Les pathologies de la paroi artérielle, et notamment l’athérosclérose, sont responsables de plus de 25% des décès dans le monde. Ces pathologies sont à l’origine de la thrombose,caractérisée par l’occlusion d’une artère par un caillot, ou thrombus. Ce travail de thèse explore l’utilisation de nanoparticules et microparticules polymères pour améliorer le diagnostic et le traitement de la thrombose. Ce type de particules permet en effet d’associer des agents de contraste et des actifs thérapeutiques à des agents de ciblage pour favoriser leur accumulation spécifique au niveau du thrombus. Les particules sont formulées à partir de polysaccharides et/ou de poly(cyanoacrylate d’isobutyle). Elles sont fonctionnalisées en surface avec du fucoïdane, un polysaccharide naturel extrait d’algues brunes. Le fucoïdane présente une affinité très forte pour la P-sélectine, molécule exprimée par les plaquettes activées qui constituent en partie le thrombus. Au cours de ce projet, un test in vitro d’adhésion en flux a été mis au point pour valider l’interaction des systèmes développés avec leur cible moléculaire, la P-sélectine, et leur cible cellulaire, les plaquettes activées. L'agent thérapeutique standard utilisé pour induire la dégradation du thrombus est l’activateur tissulaire du plasminogène. Celui-ci a été chargé sur des nanoparticules copolymères fonctionnalisées avec du fucoïdane. L’efficacité de ces systèmes a ensuite été validée dans un modèle murin de thrombose. Enfin, des nanoparticules composées exclusivement de polysaccharides et entièrement hydrophiles, dites “nanogels”, ont été synthétisées par un procédé innovant. Les résultats obtenus dans ce travail de thèse confirment le fort potentiel des approches ciblées pour le diagnostic et le traitement de la thrombose. Ils contribuent d’une manière plus générale au développement de la médecine personnalisée dans le domaine cardiovasculaire. / Arterial wall diseases, including atherosclerosis, are responsible for more than 25% of all deaths worldwide. These pathologies are at the origin of thrombotic events, characterized by the occlusion of an artery by a clot, called a thrombus. This thesis explores the use of polymer nanoparticles and microparticles for thrombosis imaging and therapy. This type of particles combines contrast agents and therapeutic agents with targeting moieties to promote their specific accumulation at the thrombus. The particles are composed of polysaccharides and/or poly(isobutyl cyanoacrylate). They are functionalized with fucoidan, a polysaccharide extracted from brown algae. Fucoidan shows a very strong affinity for P-selectin, a molecule expressed by activated platelets which form part of the thrombus. In this study, an in vitro flow adhesion assay is set up to validate the interaction of developed systems with their molecular target, the P-selectin, and with their cellular target, the activated platelets. Tissue plasminogen activator is the standard therapeutic agent used to induce thrombus degradation.This agent is loaded onto copolymer nanoparticles functionalized with fucoidan. Their efficiency is then validated in a murine model of thrombosis. Finally, nanoparticles exclusively composed of polysaccharides and entirely hydrophilic, called “nanogels”, are synthesized by an innovative process. The results of this work confirm the high potential of using targeted approach for thrombosis diagnosis and treatment and pave the way towards the development of personalized cardiovascular medicine.

During tumor progression and development of distant metastases, a subset of cancer cells undergoes transformation programs, such as epithelial-mesenchymal transition (EMT), to acquire enhanced migratory attributes to commence the metastatic cascade with the intension of achieving an active cell adhesion molecule-mediated organ-specific homing. Similarly, naive T cells reform the assemblage of their surface adhesion molecules during differentiation to activated T cells in order to successfully home to sites of inflammation and other extra-lymphoid organs for surveillance purposes. Sialyl-Lewis X (sLex) is well-known for mediating the homing of epithelial circulating tumor cellss (CTCs) and activated T cells to target sites through the interaction with endothelial selectins. Since glycan structures are not directly encoded by the genome, their expression is dependent on the glycosyltransferase (GT) expression and activity. Yet, the modulation of GTs during breast cancer transformation and in different molecular subtypes is still unknown. In addition, although the regulation of GTs during T cell activation is well-understood, the regulation at the epigenetic level is lacking. O-glycan-type sLex expression and E-selectin binding under static and flow conditions varies among molecular subtypes of breast cancer and upon the induction of EMT which is linked to the expression patterns of GTs. GTs displayed a significant prognostic value of in the association with the patients' survival profiles and in the ability to predict the breast cancer molecular subtypes from the expression data of a random patient sample. Also, GTs were able to differentiate between tumor and their normal counterparts as well as cancer types and glioblastoma subtypes. On the other hand, we studied the regulation of GTs in human CD4+ memory T cells compared to the naive cells at the epigenetic level. Memory T cell subsets demonstrated differential chromatin accessibility and histone marks within the promoters of the GTs genes. Moreover, they showed differential binding of pioneer and nonpioneer transcription factors (TFs). We proposed a model for the regulation of FUT7 during T cell activation that relies on the interplay between chromatin-remodeling and cell-fate-specifying TFs. Furthermore, we developed a fluorescent multiplex cell rolling (FMCR) assay to study the cell adhesion properties under physiological conditions. Compared to the conventional parallel plate flow chamber (PPFC) assay, the novel technique posses a high-throughput capacity which helps eliminate the inter-experimental variation problem by running multiple samples simultaneously and under competitive settings. We also developed a real-time analysis pipeline that enhanced the statistical power of the assay. Overall these modifications to the traditional parallel plate assay improves the reliability and results along with saving time and effort.

E-selectin is an endothelial adhesion molecule important in the recruitment of leukocytes to the bone marrow and inflammatory tissues; moreover, it has also been implicated in cancer metastasis and as a critical mediator of chemoresistance. This study investigated the effects triggered by the binding of E-selectin on acute myeloid leukemia (AML) cell lines regarding the activation of significant signaling pathways and their impact on biological functions. We observed that upon treatment with a recombinant E-selectin construct on AML cells, there was an activation of the AKT/NF-κB pathways, which are known to be central regulators of many cellular processes, including survival and proliferation. We found that the E-selectin-mediated activation varied in rate and amplitude among AML cell lines spanning differentiation blockage at different stages. Furthermore, we found that E-selectin binding in HL-60 and KG1-a cells sensitized the cells to daunorubicin (DNR), a chemotherapy drug commonly used to treat AML. The observed chemosensitivity could be linked to the decrease of Aldo Keto Reductases (AKR) protein levels upon E-selectin treatment, which is known to metabolize DNR and reduce its cytotoxicity. Additionally, we explored the role of exosomes as a regulator of the generation of therapy resistance by examining the effects treatment with KG1-a derived exosomes, a cell line that exhibits higher chemoresistance compared to other AML cell lines, had on viability in HL-60 cells upon chemotherapy. We found that even in the absence of KG1-a cells, exosomes were sufficient to provide an increase in chemoresistance. Overall, these studies explore properties exerted by AML cells that could lead to further understanding of AML and thus the development of potential therapeutic targets to overcome challenges currently found in the treatment of this disease.

Die Zelladhäsion von Leukozyten/ Metastasenzellen an Endothelzellen bei Entzündungsreaktionen/ Arteriosklerose und Tumormetastasierung ist ein mehrstufiger Prozess. Im ersten Schritt kommt es zu einer Interaktion zwischen E-Selektin-Rezeptoren auf den Endothelzellen und komplexen Kohlenhydraten, den Polylaktosaminen und ihren Derivaten, den Lewis-Antigenen. Im Rahmen dieser Arbeit wurden durch transiente Transfektion fünf rekombinante Glykoproteine erzeugt (hLysII/IV-FucTIII-VII). Durch hLysII/IV-FucTVI konnte die Zelladhäsion von U937 an HUVEC Zellen signifikant gehemmt werden. Fukosyltransferase VI scheint in der Lage zu sein, die Bildung von endständigen sLex-Antigenen an den Polylaktosaminketten von hLys zu ermöglichen. Auch durch eine Transfektion der Kolonkarzinomzelllinie SW480 und Colo 206 konnten Lysozymmutanten hergestellt werden, mit denen eine signifikante Adhäsionblockade möglich ist. Man kann sich hLysII/IV-FucTVI- SW480 und- Colo206 als potentielle therapeutische Substanzen vorstellen, die zum Beispiel zur Entzündungs- oder Metastasierungshemmung eingesetzt werden. / The initial step in diapedesis is the interaction between e-selectin and its ligand, the lewis antigen. In this study we examine the possibility to block this interaction to reduce metastasis and inflammatory processes. The plasmid hLysII/IV is expressed in CHO cells with active alpha1,3fucosyltransferases (III-VII). The resulting five recombinant glycoproteins are purified from cultur supernatants and static cell binding assays are performed. hLysII/IV-FucTVI was shown to bind to cells expressing e-selectin on its cell surface. It inhibited the binding of the monocytic cell line U937 to human endothelial cells activated with TNFalpha to upregulate the e-selectin expression. Alpha1,3fucosyltransferase VI seems to make possible the biosynthesis of the sLex and the hLysII/IV-FucTVI glycoprotein seems to be an interesting target to reduce inflammation and metastasis.

Während die zellulären Mechanismen der Pathogenese der Arteriosklerose intensiv untersucht worden sind, ist über die Mechanismen, die zu einer bevorzugten Lokalisation arteriosklerotischer Läsionen in bestimmten Gefäßarealen führen, weniger bekannt. Zur Untersuchung dieser Mechanismen wurden Endothelzellen aus menschlichen Koronararterien, einem Gefäßbereich, in dem häufig arteriosklerotische Läsionen beobachtete werden, isoliert und kultiviert. Endothelzellen der Mikrozirkulation menschlicher Herzen wurden unter gleichen Bedingungen kultiviert und die Reaktionen beider Zellarten verglichen. Inkubation der Zellen mit den in Bezug auf die Bildung arteriosklerotischer Plaques besonders pathogenen oxidierten LDL induzierte in makrovaskulären koronaren Endothelzellen eine stärkere Zunahme der PAI-1 Aktivität (182%, p / While the cellular mechanisms of atherosclerosis have been intensively studied, the mechanisms leading to preferential localization of atherosclerotic lesions are less well understood. To further define these mechanisms, endothelial cells from coronary arteries, i.e. vessels with frequent atherosclerotic lesions, were isolated and grown in vitro. In order to compare the reactions of both cell types, endothelial cells derived from microvessels of human hearts were isolated and cultured under identical conditions. Incubation of endothelial cells with oxidized LDL (75 µg/ml protein) induced a significant increase in PAI-1 activity (182 %, p

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Successful bone marrow (BM) transplantation requires the homing of the transplanted hematopoietic stem/progenitor cells (HSPCs) to their bone marrow niche, where they undergo differentiation to form mature cells that are eventually released into the peripheral blood. However, the survival rate of patients receiving BM transplants is poor since many of the transplanted HSPCs do not make it to their BM niches in the recipient’s body. Since the availability of HSPCs from traditional sources is limited, transplanting more number of HSPCs is not a solution to this problem. This study aims to characterize the adhesion molecules mediating cell migration in order to better understand the adhesion mechanisms of HSCs with the bone marrow endothelium. This will aid in developing future tools to improve the clinical transplantation of HSPCs. This study also aims to understand the factors that influence HSPC proliferation in the bone marrow niche. E-selectin plays an important role in the process of homing; however, its ligands on HSPCs are not well characterized. We used western blotting and immunoprecipitation to show that endomucin is expressed on HSPCs and plays a role in the binding of HSPCs to E-selectin. We also studied the effect of recombinant E-selectin on the expression of a newly characterized E-selectin ligand in our lab, CD34, in HSPCs. This will provide us insight into novel roles for endomucin and E-selectin and help us to understand the factors influencing HSPC migration to BM endothelium.

Chronic inflammation is a complex immune response linked to several diseases. The first step in the inflammatory response is the recruitment of immune cells to the endothelium of the vascular wall. This process is mediated by E/P-Selectin, for which no antagonist efficiently interacts to limit the inflammatory response. Previous work identified transcription factor GATA5 as a key regulator of endothelial homeostasis and revealed an altered expression of inflammatory genes in human endothelial cells with loss of GATA5. The objective of my project is to understand the role of GATA5 in selectin-dependent vascular inflammation and to develop selectin inhibitors. I used biochemical, cellular and in vivo approaches to evaluate a series of novel small molecules for their ability to interfere with selectin binding to their ligand, PSGL-1. The work identified a new lead candidate, LCB 2248, for the development of new E/P-Selectin antagonists and contributed to the understanding of the role of GATA5 in cell recruitment and adhesion. The mechanistic insight gathered and the identification of an E/P-Selectin antagonist will hopefully pave the way for the development of effective treatments for patients with chronic inflammation.