Browsing by Subject "Endocrinology"
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Item Open Access Broad Remodeling of the Acetylproteome by SIRT3 Manipulation Fails to Affect Insulin Secretion or β-cell Metabolism in the Absence of Dietary Overnutrition(2018) Peterson, Brett StevenSIRT3 is an NAD+-dependent mitochondrial protein deacetylase purported to influence cellular and systemic metabolism through modulation of the mitochondrial acetylproteome. Fuel-stimulated insulin secretion from pancreatic islets involves mitochondrial metabolism and might be susceptible to SIRT3-mediated effects. To investigate this idea, we used CRISPR/Cas9 technology to obtain complete SIRT3 knockout in the INS-1 832/13 insulinoma cell line. In the context of this SIRT3 knockout cell line, we re-expressed wild-type SIRT3, β-Galactosidase, or one of three enzymatically inactive mutant forms of SIRT3 to generate lines representing a wide range of SIRT3 expression and mitochondrial protein deacetylase activity. We performed large-scale acetylproteome profiling by mass spectrometry on the different lines, and observed wide-spread, SIRT3-dependent changes in acetylation of enzymes involved in fatty acid oxidation, the TCA cycle, and the electron transport chain. Remarkably, despite these broad changes, the cell lines had indistinguishable insulin secretion responses to glucose or pyruvate, and exhibited no differences in function or viability in response to metabolic or ER stress-inducing agents. Moreover, metabolomic profiling revealed that, when compared to SIRT3-null cell lines, expression of wild-type SIRT3 does not result in appreciable changes in a host of organic acid, amino acid or fatty acid-derived acylcarnitine metabolites during glucose stimulation.
We also studied mice with global SIRT3 knockout (KO) fed a standard chow (STD) or high-fat/high-sucrose (HFHS) diet. Importantly, we performed these studies in the C57Bl/6J background in which we replaced the mutant allele of nicotinamide nucleotide transhydrogenase (NNT) present in the “J” substrain, with the wild-type allele in order to restore endogenous NNT function. SIRT3 KO and wild-type (WT) mice fed a STD diet exhibited no differences in insulin secretion during oral or IP glucose tolerance tests, and the function of islets isolated from these mice was indistinguishable in islet perifusion studies conducted with a broad array of secretagogues. Only when chronically fed a HFHS diet did SIRT3 KO animals exhibit a modest impairment in insulin secretion, but without an effect on glycemic control. Our broad conclusion is that major changes in mitochondrial protein acetylation in response to manipulation of SIRT3 are not sufficient to cause changes in islet function or metabolism. However, under conditions of chronic nutritional stress (feeding of a HFHS diet for 12 weeks), a negative effect on function appears, suggesting that islets are more susceptible to nutrition-induced factors (oxidative stress, local cytokine production, etc.) when SIRT3 is absent. Further studies will be required to identify factors that may interact with SIRT3 deficiency and mitochondrial protein hyperacetylation to increase the risk of -cell dysfunction.
Item Open Access Controlled Release Systems for Treating Type 2 Diabetes and Their Application Toward Multi-Agonist Combination Therapies(2019) Gilroy, Caslin AnneOver 30 million people in the United States suffer from type 2 diabetes (T2D), and this figure is rapidly increasing. Currently available glucose-lowering drugs largely treat the symptoms of diabetes and not the underlying pathology, leaving one third of diabetes patients with improperly managed disease. Thus, there exists an urgent need for novel drugs that slow T2D progression while posing a minimal burden on the patient.
The metabolic regulatory factor fibroblast growth factor 21 (FGF21) is under investigation as a T2D therapeutic due to its favorable effects on glycemic control and body weight. However, the feasibility of native FGF21 as a drug candidate is impeded by its rapid in vivo clearance and by costly production methods associated with poor protein solubility. To address these issues, FGF21 was recombinantly expressed in E. coli as a fusion with an elastin-like polypeptide (ELP), a repetitive peptide polymer with reversible thermal phase behavior. Below their transition temperature (Tt), ELPs exist as soluble unimers, while above their Tt, they aggregate into an insoluble coacervate. The thermal responsiveness of the ELP was retained when genetically fused to FGF21, with several notably positive impacts for the synthesis and efficacy of this protein drug. First, the ELP fusion partner acted as a solubility enhancer, yielding 50 mg/L of active FGF21 protein from the soluble cell lysate fraction in shaker flask culture, and eliminating the need for protein refolding. Second, the phase transition behavior of the ELP was exploited for chromatography-free FGF21 purification. Third, the Tt of the ELP was tuned to below body temperature, such that the phase transition was initiated solely by body heat. Indeed, in vivo injection of the fusion resulted in an immiscible viscous phase in the subcutaneous (s.c.) space that dissolved at a steady rate, temporally releasing fusion unimers into circulation. The injectable FGF21 drug depot was tested in diabetic ob/ob mice, and conferred dose-dependent glucose-and weight-lowering effects that were sustained out to 5 days following a single s.c. injection.
Once an optimized ELP-based FGF21 delivery strategy was established, the fusion concept was applied to a combination therapy to afford even greater metabolic benefits, while providing controlled release properties exclusive to the ELP platform. Recent evidence supports the development of combination drug treatments that incorporate complementary mechanisms of action to more effectively treat T2D. Thus, we developed a unimolecular dual agonist by combining the incretin glucagon-like peptide-1 (GLP1) with FGF21, hypothesizing that this agent would merge the insulinotropic and anorectic effects of GLP1 with the enhanced insulin sensitivity and energy expenditure associated with FGF21 signaling. The dual agonist was designed as a single polypeptide fusion, with GLP1 located at the N terminus and FGF21 at the C terminus. This orientation allowed each peptide to activate its endogenous receptor, while the linear architecture enabled facile synthesis in a bacterial expression system. An ELP was fused between GLP1 and FGF21 to serve as both a flexible linker and a depot-forming delivery scaffold. Indeed, a single s.c. injection of GLP1-ELP-FGF21 into diabetic db/db mice resulted in potent metabolic effects that were sustained at least 7 days, indicating formation of an ELP depot with a highly controlled rate of drug release. Furthermore, dual agonist treatment outperformed a long-acting GLP1 analog in restoring glycemic control and inducing weight loss, supporting the rationale for a GLP1/FGF21 combination therapy.
With a significant proportion of T2D patients failing to properly manage their disease, there is an urgent need for novel drug and drug combinations that effectively target disease pathophysiology, while posing a minimal burden on the patient. Meanwhile, the vast – and global – prevalence of metabolic disease argues for cost-effective and scalable manufacturing methods for new drugs. An ELP-based approach to therapeutics precisely addresses these needs by providing a streamlined method for production, as well as an innovative strategy for drug delivery to reduce the frequency of administration and thereby promote patient compliance. Furthermore, the ELP platform can be utilized to unite distinct drugs into one multi-functioning molecule to more effectively treat diabetes, altogether simplifying and improving metabolic disease management.
Item Open Access Creation of Versatile Cloning Platforms for Transgene Expression and Epigenome Editing and Their Application to Pancreatic Islet Biology(2018) Haldeman, Jonathan MarkInsulin secreting β-cells within the pancreatic islets of Langerhans are vital to maintaining glycemic control. β-cell functional mass is lost during the progression to both Type 1 and Type 2 diabetes mellitus, resulting in hyperglycemia. Therefore, a major goal of diabetes research is to uncover pathways that can be exploited to induce β-cell replication while simultaneously maintaining β-cell function.
We previously reported that adenovirus-mediated overexpression of the transcription factor PDX1 is sufficient to induce β-cell replication, but underlying mechanisms remain to be resolved. Using statistical modeling, we identified the miR-17 family, a member of the miR17~92 miRNA cluster, as a candidate regulator of the PDX1-gene network. We show that PDX1 can directly regulate the MIR17HG promoter, the first example of β-cell specific regulation for this important miRNA cluster. Furthermore, the miR17~92 target PTEN is reduced in PDX1-overexpressing β-cells, and chemical inhibition of PTEN potentiates PDX1-mediated β-cell replication, supportive of the presence of a PDX1/miR17~92/PTEN regulatory node.
Recombinant adenovirus approaches pioneered by our laboratory have been the main method of genetic manipulation of primary islets in culture since 1994. Whereas adenovirus vectors have proved useful in an otherwise difficult model system, virus construction, especially for cell-type specific applications, is still laborious and time-consuming. To overcome this, we have created a new modular cloning system (pMVP) that allows a gene of interest to be rapidly recombined in the context of an array of promoters, N- or C-terminal epitope tags, inducible gene expression modalities, and/or fluorescent reporters, into 18 custom destination vectors, including adenovirus, expression plasmid, lentivirus, and Sleeping Beauty transposon, thus, permitting the creation of > 8000 unique vector permutations. Multiple features of this new vector platform, including cell type-specific and inducible control of gene expression, were validated in the setting of pancreatic islets and other cellular contexts. Furthermore, using pMVP as a foundation, we also developed an S. aureus dCas9 epigenetic engineering platform, pMAGIC, that enables the packaging of 3 guide RNAs with Sa-dCas9 fused to one of five epigenetic modifiers into a single vector. Using pMAGIC-derived adenoviruses, we functionally validated the regulation of PDX1 by Area IV, a cross-species conserved enhancer, through LSD1-mediated epigenetic modification in both INS1 832/13 cells and primary rat pancreatic islets.
In sum, my work has uncovered novel information about the role of PDX1 in regulation of the miR17~92 miRNA cluster in pancreatic islet cells. In an effort to contribute more broadly to our laboratory’s pancreatic islet research efforts, I also designed and built the pMVP and pMAGIC systems for efficient generation of purpose-built, customized vectors for manipulation of gene expression in islets and other cell types, including via targeted epigenetic modification of putative regulatory elements within their native chromatin context. Development of this novel vector platform facilitated additional discoveries about the role of Area IV in control of PDX1 expression in islet β-cells.
Item Embargo Dissecting the functional effects of non-coding gene regulatory elements(2024) Sankaranarayanan, LaavanyaOne of the most beautiful and challenging aspects in biology is deciphering the complexity of the genome, and how it functions or dysfunctions. It is this intricate complexity that is dependent on developmental stages, time of the day, and tissue types that allows for the proper development of an organism comprising of different tissue types with different functions. Amongst the many complexities, I focused on the gene-regulatory functions of the non-coding genome and its relation to diseases including disease risk, severity, and progression. Over the last few decades, there has been an increase in the research of genetic causes underlying several complex, common multifactorial diseases including metabolic and cardiovascular diseases. While these studies have identified genetic risk loci, they have not directly identified the genetic mechanisms behind what causes those diseases. Identifying genetic mechanisms for complex traits has been challenging because most of the variants are located outside of protein-coding regions, and determining the effects of such non-coding variants remains difficult. Previous studies that explore the genetic mechanism of complex diseases have underscored the need to develop new methods to study non-coding regions and to systematically identify the effects of non-coding variants towards a disease.
In this dissertation, I evaluate the hypothesis that non-coding regulatory elements can contribute to disease-relevant traits by altering gene expression levels. I will specifically focus on a common complex disease, polycystic ovary syndrome (PCOS) which is the most prevalent endocrine disorder among menstruating people. Family- and twin-studies have demonstrated a genetic basis to PCOS. Previous studies have identified non-coding genetic variation associated with PCOS risk across populations with different ancestries. However, the functional follow up of these risk loci has been limited. Therefore, there is a gap in addressing the functional effects of genetic variants and regulatory elements impacting PCOS phenotypes. We identified gene regulatory mechanisms that help explain genetic association with PCOS in several loci using high throughput reporter assays, CRISPR-based epigenome editing, and genetic association analysis. To develop approaches to study regulatory elements, I implemented reporter assays at three different scales to create a framework for the regulatory elements across PCOS risk loci. I also implemented experimental approaches that measured changes in gene expression at single cell levels to identify target genes of regulatory elements identified by the reporter assays. Specifically, we identified regulatory elements across PCOS genetic risk loci in cell models of steroidogenesis, H295R cells and COV434 cells. We then identified regulatory elements that controlled the expression of the gene DENND1A, which altered the levels of testosterone produced by the cell models upon perturbation. Lastly, we quantified the regulatory effects of allele-specific genetic variants from a population of PCOS cases and controls. Taken together, we have identified regulatory elements that could contribute to PCOS pathogenesis. More broadly, my results demonstrate the strengths of combining experimental and statistical approaches to identify molecular mechanisms of genetic risk loci contributing to disease pathogenesis.
Item Open Access Dopamine, Drugs, and Estradiol: The Roles of ERα and ERβ in the Mesencephalic Dopamine System and Dopamine-Mediated Behaviors of Mice(2012) Van Swearingen, Amanda Elyse DaySex differences in drug addiction are mediated in part by effects of the ovarian hormone estradiol (E2) within the ascending dopamine (DA) system from the midbrain to the striatum. Estradiol enhances the effects of psychostimulants, but the exact underlying mechanisms are unknown. Mice could serve as an ideal genetically-tractable model for mechanistic studies into sex and hormone effects within the DA system but have been under-utilized. This study sought to: 1) characterize psychostimulant-induced behavior in mice as an indirect but quantifiable measure of DA neurotransmission, and 2) elucidate the mechanism underlying E2's enhancement of psychostimulant effects in females using surgical, pharmacological, and genetic manipulations. The spontaneous behavior of mice during habituation to a novel environment and after the psychostimulants d-amphetamine (AMPH; 1, 2.5, and/or 5 mg/kg) and cocaine (COC; 5, 15, and/or 30 mg/kg) were assessed in open field chambers using both automated photobeam interruptions and behavioral observations. Behaviors were assessed in the following groups of mice: intact males and females; ovariectomized mice replaced with either E2 for 2 days or 30 minutes or with estrogen receptor-selective agonists; and female mice lacking either ERα (αERKO) or ERβ (βERKO) versus wildtype (WT) littermates. Brain psychostimulant concentrations and tissue content of DA and its metabolites were determined at the time of maximum behavioral stimulation. Psychostimulants induced behavioral activation in mice including both increased locomotion as detected with an automated system and a sequence of behaviors progressing from stereotyped sniffing at low doses to patterned locomotion and rearing at high doses. Intact female mice exhibited more patterned locomotion and a shift towards higher behavior scores after psychostimulants despite having lower AMPH and equivalent COC brain levels as males. Actively ovariectomized mice exhibited fewer ambulations and lower behavior scores during habituation and after psychostimulants than Sham females. Two days but not 30 minutes of E2 replacement restored COC-induced behavioral responses to Sham levels. ERα-selective PPT replacement in ovariectomized mice and genetic ablation of ERα in αERKO mice altered COC-stimulated behavior. Immunohistochemistry revealed that midbrain DA neurons in mice express ERβ but not ERα, and that non-DA cells in the midbrain and the striatum express ERα. These results indicate that E2 enhances COC-stimulated locomotion in mice through an indirect effect of ERα. ERα may alter behavior through presynaptic effects on DA neuron activity and/or through postsynaptic effects on transcription and signal transduction pathways within striatal neurons.
Item Embargo Feasibility of Kanasina Gulabi, a Pilot Peer Support Intervention for Young Adults with Type 2 Diabetes in Mysore District, South India(2024) Gopisetty, Nikhita RaniBackground: The global rise in early-onset type 2 diabetes (T2D) is impacting the lives of millions of young individuals around the world. To address the knowledge gap and emotional burden in young adults with T2D, the research team designed a four-week education and goal-based peer support intervention, Kanasina Gulabi (Kannada: My Dream Rose), implemented in Mysore, India between June and July 2023. Methods: The Reach, Effectiveness, Adoption, Implementation, Maintenance (RE-AIM) framework was used to assess feasibility. Peer navigators (n=3, mean age: 33 years) were recruited from the community and trained to deliver the intervention. Twenty-eight adults ≤ 40 years of age with T2D were recruited and quasi-randomly allocated to the intervention group (n=14, mean age: 33 years) or the control group (n=14, mean age: 33 years). Hemoglobin A1c, T2D knowledge, diabetes distress, diabetes-related stigma, depression severity, and optimism were assessed at baseline, immediately after the intervention, and at the 2-month follow-up. Attendance, fidelity, and acceptability were also assessed. Results: Intervention participants expressed high satisfaction with Kanasina Gulabi and gratitude for the guided knowledge and mentorship from their peer navigators. Participants displayed excellent attendance and adherence, with an average of 5 hours of Kanasina Gulabi exposure over 4 weeks. Peer navigators delivered the intervention with 100% fidelity. Results immediately following the intervention showed reductions in diabetes distress (Intervention: 30.9%, Control: 6.5%), diabetes-related stigma (Intervention: 25.3%, Control: 14.5%), and depression severity (Intervention: 58.3%, Control: 38.5%). Both groups presented an increase in T2D knowledge (Intervention: 19.7%, Control: 25.2%). Conclusion: Implementing Kanasina Gulabi in Mysore is feasible and acceptable, with intervention participants showing more positive changes on multiple psychosocial outcomes compared to the control group. Results support a full trial to evaluate its effectiveness and sustainability in young adults with T2D.
Item Open Access Inhibition of TGFβ Signaling Does Not Improve the Limited Proliferative Response to Nkx6.1 Or Pdx-1 Overexpression in Aged Rat Islets(2015) Rosa, TaylorA deficiency in functional pancreatic beta-cells is a defining feature in type 1 and type 2 diabetes. The development of therapeutic strategies for replacement and regeneration of beta-cell mass is a key objective of current diabetes research. The Newgard lab has had a particular focus in exploring novel beta-cell replication pathways in order to identify targets that enhance beta-cell proliferation, survival, and function. The beta-cell developmental transcription factors Nkx6.1 and Pdx-1 each have a profound proliferative effect when overexpressed in young rat islets in vitro. The unique ability of these factors and the pathways that they control to expand functional beta-cell mass while either being neutral or positive for other key functions (survival, insulin secretion) encourages further studies to better elucidate candidate target genes within these pathways.
A major limitation of the research to date is that Nkx6.1 and Pdx-1 only exert their proliferative effects in young (2 months) rodent islets and not in old (8-10 months) rodent islets. Moreover, these factors are only weakly active in human islets, most of which come from middle-aged donors. Nkx6.1 and Pdx-1 engage pathways that are upstream of the core cell cycle machinery and that have the potential to be stimulated in a beta-cell specific manner, but use of this approach will depend on a better understanding of the differences between human and rodent islets, which may be modeled by the differences between old and young rat islets.
Item Open Access Polybrominated Diphenyl Ether (PBDE) Flame Retardants: Accumulation, Metabolism, and Disrupted Thyroid Regulation in Early and Adult Life Stages of Fish(2013) Noyes, PamelaPolybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardant chemicals that are added to plastics, electronic components, furniture foam, and textiles to reduce their combustibility. Of the three commercial mixtures historically marketed, only DecaBDE, which is constituted almost entirely (~97%) of the fully brominated congener decabromodiphenyl ether (BDE-209), continues to be used in the U.S. today. While decaBDE is scheduled for phase-out in the U.S. at the end of 2013, exposures to BDE-209 and other PBDEs will continue into the foreseeable future as products that contain them continue to be used, recycled, and discarded. In addition, decaBDE use continues to be largely unrestricted across Asia, although restricted from use in electronic equipment in Europe.
Despite limits placed on PBDE uses, they are ubiquitous contaminants detected worldwide in humans and wildlife. Major health effect concerns for PBDEs come largely from evidence in laboratory rodents demonstrating neurotoxicity, reproductive and developmental impairments, and thyroid disruption. The potential for PBDEs, particularly BDE-209, to disrupt thyroid regulation and elicit other toxic outcomes in fish is less clear. Thus, the overall objective of this thesis research was to answer questions concerning how fish, as important indicators of overall environmental health, are metabolizing PBDEs and whether and how PBDEs are disrupting thyroid hormone regulation. The central hypothesis was that PBDE metabolism in fish is mediated by iodothyronine deiodinase (dio) enzymes, which are responsible for activating and inactivating thyroid hormones, and that PBDE exposures are causing thyroid system dysfunction across fish life stages.
Under the first research aim, in vitro experiments conducted in liver tissues isolated from common carp (Cyprinus carpio) suggested a role for dio enzymes in catalyzing the reductive debromination of PBDEs. Carp liver microsomes efficiently debrominated BDE-99 to BDE-47, and enzymes catalyzing this reaction were associated predominantly with the endoplasmic reticulum (i.e., microsomal fraction) where dio enzymes are located. Competitive substrate experiments in carp liver microsomes also demonstrated that rates of BDE-99 debromination to BDE-47 were significantly inhibited upon challenges with 3,3',5'-triiodothyronine (rT3) and thyroxine (T4). This finding supported the hypothesis that enzymes involved in the metabolism of PBDEs may have high affinities for thyroid hormones. Indeed, experiments to determine apparent enzymatic kinetics (apparent Vmax and Km values) of BDE-99 hepatic metabolism suggested that enzymes responsible for the catalytic activity appeared to have a higher affinity for native thyroid hormone than BDE-99.
The second and third research aims were focused on evaluating BDE-209 accumulation, metabolism, and thyroid toxicity in juvenile and adult life stages of fish using the fathead minnow (Pimephales promelas) as a model. BDE-209 bioaccumulated and was debrominated to several reductive metabolites ranging from penta- to octaBDEs in both juvenile and adult fish exposed to BDE-209. In addition, thyroid hormone regulation in juvenile and adult male fathead minnows was severely disrupted by BDE-209 at low, environmentally relevant exposures. In juvenile minnows, the activity of dio enzymes (T4-outer ring deiodination; T4-ORD and T4-inner ring deiodination; T4-IRD) declined by ~74% upon oral doses of 9.8 ± 0.2 µg/g wet weight (ww) food at 3% body weight (bw)/day for 28 days, compared to controls. Declines in dio activity were accompanied by thyroid follicle hypertrophy indicative of over-stimulation and injury. In addition to thyroid disruption, a distinctive liver phenotype characterized by vacuolated hepatocyte nuclei was measured in ~48% of hepatocytes from treated fish that was not observed in controls.
Under the third research aim, adult male fathead minnows received dietary treatments of BDE-209 at a low dose (95.3 ± 0.41 ng/g-food at 3% bw/day) and a high dose (10.1 ± 0.10 µg/g-food at 3% bw/day) for 28 days followed by a 14-day depuration period to evaluate recovery. Compared to negative controls, adult male fish exposed orally to BDE-209 at the low dose tested for 28 days experienced a 53% and 46% decline in circulating total T4 and T3, respectively, while fish at the high BDE-209 dose tested had total T4 and T3 deficits of 59% and 62%, respectively. Depressed levels of plasma thyroid hormones were accompanied by a 45-50% decline in the rate of T4-ORD in brains of all treatments by day 14 of the exposure. The decreased T4-ORD continued in the brain at day 28 with a ~65% decline measured at both BDE-209 doses. BDE-209 exposures also caused transient, tissue-specific upregulations of relative mRNA transcripts encoding dio enzymes (dio1, dio2), thyroid hormone receptors (TR&alpha, TR&beta), and thyroid hormone transporters (MCT8, OATP1c1) in the brain and liver in patterns that varied with time and dose, possibly as a compensatory response to hypothyroidism. In addition, thyroid perturbations at the low dose tested generally were equal to those measured at the high dose tested, suggesting non-linear relationships between PBDE exposures and thyroid dysfunction in adult fish. Thus, mechanisms for BDE-209 induced disruption of thyroid regulation can be proposed in adult male minnows that involve altered patterns of thyroid hormone signaling at several important steps in their transport and activation.
A growing body of evidence describing PBDE toxicity in biota, including data generated here, along with studies showing continued and rising PBDE body burdens, raises concern for human and wildlife health. Long delays in removing PBDEs from the market, their ongoing presence in many products still in use, and their active use outside the U.S. and European Union will leave a lasting legacy of rising contamination unless more concerted regulatory and policy actions are taken to reduce future exposures and harm.
Item Open Access Steroid Hormone Variation and Stress Responses in Short-finned Pilot Whales(2023) Wisse, JillianAs humans continue to introduce stressors into the marine environment, we are obligated to understand how our behaviors impact wildlife. For many cetacean populations, anthropogenic noise poses a significant conservation threat, but the ability to monitor these animals is constrained by their often-remote habitats and limited time at the surface. Researchers have developed innovative solutions to overcome these challenges, including the development of techniques that enable physiological sampling with minimal disturbance. As frontiers, these methods require careful development and validation before they can be used reliably in experimental studies. In this dissertation, I employ one of these innovative techniques, measuring steroid hormones in remote blubber biopsy samples, in short-finned pilot whales (Globicephala macrorhynchus). As modulators of reproduction and stress, steroid hormones provide information that is advantageous for wildlife monitoring. Because a validated method for measuring these compounds in short-finned pilot whale blubber did not yet exist, I adapted a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify 11 steroid hormones of interest. Before proceeding with its application, I tested its analytical and biological validity with blubber from three stranded short-finned pilot whales. Next, I applied the validated LC-MS/MS method to an archive of blubber biopsies, collected from free-ranging short-finned pilot whales in the Western North Atlantic. Leveraging the comprehensive nature of LC-MS/MS profiling, I investigated relationships between hormones and characterized steroid hormone profiles across demographic groups and seasons. In the fourth chapter, I conducted an acoustic response study, using the previously established methods to collect and quantify steroid hormones after exposure to simulated mid-frequency active sonar (MFAS). I modeled the responses of cortisol and cortisone over time to gain insight into steroid perfusion rates in cetacean blubber and asked whether demography contributed to these responses. Together, my results demonstrate the reliability of blubber for measuring steroid hormones and reflecting relevant physiological states, like stress and pregnancy. While LC-MS/MS enables extensive steroid measurement, it struggled to detect some steroids of interest in this matrix. This dissertation shows the relevance of multi-steroid profiling and offers reference points for baseline steroid concentrations in analytes relevant to behavior, reproduction, and stress. The observation of post-exposure stress responses confirms a relationship between noise and physiology in short-finned pilot whales and illustrates the ability of blubber sampling to be applied in this context.
Item Open Access The National Physicians Cooperative: transforming fertility management in the cancer setting and beyond.(Future oncology (London, England), 2018-12) Smith, Brigid M; Duncan, Francesca E; Ataman, Lauren; Smith, Kristin; Quinn, Gwendolyn P; Chang, R Jeffrey; Finlayson, Courtney; Orwig, Kyle; Valli-Pulaski, Hanna; Moravek, Molly B; Zelinski, Mary B; Irene Su, H; Vitek, Wendy; Smith, James F; Jeruss, Jacqueline S; Gracia, Clarisa; Coutifaris, Christos; Shah, Divya; Nahata, Leena; Gomez-Lobo, Veronica; Appiah, Leslie Coker; Brannigan, Robert E; Gillis, Valerie; Gradishar, William; Javed, Asma; Rhoton-Vlasak, Alice S; Kondapalli, Laxmi A; Neuber, Evelyn; Ginsberg, Jill P; Muller, Charles H; Hirshfeld-Cytron, Jennifer; Kutteh, William H; Lindheim, Steven R; Cherven, Brooke; Meacham, Lillian R; Rao, Pooja; Torno, Lilibeth; Sender, Leonard S; Vadaparampil, Susan T; Skiles, Jodi L; Schafer-Kalkhoff, Tara; Frias, Oliva J; Byrne, Julia; Westphal, Lynn M; Schust, Danny J; Klosky, James L; McCracken, Kate A; Ting, Alison; Khan, Zaraq; Granberg, Candace; Lockart, Barbara; Scoccia, Bert; Laronda, Monica M; Mersereau, Jennifer E; Marsh, Courtney; Pavone, Mary Ellen; Woodruff, Teresa KOnce unimaginable, fertility management is now a nationally established part of cancer care in institutions, from academic centers to community hospitals to private practices. Over the last two decades, advances in medicine and reproductive science have made it possible for men, women and children to be connected with an oncofertility specialist or offered fertility preservation soon after a cancer diagnosis. The Oncofertility Consortium's National Physicians Cooperative is a large-scale effort to engage physicians across disciplines - oncology, urology, obstetrics and gynecology, reproductive endocrinology, and behavioral health - in clinical and research activities to enable significant progress in providing fertility preservation options to children and adults. Here, we review the structure and function of the National Physicians Cooperative and identify next steps.Item Open Access Understanding female social dominance: comparative behavioral endocrinology in the Genus Eulemur(2015) Petty, Joseph Michael AlexanderFemale social dominance over males is unusual in mammals, yet characterizes most Malagasy lemurs, which represent almost 30% of all primates. Despite its prevalence in this suborder, both the evolutionary trajectory and proximate mechanism of female dominance remain unclear. Potentially associated with female dominance is a suite of behavioral, physiological and morphological traits in females that implicates ‘masculinization’ via androgen exposure; however, relative to conspecific males, female lemurs curiously show little evidence of raised androgen concentrations. In order to illuminate the proximate mechanisms underlying female dominance in lemurs, I observed mixed‐sex pairs of related Eulemur species, and identified two key study groups ‐‐ one comprised of species expressing female dominance and, the other comprised of species (from a recently evolved clade) showing equal status between the sexes (hereafter ‘egalitarian’). Comparing females from these two groups, to test the hypothesis that female dominance is an expression of an overall masculinization of the female, I 1) characterize the expression of female dominance, aggression, affiliation, and olfactory communication in Eulemur; 2) provide novel information about the hormonal and neuroendocrine correlates associated with the expression of female dominance; 3) investigate the activational role of the sex-steroid hormones in adult female Eulemur using seasonal correlates of hormonal and behavioral change; and 4) examine the specific role of estrogen in the regulation and expression of sex-reversed female behavior in these species. In doing so I highlight significant behavioral and physiological differences between female-dominant and egalitarian Eulemur and show that female dominance is associated with a more masculine behavioral and hormonal profile. I also suggest that these behavioral and hormonal differences may be the result of fundamental differences in the biosynthetic pathway associated with estrogen production. Moreover, I assert that these putative physiological differences could provide a parsimonious proximate mechanism explaining the evolution of female dominance and its subsequent relaxation in egalitarian Eulemur species.