Quebec platelet disorder (QPD) is a bleeding disorder characterized by a gain of function defect in fibrinolysis. The hallmark feature of QPD is the marked overexpression of urokinase plasminogen activator (uPA) in megakaryocytes (MK) and platelets. The genetic cause of QPD is a tandem duplication of a ~78 kb region that encompasses the uPA gene, PLAU. As the mechanism of PLAU overexpression is unknown, gene regulatory mechanisms specifically epigenetics were evaluated at the PLAU locus in QPD MK and granulocytes, a QPD unaffected lineage. The aims of the thesis were to assess if QPD is associated with 1) genome wide methylation changes of promoter CpG islands, particularly at PLAU and 2) genome wide changes of active histone modifications H3K27Ac, H3K36me3 and H3K4me2, particularly at the region of PLAU duplication. Methylation and active histone enrichment analysis revealed that in QPD and control subjects, PLAU promoter CpG island was characterized by unaltered hypo-methylation and changes in active histone peak enrichments that were within the realm of having one extra copy of PLAU in both MK and granulocytes. The findings imply that the PLAU CNV mutation does not induce altered promoter methylation status and/or significantly alter active histone markers as the reason for the marked PLAU overexpression in QPD MK. Instead, the rearrangement of an active enhancer element, particularly an H3K27Ac enhancer expressed in MK but not granulocytes, that is upstream of the second copy of PLAU might underlie the marked PLAU expression by differentiated QPD MK. The thesis provides novel insights into the epigenetic regulation of PLAU that will be crucial to identifying the mechanism underlying the aberrant PLAU expression in QPD. / Thesis / Master of Science (MSc)

Hantaviren sind einzelsträngige RNA-Viren, die zur Familie der Bunyaviridae gehören. In den letzten Jahren haben sie immer mehr Aufmerksamkeit als “emerging viruses” auf sich gezogen, welche zunehmend Relevanz als humanpathogene Erreger gewinnen. Sie verursachen, abhängig vom Hantavirustyp, zwei verschiedene Krankheitsbilder: das hämorrhagische Fieber mit renalem Syndrom (HFRS) und das kardiopulmonale Syndrom (HCPS). Beide Syndrome sind mit Veränderungen in der vaskulären Permeabilität, akutem Abfall der Blutplättchen (Thrombozytopenie) und Koagulationsdefekten verbunden. Die zugrundeliegenden Mechanismen sind noch immer kaum verstanden. Hantaviren infizieren zwar Endothelzellen, bisher aber konnte kein zytopathischer Effekt nachgewiesen werden. Diese Doktorarbeit untersucht in vitro, ob und auf welche Weise Hantaviren mit Zellen des hämostatischen Systems interagieren. Zu diesen gehören insbesondere Megakaryozyten, die Thrombozyten generieren und die Thrombozyten selbst. Wir zeigen, dass das pathogene Hantaan Virus (HTNV), im Gegensatz zu dem weniger pathogenen Tula Virus (TULV) und Prospect Hill Virus (PHV), megakaryozytäre Zellen infiziert. Interessanterweise erhöht sich nach Induktion der Differenzierung in den infizierten megakaryozytären Zellen die Virusrepliaktion drastisch. Das Überleben der Zellen und der Verlauf der Differenzierung wird dadurch jedoch nicht beeinflusst. Ähnlich, wie bereits früher für Endothelzellen gezeigt, haben demnach pathogene Hantaviren keinen direkten zytopathischen Effekt auf Megakaryozyten. Weiterhin verdeutlicht die vorliegende Doktorarbeit, dass HTNV mit humanen Thrombozyten interagiert. Dies resultiert in einer Herunterregulation von essentiellen Oberflächenmarkern, die wichtig für die Aggregation und das Signalling sind. / Hantaviruses are single stranded (ss) RNA viruses belonging to the family Bunyaviridae. Over the past years they attracted more and more attention as “emerging viruses”, increasingly gaining relevance as human pathogens. Depending on the hantavirus type they cause haemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS). Both syndromes are associated with changes in vascular permeability, acute thrombocytopenia, and defects in platelet function. The underlying mechanisms are still poorly understood. Although hantaviruses infect endothelial cells, no viral cytopathic effect after infection was observed. This thesis investigates in vitro whether and how hantaviruses target cells of the haemostatic system. In particular megakaryocytes (MKs), which generate platelets, and platelets themselves. We show that pathogenic Hantaan virus (HTNV), in contrast to less pathogenic Tula virus (TULV) and Prospect Hill virus (PHV), infects megakaryocytic cells. Intriguingly, after induction of differentiation megakaryocytic cells switch from low-level to high-level HTNV production without reduction in cell survival or alteration in differentiation. Thus, there is no direct viral pathogenic effect on megakaryocytic cells as previously observed for endothelial cells. Furthermore, this study demonstrates that HTNV interacts with human platelets, resulting in downregulation of essential adhesion markers, which are important for platelet aggregation and signalling.

The parasite Rangelia vitalii is the etiologic agent of rangeliosis, a disease that leads to a wide variety of clinical signs, including hemostatic disorders characterized by bleeding. However, the causes of this process have not been established. The aim of this study was to evaluate the production of megakaryocytes, platelet count, clotting time, platelet activity and to determine the activity of enzymes that hydrolyze nucleotides and adenine nucleosides in platelets of dogs experimentally infected with R. vitalii. For this study, 12 dogs were separated in two groups: Group A was composed by five healthy dogs, and group B composed by seven dogs experimentally infected with R. vitalii. After inoculation, the animals were monitored by blood smears. The parasite was found within erythrocytes, neutrophils and monocytes five days post-inoculation (PI). Blood collection to perform platelet count, coagulation tests and measurement of platelet aggregation was performed on days 0, 10 and 20 PI. On days 10 and 20 PI, after this procedure, the dogs were anesthetized for collecting bone marrow samples to evaluate the production of megakaryocytes. To measure the activity of ectonucleotidases and adenosine deaminase, blood samples were collected on days 12 and 21 PI. Blood samples were stored in tubes containing EDTA to quantification of platelets, evaluation of platelet aggregation and measurement of enzymatic activity. The blood was stored in tubes containing citrate for realization of the clotting time. This study revealed a reduction (P<0.01) of platelet numbers in the infected group when compared to control group. Prothrombin time and active partial thromboplastin time showed no significant difference between infected and control animals. There was an increase (P<0.01) in the number of megakaryocytes in infected group when compared with control group. A reduction (P<0.01) of platelet aggregation was observed in dogs infected with R. vitalii. The hydrolysis of ATP, ADP, AMP and deamination of adenosine decreased (P<0.01) on day 12 PI. On day 21 PI, the activity of adenosine deaminase remained reduced (P<0.01), whereas it was observed an increase (P<0.05) in NTPDase levels. From these results, we can conclude that rangeliosis is responsible for severe thrombocytopenia during the acute phase of infection, and this can be due to splenic sequestration and/or immune-mediated thrombocytopenia. Also, alterations in purinergic system contribute to the occurrence of hemostatic disorders observed in this disease. / O parasito Rangelia vitalii é o agente etiológico da rangeliose, uma enfermidade que cursa com uma grande variedade de sinais clínicos, incluindo desordens hemostáticas caracterizadas por sangramentos. Entretanto, as causas das alterações no processo hemostático nessa doença ainda não foram esclarecidas. O objetivo deste estudo foi avaliar os distúrbios hemostáticos através da produção de megacariócitos e contagem de plaquetas, a coagulação sanguínea, a atividade plaquetária e determinar a atividade das enzimas que hidrolisam nucleotídeos e nucleosídeo de adenina em plaquetas de cães infectados experimentalmente com R. vitalii. Para este estudo, 12 cães foram separados em dois grupos: Grupo A foi composto por cinco cães saudáveis, e grupo B com sete cães infectados experimentalmente com R. vitalii. Após inoculação, os animais foram monitorados quanto à parasitemia por esfregaço sanguíneo. O parasito foi encontrado no interior de hemácias, neutrófilos e monócitos cinco dias pós-inoculação (PI). As coletas de sangue para a realização da contagem plaquetária, testes de coagulação e mensuração da agregação plaquetária foram realizadas nos dias 0, 10 e 20 PI. Nos dias 10 e 20 PI, após esse procedimento, os cães foram anestesiados para coleta do aspirado de medula óssea com a finalidade de avaliar a produção de megacariócitos. Para avaliar a atividade das ectonucleotidases e adenosina desaminase, as coletas de sangue foram realizadas nos dias 12 e 21 PI. Este estudo demonstrou redução (P<0,01) no número de plaquetas entre o grupo infectado em relação ao controle. Os tempos de protrombina e de tromboplastina parcial ativado não apresentaram diferença significativa entre animais infectados e controles. Ocorreu aumento (P<0,01) do número de megacariócitos no grupo infectado quando comparado com o controle. Foi observado uma diminuição (P<0,01) na agregação plaquetária em cães infectados por R. vitalii. A hidrólise de ATP, ADP, AMP e desaminação da adenosina foram reduzidas (P<0,01) no dia 12 PI quando comparadas com o grupo controle. No dia 21 PI, a atividade da adenosina desaminase manteve-se reduzida (P<0,01), enquanto que ocorreu aumento (P<0,05) na atividade da ecto-nucleosídeo trifosfato difosfoidrolase (NTPDase). A partir dos resultados, pode-se concluir que a rangeliose é responsável por grave trombocitopenia na fase aguda da infecção, a qual pode estar associada ao sequestro e/ou destruição imunomediada e, que alterações no sistema purinérgico contribuem para a ocorrência das desordens hemostáticas observadas na infecção pelo parasito R. vitalii.

Les mégacaryocytes sont des cellules de la moelle osseuse, à l’origine de la production des plaquettes sanguines. Quand elles arrivent à maturité, elles grossissent et émettent des prolongements de cytoplasme à travers la paroi des vaisseaux irriguant la moelle. Dans la circulation sanguine, ces prolongements, soumis aux forces de l’écoulement, s’allongent et se rompent pour former des plaquettes. Des techniques microfluidiques capables de produire des plaquettes in vitro existent et sont une alternative prometteuse au don. Mais le rendement reste à améliorer. Pour cela, il est nécessaire de mieux comprendre la fragmentation des mégacaryocytes en plaquettes. Ce travail de doctorat s’inscrit dans ce contexte et sera développé en deux axes principaux dans ce manuscrit. Dans une première partie nous développons une méthode pour trier des cellules en fonction de leur déformabilité, afin de savoir si les propriétés mécaniques d’un mégacaryocyte sont liées à leur stade de maturité. La méthode a d’abord été mise au point avec des microcapsules. Leurs propriétés mécaniques sont déterminées par analyse inverse à partir de la mesure de leur forme en écoulement dans des constrictions droites. Puis le dispositif utilisé a été miniaturisé pour s’adapter à la taille des cellules. Pour la caractérisation de leurs propriétés mécaniques, deux outils ont été utilisés: l’analyse inverse et la microscopie à force atomique sans pointe. Une deuxième partie porte sur l’étude de l’élongation et de la rupture de mégacaryocytes soumis écoulement. Nous avons quantifié les variations spatiotemporelles du taux d’élongation et développé un protocole d’ablation laser pour étudier les mécanismes de rupture de cellules en élongation. / When they mature in the bone marrow, the precursors of platelets, called megakaryocytes, grow and extend protrusions able to join blood circulation. There these protrusions elongate and break into platelets. Microfluidic techniques for in vitro platelet production represent a promising alternative to donation. In order to enhance platelet production and match the needs of clinical applications such as transfusion, we need to better understand the fragmentation of megakaryocytes into platelets. Our contribution will be described in this manuscript in two main axes. First, in order to know if mechanical properties of megakaryocytes can indicate their maturity stage, we develop a cell sorting method based on deformability. The method is first validated with microcapsules. Their mechanical properties are determined by inverse analysis from their shape under flow in straight microchannels. Then the device is downscaled. The characterization of cell mechanical properties are performed using inverse analysis and tipless atomic force microscopy. Second, we study megakaryocyte elongation and rupture in a microfluidic device. We quantify the spatial and temporal variations of the elongation rate and develop a laser ablation protocol to trigger and study the rupture of elongating cells.

No description available.

Vetores lentivirais são ferramentas fundamentais para modificação celular. Sua utilização ganhou destaque devido à capacidade desses em integrar ao genoma de células que estão ou não em divisão. Grande parte dos vetores desenvolvidos são derivados do genoma do Vírus da Imunodeficiência Humana (HIV-1), portanto, modificações foram necessárias a fim de evitar a formação de Partículas Competentes em Replicação (RCLs) e garantir uma utilização segura. Com as modificações, foram produzidos os vetores lentivirais de terceira geração utilizados atualmente. Esses vetores podem ser usados para expressão constitutiva de genes, produção de proteínas recombinantes, produção de animais transgênicos e terapia gênica. Com isso, torna-se necessário o desenvolvimento de vetores lentivirais para aplicação em pesquisa básica e ensaios clínicos. Dessa forma, o presente estudo teve por objetivo a construção de vetores de expressão lentivirais aplicáveis à: 1- expressão constitutiva de genes de interesse e 2-vetores com promotores específicos para expressão de proteínas em megacariócitos. Esse trabalho descreve a construção desses vetores, sua importância e discute suas possíveis aplicações. As sequências selecionadas para produção dos vetores foram: os genes Runx1C e VkorC1 e os promotores proPF4 e proITGA2b. Todas as sequências encontram-se clonadas em vetor de clonagem e estoques de bactérias com esses vetores congeladas em glicerol foram confeccionados. Para a confecção dos vetores lentivirais, o gene Runx1C foi subclonado no vetor lentiviral base p1054-CIGWS sob controle do promotor forte CMV, enquanto o promotor proITGA2b foi subclonado no vetor base p1054-FVIII, em substituição ao promotor CMV, de forma a controlar a expressão de FVIII. Os dois vetores produzidos apresentam ainda o gene para proteína verde GFP precedida do sítio de ligação do ribossomo IRES, com expressão controlada pelo mesmo promotor interno do vetor. O trabalho possibilitou, portanto, a produção de dois vetores lentivirais bi-cistrônicos: p1054-Runx1C e pL-proITGA2b-FVIII. A construção p1054-Runx1C ainda não foi sequenciada, mas foi confirmada por restrição enzimática e apresenta potencial para aplicação em estudos de diferenciação hematopoética. Já a construção pL-proITGA2b-FVIII foi sequenciada, porém sem confirmação da região de ligação do proITGA2b ao vetor. Reações de PCR e de restrição enzimática confirmaram a ligação e sequenciamento mostrou 67% de similaridade entre a região sequenciada e o promotor ITGA2b depositado no banco de dados. Análise funcional foi realizada através da transfecção desse vetor em células HEK-293T. As células transfectadas apresentaram expressão positiva para GFP e secreção de FVIII no sobrenadante celular, evidenciando que o promotor proITGA2b clonado no vetor encontra-se ativo. Esse vetor apresenta potencial para aplicação em terapia gênica para hemofilias, pois apresenta expressão do fator de coagulação direcionado a megacariócitos e plaquetas, células que estão diretamente relacionadas ao processo de coagulação, representando grandes veículos para secreção desses fatores. Ainda, os dois vetores lentivirais gerados apresentam segurança e eficiência elevadas, pois são vetores de terceira geração auto-inativantes (SIN) e apresentam elementos regulatórios que melhoram o transporte e integração do DNA ao genoma hospedeiro. / Lentiviral vectors are fundamental tools for cell modification that gained prominence due to their ability to integrate the genome of non-dividing cells. Most of developed lentiviral vectors are derived from the genome of Human Immunodeficiency Virus (HIV-1), so modifications were necessary in order to avoid the formation of Competent Replication Particles (RCLs) and ensure safer operations. The modifications led to development of third generation lentiviral vectors currently used. These vectors can be used for constitutive gene expression, production of recombinant protein, production of transgenic animals and gene therapy. It\'s evident the need to develop lentiviral vectors for application in basic research and clinical trials. Thus this study aimed to construct lentiviral expression vectors applicable to: 1- constitutive expression of genes of interest and 2-vectors with specific promoters for expression of proteins in megakaryocytes and platelets. This paper describes the construction of these vectors, their importance and discuss their possible applications. Sequences were selected for production of the vectors: genes Runx1C and VkorC1 and proPF4 and proITGA2b promoters. All four sequences are cloned into cloning vectors and stocks of bacteria with these vectors frozen in glycerol were prepared. Lentiviral vectors were engineered from subcloning the sequence Runx1C into the basic lentiviral vector p1054- CIGWS under control of the strong CMV promoter, and from subcloning proITGA2b promoter into p1054-FVIII basic vector, replacing the CMV promoter in order to control the expression of FVIII. Both vectors exhibit the green fluorescence protein GFP gene preceded by a ribosome binding site IRES under control of vector\'s internal promoter. Therefore, this work resulted in the production of two bi-cistronic lentiviral vectors: p1054-Runx1C and pLproITGA2b-FVIII. The p1054-Runx1C construction has not yet been sequenced, but it was confirmed by digestion and has potential for use in hematopoietic differentiation studies. Though, pL-proITGA2b-FVIII construct was sequenced, but the technique didn\'t allow to confirm the binding region between proITGA2b and the vector. Although PCR reaction and digestion confirmed the construction. Sequence analysis showed 67% similarity between the sequenced region and ITGA2b promoter deposited in the database. Functional analysis was performed by transfection of this vector in HEK-293T cells. The transfected cells showed positive expression of GFP and FVIII secretion in cell supernatant, indicating that the proITGA2b promoter cloned into the vector is active. This vector has potential usage in gene therapy for hemophilia, since it can be used to express coagulation factors in megakaryocytes and platelets and these cells are directly related to the clotting process, representing great vehicles for secretion of these factors. Even more, the two lentiviral vectors generated have higher safety and efficiency, as they are self-inactivating (SIN) third-generation vectors and have regulatory elements that enhance transport and integration of DNA into the host genome.

Vetores lentivirais são ferramentas fundamentais para modificação celular. Sua utilização ganhou destaque devido à capacidade desses em integrar ao genoma de células que estão ou não em divisão. Grande parte dos vetores desenvolvidos são derivados do genoma do Vírus da Imunodeficiência Humana (HIV-1), portanto, modificações foram necessárias a fim de evitar a formação de Partículas Competentes em Replicação (RCLs) e garantir uma utilização segura. Com as modificações, foram produzidos os vetores lentivirais de terceira geração utilizados atualmente. Esses vetores podem ser usados para expressão constitutiva de genes, produção de proteínas recombinantes, produção de animais transgênicos e terapia gênica. Com isso, torna-se necessário o desenvolvimento de vetores lentivirais para aplicação em pesquisa básica e ensaios clínicos. Dessa forma, o presente estudo teve por objetivo a construção de vetores de expressão lentivirais aplicáveis à: 1- expressão constitutiva de genes de interesse e 2-vetores com promotores específicos para expressão de proteínas em megacariócitos. Esse trabalho descreve a construção desses vetores, sua importância e discute suas possíveis aplicações. As sequências selecionadas para produção dos vetores foram: os genes Runx1C e VkorC1 e os promotores proPF4 e proITGA2b. Todas as sequências encontram-se clonadas em vetor de clonagem e estoques de bactérias com esses vetores congeladas em glicerol foram confeccionados. Para a confecção dos vetores lentivirais, o gene Runx1C foi subclonado no vetor lentiviral base p1054-CIGWS sob controle do promotor forte CMV, enquanto o promotor proITGA2b foi subclonado no vetor base p1054-FVIII, em substituição ao promotor CMV, de forma a controlar a expressão de FVIII. Os dois vetores produzidos apresentam ainda o gene para proteína verde GFP precedida do sítio de ligação do ribossomo IRES, com expressão controlada pelo mesmo promotor interno do vetor. O trabalho possibilitou, portanto, a produção de dois vetores lentivirais bi-cistrônicos: p1054-Runx1C e pL-proITGA2b-FVIII. A construção p1054-Runx1C ainda não foi sequenciada, mas foi confirmada por restrição enzimática e apresenta potencial para aplicação em estudos de diferenciação hematopoética. Já a construção pL-proITGA2b-FVIII foi sequenciada, porém sem confirmação da região de ligação do proITGA2b ao vetor. Reações de PCR e de restrição enzimática confirmaram a ligação e sequenciamento mostrou 67% de similaridade entre a região sequenciada e o promotor ITGA2b depositado no banco de dados. Análise funcional foi realizada através da transfecção desse vetor em células HEK-293T. As células transfectadas apresentaram expressão positiva para GFP e secreção de FVIII no sobrenadante celular, evidenciando que o promotor proITGA2b clonado no vetor encontra-se ativo. Esse vetor apresenta potencial para aplicação em terapia gênica para hemofilias, pois apresenta expressão do fator de coagulação direcionado a megacariócitos e plaquetas, células que estão diretamente relacionadas ao processo de coagulação, representando grandes veículos para secreção desses fatores. Ainda, os dois vetores lentivirais gerados apresentam segurança e eficiência elevadas, pois são vetores de terceira geração auto-inativantes (SIN) e apresentam elementos regulatórios que melhoram o transporte e integração do DNA ao genoma hospedeiro. / Lentiviral vectors are fundamental tools for cell modification that gained prominence due to their ability to integrate the genome of non-dividing cells. Most of developed lentiviral vectors are derived from the genome of Human Immunodeficiency Virus (HIV-1), so modifications were necessary in order to avoid the formation of Competent Replication Particles (RCLs) and ensure safer operations. The modifications led to development of third generation lentiviral vectors currently used. These vectors can be used for constitutive gene expression, production of recombinant protein, production of transgenic animals and gene therapy. It\'s evident the need to develop lentiviral vectors for application in basic research and clinical trials. Thus this study aimed to construct lentiviral expression vectors applicable to: 1- constitutive expression of genes of interest and 2-vectors with specific promoters for expression of proteins in megakaryocytes and platelets. This paper describes the construction of these vectors, their importance and discuss their possible applications. Sequences were selected for production of the vectors: genes Runx1C and VkorC1 and proPF4 and proITGA2b promoters. All four sequences are cloned into cloning vectors and stocks of bacteria with these vectors frozen in glycerol were prepared. Lentiviral vectors were engineered from subcloning the sequence Runx1C into the basic lentiviral vector p1054- CIGWS under control of the strong CMV promoter, and from subcloning proITGA2b promoter into p1054-FVIII basic vector, replacing the CMV promoter in order to control the expression of FVIII. Both vectors exhibit the green fluorescence protein GFP gene preceded by a ribosome binding site IRES under control of vector\'s internal promoter. Therefore, this work resulted in the production of two bi-cistronic lentiviral vectors: p1054-Runx1C and pLproITGA2b-FVIII. The p1054-Runx1C construction has not yet been sequenced, but it was confirmed by digestion and has potential for use in hematopoietic differentiation studies. Though, pL-proITGA2b-FVIII construct was sequenced, but the technique didn\'t allow to confirm the binding region between proITGA2b and the vector. Although PCR reaction and digestion confirmed the construction. Sequence analysis showed 67% similarity between the sequenced region and ITGA2b promoter deposited in the database. Functional analysis was performed by transfection of this vector in HEK-293T cells. The transfected cells showed positive expression of GFP and FVIII secretion in cell supernatant, indicating that the proITGA2b promoter cloned into the vector is active. This vector has potential usage in gene therapy for hemophilia, since it can be used to express coagulation factors in megakaryocytes and platelets and these cells are directly related to the clotting process, representing great vehicles for secretion of these factors. Even more, the two lentiviral vectors generated have higher safety and efficiency, as they are self-inactivating (SIN) third-generation vectors and have regulatory elements that enhance transport and integration of DNA into the host genome.

(ENGLISH) Hematopoiesis is dependent on the actions of hematopoietic stem cells (HSCs). This process is tightly controlled through a complex array of extrinsic and intrinsic factors. Even though the hematopoiesis seems to be well conserved across the disparate vertebrate animals, erythroid and thrombocytic differentiation have changed during the evolution of mammals. Specifically, adult mammalian red blood cells have the unique feature of being enucleated, and mammalian thrombocytes are not individual cells, but fragments of megakaryocytes, instead. It is likely that these enhancements provided a survival advantage to early mammalian species; however, they also bring up the question of evolutionary origin of these cells that studied using zebrafish (Danio rerio) model. First, it was necessary to generate a toolbox of a recombinant cytokines and optimized culture media that allowed us to manipulate zebrafish hematopoietic cells ex vivo in liquid and clonal cultures. Interestingly, teleost species underwent an extra duplication event during their evolution and as a result, two copies (paralogs) of some of the genes are present in zebrafish. This was also the case for majority of the cytokines from our toolbox and here, we provide functional characterization of these paralogs. Strikingly, our results...

Indiana University-Purdue University Indianapolis (IUPUI) / The hematopoietic system is highly proliferative, making hematopoietic stem and progenitor cells (HSPC) sensitive to radiation damage. Total body irradiation and chemotherapy, as well as the risk of radiation accident, create a need for countermeasures that promote recovery of hematopoiesis. Substantive damage to the bone marrow from radiation exposure results in the hematopoietic syndrome of the acute radiation syndrome (HS-ARS), which includes life-threatening neutropenia, lymphocytopenia, thrombocytopenia, and possible death due to infection and/or hemorrhage. Given adequate time to recover, expand, and appropriately differentiate, bone marrow HSPC may overcome HS-ARS and restore homeostasis of the hematopoietic system. Prostaglandin E2 (PGE2) is known to have pleiotropic effects on hematopoiesis, inhibiting apoptosis and promoting self-renewal of hematopoietic stem cells (HSC), while inhibiting hematopoietic progenitor cell (HPC) proliferation. We assessed the radiomitigation potential of modulating PGE2 signaling in a mouse model of HS-ARS. Treatment with the PGE2 analog 16,16 dimethyl PGE2 (dmPGE2) at 24 hours post-irradiation resulted in increased survival of irradiated mice compared to vehicle control, with greater recovery in HPC number and colony-forming potential measured at 30 days post-irradiation. In a sublethal mouse model of irradiation, dmPGE2-treatment at 24 hours post-irradiation is associated with enhanced recovery of HSPC populations compared to vehicle-treated mice. Furthermore, dmPGE2-treatment may also act to promote recovery of the HSC niche through enhancement of osteoblast-supporting megakaryocyte (MK) migration to the endosteal surface of bone. A 2-fold increase in MKs within 40 um of the endosteum of cortical bone was seen at 48 hours post-irradiation in mice treated with dmPGE2 compared to mice treated with vehicle control. Treatment with the non-steroidal anti-inflammatory drug (NSAID) meloxicam abrogated this effect, suggesting an important role for PGE2 signaling in MK migration. In vitro assays support this data, showing that treatment with dmPGE2 increases MK expression of the chemokine receptor CXCR4 and enhances migration to its ligand SDF-1, which is produced by osteoblasts. Our results demonstrate the ability of dmPGE2 to act as an effective radiomitigative agent, promoting recovery of HSPC number and enhancing migration of MKs to the endosteum where they play a valuable role in niche restoration.