National Repository of Grey Literature 30 records found  1 - 10nextend  jump to record: Search took 0.00 seconds. 
Regulatory mechanisms in normal and malignant granulopoiesis
Kardošová, Miroslava ; Alberich-Jorda, Meritxell (advisor) ; Stopka, Tomáš (referee) ; Balounová, Jana (referee)
Neutrophils, known primarily as key players in defense against invading pathogens, represent an essential component of both the innate and adaptive immunity. Continuous production of large quantities of neutrophils is ensured by a complex process termed granulopoiesis. In order to maintain a stable neutrophilic population, granulopoiesis requires to be tightly regulated. Moreover, impaired granulopoiesis may lead to aberrant bone marrow function and, ultimately, give rise to acute myeloid leukemia (AML). Despite decades of research, the mechanisms regulating granulopoiesis are still unclear. In particular, the CCAAT/enhancer binding protein (C/EBP) family of transcription factors plays a critical role in this process. C/EBPα acts as a master regulator of granulopoiesis mainly by orchestrating expression of its target genes, which will mediate granulocytic differentiation. Thus, characterization of novel C/EBPα target genes is critical for a better understanding of the molecular mechanisms that regulate granulopoiesis. Previously, we showed that another C/EBP member, CEBPG, is a direct target of C/EBPα. In the first part of the present work, we addressed the unknown role of C/EBPγ in granulopoiesis. We observed that Cebpg conditional knockout (KO) mice, which have the Cebpg gene ablated specifically...
Production and analysis of cellular conditional inactivation models of the ISWI ATPase Smarca5
Tauchmanová, Petra ; Stopka, Tomáš (advisor) ; Burda, Pavel (referee)
The eukaryotic nuclear processes such as replication, DNA damage repair (DDR) and transcription are highly dependent on the regulation of chromatin structure. The dynamic changes in chromatin accessibility are controlled by a class of chromatin-remodeling factors which form multimeric complexes and use ATP as the source of their helicase activity. In this study we have established a mouse embryonic fibroblast in vitro model with conditional inactivation of chromatin remodeling ATPase Smarca5 and used this powerful tool to test the regulation of cell cycle, proliferation and DDR signaling in conditions with low Smarca5 activity. Our results show that decreased dosages lead to decreased proliferation apparent already within few days post induction of Smarca5 deletion that is accompanied with decrease of cells in S and M phases of cell cycle, increasing cell ploidy and accelerated cell senescence. Additionally, the Smarca5 depleted cells upregulated many protein markers associated with DNA damage and cellular stress. Our results thus indicate that Smarca5 has indispensable roles during cell proliferation including in the maintenance of genome integrity during S phase of cell cycle.
Epigenetic factors CTCF a SMARCA5 control expression of hematopoietic transcription factor SPI1 in cells of acute myeloid leukemia and myelodysplastic syndrome.
Dluhošová, Martina ; Stopka, Tomáš (advisor) ; Machová Poláková, Kateřina (referee) ; Kozák, Tomáš (referee)
CCCTC-binding factor (CTCF) can both activate as well as inhibit transcription by forming chromatin loops between regulatory regions and promoters. In this regard, Ctcf binding on the non-methylated DNA and its interaction with the Cohesin complex results in differential regulation of the H19/Igf2 locus. Similarly, a role for CTCF has been established in normal hematopoietic development; however its involvement, despite mutations in CTCF and Cohesin complex were identified in leukemia, remains elusive. CTCF regulates transcription dependently on DNA methylation status and can if bound block interactions of enhancers and promoters. Here, we show that in hematopietic cells CTCF binds to the imprinting control region of H19/Igf2 and found that chromatin remodeller Smarca5, which also associates with the Cohesin complex, facilitates Ctcf binding and regulatory effects. Furthermore, Smarca5 supports CTCF functionally and is needed for enhancer-blocking effect at imprinting control region. We identified new CTCF-recognized locus near hematopoietic regulator SPI1 (PU.1) in normally differentiating myeloid cells together with members of the Cohesin complex. Due to DNA methylation, CTCF binding to the SPI1 gene is reduced in AML blasts and this effect was reversible by DNA methylation inhibitor 5-azacitidine.
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...
Anaemia disease models
Vondráková, Zuzana ; Bartůněk, Petr (advisor) ; Stopka, Tomáš (referee)
Hematopoiesis is a process by which blood cells are generated. All vertebrates have two phases of hematopoiesis - primitive and definitive. The main purpose of primitive hematopoiesis is the production of red blood cells, which provide oxygenation to the developing embryo. Other blood cell lineages are established by definitive hematopoiesis. The main function of erythrocytes is oxygen transport to all tissues. When erythrocyte production is decreased or they are damaged due to the membrane, enzyme or hemoglobin impairment, the condition called anemia arises. Sickle cell disease and β-thalassemia are called hemoglobinopathies as they are caused by the damaged hemoglobin. Fanconi anemia is caused by mutations in one of 21 genes of Fanconi anemia pathway, which plays an essential role in DNA repair. Diamond Blackfan anemia is caused by mutations gene for ribosomal proteins. Human cells, Mus musculus, Gallus gallus, Xenopus laevis and Danio rerio seem to be good models for study of this diseases and they are also useful for achieving therapeutical goals.
Identification and functional characterization of C/EBPalpha targets in normal and malignant hematopoiesis
Zjablovskaja, Polina ; Alberich-Jorda, Meritxell (advisor) ; Stopka, Tomáš (referee) ; Fuchs, Ota (referee)
Thehematopoieticsystemisahighlyorganizedstructure, whichhastobetightly regulatedinordertofunctionproperly.Abnormalitiesinhematopoieticdevelopmentmaylead tohematologicaldisorders,suchasacutemyeloidleukemia(AML).Thefunctionalityofthe hematopoieticsystemlargelyreliesontranscriptionfactors.C/EBPtranscriptionfactoris knownasoneofthe majorhematopoieticregulators,requiredforthefunctionalityof hematopoieticstemcellsaswellasformyeloidlineagedevelopment.Importantly,C/EBP expressionisalteredinalargeproportionofAMLcases.C/EBPregulateshematopoiesis mainlythroughorchestratingexpressionofitstargetgenes.ManyoftheC/EBPtargetshave previouslybeenshowntoplayaroleinthehematopoieticsystemandtobeinvolvedin leukemictransformation. That makesidentificationofnovel C/EBP targetsandtheir functionalcharacterizationanexcitingsubjectofresearch.Hereweidentifiedalistofgenes whoseexpressiondependsontheactivityofC/EBPthesocalledC/EBPsignature. We demonstratedthattreatment withhistonedeacetylase(HDAC)inhibitorsreactivatesthe expressionofthesegenesincellswithnon-functionalC/EBP.Inaddition,wedemonstrated thattreatmentwiththeHDACinhibitorspromotesmyeloiddifferentiationinAMLsamples carryingbi-allelicCEBPA mutationsandcharacterizedbythereducedexpressionofthe...
Role of Smarca5 (Snf2h) chromation remodeling ATPase in hematopoitic development and erythropoiesis
Kokavec, Juraj ; Stopka, Tomáš (advisor) ; Divoký, Vladimír (referee) ; Kořínek, Vladimír (referee)
The Imitation Switch (ISWI) nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells (HSPCs) accumulated but their maturation towards erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S-18) second with CBP/p300 (K376Ac), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4- OHT-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis.
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...
Localization and function of phosphoinositides in the cell nucleus
Kalasová, Ilona ; Hozák, Pavel (advisor) ; Stopka, Tomáš (referee) ; Šolc, Petr (referee)
(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids. Their inositol head can be phosphorylated at three positions generating seven differently phosphorylated species. Cytoplasmic phosphoinositides regulate membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generate second messengers. In the nucleus, PIs are implicated in pre-mRNA processing, DNA transcription and chromatin remodelling. However, their nuclear functions are still poorly understood. Here we focus on nuclear phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). We describe their localization and interaction with proteins involved in regulation of DNA transcription. PI(4)P localizes to nuclear membrane, nuclear speckles and nucleoplasm. The majority of nuclear PI(4)P is associated with chromatin and colocalizes with H3K4me2. PI(4,5)P2 localizes to nucleoli and nuclear speckles. Besides, 30 % of nuclear PI(4,5)P2 forms small nucleoplasmic PI(4,5)P2 islets. They have carbon rich core, which is probably formed by lipids, and are surrounded by proteins and nucleic acids. The active form of RNA polymerase II associates with PI(4,5)P2 islets and DNA is actively transcribed in the vicinity of PI(4,5)P2 islets. Moreover,...
The mechanisms and regulation of lineage commitment in hematopoietic stem cell
Tichý, Marko ; Stopka, Tomáš (advisor) ; Svoboda, Ondřej (referee)
Hematopoietic stem cells (HSCs) are crucial for maintaining balanced homeostasis in the human body. HSCs are pluripotent cells, which are able to give rise to many very different cells. HSCs can be found in fetal liver initially during organismal development where they expand and move to their more definitive location, the bone marrow, shortly before birth in humans and mice. HSCs possess to not only recapitulate themselves (self-renew) or proliferate and expand, but are also the first branching point from which subsequent multipotent progenitors and eventually all blood cell lineages are formed thus establishing specific and restricted terminal differentiation pathways. The irreversible decision to initiate and follow a specific differentiation pathway is designated as lineage commitment. The drivers of lineage commitment, which are a base of this thesis, are intrinsic as well as extrinsic factors acting within the stem cell niche, such as transcription factors, chromatin remodeling factors, and cytokines, which are essential for proliferation, survival, self-renewal and lineage commitment decisions. These regulatory factors, working either independently or in mutual coordination, maintain balanced homeostasis of HSC renewal and their differentiation. The goal of this thesis will be to ascribe the...

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