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The objectives of this project were to develop a high performance liquid chromatographic method for separating the pigment esters mixture, to determine the locations of the pigment moiety in the isolated esters using pholosiphases, and to characterize the pigment-protein complex and determine its distribution in other bacteria. Saponification of the two pigment esters 1 and 2 with aqueous KOH yielded two free pigments on TLC plates developed by two solvent systems. The fasters moving of these two free pigments co-chromatographed with the one free pigment produced from each pigment ester by phospholipase A2 treatment. This suggests that the pigment molecule is a methoxy derivative of xanthomonadin and is esterified to the 2-position of the glycerol moiety of each pigment ester. No free pigment was released from phospholipases C and D treatment of the two pigment esters, indicating that pigment is not esterified to the sorbitol or phosphate moiety of pigment esters 1 or 2.

<p>  </p> <p><strong>ABSTRACT</strong></p> <p>The worldwide obesity prevalence has almost tripled since 1973 according to the World Health Organization. In the United States, the disease is especially prevalent, with a recorded prevalence of 41.9 percent in 2017, as reported by the National Health and Nutrition Examination Survey. Obesity is associated with an increased risk of heart disease, type 2 diabetes, stroke, non-alcoholic fatty liver disease (NAFLD), and certain types of cancers, including prostate cancer (PCa). While obesity is preventable and reversible, it is a relapsing disease that requires long-term intervention. Furthermore, accumulating evidence shows obesity is not simply a matter of lack of willpower but the re-wired and altered biology that may need medical treatment. Therefore, researchers have been searching for effective approaches to treat obesity and obesity-related diseases. To this end, my research focuses on exploring the role of the sterol O-acyltransferase (SOAT) enzyme and how the inhibition of the enzyme benefits the treatment of obesity and PCa. In addition, I also studied the molecular signatures of NAFLD, with a special focus on altered lipid metabolism using proteomics and determined the protein oligomerization profiles. The major lines of research are summarized in the following and discussed in greater detail in chapters 2 to 5. </p> <p>SOAT enzyme catalyzes the conversion of free cholesterol into its storage form, cholesteryl ester. Our group previously showed that increased SOAT1 expression is associated with increased adipogenesis <em>in vitro</em> and increased adiposity in adipose tissue. When SOAT1 activity was blocked using the pharmacological inhibitor avasimibe, lipid droplet formation and expansion during adipogenesis were suppressed. We further showed that non-orally administered avasimibe led to significant fat mass loss in diet-induced obese (DIO) mice with concomitant food intake suppression and decreased expression of lipogenic genes in adipose tissue. Based on the promising use of avasimibe as an anti-obesity medication, I sought to answer whether avasimibe can enhance the weight loss effect of glucagon-like peptide-1 receptor agonist (GLP-1RA) by accelerating fat mass loss (chapter 2). We found subcutaneous administration of avasimibe can significantly potentiate the weight-reducing effect of the GLP-1RA in DIO mice.</p> <p>Inspired by the lipid droplet modulatory role of the SOAT1 enzyme, I also expanded my dissertation project to cancer (chapter 3). I found that low SOAT1 expression is associated with favorable patient outcomes among PCa patients who had previously undergone anti-hormone therapy. Since the current treatment option, anti-androgen drug enzalutamide, induces mechanisms of resistance in a short period, I hypothesized that blockage of SOAT1 activity using avasimibe would enhance the enzalutamide action and help overcome the resistance. To test this hypothesis, I characterized lipidomic signatures of PCa cells in response to enzalutamide and avasimibe treatments. Then, I tested the anti-cancer effect of the combined treatment in cell cultures and in xenograft tumors in nude mice. I found the combined treatment was significantly more effective in inhibiting cancer cell proliferation and tumor growth than each drug treatment alone. These findings provide insights into molecular signatures associated with enzalutamide treatment outcomes and can serve as a prelude to developing a therapeutic regimen targeting cholesterol metabolism. </p> <p>Among the comorbidities of obesity, NAFLD is very common in obese adults and the prevalence is close to 50–90%. We launched the third project (chapter 4) where we compared the liver proteome from lean mice and DIO mice. To date, most of the omics studies on DIO have been monolithic and very few have explored the multi-omic aspects of fatty liver tissue. To address this gap, we integrated global proteomics, phosphoproteomics and lipidomics to determine molecular signatures of the fatty liver. We identified a range of biological pathways that were altered, and we showed how the alterations in lipid content and amount were correlated with the alterations in the liver proteome and phosphoproteome. The results shed light on the interrelated nature of these biological processes. This was hypothesis-generating study that provided extensive data that could guide future investigations. </p> <p>We followed up on the third project and employed extensive measures to determine the protein oligomerization profiles of the fatty liver (chapter 5). Understanding the modes of protein oligomerization is important since proteins typically exert their biological functions by interacting with other proteins to form protein complexes. We used size exclusion chromatography (SEC) to fractionate liver proteins into 32 fractions based on their size and conducted label-free quantitation of each fraction using mass spectrometry. We successfully obtained elution profiles of individual proteins for subsequent comparison. Our approach enabled the identification of 1172 proteins found common in four liver samples (two lean livers and two fatty livers) for correlation profiling. We discovered that protein elution profiles were highly conserved in the fatty livers despite the metabolic disease state. At the same time, we identified several proteins with different elution profiles between the lean and the fatty livers, which could potentially mediate the hepatic dysfunctions displayed in NAFLD. This study delivers novel pieces of information about protein complex formation in fatty livers.</p> <p>The four research projects included in this dissertation explored obesity and obesity related diseases. Cholesterol accumulation is manifested as lipid metabolism is altered during the progression of obesity in adipose tissue and in PCa. Pharmacological inhibition of SOAT is responsible for cholesterol accumulation was effective in weight management in DIO and demonstrated anti-cancer effect in PCa models together with enzalutamide. These findings suggest that SOAT could be a therapeutic target for diseases featuring cholesteryl ester accumulation. Subsequent projects explored the liver proteome and revealed. In the subsequent projects, liver proteome showed a clear distinction between lean mice and obese mice. The identified proteins in these studies could facilitate the development of targeted therapies for treating NAFLD.</p>

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Bisphenols are used in the process of polymerization of polycarbonate plastics and epoxy resins. Bisphenols can easily migrate out of plastic products and enter the gastrointestinal system. By increasing colonic inflammation in mice, disrupting the intestinal bacterial community structure and altering the microbial membrane transport system in zebrafish, bisphenols seem to interfere with the gut microbiome. The highly abundant human commensal bacterium Bacteroides thetaiotaomicron was exposed to bisphenols (Bisphenol A (BPA), Bisphenol F (BPF), Bisphenol S (BPS)), to examine the mode of action, in particular of BPF. All chemicals caused a concentration-dependent growth inhibition and the half-maximal effective concentration (EC50) corresponded to their individual logP values, a measure of their hydrophobicity. B. thetaiotaomicron exposed to BPF decreased membrane fluidity with increasing BPF concentrations. Physiological changes including an increase of acetate concentrations were observed. On the proteome level, a higher abundance of several ATP synthase subunits and multidrug efflux pumps suggested an increased energy demand for adaptive mechanisms after BPF exposure. Defense mechanisms were also implicated by a pathway analysis that identified a higher abundance of members of resistance pathways/strategies to cope with xenobiotics (i.e., antibiotics). Here, we present further insights into the mode of action of bisphenols in a human commensal gut bacterium regarding growth inhibition, and the physiological and functional state of the cell. These results, combined with microbiota-directed effects, could lead to a better understanding of host health disturbances and disease development based on xenobiotic uptake.

Seeds from the desert shrub Simmondsia chinensis (jojoba) are one of the only known natural plant sources to store a majority of its oil in the form of liquid wax esters (WE) instead of triacylglycerols (TAGs) and these oils account for ~55% of the seed weight. Jojoba oil is highly valued as cosmetic additives and mechanical lubricants, yet despite its value much is still unknown about its neutral lipid biosynthetic pathways and lipid droplet packaging machinery. Here, we have used a multi-"omics" approach to study how spatial differences in lipid metabolites, gene expression, and lipid droplet proteins influence the synthesis and storage of jojoba lipids. Through these studies mass spectrometry analyses revealed that WEs are compartmentalized primarily in the cotyledonary tissues, whereas TAGs are, surprisingly, localized to the embryonic axis tissues. To study the differences in gene expression between these two tissues, a de novo transcriptome was assembled from high throughput RNAseq data. Differential gene expression analysis revealed that the Jojoba Wax Synthase, which catalyzes the formation of wax esters, and the Diacylglycerol O-Acyltransferase1, which catalyzes the final acylation of triacylglycerol synthesis, were differentially expressed in the cotyledons and embryonic axis tissues, respectively. Furthermore, through proteomic analysis of lipid droplet proteins from lipid droplets of the cotyledons and embryonic axis, it was estimated that each of these tissues contains a different proportion of the major lipid droplet proteins, oleosins, steroleosins, caleosins, and lipid droplet associated proteins. The Jojoba Olesosin1, Lipid Droplet Associated Protein 1, and Lipid Droplet Associated Protein 3, were identified as potential lipid droplet proteins that could be important for storage of wax esters. The coding sequences of these genes were transiently expressed in N. benthamiana leaves individually, and with co-expression of Mus musculus diacylglycerol acyltransferase 2, and in all cases were able to induce neutral lipid accumulation. These data also suggest a Lipid Droplet Associated Protein 1 has a specialized role for wax ester accumulation in the cotyledons, whereas Lipid Droplet Associated Protein 3 may have a more generalized role for the storage of triacylglycerols. These differences in compartmentation suggests that the cotyledons and embryonic axis of jojoba have evolved tissue-specific sets of genes for neutral lipid assembly and lipid droplet accumulation. It may be important to consider this tissue context for genetic engineering strategies designed to introduce genes from jojoba into other oilseed crops.

Lignin-derived precursors were used in the synthesis of bio-based high-performance polymers. The project consisted of synthesizing a series of poly(ester amide)s (PEAs) from lignin building blocks and natural amino acids. In particular, the amino acid moieties were incorporated into the PEAs’ architecture to explore the effect of the side-chain size on the thermal properties and the crystallinity of the resulting materials. The polymers, which were prepared by melt polycondensation, all possessed high thermal stability in nitrogen and air with onsets of thermal degradation (Td onset) exceeding 330 °C and glass transition temperatures (Tg) ranging from 136 °C – 238 °C. It is worth noting that the Tg greatly depended on the size of the pendant R-group on the amino acid. Remarkably, the thermal stability was mostly maintained even after subjecting the polymers to various pH media (pH 1, 4 and 8) for 1 week at 50 °C. Furthermore, wide-angle X-ray scattering experiments revealed semi-crystalline polymers with identical diffraction patterns and percent crystallinity ranging from 21 – 37%. To probe the impact of chirality on the thermal properties, a meso polymer of DL-alanine was prepared and compared to the chiral version. A slight drop in the Td onset and Tg of the DL-alanine-containing polymer relative to the L-alanine counterpart occurred, signifying moderate thermal stability resulting from the chiral group. Overall, these characteristics make these bio-based PEAs potential candidates for further investigation as alternatives to petrochemical-derived thermoplastics for high-performance materials.

The effects of material/fabrication parameters on vapor-grown carbon nanofiber (VGCNF) reinforced vinyl ester (VE) nanocomposite flexural moduli and strengths were investigated. Statistically reliable empirical response surface models were developed to quantify the effects of VGCNF type, use of dispersing agent, mixing method, and VGCNF loading on flexural properties. Optimal nanocomposite formulation and processing (0.74 phr oxidized VGCNFs, dispersing agent, and high-shear mixing) resulted in predicted flexural modulus and strength values 1.18 and 1.26 times those of the neat resin. Additional flexural, tensile, and compressive tests were performed for optimally configured nanocomposites cured in a nitrogen environment. While flexural and tensile moduli significantly increased with increasing VGCNF loading, the corresponding strengths fell below those of the neat resin. In contrast, nanocomposite ultimate compressive strengths significantly exceeded the neat resin strengths. Nanocomposites prepared using aggressive high-shear mixing displayed improved elastic moduli and substantially increased strengths relative to nanocomposites prepared using baseline methods.

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