Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Our recent studies have shown a link between Adenovirus-5 (Ad-5) and elevated lipids, which prompted the hypothesis that Ad-5 infection might augment hepatic and/or adipose tissue lipid metabolism. To test our hypothesis, mice were infected with Ad-5 and screened for changes in lipogenesis and plasma markers associated with the metabolic syndrome. We observed increased expression of sterol regulatory element binding protein 1 (SREBP-1) in infected liver tissues, but not in adipose tissues and this correlated with elevated plasma and hepatic triglyceride levels. Elevated expression of adiponectin was seen in Ad-5 infected adipose tissues and this correlated with phosphorylated AMPK in infected liver tissues. These data suggested that the AMPK pathway was activated in livers of Ad-5 infected mice. Indeed, we observed reduced expression of PEPCK, a downstream target of AMPK in livers of Ad-5 infected mice. As PEPCK is an enzyme essential for gluconeogenesis, we hypothesized that Ad-5 infection would reduce blood sugar. Indeed, infected mice exhibited a transient decline in plasma glucose. The increase in SREBP1 levels in Ad-5 infected hepatic tissues was evaluated by looking at Ad-5 infected HepG2 cells. Ad-5 is thought to mimic insulin’s actions in which the PI3K pathway is activated. We hypothesized that Ad5-induced SREBP-1 expression levels are mediated through the induction of PKC λ/ζ/ι, and not through Akt because it has been shown that PKC λ/ζ/ι mediates insulin-dependent lipogenesis. To test our hypothesis, HepG2 cells were infected with Ad-5 and screened for downstream targets of the PI3K pathway. Through western blot analyses, we observed increased levels of phosphorylated PKC λ/ζ/ι. These results prompted the use of PKC pan inhibitor to see whether Ad-5 induced SREBP-1 levels would be down regulated. Indeed, with the presence of the PKC pan inhibitor, SREBP-1 expression levels were reduced. Together, these studies suggest that Ad-5 induces changes in gene expression, glucose, and lipid metabolism; which prompts the hypothesis that this common respiratory pathogen may be associated with the Metabolic Syndrome, and this may preclude its use as a vector for gene therapy.

Les peptides et protéines extracteurs de lipides (PEL) se lient aux membranes lipidiques puis en extraient des lipides en formant de plus petits auto-assemblages, un phénomène qui peut aller jusqu'à la fragmentation des membranes. Dans la nature, cette extraction se produit sur une gamme de cellules et entraîne des conséquences variées, comme la modification de la composition de la membrane et la mort de la cellule. Cette thèse se penche sur l’extraction lipidique, ou fragmentation, induite par le peptide mélittine et la protéine Binder-of-SPerm 1 (BSP1) sur des membranes lipidiques modèles. Pour ce faire, des liposomes de différentes compositions sont préparés et incubés avec la mélittine ou la BSP1. L'association aux membranes est déterminée par la fluorescence intrinsèque des PEL, tandis que l'extraction est caractérisée par une plateforme analytique combinant des tests colorimétriques et des analyses en chromatographie en phase liquide et spectrométrie de masse (LCMS). La mélittine fait partie des peptides antimicrobiens cationiques, un groupe de PEL très répandu chez les organismes vivants. Ces peptides sont intéressants du point du vue médical étant donné leur mode d’action qui vise directement les lipides des membranes. Plusieurs de ceux-ci agissent sur les membranes des bactéries selon le mécanisme dit « en tapis », par lequel ils s’adsorbent à leur surface, forment des pores et ultimement causent leur fragmentation. Dans cette thèse, la mélittine est utilisée comme peptide modèle afin d’étudier le mécanisme par lequel les peptides antimicrobiens cationiques fragmentent les membranes. Les résultats montrent que la fragmentation des membranes de phosphatidylcholines (PC) est réduite par une déméthylation graduelle de leur groupement ammonium. L'analyse du matériel fragmenté révèle que les PC sont préférentiellement extraites des membranes, dû à un enrichissement local en PC autour de la mélittine à l'intérieur de la membrane. De plus, un analogue de la mélittine, dont la majorité des résidus cationiques sont neutralisés, est utilisé pour évaluer le rôle du caractère cationique de la mélittine native. La neutralisation augmente l'affinité du peptide pour les membranes neutres et anioniques, réduit la fragmentation des membranes neutres et augmente la fragmentation des membranes anioniques. Malgré les interactions électrostatiques entre le peptide cationique et les lipides anioniques, aucune spécificité lipidique n'est observée dans l'extraction. La BSP1 est la protéine la plus abondante du liquide séminal bovin et constitue un autre exemple de PEL naturel important. Elle se mélange aux spermatozoïdes lors de l’éjaculation et extrait des lipides de leur membrane, notamment le cholestérol et les phosphatidylcholines. Cette étape cruciale modifie la composition lipidique de la membrane du spermatozoïde, ce qui faciliterait par la suite la fécondation de l’ovule. Cependant, le contact prolongé de la protéine avec les spermatozoïdes endommagerait la semence. Cette thèse cherche donc à approfondir notre compréhension de ce délicat phénomène en étudiant le mécanisme moléculaire par lequel la protéine fragmente les membranes lipidiques. Les résultats des présents travaux permettent de proposer un mécanisme d’extraction lipidique en 3 étapes : 1) L'association à l’interface des membranes; 2) La relocalisation de l’interface vers le cœur lipidique; 3) La fragmentation des membranes. La BSP1 se lie directement à deux PC à l'interface; une quantité suffisante de PC dans les membranes est nécessaire pour permettre l'association et la fragmentation. Cette liaison spécifique ne mène généralement pas à une extraction lipidique sélective. L'impact des insaturations des chaînes lipidiques, de la présence de lysophosphatidylcholines, de phosphatidyléthanolamine, de cholestérol et de lipides anioniques est également évalué. Les présentes observations soulignent la complexe relation entre l'affinité d'un PEL pour une membrane et le niveau de fragmentation qu'il induit. L'importance de la relocalisation des PEL de l'interface vers le cœur hydrophobe des membranes pour permettre leur fragmentation est réitérée. Cette fragmentation semble s'accompagner d'une extraction lipidique préférentielle seulement lorsqu'une séparation de phase est induite au niveau de la membrane, nonobstant les interactions spécifiques PEL-lipide. Les prévalences des structures amphiphiles chez certains PEL, ainsi que de la fragmentation en auto-assemblages discoïdaux sont discutées. Finalement, le rôle des interactions électrostatiques entre les peptides antimicrobiens cationiques et les membranes bactériennes anioniques est nuancé : les résidus chargés diminueraient l'association des peptides aux membranes neutres suite à l'augmentation de leur énergie de solvatation. / Lipid-extracting peptides and proteins (LEPs) bind to lipid membranes, extract lipids in the form of smaller auto-assemblies, and ultimately fragment membranes. In nature, this lipid extraction occurs in many different cell systems and causes various consequences, such as a modification of the membrane lipid composition or the cell death. This thesis focuses on the lipid extraction, or fragmentation, induced by the peptide melittin and the protein Binder-of-SPerm 1 (BSP1) on model lipid membranes. To this end, liposomes of different composition are prepared and incubated with melittin or BSP1. The association to membranes is determined by the LEPs intrinsic fluorescence, while the extraction is characterized by a combination of colorimetric phosphorus assays and liquid chromatography-mass spectrometry analyses (LCMS). Melittin is a cationic antimicrobial peptide, a very common category of LEP found in living organisms. Cationic antimicrobial peptides are interesting to medicine because they directly target membrane lipids. The action of many of these peptides is described by the carpet-like mechanism, by which they adsorb to membrane surface, induce the formation of pores and then cause the fragmentation of the membranes. In this thesis, melittin is used as a model peptide in order to study the mechanism by which cationic antimicrobial peptides fragment lipid membranes. Results show that the phosphocholine (PC) membrane fragmentation is reduced by a gradual demethylation of the ammonium group. Analysis of the fragmented material reveals that PC are preferentially extracted from membranes, due to a local enrichment in PC near melittin in the membrane. Furthermore, a melittin analogue, for which a majority of its cationic residues were neutralized, is used to investigate the role of the cationic character of native melittin. The neutralization increases the peptide affinity for neutral and anionic membranes, reduces fragmentation of neutral membranes and increases fragmentation of anionic membranes. Despite electrostatic interactions between the cationic peptide and the anionic lipids, no lipid specificity is observed in the extraction. BSP1 is the most abundant protein of the bovine seminal plasma and constitutes another example of important LEP found in nature. Upon ejaculation, it mixes with spermatozoa and extracts membrane lipids, such as cholesterol and phosphatidylcholines. This crucial process modulates the lipid composition of sperm membranes, which would then facilitate egg fertilization. However, a prolonged contact between the protein and spermatozoa could damage the semen. This thesis is looking to deepen our understanding of this delicate phenomenon by studying the molecular mechanism by which this protein fragments lipid membranes. Results of the present work suggest a 3-step mechanism for the extraction: 1) Association to membrane interface; 2) Relocation towards the lipid core; 3) Fragmentation of membranes. BSP1 binds directly to two interfacial PC; a sufficient quantity of PC in membranes is necessary for protein association and fragmentation. This specific binding generally does not lead to specificity in the lipid extraction. The impact of unsaturation of the lipid chains, of the presence of lysophosphatidylcholines, of phosphatidylethanolamines, of cholesterol and of anionic lipids is also studied. The present observations underline the complex relationship between a LEP affinity for membranes and the level of fragmentation it induces. The importance of LEP relocation, from the interface to the hydrophobic core of the membranes, for fragmentation is reiterated. This fragmentation seems to be lipid specific only when a phase separation of the lipids occurs in the membrane, notwithstanding specific LEP-lipid interactions. The prevalence of amphipathic structures in certain LEPs, as well as of the auto-assembled discoidal structures resulting from fragmentation is discussed. Finally, the role of electrostatic interactions between cationic antimicrobial peptides and anionic bacterial membranes is detailed: charged residues lower peptide association to neutral membrane due to an increase of their free energy of solvation.

<p>Advances in modern medicine have helped to prolong human life. These advancements coupled with an ever-increasing population means that diseases associated with aging will become more prevalent in the coming years. As such, it is critical to understand the pathogenesis of disease where aging is the main risk factor. While not widely known, age is in fact a large risk factor in development of obesity and metabolic syndrome. More widely known and discussed are the neurodegenerative diseases that occur late in life. While age as a risk factor is a common point between these types of pathology, there are other similarities, such as the interaction between lipid metabolism and mitochondrial health. </p><p>To study the overlap between obesity and neurodegeneration, we investigated two pathways that regulate both. First, we find that loss of cytoplasmic deacetylase HDAC6 leads to aberrant accumulation of lipid in vitro and in vivo. HDAC6 knock-out (KO) mice gain more weight than WT counterparts after a high-fat diet regimen. Additionally, the intermediary metabolism of cells lacking HDAC6 is disrupted as they increase glucose uptake while downregulating fatty acid oxidation. HDAC6 not only plays a role in lipid metabolism, but regulates mitochondrial dynamics. Upon glucose-withdrawal, HDAC6 KO cells fail to elongate their mitochondria and display increased levels of mitochondrial toxic by-products. Therefore, HDAC6 has critical roles in lipid homeostasis and mitochondrial health. </p><p>The other pathway we investigated is critical in neurodegenerative disease, Parkinson's disease. Parkin, an E3 ubiquitin ligase, flags damaged mitochondria for destruction so they do not poison the other functional organelles. We found that Parkin promotes lipid remodeling at the surface of the mitochondria. Phosphatidic acid (PA) accumulates shortly after mitochondrial damage while diacylglycerol (DAG) appears several hours later. This lipid accumulation is dependent upon Parkin's translocation and E3 ligase activity. Additionally, we found that lipin-1, a PA phosphatase, and endophilin B1 (EndoB1) are critical for DAG accumulation and effective mitochondrial clearance. </p><p>Through this work, we show that two proteins critical in quality control mechanisms also play significant roles in energy homeostasis. We aim to highlight this overlap and posit that common diseases of aging, though presenting differently, might have disruptions in the same basic process.</p> / Dissertation

ATP synthase is a ubiquitous transmembrane protein that utilises the free energy available from ion gradients across lipid membranes to synthesise adenosine triphosphate (ATP). It may be separated into two parts - the membrane-embedded (i.e. hydrophobic) FO and the hydrophilic F₁. Each undergoes a rotary motion. Single-molecule studies on the rotation of the isolated hydrophilic F₁ have been performed for many years; attempts to construct an experiment in which to view the rotation of the membrane-embedded F₁F_O complex under high space- and time- resolution (such as by attachment of a rotational probe) have not yet seen a satisfactory method emerge in the literature. Most particularly, a clear ability to generate and control a proton-motive force across the membrane in which the F₁F_O is sited is needed to probe ATP synthesis. This thesis presents the development of a candidate method for such single-molecule studies. By the use of a water-in-oil emulsion, giant unilamellar lipid vesicles are formed which entrap arbitrary components - including functionalised gold nanospheres of 60-100 nm diameter, which move freely in the internal space. A charge-based lipid fusion is developed, using mixtures of natural lipid extracts with anionic and cationic lipids. It is demonstrated that anionic giant vesicles fuse with cationic small vesicles with full content mixing and transfer of bilayer leaflets. It is shown that F₁F_O is functional in the cationic lipid mixture. Methods are shown to bind such a cationic proteoliposome to a surface and for it to fuse with an anionic giant vesicle containing functionalised gold nanospheres. Backscatter laser darkfield is used to search for rotation of the gold nanospheres under ATP hydrolysis conditions of the F₁F_O; unidirectional rotation is seen in one instance and other suggestive traces are shown with speculative analysis. Further work is proposed.

La variété de l'offre alimentaire actuelle incite à l'hyperphagie qui est en partie responsable de la prise de poids corporel. Réduire la consommation est ainsi devenu une contrainte pour une certaine partie de la population. Dans ce contexte, les fibres, qui sont des glucides non-digestibles dans l'intestin grêle, permettent de réduire la prise alimentaire, d'atténuer les sensations de faim et/ou d'augmenter les sensations de satiété. Cependant, les mécanismes comportementaux, périphériques et centraux à l'origine de ces effets sont mal-connus. Leur capacité à maintenir cet effet anorexigène en présence d'autres nutriments comme les lipides et les protéines a également été très peu étudiée.L'étude de ces mécanismes est l'objet de cette thèse. Des fibres telles que la gomme de guar (GG) fortement viscosifiante et le fructo-oligosaccharide (FOS) très fermentescible ont été administrées, en début de journée, aux souris sous forme de solution à raison de 700 µL et à une dose de 3%, 5% et 14% via un gavage intra-gastrique. Cette étude a montré que les solutions fortement visqueuses, notamment le GG 5% et le mélange GG-FOS 14% étaient les seules capables d'exercer un effet anorexigène à court terme comparées aux solutions moins visqueuses comme l'eau, le GG 3%, le FOS 14% et les solutions de nutriments comme les protéines (peptides de caséine) et les lipides (huile de colza) qui fournissent 10% de l'ingéré calorique quotidien des animaux (1.2 kcal). Pour le cas du mélange visqueux GG-FOS 14%, la baisse de la prise alimentaire est due au rassasiement (réduction de 50% de la taille et de la durée du repas dans les 30 premières minutes post-ingestion). Cette baisse, non compensée jusqu'à la fin de la journée, n'est pas associée à une aversion gustative conditionnée. Ce rassasiement est dû à l'intégration au niveau du centre de contrôle de la taille du repas (NTS), des signaux vagaux de distension gastrique et de la cholecystokinine (CCK). Quand les fibres visqueuses GG 5% et GG-FOS 14% sont mélangées avec des protéines, elles perdent leur effet anorexigène. L'action de les mélanger avec les lipides a par contre montré que seul le mélange visqueux GG-FOS 14% est capable d'exercer un effet anorexigène. L'effet anorexigène du mélange lipide et GG-FOS 14% est dû au niveau de chaque repas aux signaux mécaniques gastriques et CCK vagaux, et à l'axe PYY- système mélanocortique hypothalamique pour le maintien de l'effet sur la journée.En conclusion, les fibres visqueuses sont des nutriments qui peuvent participer au contrôle de la prise alimentaire en stimulant le rassasiement. Elles sont capables de maintenir cet effet en milieu lipidique. Elles pourraient ainsi être ajoutées aux aliments riches en lipides pour mieux contrôler la prise alimentaire et le poids corporel. / The various types of food currently marketed encourage people to eat more, thus leading to weight gain, and reducing food intake has become challenging. To help deal with this, it was shown that dietary fibers decreased food intake and/or feelings of hunger while increasing those of satiety. However, the behavioral, peripheral and central mechanisms underlying this phenomenon are not well known. This study was undertaken in order to characterize these mechanisms. Dietary fibers such as the highly viscous guar gum (GG) and the highly fermentable fructo-oligosaccharide (FOS), in doses of 3%, 5% and 14%, were given to mice by intra-gastric gavage of 700 µL volume. It appeared that highly viscous fibers such as GG 5% and the mixture GG-FOS 14% were able to decrease food intake compared to less viscous preloads such as water, FOS and nutrient solutions (protein solution with casein peptides, lipid solution with rapeseed oil supplying 1.2 kcal or 10% of daily energy intake in mice). Specifically, the mixture GG-FOS 14% induced satiation by reducing the size and duration of meals during the first 30 min post-treatment. This effect was neither compensated for by the end of the day, nor was it associated with conditioned taste aversion. The GG-FOS 14% -induced satiation was due to gastric distension and vagal CCK signaling which were integrated at the level of the NTS, a nucleus controlling meal size. Moreover, mixing GG 5% and GG-FOS 14% with protein abolished their food intake inhibitory effect. In contrast, when mixed with lipid, GG-FOS 14% maintained its anorexigenic effect. The mixture of lipid and GG-FOS 14% stimulated satiation which involved vagal CCK signaling, gastric distension and the NTS. Additionally, the communication between systemic PYY and melanocortic neurons at the level of the hypothalamus was implicated in the anorexigenic effect of this mixtureIn conclusion, viscous dietary fibers can control food intake by stimulating satiation. They are able to maintain their anorexigenic effect in lipid media such that they can be added to foods containing more fat in order to control food intake and body weight.

The effect of several fundamental reproductive 'decisions' upon reproductive success were examined over a three year period at a large, inland gullery in the Pennines. Variations in reproductive parameters in relation to timing of breeding and reproductive success were compared among years. Determinants of the degree of hatching asynchrony were identified. Eggs from two years were taken for yolk lipid analysis, using gas chromatography. Variation in micronutrient content of eggs within clutches and between years was assessed in relation to egg size and yolk size, in order to examine resource allocation decisions of individuals. Timing of breeding of individuals was experimentally manipulated through the exchange of whole clutches of eggs between early and late laying birds, whilst controlling for variation in clutch size and egg-size, in order to assess whether the seasonal decrease in reproductive success was best explained by a decrease in food supply or differences in quality among parents. I experimentally manipulated the within-brood mass hierarchy of gulls, whilst controlling for variation in both chick quality and parental quality, in order to assess the effect of hatching asynchrony per se on chick growth and survival, and whether parents optimized the degree of hatching asynchrony with respect to the prevailing food supply. Brood size was experimentally reduced in order to assess the costs and benefits of the production of supernumary young. This was acheived by comparison of chick growth, feather development and chick survival of unmanipulated three-chick broods with broods where either the a-chick or the c- chick had been removed. I present a general discussion of the results within the context of life-history theory and a model for the evolution of hatching asynchrony in the lesser black-backed gull.

Lipid peroxidation is believed to play a key role in the onset and progression of degenerative disease. Interestingly, although cholesterol is the most abundant lipid in the human body, our understanding of its autoxidation and subsequent decomposition is relatively limited. In fact, until recently, cholesterol-7-hydroperoxide was accepted as the only primary product of cholesterol autoxidation in organic solution, however, our group exhibited that the 4-, 5-, and 6-hydroperoxides are also formed. Although this work facilitated thorough investigation of the complexities of both H-atom abstraction and addition in cholesterol autoxidation in organic solution, it did not account for the dynamic environment of a cell membrane. Herein, we report on the product distribution of these primary autoxidation products in lipid bilayers and how antioxidant supplementation, H-bonding interactions, and concentration of polyunsaturated fatty acid (PUFA) substrate influence both the product distribution and efficiency of autoxidation. Indeed, not only does H-bonding of the 3β-OH of cholesterol appear to shut-down C4 H-atom abstraction, the absence of kinetic chol-5α-OOH product is likely due to the poor potency of α- tocopherol (α-TOH), also as a result of H-bonding with phosphate head group of lipid membrane phospholipids. Therefore, within a lipid membrane the 7-hydroperoxide products predominate, consistent with literature precedent, however the factors involved are more complex than previously understood. Moreover, with the authentic cholesterol hydroperoxides in hand, we sought to determine if the different regioisomers exhibit different cytotoxicity. Glutathione peroxidases (GPXs) are cytoprotective enzymes that reduce harmful hydroperoxides to benign alcohols in vivo. Using RSL3, a small-molecule inhibitor for GPX4, we were able to sensitize mammalian cells to ferroptotic cell death via administration of our exogenously prepared chol-OOHs. Surprisingly, we found that the toxicities of each of 7α-OOH, 6β-OOH and 5α-OOH were only marginally augmented by RSL3 treatment, suggesting that they do not substantially sensitize cells to ferroptosis, perhaps because their decomposition to lipid peroxidation chain-initiating species (i.e. alkoxyl radicals) is not particularly efficient. Instead their cytotoxicities may derive from other mechanisms, such as the induction of apoptosis. This inspired our investigation of the fate of lipid hydroperoxides in vivo, namely the secondary products of the predominant 7-hydroperoxide species. Acid-catalyzed Hock fragmentation, known for the industrial synthesis of phenol and acetone from cumene or implication in the generation of 4-hydroxynonenal (4-HNE), of 5α- and 6β-OOH has been shown by our group to produce highly electrophilic secosterol species; we sought to investigate the same decomposition mechanism for 7α-OOH in light of our investigations in the lipid membrane. Interestingly, we found that Hock fragmentation of 7α-OOH does not exhibit products resulting from the anticipated O-vinyl oxocarbenium intermediate, rather, the mechanism appears to funnel through an α-epoxy carbenium to produce unprecedented A-ring cleavage and epoxide products. Herein, we describe our thorough analysis of this chol-7α-OOH Hock fragmentation and attempts to investigate the presence of these products in biological samples, similar to previous analyses of similar products in atherosclerotic plaque extracts. The products isolated and characterized through this work have provided new mechanistic insight with regards to the primary and secondary oxidation products of cholesterol in vivo; through further development of these findings, we hope to provide a better understanding of the implications of cholesterol oxidation in the pathogenesis of atherosclerosis.

Thesis advisor: Thomas N. Seyfried / Thesis advisor: Charles Hoffman / Bis(monoacylglycero)phosphate, BMP, is a negatively charged glycerol-phospholipid with an unusual sn-1;sn-1’ structural configuration. BMP is primarily enriched in endosomal/lysosomal membranes. BMP is thought to play a role in glycosphingolipid degradation and cholesterol transport. It constitutes only about 1-2% of the total phospholipids in most mammalian cells, but is abundant in lung alveolar macrophages where it can comprise up to 16% of the total phospholipids. BMP also accumulates in tissues of humans and animals with lysosomal storage disorders. However, little information is available on BMP levels in gangliosidosis brain tissue. In this work, I found that total BMP content was significantly greater in cells of macrophage/microglial origin than in cells of macroglial origin (astrocyte, oligodendrocyte progenitor), whether normal or tumorigenic. I also observed that BMP in brain was significantly greater in humans and in animals (mice, cats, American black bears) with either GM1 or GM2 ganglioside storage diseases, than in brains of normal subjects. Since BMP is associated with macrophages, I also analyzed the BMP levels in relation to disease-associated inflammation in gangliosidoses. I found that BMP levels were increased due to accumulation of primary storage material gangliosides, rather than an outcome of disease-associated inflammation. In addition, in this thesis I also explored the effect of new ketogenic diet formula from Solace Nutrition (KetoGen) on the growth and metastatic spread of the VM-M3 tumor. Most current drug therapies for cancer are toxic and only marginally effective in providing long-term management. Respiratory insufficiency with compensatory aerobic fermentation (Warburg effect) is the hallmark biochemical phenotype of nearly all neoplastic cells within tumors. Calorie restriction, which lowers blood glucose and elevates ketone bodies, is known to reduce tumor growth to a certain extent, however it does not reduce systemic metastasis. Tumor bearing VM mice were fed either a standard lab chow diet in unrestricted amounts (SD-UR), a standard lab chow restricted to obtain an 18% reduction in body weight (SD-R), or the KetoGen diet restricted (KG-R) to match the body weights of the SD-R group. Tumor size was significantly smaller and organ metastasis was significantly less in the KG-R group than in the SD-UR or SD-R groups. Even though blood glucose was reduced similarly in both the SD-R and KG-R groups, blood ketones were 3-fold higher in the KG-R group than in the SD-R group. These results show that VM-M3 tumor growth and systemic metastasis were managed better with the restricted KetoGen KD than with calorie restriction of a high carbohydrate standard diet. As all human and mouse tumors cells suffer from respiratory insufficiency, my findings suggest that the restricted KetoGen diet should be an effective non-toxic therapy against tumor growth and systemic metastatic cancer. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

As an emerging field, lipidomics still encounters methodological challenges. Indeed, as it is an –omics field, analysis of data is time and labour intensive, if the necessary and appropriate bioinformatics tools are not existent. Here I present two programs I developed to address the challenges of peak identification and peak annotation and filtering for a targeted lipidomics approach, which is not supported by available software. The first program, Lipid Identification Tool (LIT), consists of a stand-alone offline search engine, which provides a robust in silico database generated by linear equations based on lipid structures, containing information lacking on presently available online databases. The second program, Lipid Identification for Targeted Lipidomics (LITL), allows the annotation of HPLC-ESI-MS/MS MRM acquired spectral data in text-based format, comparing them to a library of identities, via retention time and mass-over-charge of detected ions, and to easily export the results to a statistical analysis software. // La lipidomique est un jeune domaine encore proie à des défis quant à sa méthodologie. En effet, l’analyse de données en lipidomique demeurera longue et ardue, tant que des outils bioinformatiques appropriés ne sont pas créés. Je décris ici deux de mes programmes développés pour remédier aux défis d’identification de pics et d’annotations et de sélection de pics provenant d’une approche lipidomique dénommée ciblée, données présentement incompatibles avec d’autres programmes. Lipid Identification Tool (LIT) est un moteur de recherche hors-ligne possédant une robuste base de données in silico générée mathématiquement, et contenant des informations sur des espèces lipidiques non décrites dans les bases de données disponibles. Lipid Identification for Targeted Lipidomics (LITL) permet l’annotation de données provenant de méthodes HPLC-ESI-MS/MS MRM, en comparant leur temps de rétention ainsi que leur rapport masse-sur-charge à une bibliothèque d’identités lipidiques, et permet de les exporter facilement vers un logiciel d’analyse statistique.