Les adipocytes (cellules spécialisées dans le stockage des graisses) sont formés à partir de cellules immatures appelées cellules progénitrices lors du processus d'adipogenèse. Chez l'homme, les différentes étapes de ce processus sont mal connues ainsi que les signaux qui le régulent. La première partie de mon travail de thèse a eu pour but de caractériser la cellule intermédiaire entre la cellule progénitrice et l'adipocyte : le préadipocyte. La deuxième partie a consisté à évaluer le rôle des protéines morphogénétiques de l'os (ou BMP), des inducteurs de l'adipogenèse décrits chez la souris, dans l'adipogenèse humaine. Nous avons montré que les BMP2, 4 et 7 sont produites dans le tissu gras humain et BMP7 est modulée par l'obésité. Les BMP2 et 4 induisent l'adipogenèse des cellules progénitrices humaines mais seule la BMP7 permet la production d'adipocytes particuliers " beiges " décrits pour consommer les lipides et produire de la chaleur. Ces travaux affinent nos connaissances sur les mécanismes impliqués dans l'expansion du tissu gras et permettront d'élaborer des stratégies pour lutter contre le développement des pathologies liées à l'obésité. / Adipocytes (cells specialized in fat storage) arise from immature cells, called progenitor cells, during the process of adipogenesis. In human, the different stages of adipogenesis are not well defined as well as the signals involved in adipogenic modulation. The first part of my thesis work aimed to characterize the intermediate cell state between progenitor cell and mature adipocyte: the preadipocyte. The second part aimed to evaluate the role of bone morphogenetic proteins (BMPs) in human adipogenesis. In mice, BMP2 and BMP4 induce classical adipogenesis whereas BMP7 leads to the production of "brite" adipocytes with the capacity to use lipids to produce heat. We have shown that BMP2, 4 and 7 are produced in human fat depots and BMP7 is modulated by obesity. BMP2 and 4 induce classical adipogenesis and BMP7 only induces brite adipogenesis from human progenitor cells. These works improve our knowledge about the mechanisms involved in the expansion of fat depot and may allow the identification of new strategies to fight against the development of obesity-associated pathologies.

Conventional medical treatments fail to address the underlying problems associated with the damage inflicted by a coronary event. Thus, the long-term prognosis of patients admitted for heart failure is disheartening, with reported survival rates of 25 percent. Recent advances in stem cell research highlight the potential benefits of autologous stem cell transplantation for stimulating repair in heart tissue. However, a majority of those suffering from cardiovascular diseases are older adults whose autologous cells no longer possess optimum functional capacity. Additional work is needed to identify the optimal cell types or conditions that will promote cardiovascular regeneration across all age groups. A pretreatment, such as short-term hypoxia, and concurrent implementation of a novel progenitor, such as those that co-express Isl-1 and c-Kit, may enhance the results reported in clinical trials completed to date. However, the effects of short-term hypoxia in this novel cell type are unknown and warrant investigation in vitro. Cloned adult and neonatal Isl-1+ c-Kit+ human cardiovascular progenitor cells were characterized and expanded for study. Populations from both age groups were preconditioned using short-term hypoxia (1% O2 for six hours) and, to identify shifts in gene expression, compared to their respective control (21% O2 at 37 °C) via qRT-PCR. Flow cytometry and western blot analysis was utilized to measure phosphorylation of Akt. Progression through the cell cycle was also analyzed by flow cytometry. Cellular function was evaluated by the use of a TUNEL assay and Transwell® invasion assay. Hypoxia-mediated alterations of a genetic or functional nature in Isl-1+ c-Kit+ human cardiac progenitors are clearly age-dependent. Although both age groups accrued benefit, the neonatal progenitors procured significantly greater improvements. Short-term hypoxia significantly elevated Akt phosphorylation in neonatal Isl-1+ c-Kit+ human cardiac progenitors. Benefits afforded to both age groups by hypoxic pretreatment included significant upregulation of pro-survival transcripts, and enhanced invasion capabilities in vitro. Therefore, prior to transplantation, hypoxic preconditioning may improve the ability of transplanted stem cells to home towards damaged areas of the heart and support cardiac regeneration in vivo.

Interactions between the Eph family of receptor tyrosine kinases, and their ligands, the ephrins, are required for the normal development and maintenance of many patterns of connectivity within the nervous system. Eph receptors and ephrins are expressed widely throughout both the developing and mature nervous system where they function as important regulators of cell migration and axon guidance. The studies presented in this thesis examine the role of one particular member of the Eph receptor family, EphA4, in regulating mechanisms that underlie the development and maintenance of certain neural connections within the nervous system. This thesis demonstrates that the EphA4 receptor is expressed within specific regions of the developing and mature nervous system, some of which are associated with the control of locomotor activity. Consistent with these observations are the locomotor defects exhibited by animals with a targeted disruption of the EphA4 gene. These animals exhibit abnormal bilateral limb movements and have severe disruptions of a number of major axonal pathways. One of these disrupted axonal pathways, the corticospinal tract (CST), is a particularly important mediator of locomotor activity. This thesis reveals that EphA4 is expressed on the axons that comprise the CST. It demonstrates that although EphA4 is not required for the initial development of the CST, repulsive interactions between EphA4-bearing CST axons and ephrinB3, a ligand for EphA4 that is expressed at the midline of the spinal cord, appear to prevent CST axons from aberrantly recrossing the spinal midline during development.

Interactions between the Eph family of receptor tyrosine kinases, and their ligands, the ephrins, are required for the normal development and maintenance of many patterns of connectivity within the nervous system. Eph receptors and ephrins are expressed widely throughout both the developing and mature nervous system where they function as important regulators of cell migration and axon guidance. The studies presented in this thesis examine the role of one particular member of the Eph receptor family, EphA4, in regulating mechanisms that underlie the development and maintenance of certain neural connections within the nervous system. This thesis demonstrates that the EphA4 receptor is expressed within specific regions of the developing and mature nervous system, some of which are associated with the control of locomotor activity. Consistent with these observations are the locomotor defects exhibited by animals with a targeted disruption of the EphA4 gene. These animals exhibit abnormal bilateral limb movements and have severe disruptions of a number of major axonal pathways. One of these disrupted axonal pathways, the corticospinal tract (CST), is a particularly important mediator of locomotor activity. This thesis reveals that EphA4 is expressed on the axons that comprise the CST. It demonstrates that although EphA4 is not required for the initial development of the CST, repulsive interactions between EphA4-bearing CST axons and ephrinB3, a ligand for EphA4 that is expressed at the midline of the spinal cord, appear to prevent CST axons from aberrantly recrossing the spinal midline during development.

The discovery of neural stem cells (NSC) within the adult mammalian brain continues to fuel optimism regarding the ability of potential regenerative medicine applications to provide enhanced functional recovery from brain injuries. The adult NSC population is maintained within a complex microenvironment, referred to as the niche, where a unique cellular and extracellular environment maintains and regulates the NSC population and their progeny, enabling ongoing neurogenesis throughout adulthood. Characterization of how NSC interact with the extracellular environment and other cell subpopulations is an active area of research that will generate fundamental design parameters for biomaterial and tissue engineering strategies for neural tissue repair. A major obstacle to further progress is the lack of access to purified populations of primary NSC, a challenge which became the focus of this thesis. To address this obstacle, experimental methods were developed and optimized for isolating neural stem and progenitor cells (NSPC) from the adult NSC niche with fluorescence activated cell sorting (FACS). These methods were enhanced by the incorporation of a fluorescent reporter mouse driven by the gene Sox2, a neural stem cell associated transcription factor, which allowed NSPC enrichment within the Sox2+ population. The FACS based research approach was further developed to include additional surface antigens allowing isolation of NSPC at over 34% purity. The highly enriched population of NSPC was subjected to vital dye cell cycle analysis leading to the observation that an active and quiescent fraction exists within the NSPC pool that is delineated by β1-integrin expression. Access to enriched primary adult NSPC will lead to more a more accurate understanding of NSC dynamics with implications in fundamental biological research as well as biomaterials and tissue engineering.

Survival of transplanted neural stem/progenitor cells (NSPC) is limited after spinal cord injury (SCI). This thesis tested whether ephrin-B3 could enhance the survival of spinal cord derived NSPC because ephrin-B3 enhanced the survival of endogenous NSPC in the mouse brain. Preclustered ephrin-B3-Fc was tested, and preclustered Fc fragments and phosphate-buffered saline (PBS) were used as controls. This study showed that spinal cord derived NSPC and normal and injured rat spinal cord expressed EphA4 receptors. In culture, ephrin-B3-Fc increased the survival of NSPC at 1µg/mL (p<0.05), but Fc fragments reduced NSPC survival dose-dependently. In the injured spinal cord, infusion of ephrin-B3-Fc increased the proliferation of endogenous ependymal cells compared with infusion of PBS (p<0.05). However, in the injured cord, infusion of either ephrin-B3-Fc or Fc fragments caused a 20-fold reduction in the survival of transplanted NSPC (p<0.001). Thus, after SCI, ephrin-B3-Fc and Fc fragments are toxic to transplanted NSPC.

Survival of transplanted neural stem/progenitor cells (NSPC) is limited after spinal cord injury (SCI). This thesis tested whether ephrin-B3 could enhance the survival of spinal cord derived NSPC because ephrin-B3 enhanced the survival of endogenous NSPC in the mouse brain. Preclustered ephrin-B3-Fc was tested, and preclustered Fc fragments and phosphate-buffered saline (PBS) were used as controls. This study showed that spinal cord derived NSPC and normal and injured rat spinal cord expressed EphA4 receptors. In culture, ephrin-B3-Fc increased the survival of NSPC at 1µg/mL (p<0.05), but Fc fragments reduced NSPC survival dose-dependently. In the injured spinal cord, infusion of ephrin-B3-Fc increased the proliferation of endogenous ependymal cells compared with infusion of PBS (p<0.05). However, in the injured cord, infusion of either ephrin-B3-Fc or Fc fragments caused a 20-fold reduction in the survival of transplanted NSPC (p<0.001). Thus, after SCI, ephrin-B3-Fc and Fc fragments are toxic to transplanted NSPC.

Regenerative medicine is a rapidly developing field, merging engineering and biological life sciences to create biological replacements for damaged tissue and organ function. Development of cellular based therapies has the potential of curing present untreatable diseases and conditions, such as diabetes. The identification of protein expression patterns, that guide undifferentiated cells to different lineages, can provide important information about the progression of cellular differentiation at various stages. This research project utilizes proteomics and in vitro live-cell microscopy to investigate two distinct cellular systems: (1) the signaling pathways of calmodulin (CaM) in the differentiation of a human glioblastoma cell line; and (2) the effect of islet neogenesis associated protein (INGAP) on human islet-derived progenitor cells (hIPCs). Using a proteomic readout with a long term live-cell imagining approach, it was hypothesized that highly specific binding proteins of a CaM-mutant, and proteins in hIPCs perturbed by INGAP, could be identified and studied in vitro, characterizing specific signaling pathways which control the function of CaM in brain tumour cells and the mechanism(s) of INGAP in islet-derived progenitor cells. This thesis presents the utility of a proteomics and an in vitro cell microscopy approach to investigate therapeutic proteins, such as INGAP, on cell culture systems. The results have established the limitations and the utility of DIGE, differential binding of a CaM-mutant versus calcium-CaM, and the cell specific uptake feasibility of using the TAT-binding domain. In the hIPC system, proteomic, phenotypic, motility, proliferation and nuclear effects of INGAP were determined. Specifically, hIPCs exposed to INGAP had 50% decrease in average nuclear speed, the translocation of two identified proteins caldesmon and tropomyosin and INGAP was found to bind specifically to hIPCs. However, hIPCs had no changes in insulin specific hormone expression.

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In der vorliegenden Arbeit konnten erstmals der TGF-β-Signalweg und dessen Smad- Signalmoleküle innerhalb der MPCs nachgewiesen werden. Dieser Nachweis erfolgte sowohl auf zellulärer und Gewebeebene als auch auf Gen- und Proteinebene. Zusätzlich konnte auf Gen- und Proteinebene gezeigt werden, dass die Signalmoleküle Smad2, Smad3 und Smad4 in MPCs aus gering erkranktem Meniskusgewebe eine vermehrte Expression aufweisen im Vergleich zu den MPCs aus hochgradig erkranktem Meniskusgewebe. Diese Erkenntnis weist auf eine mögliche protektive Funktion des TGF- β-Signalwegs während degenerativer Prozesse im Meniskusgewebe hin. Um die Effekte des TGF-β-Signalwegs und dessen Smad-Signalmoleküle genauer zu verstehen und besser beschreiben zu können, wurde eine Überexpression der Smad-Signalmoleküle innerhalb von MPCs durchgeführt und die Auswirkungen dieser auf die Kollagen I- und Kollagen II-Synthese genauer beleuchtet. Infolgedessen konnte sowohl eine vermehrte Kollagen I-Synthese als auch eine vermehrte Kollagen II-Synthese festgestellt werden. Dies bestätigt die Annahme, dass dem TGF-β-Signalweg und dessen Smad-Signalmolekülen eine zentrale, protektive Funktion während der Meniskusdegeneration zukommt. Durch die vermehrte Synthese von Matrixkomponenten wird den Degenerationsprozessen innerhalb des Meniskusgewebes entgegengewirkt. Ein nicht degenerierter bzw. ein regenerierter Meniskus besitzt eine biomechanische Schutzfunktion für das Kniegelenk und wirkt somit einer Kniegelenkarthrose entgegen. In Zukunft könnte der TGF-β-Signalweg  einen möglichen Ansatzpunkt für therapeutische Behandlungen bei Meniskusläsionen darstellen. Da Meniskusdefekte häufig direkt mit einer Osteoarthrose im Kniegelenk assoziiert sind, spielt die durch den TGF-β-Signalweg induzierte Regeneration von Meniskusgewebe auch in der Prävention der Osteoarthrose eine zentrale Rolle.