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

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Alzheimer's disease (AD) is an irreversible neurodegenerative brain disease distinguished by progressive impairment of memory and decline in cognitive abilities. The hippocampus is widely recognized to play essential roles in forming and gradually transferring information from short-term memory into long-term memory, and it is involved in the onset of the neuropathological pathways of the brain to suffer neuron loss in the rise of AD. Thus, hippocampal information obtained from magnetic resonance imaging (MRI) scans have been established as crucial AD biomarkers. The hippocampus is composed of multiple subfields, and the neuron loss is not uniformly distributed on the whole hippocampus. However, this critical subfield information is not addressed by the existing surface-based morphometry (SBM) and voxel-based morphometry (VBM) studies. Due to the size, complexity, heterogeneity, and folding anatomy of the hippocampus, acquiring volumetric and morphometric measures of hippocampal subfields usually presents not only technical challenges in quantitative neuroimaging but also analytical challenges. To address these challenges and deeply understand the relationships between hippocampal shape changes and brain disorders, especially to examine the degeneration of hippocampal subfields, this thesis focuses on constructing a hippocampal subfield morphometric analysis framework including the following aspects: 1) hippocampal subfield segmentation; 2) 3D shape modeling; 3) feature formulation; 4) diffeomorphic surface registration; 5) surface shape reconstruction; and 6) association analytics. The goals include developing accurate hippocampal subfield guided registration methods, extracting useful features and identifying significant subfields on the hippocampus that are highly related to cognitive disabilities, and using such information to assist early detection of AD.

<p>This thesis covers a wide range of analytical method development for lipid analysis in complex biological samples; from sample preparation using pressurized fluid extraction (PFE) and separation with reversed phase capillary liquid chromatography (RP-LC) to detection by electrospray ionization mass spectrometry (ESI/MS) and tandem MS.</p><p>The requirements for fast, reliable and selective extraction methods with minimal usage of solvents have accelerated the development of new extraction techniques. PFE is one of the new automated, fast and efficient liquid extraction techniques which use elevated temperature and pressure with standard liquid solvents. In this thesis the reliability and efficiency of the PFE technique was investigated for the extraction of total lipid content from cod, herring muscle and human brain tissue as well as for pesticides from fatty foodstuffs. Improved or comparable efficiencies were achieved with reduced time and solvent consumption as compared to traditional methods. </p><p>A RP-LC coupled online to ESI/MS for the analysis of phosphatidylcholine (PC) and sphingomyelin (SM) molecular species was developed and used for the analysis of brain lipids from eight groups of mice treated with vehicle and various neuroleptics. The effect of postnatal iron administration in lipid composition and behavior was investigated. Whether or not these effects could be altered by subchronic administration of the neuroleptics (clozapine and haloperidol) were examined. The results support the hypothesis that an association between psychiatric disorders, behavior abnormalities and lipid membrane constitution in the brain exists.</p><p>Finally, a tandem MS precursor ion scan was used to analyze the developmental profile of brain sulfatide accumulation in arylsulfatase A (ASA) deficient (ASA -/-) as compared to wild type control (ASA +/+) mice. The ASA -/- mice were developed as a model of the monogenic disease metachromatic leukodystrophy with an established deficiency of the lysosomal enzyme ASA. The results showed that an alteration in the composition of sulfatide molecular species was observed between the ASA -/- and ASA +/+ mice.</p><p>This thesis shows that modern analytical methods can provide new insights in the extraction and analysis of lipids from complex biological samples.</p>

This thesis covers a wide range of analytical method development for lipid analysis in complex biological samples; from sample preparation using pressurized fluid extraction (PFE) and separation with reversed phase capillary liquid chromatography (RP-LC) to detection by electrospray ionization mass spectrometry (ESI/MS) and tandem MS. The requirements for fast, reliable and selective extraction methods with minimal usage of solvents have accelerated the development of new extraction techniques. PFE is one of the new automated, fast and efficient liquid extraction techniques which use elevated temperature and pressure with standard liquid solvents. In this thesis the reliability and efficiency of the PFE technique was investigated for the extraction of total lipid content from cod, herring muscle and human brain tissue as well as for pesticides from fatty foodstuffs. Improved or comparable efficiencies were achieved with reduced time and solvent consumption as compared to traditional methods. A RP-LC coupled online to ESI/MS for the analysis of phosphatidylcholine (PC) and sphingomyelin (SM) molecular species was developed and used for the analysis of brain lipids from eight groups of mice treated with vehicle and various neuroleptics. The effect of postnatal iron administration in lipid composition and behavior was investigated. Whether or not these effects could be altered by subchronic administration of the neuroleptics (clozapine and haloperidol) were examined. The results support the hypothesis that an association between psychiatric disorders, behavior abnormalities and lipid membrane constitution in the brain exists. Finally, a tandem MS precursor ion scan was used to analyze the developmental profile of brain sulfatide accumulation in arylsulfatase A (ASA) deficient (ASA -/-) as compared to wild type control (ASA +/+) mice. The ASA -/- mice were developed as a model of the monogenic disease metachromatic leukodystrophy with an established deficiency of the lysosomal enzyme ASA. The results showed that an alteration in the composition of sulfatide molecular species was observed between the ASA -/- and ASA +/+ mice. This thesis shows that modern analytical methods can provide new insights in the extraction and analysis of lipids from complex biological samples.