The nicotinamide riboside (NR) form of vitamin B3and the ketone body ß-hydroxybutyrate (BHB) are two of the most promising natural compounds yet identified for the treatment of aging and aging-related diseases. Forms of vitamin B3are precursors for the synthesis of the coenzymes nicotinamide adenine dinucleotide (NAD(H)) and nicotinamide adenine dinucleotide phosphate (NADP(H)). In aged cells levels of NAD+decline, decreasing metabolism and decreasing activity of protective sirtuin protein deacetylases. In aged cells NR, but not more common forms of vitamin B3, boost NAD+levels. BHB is naturally produced by the body when individuals fast or consume a ketogenic (KD) or calorically restricted (CR) diet. These diets have been shown to extend lifespan in mice, while they are also protective in many disease models. Caenorhabditis elegans, a roundworm with a short mean lifespan of roughly 2 to 3 weeks depending upon the temperature, is used as a model system to study aging. BHB has been previously shown to increase lifespan by roughly 20% when administered to C. elegans.We administered NR and BHB individually and together to C. elegans starting at two different developmental stages (larval stages 1 and 4) and measured lifespan. We found that administration of 20 mM DL-BHB decreased lifespan when first given at the L1 stage, while it robustly increased lifespan when first given at the L4 stage. Administration of 0.5 mM NR increased lifespan when first given at L1, with only a very slight increase when first given at L4. When initiating administration at L1, NR greatly mitigated the BHB-mediated decline in longevity, however, NR did not increase BHB-mediated lifespan extension when first administered at L4.

Breast cancer, osteoporosis, and coronary heart disease (CHD) are three major threats to women’s health. Postmenopausal women with breast cancer are also at high risk for osteoporosis and CHD. Adjuvant tamoxifen therapy is not only an effective treatment for breast cancer, but has been shown to have a beneficial effect on bone and the cardiovascular system. Although tamoxifen has been convincingly demonstrated to be able to preserve bone mineral density (BMD), an unexpected increase of risk of fractures in patients treated with tamoxifen has been reported. The findings of the association of tamoxifen and CHD from previous studies were either borderline or inconsistent. To clarify the discrepancy between BMD and fractures and test the potential beneficial effect of tamoxifen on CHD, I conducted a series of retrospective studies in postmenopausal women with breast cancer who participated in the Cancer Surveillance in HMO Administrative Data (IMPACT study) or the Study of Osteoporotic Fractures (SOF). In patients who participated in the IMPACT study, I demonstrated that the association of tamoxifen and fracture incidence varied at different skeletal sites. Although the association of tamoxifen and fractures in the spine (HR=0.40, 95% CI: 0.09-1.85), wrist (HR=2.49, 95% CI: 0.88-7.06), and total body (HR=0.87, 95% CI: 0.49-1.55) was inconclusive, tamoxifen was associated with an apparent reduction of the risk of hip fracture (HR=0.41, 95% CI: 0.17-1.03, p=0.0565). Importantly, the pattern of observed association of tamoxifen with the risks of fractures among postmenopausal women with breast cancer is consistent with its widely reported preserving effect on bone mineral density. Using SOF data, I found that the association between BMD and fractures in women with breast cancer varied at different skeletal sites, and type of BMD measured. Non-specific BMD was not associated with hip fracture (HR=1.12; 95% CI: 0.78, 1.59). Site-specific BMD was more likely linked with hip fracture (HR=1.43, 95% CI: 0.99, 2.08) while change in BMD did not predict hip fracture (HR=1.05; 95% CI: 0.63, 1.72). The association of spine morphometric fracture with either non-specific or spine-specific BMD was similar (OR=1.40; 95% CI: 1.04, 1.90; OR=1.35, 95% CI: 0.99, 1.85, respectively). Overall, the association of BMD and fracture in elderly women with breast cancer is weak. Only site-specific BMD appears to have a consistently modest association with fractures in the corresponding skeletal sites. In the IMPACT study population, compared to patients without tamoxifen, the overall incidence of CHD in tamoxifen-treated patients was lower (adjusted HR=0.60, 95% CI: 0.40-0.88). For each year of tamoxifen use, there was a statistically significant decrease in the risk of CHD (HR=0.90, 95% CI: 0.82-0.98). Further analyses categorized by length of tamoxifen use showed that an apparent association with a decreased CHD risk was found in patients who received tamoxifen for two to five years (HR=0.54, 95% CI: 0.33-0.86). No association was detected after the discontinuation of tamoxifen therapy. In summary, I detected a possible benefit associated with tamoxifen on fractures in the hip, the most common fracture site. I also found that BMD did not predict osteoporotic fractures well in postmenopausal women with breast cancer. In addition, I demonstrated that tamoxifen was associated with a reduced risk of CHD in postmenopausal women with breast cancer in a dose-dependent manner. An apparent benefit was found in those patients who received tamoxifen therapy for at least two years.

Cu, Zn superoxide dismutase (SOD1) is a dimeric, β-sandwich, metalloenzyme responsible for the dismutation of superoxide. Mutations covering nearly 50% of the amino acid sequence of SOD1 have been found to acquire a toxic gain-of-function leading to amyotrophic lateral sclerosis. A hallmark of this disease is the presence of insoluble aggregates containing SOD1 found in the brain and spinal cord. While it is unclear how these aggregates or smaller, precursor oligomeric species may be the source of the toxicity, mutations leading to increased populations of unstable, partially folded species along the folding pathway of SOD1 may be responsible for seeding and propagating aggregation. In an effort to determine the responsible species, we have systematically characterized the stability and folding kinetics of five well studied ALS variants: A4V, L38V, G93A, L106V and S134N. The effect of the amino acid substitutions was determined on a variety of different constructs characterizing the various post-translational maturation steps of SOD1: folding, disulfide bond formation and Zn binding. Zn was found to bind progressively tighter along the folding pathway of SOD1, minimizing populations of monomeric species. In contrast, ALS variants were found to have the greatest perturbation in the equilibrium populations of the folded and unfolded state for the most immature, disulfide-reduced metal-free SOD1. In this species, at physiological temperature, four out of five ALS variants were >50% unfolded. Finally the energetic barriers in the folding and unfolding reaction were studied to investigate the unusually slow folding of SOD1. These results reveal that both unfolding and refolding are dominated by enthalpic barriers which may be explained by the desolvation of the chain and provide insights into the role of sequence in governing the folding pathway and rate.

Thrombin is the key protease that regulates hemostasis; the delicate balance between procoagulation and anticoagulation of blood. In clotting disorders, like deep vein thrombosis or pulmonary embolism, procoagulation is up-regulated, but propagation of clotting can be inhibited with drugs targeting the proteases involved, like thrombin. Such drugs however, have serious side effects (e.g., excessive bleeding) and some require monitoring during the course of treatment. The reason for these side effects is the mechanism by which the drugs’ act. The two major mechanisms are direct orthosteric and indirect allosteric inhibition, which will completely abolish the protease’s activity. Herein we sought an alternative mechanism called allosteric, partial inhibition, that has shown promise to truly regulate coagulation. Partial inhibition through allosteric mechanisms are well described for membrane-bound and oligomeric proteins. However proteases, specifically monomeric proteases (i.e., thrombin), have not shown this phenomenon until now. A small library of coumarin-based sulfated allosteric modulators (CSAMs) was synthesized to target a surface region called exosite 2; mainly composed of highly positively charged residues surrounded by hydrophobic patches. Studies revealed a non-competitive mechanism of binding with a range of IC50s between 0.2-58 µM combined with inhibitory efficacies (ΔY) between 22-73%; indicative of allosteric, partial inhibition. The KD was determined for the most potent compound (3g; IC50 = 0.2 µM, ΔY = 47%) at 0.15 µM. 3g was observed to bind at exosite 2 through unfractionated heparin competition and thrombin mutant studies. Additional computational studies were in agreement with the mutant and competition studies, showing the sulfate of 3g binding within a pocket containing R126 and R233. Fluorescence quenching and antithrombin inactivation rate studies described a conformational change to thrombin’s active site in the presence of 3g, supporting reduction of thrombin’s catalytic efficiency, without complete inhibition of thrombin’s proteolytic activities. Coupled enzyme assays and gel electrophoresis showed that in the presence of 3g, hydrolysis of fibrinogen (IC50 = 0.51 µM, ΔY = 94%) and protein C activation (IC50 = 1.7 µM, ΔY = 91%) is fully inhibited. Alternatively, FXIII activation was shown to be only partially inhibited by the presence of 3g, and FXI activation did not show any significant activation or inhibition. 3g was also shown to be active in human plasma and whole blood, but requiring much higher concentrations to induce an anticoagulant effect. Mice studies looking at the effects of 3g in vivo showed that even at high concentrations, showed no abnormal bleeding or any other irregularities. This work highlights a novel occurrence regarding thrombin’s allosteric functionality against multiple endogenous substrates. This library of compounds may be useful in the future development of allosteric inhibitors and probes that pose little to no risk of bleeding events by inducing partial inhibition.

The dissertation describes a novel method for plant drug discovery based on mutation and selection of plant cells. Despite the industry focus on chemical synthesis, plants remain a source of potent and complex bioactive metabolites. Many of these have evolved as defensive compounds targeted on key proteins in the CNS of herbivorous insects, for example the insect dopamine transporter (DAT). Because of homology with the human DAT protein some of these metabolites have high abuse potential, but others may be valuable in treating drug dependence. This dissertation redirects the evolution of a native Lobelia species toward metabolites with greater activity at this therapeutic target, i.e. the human DAT. This was achieved by expressing the human DAT protein in transgenic plant cells and selecting gain-of-function mutants for survival on medium containing a neurotoxin that is accumulated by the human DAT. This created a sub-population of mutants with increased DAT inhibitory activity. Some of the active metabolites in these mutants are novel (i.e. not detectable in wild-type cells). Others are cytoprotective, and also protect DAergic neurons against the neurotoxin. This provides proof-of-concept for a novel plant drug discovery platform, which is applicable to many different therapeutic target proteins and plant species.

Neurogenin 3 and its downstream target NeuroD are basic helix-loop-helix transcription factors which promote endocrine differentiation in the gastrointestinal tract. However, mice lacking Ngn3 still produce several hormones in the stomach. Lineage tracing mouse models demonstrated that a majority of hormone cells in the corpus region of the stomach did not express Ngn3 or NeuroD during differentiation. Serotonin and histamine cells were entirely NeuroD-independently derived, and serotonin cells were additionally entirely Ngn3-independently derived. In this study, we isolated serotonin and histamine cells from the gastric corpus of transgenic mice expressing the fluorescent marker CFP. Serotonin cells expressed multiple mast cell markers by RT-PCR, and were found to be nearly absent in a mast cell-deficient mouse model. Labeled bone marrow transplant mice showed all serotonin cells derived from bone marrow. Histamine-expressing ECL cells, while lacking NeuroD, did not appear to express granulocyte or mast cell markers by analytical flow cytometry and RT-PCR, and resemble other enteroendocrine cell populations. Mouse gastric corpus serotonin cells, but not antral serotonin cells, are bone marrow-derived mast cells.

Osteoporosis is a significant public health problem in the U.S. It not only affects the physical well-being of the older women but also creates a substantial financial burden for the health care system. The mainstay of osteoporosis medications is bisphosphonate treatment of which alendronate and risedronate are the most commonly prescribed in clinical practice. However, there have been no head-to-head randomized controlled trials (RCTs) evaluating the effects of these two bisphosphonates on fracture outcomes. In the absence of RCTs, observational studies are necessary to provide alternative evidence on the comparative effectiveness between alendronate and risedronate on fracture outcomes. However, existing observational studies have provided inconclusive results partially due to residual confounding from unobserved variables such as patients’ health status or behavior. IV analysis may be one method to address unmeasured confounding bias in observational studies. While it has not been applied in bisphosphonate research, it has been used in research on a variety of other prescription medications. In this dissertation, we applied the IV approach with an IV, date of generic alendronate availability, to evaluate the comparative effectiveness between alendronate and risedronate using observational data. This dissertation improved current research in several ways. First, we extended the IV approach to research on bisphosphonates. Second, compared with the current observational studies on bisphosphonates, this dissertation may more accurately estimate the relative effects between alendronate and risedronate because IV analysis is not subject to unmeasured confounding bias. Third, the study results extended the current evidence of the comparative effectiveness between the two most commonly prescribed bisphosphonates. Finally, we proposed and provided empirical evidence of a new IV that might be used for future prescription drug research. The finding of this dissertation can be summarized from three aspects. First, we found that the evidence supported the validity of the date of generic availability as an IV in the study of bisphosphonates. Second, applying IV approach to study the comparative effectiveness of alendronate and risedronate, we found that alendronate and risedronate were comparable to reduce the risk of 12-month non-vertebral fractures in older women. Since generic alendronate is availability on the market while generic risedronate is not, promoting the use of alendronate may help reduce the healthcare cost and not sacrifice the clinical effectiveness. Finally, by comparing the proposed IV with a popular IV-physician preference, we found that both the calendar time IV based on the date of generic availability and the physician preference appeared to be valid. It might be practically easier to use the calendar time IV than the physician preference IV.

The alarming rate of increase in incidence and prevalence of the type 2 diabetes mellitus has prompted intense research on understanding the pathogenesis of the type 2 diabetes. It is observed that the development of type 2 diabetes is preceded by a state of insulin resistance and obesity. Previous studies have suggested that the obesity induced insulin resistance may be mediated by elevated levels of circulating free fatty acids (FFAs). The increase in circulating levels of FFAs may be contributed by the release of FFAs from stored triglycerides (TG) in adipocytes via lipolysis. It is hypothesized that the decrease in levels of circulating FFAs by sequestration and storage of FFAs in adipocytes may prevent deleterious effects of FFAs on insulin sensitivity. Recently our lab and others have shown that the storage of TG in adipocytes is promoted by a novel protein, Fat Specific Protein 27 (FSP27). Although, these studies also revealed FSP27 to be a lipid droplet associated protein that suppresses lipolysis to enhance TG accumulation in adipocytes, the role of FSP27 in lipolysis remains largely undetermined. Therefore, this study investigates the role and regulation of FSP27 in adipocytes in both the basal state, as well as during lipolysis. The studies presented here show FSP27 to be a remarkably short-lived protein (half-life=15 min) due to its rapid ubiquitination and proteasomal degradation. Thus, I tested the hypothesis that lipolytic agents like the cytokine, TNF-α and the catecholamine isoproterenol modulate FSP27 protein levels to regulate FFA release. Consistent with this concept, TNF-α markedly decreased FSP27 mRNA and protein along with lipid droplet size as it increased lipolysis in cultured adipocytes. Similarly, FSP27 depletion using siRNA mimicked the effect of TNF-α to enhance lipolysis, while maintaining stable FSP27 protein levels by expression of HA epitope-tagged FSP27 blocked TNF-α mediated lipolysis. In contrast, the robust lipolytic action of isoproterenol is paradoxically associated with increases in FSP27 protein and a delayed degradation rate that corresponds to decreased ubiquitination. This catecholamine-mediated increase in FSP27 abundance, probably a feedback mechanism to restrain excessive lipolysis by catecholamines, is mimicked by forskolin or 8-Bromo-cAMP treatment, and prevented by Protein Kinase A (PKA) inhibitor KT5720 or PKA depletion using siRNA. These results show that isoproterenol stabililizes FSP27 via the canonical PKA pathway and increased cAMP levels. However, the work presented here also suggests that FSP27 does not get phosphorylated in response to isoproterenol treatment, and the stabilization of FSP27 is independent of isoproterenol mediated lipolysis. The data presented in this thesis not only identifies the regulation of FSP27 as an important intermediate in mechanism of lipolysis in adipocytes in response to TNF-α and isoproterenol, but also suggests that FSP27 may be a possible therapeutic target to modulate lipolysis in adipocytes.

Supplementation with nicotinamide riboside (NR), a form of vitamin B3 and a precursor of nicotinamide adenine dinucleotide (NAD+) extends lifespan in the nematode C. elegans and delays aging-related pathologies in mammals. During aging, levels of NAD+ decline causing metabolic dysfunction and oxidative damage. Studies in C. elegans found that when NR was administered during larval development it induced the mitochondrial unfolded protein response (UPRmt), which is frequently associated with lifespan extension. Both calorie restriction (CR) and ketogenic diets (KD) have been shown to extend lifespan, in part through increasing NAD+ and through increasing levels of the pro-longevity ketone body beta-hydroxybutyrate (BHB). In a previous study from my lab, NR increased C. elegans lifespan to a much larger extent when administered starting at the L1 larval stage as compared to when started at the L4 larval stage. Conversely, a high dose of BHB greatly increased lifespan when administered starting at the L4 stage. But this same dose decreased lifespan when it was first administered at the L1 stage. I further found that NR greatly extended lifespan when only administered during larval development and that the combination of both individual pro-longevity NR and BHB treatments significantly decreased lifespan. These chemical epistasis experiments suggest that NR and BHB function in parallel pathways to extend C. elegans lifespan through a common downstream target with hormesis playing a role. Therefore, human subjects who supplement with both NAD+ precursors and ketone esters should be aware of possible negative interactions when high doses of both are administered.

In this research, a proton exchange membrane fuel cell (PEMFC) sensor was investigated for specific detection of volatile organic compounds (VOCs) for point-of-care (POC) diagnosis of the physiological conditions of humans. A PEMFC is an electrochemical transducer that converts chemical energy into electrical energy. A Redox reaction takes place at its electrodes whereas the volatile biomolecules (e.g. ethanol) are oxidized at the anode and ambient oxygen is reduced at the cathode. The compounds which were the focus of this investigation were ethanol (C2H5OH) and isoflurane (C3H2ClF5O), but theoretically, the sensor is not limited to only those VOCs given proper calibration. Detection in biosensing, which needs to be carried out in a controlled system, becomes complex in a multivariate environment. Major limitations of all types of biosensors would include poor selectivity, drifting, overlapping, and degradation of signals. Specific detection of VOCs in multi-dimensional environments is also a challenge in fuel cell sensing. Humidity, temperature, and the presence of other analytes interfere with the functionality of the fuel cell and provide false readings. Hence, accurate and precise quantification of VOC(s) and calibration are the major challenges when using PEMFC biosensor. To resolve this problem, a statistical model was derived for the calibration of PEMFC employing multivariate analysis, such as the “Principal Component Regression (PCR)” method for the sensing of VOC(s). PCR can correlate larger data sets and provides an accurate fitting between a known and an unknown data set. PCR improves calibration for multivariate conditions as compared to the overlapping signals obtained when using linear (univariate) regression models. Results show that this biosensor investigated has a 75% accuracy improvement over the commercial alcohol breathalyzer used in this study when detecting ethanol. When detecting isoflurane, this sensor has an average deviation in the steady-state response of ~14.29% from the gold-standard infrared spectroscopy system used in hospital operating theaters. The significance of this research lies in its versatility in dealing with the existing challenge of the accuracy and precision of the calibration of the PEMFC sensor. Also, this research may improve the diagnosis of several diseases through the detection of concerned biomarkers.