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1
Zetterberg, Eva. « Angiogenesis in myeloproliferative disorders / ». Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-383-3/.
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2
Shihab-El-Deen, Awatef. « Clonal development in myeloproliferative disorders ». Thesis, McGill University, 1985. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=72055.
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We assessed clonal development and extent of progression of hemopoietic malignancies (dysmyelopoietic syndrome (DMPS) and acute myelogenous leukemia) by examining in vitro growth patterns of their normal and leukemic progenitors. Additional phenotypic and cytogenetic analysis of an in vitro human myeloid leukemia model (HL-60) and its variant sublines were performed. These were aimed at determining cytogenetic abnormalities associated with phenotypic changes which accompany the derivation of these variant sublines. Our findings indicate that in vitro bone marrow cultures can be used clinically to rule out preleukemia, and that quantitations of bone marrow culture (CFU-C) can determine the potential for the development of acute leukemia in the DMPS patients. Acute leukemia developed in 48% of DMPS patients with a median transformation of 10 months.In acute leukemia, there was a preferential growth of normal karyotype in the in vitro cultures even among the phenotypically specified "blast" colonies.
Analysis of HL-60 variant sublines demonstrated the development of specific chromosomal abnormalities (1q+, iso8q, iso17q) in two cell lines (clones resistant to chemical induction) in association with loss of differentiation. These specific chromosomal abnormalities are known to be associated with tumor progression. The development of 1q+ abnormality was associated with loss of myeloperoxidase reaction and persistence of primary granules in that specific variant. A group of variant subclones was also associated with loss of differentiation, cytogenetically however, they demonstrated a revert to near diploid near normal karyotypes.
3
Singh, Rathna. « Genomic diversity in myeloproliferative neoplasms ». Thesis, The University of Sydney, 2014. http://hdl.handle.net/2123/12268.
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An exploratory study on the genetics of primary myelofibrosis was undertaken on the peripheral blood of 42 consecutive patients. Results of cytogenetic studies showed success rates with peripheral blood were superior to corresponding bone marrow analysis(P<0.001) and was preferred for ongoing patient monitoring. Several novel findings were detected in the study: A novel group of polyploid cases strongly associated with the gain of chromosome 1q was detected and showed a trend toward advancing disease. Further investigation of polyploid cases showed copy number gain around the pericentromeric region (P=0.02). One sample studied further showed unequal cell division in daughter nuclei, loss of chromosomal material from cells via micronuclei and failure of chromosome separation at mitosis. This provided some evidence of genetic instability and a pathogenic mechanism for the polyploidy. A subset of independent cases showed somatic mosaicism for copy number gain of the MAPT/KANSL1 locus on chromosome 17q21.31, involved commonly with neurodevelopmental defects. Other regions containing neurodevelopmental genes pertain to the NF1 locus on 17q11.2 suggestive of a role in aging and cancer development. The profile of DNA aberrations detected by SNP array confirmed similarities to t-MDS/AML and CN-LOH occurred commonly in regions associated with DNA repair. In addition massively parallel targeted sequencing complemented cytogenetic studies in detecting known mutations in MPN.
4
Macdonald, Donald Hugh Charles. « Chromosome 13 abnormalities in myeloproliferative diseases ». Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411308.
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Basu, Titiksha [Verfasser], et Heike L. [Akademischer Betreuer] Pahl. « Myeloproliferative neoplasms : cause, mechanism and treatment ». Freiburg : Universität, 2017. http://d-nb.info/1162443340/34.
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6
Jones, Amy Victoria. « The molecular pathogenesis of myeloproliferative neoplasms ». Thesis, University of Southampton, 2010. https://eprints.soton.ac.uk/162665/.
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Myeloproliferative neoplasms (MPNs) are a heterogeneous group of haematological stem cell malignancies characterised by proliferation of one or more cells of the myeloid lineage. The molecular investigation of MPN was revolutionized in 2005 by the finding that approximately 95% of cases with polycythaemia vera (PV) and 50-60% of cases of essential thrombocythaemia (ET) and primary myelofibrosis (PMF) are characterised by a single acquired mutation, JAK2 V617F. My study has focused on four principal areas: (i) Involvement of V617F in other myeloid disorders. After developing sensitive methods to detect and quantify V617F, this mutation was identified in 17% of cases of atypical chronic myeloid leukaemia (17/99) as well as other atypical MPN, thus demonstrating that it was more widely involved in myeloid disorders that initially thought. Homozygosity of V617F was shown to have arisen by acquired uniparental disomy (UPD) and examination of two cases with V617F plus either KIT D816V or BCR-ABL demonstrated that the mutations had arisen in independent clones. (ii) In vitro assays to predict imatinib sensitivity. Haemopoietic colony and liquid cultures were used to determine if peripheral blood or bone marrow cells from atypical MPN cases (n=200) were sensitive to imatinib. Of those that responded in one or both cultures (n=185) some had known abnormalities of PDGFRA or PDGFRB, but a significant minority proved negative for all molecular tests suggesting the presence of uncharacterised imatinib-sensitive mutations. (iii) V617F as a marker of response to therapy. JAK2 V617F was used as a molecular marker to monitor the response of PV patients (n=21) to therapy with imatinib and interferon-α. Neither therapy eradicated V617F but there was a modest reduction in %V617F which correlated with haematological response. By contrast, in those patients that did not respond (n=13) the %V617F marginally increased. (iv) Genetic predisposition to MPN. Whilst investigating the possible contribution of JAK2 single nucleotide polymorphisms to the phenotypic diversity associated with V617F, marked skewing of alleles associated with the mutation was observed. Further investigation revealed that V617Fassociated disease is strongly associated with a specific constitutional JAK2 haplotype, designated 46/1, in all three disease entities compared to healthy controls (PV, n=192, P=2.9x10-16; ET, n=78, P=8.2x10-9 and MF, n=41, P=8.0x10-5). Furthermore, allele-specific PCR demonstrated that V617F specifically arises on the 46/1 allele in most cases. The 46/1 JAK2 haplotype thus predisposes to the development of V617F associated MPNs (OR=3.7; 95% CI 3.1-4.3) and provides a model whereby a constitutional genetic factor is associated with an increased risk of acquiring a specific somatic mutation.
7
`Arnold, Claire. « Intracellular signalling pathways in myeloproliferative neoplasms ». Thesis, Queen's University Belfast, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.680884.
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8
Lynch, Susan Fraser. « Platelet and vascular studies in myeloproliferative disorders ». Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/24860.
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We have studied a cohort of patients with polycythaemia vera (PV) primary thrombocythaemia (PT) and primary myelofibrosis (PMF) and described their clinical and laboratory features in comparison to other published observations. The demographic, haematological and molecular characteristics of our cohort were similar to other retrospective analyses, but the occurrence of thrombo-haemorrhagic complications was lower. The presence of vascular abnormalities in these patients was investigated using both established markers and an assay was devised to measure platelet, endothelial, leucocytes and red cell microparticles in platelet poor plasma using flow cytometry techniques. This assay was optimised for pre-analytical variables, the most important of which was found to be sample centrifugation. In keeping with previous studies, increased platelet activation was observed in PV and PT patients compared to healthy controls using both established markers and as evidenced by increased numbers of platelet microparticles. There was no evidence of endothelial disturbance using the soluble endothelial marker E-selectin but we did observe elevated endothelial microparticles in patients compared to controls. Microparticles may therefore be useful as a marker of vascular abnormalities in these disorders and in view of their prothrombotic properties may be an additional pathogenic mechanism in the prothrombotic state and a potential therapeutic target. In relation to bone marrow fibrosis, plasma levels of platelet α-granule contents, including the pro-fibrotic cytokine transforming growth factor β (TGFβ), were studied. We observed elevated levels of TGFβ in patients compared to controls, with the highest levels in patients with PMF and in those with PT or PV who had more marked fibrosis. Further, levels of TGFβ were strongly associated with another α-granule protein beta-thromboglobulin, suggesting that platelet α-granules may be an important source for this pro-fibrotic cytokine.
9
Boyd, M. T. « Detection of retroviral indicators in myeloproliferative diseases ». Thesis, University of Southampton, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.234379.
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10
Vassiliou, George Steliou. « The molecular pathogenesis of the myeloproliferative disorders ». Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614681.
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11
Godfrey, Anna Louise. « Mechanisms determining phenotype in JAK2-mutated myeloproliferative neoplasms ». Thesis, University of Cambridge, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708184.
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Sohal, Jastinder. « The molecular analysis of the BP11 myeloproliferative syndrome ». Thesis, Imperial College London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.395537.
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13
Beer, Philip. « The human myeloproliferative disorders : molecular pathogenesis and clonal heterogeneity ». Thesis, University of Cambridge, 2009. https://www.repository.cam.ac.uk/handle/1810/226756.
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The classical myeloproliferative disorders (MPD), comprising essential thrombocythaemia (ET), polycythaemia vera (PV) and idiopathic myelofibrosis (IMF), are clonal premalignant haematopoietic neoplasms associated with activating mutations in signalling pathway molecules and a variable tendency to develop acute myeloid leukaemia (AML). This thesis examined genotype-phenotype associations of JAK2 and MPL mutations, the presence of clonal diversity in the MPD and the genetic events associated with progressive disease. Mutations in MPL were identified in 4% of ET and 7% of IMF but not in PV. Three different acquired MPL mutations were identified, one of which had been reported as an inherited allele. Although MPL mutations did not delineate a distinct clinical or histopathological subtype of ET, molecular testing provides an important new tool in the diagnostic armamentarium. Clones homozygous for the JAK2 V617F mutation were identified in female but not male patients with ET, suggesting that gender differences may be important in the determination of disease phenotype. In patients with two acquired genetic alterations, a signalling pathway mutation and a cytogenetic abnormality were usually present within the same clone. By contrast, coexistence of two signalling pathway mutations indicated the presence of biclonal disease that in two patients had arisen independently and not from a shared founder clone. RAS mutations were identified as potential cooperating events in patients with JAK2 or MPL mutant IMF. In patients developing AML following a JAK2 V617F-positive MPD, those with V617F-positive leukaemia had progressed via an accelerated phase of disease and harboured acquired alterations of RUNX1 or EVI1. V617F-negative leukaemias tended to follow directly from ET or PV, and loss of the JAK2 mutation by reversion to wild-type due to mitotic recombination, gene deletion or gene conversion was excluded. The thesis concludes with a discussion of how clonal heterogeneity can be integrated into current models of MPD disease pathogenesis.
14
Vaughan, Beverley Rebecca. « A molecular cytogenetic profile of the chronic myeloproliferative disorders ». Thesis, De Montfort University, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485624.
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The Chronic Myeloproliferative Disorders (CMPD) form a continuum of malignancies, the molecular pathogenesis of which remains obscure. Chronic Myeloid Leukaemia (CML) is the only disorder with a recognisable pathogenomic abnormality, the Ph chromosome. The remaining disorders have as yet, no defined disease markers although up to 30% display recurrent cytogenetic aberrations. Historically, conventional cytogenetic methods have relied on culturing of bone marrow (BM) aspirates to obtain suitable and sufficient mitotic figures for G-banded analysis. CMPD samples routinely have increased failure rates due to reduced growth and poor chromosome morphology. However, the application of Growth Factor (GF) stimulants to short term (24-48hr) BM cultures significantly improved both the quality and quantity of metaphases. BM samples were stimulated with three GF combinations namely, Bone Marrow Growth Supplement (BMGS), Myeloid Expansion Medium (MEM) and Conditioned Supernatant (CS) from human bladder carcinoma cell line 5637{ To assess whether application ofGF stimulants leads to clonal selection, cultured samples from 15 patients were analysed by Fluorescence In situ Hybridisation (FISH), which supported the theory that clonal selection remained unaltered in stimulated samples. In addition to this, immunophenotyping ofcells demonstrated that the lineage ofpropagated cells remained unaltered. By applying these improved techniques, CMPD samples and immortalised cell lines were then investigated for cryptic aberrations. Array comparative genomic hybridisation (CGH) investigations performed on CML cell lines revealed a complex pattern of imbalances affecting the short arm of chromosome 9, on the background of a 2-3 fold amplification. To clarify this observation a series of Bacterial Artificial Chromosome (BAC) Deoxyribonucleic Acid (DNA) probes sequenced to regions along 9p were selected. These probes were then applied to CMPD patient samples with both normal and abnormal karyotpyes, focusing on the region containing Janus Kinase 2 Gene (JAK2) at 9p24.1. In this ongoing study 18% of patients have been shown to carry a relative loss in this region. The pathogenomic significance of imbalances involving 9p remains unclear. It may however, represent an acquired homozygousity as a consequence ofmitotic recombination, with a 'second hit' removing a wild type allele, leading on to further, as yet, un-clarified but clinically significant changes.
15
Campbell, P. J. « The molecular pathogenesis and management of the myeloproliferative disorders ». Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597260.
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The myeloproliferative disorders (MPDs) are clonal haematological malignancies comprising three main entities: polycythaemia vera (PV), essential thrombocythaemia (ET) and idiopathic myelofibrosis (IMF). The disorders are characterised by overactive haematopoiesis with a propensity to thrombosis, haemorrhage and leukaemic transformation. One chapter of this thesis covers the PT-1 trial, the largest randomised clinical trial in the MPDs. Patients with ET at high risk of thrombosis were randomised between hydroxyurea + aspirin and anagrelide + aspirin. The primary end-point of arterial or venous thrombosis, major haemorrhage or vascular death was increased in the anagrelide arm. The three secondary end-points of arterial thrombosis, major haemorrhage and myelofibrotic transformation were more common in the anagrelide arm, whereas venous thrombosis was more frequent in patients on hydroxyurea. Three chapters cover the molecular pathogenesis of the MPDs. Firstly, two balanced chromosomal translocations in patients with ET are characterised. The breakpoints of a t(X;5) translocation were shown to lie close to the X inactivation centre on the X chromosome and the common deleted region for the 5q-syndrome on chromosome 5. The inactive X chromosome was translocated, leading to monoallelic expression of genes in the 5q-region. This suggests spread of X inactivation as a novel mechanism of oncogenesis. A t(1;22) translocation was also characterised and shown to lead to synthesis of a Rho guanine nucleotide exchange factor with aberrant N terminus. The other two chapters describe the JAK2 V617F mutation. With an appropriately sensitive method for its detection, the mutation is found in virtually all patients with PV, and about half those with ET and IMF, 776 DNA samples from patients in the PT-1 trials were tested for the mutation, and clinical and laboratory findings compared. Compared to those without the mutation, V617F-positive patients with ET had higher haemoglobin levels and white cell counts, greater marrow cellularity, more venous thromboses, greater sensitivity to hydroxyurea (but not anagrelide) and lower erythropoietin levels and iron stores.
16
Fourouclas, Nasios. « The investigation of the molecular pathogenesis of myeloproliferative disorders ». Thesis, Anglia Ruskin University, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.440250.
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17
Hidalgo-Curtis, Claire. « Abnormalities affecting tyrosine kinase signalling in atypical myeloproliferative disorders ». Thesis, University of Southampton, 2009. https://eprints.soton.ac.uk/72798/.
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The myeloproliferative disorders (MPDs) are a group of haematopoietic stem cell diseases, characterised by proliferation of one or more cells of the myeloid lineage. Several lines of evidence have highlighted the importance of aberrant tyrosine kinase signalling in the pathogenesis of these disorders. Cloning of rare chromosomal translocations and point mutation analysis in the MPDs has identified diverse deregulated tyrosine kinase genes, notably PDGFRA, PDGFRB, FGFR1 and JAK2. However the majority of atypical MPDs still remain to be characterised and identification of patients harbouring fusions, particularly those involving the PDGF receptors is of increasing importance, as they are likely to be responsive to targeted therapy with imatinib. I am investigating MPD patients for abnormalities affecting tyrosine kinase signalling, and have used three approaches, translocation cloning, expression analysis and SNP array analysis to detect regions of loss of heterozygosity (LOH). Thus far, by translocation cloning I have identified a previously undescribed partner gene fused to PDGFRB and two new PDGFRA fusion genes. I have also designed two reverse transcriptase PCR (RT-PCR) assays and a cDNA MLPA assay to detect over-expression of specific tyrosine kinases screening approximately 200 patients. Each assay identified all patients previously diagnosed with known fusions. Additionally, two patients identified with overexpression of PDGFRB have been found to have cryptic ETV6-PDGFRB fusions and overexpression of PDGFRA in one patient lead to the discovery of a previously undescribed fusion involving a novel partner gene (KIF5B). Recent evidence has indicated that acquired isodisomy is a novel mechanism by which mutations in cancer may be reduced to homozygosity. Typically, acquired isodisomy is associated with oncogenic changes rather than tumour suppressor genes, eg. the activating JAK2 V617F mutation and 9p aUPD. I have undertaken a screen using Affymetrix 50K SNP arrays for regions of acquired isodisomy as a means to identify genomic regions that may harbour novel oncogenes in different subgroups of MPD patients. Large tracts of homozygosity (defined as >20Mb running to a telomere), strongly suggesting acquired isodisomy, were seen in 40% aMPD patients. The homozygous tracts encompassed diverse genomic regions in aMPD, but two common regions (3 cases for each region) were identified at 7q and 11q. Mutations in the CBL ubiquitin ligase gene were discovered in all three aCML patients with 11q aUPD as well as in an additional 23 MPD patients following further screening.
18
CASETTI, ILARIA CAROLA. « Genomic profiling and genotype-phenotype correlations in myeloproliferative neoplasms ». Doctoral thesis, Università degli studi di Pavia, 2021. http://hdl.handle.net/11571/1448465.
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Myeloproliferative neoplasms (MPNs) are disorders of the stem cell, due to acquired mutations causing a clonal proliferation of one or more hemopoietic progenitor in the bone marrow. According to the World Health Organization (WHO) 2016 classification of myeloid neoplasms, Ph-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and prefibrotic myelofibrosis (prePMF). A somatic mutation in JAK2, CALR or MPL genes is found in the great majority of patients with MPNs. These driver mutations constitutively activate the JAK/STAT pathway, resulting in increased phosphorylation of its substrates and leading to increased cytokine responsiveness of myeloid cells. MPNs may progress to more aggressive diseases. This evolution is typically associated with the acquisition of somatic variants in genes involved in different pathways, including DNA methylation and regulation of chromatin structure, transcriptional regulators, signaling pathway and splicing factors. The phenotype of MPNs seems to be related to the combination of driver and subclonal variants and their order of acquisition but the molecular and clinical correlations are not yet completely understood.
This work is a genotype-phenotype study aimed to correlate biological and clinical features in a cohort of 509 MPN patients, diagnosed with ET, prePMF and PMF at the UOC Ematologia, Fondazione IRCCS Policlinico San Matteo, between 1985 and 2019. DNA sequence variants were studied through a NGS approach using the Illumina Nextera Rapid Capture Custom Enrichment Kit and HiSeq2500 platform. The panel targeted the coding sequence of 81 genes known to be involved in myeloid neoplasms.
Overall, 589 additional somatic variants were detected. Compared to ET and prePMF, PMF showed a larger proportion of patients carrying at least one additional somatic variant, a higher average number of variants per patient and a greater involvement of high molecular risk genes. ET, prePMF and PMF showed different mutational landscapes: in ET the most commonly involved pathway was DNA methylation genes, while in prePMF RNA splicing genes were often affected, together with DNA methylation; in PMF chromatin structure, DNA methylation and RNA splicing were the most recurrent mutated pathways. This finding suggests that prePMF and PMF share molecular features with MDS, since RNA splicing is often involved in MDS development. No significative association between driver mutation and additional variants was found, except for mutations in the spliceosome genes, which did not occur in CALR- mutated patients. The correlations of the additional variants with the clinical picture highlighted a negative impact of high-risk genes on OS and on the progression of the disease.
In conclusion, these data suggest that not only clinical and histopathological criteria, but also distinct mutation patterns might differentiate ET, prePMF and PMF. In the era of precision medicine, an accurate diagnosis and prognostic stratification will be useful in the choice of a proper treatment for each patient.
19
Champion-Suntharalingam, K. M. « Aspects of molecular analysis in myeloproliferative disorders and myelodysplastic syndromes ». Thesis, Anglia Ruskin University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342919.
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20
White, Helen Elizabeth. « Detection of erythropoietin receptor mutations in patients with myeloproliferative disorders ». Thesis, University of Southampton, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242216.
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Cheung, Manyee. « Investigation of megakaryocytes from normal and myeloproliferative bone marrow biopsies ». Thesis, University of Southampton, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343012.
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22
Baxter, E. Joanna. « Molecular characterisation of receptor tyrosine kinases in chronic myeloproliferative disorders ». Thesis, University of Southampton, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400503.
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23
Sangkhae, Veena. « The role of thrombopoietin signalling in JAK2V617F-positive myeloproliferative neoplasms ». Thesis, University of York, 2015. http://etheses.whiterose.ac.uk/9669/.
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Thrombopoietin (TPO) is the primary regulator of megakaryocyte development, regulating proliferation and differentiation in addition to the number of circulating platelets through binding to and stimulation of the cell surface receptor MPL. Activating mutations in MPL constitutively stimulate downstream signalling pathways, leading to aberrant haematopoiesis and contribute to development of myeloproliferative neoplasms (MPNs). Several studies have mapped the tyrosine residues within the cytoplasmic domain of MPL that mediate these cellular signals; however, secondary signalling pathways are incompletely understood. Additionally, the identification of the JAK2V617F mutation has profoundly increased our understanding of MPNs and although a role has been implicated in vitro, the in vivo role of MPL in JAK2V617F-positive MPNs has yet to be determined. In this thesis, a novel signalling pathway for the negative regulation of TPO signalling was identified whereby MPLY591 is phosphorylated resulting in association of SYK which negatively regulates TPO-mediated ERK1/2 signalling. Additionally, genetic manipulation of an in vivo JAK2V617F-positive MPN mouse model led to the identification of MPL as an essential molecular component for development of JAK2V617F-postive MPNs. In the absence or reduction of MPL, the disease fails to develop. However, removal of the cytokine, TPO, was unable to prevent the disease from developing. These findings provide novel insights not only into regulation of TPO-signalling but also the role of TPO and MPL in JAK2V617F-positive MPN disease pathogenesis. Identification of the role of MPL in MPN pathogenesis, as well as insights into additional regulatory pathways, contributes to our understanding of normal and pathological TPO signalling. These new insights also provide a basis for development of novel therapeutics for the treatment of MPNs and other diseases resulting from aberrant of TPO signalling.
24
Leung, Kin-sang, et 梁建生. « A rapid molecular testing system for differential diagnosis of myeloproliferative neoplasms ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48334145.
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Myeloproliferative neoplasms include a heterogeneous group of stem cell disorders with overproduction of myeloid cells. They have very different clinical courses and prognosis and are amenable to specific targeted therapy. A prompt and accurate diagnosis is therefore very important. Genetic characterisation plays an important role in diagnosis and classification of these disorders. BCR-ABL1fusion gene and JAK2V617F mutation are the particular major molecular markers to be detected because of availability of targeted therapy.
In this study, a new molecular testing system was developed for the differential diagnosis of myeloproliferative neoplasms. A multiplex reverse-transcriptase polymerase chain reaction was developed for fast detection of JAK2 V617F mutation and BCR-ABL1fusion simultaneously. It was demonstrated to be fast and highly sensitive and specific for the mutations as validated by analysis of clinical samples. The sensitivity limit was well suited for clinical diagnosis. There was great potential saving in consumables and manpower with a much shortened turn-around-time in most cases when compared to conventional diagnostic protocol.published_or_final_version
Pathology
Master
Master of Medical Sciences
25
Johan, Muhammad Farid. « Pathogenesis of core binding factor in acute and chronic myeloproliferative disorders ». Thesis, University of Sheffield, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.490188.
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Pathogenesis of leukaemia is a multistep process that involves multiple genetic alterations of normal cells to transform into a malignant phenotype. Mutations in the receptor tyrosine kinase (RTK/RAS) signalling pathway frequently provide a proliferative signal particularly the core binding factors leukaemia. In addition, DNA methylation may playa major role in the pathogenesis of myeloid malignancies where aberrant methylation of the promoter region is a major mechanism for silencing of tumour suppressor genes in cancers. In this study, the role of downstream effectors of RTK/RAS signalling; NRAS, KRAS and PTPNll, two RTKs; PDGFRA and PDGFRB and effectors of JAK/STAT pathway; JAK2 has been investigated to determine if point mutation are involved in pathogenesis of myeloid leukaemia. The role of negative regulators of RTK/RAS signalling; RASSF1A, SHPl and SaCSl was also investigated to determine if aberrant promoter methylation are involved in the pathogenesis of myeloid leukaemia. A total of 326 DNAs from AML, CMML, MDS and JMML patients were screened for mutations . and DNA promoter methylation in these genes. The relative expression of SaCS1 was also analysed using RQ-PCR to confirm the role of saCSl methylation in haematopoietic cell lines. NRAS mutation was found in 20%(8/40) inv(16) AML and 19%(4/21) of t(8;21). KRAS mutation was detected in 3%(1/40) inv(16) AML. PTPNll mutation was detected in 1/67 AML and 1/5 JMML. 1/50 CMML patients possessed a JAK2 p.VaI617Phe. Aberrant RASSF1A methylation was found in 9%(5/55) MDS and in 1/5 JMML. Aberrant SHPl methylation was present in 11%(13/121) AML. SaCSl 'promoter' methylation was present in 11%(8/74) MDS patients. SaCSl 'exon 2' methylation was found in 40%(19/47) AML and is associated with the transcriptional silencing of SaCSl gene in haematopoietic cell lines. Interestingly, 68% of patients with AML arid inv(16) possessed either NRAS or KRAS or RTK mutation (KIT or FLT3) or SHPl aberrant methylation. 92% of class I mutations in AML and inv(16) were not associated with one another, thus suggesting the mutual exclusivity of RTK/RAS signalling pathway mutations. The findings lead to support the two-hit model of leukaemogenesis and moreover for the possibility of targeted therapy for patients with CBF AML especially inv(16).
26
Cashman, Johanne. « Analysis of hematopoietic progenitor cell cycle control in the myeloproliferative disorders ». Thesis, University of British Columbia, 1986. http://hdl.handle.net/2429/27036.
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The myeloproliferative disorders (MPD) comprise an interesting group of hematological neoplasms in which clonal expansion is initiated at the level of the pluripotent stem cell compartment but differentiation proceeds essentially normally. Although evidence for the involvement of specific genetic changes exist in one of these diseases (chronic myeloid leukemia, CML), the nature of the lesion that permits the progeny of a single stem cell to dominate the mature cell compartment has not been elucidated. Application of clonal assay systems to the study of the MPD has provided information about the numbers, proliferative capacity, physical properties and the responsiveness to regulatory factors of hemopoietic progenitors from all cell lineages. However, clonal assays can offer only limited information about the processes that underly stem cell regulation. Some of the limitations imposed by these assays may be overcome by the use of long-term cultures in which primitive and pluripotent progenitors may be maintained for at least 2 months.
The purpose of this thesis was to determine if consistent alterations in cell cycle activity were characteristic of progenitors in MPD patients, and to evaluate the potential of the long-term culture system for further investigations of any changes observed. The proliferative behaviour of clonogenic progenitors from the blood and bone marrow of a large number of MPD patients was compared with that of normal individuals using the ³H-thymidine cell suicide technique. These experiments showed that all progenitor classes in the blood and marrow of patients with CML and polycythemia vera (PV), which in normal individuals are quiescent, had a significant component of cycling cells. In addition, a consistent association of this abnormality in cycling control with expression of erythropoietin (EP)-independence in patients with essential thrombocytosis (ET) was revealed. Further studies were then undertaken to determine if these abnormalites could be reproduced in vitro. Experiments with normal marrow showed that the most primitive progenitor classes located in the adherent fraction of standard long-term cultures undergo cyclic changes of proliferative activity with each weekly addition of new growth medium. These studies suggested that the proliferative activity of normal primitive hemopoietic cells may be both positively and negatively regulated by close range interactions with marrow stromal elements. In contrast, in similar experiments with long-term cultures established with PV marrow, where maintenance of neoplastic cells could be documented, analogous primitive progenitor cells in the adherent layer failed to return to a quiescent state and remained continuously in cycle.
From previous experiments with CML patients, it was already known that Ph¹ -positive progenitors usually disappeared rapidly in long-term cultures established with CML marrow. Therefore, as an alternative approach CML peripheral blood cells were seeded onto preestablished normal marrow adherent layers, since preliminary studies had suggested that this would allow sufficient numbers of primitive Ph¹-positive progenitors to be maintained for cycling studies. Analysis of such cultures together with studies of appropriate normal controls, revealed the same lack of cycling regulation in CML as previously shown for PV. In addition, studies of control cultures showed that in the absence of an adherent layer, normal peripheral blood progenitors cycle continuously, again suggesting that one regulatory function of the adherent layer is to maintain normal progenitors in a quiescent state.
These studies demonstrate that consistent abnormalities of cell cycle control characterize the primitive progenitor compartments in MPD patients, and that these abnormalities can be reproduced in vitro. Initial findings with the long-term marrow system suggest that these abnormalities may be due to the in-sensitivity of primitive neoplastic cell types to respond to factors that arrest their normal counterparts from further progression through the cell cycle.Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
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Wood, Andrew David. « The role of JAK2-STAT5 signalling in the human myeloproliferative neoplasms ». Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611834.
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Morlan-Mairal, M. « New molecular and cellular aspects of mutant calreticulin in myeloproliferative neoplasms ». Thesis, University of Salford, 2018. http://usir.salford.ac.uk/47300/.
Texte intégralRésumé :
Calreticulin (CALR) is an endoplasmic reticulum (ER) protein that plays an important role as a calcium (Ca2+) buffering chaperone. Mutations in CALR exon 9 have been identified in essential thrombocythemia and primary myelofibrosis, two myeloproliferative neoplasms (MPNs) characterised by megakaryocyte hyperplasia. Despite the large body of research built around CALR mutations, many aspects of the oncogenic mechanisms of CALR in MPNs remain unanswered. This investigation aims to investigate whether CALR mutations affect the nature of the C-terminal domain of this protein, its sub-cellular compartmentalisation and its Ca2+ buffering activity during megakaryocyte hyperplasia. Additionally, this study establishes a new cellular model to investigate megakaryocyte differentiation in presence of CALR mutations. In silico analysis of the structural characteristics of CALR mutant C-terminal domain revealed that CALR mutations lead to changes in its secondary structure, its protein binding properties and changes the acidity of CALR mutant ́s C-terminal domain. These physical alterations could affect CALR cellular behaviour by leading to inefficient ER Ca2+ buffering activity and lead to a novel oncogenic network of protein interactions. This study revealed that MARIMO leukemic cell line, which harbours a CALR mutation, has in vitro megakaryocyte differentiation potential. Importantly, this discovery was useful for further studies aiming to analyse CALR mutant cells during megakaryocyte commitment. Moreover, study of CALR mutant cellular localisation showed that this protein is localised within the ER, but it is also mislocalised within the cytoplasm and cell membrane, where it co-localised with thrombopoietin receptor. Interestingly, CALR cell surface expression increased during megakaryocyte commitment in CALR mutant cells, showing a dynamic process of CALR compartmentalisation during megakaryocyte differentiation. One of the more significant findings shown in this study is the emergence of intracellular Ca2+ concentrations ([Ca2+I]) as an important element during megakaryopoiesis. Importantly, CALR mutations impaired the cellular ER Ca2+ buffering activity and led to changes in the [Ca2+I] during the process of megakaryocyte differentiation. In addition, initial experimentsrevealed that physical manipulation of [Ca2+I] leads to the emergence of a megakaryocyte phenotype in leukemic cells, showing the relevance of this factor during megakaryocyte commitment. All together, these findings elucidate novel effects of CALR mutations into the physical and functional characteristics of CALR mutant in MPNs, describing new aspects of this driver mutation during the oncogenesis of these diseases. Finally, the current data highlight the importance of studying the effects of CALR mutations during the process of megakaryocyte differentiation, as CALR mutant sub-cellular compartmentalisation and ER Ca2+ buffering activity variate during the process of megakaryopoiesis.
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Hookham, Michelle Bernadette. « The myeloproliferative disorder-associated JAK2V617F mutant escapes negative regulation by SOCS-3 ». Thesis, Queen's University Belfast, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491953.
Texte intégralRésumé :
The somatic JAK2 valine-to-phenylalanine (V617F) mutation has been detected in more than 90% of patients with polycythaemia vera (PV) and in a sizeable proportion of patients with other myeloproliferative disorders such as essential thrombocythaemia (ET) and idiopathic myelofibrosis (IMF). Suppressor of cytokine signalling 3 (SOCS-3) is known to be a strong negative regulator of erythropoietin (EPO) signalling through interaction with both the EPO receptor (EPOR) and JAK2. We report that JAK2V617F phosphorylation can be actually potentiated in the presence of SOCS-3. Instead of acting as a suppressor, SOCS-3 enhanced the proliferation of cells expressing both JAK2V617F and EPOR. Additionally, turnover of SOCS-3 was inhibited by the JAK2 mutant kinase and this correlated with marked tyrosine phosphorylation and stabilization ofSOCS-3 protein. We also observed constitutive tyrosine phosphorylation of SOCS-3 in granulocytes and peripheral blood mononuclear cells (PBMCs) derived from patients homozygous for the JAK2V617F mutation. We also discovered that other recently identified MPD-associated mutations were also able to hyper-phosphorylate SOCS-3. These findings suggest that JAK2V617F and the other MPD-associated JAK2 mutations have overcome normal regulation by aberrantly phosphorylating SOCS-3, rendering it unable to inhibit these mutant kinases.
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Griner, Lori Nicole. « The Mevalonate Pathway : A Potential Therapeutic Target for JAK2-driven Myeloproliferative Neoplasms ». Scholar Commons, 2013. http://scholarcommons.usf.edu/etd/4497.
Texte intégralRésumé :
The Mevalonate Pathway: A Potential Therapeutic Target for JAK2-driven Myeloproliferative Neoplasms
Lori Nicole Griner
Abstract
Myeloproliferative neoplasms (MPNs) are diseases of hematopoietic stem cell origin and are characterized by uncontrolled growth of cells of the myeloid compartment. The Philadelphia chromosome negative classical MPNs, including polycythemia vera, essential thrombocythemia, and myelofibrosis, are diseases of dysregulated JAK2 signaling. In fact, the majority of MPN patients have activating mutations in JAK2 (e.g JAK2-V617F), a tyrosine kinase that contributes to the growth and survival of myeloid cells. While MPNs were first described over sixty years ago, a significant need remains to develop therapeutic strategies for them. Inhibitors of JAK2 are currently being developed, and one inhibitor, ruxolitinib, was recently approved for certain MPN patients. Ruxolitinib has made profound impacts on improving splenomegaly and constitutional symptoms in MPN patients, but it and other JAK2 inhibitors have not significantly reduced the JAK2 mutant allele burden, and thus such inhibitors have not induced remission in these patients. The current consensus in the MPN field supports JAK inhibition for the treatment of patients, but a further understanding of MPNs and JAK2 signaling, as well as improved JAK2 inhibitors, may be necessary for treating MPN patients.
The work described in this dissertation has uncovered novel requirements for JAK2-V617F-driven signaling and transformation. We demonstrate that JAK2-V617F co-localizes with lipid rafts, cholesterol-rich microdomains within the plasma membrane that function to serve as platforms for signaling complex formation. Signaling complex formation is a necessary component for dysregulated signaling induced by JAK2-V617F. We provide evidence that cholesterol altering-lipid raft disrupting agents attenuate JAK2-V617F-driven signaling. We also show that cholesterol-lowering statins are effective at downregulating JAK2 signaling and inducing apoptosis in JAK2-V617F-driven cell lines. Importantly, we show that statins, inhibitors of the mevalonate pathway, inhibit the growth of primary MPN cells, while the same statin doses have no effect on healthy controls. Impressively, we demonstrate that statins cooperate with multiple JAK inhibitors, including ruxolitinib, to inhibit cell growth and induce apoptosis of JAK2-V617F-driven cells.
This report establishes statin-mediated inhibition of the mevalonate pathway as a potential approach to improve MPN therapeutics. We propose future studies with statins and JAK2 inhibitors in the treatment of MPNs.
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Schoenwandt, Elias [Verfasser], Heike L. [Akademischer Betreuer] Pahl et Jonas Samuel [Akademischer Betreuer] Jutzi. « The role of histone demathylase JMJD1C in the pathophysiology of myeloproliferative neoplasms ». Freiburg : Universität, 2019. http://d-nb.info/1223849279/34.
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AMARU, CALZADA ARIEL. « Mechanism of action of Histone Deacetylase inhibitor. Givinostat in Chronic Myeloproliferative neplasm ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2013. http://hdl.handle.net/10281/44363.
Texte intégralRésumé :
We investigated the mechanism of action of the histone deacetylase inhibitor Givinostat (GVS) in Janus kinase 2 (JAK2)V617F myeloproliferative neoplasm (MPN) cells. GVS inhibited colony formation and proliferation and induced apoptosis at doses two- to threefold lower in a panel of JAK2V617F MPN compared to JAK2 wild- type myeloid leukemia cell lines. By global gene expression analysis, we observed that GVS modulated 293 common genes in the JAK2V617F cell lines HEL and UKE1. In particular, the hematopoietic transcription factors NF-E2 and C-MYB were downmodulated by the drug specifically in JAK2V617F cells at both the RNA and protein level. GVS had a direct effect on the NF-E2 promoters, as demonstrated by specific enrichment of associated histone H3 acetylated at lysine 9. Modulation by GVS of NF-E2 was also observed in freshly isolated CD34+ cells from MPN patients, and was accompanied by inhibition of their proliferation and differentiation toward the erythroid lineage. We conclude that GVS acts on MPN cells through dual JAK2-STAT 5-extracellular signal- regulated kinase 1/2 inhibition and downmodulation of NF-E2 and C-MYB transcription. In the second part of the thesis we investigated whether clinically achievable concentrations of the histone deacetylase (HDAC) inhibitors givinostat and hydroxyurea induce synergistic cytotoxicity in Jak2V617F cells in vitro and through which possible mechanism. The results suggest that combined treatment with givinostat and hydroxyurea is a potential strategy for the management of Jak2V617F myeloproliferative neoplasms.
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Edelmann, Anja. « Meilensteine in der Verlaufskontrolle von Patienten mit JAK2 p.V617F positiver myeloproliferativer Neoplasie nach Stammzelltransplantation ». Doctoral thesis, Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-144369.
Texte intégralRésumé :
Das Ziel der vorliegenden Arbeit war die Quantifizierung JAK2 p.V617F mutierter Allele zur Verlaufskontrolle von Patienten mit JAK2 p.V617F positiven MPN nach allogener Stammzelltransplantation (SCT). Dabei sollte insbesondere untersucht werden, ob sich frühzeitig nach SCT ein höheres Rezidivrisiko der MPN vorhersagen lässt und zu welchen Zeitpunkten molekulare Untersuchungen nach SCT sinnvoll sind.
Wir analysierten retrospektiv den Krankheitsverlauf von 30 Patienten. Dafür verwendeten wir die ARMS-QPCR und WTB-AS QPCR als zwei allel-spezifische Amplifikationsmethoden und untersuchten 142 Proben der ersten Kohorte (n=14) und 32 Proben einer zweiten Kohorte (n=16) im direkten Vergleich.
Aus unseren Ergebnissen konnten folgende Rückschlüsse gezogen werden:
1. Die beiden allel-spezifischen Amplifikationsmethoden ARMS-QPCR und WTB-AS QPCR zur Quantifizierung der JAK2 p.V617F Mutation sind vergleichbar.
2. Als Ausgangsmaterial sind antikoaguliertes Vollblut oder auch Beckenkammbiopsien gleichermaßen geeignet.
3. Der Nachweis von > 1% JAK2 p.V617F Allele 28 Tage nach allogener SCT ist assoziiert mit einem signifikant höheren Rezidivrisiko einer JAK2 positiven MPN und einem schlechteren Gesamtüberleben.
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黃庭欣 et Ting-yan Cybil Wong. « The use of JAK2 quantitative polymerase chain reaction for the diagnosis and monitoring of patients with chronic myeloproliferativediseases ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B40738279.
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Wong, Ting-yan Cybil. « The use of JAK2 quantitative polymerase chain reaction for the diagnosis and monitoring of patients with chronic myeloproliferative diseases ». Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B40738279.
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Hasan, Salma. « JAK2V617F-positive Myeloproliferative Neoplasms : KI mouse models, Interferon-α therapy and clonal architecture ». Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-00918966.
Texte intégralRésumé :
This work concerns malignant myeloid hemopathies called classical BCR-ABL-negative Myeloproliferative Neoplasms (MPN) and include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). They result from the transformation of a multipotent hematopoietic stem cell (HSC) with hyperproliferation but no blockade of differentiation. The most common molecular defect is the acquired point mutation JAK2V617F resulting into the activation of the cytokine receptor/JAK2 pathway. We have developed a mouse constitutive and a conditional JAK2V617F knock-in (KI) mouse models. These animals developed a disease mimicking human PV evolving into secondary MF. They also displayed an age dependent increase in the total numbers of early hematopoietic cells (phenotype LK, LSK and SLAM: LSK/CD48-/CD150+). Using In vivo competitive repopulation assays we demonstrated that cells from KI origin outcompeted their WT counterparts and that a low number of JAK2V617F KI SLAM cells propagates the disease. These results show that the sole JAK2V617F mutation, without any additional mutations, is sufficient for disease phenotype and emergence. Using this KI mouse model, we tested the effect of interferon-a (IFNa) treatment on MPN development. We found that IFNa treats the disease phenotype by blocking the propagation of early JAK2V617F cells and eradicates disease-initiating cells, showing that IFNα could cure the disease in mice, as shown in some PV patients. Finally, we developed a new method combining the measurement of 46/1 SNPs and JAK2V617F allele burdens in blood predicting the frequency of normal, heterozygous and homozygous JAK2V617F clones in PV patients. This study suggested that IFNa preferentially targets the homozygous JAK2V617F clone in PV patients suggesting a link between the levels of JAK2 signaling and the success of the IFNa response.
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Asimakopoulos, Fotios A. « Molecular analysis of chromosome 20q deletions associated with myeloproliferative disorders and myelodysplastic syndromes ». Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.388490.
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Gari, Mamdooh Abdullah Mahmoud. « Pathogenesis of c-kit proto-oncogene mutations in acute and chronic myeloproliferative disorders ». Thesis, University of Sheffield, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341830.
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Huntly, Brian James Patrick. « The role of chromosomal deletions and translocations in the pathogenesis of the myeloproliferative disorders ». Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619532.
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Abu-Duhier, Faisel Mohammed. « Pathogenic role of C-FMS and FLT3 mutations in acute and chronic myeloproliferative disorders ». Thesis, University of Sheffield, 2003. http://etheses.whiterose.ac.uk/14753/.
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Montazeri, Ghahjavarestani Maryam [Verfasser], Andreas [Akademischer Betreuer] Schuppert et Steffen [Akademischer Betreuer] Koschmieder. « Modelling of disease progression in myeloproliferative neoplasms / Maryam Montazeri Ghahjavarestani ; Andreas Schuppert, Steffen Koschmieder ». Aachen : Universitätsbibliothek der RWTH Aachen, 2019. http://d-nb.info/1211963721/34.
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Wahlström, Annika. « Defining RCE1 and ICMT as therapeutic targets in K-RAS-induced cancer / ». Göteborg : The Wallenberg Laboratory, Dept.of Molecular and Clinical Medicine, Institute of Medicine at Sahlgrenska Academy, University of Gothenburg, 2009. http://hdl.handle.net/2077/19643.
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Halank, Michael, C. Marx, Gustavo B. Baretton, K. M. Müller, Gerhard Ehninger et Gerd Höffken. « Severe Pulmonary Hypertension in Chronic Idiopathic Myelofibrosis ». Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-135018.
Texte intégralRésumé :
Background: Chronic myeloproliferative disorders (CMPD) seem to be associated with an increased risk for pulmonary hypertension (PH). Case Report: A patient with history of chronic idiopathic myelofibrosis (CIMF) presented with progressive dyspnea (New York Heart Association class III). Until this time he had not received specific treatment for CIMF. Echocardiography and rightheart catheterization confirmed PH. Further diagnostic procedures excluded a specific cause of PH. Therefore, primary PH was assumed. 2 years later he presented again with progressive dyspnea due to a progress of PH. A few days later the patient died from acute posterior myocardial infarction. Pathologic examination of the lung showed an obstruction of the small vessels by conglomerates of megakaryocytes. Discussion: We conclude that PH developed secondarily due to CMPD. PH should be suspected in patients with CMPD and should influence the decision for treatment of CMPDHintergrund: Chronische myeloproliferative Erkrankungen (CMPD) scheinen mit einem erhöhten Risiko für pulmonale Hypertonie (PH) assoziiert zu sein. Kasuistik: Ein Patient mit chronisch idiopathischer Myelofibrose (CIMF) wurde aufgrund einer progressiven Belastungsdyspnoe (New York Heart Association Stadium III) überwiesen. Bis zu diesem Zeitpunkt erhielt er keine spezifische Behandlung seiner CIMF. Echokardiographie und Rechtsherzkatheter ergaben das Vorliegen einer PH. Eine spezifische Ursache der PH konnte zunächst ausgeschlossen werden. Somit wurde das Vorliegen einer primären PH vermutet. 2 Jahre später wurde der Patient mit erneut verschlechterter Belastungsdyspnoe vorgestellt, wobei ein Progress der PH feststellbar war. Einige Tage später verstarb der Patient an einem Hinterwandinfarkt. Die Autopsie des Lungengewebes zeigte einen Verschluss der kleinen Lungengefäße durch Konglomerate von Megakaryozyten. Diskussion: Die Entwicklung der PH ist bei diesem Patienten als Folge der CMPD einzuschätzen. Das Vorliegen einer PH bei Patienten mit CMPD sollte die Entscheidung zu spezifischen therapeutischen Maßnahmen hinsichtlich der CMPD beeinflussen
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Poulet, Erma. « Implementation of the JAK2V617F mutation analysis in the pathway of suspected myeloproliferative neoplasms in Groote Schuur Hospital ». Master's thesis, University of Cape Town, 2016. http://hdl.handle.net/11427/23660.
Texte intégralRésumé :
We studied the implementation of JAK2 mutation analysis in conjunction with the World Health Organisation (WHO) guidelines in the pathway to MPN diagnosis in 279 patients presenting with one of three clinical scenarios: erythrocytosis, OR leukocytosis and/or thrombocytosis and/or splenomegaly; OR patients with thrombosis without cytoses. Patients were investigated for MPN and managed in the haematology clinic of Groote Schuur Hospital. We studied the association of clinical and laboratory variables with clonal vs non-clonal diagnoses. In 120/297 patients MPN was confirmed: Polycythemia vera (PV), (n=51, 100% JAK2 mutated); essential thrombocytosis, (n=41, 42% JAK2 mutated); primary myelofibrosis (n=28, 57% JAK2 mutated). The 2016 WHO haemoglobin/haematocrit thresholds in PV were validated. Idiopathic erythrocytosis (IE) found in 44 patients. Bone marrow histology, but not serum EPO level, was essential to differentiate between clonal and non-clonal erythrocytosis. Both PV and IE patients complied with the criteria of absolute erythrocytosis on peripheral blood, yet nuclear red cell mass identified critical differences between clonal and non-clonal erythrocytosis. No patient venesected for nonclonal erythropoiesis developed thrombocytosis. JAK2 mutation analysis applied with the WHO diagnostic algorithm efficiently differentiated true clonal myeloproliferation from reactive cytoses. Lifestyle and metabolic factors such as smoking and thrombosis were not associated with either clonal or non-clonal erythrocytosis, and were equally present in mutated and unmutated essential thrombocytosis.
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Tanaka, Hiroki. « Increased c-Myc activity and DNA damage in hematopoietic progenitors precede myeloproliferative disease in Spa-1-deficiency ». Kyoto University, 2011. http://hdl.handle.net/2433/142075.
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Siebolts, Udo [Verfasser], Joachim [Akademischer Betreuer] Schultze et Jens [Akademischer Betreuer] Brüning. « New aspects into pathophysiology and molecular diagnostics of myeloproliferative neoplasms / Udo Siebolts. Gutachter : Joachim Schultze ; Jens Brüning ». Köln : Universitäts- und Stadtbibliothek Köln, 2011. http://d-nb.info/1038224616/34.
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Toppaldoddi, Katte Rao. « Role of rare calreticulin mutants and of the endoplasmic reticulum stress in the pathogenesis of myeloproliferative neoplasms ». Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC322/document.
Texte intégralRésumé :
Après la découverte des mutations de la calréticuline dans les néoplasmes classiques myéloproliferatifs négatifs pour le Ph1, les travaux se sont focalisés sur les deux mutations les plus fréquentes, c'est-à-dire la calréticuline del52 et l’ins5, mais il existe environ 20% de mutants rares de la calréticuline (une cinquantaine), qui ont été classés en type-1 « like » et type-2 « like », classification basée sur leur structure. Cependant il reste à déterminer si cette classification est pertinente du point de vue fonctionnel, ce qui pourrait avoir des conséquences pour la prise en charge des patients et leur traitement. Ici, nous démontrons que deux mutants rares de type-1 (del34 et del46) et un de type-2 (del19) se comportent de manière similaire aux deux mutations fondatrices de cette classification, del52 et ins5, respectivement. Ces résultats ont été validés par des expériences in vivo chez la souris. Tous les mutants de la calréticuline (del19, del34 et del46) nécessitent absolument le récepteur de la thrombopoïétine, appelé MPL, pour induire une transformation cellulaire en provoquant une activation indépendante de la thrombopoïétine de la voie MPL / JAK2-STAT, comme les mutants del52 et ins5. Dans les expériences de transplantation de moelle osseuse de souris, les mutants rares de type-1 sont associés à une progression fréquente de la maladie d’un tableau proche d’une thrombocytémie essentielle à une myélofibrose, tandis que le mutant rare de type 2 est associé à une légère thrombocytose. Du point de vue hématopoïétique, les mutants rares de type-1 provoquent une amplification au niveau des cellules souches hématopoïétiques donc à un stade précoce tandis que les mutants rares de type-2 provoquent une amplification tardive de la mégacaryopoïèse. Grâce à une modélisation protéique basée sur l'homologie des mutants de calréticuline, nous avons identifié des domaines oncogènes qui seraient potentiellement responsables de l'interaction pathologique de la calréticuline et de MPL pour conduire à une activation indépendante de la thrombopoïétine. Maintenant, ces résultats in silico doivent être absolument validés par des études structure fonction. Enfin, nous avons modélisé un nouveau mécanisme de signalisation dans la leucémie myéloïde chronique comprenant IRE-1alpha, un bras de la voie de réponse des protéines mal repliées (UPR), qui pourrait être responsable de la perte de la fonction de la p53 pendant la progression de la leucémie myéloïde chronique vers une leucémie aiguë. Un tel mécanisme pourrait être impliqué dans les autres MPNAfter the discovery of calreticulin mutations in classical Ph1- Myeloproliferative Neoplasms, extensive investigation is underway on the two most frequent mutations, i.e., del52 and ins5, but it remains that the rare calreticulin mutants, which include both type-1 like and type-2 like require a similar investigation for ascertaining whether the classification of type-1 and type-2 has a functional relevance as well as for therapeutic intervention and patient management. Here we demonstrate that type-1 like (del34 and del46) and type-2 like (del19) mutants behave similarly as del52 and ins5 mutants, respectively. Moreover, we validate our findings with in vivo experiments. All the calreticulin mutants (del19, del34 and del46) absolutely require the thrombopoietin receptor, MPL, to induce cell transformation by causing ligand independent activation of the MPL/JAK2-STAT pathway. In mouse bone marrow transplantation experiments, type-1 like mutants are associated with frequent progression from an essential thrombocythemia-like phenotype to myelofibrosis whereas type-2 like mutant is associated with mild thrombocytosis. Type-1 like mutants cause clonal amplification of early hematopoetic stem cells whereas the type-2 like mutant causes late platelet amplification. Further, by homology based protein modeling of calreticulin mutants, we have identified possible oncogenic domains responsible for pathologic interaction of CALR and MPL leading to ligand independent activation of MPL. Now they must be validated by structural-functional studies Finally, we have modelled a novel signaling mechanism in chronic myeloid leukemia comprising of IRE-1alpha, an unfolded protein response (UPR) pathway arm, which may be responsible for loss of the WT p53 function during leukemic development and progression. Such a mechanism may be involved in the other MPNs
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El, khoury Mira. « Rôle de la calréticuline dans les néoplasmes myéloprolifératifs ». Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC227.
Texte intégralRésumé :
Les néoplasmes myéloprolifératifs (NMPs) classiques BCR-ABL négatifs regroupent la Polyglobulie de Vaquez, la Thrombocytémie Essentielle et la Myélofibrose Primaire. Ce sont des pathologies malignes clonales entraînées par la signalisation constitutive de la voie JAK2/STAT en raison de mutations somatiques acquises qui affectent trois gènes, JAK2, CALR et MPL. Il s’agit des mutations “motrices” de la maladie responsable du syndrome myéloprolifératif et du phénotype. Cependant CALR n’est pas une molécule de signalisation mais une chaperonne du réticulum endoplasmique. En utilisant des lignées dépendantes de facteurs de croissance soit murines (Ba/F3) soit humaines (UT-7), des cellules primaires de patients et des modèles murins nous avons montré que les mutants CALRdel52 et CALRins5 avaient acquis de nouvelles propriétés qui en font des molécules de signalisation en induisant: - une indépendance aux facteurs de croissance uniquement lorsque MPL, le récepteur de la thrombopoïétine est exprimé ; - une phosphorylation constitutive de JAK2, des STAT1, 3 et 5 et une activation faible des voies PI3K/AKT et ERK1/2, suggérant une activation de MPL/JAK2 par les mutants CALR différente de celle induite par JAK2V617F. De manière intéressante un mutant de la CALR ayant une délétion entière de l’exon 9 n’est pas transformant, suggérant que l’activité oncogénique est liée à la présence de la nouvelle séquence C-terminale. L’activation de JAK2 uniquement par MPL en présence des mutants CALR pourrait expliquer le phénotype mégacaryocytaire/plaquettaire de ces NMPs. Cette activation de MPL au contraire de celle exercée par JAK2V617F a lieu non seulement à la membrane mais aussi dans le cytoplasme.Les modèles murins ont montré que les mutants CALR étaient responsables de la maladie et que celle-ci était dépendante de MPL, validant les résultats obtenus sur les lignées.Nous avons également montré que contrairement à JAK2V617F, les mutants de la CALR induisent chez l’homme une dominance clonale très tôt au niveau du compartiment des cellules souches. L’ensemble de ces résultats contribue à une meilleure compréhension du rôle des mutations CALR dans les NMPs. La démonstration que les molécules mutées sont présentes à la surface cellulaire ouvre la voie à des immunothérapies ciblant le nouveau peptide C-terminalClassical BCR-ABL negative myeloproliferative neoplasms (MPNs) include three disorders: Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis. They are clonal malignant diseases driven by the constitutive JAK2/STAT signaling pathway due to acquired somatic mutations affecting three genes: JAK2, CALR and MPL. These are the "driver" mutations of the disease responsible of the myeloproliferation and of the disease phenotype. However, CALR is not a signaling molecule, but a chaperonne of the endoplasmic reticulum. Using murine (Ba/F3) and human (UT-7) cell lines dependent on growth factors and primary patient cells and mouse model, we have shown that the CALRdel52 and CALRins5 mutants have acquired new signaling properties and induce:- growth factor independence only when MPL, the thrombopoietin receptor, is expressed;- constitutive phosphorylation of JAK2, of STAT1, 3 and 5 and a low activation of the PI3K/AKT and ERK1/2 pathways, suggesting an activation of MPL/JAK2 by a different manner than JAK2V617F. Interestingly, a CALR mutant deleted for the entire exon 9 has not transformation properties suggesting that the oncogenic activity is related to the presence of the new C-terminal sequence. This JAK2 activation only by MPL in presence of CALR mutants could explain the megakaryocytic/platelet phenotype of these MPNs.The use of a mouse modeling using retroviral vectors and bone marrow transplantation has shown that CALRdel52 and ins5 were really the drivers of the disease and that in vivo the thrombocytosis was dependent of MPL validating the results obtained in vitro.In addition, we have shown that in human, CALR mutants induce a clonal dominance early in the stem cell compartment in ET. This is in sharp contrast with JAK2V617F in ET. Overall, these results contribute to a better comprehension of the role of CALR mutations in MPNs. Furthermore, the demonstration that the CALR mutants are expressed at the cell surface open the way to the development of new immunotherapy targetting the new C-terminus peptide
49
Zhao, Rui. « Bcl-xL deamidation in oncogenic tyrosine kinase signalling ». Thesis, Anglia Ruskin University, 2011. http://arro.anglia.ac.uk/213610/.
Texte intégralRésumé :
I have been interested in the molecular mechanisms of Haematopoietic malignant diseases such as leukaemia and lymphoma, especially those involving oncogenic tyrosine kinases. About 30 of the 90 tyrosine kinases in the human genome have been implicated in cancer (Blume-Jensen P, 2001). The oncogenic tyrosine kinases (OTKs), such as Bcr-Abl (product of chromosomal translocations of two genes bcr and abl) in Chronic Myelogenous Leukaemia, and Erythroblastic leukaemia viral oncogene homolog 2(Erb-B2) in mammary and other cancers, mediate their transforming effects via a diverse array of signalling pathways involved in DNA damage, cell survival and cell cycle regulation (Deutsch E, 2001; Skorski T, 2002; Kumar R, 1996). My work has been centred around the analysis of a mouse cancer model that is driven by an oncogenic tyrosine kinase – p56 Lck-F505 expressed on CD45 knock- out background (Baker M, 2000). The investigation of this mouse model has revealed that oncogenic inhibition of deamidation of the Bcl-xL survival protein plays a critical role in protecting thymocytes from DNA-damage induced apoptosis. Cells that would normally be eliminated due to accumulating DNA damage are instead preserved with an increasing load of double-stranded breaks, leading to genomic instability, chromosomal abnormalities and transformation. This work was published in Cancer Cell (An oncogenic tyrosine kinase inhibits DNA repair and DNA-damage-induced BclxL deamidation in T cell transformation. Zhao R, 2004). Following that I have tried to elucidate the different roles of the two deamidated species of Bcl-xL in apoptosis, and also the molecular mechanisms of DNA damage- induced Bcl-xL deamidation in order to understand the inhibition of Bcl-xL deamidation by oncogenic tyrosine kinases. Recently I have shown that Bcl-xL deamidation, whereby two critical Asn residues are converted to iso-Asp, cripples the ability of the protein to sequester pro-apoptotic BH3-only proteins such as Bim and p53- upregulated modulator of apoptosis (PUMA), thereby explaining its loss of pro-survival functionality. In vivo, DNA damage causes intracellular alkalinisation that is both necessary and sufficient to deamidate Bcl-xL, promoting apoptosis: no enzyme is necessary for this process. In pre-tumourigenic thymocytes alkalinisation is blocked, so preserving Bcl-xL in its pro-survival mode. Furthermore murine tumours are protected from genotoxic attack by native Bcl-xL, but enforced alkalinisation and consequent Bcl-xL deamidation promotes apoptosis. This part of work was published in Plos Biology (DNA damage-induced Bcl-xL deamidation is mediated by NHE-1 antiport regulated intracellular pH. Zhao R, 2007). Through collaboration with Prof AR Green’s research group at the Department of Haematology of the University of Cambridge, I have also analysed the Bcl-xL deamidation pathway in human myeloproliferative disorders, e.g. Polycythemia vera(PV) and Chronic Myelogenous Leukaemia (CML). We found that the oncogenic tyrosine kinases involved in these disorders, i.e. Jak2V617F and Bcr-Abl also inhibit the Bcl-xL deamidation pathway in DNA damage responses. These findings shed light on potential therapeutic application of the Bcl-xL deamidation pathway in human malignancies. This piece of work was recently published in the New England Journal of Medicine (Inhibition of the Bcl-xL deamidation pathway in myeloproliferative disorders. Zhao R, 2008). Overall the cited work has led to several important new insights into the molecular mechanisms involved in oncogenesis: first, that Bcl-xL deamidation is important in the cascade of events leading from DNA damage to apoptosis; second, that oncogenic tyrosine kinases inhibit these events in both the murine and human context; third, that up-regulation of the NHE-1 antiport and consequent intracellular alkalinisation are critical events in this DNA damage-induced cascade leading to apoptosis. In the process I have demonstrated the first in vivo mechanism for the deamidation of an internal protein Asn. Essentially, a completely new and unexpected signalling pathway has been uncovered that seems to pertain to all murine and human haematopoietic cell lineages that have been investigated so far.
50
Zhao, Rui. « Bcl-xL deamidation in oncogenic tyrosine kinase signalling ». Thesis, Anglia Ruskin University, 2011. https://arro.anglia.ac.uk/id/eprint/213610/1/Rui_Thesis_2011.pdf.
Texte intégralRésumé :
I have been interested in the molecular mechanisms of Haematopoietic malignant diseases such as leukaemia and lymphoma, especially those involving oncogenic tyrosine kinases. About 30 of the 90 tyrosine kinases in the human genome have been implicated in cancer (Blume-Jensen P, 2001). The oncogenic tyrosine kinases (OTKs), such as Bcr-Abl (product of chromosomal translocations of two genes bcr and abl) in Chronic Myelogenous Leukaemia, and Erythroblastic leukaemia viral
oncogene homolog 2(Erb-B2) in mammary and other cancers, mediate their transforming effects via a diverse array of signalling pathways involved in DNA damage, cell survival and cell cycle regulation (Deutsch E, 2001; Skorski T, 2002; Kumar R, 1996).
My work has been centred around the analysis of a mouse cancer model that is driven by an oncogenic tyrosine kinase – p56 Lck-F505 expressed on CD45 knock- out background (Baker M, 2000). The investigation of this mouse model has revealed that oncogenic inhibition of deamidation of the Bcl-xL survival protein plays a critical role in protecting thymocytes from DNA-damage induced apoptosis. Cells that would normally be eliminated due to accumulating DNA damage are instead preserved with an increasing load of double-stranded breaks, leading to genomic instability, chromosomal abnormalities and transformation. This work was published in Cancer Cell (An oncogenic tyrosine kinase inhibits DNA repair and DNA-damage-induced BclxL deamidation in T cell transformation. Zhao R, 2004). Following that I have tried to
elucidate the different roles of the two deamidated species of Bcl-xL in apoptosis, and also the molecular mechanisms of DNA damage- induced Bcl-xL deamidation in order
to understand the inhibition of Bcl-xL deamidation by oncogenic tyrosine kinases.
Recently I have shown that Bcl-xL deamidation, whereby two critical Asn residues are converted to iso-Asp, cripples the ability of the protein to sequester pro-apoptotic BH3-only proteins such as Bim and p53- upregulated modulator of apoptosis (PUMA), thereby explaining its loss of pro-survival functionality. In vivo, DNA damage causes intracellular alkalinisation that is both necessary and sufficient to deamidate Bcl-xL, promoting apoptosis: no enzyme is necessary for this process. In pre-tumourigenic thymocytes alkalinisation is blocked, so preserving Bcl-xL in its pro-survival mode. Furthermore murine tumours are protected from genotoxic attack by native Bcl-xL, but enforced alkalinisation and consequent Bcl-xL deamidation promotes apoptosis. This part of work was published in Plos Biology (DNA damage-induced Bcl-xL deamidation is mediated by NHE-1 antiport regulated intracellular pH. Zhao R, 2007). Through collaboration with Prof AR Green’s research group at the Department of Haematology of the University of Cambridge, I have also analysed the Bcl-xL deamidation pathway in human myeloproliferative disorders, e.g. Polycythemia vera(PV) and Chronic Myelogenous Leukaemia (CML). We found that the oncogenic tyrosine kinases involved in these disorders, i.e. Jak2V617F and Bcr-Abl also inhibit the Bcl-xL deamidation pathway in DNA damage responses. These findings shed light on potential therapeutic application of the Bcl-xL deamidation pathway in human malignancies. This piece of work was recently published in the New England Journal of Medicine (Inhibition of the Bcl-xL deamidation pathway in myeloproliferative disorders. Zhao R, 2008). Overall the cited work has led to several important new insights into the molecular mechanisms involved in oncogenesis: first, that Bcl-xL deamidation is important in the cascade of events leading from DNA damage to apoptosis; second, that oncogenic tyrosine kinases inhibit these events in both the murine and human context; third, that up-regulation of the NHE-1 antiport and consequent intracellular alkalinisation are critical events in this DNA damage-induced cascade leading to apoptosis. In the process I have demonstrated the first in vivo mechanism for the deamidation of an internal protein Asn. Essentially, a completely new and unexpected signalling pathway has been uncovered that seems to pertain to all murine and human haematopoietic cell lineages that have been investigated so far.