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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
51

Caractérisation moléculaire des syndromes myéloprolifératifs non leucémie myéloïde chronique / Molecular characterization of myeloproliferative neoplasms non-Chronic Myeloid Leukemia

Brecqueville, Mandy 27 September 2013 (has links)
Les syndromes myéloprolifératifs (SMP) non leucémie myéloïde chronique (LMC) sont des hémopathies myéloïdes chroniques affectant la cellule souche hématopoïétique, pouvant évoluer en leucémie aigüe myéloïde (LAM). Les SMP non LMC incluent la polyglobulie de Vaquez (PV), la thrombocytémie essentielle (TE) et la myélofibrose (MF). La mutation JAK2V617F est retrouvée dans 97% des cas de PV et dans 50% des cas de TE et MF ; elle n'est pas indispensable à la physiopathologie des SMP car JAK2 n'est pas muté à 100%. Afin de progresser dans la compréhension de la physiopathologie des SMP et afin d'identifier de nouveaux marqueurs moléculaires pour le diagnostic, le suivi et le pronostic; nous avons étudié des échantillons de PV, TE, MF ainsi que des LAM post-SMP. Nous avons utilisé des approches moléculaires complémentaires: séquençage, hybridation génomique comparative (CGH-array) et profils d'expression génique. Nous avons identifié des mutations de gènes régulateurs de l'épigénétique (ASXL1, TET2, DNMT3A, SUZ12) et des gènes de la machinerie de l'épissage de l'ARN (SF3B1, SRSF2). Nous avons également identifié que les co-mutations des gènes JAK2 et ASXL1 étaient associées à un mauvais pronostic. Au sein du sous-type MF, nous avons identifié par CGH-array des aberrations du nombre de copies des gènes. Celles-ci contiennent plusieurs gènes candidats susceptibles de participer à la physiopathologie des MF et à l'évolution en LAM (délétion 20q, NF1, ETV6). Nos travaux sur la caractérisation moléculaire des SMP contribuent à l'évolution vers une classification moléculaire avec l'objectif d'une médecine de précision où chaque SMP sera traité en fonction de ses altérations. / Myeloproliferative neoplasms (MPN) are chronic and clonal stem cell myeloid disorders, which can evolve to acute myeloid leukemia (AML). MPN non-chronic myeloid leukemia (CML) include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF) (primary or secondary to PV/ET). JAK2V617F mutation is found in 97% of PV and in around half of patients with ET or MF. Nevertheless, this mutation is not essential for MPN physiopathology, because in half of ET/MF cases, JAK2 is not mutated. To progress in the knowledge of MPN physiopathology and in particular of MF; and to find new molecular markers for MPN diagnosis, disease course, and prognosis, we studied several samples of PV, ET, MF and post-AML. We used gene sequencing, array-Comparative Genomic Hybridization (aCGH) and gene expression analyses. We identified several mutations in genes implicated in Epigenetic regulation (ASXL1, TET2, DNMT3A, SUZ12) and in genes implicated in the RNA splicing machinery (SF3B1, SRSF2). We also found that JAK2 and ASXL1 co-mutation is associated with a poor prognosis. In MF, we found by aCGH several copy number aberrations that involve potential leukemogenic genes. Our gene expression data support the hypothesis that PV, ET and MF are a continuum of the same pathology. Our results on molecular characterization help establish a new molecular classification of MPNs with the objective personalized treatment where each MPN will be treated depending on the alterations present in the myeloid cell genome.
52

Alterações da expressão de apoptomirs, de genes e proteínas pró- e anti-apoptóticos em Mielofibrose e Trombocitemia Essencial / Deregulated expression of apoptomirs and apoptosis-related genes and proteins in Primary Myelofibrosis and Essential Thrombocythemia

Raquel Tognon-Ribeiro 11 October 2011 (has links)
As Neoplasias Mieloproliferativas Crônicas (NMPC) Trombocitemia Essencial (TE), Mielofibrose (MF) - são desordens hematopoéticas resultantes da expansão clonal da célula tronco hematopoética alterada. Essas doenças caracterizam-se pela independência dos progenitores hematopoéticos aos estímulos dos fatores de crescimento e citocinas e pela proliferação exacerbada das células da linhagem mielóide com maturação preservada. Os mecanismos moleculares e celulares envolvidos na patogênese e progressão da TE e MF não foram ainda esclarecidos, mas o mieloacúmulo presente nessas doenças parece estar associado à alteração de proliferação e apoptose celular. Nesse contexto, os objetivos deste trabalho foram avaliar em células CD34+ da medula óssea e leucócitos dos pacientes com TE e MF: (1) a expressão de apoptomirs e de genes pró- e anti-apoptóticos pertencentes à via intrínseca e extrínseca da apoptose pela metodologia de RT-PCR em tempo real; (2) expressão de proteínas pró- e anti-apoptóticas por Western-blot; (3) o perfil de sensibilidade das células mononucleares à apoptose induzida por diferentes agentes apoptogênicos pela técnica de citometria de fluxo e (4) correlacionar os resultados obtidos com dados clínico-laboratoriais dos pacientes e com a expressão do gene PRV-1. Em TE, nas células CD34+, foi detectado aumento da expressão de a1, mcl-1, bid, bok e noxa e diminuição de bax em relação aos controles. Nos leucócitos, foi detectada a elevação da expressão de a1, bcl-2, bcl-w e bcl-xL, bad e bok e diminuição de bid e bimEL. Em comparação com o grupo controle, os pacientes com MF apresentaram maior expressão dos genes a1, bcl-w bak, bok e noxa e menor de bcl-2 nas células CD34+. Nos leucócitos dos pacientes com MF foi verificado aumento da expressão dos genes bcl-2, bcl-w e bcl-xL, bad e bok. O c-iap-1 apresentou maior expressão nas células CD34+ dos pacientes com MF e TE, enquanto que o c-iap-2 estava elevado nos leucócitos e células CD34+. Foi ainda detectada a expressão diferencial em TE e MF dos genes pertencentes a via de receptor de morte, como a maior expressão dos genes fas, fas-L, faim e dr4 nas células CD34+ dos pacientes e menor expressão do fas-L e trail nos leucócitos dos pacientes com TE e MF. Os resultados indicaram associação da expressão do gene PRV-1 com a mutação JAK2V617F e com a expressão dos genes a1, bcl-w, bik, bax, c-iap-2 e trail. Com relação à expressão proteica, BCL-XL estava aumentada e TRAIL diminuída em leucócitos de pacientes com MF enquanto que a molécula BID estava diminuída em leucócitos de pacientes com TE. Os miRNA26a, let 7d e miR15a estavam mais expressos nos leucócitos de pacientes com TE e MF. Em pacientes com TE o miR130b estava elevado e o mIR16 diminuído, enquanto que nos pacientes com MF o miR198 estava aumentado. Os linfócitos dos pacientes com TE e MF apresentaram maior resistência à apoptose estimulada por: actinomicina, etoposídeo, teniposídeo, citarabina e estaurosporina do que os linfócitos dos controles. Em conclusão, os dados obtidos sugerem a ligação da alteração do processo de apoptose celular, com a expressão do gene PRV-1 e status da mutação JAK2V617F. Esses resultados contribuem para o melhor entendimento da fisiopatologia das NMPC visto que associam a fisiopatologia da TE e MF com a resistência das células alteradas à apoptose. Essas informações serão úteis no futuro, possibilitando o desenho de novos alvos terapêuticos e a descrição de novos marcadores de prognóstico. / Chronic Myeloproliferative Neoplasms (cMPN) - Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF) and Polycythemia Vera (PV) - are clonal hematopoietic stem cell malignancies characterized by an accumulation of mature myeloid cells in bone marrow and peripheral blood. It seems that apoptotic machinery deregulation contribute to ET and PMF pathogenesis. Despite the advances in the molecular knowledge, the physiopathology of these diseases remains unknown. This study investigated cellular and molecular mechanisms involved in apoptosis process regulation in bone marrow haematopoietic progenitor CD34+ cells and leukocytes from ET and PMF patients. The specifics aims were: (1) to evaluate death receptors family members and Bcl-2 related genes expression as well apoptosis-related microRNAs by Real Time PCR; (2) apoptosis-related protein expression by Western Blot; (3) access mononuclear cells apoptosis resistance by flow cytometry and (4) to correlate the results with JAK2V617F mutation, PRV-1 gene expression and as well clinical data. In ET CD34+ cells, we found overexpression of a1, mcl-1, bid, bok e noxa and a decrease of bax compared to controls, while in leukocytes a1, bcl-2, bcl-w e bcl-xL, bad e bok expression was increased and bid and bimEL expression was lower than in controls. In PMF, a1, bcl-w bak, bok e noxa were overexpressed and bcl-2 downregulated in CD34+ cells. In PMF leukocytes bcl-2, bcl-w e bcl-xL, bad e bok mRNA levels were increased. c-iap-1 was increased in ET and PMF CD34+ cells and c-iap-2 expression elevated in ET and PMF CD34+ cells and leukocytes. Death receptor related genes showed overexpression of fas, fas-L, faim and dr4 in patients CD34+ cells and downregulation of fas-L and trail in ET and PMF leukocytes. We found differential expression of several genes between patients JAK2V617F positive and negative, as well we found correlation between gene expression and JAK2V617F allele burden, white blood cells and platelets count and splenomegaly. PRV-1 gene was overexpressed in ET and PMF leukocytes and showed correlation with JAK2V617F mutation and a1, bcl-w, bik, bax, c-iap-2 and trail gene expression. Regarding protein expression, BCL-XL was increased and TRAIL decreased in PMF leukocytes and BID was decreased in ET leukocytes. miRNA26a, let 7d e miR15a was overexpressed in ET and PMF leukocytes, while miR130b was increased only in ET and miR198 only in PMF. miR16 was downregulated in ET leukocytes comparing to controls. We also detected a resistance to apoptosis-inducers in ET and PMF lymphocytes and we observed correlation between apoptosis percentage and the expression of many studied genes. In conclusion, the results indicate the participations of Bcl-2 family genes and Death Receptor pathway genes, as well PRV-1 and JAK2V617F mutation in these disorders, which contribute to elucidate cMPN physiopathology and might lead to the discovery of new cMPN therapies and molecular markers.
53

Expressão de microRNAs em leucócitos e células CD34+ em Policitemia Vera / microRNA expression in leukocytes and CD34+ cells in Polycythemia Vera

Natália de Souza Nunes 25 January 2012 (has links)
A Neoplasia Mieloproliferativa Crônica (NMPC)- Politemia Vera é uma desordem clonal caracterizada pelo acúmulo de eritrócitos, leucócitos, plaquetas e progenitores normais na ausência de um estímulo definido. Apesar dos avanços no diagnóstico de PV e da descrição de mecanismos envolvidos no estabelecimento da doença sua patogênese permanece desconhecida entretanto, alterações no mecanismo regulador da apoptose parecem estar envolvidos em sua fisiopatologia. A compreensão acerca do funcionamento da maquinaria apoptótica e sua possível regulação por microRNAs em pacientes com Policitemia Vera parece revelar novos alvos para estudo e consequentemente transformar-se em novas terapias para a doença. Neste contexto os objetivos deste trabalho foram avaliar em leucócitos de sangue periférico e células CD34+ de medula óssea dos pacientes de PV: (1) a quantificação da expressão de microRNAs cujos alvos são RNAs associados a regulação da apoptose; (2) Correlação dos níveis de expressão dos miRNAs com os de RNAm das moléculas pró e antiapoptóticas da família Bcl-2 e dos receptores de morte; (3) Correlação dos níveis de expressão dos miRNAs com os seguintes dados clínico-laboratoriais dos pacientes: concentração de hemoglobina, hematócrito e percentagem da mutação JAK2. Os pacientes com Policitemia Vera apresentam aumento de expressão dos microRNAs 29c, 16, 21, 26a, 130b e let-7d e diminuição de miR15a e 34c em leucócitos de sangue periférico; Aumento de expressão dos miRs 29c, 16 e 21; diminuição na expressão de miR 130b e let-7d em células CD34+ ; alteração na expressão de genes pró e anti-apoptóticos em leucócitos de sangue periférico com aumento de a1, mcl-1 e diminuição de bcl-2, ciap-2, bax e fas-L,alteração na expressão de genes pró e anti-apoptóticos em células CD34+ com aumento de expressão de fas, bid, mcl-1, bcl-xl e c-flip; diminuição na expressão de bik. Observamos diminuição na expressão protéica de BCL-2 em pacientes quando comparado aos indivíduos controle.Notamos também a correlação entre a alteração na expressão de microRNAs e seus respectivos genes alvo e destes com parâmetros hematológicos analisados. Os linfócitos dos pacientes de PV apresentam maior resistência a apoptose do que os indivíduos controles quando induzidos por: Actinomicina, etoposídeo, citarabina e cicloheximida. Concluindo, os dados obtidos sugerem a participação dos microRNAs na regulação da maquinaria apoptótica e a participação desta desregulação na fisiopatologia da PV. Estes resultados contribuem para o melhor entendimento da fisiopatologia da PV e serão úteis futuramente para o desenho de novos alvos terapêuticos e descrição de marcadores de prognóstico. / Polycythaemia vera (PV) is a clonal disorder characterized by an accumulation of normal red and white cells, platelets, and their progenitors in absence of a definable stimulus. Despite the advances in PV diagnosis and the description of the mechanisms involved in disease establishment its pathogenesis remains unclear. The apoptosis deregulation might have a role in PV physiopathology. Fully understanding the basic apoptotic pathway and its potential regulation by microRNA in PV patients cells might unveil targets for manipulation, which may be translated into novel therapies for disease.The aims of this study were to avaluate in leukocytes and CD34+ cells: (1) The microRNA expression whose RNA target are associated with apoptosis regulation (2) Correlation between microRNA expression and the mRNA levels of anti and pro-apoptotic family Bcl-2 expression and death receptors; (3) Correlation between microRNA expression and the clinical data of patients: hemoglobin concentration, hematocrit and JAK2 percentage. Patientes with Polycythemia Vera shows increased expression of microRNAs 29c, 16, 21, 26a, 130b and let-7d and 34c and 15a decrease in peripheral leukocytes; incresead expression of miRs 29c, 16 and 21, decrease in miR130b and let-7d expression in CD34+ cells; deregulation in pro and anti-apoptotic gene expression in peripheral leukocytes with increase in a1, mcl-1 and decrease in bcl-2, ciap-2, bax and fas-L, deregulation in pro and anti-apoptotic gene expression in CD34+ cells with increased expression of fas, bid, mcl-1, bcl-xl and c-flip and decreased expression of bik. Decreased in proteic expression of BCL-2 in peripheral leukocytes of patients when compared to controls. Correlation between de deregulated expression of microRNA and their genes target and those with hematological parameters. Lymphocytes from PV patients shows higher resistance to apoptosis than controls when induced by: Actinomicin, etoposide, cytarabine and cycloheximide. In conclusion the data suggest the involvement of microRNA in apoptosis regulation and the involvement of apoptosis machinery in the PV pathophysiology. These results will contribute for a better understanding of PV pathophysiology and will be useful for the discover of future therapies.
54

Estudo do perfil genético de pacientes com Neoplasias Mieloproliferativas (NMP) cromossomo Filadélfia negativo / Study of genetic profile of patients with Philadelphia-negative Myeloproliferative Neoplasms (MPN)

Marchiani, Mariana 26 February 2016 (has links)
As neoplasias mieloproliferativas (NMPs) Filadelfia negativo como a policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primária (MFP) são desordens clonais da célula tronco hematopoiética caracterizadas pela produção excessiva de células mielóides diferenciadas. Este fenômeno ocorre devido à uma mutação somática (JAK2V617F) que ativa a via JAK-STAT de transdução de forma constitutiva. Esta mutação é mais frequente na PV, ocorrendo em 95% dos casos, e em 50% dos casos de TE e MFP. Outro defeito genético que ocorre é a mutação no receptor de trombopoetina, MPL. As mutações em MPL podem ser germinativas ou somáticas e menos de 10% dos pacientes com TE e MFP apresentam essa alteração genética. Entretanto, grande parte dos pacientes com TE e MFP que não apresentam mutação em JAK2V617F ou MPL podem apresentar mutações somáticas no gene CALR. Em adição às mutações somáticas que causam mieloproliferação, outras alterações genéticas em genes que funcionam como reguladores epigenéticos são encontrados nas NMPs nos genes TET2, IDH1, IDH2 e ASLX1. Objetivo: Estabelecer um perfil genético em pacientes com NMP através da avaliação de mutações nos genes JAK2, CALR, MPL, IDH1, IDH2, TET2 e ASXL1 assim como estabelecer uma correlação laboratorial destas na PV, TE e MFP. Casuística e Métodos: Foram utilizadas amostras de sangue periférico de 104 pacientes que foram enviadas para o Laboratório de Biologia Tumoral do Serviço de Hematologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo para avaliação diagnóstica. Quinze dos 104 pacientes são de pacientes com PV (14,4%), 20/104 (19,2%) com MFP, 20/104 (19,2%) com TE e 49/104 (47,1%) com outras doenças hemtológicas. Foi feita a avaliação da prevalência de mutações somáticas, seja por sequenciamento ou análise de fragmentos, nos genes JAK2 (exon 12 e Y931C), MPL, IDH1, IDH2, CALR, TET2 e ASXL1. PCR-RFLP foi realizada para identificação de mutações em JAK2V617F. Resultados: A mutação JAK2V617F foi observada em 30 (28,8%) pacientes (12 PV, 11 TE e 7 MFP), a mutação JAK2 exon 12 foi observada em apenas um (0,96%) paciente com PV, mutação JAK2Y931C em 4 (3,8%) pacientes (1 PV, 2 TE e 1 MFP) e 8 (7,7%) pacientes apresentaram mutações em CALR (3 TE e 5 MFP). Mutações nos genes epigenéticos como IDH1 foram observadas em 9 (8,7%) pacientes (2 TE, 2 MFP, 1 SMD, e 4 pacientes com suspeita de NMP), mutações em IDH2 estão presentes em 5 (4,8%) pacientes (2 TE, 1 SMD/leucemia e 4 pacientes com suspeita de NMP), mutações em ASXL1 foram identificadas em 13 (12,5%) pacientes (1 PV, 3 TE, 2 MFP, 3 SMD/leucemias e 4 com suspeita de NMP) e finalmente, mutações em TET2 foram encontradas em 33 (31,7%) pacientes (3 PV, 5 TE, 4 MFP, 8 SMD/leucemias e 13 pacientes com suspeita de NMP). Além disso, no caso da PV, os pacientes que apresentam mutações em JAK2V617F apresentam valores aumentados de plaquetas (mediana de 5,41 x 105/mm3 plaquetas) em relação aos pacientes sem a mutação (mediana de 2,06 x 105/mm3 plaquetas), com diferença estatística (p=0,031). Pacientes do mesmo grupo que apresentam mutações em TET2 apresentam, opostamente aos com mutações em JAK2V617F, menores valores de plaquetas (mediana de 1,75 x105/mm3 plaquetas) em relação aos pacientes sem mutações no gene TET2 (mediana de 5,41 x 105/mm3 plaquetas), com diferença estatística (p=0,048). No caso da MFP, os pacientes que apresentam mutações em JAK2V617F apresentam valores maiores de leucócitos (mediana de 1,09 x104/mm3 leucócitos) do que os pacientes que não apresentam a mutação (mediana de 6,99 x103/mm3 leucócitos) com diferença estatística (p=0,046), já os pacientes que apresentam mutações no gene ASXL1 apresentam valores menores de hemácias (mediana de 2,43 x106/mm3 hemácias) em relação aos pacientes que não apresentam mutação (mediana de 3,71 x106/mm3 hemácias) com diferença estatística (p=0,042). Conclusão: O trabalho permitiu fornecer um perfil genético dos pacientes com NMP estudados. Além disso, é possível observar que algumas mutações epigenéticas podem influenciar em diferenças clínicas / Myeloproliferative neoplasms (MPNs) Philadelphia (Ph) chromosome negative, such as polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are clonal disorders of hematopoietic stem cell characterized by increased proliferation of differentiated myeloid cells. This phenomenon occurs due somatic mutation (JAK2V617F) that constitutively stimulates the JAK-STAT signaling pathway. This mutation is more frequent in PV, around 95%, and between 50% in ET and PMF. Other genetic aberration can be observed in the thrombopoietin (TPO) receptor MPL. Mutations in MPL can be in the germline line or somatic and less than 10% of patients with TE or PMF would harbor this genetic alteration. Otherwise, patients with TE or PMF without JAK2V617F or MPL mutation could present somatic mutations in calreticulin (CALR). In addition to somatic mutations that cause myeloproliferation, other genetic alterations that function as epigenetic regulators were identified in genes as TET2, IDH1, IDH2 e ASLX1 in MPN. Objective: Establish genetic profile in patients with diagnosis of PV, ET, and PMF through genetic alterations in the following genes: JAK2, MPL, CALR, IDH1, IDH2, TET2 e ASXL1, and correlate those alterations with demographic characteristic of the study population. Casuistic and Methods: Peripheral blood samples from 104 patients referred to the Tumor Biology Laboratory of the Department of Hematology of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo for diagnostic investigation were analyzed. Fifteen out 104 samples were from PV patients (14.4%), 20/104 (19.2%) in PMF, 20/104 (19.2%) in ET and 49/104 (47.1%) with other hematologic diseases. Identification of somatic mutations was made, either by direct sequencing or fragment analysis in JAK2 (exon 12 and Y931C), MPL, IDH1, IDH2, CALR, TET2 and ASXL1. PCR-RFLP was performed to identify JAK2V617F mutation. Results: JAK2V617F mutation was observed in 30 (28.8%) patients (12 PV, 11 ET and 7 PMF), JAK2 exon 12 in only one (0.96%) patient with PV, JAK2Y931C in 4 (3.8%) patients (1 PV, 2 ET and 1 PMF), and 8 patients (7.7%) presented CALR mutation (3 ET and 5 PMF). Mutations in the epigenetic genes as IDH1 were observed in 9 (8.7%) patients (2 ET, 2 PMF, 1 MDS and 4 patients with suspected MPN), IDH2 mutations were present in 5 (4.8%) patients (2 ET, 1 MDS/leukemia, and 4 patients with suspected MPN), ASLX1 mutations were identified in 13 (12.5%) patients (1 PV, 3 ET, 2 PMF, 3 MDS/leukemia and 4 with suspected MPN) and finally, TET2 mutations were present in 33 (31.7%) patients (3 PV, 5 ET, 4 PMF, 8 MDS/leukemia, and 13 with suspected MPN). In addition, patients with PV who harbor JAK2V617F have increased platelet counts (median 5.41 x 105/mm3 platelets) compared to those without the mutation (median 2.06 x 105/mm3 platelets, p=0.031). Patients in the same group with TET2 mutation, as opposed to those with JAK2V617F, presented low platelets counts (median of 1.75 x 105/mm3 platelets) compared to those without TET2 mutation (median 5.41 x 105/mm3 platelets, p=0.048). Presence of JAK2V617F in patients diagnosed with PMF have a greater number of leukocytes (median 1.09 x104/mm3 leukocytes) when compared to patients without the mutation (median 6.99 x 103/mm3 leukocytes, p=0.046). Patients with PMF who presented mutations in ASXL1 gene have a lower number of red blood cells (median of 2.43 x 106/mm3) compared to patients without mutations in the same gene (median 3.71 x 106/mm3, p=0.042). Conclusion: The present study allows us to provide a genetic profile of patients with MPN. Furthermore, it is possible to observe that some epigenetic mutations could influence in some clinical differences
55

Molecular and cellular analysis of Lhx2 function in hematopoietic stem cells

Richter, Karin January 2007 (has links)
The formation of blood, hematopoiesis, is a dynamic process originating from a small number of hematopoietic stem cells (HSCs). To sustain hematopoiesis throughout life HSCs have the unique capacity to differentiate into all mature hematopoietic lineages as well as generating more HSCs by a mechanism referred to as self-renewal. However, the regulation of these processes is largely unknown. During embryonic development HSCs expand in the fetal liver, indicating that this environment supports HSC self-renewal. The LIM-homeobox gene Lhx2 is expressed in the fetal liver during this period and Lhx2 null mutant mice die in utero due to severe anemia caused by an environmental defect in the fetal liver. Embryonic stem cells differentiate in vitro, forming embryoid bodies (EBs) containing various tissues including hematopoietic progenitor cells. Introduction of Lhx2 into this system by retroviral transfer led to the generation of cytokine dependent HSC-like cell lines that were multipotent and expressed surface markers similar to embryonic HSCs. However, the specificity and efficiency of this event could not be elucidated. To further evaluate the function of Lhx2 expression during hematopoietic development, Lhx2 was introduced into an ES cell system where expression could be efficiently turned on. This approach revealed that Lhx2 induce self-renewal of distinct multipotent hematopoietic progenitor/stem cells present in the EB, with the ability to form HSC-like cell lines. The Lhx2 induced self-renewal is growth factor specific since stem cell factor and interleukin-6 are necessary and sufficient for this process. However, Lhx2 expression blocked erythroid differentiation and interfered with early ES cell commitment, indicating that the effect of Lhx2 is cell type specific. Since HSCs of early embryonic origin are inefficient in engrafting adult recipients upon transplantation, we wanted to address whether we could generate cell lines retaining this capacity by expression of Lhx2 in hematopoietic cells from adult bone marrow. This led to the generation of clonal and cytokine dependent HSC-like cell lines capable of generating erythroid, myeloid and lymphoid cells upon transplantation into lethally irradiated recipients. When transplanted into stem cell-deficient mice, they contributed to circulating erythrocytes for at least 18 months, revealing a remarkable potential for self-renewal and differentiation in vivo. However, expression of Lhx2 was maintained in vivo and most engrafted mice developed a transplantable myeloproliferative disorder resembling human chronic myeloid leukemia. Thus, elucidation of the mechanism for Lhx2 function in HSC-like cell lines would give insights into both normal and pathological regulation of HSCs. Down-regulation of Lhx2 expression in HSC-like cell lines with inducible Lhx2 expression led to rapid loss of stem cell characteristics and differentiation into various hematopoietic cell types. Thus, global gene expression analysis comparing Lhx2+ HSC-like cell lines to their Lhx2- progeny would give insights into the molecular basis for Lhx2 function in stem cells. A number of differentially expressed genes overlapped with previously reported HSC enriched genes, further emphasizing the resemblance between HSCs and the HSC-like cell lines also at the molecular level. Moreover, a number of genes were identified with functions or expression patterns related to Lhx2 in other organs. Collectively, these data suggest that these HSC-like cell lines represent a relevant model system for normal HSCs on the molecular and the functional level as well as for evaluating Lhx2 function in the development of various tissues in the embryo as well as in disease.
56

JAK2V617F-positive Myeloproliferative Neoplasms : KI mouse models, Interferon-α therapy and clonal architecture

Hasan, Salma 27 November 2013 (has links) (PDF)
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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Néoplasies myéloprolifératives et thromboses : épidémiologie et identification des facteurs de risque / Myeloproliferative neoplasms and thromboses : epidemiology and identification of thrombotic risk factors

Ianotto, Jean-Christophe 16 April 2018 (has links)
Les néoplasies myéloprolifératives (NMP) sont des hémopathies myéloïdes clonales, chroniques et prolifératives. Les plus fréquentes sont la polyglobulie de Vaquez et la thrombocytémie essentielle. Elles s’accompagnent de risques importants de thrombose (artérielles et veineuses) et de transformation en pathologies plus agressives (myélofibrose secondaire et leucémie aigüe). Les thromboses peuvent être la situation diagnostique de ces maladies, ou survenir au cours de la prise en charge. Le sujet de cette thèse est d’étudier la relation clinique entre NMP et thrombose. Dans un contexte de survenue de thrombose veineuse idiopathique, sans antécédent NMP, nous nous sommes intéressés à la recherche de mutation clonale chez les patients comme moyen diagnostique d’une NMP. Nous avons ainsi exploité la cohorte EDITH du CIC en prenant les patients ayant expérimentés un puis une récurrence thrombotique. A l’inverse, nous avons constitué une base de données (OBENE) des patients pris en charge pour une NMP au CHRU de Brest.Nous avons ensuite exploité cette base, en analysant la fréquence et l’impact des arythmies cardiaques auriculaires, la balance bénéfice-risque à l’utilisation des NACO, l’impact des statines sur la réduction du risque de thrombose ainsi que la fréquence et l’impact de la nonadhérence aux traitements dans les PV et TE.NMP et thromboses sont liées, il est donc nécessaire d’approfondir les connaissances de leur physiopathologie spécifique pour améliorer la prévention et le traitement des épisodes. Cette thèse amène quelques réponses àcertaines questions mais elle est surtout le point de départ de réflexion commune entre les praticiens et biologistes intéressés par ces domaines. / The myeloproliferative neoplasms (MPN) are clonal myeloid, chronic and proliferative disorders. The most frequent are polycythemia vera and essential thrombocythemia. The more frequent complications are thromboses (arterial and venous) and phenotypic evolutions (secondary myelofibrosis and acute leukemia). Thromboses can be a situation of diagnosis or observed during the followup of a MPN. This thesis is focused on the clinical link between MPN and thromboses.In a context of idiopathic venous thromboses (first event or recurrence), without medical history of MPN, we have tested patients for the most frequent MPN clonal mutations. So, we have used the informations and patients of the dedicated EDITH cohort.On the other hand, we have constituted a MPN database (OBENE) of the patients diagnosed for MPN in our Hospitalcentre. By this way, we have analysed the frequency and impact of atrial arrhythmias, the benefit-risk balance of the use of DOAC, the impact of statins to reduce the thrombotic risk and the frequency and impact of the treatment nonadherence in this population.MPN and thromboses are linked, so it is necessary to increase our knowledge of their physiopathology to improve prevention and treatment of the events. This thesis brings some answers to some questions but, she is almost the starting point of common reflexion between clinicians and biologists interested in these domains.
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Estudo do perfil genético de pacientes com Neoplasias Mieloproliferativas (NMP) cromossomo Filadélfia negativo / Study of genetic profile of patients with Philadelphia-negative Myeloproliferative Neoplasms (MPN)

Mariana Marchiani 26 February 2016 (has links)
As neoplasias mieloproliferativas (NMPs) Filadelfia negativo como a policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primária (MFP) são desordens clonais da célula tronco hematopoiética caracterizadas pela produção excessiva de células mielóides diferenciadas. Este fenômeno ocorre devido à uma mutação somática (JAK2V617F) que ativa a via JAK-STAT de transdução de forma constitutiva. Esta mutação é mais frequente na PV, ocorrendo em 95% dos casos, e em 50% dos casos de TE e MFP. Outro defeito genético que ocorre é a mutação no receptor de trombopoetina, MPL. As mutações em MPL podem ser germinativas ou somáticas e menos de 10% dos pacientes com TE e MFP apresentam essa alteração genética. Entretanto, grande parte dos pacientes com TE e MFP que não apresentam mutação em JAK2V617F ou MPL podem apresentar mutações somáticas no gene CALR. Em adição às mutações somáticas que causam mieloproliferação, outras alterações genéticas em genes que funcionam como reguladores epigenéticos são encontrados nas NMPs nos genes TET2, IDH1, IDH2 e ASLX1. Objetivo: Estabelecer um perfil genético em pacientes com NMP através da avaliação de mutações nos genes JAK2, CALR, MPL, IDH1, IDH2, TET2 e ASXL1 assim como estabelecer uma correlação laboratorial destas na PV, TE e MFP. Casuística e Métodos: Foram utilizadas amostras de sangue periférico de 104 pacientes que foram enviadas para o Laboratório de Biologia Tumoral do Serviço de Hematologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo para avaliação diagnóstica. Quinze dos 104 pacientes são de pacientes com PV (14,4%), 20/104 (19,2%) com MFP, 20/104 (19,2%) com TE e 49/104 (47,1%) com outras doenças hemtológicas. Foi feita a avaliação da prevalência de mutações somáticas, seja por sequenciamento ou análise de fragmentos, nos genes JAK2 (exon 12 e Y931C), MPL, IDH1, IDH2, CALR, TET2 e ASXL1. PCR-RFLP foi realizada para identificação de mutações em JAK2V617F. Resultados: A mutação JAK2V617F foi observada em 30 (28,8%) pacientes (12 PV, 11 TE e 7 MFP), a mutação JAK2 exon 12 foi observada em apenas um (0,96%) paciente com PV, mutação JAK2Y931C em 4 (3,8%) pacientes (1 PV, 2 TE e 1 MFP) e 8 (7,7%) pacientes apresentaram mutações em CALR (3 TE e 5 MFP). Mutações nos genes epigenéticos como IDH1 foram observadas em 9 (8,7%) pacientes (2 TE, 2 MFP, 1 SMD, e 4 pacientes com suspeita de NMP), mutações em IDH2 estão presentes em 5 (4,8%) pacientes (2 TE, 1 SMD/leucemia e 4 pacientes com suspeita de NMP), mutações em ASXL1 foram identificadas em 13 (12,5%) pacientes (1 PV, 3 TE, 2 MFP, 3 SMD/leucemias e 4 com suspeita de NMP) e finalmente, mutações em TET2 foram encontradas em 33 (31,7%) pacientes (3 PV, 5 TE, 4 MFP, 8 SMD/leucemias e 13 pacientes com suspeita de NMP). Além disso, no caso da PV, os pacientes que apresentam mutações em JAK2V617F apresentam valores aumentados de plaquetas (mediana de 5,41 x 105/mm3 plaquetas) em relação aos pacientes sem a mutação (mediana de 2,06 x 105/mm3 plaquetas), com diferença estatística (p=0,031). Pacientes do mesmo grupo que apresentam mutações em TET2 apresentam, opostamente aos com mutações em JAK2V617F, menores valores de plaquetas (mediana de 1,75 x105/mm3 plaquetas) em relação aos pacientes sem mutações no gene TET2 (mediana de 5,41 x 105/mm3 plaquetas), com diferença estatística (p=0,048). No caso da MFP, os pacientes que apresentam mutações em JAK2V617F apresentam valores maiores de leucócitos (mediana de 1,09 x104/mm3 leucócitos) do que os pacientes que não apresentam a mutação (mediana de 6,99 x103/mm3 leucócitos) com diferença estatística (p=0,046), já os pacientes que apresentam mutações no gene ASXL1 apresentam valores menores de hemácias (mediana de 2,43 x106/mm3 hemácias) em relação aos pacientes que não apresentam mutação (mediana de 3,71 x106/mm3 hemácias) com diferença estatística (p=0,042). Conclusão: O trabalho permitiu fornecer um perfil genético dos pacientes com NMP estudados. Além disso, é possível observar que algumas mutações epigenéticas podem influenciar em diferenças clínicas / Myeloproliferative neoplasms (MPNs) Philadelphia (Ph) chromosome negative, such as polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are clonal disorders of hematopoietic stem cell characterized by increased proliferation of differentiated myeloid cells. This phenomenon occurs due somatic mutation (JAK2V617F) that constitutively stimulates the JAK-STAT signaling pathway. This mutation is more frequent in PV, around 95%, and between 50% in ET and PMF. Other genetic aberration can be observed in the thrombopoietin (TPO) receptor MPL. Mutations in MPL can be in the germline line or somatic and less than 10% of patients with TE or PMF would harbor this genetic alteration. Otherwise, patients with TE or PMF without JAK2V617F or MPL mutation could present somatic mutations in calreticulin (CALR). In addition to somatic mutations that cause myeloproliferation, other genetic alterations that function as epigenetic regulators were identified in genes as TET2, IDH1, IDH2 e ASLX1 in MPN. Objective: Establish genetic profile in patients with diagnosis of PV, ET, and PMF through genetic alterations in the following genes: JAK2, MPL, CALR, IDH1, IDH2, TET2 e ASXL1, and correlate those alterations with demographic characteristic of the study population. Casuistic and Methods: Peripheral blood samples from 104 patients referred to the Tumor Biology Laboratory of the Department of Hematology of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo for diagnostic investigation were analyzed. Fifteen out 104 samples were from PV patients (14.4%), 20/104 (19.2%) in PMF, 20/104 (19.2%) in ET and 49/104 (47.1%) with other hematologic diseases. Identification of somatic mutations was made, either by direct sequencing or fragment analysis in JAK2 (exon 12 and Y931C), MPL, IDH1, IDH2, CALR, TET2 and ASXL1. PCR-RFLP was performed to identify JAK2V617F mutation. Results: JAK2V617F mutation was observed in 30 (28.8%) patients (12 PV, 11 ET and 7 PMF), JAK2 exon 12 in only one (0.96%) patient with PV, JAK2Y931C in 4 (3.8%) patients (1 PV, 2 ET and 1 PMF), and 8 patients (7.7%) presented CALR mutation (3 ET and 5 PMF). Mutations in the epigenetic genes as IDH1 were observed in 9 (8.7%) patients (2 ET, 2 PMF, 1 MDS and 4 patients with suspected MPN), IDH2 mutations were present in 5 (4.8%) patients (2 ET, 1 MDS/leukemia, and 4 patients with suspected MPN), ASLX1 mutations were identified in 13 (12.5%) patients (1 PV, 3 ET, 2 PMF, 3 MDS/leukemia and 4 with suspected MPN) and finally, TET2 mutations were present in 33 (31.7%) patients (3 PV, 5 ET, 4 PMF, 8 MDS/leukemia, and 13 with suspected MPN). In addition, patients with PV who harbor JAK2V617F have increased platelet counts (median 5.41 x 105/mm3 platelets) compared to those without the mutation (median 2.06 x 105/mm3 platelets, p=0.031). Patients in the same group with TET2 mutation, as opposed to those with JAK2V617F, presented low platelets counts (median of 1.75 x 105/mm3 platelets) compared to those without TET2 mutation (median 5.41 x 105/mm3 platelets, p=0.048). Presence of JAK2V617F in patients diagnosed with PMF have a greater number of leukocytes (median 1.09 x104/mm3 leukocytes) when compared to patients without the mutation (median 6.99 x 103/mm3 leukocytes, p=0.046). Patients with PMF who presented mutations in ASXL1 gene have a lower number of red blood cells (median of 2.43 x 106/mm3) compared to patients without mutations in the same gene (median 3.71 x 106/mm3, p=0.042). Conclusion: The present study allows us to provide a genetic profile of patients with MPN. Furthermore, it is possible to observe that some epigenetic mutations could influence in some clinical differences
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Rôle des cellules endothéliales JAK2V617F dans l’augmentation de l’angiogenèse des néoplasies myéloprolifératives. / Role of JAK2V617F endothelial cells in the increase of angiogenesis in myeloproliferative neoplasms.

Kilani, Badr 18 December 2015 (has links)
Les néoplasies myéloprolifératives (NMP) sont des maladies hématologiques acquises de la cellule souche hématopoïétique. Une mutation activatrice de la protéine de signalisation JAK2, JAK2V617F, a été identifiée chez la moitié des patients atteints de NMP Philadelphie négatives. Il a été rapporté que les patients avec des NMP avaient une augmentation du risque thrombotique et de la densité microvasculaire dans la rate et la moelle osseuse, sans explication physiopathologique claire. Des travaux récents ont mis en évidence la présence de la mutation JAK2V617F non seulement dans les cellules sanguines mais également dans les cellules endothéliales (CE) de ces patients. Nous faisons l’hypothèse que la présence de JAK2V617F dans les CE pourrait modifier leurs propriétés expliquant l’augmentation de l’angiogenèse dans les NMP. Pour répondre à cette hypothèse, nous avons voulu étudier le phénotype angiogénique des cellules endothéliales portant la mutation JAK2V617F. In vitro, nous disposons des particules lentivirales permettant d’obtenir des CE JAK2V617F par transduction lentivirale. In vivo, nous disposons des souris transgéniques exprimant la mutation JAK2V617F de manière conditionnelle (JAK2V617F/WT) grâce à la stratégie Cre-lox. Pour répondre à notre hypothèse, il été nécessaire de travailler avec des modèles murins exprimant la mutation JAK2V617F spécifiquement dans les CE sans atteinte concomitante de la lignée hématopoïétique. Dans un premier temps, nous avons voulu caractériser deux modèles endothéliaux inductibles couramment utilisés, Cdh5(PAC)-CreERT2 et Pdgfb-iCreERT2, en termes d’efficacité et de spécificité de recombinaison dans les cellules endothéliales vis-à-vis du compartiment hématopoïétique. Nous avons démontré que les souris adultes Cdh5(PAC)-CreERT2 pouvaient être utilisées comme modèles endothéliaux spécifiques, avec toutefois la mise en garde que la recombinaison est très variable entre les souris. Nous avons constaté que les souris PDGFB-iCreERT2 sont appropriées pour cibler les cellules endothéliales dans une large gamme d’organes à l'exception du foie, et devraient être utilisées dans les quatre premières semaines qui suivent l'induction, pour cibler un gène d’intérêt au niveau des cellules endothéliales, sans qu’il ait une atteinte concomitante dans la lignée hématopoïétique. Nous avons ensuite étudié les propriétés angiogéniques des cellules endothéliales JAK2V617F, in vitro en utilisant des HUVEC transduites avec un lentivirus permettant l’expression de JAK2V617F, et in vivo avec les souris Pdgfb-iCreERT2;JAK2V617F/WT. Nous avons démontré que les HUVEC JAK2V617F avaient un profil proangiogénique lié à une capacité proliférative élevée, résultant de l’activation de la voie JAK2/STAT3/PI3K. L’avantage hyperprolifératif que confère la mutation JAK2V617F aux cellules endothéliales a été confirmé in vivo avec le modèle de la vascularisation post-natale de la rétine, avec toutefois une diminution de la densité du réseau vasculaire due à une augmentation de la régression vasculaire au niveau de la rétine des souris Pdgfb-iCreERT2;JAK2V617F/WT. / Myeloproliferative neoplasms (MPNs) are acquired hematopoietic stem cell disorders. An activating mutation in the JAK2 signaling protein, JAK2V617F, was identified in half of the patients with Philadelphia chromosome-negative MPNs. It has been reported that patients with MPN had an increased risk of thrombosis but also an increased microvessel density in the spleen and bone marrow with no clear pathophysiological explanation. Several recent studies have demonstrated the presence of JAK2V617F mutation not only in blood cells but also in endothelial cells (EC) in MPN patients. We hypothesized that the presence of JAK2V617F in EC could change their properties leading to an increased angiogenesis process in MPNs. To address this question, our aim was to study the angiogenic phenotype of endothelial cells carrying the JAK2V617F mutation. For the in vitro experiments, we used lentiviral transduction of human JAK2V617F in EC. For the in vivo approach, we used transgenic mice (JAK2V617F/WT) that conditionally express JAK2V617F through Cre-lox strategy. To investigate our hypothesis, it was necessary to work with mice that express JAK2V617F specifically in EC without concomitant expression in hematopoietic cells. We first characterized two commonly-used inducible endothelial models, Cdh5(PAC)-CreERT2 and Pdgfb-iCreERT2, in terms of efficiency and specificity of recombination in endothelial cells. We showed that adult Cdh5(PAC)-CreERT2 mice can be used as specific endothelial model with however the wariness that recombination is highly variable among mice. We found that Pdgfb-iCreERT2 mice are appropriate to target endothelial cells in a wide range of organs except liver, and should be used within the four weeks after induction of Cre-mediated recombination to target a gene of interest in endothelial cells, without having a concomitant expression in hematopoietic lineage. We then studied the angiogenic properties of JAK2V617F endothelial cells, in vitro using JAK2V617F transduced HUVECs, and in vivo using Pdgfb-iCreERT2;JAK2V617F/WT mice. We observed that JAK2V617F HUVECs had a proangiogenic profile that was related to a highly proliferative potency, and that this phenotype results from a constitutive activation of JAK2/STAT3/PI3K pathway. The hyperproliferative advantage conferred by JAK2V617F to endothelial cells was confirmed in vivo using the postnatal vascularization model of the retina, with however a decrease in the density of the vascular network due to an increased vascular regression in Pdgfb-iCreERT2;JAK2V617F/WT mice’s retinas.
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Cooperative Drug Combinations Target Oncogenes and Tumor Suppressors in Cancer

Tyler John Peat (11790659) 19 December 2021 (has links)
<p>Multiple myeloma (MM) is a neoplasm involving plasma cells in the bone marrow. Drug resistance and progression are common, underscoring the need for new drug combinations. Utilizing a high-throughput screen of tool compounds to limit gro­­­­wth of human MM cell lines and <i>i</i><i>n silico</i> robust regression analysis of drug responses, potential synergistic combinations were identified. Further selection of effective combinations that reduce oncogenic MYC expression and enhance tumor suppressor p16 activity was based on earlier genetic and drug studies that identified MYC and p16 as appropriate targets in MM. Furthermore, the top three combinations synergistically reduced drug sensitive and resistant cell viability <i>in vitro</i> and the were effective in <i>ex vivo</i> treated patient cells Combination-associated survival was also prolonged in a transplantable Ras-driven allograft model of advanced MM that closely recapitulates MM in humans. One top drug combination was selected for further preclinical development. Targets, mechanism of action, and efficacy of the combination were evaluated through several <i>in vitro</i> and <i>in vivo </i>models, as well as <i>ex vivo</i> in myeloma patient cells. Effective targeting of the combination resulted in synergistic inhibition of proteasome inhibitor (PI) sensitive MM cells, as well as cell with induced PI resistance. Additionally, the combination was effective at delaying L363 MM xenograft growth in NSG mice and prolonging survival compared to single agent therapy. Finally, a cooperative signature of combined targeting was elucidated via RNA sequencing. These data identify potentially useful drug combinations for preclinical evaluation in drug-resistant MM and may ultimately reveal novel mechanisms of combined drug sensitivity.</p>

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