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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,...
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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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Generation of the Mouse Model to Delineate Function of Chromatin Remodeling Gene Smarca5 (Snf2h)
Turková, Tereza ; Stopka, Tomáš (advisor) ; Dobeš, Jan (referee)
The chromatin structure, consisting of DNA and histones, changes dynamically during the cell cycle and cell differentiation. DNA can only be transcribed and replicated when it is packaged loosely, whereas tight packaging allows for more efficient storage. Chromatin remodelling is therefore one of the tools of gene expression control. The chromatin remodelling factors recognise chromatin with varying specificity and have an effect on the interaction between DNA and the histones. One of these factors is the Smarca5 protein. This study investigates the role of Smarca5; its goal is to create a mouse model with the ability to trigger Smarca5 overproduction in specific tissues. This model will be used to study the effect of a high, unregulated dose of Smarca5 on the physiological function of the protein. Previous studies have shown that non-physiological expression of a chromatin-remodelling factor can lead to malignant transformation. Our model can help to understand this process. Another goal of this study is to investigate some phenotype aspects of the mouse model with conditional deletion of Smarca5 in T and B cells, in particular the effects of this deletion on progenitor cell differentiation. Our results show that Smarca5 has an important role in lymphocyte development, and we have observed that...
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SWI2/SNF2 ATPases with a focus on the ISWI subfamily: protein complexes and mouse models for their study
Turková, Tereza ; Stopka, Tomáš (advisor) ; Janoštiak, Radoslav (referee)
In the nucleus the DNA is packed along with proteins into a dynamic structure called chromatin. During cell cycle the chromatin structure becomes a subject to various changes. During interphase chromatin structure becomes loose while shortly before cell division it undertakes the form of highly condensed mitotic chromosomes. Structure of chromatin influences significantly mode of gene expression and its pattern. DNA-binding proteins interacting within chromatin are also necessary during this process. To gain the access to the DNA binding factors, the chromatin has to be in a loosened form. As long as the structure of the chromatin is more condensed it creates a barrier for the DNA binding proteins. Therefore it becomes obvious that the remodeling of the chromatin structure is one of the important regulators of gene expression and that the enzymes, which execute remodeling, are of great importance. One of them is ATPase Smarca5, which belongs to the protein subfamily ISWI and which creates the catalytic subunit for several different ATP-dependent chromatin remodeling complexes. Mutations of members of those complexes disturb regulation of transcription and cellular differentiation. In some cases the incorrect function of these complexes can lead to cellular transformation into a tumours state. This...
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Pathophysiologic aspects of myelodysplastics syndromes in relation to the effect of targeted imunomodulation and demetylation therapy
Jonášová, Anna ; Stopka, Tomáš (advisor) ; Maisnar, Vladimír (referee) ; Faber, Edgar (referee)
Myelodysplastic syndromes (MDS) represent a group of clonal stem cell disorders characterized by ineffective hematopoiesis, peripheral cytopenia, morphological dysplasia and the risk of transformation to acute myeloid leukemia (AML). MDS belongs to one of the most common hematological diseases in patients over 60 years old. MDS incidence is still increasing. Appropriate therapy of MDS remains challenging. There is no curative approach besides peripheral stem cells transplantation, which is regretfully appropriate only for a small group of patients due to a higher median age of the MDS population. This is why the search for therapeutic alternatives remains paramount. MDS treatment was rather frustrating until the recent introduction of two new therapeutic approaches: immunomodulation therapy with lenalidomide and epigenetic or demethylating therapy with 5-azacytidine. Both new drugs have significantly higher effect than standard therapy. However, the precise mechanism of this effect remains unknown. As a result, we decided to initiate several research projects while introducing this promising treatment to our patients. Our aim is to investigate the mechanism of both agents in relation to disease pathogenesis by examining changes of certain occurrences and factors prior to and during the course of...
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Adjusting Wnt signaling, new regulatory mechanisms of the Wnt pathway
Fafílek, Bohumil ; Kořínek, Vladimír (advisor) ; Stopka, Tomáš (referee) ; Machoň, Ondřej (referee)
4 Abstract The Wnt pathway is one of the major signaling cascades contributing to multiple cellular processes during embryogenesis, and adult tissue homeostasis and regeneration. Moreover, aberrant activation of the Wnt signaling pathway is connected with development of neoplasia, notably colorectal cancer. The aim of the thesis was to identify new ways of the Wnt pathway regulation to understand better physiological as well as non-physiological mechanisms of Wnt signaling. The results are summarized in four publications. The first article deals with TROY, a member of tumor necrosis factor receptor family. We identified TROY as a Wnt target gene during our search for Wnt responsive genes in colorectal cancer cell lines. Additionally, we detected expression of Troy in tumors of two mouse models of intestinal cancer. In the healthy gut, Troy is produced in fast cycling intestinal stem cells where negatively regulates the Wnt pathway. The second study focuses on processing and posttranslational modification of murine Wnt1 and Wnt3a. Wnts are glycosylated and double acetylated by lipid adducts and our results revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. Moreover, acylation of Wnts is connected with their signaling activity which is related to Wnt1 and...
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Targeted therapy of AML1-ETO positive acute myeloid leukemia with histone deacetylase inhibitors
Zápotocký, Michal ; Trka, Jan (advisor) ; Stopka, Tomáš (referee) ; Trbušek, Martin (referee)
In t(8;21) acute myeloid leukaemia (AML), the leukemogenesis is supposed to be promoted by interference with expression of AML1 target genes. Repressor complex associated with AML1-ETO fusion protein recruits class I histone deacetylases (HDAC). Valproic acid (VPA) was found to have an extensive effect on AML blasts, via inhibition of class I HDAC. We aimed to characterize the differentiation effect of VPA on AML1-ETO-positive leukemic cells and to determine the expression pattern of AML1 target genes. Kasumi-1 (M2 AML1-ETO-positive), Kasumi-6 (M2 AML1-ETO- negative), MV4-11 (MLL-AF4-positive) and K562 cells were treated with VPA and 12-0-tetra- decanoylphorbol-13-acetate (TPA) and examined by flow cytometry and qRT-PCR. Two AML1-ETO- positive and two negative patients' bone marrow diagnostic samples were treated with VPA and TPA to confirm in vitro findings. Valproic acid induced apoptosis in AML1-ETO-positive and MLL- AF4-positive cells in dose dependent manner. But changes of immunophenotype proving the differentiation were observed purely in AML1-ETO-positive cell line (decreased CD33/34/117 and increased CD11a/11b expression). However, differentiated cells exhibited positivity of AnnexinV; hence the relationship between cell death and differentiation had to be evaluated. Apoptosis was blocked by...
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Role onkogenní mikroRNA-155 a proto-onkogenu MYB u chronické lymfatické leukémie
Vargová, Karina ; Stopka, Tomáš (advisor) ; Móciková, Heidi (referee) ; Trka, Jan (referee)
(EN) Chronic lymphocytic leukemia (B-CLL) represents a disease of mature-like B-cells. Due to failed apoptosis but also due to enhanced proliferative signals, the leukemic B-cells accumulate in the peripheral blood, bone marrow, lymph nodes and spleen. The clinical course of B-CLL is very heterogeneous; in some patients B-CLL progresses very rapidly into an aggressive form. Such patients need therapy sooner while in other patients with indolent B-CLL the onset of therapy takes years. Several standard prognostic and disease progression markers are used for disease staging and monitoring, however a reliable marker that will suggest when to start therapy is unknown. Expression of small, non-coding microRNAs is often deregulated and represent important prognostic markers in variety of cancers including leukemia. Hence in our study we concentrated to miR-155, an important molecule regulating differentiation of hematopoietic cells, inflammation process and antibody production. Its aberrant expression was described in Hodgkin`s as well as in non-Hodgkin`s lymphoma, including indolent lymphoproliferations like B- CLL. Our results confirmed elevated levels of both, primary miR-155 transcript and mature form of miR-155 in our B-CLL patient samples (N=239). The aberrant expression of miR-155 in B-CLL samples...
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The functional in vitro analysis of the BRCA1alternative splicing variants
Ševčík, Jan ; Kleibl, Zdeněk (advisor) ; Stopka, Tomáš (referee) ; Macůrek, Libor (referee)
BACKGROUND: The inactivation of the tumor suppressor gene BRCA1 is a predisposing factor for a breast/ovarian cancer development. Formation of cancer-specific alternative splicing variants with aberrant biological properties can represent additional mechanism decreasing the overall BRCA1 activity in DNA double strand break (DDSB) repair. In this study, we analyzed BRCA1 alternative splicing variants BRCA114-15 and 17-19 ascertained previously during the screening of high-risk breast cancer individuals. METHODS: We established a stable MCF-7 cell line-based model system for an in vitro analysis of BRCA1 variants. Using this system, we analyzed the impact of BRCA114-15 and 17-19 variants on DNA repair kinetics using comet assay and confocal immunomicroscopy. The capacity of DNA repair was assessed directly by an in vitro NHEJ assay and indirectly by a mitomycin C sensitivity test. The proliferation activities were determined by a clonogenic assay and growth curves. RESULTS: Overexpression of BRCA114-15 and 17-19 increases the endogenous level of DNA damage, slows down the DDSB repair, and decelerates the initial phase of radiation-induced foci formation and prolongs their persistence. Moreover, BRCA114-15 and 17-19 differentially influence the activity of HR and NHEJ and sensitivity of MCF-7 cells to ionizing...
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Delineating aggressiveness of acute myeloid leukemia in a mouse model carrying mutations of Spil (PU.1) and Trp53.
Bašová, Petra ; Stopka, Tomáš (advisor) ; Machová Poláková, Kateřina (referee) ; Zuna, Jan (referee)
PU.1 downregulation within haematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well known tumor suppressor that is often mutated in human haematologic malignancies including AML and adds to their aggressiveness; however its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1ure/ure mice (PU.1ure/ure p53-/- ) results in more aggressive AML with shortened overall survival. PU.1ure/ure p53-/- progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in absence of p53 contribute to decreased PU.1 levels in PU.1ure/ure p53-/- mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved...
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