National Repository of Grey Literature 16 records found  1 - 10next  jump to record: Search took 0.00 seconds. 
Genomic instability in patient tumors due to excesive AID activity
Vaníčková, Karolína ; Drbal, Karel (advisor) ; Macůrek, Libor (referee)
AID is a member of APOBEC family of mutational enzymes. AID generates U:G mismatches in ssDNA by deaminating cytosine to uracil. In B cells error-prone repair of these mismatches induces a mutational burden in the process of somatic hypermutation of Ig locus during affinity maturation of immunoglobulins (Ig). AID also induces double-strand breaks during Ig class switch recombination or primary Ig diversification through templated gene conversion in some vertebrate species. AID might gain tumorigenic potential in case of insufficient regulation of induction and repair processes, causing genomic instability and possibly leading to tumorigenesis. AID is induced in epithelial tissues by proinflammatory cytokines via canonical NF-B pathway. Both exogenous factors (pathogens Helicobacter pylori or HCV), endogenous factors (bile acid) or even physiological state such as ovulation are the initiating factors. Thus, AID might be the link between inflammation and carcinogenesis. AID is expressed in different stages of carcinomas, mostly during the initial oncogenic transformation. Mice with ectopic AID expression develop lung, gastric, oral and hepatic carcinomas as well as melanomas. AID also regulates epithelial-mesenchymal transition in other tumors. AID is responsible for treatment resistance in both CML...
Role of Rad18 in genome stability
Palek, Matouš ; Macůrek, Libor (advisor) ; Šolc, Petr (referee)
Rad18 is an E3 ubiquitin ligase well-known for its function in DNA damage tolerance (DDT). Especially, its role in translesion DNA synthesis, one of two DDT branches, was extensively studied in the past. Recently, Rad18 was shown to be involved in the repair of DNA double- strand breaks (DSBs) in mammalian cells. The role of Rad18 in human cells seems to be important since DSB repair as well as DDT pathway are essential for maintenance of genome stability. In this work, I introduce the function of Rad18 in both DDT pathways, translesion DNA synthesis (TLS) and template switching (TS). Then I summarize current knowledge about the role of human Rad18 in DSB repair. Finally, I describe potential involvement of Rad18 dysregulation in human cancer, since loss of genome integrity is an important driving force for tumorigenesis. Keywords: Rad18, genome stability, DNA double-strand break repair, tumorigenesis, DNA damage tolerance, translesion DNA synthesis, template switching.
Molecular characterization of γ -tubulin interactions with signalling molecules
Macůrek, Libor ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Svoboda, Augustin (referee)
52 V. CONCLUSIONS The results of presented PhD thesis can be summarized as follows: For the first time it has been demonstrated that γ-tubulin forms complexes with αβ-tubulin dimers in brain tissue as well as in other models of neuronal differentiation. Two forms of γ- tubulin have been identified in complexes of various sizes. It has been shown that γ-tubulin is posttranslationally modified. One of the identified posttranslational modifications of γ-tubulin is phosphorylation that appears to depend on Src family kinase activity. It has been proposed that posttranslational modifications of γ-tubulin may regulate interactions of γ-tubulin with αβ-tubulin heterodimers or other associated proteins during neurogenesis. It has been shown that γ-tubulin associates with protein tyrosine kinases involved in signal transduction events. γ-Tubulin interaction with Src family kinases significantly increased after long-term RA-activation embryonal carcinoma P19 cells. A similar increase has been observed after rapid activation of mast cells, indicating that this regulatory mechanism is not restricted to a particular model system. In both models, Src family kinases bound to γ-tubulin are active and phosphorylate proteins present in γ-tubulin complexes. Fyn kinase interacts with γ-tubulin through its SH2 domain in a...
Investigating critical mechanisms of oncogenesis using cell model systems
Hušková, Hana ; Stopka, Tomáš (advisor) ; Macůrek, Libor (referee) ; Vojtěšek, Bořivoj (referee)
(EN) Humans and cells in their bodies are exposed to various mutagens in their lifetime that cause DNA damage and mutations, which affect the biology and physiology of the target cell, and can lead to the expansion of an immortalized cell clone. Genome-wide massively parallel sequencing allows the identification of DNA mutations in the coding sequences (whole exome sequencing, WES), or even the entire genome of a tumour. Mutational signatures of individual mutagenic processes can be extracted from these data, as well as mutations in genes potentially important for cancer development ('cancer drivers', as opposed to 'passengers', which do not confer a comparative growth advantage to a cell clone). Many known mutational signatures do not yet have an attributed cause; and many known mutagens do not have an attributed signature. Similarly, it is estimated that many cancer driver genes remain to be identified. This Thesis proposes a system based on immortalization of mouse embryonic fibroblasts (MEF) upon mutagen treatment for modelling of mutational signatures and identification and testing of cancer driver genes and mutations. The signatures extracted from WES data of 25 immortalized MEF cell lines, which arose upon treatment with a variety of mutagens, showed that the assay recapitulates the...
Role of genetic factors in the prognosis and prediction of efficacy of chemotherapy in breast carcinoma patients
Brynychová, Veronika ; Souček, Pavel (advisor) ; Macůrek, Libor (referee) ; Tichá, Ivana (referee)
Changes in the regulation of apoptosis and cell cycle are involved in tumor development, progression, and resistance to antitumor therapy. The aim of this work was to evaluate the importance of apoptotic caspases and regulators of cytokineses as possible prognostic and predictive markers in breast carcinoma patients. In addition to determining the transcript levels of selected genes in tumor and control tissues obtained from breast carcinoma patients, we have also focused on the importance of alternative splice variants of caspases and their potential genetially determined regulation. We analysed the obtained data in relation to the clinical-pathological characteristics of the tumors, the progression-free survival of patients and to the response of the patients to the neoadjuvant chemotherapeutic treatment. Part of the work was determination of protein expression levels and verification of the importance of selected candidates for the effect of chemotherapy by functional study. The transcript levels of caspase 2, 3, 7, 8, 9, 10, the specifically detected splice variants caspase 2S, 2L, 3A and B, 3S, 9A, 9B, 8L, and the transcript levels of KIF14 and CIT in breast carcinomas were unrelated to the progression-free survival of patients, or to the response of patients to neoadjuvant treatment. The...
Potenciální využití WIP1 fosfatasy v terapii nádorového onemocnění prsu
Pecháčková, Soňa ; Macůrek, Libor (advisor) ; Souček, Pavel (referee) ; Krejčí, Lumír (referee)
Cells in our body respond to genotoxic stress by activation of a conserved DNA damage response pathway (DDR). Depending on the level DNA damage, DDR signaling promotes temporary cell cycle arrest (checkpoint), permanent growth arrest (senescence) or programmed cell death (apoptosis). Checkpoints prevent progression through the cell cycle and facilitate repair of damaged DNA. DDR represents an intrinsic barrier preventing genome instability to protect cells against cancer development. WIP1 (encoded by PPM1D) phosphatase is a major negative regulator of DDR pathway and is essential for checkpoint recovery. This thesis contributed to the understanding of molecular mechanisms of WIP1 function and revealed how WIP1 can be involved in tumorigenesis. Firstly, we described that WIP1 protein levels decline during mitosis by APC-Cdc20 dependent proteasomal degradation. WIP1 is phosphorylated at multiple residues which inhibit its enzymatic activity. We propose that inhibition of WIP1 in mitosis allows sensing of low levels of DNA damage that appear during unperturbed mitosis. Further, we identified novel gain-of-function mutations of PPM1D which result in expression of C-terminally truncated WIP1. These truncated WIP1 variants are enzymatically active and exhibit increased protein stability. As result, cells...
Regulation of cell cycle and DNA damage response in mouse oocytes.
Mayer, Alexandra ; Šolc, Petr (advisor) ; Macůrek, Libor (referee) ; Macek, Milan (referee)
A specific feature of mammalian oocytes is a long prophase I arrest, which can be maintained for many years in humans. The oocytes must ensure robust mechanisms, which can keep them in prophase I, but effectively trigger meiotic resumption when required. Consequently, throughout the maturation of an oocyte, non-erroneous chromosome segregation is a prerequisite for the generation of healthy offspring. In this study we aimed to investigate the new roles of Aurora A (AURKA) and polo-like kinase 1 (PLK1) in the regulation of the cell cycle progression. For this purpose, we used transgenic mice that specifically overexpress wild type (WT-) or kinase-dead (KD-) AURKA in oocytes only, and to study PLK1 we treated oocytes with BI2536, a small molecule inhibitor known to specifically inhibit PLK1 in somatic cells. Our data show, that both AURKA and PLK1 are not essential for meiotic resumption, however they participate in this process. Active AURKA regualtes the increase in microtubule organizing centers (MTOC) in prophase I, which is the first visible marker of resumption of meiosis in oocytes. AURKA activation is biphasic, and the initial increase in MTOC is transient, while full AURKA activation needed for the stability of MTOC requires the activity of Cyclin-dependent kinase 1 (CDK1). We show that PLK1...
Molecular mechanisms of G2/M checkpoint regulation
Kořínková, Klára ; Macůrek, Libor (advisor) ; Forman, Martin (referee)
Cell division is necessary for maintaining tissue homoeostasis, but at the same time its defects are closely related to the development of many diseases including cancer and premature ageing. Activation of oncogenes leads to replication stress and directly threatens genome stability. The right control of transition between interphase and mitosis is an important mechanism for the protection of genome integrity. Nuclear division is only possible with those cells in which flawless duplication of genetic information occurred. By contrast, cells with damaged DNA structure remain temporarily or permanently stopped at G2 phase of the cell cycle. The topic of this thesis is a detailed literature overview with the subject of molecular mechanisms of the G2/M transition regulation under unperturbed conditions and in the presence of damaged DNA.
Characterisation of the mechanisms regulating 53BP1 nuclear transport
Liďák, Tomáš ; Macůrek, Libor (advisor) ; Brábek, Jan (referee)
Tumor suppressor p53-binding protein 1 (53BP1) is an integral part of a sophisticated network of cellular pathways termed as the DNA damage response (DDR). These pathways are specialized in the maintenance of genome integrity. Recently, it was reported that nuclear import of 53BP1 depends on importin ß. Here, I used fluorescence microscopy and co-immunoprecipitation experiments to identify its nuclear localization signal (NLS). Clusters of basic amino acids 1667-KRK-1669 and 1681-KRGRK- 1685 were required for 53BP1 interaction with importin ß and for its nuclear localization. Short peptide containing these two clusters was sufficient for interaction with importin ß and targeting EGFP to the nucleus. Additionally, the effect of 53BP1 phosphorylation at S1678 on its nuclear import was examined. Mimicking the phosphorylation in the 53BP1-S1678D mutant decreased the binding to importin ß and resulted in a mild defect in 53BP1 nuclear import. However, 53BP1 entered the nucleus continuously during the cell cycle, suggesting that CDK-dependent phosphorylation of S1678 probably does not significantly contribute to the regulation of 53BP1 nuclear transport. Taken together, 53BP1 NLS meets the attributes of a classical bipartite NLS. Although no cell cycle-dependent regulation of its import was observed, the...
Role of Polo-like kinases in the cell cycle and DNA damage response
Kudláčková, Radmila ; Macůrek, Libor (advisor) ; Šolc, Petr (referee)
Within the process of cell division, genetic material must be equally distributed between the two daughter cells. In the next phase, the missing portion of the genome must be synthesized. The entire cycle is regulated by cyclin-dependent kinases (Cdks) in cooperation with cyclins. If the DNA is damaged during the cell cycle, signaling pathways of checkpoints supress cycle progression and enforce the cell cycle arrest until the damage is repaired. Malfunction of the checkpoints results in tumorigenesis. Polo-like kinases (Plks) are, much like Cdks, important regulators of the cell cycle. Plks play significant role mainly in the mitosis and also in a response to the DNA damage. This thesis is focused on human homologues, nevertheless conservation of homologues among organisms is considerable, thus presented findings are of general relevance. Human cells express five proteins from the family of Polo-like kinases, from which Plk1 corresponds to Polo-like kinases of lower eukaryotes. Knowledge on the remaining four kinases is still on the rise.

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