signalling and repair complexes at sites of DNA damage. We identified nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import and DDR. We showed that NUP153 knockdown prevents 53BP1 from entering the nuclei in newly forming daughter cells, which translates to decreased IR-induced 53BP1 foci formation, delayed DNA repair, impaired survival after IR and exacerbates replication stress-induced DNA damage. Furthermore, we identified ubiquitin-activating enzyme UBA1 as the apical E1 enzyme required for response to IR and replication...
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Posttranlational protein modifications in response to DNA damage
Kroupa, Michal ; Hodný, Zdeněk (advisor) ; Novotný, Marian (referee)
- 5 - Abstract Thousands of DNA lessions occur in each cell every day of which the most toxic are double-strand breaks (DSBs). Signaling of their presence and subsequent repair are mediated by so-called DNA-damage response mechanism (DDR), which involves accumulation of many effector proteins into DSBs sites. These molecular accumulation at DSBs are termed DNA damage foci. Depending on presence of sister chromatid, DSBs are repaired by two major mechanisms: by homologous recombination and by non-homologous end joining. Both pathways lead to activation of checkpoint kinases (Chk1 or Chk2) which iniciate checkpoints in cell cycle and allow repair of damaged DNA. Signaling of DNA damage and activation of these pathways are regulated by posttranslational protein modifications. These enzymatic reactions involve mainly phosphorylation, ubiquitination and sumoylation. Recently it was shown that ubiquitination of damaged chromatin is a prerequisite for sumoylation of tumor supressors BRCA1 and 53BP1. Failure in DNA damage recognizing mechanisms caused by disorders such as modifications or mutations of 53BP1 and BRCA1 genes can lead to subsequent disruption of genomic integrity and then a high risk for selection of cell clones with tumorigenic potencial. Current research is focused on regulation of posttranslational...
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Regulation of promyelocytic leukemia protein expression
Dobrovolná, Jana ; Hodný, Zdeněk (advisor) ; Novotný, Jiří (referee) ; Kovarik, Pavel (referee)
4. CONCLUSIONS Major resultsof this PhD thesiscanbe summarizedas follows: |. For full tÍanscTiptionalactivationof ISG @ML' Sp100'IRFI) by inteďeronor'the deaceýlationof unknownfactor(s)is necessary.IFNer.inducedexpressionof PML at mRNA andproteinlevel and zubsequentincreaseof PML NBs are suppressedby all tested HDACIs independentlyon cellular origin. Importantly,baml (i.e. IFNc- nonstimulated)expressionof PML is not influencedby ÍIDACIs. The classical Jak. STAT pathwaymediatingtransitionof IFNo signalremainsunaffectedby HDACIs and the targetof HDACIs lies downstreamof ISGF3 binding to PML promoter. AdverseaffectsofHDACIs usedin clinical praxisarepredicted. 2. Human mesenchymalstem cells (gfowing, confluent or terminally differentiated) expressPML protein,form PML nuclearboďes and respondto IFNcr teaEnent by dramaticelevationof PML mRNA andproteinlevels and mďerate increaseof PML NBs. Numberof PML NBs per cell nucleusis depende,nton thecultureproliferative age. ln normal diploid but not in immorbl cells PML forms various nucleoli- associatedshuctuÍes.The localization of PML into nucleoli is teestablishedin senescentcells. The importanc€ of PML association with nucleoli for its fumor suppressoractivitiesis hypothesized. 3. Drug-induced senescencein several human cell lines leads to increased PML...
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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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The role of sumoylation in cellular senescence
Kopová, Ivana ; Švadlenka, Jan (referee) ; Hodný, Zdeněk (advisor)
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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The role of DNA damage in cellular senescence
Dušková, Pavlína ; Vondrejs, Vladimír (referee) ; Hodný, Zdeněk (advisor)
Cells grown in vitro may be introduced into terminal growth arrest termed cellular senescence. In normal cells, it always arises when they reach the end of their proliferative capacity. However, this process may be also caused by various means anytime during the cultivation of both normal and at some circumstances also in immortal cells. Current view of cellular senescence places this phenomenon into direct relationship with DNA damage. The first aim of this study is to elucidate the role of DNA damage in chemically induced senescence. Until recently, DNA damage was not studied in this particular model. All chemicals used in this work are routinely used to unveil unstable parts of the human genome, so called fragile sites, which are inherently prone to DNA breakage. The second aim of this study is to asses the possibility that these sensitive regions of DNA might be also involved in senescent phenotype. Two of the three chemicals used in this study, 5-bromodeoxyuridine and distamycin A, were commonly used to accomplish senescence, and their effect on main senescence-associated changes in cellular proteome is thus relatively well defined. The changes caused by the third compound, thymidine, were so far poorly investigated. Therefore, the last aim of this work is to find out, if the changes of protein...
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The role of JAK-STAT signalling pathway in cellular senescence
Černohorská, Markéta ; Schierová, Michaela (referee) ; Hodný, Zdeněk (advisor)
Proliferating human cells cultivated in vitro after certain number of population doublings withdraw from the cell cycle and enter a specific state termed replicative cellular senescence. Lately, several other forms of senescence independent of the proliferative history and telomere shortening were described. This is called premature senescence, and can be elicited by exposure of cells to aberrant mitogenic or oncogenic signals, to oxidative stress or to variety of chemically and functionally unrelated DNA damaging agents. Senescent cells alter their morphology and expression pattern. This complex phenotype is characterized by enlarged cytoplasm, activation of cell cycle inhibitors, expression of tumor supressors and profound changes in cell secretory phenotype. These cytokines/chemokines induce many different cascades, for example Jak/STAT signaling pathway, that are activated in response to viral infection or inflammation. Senescent cells were found also in vivo in the tumor tissue that produces altered cytokines itself. This diploma thesis inquires into the role of interferon-Jak/STAT signaling pathway in premature cellular senescence induced by genotoxic agents that are often used in chemotherapy. Obtained results might help to understand the complexity of tumorogenesis and senescence. Powered by TCPDF...
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