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Differentiation plasticity of hematopoietic cells
Polgárová, Kamila ; Stopka, Tomáš (advisor) ; Otáhal, Pavel (referee) ; Šálek, Cyril (referee)
Hematopoiesis has been for many years seen as a straightforward process based on sequential restriction of cell fate potential leading to production of mature blood cells. In the last decade, however, several works documented an unexpected plasticity of hematopoietic cells with expanded potential of myeloid development from lymphoid progenitors and vice versa. Under physiologic conditions hematopoiesis is tightly controlled and the definite cell fate is denominated by multiple factors that all lead to changes in regulatory networks that include transcription factors, epigenetic changes and post-transcriptional modulations. Any disruption of this strict regulation, caused by mutations or other events, affects the proliferation and lineage fidelity of hematopoietic precursors. This may lead to clonal growth of variable significance or leukemogenesis and may possibly affect the treatment sensitivity of the hematological malignancies. For better understanding of hematopoietic regulation we described gene expression changes during physiological development of lymphoid and myeloid lineages and in leukemic specimens using our own simplified real-time PCR based platform. We investigated expression of 95 genes connected with lymphoid and myeloid differentiation or with leukemogenesis in sorted hematopoietic...
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Characterization of hematopoietic cells in patients with mature B-cell malignancies
Maswabi, Bokang Calvin ; Živný, Jan (advisor) ; Otáhal, Pavel (referee) ; Alberich Jorda, Meritxell (referee)
(English) Using flow cytometry we analyzed absolute and relative proportions of hematopoietic stem and progenitors cells (HSPC) populations including hematopoietic stem cells (HSC), multipotent progenitors (MPP), multilymphoid progenitors (MLP) and pro B cells from bone marrow of patients with mature B cell malignancies and in healthy controls. We found lower absolute and relative numbers of MLP and higher relative numbers of HSC were observed in patients when compared to age-matched controls irrespective of bone marrow (BM) involvement. On the other hand significantly decreased absolute numbers of MPP were observed only in patients who had their BM infiltrated by disease. We also confirmed published data showing increasing absolute and relative percentages of MLP with increasing age, decreasing relative percentages of HSC with increasing age, and decreasing absolute and relative pro B cell frequencies with increasing age in healthy subjects. While decreased absolute and relative pro B cell numbers were also found in patient samples as age increased, no significant correlations were detected in patients HSC, MPP or MLP populations. Age-related sub-analysis of PTs samples demonstrated that most of the disease associated changes in HSPC frequencies were observable more prominently in the elderly (>45...
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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.
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Haematopoiesis in Sea lamprey
Kovář, Martin ; Bartůněk, Petr (advisor) ; Živný, Jan (referee)
To find out if the haematopoietic system is common feature of vertebrates, we decided to examine haematopoiesis in a sea lamprey (Petromyzon marinus). All blood cells arises from the haematopoietic stem cells in higher vertebrates. We assume that this is common for the higher vertebrates and a jawless vertebrates, but nobody was interested in the jawless haematopoiesis since 1970. Using a reverse genetic, we identify homologues of important hematopoietic of higher vertebrates in transcriptome of the sea lamprey with emphasis on important receptors or transcription factors, because they can be used as the specific markers of different blood cells and their progenitors. Then we use those sequences for cloning, expression measurements and other work. We picked up sea lamprey as model organism because its unique phylogenetic position, important foe evo-devo studies, but also because lack of elementary knowledge about sea lamprey haematopoiesis. Key words: Petromyzon marinus, haematopoiesis, HSC, evo-devo
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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.
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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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Lineage plasticity in normal and malignant lymphocyte precursors
Rezková Řezníčková, Leona
The classical scheme of hematopoiesis presumes early separation of lymphoid and myeloid precursors. Recently, more complex models are put forward, suggesting greater flexibility of hematopoiesis with progenitors sharing lymphoid and myeloid potential. Acute hybrid leukemia is a malignancy, in which it is not possible to assess unambiguously myeloid or lymphoid lineage of origin. The behaviour of those malignancies favors new models of hematopoiesis. Our work concentrated mainly on the research of childhood leukemias with lineage switch from lymphoid to myeloid lineage during induction treatment. Our task within this extensive project was to determine lineage assignment of leukemic blasts using detection of immunoglobulin and T-cell receptor gene rearrangements. We confirmed that myeloid cells derived during the treatment in all patients descend from the original lymphoid clone. We also investigated the expression of selected genes in those cases compared to common leukemia types. Lastly, we explored prognostic impact of TCR rearrangements (and thus lymphoid lineage commitment) in T-lineage leukemia.
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Comparing results of cell blood count and leukocyte differential count between different series of analyzers Sysmex
Opletalová, Monika ; Blažková, Hana (advisor) ; Bártů, Iva (referee)
In this bachelor thesis I focused on comparing results of cell blood count and leukocyte differential count between three types of Sysmex analyzers. XS - 1000 i, XT - 4000i and XE - 5000 are automatic analyzers, measuring on principles of flow cytometry and impedance methods using hydrodynamic focusation. These methods provide very accurate blood cell an alysis. The theoretical part of bachelor thesis is focused on description of hematopoesis, options for measuring cell blood counts and leukocyte differential counts and general description of measuring methods used by Sysmex analysers. In the experimental part I described the composition of mentioned analysers, their measuring channels and reagents which are used for peripheral blood cells analysis. Every sample from 50 patients was measured on each analyser and results were statistically compared by using ANOVA test. Graphs showing average values of each parameter for negative and positive patient ́s samples were made. The aim of this study was to summarize the main advantages and disadvantages of all three analyzers and determine whether differences in the results of samples between analyzers are statistically significant or not. Powered by TCPDF (www.tcpdf.org)
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Transplantace kostní dřeně příjemcům s regenerující krvetvorbou: účinnost transplantace a stav regenerující kostní dřeně
Forgáčová, Katarína ; Nečas, Emanuel (advisor) ; Vávrová, Jiřina (referee) ; Hofer, Michal (referee)
Hematopoietic stem cells (HSCs) have the ability of both self-renewal and differentiation. After bone marrow damage, surviving host HSCs or transplanted donor HSCs are able to restore hematopoiesis and maintain it for a long time due to the self-renewal potential. HSCs reside in a specific microenvironment in the bone marrow, in stem cell niche, which supports their survival and controls their functioning. In this study, we investigated the impact of bone marrow damage induced by increasing doses of irradiation on engraftment efficiency of transplanted donor repopulating cells. Using the CD45.1/CD45.2 congenic mouse model, we developed a new approach enabling estimation of surviving HSCs in damaged hematopoietic tissue. Its principle is in measuring of the donor chimerism resulting from transplantation of a defined dose of normal congenic bone marrow cells. The transplanted donor cells contain repopulating cells, progenitors (STRCs) and HSCs (LTRCs) that give rise to blood cell production which proceeds in parallel with that present in the host hematopoietic tissue. We applied this approach to monitor spontaneous regeneration of repopulating cells, including HSCs, in mice irradiated with a sublethal dose of 6 Gy or by a lethal dose of 9 Gy and rescued by syngenic bone marrow cells. This was...
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B- and T- lymphocyte subpopulations in lymphocyte-associated immunodeficiencies
Šinkorová, Vendula ; Kalina, Tomáš (advisor) ; Javorková, Eliška (referee)
The antigen-specific immunity consists of cells called T and B lymphocytes. These cells together with cells of non-specific (innate) immunity begin their development in fetal liver and later in bone marrow from the common progenitor, the hematopoietic stem cell. Both B and T lymphocyte lineages then undergo differentiation which is regulated by many cytokines and transcriptional factors and leads to very heterogeneous cohort of subsets. Because the immune system is not only protecting the organism from infections and malignant growth but also from itself, lymphocyte differentiation must pass many checkpoints where B and T clones are strictly selected. Cells of both lineages closely communicate with each other and also with cells of innate immunity. If, due to mutation of protein encoding genes, disturbance of differentiation or malfunction of effector activities providing some of these functions occurs, an immune system malfunction called immunodeficiency arises. Multiparametric immunophenotyping followed by flow cytometry examination has been proven one of the most suitable techniques for studying lymphocyte subsets and lymphocyte- associated immunodeficiencies. Here we describe examples of primary lymphocyte- associated immunodeficiencies, how they affect individual lymphocyte subsets, what it...
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