Thèse Médecine Paris, 1857 numéro 156.

Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

Le cordon ombilical humain suscite beaucoup d’intérêt comme source de cellules à des fins de recherche et de thérapie. Quatre types cellulaires majeurs - les cellules épithéliales, stromales, musculaires lisses et endothéliales - composent les tissus solides du cordon ombilical. Quelques-uns de ces types cellulaires ont été utilisés en recherche scientifique depuis longtemps, alors que d’autres commencent à peine à dévoiler leur potentiel. Nous avons développé un protocole unique pour l’extraction séquentielle de tous ces types cellulaires d’un seul cordon ombilical, permettant ainsi la reconstruction à partir d’une même source. La combinaison des techniques de perfusion, immersion et explants a mené à la mise en culture et à l’expansion de ces cellules, dont les cellules épithéliales et les cellules stromales de la gelée de Wharton qui ont été caractérisées plus en détail par l’immunomarquage de protéines spécifiques. Leur potentiel pour la médecine régénératrice a été démontré par la production de tissus par génie tissulaire. Un vaisseau sanguin composé de cellules stromales et de cellules musculaires lisses du cordon ombilical démontra une résistance substantielle à l’éclatement. Les capacités de différenciation des cellules épithéliales ont été étudiées dans le contexte d’une peau bilamellaire reconstruite en combinaison avec des kératinocytes, des fibroblastes dermiques, et des cellules stromales de la gelée de Wharton. Les cellules épithéliales ont montré une différenciation similaire à celle des kératinocytes lorsque cultivées sur des fibroblastes dermiques et exposées à l’air, tandis que sur des cellules stromales du cordon, elles ont subi une désorganisation. Finalement, la différenciation des cellules stromales a été induite en culture vers plusieurs types cellulaires afin de compléter cette étude. L’ensemble des résultats fait ressortir l’importance non seulement de l’influence du milieu physique sur la croissance et la différenciation des cellules, mais également de l’impact de la provenance des cellules sur la qualité des tissus reconstruits. / The human umbilical cord has received increasing attention as a source of cells for both research and therapeutic purposes. Four main cell types – epithelial, stromal, smooth muscle and endothelial cells – make up the solid tissues of the umbilical cord. Some of these cell types have been used in research for decades, while the potential of others is just being recognised. We have developed a unique protocol for the sequential extraction of all four cell types from a single umbilical cord, thus allowing the reconstruction of tissues and organs with cells from the same source. A combination of perfusion, immersion and explant techniques allows the successful extraction and expansion in culture of these cells. Further characterisation of the epithelial and Wharton’s jelly cells was carried out by immunofluorescent staining of specific proteins. The potential of these cells for use in regenerative medicine was demonstrated through the production of tissue-engineered constructs, including a blood vessel composed of umbilical cord stromal and smooth muscle cells which showed a substantial burst resistance under pressure. The capacity for differentiation of cord epithelial cells was studied in the context of a bilayered reconstructed skin substitute, in combination with keratinocytes, dermal fibroblasts, and Wharton’s jelly cells. These epithelial cells differentiated in a manner similar to keratinocytes when cultured on dermal fibroblasts and exposed to air, but under the same conditions on cord stromal cells they degenerated. Finally, to complete our study the Wharton’s jelly cells were induced to differentiate in vitro into various mesenchymal cell types. Globally, this work shows the importance of not only the culture conditions on the growth and differentiation of the various cell types, but also the important effect of the cell source on the resulting reconstructed tissues.

Les ectonucléotidases sont des familles d’enzymes présentes au niveau de la membrane cellulaire et ayant pour rôle d’hydrolyser les nucléotides en nucléosides. L’objectif principal de cette étude était d’abord de caractériser des anticorps produits dans le laboratoire contre les ectonucléotidases afin de les utiliser pour définir l’expression de ces enzymes dans le cordon ombilical et dans le rein humain par immunohistochimie. Les ectonucléotidases qui nous ont intéressées dans ce travail sont les Nucléosides Triphosphates Diphosphohydrolases (NTPDases) de la membrane cytoplasmique, soient les NTPDase1, -2, -3, -8, ainsi que l’ecto-5'-Nucléotidase (CD73). Nous avons aussi localisé ces enzymes dans ces tissus par histochimie enzymatique pour vérifier l’immunolocalisation obtenue et aussi pour vérifier si ces enzymes sont bel et bien actives dans les structures détectées par immunohistochimie. Nos résultats suggèrent que, dans le cordon ombilical, les NTPDase1, -2, -3, et l’ecto-5'-Nucleotidase sont localisées au niveau de l’endothélium des artères et de la veine et au niveau de la gelée de Wharton. Au niveau du rein, la NTPDase1 est localisée dans les vaisseaux sanguins, la NTPDase2 est localisée sur la face apicale des tubules rénaux, la NTPDase3 ainsi que la NTPDase8 sont localisées dans les tubules rénaux. L'identification de la localisation cellulaire de ces enzymes dans le cordon ombilical et dans le rein humain pourra nous aider à définir le rôle de ces enzymes dans ces organes et par extension pourra nous informer sur le rôle potentiel des nucléotides extracellulaires dans ces tissus. / Ectonucleotidases are families of enzymes present in the cell membrane and whose role is to hydrolyse the nucleotides into nucleosides. The main objective of this study was to characterize antibodies produced in the laboratory against ectonucleotidases and then to evaluate the expression of these enzymes in the umbilical cord and in the kidney by immunohistochemistry. The ectonucleotidases that we have selected for this work were the Nucleosides Triphosphates Diphosphohydrolases (NTPDases) of the cytoplasmic membrane, namely NTPDase1, -2, -3, -8, as well as ecto-5'-Nucleotidase (CD73). In addition, we have performed enzymatic histochemistry to verify the immunolocalization obtained and also to verify if these enzymes are active in the structures detected by immunohistochemistry. Our results suggest that, in the umbilical cord, NTPDase1, -2, -3, and ecto-5'-Nucleotidase are located at the endothelium of the arteries and vein and in Wharton frost. In the kidney, NTPDase1 is located in the blood vessels, NTPDase2 is located on the apical surface of the renal tubules, NTPDase3 as well as NTPDase8 are located in the renal tubules. The current identification of these enzymes at the protein and activity level in the umbilical cord and in the kidney is a prerequisite with the goal to define the role of these enzymes and in extension of extracellular nucleotides in these tissues.

Distributed teams of air and ground robots have the potential to be very useful in a variety of application domains, and much work is being done to design distributed algorithms that produce useful behaviors. This thesis presents a set of distributed algorithms that operate under minimal human input for patrol and cordon tasks. The algorithms allow the team to surround and travel between objects of interest. Empirical analyses indicate that the surrounding behaviors are robust to variations on the shape of the object of interest, communication loss, and robot failures.

<p>The actin cytoskeleton is a fundamental component of the cell and is involved in many processes, including cell division, cell migration, vesicle trafficking and cell polarity. The actin cytoskeleton has a very important role in embryogenesis as the cells within developing tissues proliferate, migrate, interpret extracellular cues, and shape complex tissues. The molecules that help the cell to interpret their environment and turn those cues into morphological changes are of great interest. One protein which may be involved in this manner is Cordon-bleu (Cobl). </p><p>In mouse embryos, <italic>Cobl's</italic> expression pattern resembles that of important developmental genes, is restricted to distinct domains, and changes dynamically throughout development as tissues are formed. While it is known that <italic>Cobl</italic> expression is regulated by developmental signaling pathways such as Shh and BMP, its molecular function at the cellular level remains elusive. In this study, we have identified molecular functions of Cobl. Cobl has C-terminal Wasp Homology-2 (WH2) domains which bind actin and nucleate new actin filaments in <italic>in vitro</italic> polymerization assays. Using cultured cells, we have determined that Cobl is involved in cytoskeletal remodeling during neurite branching and epithelial cell migration. We also demonstrate that Cobl interacts with the Syndapin family of adaptor proteins that link endocytosis and vesicle trafficking. Cobl colocalizes with Sdp2 in cultured epithelial cells and similarly localizes with Sdp1 and Sdp2 in developing mouse embryos. The localization of Cobl or Sdp2 in cultured epithelial cells is dependent on the other, as demonstrated using shRNA knockdown. </p><p>Previous studies demonstrated that a hypomorphic allele of <italic>Cobl</italic> interacts genetically with <italic>Looptail</italic>, in midbrain neurulation. <italic>Looptail</italic> mutants are deficient in the gene <italic>Vangl2</italic>, a member of the planar cell polarity pathway that coordinates the morphogenesis of a sheet of cells. To discover other roles for <italic>Cobl</italic> in the developing mouse, we have generated a conditionally null allele of <italic>Cobl</italic>. We find that outbred <italic>Cobl</italic> homozygous mutants are viable, but that they have inner ear defects. Together, our studies demonstrate that Cobl is a tissue-specific actin nucleator whose localization is regulated by its interaction with Syndapins and which functions in the development of sensory epithelia.</p> / Dissertation

Les cellules souches hématopoïétiques (CSH) sont greffées à des patients dont le système immunitaire est affaibli ou déficient, afin de reconstituer leur système hématopoïétique. En raison de leurs nombreux avantages, les CSH provenant du sang de cordon ombilical sont de plus en plus utilisées. Cette hausse de la demande a mené à l’implantation de plusieurs banques publiques, dont celle d’Héma-Québec, opérant selon les lignes directrices d’organismes réglementaires. Malgré la standardisation des protocoles de mise en banque, certains paramètres ne sont pas règlementés, telle la température d’entreposage des sangs de cordon avant leur cryopréservation. À Héma-Québec, le transport et la conservation des prélèvements avant la mise en banque sont faits à température pièce (TP) en respectant un délai maximal de 48 heures entre le moment du prélèvement et celui de la mise en banque. De récents travaux rapportés dans la littérature ont montré que les CSH provenant de sang de cordon conservé à TP pendant une période de 72 heures avant la mise en banque perdaient complètement leur capacité de reconstitution hématopoïétique alors que celle-ci était préservée si la conservation était faite à 4°C. Nous avons donc entrepris la présente étude afin de déterminer l’impact de l’entreposage à 4oC ou TP allant jusqu’à 48 heures sur plusieurs paramètres fonctionnels des CSH de sang de cordon ombilical, soit la viabilité, la capacité à se différencier et le potentiel de reconstitution hématopoïétique à l’aide d’un modèle animal de greffe. Les essais de différenciation ont permis de prédire les résultats des greffes, et ces derniers ont mis en évidence une grande variabilité entre les différents sangs de cordon. À ce stade, l’impact de la température d’entreposage avant la mise en banque sur le potentiel de reconstitution hématopoïétique des CSH n’est pas encore déterminé et des travaux supplémentaires devront être effectués. / Umbilical cord blood (UCB) has been proven to be an important alternative source of hematopoietic stem cells (HSCs) mostly for pediatric patients suffering from hematologic disorders. Because of its many advantages over other sources of HSCs, such as ease of collection, less stringent HLA restrictions and lower risks of developing GVHD, the use of UCB has expanded in recent years and led to a growing number of public cord blood banks (CBB) that operate under guidelines established by the regulatory organisms such as Netcord FACT, AABB or FDA. Despite the standardization of CBB procedures, some parameters remain unregulated, such as the pre-processing storage temperature. At Héma-Québec Public CBB, the units are kept at room temperature (RT) before being processed and cryopreserved within 48 hours after collection. Recently, a study using a mouse model of engraftment revealed that a pre-processing storage of 72 hours at room temperature might have deleterious effects on the HSC reconstitution capacity. The aim of our study was to evaluate the impact of pre-processing storage temperature on HSCs, by evaluating their viability, differentiation capacity and in vivo hematopoietic reconstitution in a mouse engraftment model. Results show that hematopoietic reconstitution potential measured in NSG mice differed for each of CBUs tested and that, in contrast to the previously published study, the in vivo reconstitution could be predicted by CFU assays. At this stage, the impact of preprocessing temperature on HSCs has not been confirmed, and to do so will require additional experiments.

Le nombre d’enfants nés par Assistance Médicale à la Procréation (AMP) dans le monde est estimé à plus de 5 millions, représentant jusqu’à 4% des naissances. Environ 10% des couples en âge de procréer sont actuellement infertiles, et leur apporter des techniques pour devenir parents est devenu un problème de santé publique. Cependant, l’innocuité de ces techniques n’a pas été totalement démontrée. Notamment, le risque de pathologies d’origine placentaire pourrait être augmenté. De plus, des issues périnatales défavorables, un risque majoré de malformations majeures et de pathologies liées à l’empreinte ont été rapportés chez ces enfants. Ceci soulève la question d’une éventuelle vulnérabilité épigénétique induite par l’AMP. L’objectif de ce travail de thèse était d’étudier la régulation épigénétique des gènes soumis à empreinte (GSE) et des éléments transposables (TE) dans le placenta et le sang de cordon d’enfants conçus par AMP comparés à des enfants conçus naturellement. En guise d’introduction, nous avons rédigé une revue détaillée des modifications phénotypiques et épigénétiques induites par l’AMP dans les embryons, le placenta et le sang de cordon chez l’Homme et sur les modèles animaux.Au cours de cette thèse, une cohorte de presque 250 patientes a été incluse prospectivement, répartie en 4 groupes de patientes selon la technique d’AMP et 4 groupes de témoins selon la durée d’infertilité.A partir de cette cohorte, la première question posée a été l’effet de la Fécondation in vitro (FIV) sur la méthylation de l’ADN et/ou la transcription des GSE et TE dans le sang de cordon et le placenta à la naissance. Pour cela, nous avons sélectionné 51 patientes enceintes après FIV avec ou sans ICSI avec transfert d’embryon frais à J2 et les avons comparées à 48 témoins enceintes dans l’année après l’arrêt de la contraception. Nous avons étudié la méthylation de l’ADN et l’expression de 3 GSE et 4 TE. Les niveaux de méthylation de l’ADN placentaire pour H19/IGF2, KCNQ1OT1, LINE-1 et ERVFRD-1 et le niveau d’expression placentaire d’ERVFRD-1 étaient plus bas dans le groupe FIV/ICSI que dans le groupe contrôle. Ces modifications épigénétiques pourraient faire partie des mécanismes de compensation développés pendant la grossesse après AMP, comme discuté dans notre revue.Ensuite, nous avons voulu déterminer si ces changements de méthylation de l’ADN des GSE pouvaient être associés à des modifications des histones. A partir de la cohorte précédente, nous avons sélectionné 16 patientes du groupe FIV/ICSI avec des niveaux de méthylation dans le placenta inférieurs au 5ème percentile pour au moins un des GSE étudiés. Elles ont été appariées à 16 témoins sur la parité, le sexe du nouveau-né et l’âge gestationnel à l’accouchement. Des marques permissives (H3K4me2 et me3 et H3K9ac) et répressives (H3K9me2 et me3) ont été étudiées. Les résultats ont révélé une quantité significativement augmentée de H3K4me2 dans le groupe FIV/ICSI pour H19/IGF2 et KCNQ1OT1. La quantité des deux marques répressives pour H19/IGF2 et SNURF était significativement abaissée dans le groupe FIV/ICSI.Ces données montrent que l’hypométhylation de l’ADN au niveau des GSE pourrait être associée à une augmentation des marques permissives et une diminution des marques répressives des histones, ce qui permettrait de favoriser un état « actif » de la chromatine au niveau de l’allèle normalement réprimé.Nos résultats, ainsi que les données de la littérature, renforcent l’hypothèse de potentiels mécanismes mis en place dans le placenta après AMP, utiles pour compenser des anomalies précoces de la placentation, qui seraient écrits à travers des modifications épigénétiques comme la méthylation de l’ADN mais aussi les modifications des histones.Bien que certaines questions restent en suspens, cette thèse a permis de bâtir les fondations de travaux futurs, notamment pour étudier l’impact de la congélation/décongélation des embryons et le rôle joué par l’infertilité en elle-même. / It is estimated that more than five million children have been born by Assisted Reproductive Technologies (ART) worldwide, representing up to 4% of all births. As around 10% of reproductive-aged couples are currently infertile, providing them with treatment options is a public health issue. However, the safety of these techniques has not been fully demonstrated. Notably, the rate of placenta-related adverse pregnancy outcomes could be increased after ART. Moreover, adverse perinatal outcomes, a higher risk of major malformations and imprinting disorders have also been reported in children born following ART. These issues combined raise the question of a potential ART-induced epigenetic vulnerability.The aim of this thesis was to investigate the epigenetic regulation of imprinted genes (IGs) and transposable elements (TEs) in the placenta and cord blood of children conceived by ART and to compare them to children conceived naturally.By way of introduction, we wrote a comprehensive review about phenotypic and epigenetic modifications induced by ART in embryos, placenta and cord blood either in human or animal models.Then, an extensive cohort of almost 250 patients was prospectively included, resulting in 4 groups of ART techniques and 4 groups of controls stratified on the time to pregnancy.From this cohort, the first question we investigated was the effect of in vitro fertilization (IVF) on DNA methylation and/or transcription of TEs and IGs in cord blood and placenta collected at birth. For this purpose, we selected 51 pregnant women after IVF with fresh embryo transfer at day -2 and compared them with 48 controls pregnant within 1 year of stopping contraception. We studied the DNA methylation and expression of 3 imprinted DMRs and 4 TEs. DNA methylation levels for H19/IGF2 and KCNQ1OT1 DMRs, LINE-1 and ERVFRD-1 in the placenta were lower in the IVF/ICSI group than in the control group. The expression level of ERVFRD-1 in the placenta was also lower in the IVF/ICSI group than in the control group. These modifications in epigenetic regulation may influence some compensation mechanisms developed throughout pregnancy after ART, as discussed in our review.We then intended to determine if these DNA methylation changes in IGs were associated with histone modifications. From the previously mentioned cohort, we selected the 16 patients from the IVF/ICSI group who presented with below the 5th percentile of percentage placenta DNA methylation for at least one of the previously studied DMRs. These patients were compared with 16 controls matched for parity, new-born sex, and gestational age at delivery. Permissive (H3K4me2 and me3 and H3K9ac) and repressive (H3K9me2 and me3) histone marks were studied. The results revealed a significantly higher quantity of H3K4me2 in the IVF/ICSI group than in the natural conception group for H19/IGF2 and KCNQ1OT1 DMRs. The quantity of both repressive marks at H19/IGF2 and SNURF DMRs was significantly lower in the IVF/ICSI group than in the natural conception group.These data demonstrate that DNA hypomethylation at imprinted DMRs may be associated with an increase in permissive histone marks and a decrease in repressive histone marks. This is consistent with a more “active” chromatin conformation on the normally repressed allele.Our findings, together with the literature data, reinforce the hypothesis that some mechanisms are established in the placenta after ART, probably to mediate placental plasticity and compensate primary disorders in trophoblastic invasion, and written through epigenetic changes such as DNA methylation but also histone modifications.Although some questions remain unanswered, this thesis paves the way for further original studies, notably to assess the impact of frozen-thawed embryo transfer and to decipher the role of infertility per se.

<p>The means by which cells, tissues, and organisms undergo morphogenesis are variable and highly regulated, and the mechanisms that govern cellular changes in response to signaling cues are poorly understood. This study seeks to address the role of a newly characterized protein in zebrafish in translating signaling cues into physical changes within a cell.</p><p>The <italic>Cordon&ndash;bleu (Cobl)</italic> gene is widely conserved in vertebrates, with developmentally regulated axial and epithelial expression in mouse and chick embryos. <italic>In vitro</italic>, Cobl can bind monomeric actin and nucleate formation of unbranched actin filaments, while in cultured cells it can modulate the actin cytoskeleton. However, an essential role for Cobl <italic>in vivo</italic> has yet to be determined. We have identified the zebrafish <italic>cobl</italic> ortholog and have used zebrafish as a model to assess the requirements for Cobl in embryogenesis. We find that cobl shows enriched expression in ciliated epithelial tissues during zebrafish organogenesis. The utilization of antibodies developed against Cobl shows that the protein is concentrated along the apical domain of ciliated cells, in close proximity to the apical actin cap. </p><p>Reduction of <italic>cobl</italic> by antisense morpholinos reveals an essential role in embryonic morphogenesis and organ development. A requirement for Cobl was shown for the proper function of various and ciliated epithelial organs. Cobl appears to direct the elongation of motile cilia in organs such as Kupffer&rsquo;s vesicle and the pronephros. In Kupffer&rsquo;s vesicle, the reduction in Cobl coincides with a reduction in the amount of apical F-actin. Additionally, Cobl may play a role during gastrulation cell movements and convergence and extension morphogenesis during early embryonic development. Thus, Cobl may represent a molecular activity that couples developmental patterning signals with local intracellular cytoskeletal dynamics to support elongation of motile cilia and tissue morphogenesis.</p> / Dissertation

The City of Toronto has done much to reduce congestion through transportation system management and travel demand measures. Yet, while measures to eliminate the traffic congestion problem have been necessary, they simply have not been sufficient to accommodate over 2.5 million residents and the many more who find their way into the area from points beyond particularly from other regions in the Greater Toronto Area (GTA). In addition, the transportation improvements certainly do not provide capacity adequate to address the needs of the future predicted residents and added economic activity. Congestion pricing is an untapped transportation strategy that can reduce traffic congestion, improve air quality, and raise the revenue essential to implement needed transportation measures that are effective in improving transportation services and facilities. While experience with congestion pricing is limited, there are sufficient examples and experiences around the world to demonstrate that, when implemented properly, it virtually never fails to be an effective tool to curb congestion. Yet, when initially proposed, it never fails to be controversial. This is due in part to the lack of research on the equity impacts on different socio-economic groups. This is the dichotomy and the dilemma of congestion pricing that every city must face in seeking this new approach to congestion management. The main goal of the research is to provide empirical research that enhances our understanding of the equity implications of cordon pricing for the urban region of Toronto, Canada. Three research objectives are identified to address the research goal. The first objective is to examine the ways that the GTA is moving toward or away the principles of sustainable transportation, and thus to make a case that Downtown Toronto is a candidate for cordon pricing. The second objective is to investigate if particular socio-economic groups would be disproportionately affected by the implementation of cordon pricing in Downtown Toronto, as one way of approaching the equity dimensions of such a policy. The third objective is to explore some of the policy aspects associated with implementing cordon pricing in Toronto, including public perceptions of such a policy as well as probable responses to the policy. The major findings of this analysis are that the GTA is not moving in the direction of sustainable transportation, which provides a concrete justification for demand-management interventions and that Downtown Toronto is a candidate for cordon pricing. A Downtown Toronto cordon pricing scheme would be progressive in its effects on the various socio-economic groups, and that the progressivity holds up even when travel is disaggregated by demographic factors such as age, gender, household size and occupational category. Full-time workers account for a larger proportion of the affected trips and the percentage of trips that would be affected is highest for those in the full-time high-income neighborhoods. The analyses show that toll charge is an important factor that would trigger some income groups to change their travel behaviour. People from high-income neighborhoods are more willing to pay the charges and drive as usual than people from other income neighborhoods. Revenue redistribution is critical to assess and achieve equity of congestion pricing.