Breast cancer is one of the most common malignancy diagnosed in women and is the primary cause of cancer-related deaths in women worldwide. It is a heterogeneous group of diseases that have a different response, prognosis, and clinical outcomes. Estrogen, progesterone and HER2 negative breast cancer, known as triple negative breast cancer (TNBC), does not respond to hormonal therapy. Basal-like breast cancer (BLBC) has shorter overall survival rate among other subtypes. Tumors sharing both TNBC and BLBC are considered less responsive to currently available treatment. Chemoresistance to treatment has been a challenge in cancer biology and force investigation toward developing new targeted therapies, which selectively target specific subtypes. Sphingolipid metabolites have an important physiological role in determining cell fate. Sphingolipid metabolites, ceramide, sphingosine, and sphingosine-1-phosphate (S1P), are implicated in cancer. S1P exerts its functions via extracellular and intracellular targets. S1P synthesized inside the cell is exported outside and binds to G-protein coupled receptors, the sphingosine-1-phosphate receptors 1-5 (S1PR1-5). Although the intracellular function is not well defined, its suggested intracellular S1P promotes cell apoptosis. The S1P pathway has received great attention recently due its function in cell survival and death. This effect was reported to be concentration dependent. In this research, I focused on S1P effect on nine TNBC/BLBC cell lines. I examined the in-vitro effects of S1P on apoptosis, proliferation, and cytotoxicity in triple negative/ basal-like breast cancer cell lines. Moreover, I studied the co-administration of S1P with currently used chemotherapeutic agents in these cell lines. Data show that S1P can selectively induce cell death in TNBC/BLBC cell lines at a specific concentration. In this research, I found that the mechanism of cell death following treatment with different S1P concentrations was mainly due to apoptosis. Results show that S1P leads to cell shrinkage, rounding and detachment in the nine TNBC/BLBC cell lines. S1P combination with doxorubicin and docetaxel at different concentrations shows no beneficial effect of the combination compared to the chemotherapeuitc agent alone. In some cell lines, the combination showed a protective effect. Further studies are required to determine the mechanism by which S1P induces cell apoptosis, inhibits cell growth, and demonstrates lack of responsiveness in combination studies.

Platelet exocytosis is essential for hemostasis and for many of its sequelae. Platelets release numerous bioactive molecules stored in their granules enabling them to exert a wide range of effects on the vascular microenvironment. Are these granule cargo released thematically in a context-specific pattern or via a stochastic, kinetically-controlled process? My work describes platelet exocytosis using a systematic examination of platelet secretion kinetics. Platelets were stimulated for increasing times with different agonists (i.e. thrombin, PAR1-agonist, PAR4-agonist, and convulxin) and micro-ELISA arrays were used to quantify the release of 28 distinct α-granule cargo molecules. Agonist potency directly correlated with the speed and extent of release. PAR4-agonist induced slower release of fewer molecules while thrombin rapidly induced the greatest release. Cargo with opposing actions (e.g. pro- and anti-angiogenic) had similar release profiles, suggesting limited thematic response to specific agonists. From the release time-course data, rate constants were calculated and used to probe for underlying patterns. Probability density function and operator variance analyses were consistent with three classes of release events, differing in their rates. The distribution of cargo into these three classes was heterogeneous suggesting that platelet secretion is a stochastic process potentially controlled by several factors such as cargo solubility, granule shape, and/or granule-plasma membrane fusion routes. Sphingosine 1 phosphate (S1P) is a bioactive lipid that is stored in platelets. S1P is essential for embryonic development, vascular integrity, and inflammation. Platelets are an abundant source of S1P due to the absence of the enzymes that degrade it. Platelets release S1P upon stimulation. My work attempts to determine how this bioactive lipid is released from platelets. Washed platelets were stimulated with agonists for defined periods of time and the supernatant and pellet fractions were separated by centrifugation. Lipids were separated by liquid phase extraction and S1P was quantified with a triple quadrapole mass spectrometer. A carrier molecule (BSA) is required to detect release of S1P. Further, there is a dose-dependent increase in total S1P with increasing BSA. S1P release shows characteristics similar to other platelet granule cargo e.g. platelet factor IV (PF4). Platelets from Unc13-d Jinx mice and VAMP8-/- mice, which are secretion-deficient (dense granule, alpha granule and lysosome), were utilized to understand the process of S1P release. S1P release was more affected in Unc13-d Jinx mice mirroring their dense granule secretion defect. Fluorescence microscopy and sub-cellular fractionation were used to examine localization of S1P in platelets. S1P was observed to be enriched in a granule population. These studies indicate the existence of two pools of S1P, a readily extractable agranular pool, sensitive to BSA, and a granular pool that requires the secretion machinery for release. The secretion machinery of platelets in addition to being involved in the release of normal granule cargo is thus proved to be involved in the release of bioactive lipid molecules like S1P.

Breast cancer is the most common malignancy diagnosed among women and is the first cause of neoplastic death in women globally. In the last decade our understanding of breast cancer biology has increased and led to the development of a number of targeted therapies, one of which is targeting the cell apoptosis pathway. One of the new targeting pathways under investigation, which was found to be involved in both cell apoptosis and cell proliferation processes, is the sphingolipid signalling pathway. The sphingolipid pathway represents a group of intracellular and extracellular bioactive lipid molecules, including ceramide, ceramide- 1-phosphate, sphingosine, and sphingosine-1-phosphate (S1P). In my research, I focused on the role S1P plays in breast cancer and its potential application as a therapeutic agent. I examined the effects of S1P on the apoptosis, proliferation, and cytotoxicity of different types of breast cancer cell lines in vitro. In addition, I evaluated the effect of both low and high doses of S1P when co-administrated with anticancer drugs commonly used in breast cancer treatment in vitro and in vivo. Moreover, I studied the S1P cellular distribution following exogenous administration. My results demonstrate that S1P can selectively induce apoptosis in breast cancer cells without harming normal breast cells and that S1P is more effective against aggressive breast cancer cells. Another major finding of my study is that S1P can increase the efficacy of chemotherapies against human breast cancer cells. Although S1P cannot directly substitute the current chemotherapies, S1P may function as a good candidate for combination therapy. Furthermore, my work showed that the pro-apoptotic and anti-proliferative effect of S1P is correlated with its intracellular action and that chronic exposure of exogenous S1P in vivo is not toxic to the major organs. Certainly, S1P inclusion in breast cancer treatment modalities may decrease the morbidity and mortality of breast cancer patients and improve clinical outcomes. Further investigations are required to understand the mechanism by which S1P induces apoptosis and inhibits cell proliferation.

There is significant sequence homology and anatomical co-distribution between cannabinoid (CB1) and sphingosine-1-phosphate (S1P) receptors in the CNS, but potential functional relationships between these lysolipid receptors have not been examined. Therefore, to investigate possible relationships between these two systems at the level of G-protein activation, agonist-stimulated [35S]GTPγS binding and autoradiography were conducted. Autoradiographic studies were first performed to localize receptor-mediated G-protein activation in mouse brain. Coronal brain slices were processed for stimulation of [35S]GTPγS binding using the synthetic cannabinoid agonist WIN 55,212-2 (WIN) or SIP. High levels of WIN- and S1P-stimulated [35S]GTPγS binding were observed in the caudate putamen, hippocampus, substantia nigra, and cerebellum. To further characterize the relationship between S1P-and CB1-mediated G-protein activation, spinal cords from adult male CB1 receptor knockout mice, CNS-deleted S1Pl receptor knockout mice and wild type C57 mice were collected, and assessed using agonist-stimulated [35S]GTPγS binding. Results from this experiment revealed that the S1Pl receptor is predominant in mouse spinal cord. To further investigate potential CBl and SIP receptor interactions spinal cords were collected from adult male ICR mice. Additivity studies were preformed using agonist-stimulated [35S]GTPγs binding. Results showed significantly less than additive stimulation when spinal cord tissue was treated with both WIN and SIP. These results suggest an interaction between the CB1 and S1P receptors in the mouse spinal cord. The effect of cannabinoid antagonists, SR141716A (CB1) and SR144528 (CB2) on S1P-and WIN-stimulated [35S]GTPγS binding were also examined in mouse spinal cord homogenates. These results showed that there was no significant difference between S1P-stimulated [35S]GTPγS binding in the presence of SR141716A or SR144528 compared to vehicle control. This shows that S1P produced stimulation independent of the CBl or CB2receptor. In addition WIN-stimulated [35S]GTPγS binding was not affected by SR144528, but was inhibited by SR141716A, confirming that this action is due to the CB1 receptor. The combined results of this project demonstrate an interaction between CB1 and S1P receptors in certain CNS regions where they are co-distributed, such as the caudate putamen, hippocampus, substantia nigra, cerebellum and spinal cord. These results may be due to convergence on a common pool of G-proteins via dimerization or co-localization in lipid rafts, or a possible direct ligand-receptor interaction.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

In biological systems, small changes can have significant impacts. It is, therefore, very important to be able to identify these changes in order to understand what is occurring in the organism. In many cases, this is not an easy task. Mass spectrometry has proven to be a very useful tool in elucidating biological changes even at a very small scale. Several different mass spectrometry based techniques have been developed to discover and investigate complex biological changes. Some of these techniques, such as proteomics, have been through years of development and have advanced to the point that anyone can complete complex analyses of global protein identification and measurement with relative ease. Other techniques are still developing and still have some ground to cover in terms of experimental outcome and ease of execution. Herein we show improvements we have made in high-throughput high-resolution mass spectrometry based techniques to identify and quantify small molecules that are involved in significant biological changes. To begin, we show that our improved high-resolution mass spectrometry based lipidomics techniques are capable of identifying small changes in diseased states that are associated with inflammation, mitochondrial shape and function, and cancer. With our techniques we have been able to extract, identify, and quantify several thousand unique lipid species from complex samples with confidence. Our initial studies looked at global lipidome profiles of differing tissue types from human and mouse biopsies. This was then adapted to compare the global lipidomes of diseased states against healthy states in asthmatic lung tissue, cigarette smoke treated cells, high fat high sugar (HFHS) stressed animals (with and without additional treatment), and in signaling lipids associated with cell death resistance and growth signaling in pancreatic cancer. As a result of our success with lipidomic method improvement we then adapted our techniques and knowledge for use in elucidating small molecule signaling peptides and oxidation changes in proteins. We were able to show that our improved liquid chromatography mass spectrometry based small molecule assays are capable of identifying and quantifying small peptides and protein modifications that would otherwise be undetectable using traditional techniques. This work resulted in the development of a scalable method to detect and quantify the small iron-regulatory hormone known as hepcidin from a variety of samples such as blood, urine, and cell-culture media. We were also instrumental in evaluating and revising a new ultra-high pressure liquid chromatography (UHPLC) system that allows for better separation of analytes from complex mixtures for identification and quantification. Through these advances we hope to aid researchers and clinicians to enable them to use mass spectrometry to further our knowledge about the small but significant changes that regulate complex biological systems.

Many experiments are being conducted to find compounds that offer radioprotection against radiation damage and that are also non-toxic. It is hopeful that in the future, research for this technology will benefit patients undergoing cancer treatment by reducing radiation damage to normal cells and therefore reducing short and long term side effects experienced from treatments. Hamster cells were irradiated at doses of 60 and 120 rad, with and without Sphingosine-1-Phosphate mixed in with their growth medium. Post irradiation, it was observed that the S1P molecule seemed to have a radioprotective effect by decreasing the amount of cell death compared to the amount of cell death that occurred with the absence of the molecule. The results of this experiment will sent to Dr. Jon Tilly at Massachusetts General Hospital. Dr. Tilly is currently researching S1P as a possible radioprotector. / Graduation date: 2003

The first part of this thesis describes a synthetic strategythat provides all eight possible isomers of a given vic-aminoalcohol starting from vinylepoxides. The value of a generalroute is evident, as several isomers are needed ininvestigations of structure-activity relationships forpharmacologically active derivatives, and for optimizing theperformance of chiral ligands containing the amino alcoholmoiety. Vinylepoxides, obtained in high enantiomeric excess, werering-opened both with inversion and retention ofstereochemistry, delivering two diastereomeric amino alcoholswith high regio- and stereoselectivity. Via ring-closure toaziridines and subsequent regioselective ring-opening withsuitable oxygen nucleophiles, the two remaining amino alcoholswere selectively achieved. Within this study, two efficient protocols for theregioselective and stereospecific aminolysis of vinylepoxideshave been presented. Comparedto previous methods, theseprocedures use milder reaction conditions, shorter reactiontimes, generally give higher yields and are applicable to alarger set of substrates. Furthermore, the ring-closure ofvic-amino alcohols to the corresponding N-H vinylaziridines hasbeen investigated. Three routes have been found useful, whichone is preferred depends on substrate and scale. In the second part of the thesis, the synthetic strategy isapplied on the synthesis of Sphingosine and its regio- andstereoisomers. Moreover, a rapid way of determining relativeconfiguration of vic-amino alcohols is described, which shouldbe of substantial use when amino alcohols are formed bydiastereoselective reactions. amino alcohols, vinylepoxides, vinylaziridines, oxazolines,oxazolidinones, ring-opening, regioselective,diastereoselective, sphingosine, configuration, NMRspectroscopy.

La contribution des cellules souches au développement tumoral est une percée conceptuelle récente dans notre compréhension des mécanismes moléculaires et cellulaires impliqués dans la carcinogenèse. En ce sens, il est reconnu depuis quelques années qu'une sous-population de cellules souches mésenchymateuses (MSC) mobilisables en réponse à des facteurs de croissance tumoraux pourrait contribuer au développement tumoral. Les recherches rapportées dans ce mémoire nous ont permis d'étudier certains partenaires clé dans la régulation de la migration et de la survie cellulaire des MSC. L'observation préalable d'une modulation conjointe de l'expression d'une métalloprotéase matricielle de type membranaire (MT1-MMP) et du transporteur microsomal de glucose-6-phosphate (G6PT) nous a permis d'évaluer la contribution respective de ces joueurs dans la signalisation affectant la chimiotaxie des cellules souches ainsi que des cellules tumorales cérébrales. De plus, nous avons évalué l'impact de certains « mannosides » synthétisés en vue de cibler spécifiquement les fonctions de surfaces de MT1-MMP et qui pourraient être à l'origine de nouvelles approches thérapeutiques anticancéreuses affectant le recrutement des cellules souches au foyer tumoral. Finalement, l'importance de l'axe de signalisation MT1-MMP/G6PT dans la mobilisation du calcium intracellulaire en réponse à la sphingosine-1-phosphate, un lipide bioactif synthétisé par des niveaux d'expression élevés de sphingosine-kinase retrouvé au niveau tumoral, permet également de concevoir le ciblage effectif de l'un ou l'autre de ces partenaires dans la progression tumorale. L'ensemble de nos résultats permettra de mieux comprendre les phénomènes régulant la survie et le recrutement des MSC aux sites de foyers tumoraux, en plus de fournir de précieux renseignements sur un nouvel axe original de signalisation liant les fonctions de MT1-MMP à celles, inattendues, de G6PT. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Cellules souches, Cancer, MT1-MMP, G6PT, Sphingosine-1-phosphate.

<p>The first part of this thesis describes a synthetic strategythat provides all eight possible isomers of a given vic-aminoalcohol starting from vinylepoxides. The value of a generalroute is evident, as several isomers are needed ininvestigations of structure-activity relationships forpharmacologically active derivatives, and for optimizing theperformance of chiral ligands containing the amino alcoholmoiety.</p><p>Vinylepoxides, obtained in high enantiomeric excess, werering-opened both with inversion and retention ofstereochemistry, delivering two diastereomeric amino alcoholswith high regio- and stereoselectivity. Via ring-closure toaziridines and subsequent regioselective ring-opening withsuitable oxygen nucleophiles, the two remaining amino alcoholswere selectively achieved.</p><p>Within this study, two efficient protocols for theregioselective and stereospecific aminolysis of vinylepoxideshave been presented. Comparedto previous methods, theseprocedures use milder reaction conditions, shorter reactiontimes, generally give higher yields and are applicable to alarger set of substrates. Furthermore, the ring-closure ofvic-amino alcohols to the corresponding N-H vinylaziridines hasbeen investigated. Three routes have been found useful, whichone is preferred depends on substrate and scale.</p><p>In the second part of the thesis, the synthetic strategy isapplied on the synthesis of Sphingosine and its regio- andstereoisomers. Moreover, a rapid way of determining relativeconfiguration of vic-amino alcohols is described, which shouldbe of substantial use when amino alcohols are formed bydiastereoselective reactions.</p><p>amino alcohols, vinylepoxides, vinylaziridines, oxazolines,oxazolidinones, ring-opening, regioselective,diastereoselective, sphingosine, configuration, NMRspectroscopy.</p>