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Erythroid progenitor cells of the abnormal clone in polycythemia vera (PV) are capable of colony formation in vitro without the addition of erythropoietin, the regulatory hormone required for normal in vivo and-in vitro erythropoiesis. This property of "erythropoietin-independent" colony formation has been considered a marker for the abnormal clone in PV, although recent studies indicate that not all erythropoietic members of the clone may be capable of exhibiting this abnormal phenotype. The present studies were undertaken to investigate the level of maturation at which establishment of an erythropoietin-independent phenotype might be determined. A series of experiments was performed on the replated progeny of single primitive hemopoietic cells already committed to erythropoiesis (primitive BFU-E). First, conditions were established to maximize the number of erythroid colonies obtainable in secondary assays of replated primary colonies of primitive BFU-E origin. Time course studies and experiments with irradiated peripheral blood "feeder" cells treated in different ways established that results were best when primary colonies were allowed to grow for 9 days prior to replating and when 9 day old feeders stored at 4°C were included in the secondary assay medium. Second, a technique was developed for dividing such colonies between 2 secondary assay cultures. Experiments with normal primary colonies transferred to 2 secondary assays, both containing erythropoietin, showed that the variation between true replicates was random, indicating that the procedure used divided each primary colony equally. Third, it was shown that secondary assays to which no erythropoietin was added failed to support erythroid colony formation by progenitors present in normal 9 day old primary colonies. Finally, the distribution of erythropoietin-dependent and erythropoietin-independent phenotypes in individual colonies derived from primitive BFU-E from 5 patients with. PV was assessed by replating experiments. Most of the replated colonies from PV cultures that yielded erythroid colonies in secondary assays containing 3 units of erythropoietin per ml also produced some erythroid colonies in the paired replicate that contained < 0.01 units of erythropoietin per ml. However, fewer colonies were consistently obtained in the low erythropoietin cultures. These results indicate that in PV, most of the primitive erythroid bursts that generate phenotypically abnormal progeny capable of erythroid colony formation under conditions which are non-permissive for normal cells also produce significant numbers of progeny that are phenotypically normal in this respect. It is concluded that the capacity for erythropoietin-independent growth and maturation exhibited in vitro by terminally differentiating members of the abnormal clone in PV is not commonly fixed at or prior to the primitive BFU-E stage of erythropoietic cell development. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Thesis (M.S.)--University of Wisconsin. School of Nursing, 1976. / eContent provider-neutral record in process. Description based on print version record.

An underlying myeloproliferative disorder (MPD), especially polycythemia vera (PV) or essential thrombocythemia (ET), is a risk factor for thrombosis. Considering large selected studies, prevalence rates for major thrombosis, at time of diagnosis, range from ∼34 to 39% for PV and 10 to 29% for ET; the corresponding figures for thrombosis at follow-up are ∼8 to 19% for PV and 8 to 31% for ET. In all instances, arterial events were more frequent than venous events. In both PV and ET, advanced age and history of thrombosis are independent predictors of recurrent thrombosis. In addition, leukocytosis, but not thrombocytosis, has been identified as a potential risk factor for thrombosis in both diseases. The particular observation is consistent with the laboratory demonstration, in these disorders, of increased number of activated granulocytes and granulocyte-platelet aggregates, upregulation of platelet P-selectin and tissue factor expression by granulocytes, and the antithrombotic value of hydroxyurea therapy. Most recently, a JAK2 gain-of-function mutation (JAK2V617F) was described in virtually all patients with PV and ∼50% of those with ET. Whether the presence of this specific mutation or its allele burden modifies the risk of thrombosis in patients with MPDs currently is under investigation.

Thesis (M.A.)--Boston University

All red blood cells, erythrocytes, originate in bone marrow. The process of their differentiation and maturation is called erythopoiesis and is regulated through hormone erythropoietin (EPO), which functions as a stimulatory factor for erythropoiesis. EPO is produced in kidney and its production is regulated by oxygen supplementation. EPO is transported to bone marrow via blood vessels. Chronic overproduction of erythrocytes leads to disease called polycythemia. Polycythemia may be diagnosed for example by measurement of haematocrit or haemoglobin concentration in blood. EPO level may or may not be increased. Patients suffering from polycythemia may or may not have any symptoms. It depends on manifestation level of the disease. The most common symptoms are higher blood pressure, headaches, dizziness, swelling and epistaxis. Recently, the most common treatment of polycythemia is phlebothomy. The aim of this master thesis is to unravel the role of a newly described mutation, which was found among members of one family suffering from polycythemia with increased EPO level. It is single substitution mutation -136 G > A in 5' UTR region of EPO gene. The clones of EPO producing cell lines bearing this mutation were prepared using CRISPR/Cas9 technology. Several experiments performed not only on those cell...

Polycythemia Vera är en livslång blodsjukdom som innebär en överproduktion av blodceller. Vid noggrannhet i behandlingen har sjukdomen god prognos. Att leva med långvarig sjukdom innebär en långvarig och regelbunden kontakt med sjukvården. Det är viktigt att möjliggöra delaktighet för dessa patienter då detta skulle kunna medföra att egenvårdsförmågan ökar och beroendet av sjukvården minskar. För att främja långvarigt sjukas delaktighet har e-hälsotjänsten PODS-Patient Online Decision System tagit fram. PODS är utformat för patienter med Polycythemia Vera. För att tjänsten ska bli patientanpassad bör patienter få komma till tals angående tjänstens utformning och innehåll. Syftet med denna studie är att beskriva patienters uppfattning av e- hälsotjänsten PODS vid en introduktion. Semistrukturerade intervjuer genomfördes med sex informanter som är diagnostiserade med Polycythemia Vera. Intervjuerna bearbetades och tolkades genom innehållsanalys enligt Lundman och Hällgren Graneheim (2012 ss. 194-195). Resultatet i studien visar att patienter sätter stort värde på en välfungerande vårdkontakt, då detta inger en känsla av trygghet. PODS meddelandefunktion ansågs kunna bidra till en smidig kontakt med vården. I dagsläget uppfattas PODS vara användarvänligt i sin utformning. Det fanns dock frågetäcken gällande praktiska lösningar. Överskådligheten av blodvärdena som erbjuds i programmet uppskattas då det gav möjlighet till en fördjupad förståelse för den egna hälsan. PODS har potential att fungera för andra patientgrupper. Det erfordras förbättringar och utveckling av programmets samt organisationen omkring det. Om detta kommer till stånd finns det behov av att vidare studera hur en längre tids användning av PODS upplevs av patienterna.

Marking proteins for degradation by the proteasome is a classical function of ubiquitination. This process of covalent attachment of a chain of ubiquitin molecules to target proteins is governed by the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin ligase (E3). The von Hippel-Lindau (VHL) tumour suppressor protein forms an E3 ubiquitin ligase, ECV (Elongins BC/Cul2/VHL), which targets the alpha subunit of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction under normal oxygen tension. Tumour hypoxia promotes accumulation of HIFalpha, whose expression is associated with cancer progression, poor prognosis and resistance to conventional therapies, thus establishing HIF as a therapeutic target. Notably, VHL is functionally inactivated in VHL disease, a hereditary cancer syndrome characterized by the formation of tumours in multiple organs, as well as in the majority of sporadic clear-cell renal cell carcinomas (CCRCC) and haemangioblastomas. Recently, certain VHL mutations have been shown to cause the congenital disorder Chuvash polycythemia. Work contained in this thesis describes the temporally coordinated activation of the ECV, whereby oxygen-dependent recognition of HIFalpha by VHL triggers Cul2 modification by the ubiquitin-like molecule NEDD8, which enhances ECV ubiquitin ligase activity by recruiting the E2. In addition, the feasibility of ‘bio-tailored’ enzymes in the treatment of cancer is introduced by creating a bioengineered VHL capable of targeting HIFalpha for degradation irrespective of oxygen tension, which leads to the dramatic inhibition of CCRCC tumour growth and angiogenesis in a xenograft model. Furthermore, a ubiquitin ligase composed of two F-box proteins, VHL and suppressor of cytokine signalling 1 (SOCS1), was identified and shown to be paramount for the negative regulation of erythropoiesis by targeting phosphorylated Janus kinase 2 (JAK2) for ubiquitin-mediated destruction. The malfunction of this ubiquitin ligase explains the excessive erythrocytosis observed in Chuvash polycythemia patients and reveals a novel genetic link between the seemingly distinct genes VHL and JAK2 in the development of polycythemia.

Marking proteins for degradation by the proteasome is a classical function of ubiquitination. This process of covalent attachment of a chain of ubiquitin molecules to target proteins is governed by the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin ligase (E3). The von Hippel-Lindau (VHL) tumour suppressor protein forms an E3 ubiquitin ligase, ECV (Elongins BC/Cul2/VHL), which targets the alpha subunit of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction under normal oxygen tension. Tumour hypoxia promotes accumulation of HIFalpha, whose expression is associated with cancer progression, poor prognosis and resistance to conventional therapies, thus establishing HIF as a therapeutic target. Notably, VHL is functionally inactivated in VHL disease, a hereditary cancer syndrome characterized by the formation of tumours in multiple organs, as well as in the majority of sporadic clear-cell renal cell carcinomas (CCRCC) and haemangioblastomas. Recently, certain VHL mutations have been shown to cause the congenital disorder Chuvash polycythemia. Work contained in this thesis describes the temporally coordinated activation of the ECV, whereby oxygen-dependent recognition of HIFalpha by VHL triggers Cul2 modification by the ubiquitin-like molecule NEDD8, which enhances ECV ubiquitin ligase activity by recruiting the E2. In addition, the feasibility of ‘bio-tailored’ enzymes in the treatment of cancer is introduced by creating a bioengineered VHL capable of targeting HIFalpha for degradation irrespective of oxygen tension, which leads to the dramatic inhibition of CCRCC tumour growth and angiogenesis in a xenograft model. Furthermore, a ubiquitin ligase composed of two F-box proteins, VHL and suppressor of cytokine signalling 1 (SOCS1), was identified and shown to be paramount for the negative regulation of erythropoiesis by targeting phosphorylated Janus kinase 2 (JAK2) for ubiquitin-mediated destruction. The malfunction of this ubiquitin ligase explains the excessive erythrocytosis observed in Chuvash polycythemia patients and reveals a novel genetic link between the seemingly distinct genes VHL and JAK2 in the development of polycythemia.

Orientador: Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T12:54:32Z (GMT). No. of bitstreams: 1 Monte-Mor_BarbaradaCostaReis_D.pdf: 7171978 bytes, checksum: 5c73cb6c7edd6138186b17d972fce769 (MD5) Previous issue date: 2007 / Resumo: Policitemia vera (PV) é uma síndrome mieloproliferativa (SMP) crônica que ocorre por proliferação clonal de progenitores hematopoéticos multipotenciais. Pacientes com PV apresentam expansão das três principais linhagens mielóides na medula óssea, levando à produção aumentada de hemácias, granulócitos e plaquetas. Características importantes de PV incluem elevação de hematócrito em presença de níveis normais ou diminuídos de eritropoetina (EPO) e a formação de colônias eritróides endógenas (CEE). A mutação JAK2 V617F, presente na maioria dos pacientes com PV, causa ativação constitutiva de JAK2 e parece ser responsável pelo fenótipo observado. Apesar disso, as mudanças transcricionais desencadeadas pela mutação ainda não foram completamente caracterizadas. Neste trabalho, utilizou-se Serial Analysis of Gene Expression (SAGE) para realizar um estudo de expressão gênica global em células da medula óssea de um paciente com PV portador da mutação, ao diagnóstico e em células normais de doadores saudáveis. Genes com expressão aumentada em PV estão envolvidos em processos biológicos importantes, como transdução de sinal, diferenciação e proliferação celular, ciclo celular, apoptose, resposta imune e regulação da transcrição. JUNB foi um dos genes identificados e, usando reação em cadeia da polimerase quantitativa em tempo real (qRT-PCR), detectou-se expressão aumentada de JUNB em dois outros pacientes com PV JAK2 V617F-positivos. Por meio de linhagens murinas Ba/F3-REPO e culturas primárias de eritroblastos humanos, observou-se que JUNB é expresso após adição de EPO e que a proteína JunB é constitutivamente induzida por JAK2 V617F. Além disso, interferência na expressão de JUNB diminuiu o potencial clonogênico e proliferativo de progenitores eritróides humanos Ainda, em PV, a eritropoese causada por JAK2 V617F mostrou-se mais sensível à diminuição de expressão de JunB do que a eritropoese normal. Assim, esses resultados sugerem que JunB tenha um papel fundamental no desenvolvimento de SMPs causadas por JAK2 V617F / Abstract: Polycythemia vera (PV) is a chronic myeloproliferative disorder that arises through clonal proliferation of multipotent hematopoietic progenitors. PV patients present bone marrow trilineage expansion, leading to increased production of mature red cells, granulocytes and platelets. Important PV features are elevated red cell mass, despite normal or subnormal erythropoietin (EPO) levels, and endogenous erythroid colony (EEC) formation. The JAK2 V617F mutation, present in the majority of polycythemia vera (PV) patients, causes a JAK2 constitutive activation and seems to be responsible for the PV phenotype. However, the transcriptional changes triggered by the mutation have not yet been totally characterized. Serial analysis of gene expression (SAGE) was used to perform a large scale gene expression study in bone marrow cells of a newly-diagnosed PV patient harboring the JAK2 V617F mutation and in normal bone marrow cells from healthy donors. Genes overexpressed in PV are involved in important biological processes, such as signal transduction, cellular differentiation, cellular proliferation, cell cycle, apoptosis, immune response and transcriptional regulation. JUNB was one of the genes up-regulated in PV and overexpression of JUNB was also detected by quantitative real time polymerase chain reaction (qRT-PCR) in hematopoietic cells of another two JAK2 V617F PV patients. Using Ba/F3-EPOR cell lines and primary human erythroblast cultures, JUNB was found to be expressed after Epo addition and JunB protein was shown to be constitutively induced by JAK2 V617F. In addition, JUNB knock down reduced the clonogenic and proliferative potential of human erythroid progenitors. Furthermore, in PV, the JAK2 V617F driven erythropoisis was more sensitive to JunB downregulation than normal erythropoiesis. Hence, these results demonstrate that JunB may play a major role in the development of JAK2 V617F myeloproliferative disorders / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica