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 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.

Cell-free transcription and translation systems promise to accelerate and simplify the engineering of synthetic proteins, biological circuits or metabolic pathways. Microfluidic droplet platforms can generate millions of reactions in parallel. This allows cell-free reactions to be miniaturized down to picoliter volumes. Nevertheless, the true potential of microfluidics have not been reached for cell-free bioengineering. Better approaches are needed for reaching sufficient in-drop expression levels while efficiently creating DNA diversity among droplets. This work develops a droplet microfluidic platform for single or multiple protein expression from a single DNA coated bead per droplet. This opens up the possibility to diversify a million droplets for synthetic biology applications.

In this work the differences between and characteristics of water and high solids, heated black liquor sprays from air-assist atomizers are examined. Sprays were imaged with a high speed camera and the images analyzed with computer code to produce droplet size data and macroscopic spray characteristics such as mass distribution. Fluid flow rates were measured to allow relevant dimensionless groups for the spray to be calculated. A 1000 degree C tubular furnace was placed around the spray to determine the effect of industrially relevant temperatures on the droplet formation process, relative to room-temperature conditions. It was found that high solids black liquor forms long, thin ligaments rather than droplets. In high-temperature surroundings the size of these ligaments increases, which from a comparison with theory in the literature was attributed to enhanced skin-formation driven by heat transfer. The data suggest that this skin formation may prevent secondary breakup. All sprays for both fluids produced droplet size mass distributions that were well described by the square-root normal distribution. The normalized width (s*) of these distributions was similar for all sprays and consistent with literature data for other nozzle designs (0.24 < s* < 0.38). The image analysis method assumed droplets were spheres with the same projected area. When this assumption was changed for black liquor sprays to a cylindrical droplet assumption, the shape and normalized width of the resulting mass distributions remained the same, but the representative diameter (calculated from surface area to volume ratios) decreased. Based on the agreement between the normalized distribution width in this work and that in the black liquor spraying literature it was concluded that the addition of atomizing air cannot be considered a means to narrow a droplet size distribution independent of droplet size. The results also indicate the importance of including the effects of skin formation and temperature- and time-dependent fluid properties in spray modeling. It is intended that these results contribute to increased understanding of the black liquor atomization process and lead to improved computational modeling of the same.

Three-dimensional spheroid arrays represent in vivo activity better than conventional 2D cell culturing. A high-throughput microfluidic chip may be capable of depositing cells into spheroid arrays, but it is difficult to regulate the path of individual cells for deposition. Droplets that encapsulate cells may aid in facilitating cell delivery and deposition in the return bend of a microfluidic chip. In this study, a low-cost method for fabricating polymer-cast microfluidic chips has been developed for rapid device prototyping. Computational fluid dynamic (CFD) simulations were conducted to quantify how a change in geometry or fluid properties affects the dynamics of a droplet. These simulations have shown that the deformation, velocity, and trajectory of a droplet are altered when varying the geometry and fluid properties of a multiphase microfluidic system. This quantitative data will be beneficial for the future design of a microfluidic chip for cell deposition into 3D spheroid arrays.

Many industrial processes such as printing and cleaning, as well as products like adhesives, coatings, and biological testing devices, rely on the wetting of liquids on a surfaces. Wetting is commonly controlled through material selection, coatings, and/or surface texture, but these means are sensitive to environmental conditions. Wetting is influenced by variables like surface tension, density, the surface chemistry, local energy barriers like surface roughness, and how the droplet is placed on the surface. Wetting of droplets can also be influenced externally in many ways such as introducing surfactants, applying electrical fields, or by dynamically excitation. Low-frequency, high amplitude vibration can initiate wetting changes prompted by droplet contact line oscillations that exceed the range of stable contact angles inherent of a droplet on a solid surface. The study of ultrasonic vibration wetting and spreading effects is sparse [1, 2], and is usually only qualitatively analyzed. Therefore, the specific goal of this thesis is somewhat unique, but also has potential as a means to controllably reverse surface adhesion. High frequency vibration effects and the governing mechanisms are relatively uncharacterized due to difficulties posed by the spatial and temporal scales. To investigate, droplets of 10, 20, and 30 µL are imaged as they vibrate on a hydrophobic surface forced via a piezoelectric transducer over different high frequencies (>10 kHz). Wetting transitions occur abruptly over a range of parameters, but coincide with transducer resonance modes. The magnitude of contact angle change is dependent on droplet volume and surface acceleration, and remains after cessation of vibration, however new droplets wet with the original contact angle. A more detailed investigation of this phenomenon was necessary to obtain a better understanding. This required repeatable testing conditions, which relies heavily on surface integrity. However, some “hydrophobic” coatings are sensitive to extended water exposure. To determine which hydrophobic coatings may be appropriate for investigating dynamic wetting phenomena, samples of glass slides coated with a series of fluoropolymer coatings were tested by measuring water contact angle before, during, and after extended submersion in deionized water and compared to the same coatings subjected to ultrasonic vibration while covered in deionized water. Both methods caused changes in advancing and receding contact angle, but degradation rates of vibrated coatings, when apparent, were significantly increased. Prolonged soaking caused significant decreases in the contact angle of most coatings, but experienced significant recovery of hydrophobicity when later heat-treated at 160 C. Dissimilar trends apparent in receding contact angles suggests a unique degradation cause in each case. Roughening and smoothing of coatings was noted for coatings that were submerged and heat-treated respectively, but this did not correlate well with the changing water contact angle. Degradation did not correspond to surface acceleration levels, but may be related to how well coatings adhere to the substrate, indicative of a dissolved coating. Most coatings suffered from contact angle degradation between 20-70% when exposed to water over a long period of time, however the hydrophobic fluoropolymer coating FluoroSyl was found to remain unchanged. For this reason it was found to be the most robust coating for providing long term wetting repeatability of vibrated droplets. Droplets (10 to 70 µL) were imaged on hydrophobic surfaces as they were vibrated with ultrasonic piezoelectric transducers. Droplets were vibrated at a constant frequency with ramped amplitude. Spreading of droplets occurs abruptly when a threshold surface acceleration is exceeded of approximately 20,000 m/s2. Droplet contact area (diameter) can be controlled by varying acceleration levels above the threshold. The threshold acceleration was relatively independent of droplet volume, while initial contact angle impacts the extent of spreading. Wetting changes remain after cessation of vibration as long as the vibrated droplet remained within the equilibrium contact angle range for the surface (> the receding contact angle), however new droplets wet with the original contact angle except for some cases where vibration of liquid can affect the integrity of the coating. Reversible wettability of textured surfaces is a desired effect that has various industry applications where droplet manipulation is used, like biomedical devices, coating technologies, and agriculture [3-5].

Adipose tissue is a key regulator of energy metabolism and glucose homeostasis by promoting triglyceride storage and breakdown in various physiological states. Obesity, however, alters adipose tissue metabolism, inducing chronic inflammation, followed by excessive lipolysis. This results in higher systemic free fatty acid (FFA) levels, leading to desensitization of insulin signaling and ultimately to insulin resistance. Although the link between obesity and progression of insulin resistance and type 2 diabetes mellitus (T2DM) remains unclear, tumor necrosis factor-alpha (TNF-alpha) has been proposed to be a key player in promoting obesity-related development of T2DM through chronic inflammation of adipose tissue. TNF-alpha has direct and indirect mechanisms by which it elicits insulin resistance in adipocytes. TNF-alpha attenuates insulin signaling by directly inhibiting insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1). Indirectly, TNF-alpha activates signaling pathways to increase lipolysis and FFA release into circulation, leading to insulin resistance. Lipid droplet-associated fat specific protein 27 (FSP27) protects adipocytes from lipolysis by regulating the lipolytic capacity as well as transcription of adipose triglyceride lipase (ATGL). It has been observed that TNF-alpha promotes lipolysis by reducing the expression of FSP27 in murine adipocytes. The effect of TNF-alpha on lipolysis human adipocytes has also been studied; yet its effect on promoting insulin resistance in human adipocytes still remains elusive. In the present study, we examined the effect of FSP27 on TNF-alpha induced lipolysis and insulin resistance in human adipocytes. TNF-alpha enhanced lipolysis in cultured human adipocytes. In addition, TNF-alpha reduced the expression of endogenous FSP7 and the phosphorylation of AKT, inhibiting the activation of insulin signaling pathway in cultured human adipocytes. FSP27 overexpression, however, attenuated TNF-alpha induced lipolysis and restored activation of insulin signaling through phosphorylation of AKT in cultured human adipocytes. Taken together, these data suggest that FSP27 has a protective effect against TNF-alpha induced lipolysis and insulin resistance through regulating lipolysis and insulin signaling in human adipocytes.

Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues.

At the cutting edge of the chemical and biological research, innovation takes place in a field referred to as Lab on Chip (LoC), a multi-disciplinary area that combines biology, chemistry, electronics, microfabrication, and fluid mechanics. Within this field, droplets have been used as microreactors to produce advanced materials like quantum dots, micro and nanoparticles, active pharmaceutical ingredients, etc. The size of these microreactors offers distinct advantages, which were not possible using batch technologies. For example, they allow for lower reagent waste, minimal energy consumption, increased safety, as well as better process control of reaction conditions like temperature regulation, residence times, and response times among others. One of the biggest drawbacks associated with this technology is its limited production volume that prevents it from reaching industrial applications. The standard production rates for a single droplet microfluidic device is in the range of 1-10mLh-1, whereas industrial applications usually demand production rates several orders of magnitude higher. Although substantial work has been recently undertaken in the development scaled-out solutions, which run in parallel several droplet generators. Complex fluid mechanics and limitations on the manufacturing capacity have constrained these works to explore only in-plane parallelization. This thesis investigates a three-dimensional parallelization by proposing a microfluidic system that is comprised of a stack of droplet generation layers working on the liquid-liquid ow regime. Its realization implied a study of the characteristics of conventional droplet generators and the development of a fabrication process for 3D networks of microchannels. Finally, the combination of these studies resulted in a functional 3D parallelization system with the highest production rate (i.e. 1 Lh-1) at the time of its publication. Additionally, this architecture can reach industrially relevant production rates as more devices can be integrated into the same chip and many chips can compose a manufacturing plant. The thesis also addresses the concerns about system reliability and quality control by proposing capacitive and radio frequency resonator sensors that can measure accurately increments as small as 2.4% in the water-in-oil volume fraction and identify errors during droplet production.

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