The central properties of stem cells are the pluripotency and the capacity of self-renewal. Hematopoietic stem cells (HSCs) posses such common features that allows them to generate all the cells of the hematopoietic compartments, maintaining in the same time the HSC pool. We develop approaches focused on ex vivo HSC expansion through activation by exogenous HOXB4 (human HSCs) or Notch/Dll-4 ligand (murine HSCs). Two independent transcriptomic analyses surprisingly converged toward an increased expression of two genes never identified sofar as crucial for HSC functions: Pumilio1 (Pum1) and Pumilio2 (Pum2). Pum1 and Pum2 are posttranscriptional regulators belonging to the Pumilio-FBF (PUF) family of RNA-binding proteins. Although it was established that the primordial role of PUF proteins is to sustain mitotic proliferation of stem cells in Invertebrates, so far nothing is known about the role of Pum1 and Pum2 in human and murine HSCs.For these reasons, we have investigated the roles and mechanisms of action of Pum1 and Pum2 in murine and human HSCs through shRNA strategy. Pum1 and Pum2 knockdown (KD) in murine HSCs led to a decreased HSC expansion and clonogenic potential ex vivo, associated with an increased apoptosis and a cell cycle arrest in G0/G1 phase. KD of both Pum1 and Pum2 enhanced these effects, suggesting a cooperative effect. Expansion and clonogenic potential of KD Pum1 HSCs were rescued by enforced expression of Pum1 (insensitive to our shRNA), thus validating the specificity of our shRNA. Enforced expression of Pum1 could not rescue the functions of Pum2 KD HSCs, highlighting the non-redundant role of these proteins. Furthermore, when Pum1 or Pum2 KD HSCs were inoculated into lethally irradiated mice to follow the long-term hematopoietic potential, only rare bone marrow cells derived from Pum1 and Pum2 KD HSCs were evidenced after 4 months, contrary to control HSCs. Identical results were obtained with human Pum1 or Pum2 KD HSCs.In conclusion, our results demonstrate the involvement of Pumilio factors in stemness maintenance, expansion and survival of murine and human HSCs. Identification of Pumilio factors and their targets as new regulators of HSCs expansion will allow consider them as new tools for therapeutic perspectives.

La mégacaryopoïèse représente le processus de différenciation des cellules souches hématopoïétiques (CSH) en mégacaryocytes (MK). Ce processus précède la thrombopoïèse qui aboutira à la formation des plaquettes sanguines. Ces processus complexes ont lieu 1) au sein de la structure tridimensionnelle (3D) de la moelle osseuse, 2) dans les vaisseaux sinusoïdes de la moelle et 3) dans la circulation sanguine. Le but général de ce travail a été de comprendre le mécanisme de chaque étape. Le premier objectif a été d’étudier les effets d’une structure poreuse 3D mimant celle de la moelle osseuse, sur la différenciation mégacaryocytaire et la production plaquettaire in vitro. Cette étude a permis de démontrer que la synergie entre l’organisation spatiale et les signaux du microenvironnement améliore la production en MK et en plaquettes. Par la suite, nous avons souhaité caractériser in vitro et in vivo les plaquettes produites en conditions de flux. Nous avons notamment mis en évidence la capacité des plaquettes produites in vitro dans un système de microfluidique, à s’incorporer et à participer à la formation d’un thrombus in vitro et in vivo contrairement aux plaquettes obtenues en statique. Ces travaux prouvent donc l’intérêt d’une part, de mimer le microenvironnement de la moelle osseuse et d’autre part, de reproduire les forces de cisaillement du sang afin d’améliorer et d’augmenter la production de plaquettes in vitro pour de futures applications en thérapeutique. / Megakaryopoiesis is a process allowing hematopoietic stem cell (HSC) to proliferate and differentiate into megakaryocytes (MK). It is followed by thrombopoiesis allowing blood platelet production. These processes occur 1) in the bone marrow three-dimensional (3D) structure, 2) in the bone marrow sinusoid vessels and 3) in the blood flow. Our general aim was to decipher the mechanism associated to each process. The first objective was to study the effects of porous 3D structure on MK differentiation and platelet production. This study demonstrated that the synergy between spatial organization and biological cues improved MK and platelet production. We also characterized platelets produced from mature MK in flow conditions, with respect to their in vitro and in vivo properties. We highlighted the capacity of flow-derived platelets to incorporate in a thrombus in vitro and in vivo, compared to static-derived platelets. These works represent some new developments for mimicking the bone marrow structure and to reproduce blood shear forces in order to improve and increase in vitro platelet production for therapeutic use.

In a particle based geometallurgical model, the behavior of the particles can be used for forecast the products and quantify the performance of the different ore types within a deposit. The particle tracking is an algorithm developed by Lamberg and Vianna 2007 whose aim is to balance the liberation data in a mineral processing circuit composed by several processing units. Currently, this tool is being developed for the HSC Chemistry software by Outotec.The objective of this study is to understand and evaluate the particle tracking algorithm in a geometallurgical test for iron ore. To achieve this objective, the liberation data is balanced in a Davis tube test circuit. A total of 13 samples from Leveäniemi iron ore were process in a Davis tube circuit.The magnetite is the main mineral in the Leveäniemi iron ore samples. Its high recovery in the Davis tube circuit along with the V, Ti and Mn suggest that these elements are present in the magnetite lattice. These penalty elements in the iron concentrates cannot be avoided at the stage of mineral concentrations.The washing effect of the Davis tubes controlled by the rotational and longitudinal agitation of the tube perturb the particles agglomeration between the pole tips of the electromagnet. A higher agitation frequency and amplitude will wash away most of the gangue minerals and also fine grained magnetite.In this work, the particle tracking is depicted and implemented in a magnetic separation circuit for high liberated material. The liberation data was balanced in a way that the particle classes can be followed through circuit and their recoveries can be calculated. Nevertheless, the algorithm requires further validation and analysis of its limitations in terms of resolution and reproducibility.

This Master's thesis deals with the issue of high-speed turning of the 100Cr6 material. The aim is to identify and experimentally validate an innovative production process for a particular component. The thesis includes theoretical basis of high-speed machining, analysis of the current state of the manufacturing process, innovation design and experimental verification. At the end of the thesis, parameters of the machined surfaces of specimens are evaluated.

This work is devoted to study the influence of the composition on mechanical properties of high performance concret based on portland cement. 29 samples of high performance concrete (HPC) warying in composition were prepared. The constituents used for HPC preparation were: cement Aalborg White, silica fume, finelly ground blast furnace slag, finelly ground silica, calcinated bauxite and polycarboxylate based superplasticizer. The mechanical parameters (flexural and compressive strength) of the samples were observed after 7 and 28 days of moist curing. Compressive strength values after 28 days were in the range of 92 to 194 MPa and the flexural strength values were in the range of 7 to 23 MPa (without using of fiber reinforcement). The graphs showing mechanical parameters depending on the mixture composition were constructed and consequently evaluated.

La greffe de cellules souches hématopoïétiques est parfois le seul traitement efficace contre les cancers hématologiques ainsi que plusieurs autres désordres reliés au système hématopoïétique. La greffe autologue est souvent le traitement de choix pour les patients atteints de lymphome ou de myélome. Dans ce cas, les cellules souches hématopoïétiques (CSH) du patient sont récoltées et congelées. Le patient subit ensuite des traitements de chimiothérapie et/ou radiothérapie qui éliminent les cellules malignes, mais détruisent aussi son système hématopoïétique. Ce dernier sera ensuite reconstitué par la greffe de CSH. Ces traitements ont pour conséquence de plonger le patient en état d’aplasie pour une période variant de 2 à 4 semaines. La thrombocytopénie (faible taux de plaquettes) est une complication majeure nécessitant des transfusions plaquettaires répétées et associée à une augmentation de la mortalité hémorragique post-transplantation. Il serait particulièrement intéressant de développer une thérapie accélérant la reconstitution des mégacaryocytes (MK), ce qui aurait pour effet de raccourcir la période de thrombopénie et donc de diminuer les besoins transfusionnels en plaquettes et potentiellement augmenter la survie. HOXB4 est un facteur de transcription qui a déjà démontré sa capacité à expandre les CSH et les progéniteurs multipotents (CFU-GEMM) donnant naissance aux MK. Il est donc un bon candidat pour l’expansion des progéniteurs MK. Comme la protéine HoxB4 a par contre une courte demi-vie (~1.1h), des protéines HoxB4 de deuxième génération avec une plus grande stabilité intracellulaire ont été créées (1423 (HoxB4L7A), 1426 (HoxB4Y23A) et 1427 (HoxB4Y28A)). Nous avons donc étudié la capacité d’HoxB4 sauvage et de deuxième génération à expandre les CSH, ainsi que les MK donnant naissance aux plaquettes. La surexpression rétrovirale de ces protéines HoxB4Y23A et HoxB4Y28A conduit à une expansion des progéniteurs MK murins in vitro supérieure à HoxB4-wt, 1423 et au contrôle GFP. La reconstitution plaquettaire in vivo dans un modèle murin a ensuite été évaluée par des transplantations primaires et secondaires. Les résultats révèlent que la surexpression rétrovirale des différents HoxB4 n’apporte pas de bénéfice significatif à la reconstitution plaquettaire des souris. Lorsque cultivées dans un milieu favorisant la différenciation mégacaryocytaire, le traitement de cellules CD34+ dérivées du sang de cordon ombilical avec les protéines recombinantes TATHoxB4WT ou de seconde génération n’a pas augmenté la production plaquettaire. Par contre, de manière intéressante, les cellules CD34+ provenant de sang mobilisé de patients atteints de myélome et mises en culture dans un milieu favorisant l’expansion des CSH ont montré des différences significatives dans la différenciation des progéniteurs MK en présence de la protéine recombinante TATHoxB4. La protéine HOXB4 possède donc un avenir prometteur quant à une amélioration de l’état thrombocytopénique chez les patients. / Haematopoietic stem cell (HSC) transplantation is the most efficient treatment against a number of cancers or other disorders of the hematologic system. Prior to HSC transplantation, patients are exposed to high doses of radiotherapy and/or chemotherapy to eliminate malignant cells. However, these treatments result in a state of aplasia, particularly in thrombocytopenia, which is characterised by very low blood platelet counts. Platelets produced by megakaryocytes (MK) are essential components of the blood system and play a critical role in the prevention of bleeding. Thus a low platelet blood level is a major complication and contributes significantly to transplant related mortality. At present, regular infusion of platelets isolated from healthy donors is the treatment of choice for thrombocytopenia. However, this is cumbersome for patients as well as donors and, in many instances results in platelet refractoriness due to the generation of auto-antibodies against disparate HLA molecules expressed on donor platelets. Therefore, the development of strategies to accelerate MK production and thus platelet reconstitution post HSC transplant would represent a major advance. It has already been shown that HoxB4 expands HSC and multipotent progenitors (CFU-GEMM) that give rise to megakaryocytes (MK). Thus HoxB4 is a great candidate for in vitro MK progenitor expansion. However, the short half-life of HoxB4 protein prompted us to generate a second generation of HoxB4 proteins with greater intracellular stability. We therefore studied the capacity of wild type (WT) and HoxB4 with 3 substitutions (1423 (HoxB4L7A), 1426 (HoxB4Y23A) and 1427 (HoxB4Y28A) resulting in a longer protein half-life to expand HSC as well as MK progenitors. Retroviral-mediated expression of HoxB4Y23A and HoxB4Y28A proteins showed a greater expansion of murine MK progenitors, in comparison with HoxB4WT or HoxB4L7A proteins or GFP control. We also evaluated the ability of HSC expressing second generation HoxB4 to generate platelets in a murine model. Our results show that retroviral-mediated transduction of second generation HoxB4 in murine HSC does not provide a significant advantage over HoxB4WT in platelet reconstitution in mice. Interestingly, treatment of CD34+ cells derived from cord blood showed only marginal effect of HoxB4WT or second generation HoxB4 soluble recombinant proteins when cultured under conditions optimized for megakaryocyte differentiation. Unexpectedly, CD34+ cells derived from mobilized peripheral blood of myeloma patients showed a significant increase in MK progenitor differentiation in the presence of TAT-HoxB4WT when cultured in expansion medium for HSC. Thus, HoxB4 holds promise in autologous HSC transplantation for the treatment of thrombocytopenic patients.

The first definitive haematopoietic stem cells (HSCs) produced during embryonic development are generated from a specialised subset of endothelial cells known as haemogenic endothelium. Recently, it was reported that Gata3 plays a dual role in the development of sympathetic nervous system and haematopoietic system. In fact, Gata3 has proven to be crucial for the production of HSCs through regulation of catecholamine production from the co-developing sympathetic nervous system. Also, it was recently shown that Gata3 is expressed in the haemogenic endothelium and haematopoietic progenitor cells. Here, I will specifically examine the role of Gata3 in the production of HSCs; if it is expressed and plays a role in the precursors from which HSCs arise. Using a Gata3-GFP reporter mouse line, we found that Gata3 is expressed in various cell types in the HSCs microenvironment, including mesenchymal cells, endothelial cells, haematopoietic cells and sympathetic nervous system, and this expression was stage dependant. In the endothelial cells, we have found that the haemogenic endothelium activity is enriched in Gata3 expressing cells. Within the haematopoietic cells, we have found that Gata3 marks a specific stage along the developmental pathway towards the generation of definitive haematopoietic stem cells, and that Gata3 expressing haematopoietic cells are enriched for the most immature and stem cell like progenitors. Moreover, Gata3 will be specifically knocked out in haemogenic endothelial cells to determine whether it plays an essential role in the production of HSCs from the endothelium using the Vec-Cre system. We found that Gata3 within the haemogenic endothelium plays a major role in haematopoietic progenitors formation, and possibly haematopoietic stem cell formation. Finally, we used molecular assay (RNA seq) to identify the role of Gata3 in the haematopoietic stem cell microenvironment and found that Gata3 plays a major role in the development and differentiation of various cells and systems, and implicated Gata3 as cell cycle regulator. In summary, we found that Gata3 expressing cells is enriched for haemogenic endothelium, crucial for the haematopoietic progenitors formation, plays and important role in endothelial to haematopoietic transition, and plays a key developmental role in both haematopoietic stem cell and its microenvironment.

La leucémie myélomonocytaire chronique (LMMC) est une hémopathie myéloïde rare du sujet âgé. Les caractéristiques cliniques, génétiques et moléculaires de la maladie sont bien connues. L’expression très hétérogène de la maladie ne peut être expliquée par la seule hétérogénéité génétique du clone leucémique. Les altérations épigénétiques jouent manifestement un rôle important. Le rôle de facteurs extrinsèques issus du microenvironnement est plus obscur. La niche hématopoïétique est le siège d’interactions entre cellules. Deux schémas non-exclusifs d’altération primaire ou secondaire de la niche sont proposés. Le premier implique que l’émergence d’un clone hématopoïétique modifie son environnement. Le second postule que le premier évènement dans l’émergence d’une hémopathie clonale est une altération de l’environnement. Mon travail de thèse a étudié les altérations du microenvironnement médullaire chez les patients et leur impact sur la physiopathologie de la maladie selon 2 axes: 1) la mise au point d’un modèle murin de reconstitution de la niche hématopoïetique humaine et 2) la caractérisation des cellules souches mésenchymateuses des patients. Dans une première partie, j’ai transposé un modèle rapporté en 2016 à l’étude de la LMMC. Ce modèle de greffe de cellules médullaires humaines chez la souris immunodéprimée s’est avéré difficilement reproductible. Dans la seconde partie, j’ai analysé les cellules souches mésenchymateuses de patients atteints de LMMC. J’ai identifié la production excessive d’IGFBP2 (Insuline-like Growth Factor Binding Protein 2), conséquence probable d’une dérégulation épigénétique. Le séquençage des CSM à l’échelle unicellulaire a révélé une restriction de l’hétérogénéité de ces cellules dont une fraction seulement produit IGFBP2. Finalement, j’ai montré qu’IGFBP2 favorise la différenciation des progéni-teurs myéloïdes vers la lignée monocytaire. IGFBP2 pourrait donc contribuer à amplifier la monocytose caractéristique de cette maladie.En conclusion, la LMMC s’accompagne de modifications des cellules de la niche hématopoÏétique dont certaines produisent des quantités excessive d’IGFBP2. La recherche de l’origine de ce dérèglement et de son importance dans la progression de la maladie permettra d’évaluer l’intérêt potentiel d’une neutralisation de cette cytokine à des fins thérapeutiques. / Chronic myelomonocytic leukemia (CMML, is a rare myeloid hemopathy of the elderly. Clinical, genetic and molecular characteristics of the disease are well-known. The highly heterogeneous expression of the disease can’t be solely explained by genetic heterogeneity of the leukemic clone. Epigenetic alterations obviously play an important role. However, the role of extrinsic factors from the medullar microenvironment in CMML physiopathology is still poorly understood. The hematopoietic niche hosts a lot of bi-directionnal interactions between cells. Two non-exclusive schemes of primary and secondary alterations of the niche can be proposed. First postulate implies that the emergence of a hematopoietic clone alters its environment. The second one supposes that the first event causing the emergence of a clonal hemopathy is an alteration of the environment. My PhD work consisted of studying medullar alterations in patients and their impact on CMML physiopathology upon 2 axes: 1) to set up a murine model of human hematopoietic niche reconstitution 2) to caracterise mesenchymal stem cells from CMML patient ex vivo. During the first part of my PhD, I adapted a model published in 2016 to CMML. This model of human MSC graft in immunodeficient mice proved to be hardly reproducible. During the second part, I analysed of CMML patients MSC. I identified an excessive production of IGFBP2 (Insuline-like Growth Factor Binding Protein 2) probably secondary to an epigenetic disregulation. Single cell RNA sequencing revealed a restriction of MSC heterogeneity of which only a fraction produces IGFBP2. Finally, I showed that IGFBP2 favors myeloid progenitors differenciation towards monocytic lineage. IGFBP2 could therefore contribute to the amplification of CMML characteristic monocytosis.To conclude, CMML goes along with modifications of hematopoietic niche cells, some of which produce excessive amounts of IGFBP2. Investigation on the origin of this alteration and its significance in disease progression should allow to evaluate the potential interest of its neutralization for therapeutic strategies.

Die Verwendung von lang kragenden Schaftfräsern im Bereich der Hochgeschwindigkeitsbearbeitung birgt besondere Herausforderungen bezüglich der Prozessdynamik. In diesem Zusammenhang werden verschiedene kontinuumsmechanische Berechnungsmodelle für Werkzeuge mit Hohlschaft vorgestellt. Dabei wird eine teilweise Füllung des Schaftes mit einer fließfähigen Ausgleichsmasse zum Zweck des automatischen Wuchtens berücksichtigt. Ausgehend von der Verformungskinematik wird die systembeschreibende Variationsformulierung mit Hilfe des Hamilton'schen Prinzips hergeleitet. Dabei wird auch auf den Einfluss von stochastisch verteilten Unwuchten, geometrischen Nichtlinearitäten und Schubdeformationen eingegangen. Zur Ortsdiskretisierung werden sowohl lokale als auch globale Methoden angewendet und miteinander verglichen. Die Auswertung stellt den Einfluss von verschiedenen geometrischen sowie prozessbedingten Parametern auf die Eigenfrequenzen, stationäre Deformation, Stabilität sowie Zeitlösung dar.:1. Einleitung 1.1. Problemstellung und Motivation der Arbeit 1.2. Stand der Technik 1.2.1. Hochgeschwindigkeitsfräsen 1.2.2. Verwendung lang kragender Schaftfräser 1.3. Thema und Aufbau der Arbeit 2. Theoretische Grundlagen 2.1. Kontinuumsmechanische Grundbegriffe 2.2. Spannungen und konstitutive Gleichungen 2.3. Prinzip von Hamilton 2.4. Lösungstheorie 2.4.1. Anfangswertprobleme 2.4.2. Randwertprobleme 2.5. Stochastische Grundbegriffe 3. Balkenmodelle 3.1. Verformungskinematik des Balkens 3.2. Variationsformulierung 3.3. Modellierung der Unwucht 3.4. Globale Diskretisierung 3.4.1. Stationäre Lage und Linearisierung 3.4.2. Ortsfunktionen 3.5. Lokale Diskretisierung 3.6. Anmerkungen zur schubweichen Formulierung 3.7. Berechnungsergebnisse 3.7.1. Ruhendes Werkzeug 3.7.2. Rotierendes Werkzeug 4. Schalenmodelle 4.1. Verformungskinematik der Schale 4.2. Variationsformulierung 4.3. Globale Diskretisierung 4.3.1. Stationäre Lage und Linearisierung 4.4. Lokale Diskretisierung mittels FEM 4.4.1. Konforme flache Schalenelemente 4.5. Anmerkungen zur schubweichen Formulierung 4.6. Berechnungsergebnisse 4.6.1. Ruhender Schaft 4.6.2. Rotierender Schaft 5. Zusammenfassung und Ausblick 6. Verzeichnisse 6.1. Quellenverzeichnis 6.2. Symbolverzeichnis 6.3. Abbildungsverzeichnis 6.4. Tabellenverzeichnis A. Feldgleichungen und Ableitungen der Ansätze für die Balkenmodelle B. Anmerkungen zum Timoshenko-Balken C. Feldgleichungen und Ableitungen der Ansätze für die Schalenmodelle D. Anmerkungen zur Mindlin-Reissner-Schale / The use of long slender end mills for high-speed-cutting (HSC) holds special requirements with respect to the system dynamics. In this context, several tool models in the area of continuum mechanics are presented. Especially hollow tool shafts, with a fluid medium inside, for the purpose of automatic balancing are considered. Starting with the kinematics of deformation, Hamilton's principle is used to evaluate the variational formulation. Therefore, also the influence of a stochastic distributed unbalance, geometrical nonlinearities and shear deformations are discussed. For space discretisation local as well as global approaches are used and compared with each other. Following up on this, results are presented, which show the influence of different geometrical and process-related parameters due to the eigenfrequencies, stationary deformation, stability and time solution.:1. Einleitung 1.1. Problemstellung und Motivation der Arbeit 1.2. Stand der Technik 1.2.1. Hochgeschwindigkeitsfräsen 1.2.2. Verwendung lang kragender Schaftfräser 1.3. Thema und Aufbau der Arbeit 2. Theoretische Grundlagen 2.1. Kontinuumsmechanische Grundbegriffe 2.2. Spannungen und konstitutive Gleichungen 2.3. Prinzip von Hamilton 2.4. Lösungstheorie 2.4.1. Anfangswertprobleme 2.4.2. Randwertprobleme 2.5. Stochastische Grundbegriffe 3. Balkenmodelle 3.1. Verformungskinematik des Balkens 3.2. Variationsformulierung 3.3. Modellierung der Unwucht 3.4. Globale Diskretisierung 3.4.1. Stationäre Lage und Linearisierung 3.4.2. Ortsfunktionen 3.5. Lokale Diskretisierung 3.6. Anmerkungen zur schubweichen Formulierung 3.7. Berechnungsergebnisse 3.7.1. Ruhendes Werkzeug 3.7.2. Rotierendes Werkzeug 4. Schalenmodelle 4.1. Verformungskinematik der Schale 4.2. Variationsformulierung 4.3. Globale Diskretisierung 4.3.1. Stationäre Lage und Linearisierung 4.4. Lokale Diskretisierung mittels FEM 4.4.1. Konforme flache Schalenelemente 4.5. Anmerkungen zur schubweichen Formulierung 4.6. Berechnungsergebnisse 4.6.1. Ruhender Schaft 4.6.2. Rotierender Schaft 5. Zusammenfassung und Ausblick 6. Verzeichnisse 6.1. Quellenverzeichnis 6.2. Symbolverzeichnis 6.3. Abbildungsverzeichnis 6.4. Tabellenverzeichnis A. Feldgleichungen und Ableitungen der Ansätze für die Balkenmodelle B. Anmerkungen zum Timoshenko-Balken C. Feldgleichungen und Ableitungen der Ansätze für die Schalenmodelle D. Anmerkungen zur Mindlin-Reissner-Schale

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