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The role of Kit ligands in hematopoiesis of Danio rerio
Oltová, Jana ; Bartůněk, Petr (advisor) ; Živný, Jan (referee) ; Divoký, Vladimír (referee)
Hematopoiesis is a precisely regulated process, dependent on the activity of hematopoietic cytokines and their receptors. Due to an extra round of whole genome duplication in teleost fish, two paralogs of many important genes, including some hematopoietic cytokines and their receptors, are present in the zebrafish (Danio rerio) genome. In this project, we have been investigating the role of zebrafish Kit ligands in hematopoiesis. Kit ligand is a pleiotropic cytokine, which is essential for vertebrate erythropoiesis; however, in zebrafish, no such role has been reported so far. To determine the function of zebrafish paralogs of Kit ligand (Kitlga and Kitlgb) in hematopoiesis, we performed in vivo and ex vivo gain- and loss-of-function experiments. Strikingly, we were the first to report the synergistic cooperation of zebrafish Kitlga with erythropoietin and dexamethasone, enabling the growth of kidney marrow-derived suspension cells and providing optimal conditions for the expansion of adult erythroid progenitors. We assume that by using different cytokine combinations, optimal conditions for the growth of other hematopoietic cell types can be established, and therefore, this new approach now available for the...
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Role of Rnf207b in zebrafish hematopoiesis
Vondráková, Zuzana ; Bartůněk, Petr (advisor) ; Živný, Jan (referee)
Hematopoiesis is the process of proliferation, differentiation and self-renewal of hematopoietic stem cells. Regulation of hematopoiesis is a complex process, which takes place on many different levels and is directed by many signals. RNF207 is one of the perspective genes chosen based on a screen in chicken model, where obtained data show its role in hematopoiesis. The aim of this work was to confirm the role of rnf207b as a new regulator of hematopoiesis in Danio rerio and to find out on which level of hematopoiesis is active. Danio rerio is an excellent model to study the function of genes in vivo, thanks to the easy manipulation of genetic expression and wide range of phenotypes during the development. To study the effect of rnf207b in hematopoiesis of Danio rerio we performed the knock-down of this gene by microinjection of morpholino oligonucleotides into one cell stage embryos. In these injected fish, we saw the effect in both thrombocyte and erythroid lineage, suggesting that rnf207b could be a regulator at the hierarchical level of progenitors or even more upstream. The results of developmental and tissue specific expression analysis then show that expression of rnf207b begins as early as 18 hpf, at the time of primitive hematopoiesis. Although rnf207b is expressed in the kidney (an...
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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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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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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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Application of Haematological Methods in Zoological Studies
Poplová, Jitka ; Vinkler, Michal (advisor) ; Krulová, Magdaléna (referee)
Haematological methods are widely used in zoological researches. Unfortunately, especially in ecological studies there is often much imperfection in techniques adopted, and misinterpretation of results is also common. This is particularly the case of methods serving to assessment of the peripheral blood cellular composition. In this thesis I therefore decided to describe basic haematological methods, their mechanisms, advantages and disadvantages. I also highlighted possible application potential in several less widely utilised haematological techniques. In the practical part of this thesis I have shown that the examination of immature erythrocyte differential count may serve as a meaningful indicator of health state in birds.
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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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Lineage plasticity in normal and malignant lymphocyte precursors
Rezková Řezníčková, Leona ; Froňková, Eva (advisor) ; Otáhal, Pavel (referee)
Klasické schéma vývoje hematopoetických buněk předpokládá časné oddělení lymfoidního a myeloidního prekurzoru. V poslední době jsou navrhovány složitější modely, které předpokládají větší flexibilitu hematopoezy a navrhují existenci progenitorů s lymfoidním i myeloidním potenciálem. Akutní hybridní leukémie jsou malignity, které podle různých kritérií nelze jednoznačně zařadit k lymfoidní nebo k myeloidní linii a jejichž chování spíše dává za pravdu novým modelům hematopoezy. Předkládaná práce se zabývala především výzkumem dětských leukémií s přesmykem z lymfoidní do myeloidní linie během indukční léčby. Jedná se o rozsáhlý projekt, v jehož rámci si diplomová práce si kladla za úkol určit liniové zařazení leukemických blastů pomocí detekce přestaveb genů pro imunoglobuliny a T-buněčné receptory (TCR). Potvrdili jsme, že myeloidní buňky derivované v průběhu léčby pochází u všech pacientů z původního lymfoidního klonu. Dále jsme u těchto případů zkoumali expresi vytipovaných genů ve srovnání s běžnými druhy leukémií. Třetí částí práce byl výzkum prognostického významu přítomnosti přestaveb TCR (a tedy příslušnosti k lymfoidní linii) u leukémií z T-lymfoidní řady.
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The role of HOXA9 gene in leukemogenesis
Rejlová, Kateřina ; Starková, Júlia (advisor) ; Fraiberk, Martin (referee)
The evolutionarily conserved family of homeobox genes plays an important role in the development of the anterior-posterior body axis of vertebrates. These genes significantly affect hematopoiesis, the development of blood cells. Extensive studies on homeobox genes in normal hematopoiesis confirmed their role also in leukemogenesis. Since the neoplastic transformation of blood cells, i.e. leukemia, is the most frequent malignancy in children, it has become a major subject of research for many scientists. Precisely in what stage of the malignant transformation the homeobox genes take part has not been shown yet. Neither is it known whether HOX genes are crucial in pathogenesis or whether their deregulation is only a side effect of leukemogenesis. The most studied homeobox gene in leukemogenesis is the HOXA9 gene, which showed correlation with the prognosis of patients with certain leukemias. Many studies describe the effect of HOXA9 in leukemic cell transformation, suggesting this gene could be a promising future target in leukemia therapy. This work is focused on the HOXA9 gene and its association with leukemic transformation of blood cells.
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