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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/EBPtranscriptionfactoris knownasoneofthe majorhematopoieticregulators,requiredforthefunctionalityof hematopoieticstemcellsaswellasformyeloidlineagedevelopment.Importantly,C/EBP expressionisalteredinalargeproportionofAMLcases.C/EBPregulateshematopoiesis mainlythroughorchestratingexpressionofitstargetgenes.ManyoftheC/EBPtargetshave previouslybeenshowntoplayaroleinthehematopoieticsystemandtobeinvolvedin leukemictransformation. That makesidentificationofnovel C/EBP targetsandtheir functionalcharacterizationanexcitingsubjectofresearch.Hereweidentifiedalistofgenes whoseexpressiondependsontheactivityofC/EBPthesocalledC/EBPsignature. We demonstratedthattreatment withhistonedeacetylase(HDAC)inhibitorsreactivatesthe expressionofthesegenesincellswithnon-functionalC/EBP.Inaddition,wedemonstrated thattreatmentwiththeHDACinhibitorspromotesmyeloiddifferentiationinAMLsamples carryingbi-allelicCEBPA mutationsandcharacterizedbythereducedexpressionofthe...
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Apoptosis Signating Pathways and Biological Effects of TGFB
Šimáková, Olga ; Fuchs, Ota (advisor) ; Brdička, Radim (referee) ; Holada, Karel (referee)
Apoptosis is necessary for maintaing the integrity of all alive multicellular organisms and therefore needs to be precisely regulated. Very important regulators of apoptosis are pleiotropic cytokines from TFGβ superfamily (e.g. TGFβ, BMP, aktivins), whose signals are transduced by SMAD proteins. Patients with secondary myelodysplasias and acute myeloid leukemias (MDS/AML) frequently exhibit interstitial deletions of the chromosome-5q resulting in hemizygous loss of the transcription transactivator SMAD5. SMAD5 is a member of the signal transducer family conveying the pleiotropic TGFβ/BMP cytokine signals with roles in development, cell growth control, and tumor progression. Consistent Smad5 gene expression in these cell types and the gradual increase in its mRNA and protein levels in a model of induced erythroid differentiation of murine erythroleukemia (MEL) cells suggest a role of the gene in hematopoiesis. We show that bone morphogenetic protein 4 (BMP4) directs Smad5 activation in human hematopoietic cells, as monitored at the levels of protein phosphorylation, nuclear translocation, and specific transcription response. In vitro induction of normal human CD34+ cells by BMP4 results in significantly increased proliferation of erythroid progenitors (BFU-E) and formation of glycophorin- A+ cells, whereas...
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The role of cereblon in lenalidomide therapy of del(5q) myelodysplastic syndrome
Bokorová, Radka ; Fuchs, Ota (advisor) ; Lukačková, Renata (referee) ; Krijt, Jan (referee)
Myelodysplastic syndrome (MDS) with deletion of the long arm of the chromosome 5 (5q - syndrome, del( 5q)) can be characterized by anemia, macrocytosis, a normal or high platelet count, and hypolobulated megakaryocytes in the bone marrow. 5q - syndrome belongs to low - risk MDS, which means low risk to transform to acute myeloid leukemia. 5q - syndrome is ass ociated with female predominance and older age. Another sign is transfusion burden that is treated by erythropoiesis - stimulating agents (ESA) as erythropoietin (EPO). Moreover, the response of MDS patients is around 30 - 60% with the median of the response b eing ~ 24 months. The second line of treatment is lenalidomide (LEN) which is a derivate of teratogenic analog thalidomide. LEN increases erythropoiesis and inhibits the growth of del(5q) erythroid progenitors in vivo and it does not have a significant effe ct on the growth of normal CD34+ progenitors or cytogenetically normal progenitors in MDS with del(5q) clones. LEN is used as therapy in multiple myeloma, myelodysplastic syndrome, and lymphoma. LEN is an expensive agent and not every MDS patient re sponds to this therapy. This is a reason why is a need to find a biomarker for the determination of successful treatment. Some multiple myeloma studies showed that cereblon can be the biomarker...
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The role of TGFß and study of prognostic factors of patients with MDS and AML
Provazníková, Dana ; Fuchs, Ota (advisor) ; Pohlreich, Petr (referee) ; Martásek, Pavel (referee)
We did not find mutation in coding areas of genes for components of TGFbeta1 signaling pathway but we detected decreased or undetectable expression of these analysed genes.The decreased expression is probably caused by epigenetic changes, so by hypermethylation and deacetylation of promoter regionsof these genes.Antiproliferative and apoptotic effect of TGF1 was analysed in AML cell lines (ML1, ML2, CTV1 and Kasumi1). ML2 cells rezistence to inhibition of DNA synthesis by TGFβ1 is not caused by mutations of genes for components of TGFβ1 signaling pathway. We found that increased SnoN (Ski-like novel gene) expression on the level of coresponding mRNA and protein is probably accountable for this rezistence. Kasumi1 and M2 cells were sensitive to induction of apoptózis caused by TGFβ1 treatment but in less extent than by proteazome inhibitor bortezomib. The difference of AML cells of different lines answers shows a great heterogeneity AML in AML patients. Prognostic factors analysis in AML with normal karyotype confirmed that CEBPA (CCAAT/enhancer binding protein alpha) mutations predict favourable prognosis but the elevated EVI1 ("Ecotropic Virus Integration Site 1") and ERG ("ETS-related gene") expression are connected with unfavourable prognosis. EVI1 is a negative marker for MDS as well. We did not confirm...
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Identification and characterisation of novel mechanisms regulating steady state and emergency granulopoiesis
Daněk, Petr ; Alberich Jorda, Meritxell (advisor) ; Fuchs, Ota (referee) ; Balounová, Jana (referee)
ABSTRACT Neutrophils are essential cells of the immune system. They engage in pathogen clearance, inflammatory response, and wound healing. Proper production and activation of neutrophils is critical for the health of an individual, since several disorders are related to neutrophilic alterations. In this thesis, we explore three previously uncharacterized mechanisms that might be involved in the regulation of neutrophilic differentiation. First, we addressed the role of the canonical Wnt signaling pathway. This signaling is executed by interaction of -catenin with TCF/LEF transcription factors. We employed a murine model that specifically inactivates -catenin-TCF/LEF-mediated transcription by expressing a dominant negative form of TCF4 (dnTCF4). Using this model in combination with several in vitro and in vivo assays we demonstrated that -catenin-TCF/LEF signaling directly upregulates expression of G-CSF receptor in hematopoietic progenitors, imposing myeloid commitment and favoring neutrophilic differentiation. This appeared to be especially important during the response to systemic infection, termed emergency granulopoiesis, as dnTCF4-expressing mice showed high susceptibility to Candida albicans infection. Remarkably, the critical role of -catenin-TCF/LEF signaling for neutrophil differentiation...
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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/EBPtranscriptionfactoris knownasoneofthe majorhematopoieticregulators,requiredforthefunctionalityof hematopoieticstemcellsaswellasformyeloidlineagedevelopment.Importantly,C/EBP expressionisalteredinalargeproportionofAMLcases.C/EBPregulateshematopoiesis mainlythroughorchestratingexpressionofitstargetgenes.ManyoftheC/EBPtargetshave previouslybeenshowntoplayaroleinthehematopoieticsystemandtobeinvolvedin leukemictransformation. That makesidentificationofnovel C/EBP targetsandtheir functionalcharacterizationanexcitingsubjectofresearch.Hereweidentifiedalistofgenes whoseexpressiondependsontheactivityofC/EBPthesocalledC/EBPsignature. We demonstratedthattreatment withhistonedeacetylase(HDAC)inhibitorsreactivatesthe expressionofthesegenesincellswithnon-functionalC/EBP.Inaddition,wedemonstrated thattreatmentwiththeHDACinhibitorspromotesmyeloiddifferentiationinAMLsamples carryingbi-allelicCEBPA mutationsandcharacterizedbythereducedexpressionofthe...
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The role of TGFß and study of prognostic factors of patients with MDS and AML
Provazníková, Dana ; Fuchs, Ota (advisor) ; Pohlreich, Petr (referee) ; Martásek, Pavel (referee)
We did not find mutation in coding areas of genes for components of TGFbeta1 signaling pathway but we detected decreased or undetectable expression of these analysed genes.The decreased expression is probably caused by epigenetic changes, so by hypermethylation and deacetylation of promoter regionsof these genes.Antiproliferative and apoptotic effect of TGF1 was analysed in AML cell lines (ML1, ML2, CTV1 and Kasumi1). ML2 cells rezistence to inhibition of DNA synthesis by TGFβ1 is not caused by mutations of genes for components of TGFβ1 signaling pathway. We found that increased SnoN (Ski-like novel gene) expression on the level of coresponding mRNA and protein is probably accountable for this rezistence. Kasumi1 and M2 cells were sensitive to induction of apoptózis caused by TGFβ1 treatment but in less extent than by proteazome inhibitor bortezomib. The difference of AML cells of different lines answers shows a great heterogeneity AML in AML patients. Prognostic factors analysis in AML with normal karyotype confirmed that CEBPA (CCAAT/enhancer binding protein alpha) mutations predict favourable prognosis but the elevated EVI1 ("Ecotropic Virus Integration Site 1") and ERG ("ETS-related gene") expression are connected with unfavourable prognosis. EVI1 is a negative marker for MDS as well. We did not confirm...
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Apoptosis Signating Pathways and Biological Effects of TGFB
Šimáková, Olga ; Fuchs, Ota (advisor) ; Brdička, Radim (referee) ; Holada, Karel (referee)
Apoptosis is necessary for maintaing the integrity of all alive multicellular organisms and therefore needs to be precisely regulated. Very important regulators of apoptosis are pleiotropic cytokines from TFGβ superfamily (e.g. TGFβ, BMP, aktivins), whose signals are transduced by SMAD proteins. Patients with secondary myelodysplasias and acute myeloid leukemias (MDS/AML) frequently exhibit interstitial deletions of the chromosome-5q resulting in hemizygous loss of the transcription transactivator SMAD5. SMAD5 is a member of the signal transducer family conveying the pleiotropic TGFβ/BMP cytokine signals with roles in development, cell growth control, and tumor progression. Consistent Smad5 gene expression in these cell types and the gradual increase in its mRNA and protein levels in a model of induced erythroid differentiation of murine erythroleukemia (MEL) cells suggest a role of the gene in hematopoiesis. We show that bone morphogenetic protein 4 (BMP4) directs Smad5 activation in human hematopoietic cells, as monitored at the levels of protein phosphorylation, nuclear translocation, and specific transcription response. In vitro induction of normal human CD34+ cells by BMP4 results in significantly increased proliferation of erythroid progenitors (BFU-E) and formation of glycophorin- A+ cells, whereas...
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