Macrophage foam cells are the major culprits in atherosclerotic lesions, having a prominent role in both lesion initiation and progression. With atherosclerosis being the main factor underlying cardiovascular complications, there is a long-standing interest on finding ways to reverse lipid buildup in plaques. Studies have shown that promoting reverse cholesterol transport (RCT) from macrophage foam cells is anti-atherogenic because it alleviates the cholesterol burden of the plaques. The goal of this thesis was to gain insight into the mechanisms that govern cholesterol efflux from macrophage foam cells. The first part of this study looked at the ability of different oxysterols to promote cholesterol efflux in unloaded as compared to lipid-loaded macrophages, and our major finding here is that epoxycholesterol decreases efflux in lipid-loaded macrophages. It appears that epoxycholesterol does so by impairing the release cholesterol from its cellular storage site, the lipid droplet (LD), where it accumulates in the form of cholesteryl esters (CE). These results highlighted the importance of cholesterol release from LDs for efflux; indeed, this process is increasingly being recognized as the rate-limiting step for RCT in vivo. Subsequent experiments aimed at elucidating the mechanisms that govern LD CE hydrolysis in macrophage foam cells lead to the discovery of a novel pathway involved in cholesterol efflux. Macrophage CE hydrolysis is classically defined as being entirely dependent on neutral CE hydrolases. In the second part of this study, we demonstrate that in addition to the canonical CE hydrolases, which mediate neutral lipid hydrolysis, lysosomal acid lipase (LAL) also participates in the hydrolysis of cytoplasmic CE. Autophagy is specifically triggered in macrophages by atherogenic lipoproteins and delivers LD CE to LAL in lysosomes, thus generating free cholesterol for efflux. This autophagy-mediated cholesterol efflux is a process that is primarily dependant on the ABCA1 transporter and, importantly, is important for whole-body RCT. Overall, the studies presented in this thesis support that macrophage LD CE hydrolysis is rate-limiting for cholesterol efflux and shed light on the mechanisms of cholesterol mobilization for efflux in macrophage foam cells.

In the last few decades, an increasing number of physicists specialized in soft matter, including polymers, have turned their attention to biologically relevant materials. The properties of various molecules and fibres, such as DNA, RNA, proteins, and filaments of all sorts, are studied to better understand their behaviours and functions. Self-assembled biological membranes, or lipid bilayers, are also the focus of much attention as many life processes depend on these. Small lipid bilayers vesicles dubbed liposomes are also frequently used in the pharmaceutical and cosmetic industries. In this thesis, work is presented on both the elastic properties of polymers and the response of lipid bilayer vesicles to extrusion in narrow-channels. These two areas of research may seem disconnected but they both concern deformed soft materials. The thesis contains four articles: the first presenting a fundamental study of the entropic elasticity of circular chains; the second, a simple universal description of the effect of sequence on the elasticity of linear polymers such as DNA; the third, a model of the symmetric thermophoretic stretch of a nano-confined polymer; the fourth, a model that predicts the final sizes of vesicles obtained by pressure extrusion. These articles are preceded by an extensive introduction that covers all of the essential concepts and theories necessary to understand the work that has been done.

In vitro studies have shown that the major membrane phospholipid phosphatidylcholine (PC) can positively influence the incorporation of cholesterol in lipid membranes. The influence of PC on the cellular trafficking of LDL-derived free cholesterol was investigated. Sterol regulatory-defective (SRD)-4 cells are Chinese hamster ovary (CHO)-derived fibroblasts that display vastly elevated rates for the synthesis and catabolism of PC. SRD-4 cells harbor two known gene mutations: a mutation in the functional allele for SCAP, resulting in defective feedback suppression of cholesterol biosynthesis; and a loss-of-function mutation in the functional allele for acyl-CoA:cholesterol acyl transferase (ACAT), an endoplasmic reticulum (ER)-localized enzyme that esterifies free cholesterol. Incubation of SRD-4 cells with 50 µg/ml low density lipoprotein (LDL) for 18 h resulted in lysosomal accumulation of free cholesterol as revealed by filipin staining. This accumulation was not evident following LDL treatment of parental CHO7 cells, and was blunted in SRD-2 cells that express a constitutively-active form of SREBP-2 and overproduce cholesterol but have functional ACAT activity. Treatment of SRD-2 cells with LDL in the presence of an ACAT inhibitor 58-035 resulted in robust lysosomal cholesterol accumulation that was reversible upon drug washout, supporting that cholesterol trafficking in cholesterol-loaded cells is dependent on ACAT activity and, more specifically, ER free cholesterol levels. Lysosomal accumulation of LDL-derived cholesterol was prevented in SRD-4 cells supplemented with lyso-PC (50 µM), a substrate for PC synthesis through the reacylation pathway, and also in cells treated with bromoenol lactone (BEL), an inhibitor of phospholipase A2 implicated in bulk PC turnover. In a counter study, lysosomal LDL-derived cholesterol accumulation was induced in parental CHO-7 cells using R-propranolol, which inhibits the conversion of phosphatidic acid to diacylglycerol (DAG), a substrate in the CDP-choline pathway. This blockage was also relieved through co-treatment with lyso-PC. These studies support that PC to free cholesterol ratios in downstream organellar membranes can influence cholesterol trafficking out of lysosomal compartments in cholesterol-loaded cells.

Heat exposure causes several physiological and metabolic alterations. Although lipids are vital in sustaining energy production, heat-induced alterations in lipid metabolism have not been clearly established. CHAPTER 1 reviews the known metabolic alterations resulting from heat stress, with a specific focus on changes in whole-body lipid utilization and plasma lipids. CHAPTER 1also outlines the physiological changes caused by heat stress, and their role in reducing exercise performance. The study presented in CHAPTER 2 has shown that, compared to thermoneutral conditions, NEFA concentrations were 37% higher following passive heating and 34% higher following exercise in the heat, without significant changes in whole-body lipid utilization. In addition, the level of hyperthermia attained during passive pre-heating and exercise in the heat resulted in a 13% decrease in total external work and a significantly higher rate of perceived exertion. CHAPTER 3 summarizes the study results and presents the limitations and applications of the study.

This thesis describes the development of novel fluorescent and photocaged lipids, and their application as tools to probe the morphological effects of ceramide (Cer)-mediated membrane reorganization in supported lipid bilayers. Cer is a sphingolipid found in eukaryotic cells that plays a key role in regulating biological processes such as apoptosis, cell-to-cell communication, differentiation and some types of pathogenesis. Sphingolipid and cholesterol-rich lipid rafts in the plasma membrane are thought to be the point of origin for many of this lipid second messenger’s effects. Cer is formed in the exoplasmic leaflet of the plasma membrane via the enzymatic hydrolysis of sphingomyelin. The compositional complexity of biological membranes has prompted the adoption of simpler model systems to study the effects of Cer generation. When it is directly incorporated into model membranes, Cer segregates into highly ordered domains with physical properties that are distinct from those of the surrounding fluid environments. However, enzymatic generation of Cer induces complex and dynamic membrane heterogeneity that is difficult to interpret and reconcile with its direct incorporation. Here I describe the synthesis of 4-nitrobenzo-2-oxa-1,3-diazol-7-yl (NBD)-labelled cholesterol (Chol) and Cer analogs, and their use as probes in model membranes exhibiting liquid-disordered (Ld) and liquid-ordered (Lo) phase coexistence. The Chol probes reproduce the modest enrichment of Chol in Lo membrane domains as well as the Cer-induced displacement of cholesterol. One of the NBD Chol probes is used to provide direct visualization of Chol redistribution during enzymatic Cer generation, and assists in identifying new features as Cer-rich regions. The NBD-labelled Cer quantifies membrane order using orientational order parameter measurements derived from polarized total internal reflection fluorescence microscopy (pTIRFM) images. The probe reports on changes in membrane order upon enzymatic generation of Cer, and indicates a significant increase in the molecular order of Ld membrane regions that is consistent with the redistribution of Chol into these areas. The probe also identifies de novo Cer-rich domains as areas of particularly high molecular order. In the final project area, 6-Bromo-7-hydroxycoumarin-4-ylmethyl (Bhc)-caged Cers are shown to release Cer rapidly and efficiently upon irradiation with near-visible UV light. The caged lipids are then incorporated into supported membranes and photolyzed to release Cer with a high degree of spatial and temporal control. Controlled Cer generation is then used to drive protein-ganglioside clustering in lipid bilayers.

Membranes are essential components of all living organisms forming the borders of cells and their organelles. Planar lipid membranes deposited on solid substrates (solid supported membranes) provide models to study the functions of membrane proteins and are used as biosensing platforms. However, despite remarkable progress, solid supported membranes are not stable to harsh conditions such as dehydration, high temperature and pressure, and mechanical stress. In addition, the direct deposition of membranes onto a solid substrate often causes restricted mobility and denaturation of reconstituted membrane proteins. Membrane stability can be addressed by altering the structure of the component lipids. Bolalipids are an interesting class of bipolar lipids that have been proposed for biosensing applications. Membranes formed from mixtures of a bolalipid, C20BAS, and dioleoylphosphaphatidylcholine, POPC, were characterized by atomic force spectroscopy (AFM). The lipid mixtures produced a phase separated membrane consisting of thinner bolalipid-rich and thicker monopolar-rich POPC regions, with a height difference of approximately 1-2 nm. This confirmed an earlier prediction that some bolalipid/PC membranes would phase separate due to the hydrophobic mismatch between the two lipids. Interestingly, the surface coverage of the two phases was inconsistent with what one would expect from the initial starting lipid ratios. The complex membrane morphologies observed were accredited to the interplay of several factors, including a compositionally heterogeneous vesicle population, exchange of lipid between the vesicle solution and solid substrate during formation of the supported membrane, and slow equilibration of domains due to pinning of the lipids to the solid support. Decoupling the membrane from its underlying surface is one strategy to maintain the structure and mobility of membrane proteins. This decoupling can be achieved by depositing the membrane on a soft cushion composed of a water swelling hydrophilic polymer. A polyelectrolyte multilayer (PEM) and a tethered poly(ethylene) glycol (PEG) polymer are the two types of polymer cushions used in this study. The PEMs consist of the charged polysaccharides, chitosan (CHI) and hyaluronic acid (HA) which offer the advantage of biocompatibility over synthetic PEMs. DOPC lipid bilayers were formed at pH 4 and 6.5 on (CHI/HA)5 films. At higher pH adsorbed lipids had low mobility and large immobile lipid fractions; fluorescence and AFM showed that this was accredited to the formation of poor quality membranes with defects and pinned lipids rather than to a layer of surface-adsorbed vesicles. However, more uniform bilayers with mobile lipids were produced at pH 4. Measured diffusion coefficients were similar to those for bilayers on PEG cushions and considerably higher than those measured on other polyelectrolyte films. The results suggest that the polymer surface charge is more important than the surface roughness in controlling formation of mobile supported bilayers. The suitability of polymer supported membranes for the incorporation of integral membrane proteins was also assessed. The integral membrane protein Ste14p, a 26 kDa methyltransferase enzyme, was reconstituted into POPC membranes on PEM and PEG supports. A combination of fluorescence microscopy, FRAP, AFM and an in situ methyltransferase activity assay were utilized to characterize the protein incorporated polymer supported membranes. Fluorescence measurements showed that more protein was incorporated in model membranes formed on the PEG support, compared to either glass or PEM cushions. However, the protein activity on a PEG support was comparable to that of the protein in a membrane on glass. FRAP measurements showed that the lipid mobilities of the POPC:Ste14p bilayers on the various supports were also comparable. Lastly, as a new platform for manipulating and handling membrane proteins, nanodiscs containing reconstituted Ste14p were studied. Nanodiscs are small, soluble and stable bilayer discs that permit the study of membrane proteins in a uniform phospholipid bilayer environment. Empty and protein containing nanodiscs were deposited on a mica surface and imaged by AFM. AFM showed that protein containing samples possessed two subpopulations of nanodiscs with a height difference of ~1 nm. The taller discs, ~20% of the population, contained protein. Other experiments showed that the packing of the nanodisc samples was influenced by their initial stock concentration and that both imaging force and the addition of Mg2+ caused formation of larger bilayer patches.

Lipid peroxidation has been implicated in the onset and progression of many degenerative diseases, including cardiovascular disease, Alzheimer’s disease and cancer. Accordingly, for more than 50 years, considerable effort has been devoted to the design of synthetic compounds or the discovery of natural products that can slow lipid peroxidation. Despite the enormous investments made to date, no clear antioxidant strategies have emerged for the treatment and/or prevention of degenerative disease. We argue that this is because of a lack of fundamental understanding of the chemical reactivity of these compounds in relevant contexts. Herein, we describe studies of our optimized synthetic radical-trapping antioxidant (RTA) – the tetrahydronaphthyridinols (THNs). We first present the synthesis of a series of THN analogs of α-tocopherol (Nature’s premier lipid-soluble radical-trapping antioxidant) with varying sidechain substitution and then demonstrate how systematic changes in the lipophilicity of these potent antioxidants impact their peroxyl radical-trapping activities in lipid bilayers and mammalian cell culture. Their regenerability by water-soluble reductants in lipid bilayers, binding to human tocopherol transport protein (hTTP), and cytotoxicity were also evaluated to provide insight on whether this type of antioxidant can be potentially pushed toward animal studies. We also describe analogous studies of natural products such as the garlic-derived thiosulfinate allicin and the grape-derived polyphenol resveratrol. These compounds have attracted significant attention in the past 20 years due to their purported health benefits, which are often ascribed to their purported radical-trapping activities. To date, systematic studies on their radical-trapping activities in solution, lipid bilayers and mammalian cells have been lacking. We have determined that allicin and petivericin, while effective RTAs in solution, are not so in lipid bilayers. Moreover, the compounds are not antioxidants in cell culture, but instead kill the cells. Similarly, resveratrol and its dimers pallidol and quadrangularin A, are found to be inefficient RTAs in lipid bilayers. Our studies to date rather suggest that they autoxidize readily to produce hydrogen peroxide, which may induce expression of phase 2 antioxidant enzymes, affording cytoprotection. Our insights underscore the need for systematic studies of antioxidant activity in multiple contexts.

Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and hepatocellular carcinoma worldwide. HCV, like all obligate parasites, relies on host pathways to facilitate its pathogenesis. In particular, the virus possesses an intimate link with hepatic lipid metabolism, promoting a lipid-rich cellular environment conducive to HCV propagation. Clinically, these metabolic perturbations manifest as steatosis in over 50% of patients. The majority of research to-date examining how the virus co-opts hepatic lipid pathways has been focused on coding genes and their protein products. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression, which have been implicated in virtually every cellular process. Through interactions with partially complementary mRNAs, each individual miRNA has the capacity to repress the expression of hundreds of genes and induce significant regulatory effects. Herein, we demonstrate that hepatic miRNAs, including miR-7, miR-27a/b, miR-130b, and miR-185, act as crucial regulatory molecules to the maintenance of hepatic lipid homeostasis. These miRNAs cooperate to regulate fatty acid and cholesterol metabolism. HCV modulates the expression of a subset of these miRNAs (miR-27a/b, miR-130b, and miR-185) to promote hepatocellular lipid accumulation and the HCV life cycle. There appears to be a broad viral requirement for lipids, and the mammalian innate immune response strategically targets host metabolic pathways to restrict virus’ access to key lipid species. We demonstrate that 25-hydroxycholesterol, a broadly anti-viral oxysterol produced as part of the innate anti-viral response, activates miR-185 expression in the liver to deplete virus infected cells of lipids. HCV appears to actively counteract this anti-viral response by suppressing miR-185 expression. Collectively, our results highlight the role of microRNAs in hepatic lipid metabolism and the immunometabolic response to viral infection.

Adipose tissue plays a central role in the regulation of lipid storage and mobilization. A tight control between adipose tissue lipid storage and mobilization functions must be exerted to prevent an overload of lipids at other organs such as the heart, liver and skeletal muscles, and favor the risk of developing metabolic disorders, such as Type 2 diabetes and cardiovascular diseases (CVD). There is strong evidence from animal studies that low oxygen levels (hypoxia) are noted in adipose tissue as the mass of the organ excessively expands and, in turn, exacerbates some adipose tissue functions. Whether hypoxia exposure, which could be derived from reduced environmental oxygen availability, disease or a combination of both, affects adipose tissue lipid storage and mobilization functions in humans is not well known. Using in vitro and in vivo approaches, this thesis aimed at characterizing the effects of hypoxia on human adipose tissue lipid storage and lipid mobilization functions. Study I investigated how hypoxia can modulate human adipose functions such as lipid storage and lipid mobilization in vitro. Study II examined whether acute intermittent hypoxia, which simulates obstructive sleep apnea, affects adipose tissue lipid storage/mobilization functions and triglyceride levels in healthy young men in postprandial state. Study III tested the effect of an acute 6-hour continuous exposure to hypoxia (fraction of inspired oxygen (FIO2) = 0.12)) on plasma triglyceride levels in healthy young men in the fasting state. Study I indicates that both acute (24h) and chronic (14d) hypoxia (3%, and 10% O2) modulate human adipose tissue lipid storage and mobilization functions in a different manner. Study II demonstrates that acute exposure to intermittent hypoxia (6h) is sufficient to increase plasma non-esterified fatty acids (NEFA) levels, as well as insulin levels, but does not alter circulating triglyceride or subcutaneous adipose tissue lipid storage and/or mobilization capacity ex vivo in healthy men. Study III shows that acute exposure to normobaric hypoxia increases circulating NEFA and glycerol concentrations but did not translate in altering circulating triglycerides in fasting healthy men. In conclusion, our observations suggest that an exposure to reduced oxygen levels impairs human adipose tissue storage and/or mobilization functions, a phenomenon known in the development of metabolic disorders, such as Type 2 diabetes and CVD.

Hepatitis C virus (HCV) is remarkably capable of efficiently hijacking host cell pathways including lipid metabolism in the liver in order to create pro-viral environments for pathogenesis. It is becoming increasingly clear that identifying small molecule inhibitors that target host factors exploited by the virus will expand available HCV treatment options. As such, a thorough understanding of host-virus interactions is critical to the development of alternative therapeutic strategies. Hepatic lipid droplets (LDs) are recruited by HCV to play essential roles in the viral lifecycle. The intracellular location of LDs is modified upon interacting with viral structural core protein. This enables formation of platforms that support viral particle assembly. Because these interactions are non-static, capturing its dynamic processes in order to better understand viral assembly can be achieved with label-free molecular imaging enhanced with live-cell capabilities. Chemical biology approaches that includes CARS microscopy employed in a multi-modal imaging system was used to probe interactions between HCV and host LDs. By successfully tracking LD trajectories, we identified core protein’s ability to alter LD speed and control for LD directionality. Using protein expression model systems that allowed for simultaneous tracking of core protein and LDs, our data revealed that mutations in the core protein region that vary in hydrophobicity and LD binding strengths, are factors that control for differential modulation of LD kinetics. Furthermore, we measured bidirectional LD travels runs and velocities, and observed critical properties by which core protein induces LD migration towards regions of viral particle assembly. Given that many steps in the HCV lifecycle are directly linked to host lipid metabolism, it is not surprising that disrupting lipid biosynthetic pathways would negatively affect viral replication. From this outlook, we explored small molecule inhibitors that targeted several lipid metabolic pathways to study its antiviral properties. Using fluorescent probes covalently labeled to viral RNA, we captured the visualization of disrupted replication complexes upon antagonizing nuclear hormone receptors that are linked to regulating lipid homeostasis. Correspondingly, biochemistry and molecular imaging techniques were also employed to identify novel antiviral mechanisms of small molecule inhibitors that target additional HCV-dependent lipid metabolic pathways.