<p> It is well understood that compared with men, women are better able to withstand starvation, have better ultra-endurance capacity, oxidize more fat during endurance exercise, and are more resistant to fat oxidation defects i.e. diet-induced insulin resistance. However, the mechanism(s) for the observed sex differences are unknown. It was my hypothesis that women have greater fat oxidation capacity in skeletal muscle than men.</p> <p> The objectives of my thesis were to determine the mechanism(s) by which women oxidize more lipids; including the role of estrogen as a possible regulator. The most significant findings were that: 1) mRNA for fatty acid oxidation genes are higher in women compared with men, which was confirmed by Stringent Affymetrix GeneChip array analysis, combined with RT-PCR (chapter 2); 2) long-chain acyl-CoA dehydrogenase in human skeletal muscle is not quantifiable despite the majority (90%) of fatty acids oxidized during exercise are long-chain fatty acids (chapter 3); 3) β-oxidation enzymes: tri-functional protein alpha, very long chain acyl-CoA dehydrogenase, and medium chain acyl-CoA dehydrogenase are significantly higher in women compared with men (chapter 4); 4) Acute (8 days) 17β-estradiol supplementation in men significantly increased protein content of β-oxidation enzymes in skeletal muscle, possibly through the regulation of PGC-1α and microRNA (chapter 5).</p> <p> In conclusion, my data provided novel insights into the enhanced ability of women to oxidize fat under periods of metabolic stress by showing that: 1) women are transcriptionally (mRNA) "primed" for known physiological differences in metabolism; 2) women have more protein content of the major enzymes involved in long and medium chain fatty acid oxidation; 3) E2 partially regulates lipid metabolism in skeletal muscle by pre-translational modifications of factors involved in β-oxidation. These findings contribute to the molecular understanding of sex differences in substrate utilization.</p> / Thesis / Doctor of Philosophy (PhD)

Despite significant advances in chemotherapeutics since their initial application in the early 1900s, cancer remains a leading cause of death worldwide. Furthermore, issues encountered with the first chemotherapeutics, off-target toxicity, limited effective dosing time in the therapeutic window, multidrug resistance, and poor uptake in solid tumors persist today. This dissertation investigates two platforms to enhance chemotherapy efficacy, an implantable drug delivery depot affording tumor drug levels unachievable through systemic delivery, and a platform utilizing CRISPR knockout screening (CRISPRKO) to identify microRNA (miRNA) targets that synergize with chemotherapeutics using RNA interference (RNAi). First, we developed a flexible, implantable surgical buttress coated with a free and covalently-bound paclitaxel polymer blend with poly(1,2-glycerol carbonate)-graft-succinic acid-paclitaxel (PGC-PTX). Drug release is tunable between burst release of free paclitaxel and delayed, extended release of paclitaxel from PGC-PTX, delivering supratherapeutic levels of PTX locally at the tumor resection bed while avoiding systemic toxicity. Fabrication of paclitaxel-loaded film is scalable up to 8”x11” and well-tolerated in a porcine model wherein surgical technique and optimized film formulations reduce foreign body response and bowel adhesions. We test a similarly-designed film formulation with the novel chemotherapeutic, eupenifeldin, for treatment of lung cancer. Eupenifeldin-loaded films significantly prolong mice survival, although 60% of mice present issues with drug-related toxicity and wound healing. Second, despite the formidable ability of pooled CRISPRKO libraries to screen thousands of single-gene knockouts against a selection agent, minimal has been published on their use to determine novel targets for RNAi-based sensitization of chemotherapeutics. We utilize whole-genome CRISPRKO screening against a panel of lung cancers to identify miRNAs capable of synergizing with paclitaxel as well as two novel chemotherapeutics, eupenifeldin and verticillin A. Identified targets are validated for synergy with their respective chemotherapeutic in vitro, demonstrating a 50% or greater increase in cell death compared to drug-only treatment. In summary, this work presents two successful treatment platforms that address key issues preventing translation of chemotherapeutics to the clinic: 1) dose-limiting toxicity and 2) limited efficacy with poor safety/benefit ratio. Implantable, drug-loaded films serve as a platform to deliver increased doses of chemotherapeutic to tumor while avoiding off-target toxicity. As a second platform, we enhance tumor-specific chemotherapy efficacy utilizing miRNA targets identified in a CRISPRKO screening pipeline.

Efficient use of renewable feedstocks for production of chemicals and intermediates is necessary to reduce dependence upon petroleum. A large portion of these chemicals could be produced using lipids from renewable feedstocks such as vegetable oils, animal fats, and bacterial lipids. For example, many lipid sources contain unsaturated fatty acids, which can be oxidized to form a variety of products such as diacids and epoxides. These chemicals are used to formulate herbicides, detergents, plasticizers, lubricants, paints, and other useful products. One of the most common unsaturated fatty acids is oleic acid, and it can be oxidized with an ozone/oxygen mixture to produce azelaic acid and pelargonic acid. Since the ozone/oxygen mixture is a gas and oleic acid is a liquid under reaction conditions, mass transfer limitations exist. However, a reduction of the mass-transfer limitations can be achieved if the reactants coexist in a single phase. When supercritical carbon dioxide (SC-CO2) is used as the reaction medium, it is possible for both oleic acid and the ozone/oxygen mixture to both exist in the same phase at the same time. Use of supercritical carbon dioxide also provides the possibility of product fractionation, depending upon the solubility of the products in SC-CO2. The overall goal of this research was to determine if any advantages could be realized by conducting the oleic acid oxidation in a supercritical fluid medium. First, the solubility of azelaic acid and pelargonic acid in supercritical carbon dioxide was determined over a range of temperatures and pressures. Pelargonic acid was found to have a significantly higher solubility than azelaic acid, which indicated the potential for product separation with supercritical carbon dioxide. Second, the impact of the solvent medium on reaction kinetics and product formation was determined using two oxidizers: ozone and potassium permanganate. Due to experimental limitations, no reaction was observed in the case of ozone in supercritical carbon dioxide. However, oxidation of oleic acid with potassium permanganate in supercritical carbon dioxide resulted in higher oleic acid conversion and increased yields of azelaic acid and pelargonic acid compared to the oxidation without SC-CO2.

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The nematode, Caenorhabditis elegans is an ideal animal model to study conserved mechanisms of developmental and postdevelopmental processes. Here, I describe the role of an Axin family member, pry-1, in aging, lipid metabolism, and seam cell development. Our analysis of pry-1 animals showed a catastrophic collapse of adult lifespan, which was accompanied with hallmarks of accelerated aging. Transcriptome profiling of pry-1 mutants revealed altered expression of genes associated with aging and lipid metabolism such as vitellogenins, fatty acid desaturases, lipases, fatty acid transporters and genes involved in cuticle synthesis. Consistent with this, pry-1 animals display significantly reduced levels of somatic lipids. Knockdowns of vitellogenins in the pry-1 background restored lifespan and lipid levels, suggesting that vitellogenins are necessary to mediate pry-1 function in aging and lipid metabolic processes. Additionally, lowered expression of desaturases and lipidomics analysis provided evidence of reduced fatty acid synthesis in pry-1 animals. In agreement with this, an exogenous supply of oleic acid restored depleted lipids in somatic tissues in addition to suppressing the short-lived phenotype of worms. In addition, transcriptome profiling for differentially expressed miRNAs in pry-1(mu38) identified heterochronic miRNAs (lin-4 and let-7 -family members) to act downstream of pry-1 /Axin. In C. elegans, these miRNAs are known to robustly regulate the stem-like, seam cell division. Loss of pry-1 function caused heterochronic defects such that the seam cells divide precociously to produce additional cells. The pry-1-miRNAs are involved in mediating silencing of the heterochronic gene, hbl-1, a C. elegans hunchback homolog, to regulate seam cell division. Furthermore, I report identification of novel miRNAs from C. elegans and C. briggsae. Overall, our findings demonstrate a novel role of the Wnt signaling regulator, pry-1/Axin, in the maintenance of adult lifespan that involves lipid homeostasis and regulation of heterochronic miRNA to control the developmental timing of seam cell division in C. elegans. / Thesis / Doctor of Philosophy (PhD)

The proprotein convertase subtilisin/kexin type 9 (PCSK9) was first characterized in 2003 by Seidah and colleagues and marked the beginning of what is now considered by many as the greatest advancement in the field of cardiovascular disease (CVD) research since the discovery of the LDLR nearly half of a century ago. Since its discovery, PCSK9 was shown to enhance the degradation of cell-surface low-density lipoprotein (LDL) receptor (LDLR) and gain-of-function (GOF) mutations were shown to correlate with CVD risk. In contrast, patients carrying loss-of-function (LOF) mutations in PCSK9 highlighted a novel therapeutic approach for LDL lowering as they exhibit a life-long state of hypocholesterolemia and reduced CVD risk. A decade after the cloning of the PCSK9 gene, pharmaceutical companies have now developed a variety of PCSK9 inhibitors, ranging from monoclonal antibodies (mAbs) to small interfering RNA (siRNA) and vaccines, which have been shown to markedly reduce LDL cholesterol levels in pre-clinical models, as well as in patients at high risk of CVD. Despite these advances, there remained several unanswered questions regarding the mechanisms by which PCSK9 expression and secretion is regulated in the liver; the tissue from which the circulating pool of PCSK9 almost exclusively originates. The thought that further development of our understanding of PCSK9 biology may lead to the discovery of a signaling cascade that could be targeted by small molecules, the only class of inhibitor that has not yet been developed, has now merited additional research attention. The focal point of my doctoral studies represents the axis between a cellular process known as endoplasmic reticulum (ER) stress and PCSK9 expression/biosynthesis. ER stress is a deleterious cellular process that is known to occur in secretory cell types, such as liver hepatocytes, and is a well-established causative driver of an array of human diseases ranging from CVD to neurodegenerative diseases. ER stress is prevalent in the livers of patients with metabolic disease and is also known to activate the transcription factor capable of regulating PCSK9 levels, the sterol regulatory element-binding protein 2 (SREBP2). Based on this information, the first aim during the course of my PhD studies was to determine whether ER stress affected the expression and secretory status of PCSK9. In the past several years, I demonstrated that ER stress caused by ER Ca2+ depletion led to a marked increase in PCSK9 protein expression, but blocked its secretion as a result of its retention in the ER. Such a result was also associated with heightened hepatic LDLR expression and reduced LDL cholesterol levels in mice. Additional studies also characterized a variety of agents, including caffeine, as potent inhibitors of PCSK9 expression via increasing ER Ca2+ levels, which antagonized SREBP2 activity. As our initial studies revealed ER PCSK9 retention as a viable strategy for PCSK9 inhibition and LDL lowering, follow-up studies were also carried out to determine the outcome of such a strategy on liver function and injury. Given that heritable mutations in proteins that transit the ER can accumulate in this compartment and cause ER storage disease (ERSD), it was critical to further evaluate whether ER PCSK9 retention would lead to a similar outcome. In a series of experiments with rather surprising outcomes, we observed that the retention of the LOF Q152H PCSK9 mutant in the ER failed to cause ER stress; even in mice overexpressing the protein. Interestingly, tissue culture and mouse models demonstrated that the retention of PCSK9 in this cellular compartment increased the cellular abundance of ER stress response chaperones, such as the glucose-regulated proteins of 78- and 94-kDa (GRP78 and GRP94, respectively), but did not activate transducers of the ER stress signaling cascade. Strikingly, mice expressing the ER-retained PCSK9 Q152H mutant were protected against ER stress, suggesting a novel co-chaperone-like role of intracellular PCSK9. Collectively, the ER environment including secondary messengers like Ca2+ as well as its chaperones, plays a critical regulatory role on PCSK9 expression and secretion. Agents that increase ER Ca2+ levels can be utilized to block PCSK9 expression at the mRNA level to increase hepatic LDL clearance, and ER PCSK9 retention may also represent a safe avenue with a similar LDL lowering outcome. Beyond LDL lowering, hepatic ER PCSK9 retention may also serve as a novel strategy to enhance ER function and protect against ER stress-driven diseases of the liver. / Thesis / Doctor of Philosophy (Medical Science)