Thrombosis, the major cause of heart attack and strokes,1 is triggered by localized clotting of the blood as the result of deregulated platelet aggregation. During the repair of vascular injury, clotting usually occurs when platelets adhere to each other at the site of vascular injury in order to stop bleeding.2 Distinct protein receptors and adhesive ligands together with the blood flow conditions govern this process. One of the negative regulators in platelet aggregation is Disabled-2 (Dab2), a modular protein that is released upon platelet activation to the extracellular platelet surface.3 Dab2 inhibits platelet aggregation through its phosphotyrosine-binding (PTB) domain by competing with fibrinogen for ï ¡IIï ¢3 integrin binding on the activated platelet surface.3 Sulfatides are also found on the platelet surface,4 interacting with adhesive and coagulation proteins5-7 and, thus, they are thought to play a major role in haemostasis and thrombogenesis. Here, we show that the Dab2 PTB domain specifically interacts with sulfatides through two conserved basic motifs. The sulfatide-binding site overlaps with that of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2) in the PTB domain. Whereas sulfatides recruit the Dab2 PTB domain to the platelet surface, thus sequestering the protein from thrombin-mediated platelet aggregation, the phosphoinositide mediates its internalization. Experimental data support the hypothesis that two pools of Dab2 co-exist at the platelet surface and that the balance between them controls the extent of the clotting response. / Master of Science

At the site of vascular injury platelet aggregation serves to stem blood flow, initiate the inflammatory response, and stimulate wound healing. Platelets become stimulated, release their granule contents, and become adherent to one another. Platelet granules contain important clotting factors and regulators of aggregation. Disabled-2 (Dab2) is a negative regulator of platelet aggregation released from platelet α-granules. Dab2 binds to the αIIbβ3 integrin, through the PTB domain, and blocks fibrin binding to the integrin which serves as the major cause of platelet-platelet interactions. Dab2 is also capable of binding to sulfatides, through the N-PTB region, expressed on the outer leaflet of adjacent cells. Dab2-sulfatide binding decreases Dab2's ability to interact with the αIIbβ3 integrin, however sulfatides activate and stimulate platelet-platelet and platelet-leukocyte aggregation. Sulfatide addition to platelets stimulates increased αIIbβ3 integrin and P-selectin expression through stimulation of continued platelet degranulation, and these surface receptors mediate platelet heterotypic and homotypic aggregation. Here, we show that Dab2 N-PTB binding of sulfatides serves to increase the inhibitory affect of Dab2. Sulfatide stimulation of platelet degranulation can be blocked by the addition of N-PTB. Inhibition of sulfatide induced αIIbβ3 integrin and P-selectin expression result in decreased platelet-platelet aggregation under flow. N-PTB also blocks sulfatide induced platelet-leukocyte interactions and aggregation. Experimental data supports the hypothesis that Dab2-sulfatide binding serves to increase the inhibition of platelet aggregation. / Master of Science

Disabled-2 (Dab2) is an adaptor protein that plays critical roles in various biological processes, including protein endocytosis, platelet activation and aggregation, tumor growth, and development. In platelets, Dab2 associates with membrane sulfatide at the platelet surface, modulating platelet inside-out and outside-in signaling pathways. A Dab2-derived peptide, named the sulfatide-binding peptide (SBP), is the minimal unit of Dab2 to exert its function as a negative regulator of platelet activation and aggregation. The work of this thesis refines the model of Dab2 SBP binding to sulfatide and provides structural and functional insights into the mechanism by which Dab2 SBP modulates platelet activation. Using molecular docking, lipid-protein overlay assay, nuclear magnetic resonance, and surface plasma resonance tools, this work identifies the critical residues within two major regions responsible for sulfatide interaction. First, docking a sulfatide to Dab2 SBP, a hydrophilic region, primarily mediated by Arg42, is thought to be responsible for the association with the sulfatide headgroup. We observed that Arg 42 could directly interact with sulfatide by forming hydrogen bonds with the OS atoms in the sulfatide head group. Further lipid-protein overlay assay and surface plasma resonance experiments confirmed that both the positive charge and stereochemistry of the side chain of Dab2 SBP Arg42 are required for the sulfatide binding. Moreover, Arg42 is found to be critical in the inhibition of P-selectin expression on activated platelets. The residues nearby Arg42 (i.e., Glu33, Ty38, and Lys 44) also contribute to sulfatide interaction. Second, the second polybasic motif located at the C-terminal -helix 2 is considered to interact with the acyl chain through hydrophobic interactions rather than direct binding to the charged sulfatide head group. Lysine residues in this region are suggested to exert a dual role in sulfatide association, that is, by favoring electrostatic interactions with the negatively-charged sulfatide and/or by employing their flexible hydrocarbon spacers for hydrophobic interactions with membrane lipids. Consistent with this suggestion, we found a hydrophobic patch in the wild type Dab2 SBP structure surrounded by Lys49, Lys51, and Lys53. Furthermore, the role of the second sulfatide binding motif in sulfatide binding is confirmed by mutagenesis analysis and lipid-protein overlay assays, highlighting the ability of molecular docking to accurately predict critical residues responsible for sulfatide binding. In summary, this work provides a detailed structural basis for Dab2 recognition by sulfatide through multiple biophysical methods. The corresponding biological implications in the inhibition of platelet activation are also evaluated by flow cytometry. By elucidating the underlying mechanisms of Dab2 mediating platelet activation through sulfatide binding, we provided structural and functional insights for designing a Dab2-derived peptide with altered sulfatide recognition features in platelets, which can be further employed in antiplatelet therapy. / Doctor of Philosophy / Platelets are blood cells that are fundamentally intended to help form clots to stop bleeding. They do so by being activated after getting signals from damaged blood vessels and reaching the injury site. Consequently, they form aggregates by attracting more platelets to clump on the clot. However, platelet activation induced by a tumor cell can, in turn, protect the tumor cell from immune system elimination and facilitates their growth and spread. This platelet-tumor complex formation suggests platelets as a therapeutic target for reducing tumor migration out of the bloodstream. Our study investigates the mechanism of a Disabled-2-derived peptide, named Dab2 SBP, which upon binding to a sulfatide lipid, can reduce the platelet activation extent, using molecular and cellular approaches. The results of this study may be instrumental in the generation of Dab2 SBP-derived peptides with altered sulfatide binding ability and selectivity, which may lead to a design of an antiplatelet drug that can limit the ability of tumor cells to invade other tissues.

The initiating events of ovarian cancer remain unknown, but an established risk factor is use of estrogen therapy by post-menopausal women where there is a positive correlation between duration of use and risk for disease. Mouse models of ovarian cancer have shown that exposure to exogenous 17β-estradiol (E2) accelerates tumour onset so this study aims to investigate the E2 signalling mechanisms responsible for sensitizing ovarian epithelial cells to transformation. By developing model systems that are responsive to E2 manipulation, we showed that E2 induces the formation of epithelial dysplasias both in vitro and in vivo. microRNA microarray was used to discover that E2 up-regulates microRNA-378 via the ESR1 pathway, resulting in the down-regulation of a tumour suppressor gene called Disabled-2 (Dab2). E2 suppression of Dab2 was found to result in increased proliferation, loss of contact inhibition, epithelial dysplasia, and increased sensitivity to transformation. This mechanism was also found to be active in mouse fallopian tube epithelium and human ovarian cancer cells. Single-cell RNA sequencing and trajectory analysis was subsequently used to explore additional signalling mechanisms that might contribute to the emergence of dysplastic lesions induced by E2. Multiple molecular signalling pathways dysregulated by E2 were identified and this revealed several possible biomarkers to be investigated for early detection of ovarian cancer. In the context of a current lack of strategies for ovarian cancer prevention or early detection, this work represents a significant advance in our understanding of how E2 promotes ovarian cancer initiation.

Disabled-2 (Dab2) is an adapter protein that interacts with cell membranes and it is involved in several biological processes including endocytosis and platelet aggregation. During endocytosis, the Dab2 phosphotyrosine-binding (PTB) domain mediates protein binding to phosphatidylinositol 4,5-bisphosphate (PIP2) at the inner leaflet of the plasma membrane and helps co-localization with clathrin coats. Dab2, released from platelet alpha granules, inhibits platelet aggregation by binding to the °IIb? integrin receptor on the platelet surface through an Arg-Gly-Asp (RGD) motif located within the PTB domain. Alternatively, Dab2 binds sulfatides on the platelets surface, and this binding partition Dab2 in two pools (sulfatide and integrin receptor-bound states), but the biological consequences of lipid binding remain unclear. Dab2 binds sulfatides through two basic motifs located on its N-terminal region including the PTB domain (N-PTB). We have characterized the binding of Dab2 to micelles, which are widely used to mimic biological membranes. These micellar interactions were studied in the absence and presence of Dab2 lipid ligands, sulfatides and PIP2. By applying multiple biochemical, biophysical, and structural techniques, we found that whereas Dab2 N-PTB binding to PIP2 stabilized the protein but did not contribute to the penetration of the protein into micelles, sulfatides induced conformational changes and facilitated penetration of Dab2 N-PTB into micelles. This is in agreement with previous observation that sulfatides, but not PIP2, protect Dab2 N-PTB from thrombin cleavage. By studying the mechanism by which Dab2 targets membranes, we will have the opportunity to manipulate its function in different lipid-dependent biological processes. / Master of Science

L’hyperaldostéronisme primaire [HAP] résulte d’une hypersécrétion d’aldostérone d’origine surrénale. La compréhension de la pathogénie de cette maladie, dont la prévalence est estimée à 10% de la population hypertendue, est essentielle pour le développement de nouveaux outils diagnostiques et thérapeutiques. Dans ce contexte, ce travail de doctorat avait pour but d’identifier de nouvelles orientations thérapeutiques en testant un inhibiteur de l’aldostérone synthase et de rechercher de nouveaux marqueurs diagnostiques par l’étude du profil d’expression des microARN [miRs]. Dans une étude de phase II, 14 patients présentant un HAP ont reçu un inhibiteur de l’aldostérone synthase : le LCI699 pendant 4 semaines. Nous avons ainsi pu montrer que le LCI699 permet de diminuer les concentrations d’aldostérone de 70 à 80% et de normaliser la kaliémie chez tous les patients. En revanche, il n’a qu’un effet modéré sur la pression artérielle et sur l’élévation des concentrations de rénine, et n’est que partiellement sélectif pour l’aldostérone synthase. De plus son efficacité est moindre que celle de l’éplérénone, antagoniste minéralocorticoide administré aux mêmes patients au décours du LCI699. Nous avons ensuite étudié l’expression de 754 miRs dans des adénomes produisant de l’aldostérone [APA] et dans des surrénales contrôles. L’hypothèse était qu’une dérégulation de leur expression pouvait être impliquée dans la tumorigénèse et la surproduction d’aldostérone. L’objectif secondaire était d’identifier des miRs utilisables en tant que biomarqueurs. Cette analyse par carte microfluidique a révélé que 27 miRs sont significativement sous exprimés dans les APA et un seul miR est surexprimé. L’expression différentielle de deux de ces miRs : miR 137 et miR 375 a pu être confirmée dans une cohorte de validation de 36 APA: Des résultats préliminaires in vitro indiquent que le miR 375 pourrait induire une diminution de la synthèse d’aldostérone. Enfin, l’analyse de l’expression de ces miRs dans le plasma a permis de mettre en évidence une sous-expression du miR 375 chez les patients atteints d’HAP en comparaison à des sujets sains. En conclusion, le blocage de la biosynthèse de l’aldostérone représente une nouvelle option thérapeutiques, cependant il est nécessaire de développer une seconde génération de molécules : plus puissantes et plus sélectives. Les analyses effectuées sur les APA ouvrent de nouvelles perspectives pour l’identification de nouveaux biomarqueurs tels que les miRs circulants / Primary aldosteronism [PA] results from the hypersecretion of aldosterone by the adrenals. Understanding the pathogenesis of the disease is essential for identifying new diagnostic and therapeutic tools. In this context the purpose of my PHD was to investigate the effects of an aldosterone synthase inhibitor and second to investigate new diagnostic options by the extensive study of microRNA [miRNA]. In a phase II clinical study, 14 patients with PA were administered an aldosterone synthase inhibitor: LCI699. Four weeks of treatment lead to a 70 to 80% decrease in aldosterone concentration, associated with the cure of hypokalemia. However, there was only a mild effect on blood pressure and volemia (reflected by renin concentration). In addition, these results demonstrated an incomplete selectivity of LCI699 for aldosterone synthase in vivo, and showed that LCI699 is less potent than the blocker of the mineralocorticoid receptor: eplerenone . We also characterized the miRNA profile of Aldosterone producing adenomas [APA]. The hypothesis was that a dysregulation of the expression of miRNA could induce tumorigenesis and increase the production of aldosterone. The secondary aim of the study was to identify miRNA that could be measured in plasma as biomarkers. miRNA profiling of 754 miRNA using quantitative PCR Low Density array, revealed 28 miRNA whose expression was significantly different in APA. The differential expression of two miRNA: miRNA 137 and miRNA 375 was confirmed in a validation cohort of 36 APA. Preliminary in vitro studies showed that up-regulation of intracellular levels of miR 375 may reduce aldosterone secretion in H295R cells. Lastly, circulating plasma levels of miR 375 are differentially expressed between patients with PA and healthy volunteers. In conclusion, the blocking of the aldosterone pathway in hypertensive patients is a novel therapeutic option but second-generation drugs more potent and more selective of aldosterone synthase are required. Profiling miRNA in APA offers new prospect for the development of biomarkers, such as measuring circulating miRNA in plasma