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The thematic research areas of Wyoming INBRE 3 are structured around what are perceived as strengths and as having significant health relevance to make maximum use of existing research expertise. Cardiometabolic syndrome and technology for chronic disease research and therapeutics relate to the leading causes of morbidity and mortality in the United States, including rural areas like Wyoming. Effective management of cardiovascular related syndromes, especially cardiometabolic diseases, to improve the quality of life and reduce the overwhelming health care costs has become a burning issue for NIH and the American Heart and American Diabetes Associations.
Eunsook Park, Assistant Professor Molecular Biology. Autophagy: a new target biological process to develop effective antifungal drugs for human fungal diseases. Cryptococcus neoformans causes morbidity and mortality in immunocompromised people, yet there still is no effective therapy to prevent this infection. About 4 million death associated with AIDS-related death has been attributed to the fungal infection in the past decades. Many fungal pathogens of clinical importance, including C. neoformans, are dimorphic, and the morphological status often associates with their virulence. C. neoformans have a striking life cycle depending on the environmental niche. The sexual life is observed on C. neoformans-plant interaction, while it has asexual morphology inside vertebrate hosts. Although basidiospores at the end of the sexual life are infectious propagules and capable of colonization in human lung, current knowledge for the pathogenesis of this fungus has been limited. Thus, it is necessary not only to understand the molecular mechanism of the life cycle of C. neoformans adapted to dual compatible hosts of both animal and plant but also to identify effective fungicides against the infectious fungal pathogen for the prevention of diseases and improvement of healthcare. Preliminary results from other fungal pathogens indicated the importance of autophagy in several key developmental processes of fungi in their sexual life cycle. In addition, autophagy mutant of C. neoformans showed compromised pathogenicity, suggesting the tight connection of autophagy and pathogenicity and dimorphic life cycles of C. neoformans. Therefore, in this study, we will identify fungal autophagy-specific inhibitor by an innovative high throughput chemical screening using versatile Bioluminescence Resonance Energy Transfer (BRET)-based sensors. In addition, we will dissect the role of autophagy at the host-fungus interface in Cryptococcosis disease development. The proposed study will not only expand our limited knowledge on the molecular mechanisms of autophagy at the interface of fungal pathogen and host interaction but also envision developing novel therapies to combat C. neoformans for human health.
Emily Schmitt, Assistant Professor of Kinesiology and Health. The mammalian circadian clock coordinates many biological processes from behaviors to cellular metabolism and mitosis and is coordinated in a 24-hr rhythmic pattern. A central molecular clock known as the suprachiasmatic nucleus (SCN), located in the hypothalamus, acts as the pacemaker to multiple circadian oscillators including those in peripheral tissues where key circadian rhythm genes are expressed. Frequent jet lag or shifts of daily rhythms as a result of rotating shift work disrupt the circadian clock and can lead to many deleterious health outcomes including breast cancer and reproductive disorders in women, as well as cardiovascular disease, stroke, and metabolic syndrome in both men and women. Therefore, identifying ways to mitigate the harmful consequences that arise from circadian disruption that negatively impact human health is imperative. Little is known about the mechanisms that control physiological consequences from shift work. The proposed project is designed to understand how exercise can mitigate circadian disruption. Determining the harmful impacts of circadian disruption and using physical activity to mitigate the harmful effects from circadian misalignment is a novel solution to combat chronic diseases that arise from shift work. The overall approach is to use exercise as a treatment to restore molecular clock disruption, either through a chrono-timed treadmill exercise protocol experiment or lifelong exercise on a running wheel. Our goal is to determine how exercise can re-entrain the central and peripheral clocks in both male and female mice. Specifically, we aim to test how chrono-timed exercise can re-entrain the misaligned molecular clock (Aim 1) and how lifelong exercise can provide protection from circadian disruption (Aim 2).
Thomas Boothby, Assistant Professor Molecular Biology. Developing strategies for the long-term preservation of Drosophila stocks. Laboratory animal models, such as the fruit fly Drosophila melanogaster, are essential to understanding human health and disease and facilitating the development of diagnostic approaches and therapeutic interventions. A large number of valuable genetic fly strains are being generated at an unprecedented pace due to the rapid advances in genome editing tools. However, this increase in genetic stocks creates challenges in maintenance, preservation and sustainability of these resources. Currently, there are over 60,000 Drosophila stocks at the NIH-supported Bloomington Drosophila Stock Center all of which must be maintained as labor intensive live cultures. Therefore, there is an urgent need to achieve simple, reliable and cost-effective methods for long-term preservation and revival of Drosophila. To address this problem, we will develop technology for the long-term storage of Drosophila embryos in a dry state. While water is essential for the viability of most living systems, there are some organisms that can survive being desiccated. One such system is the tardigrade, a tiny animal, which possesses three families of intrinsically disordered proteins (IDPs) that are necessary for them to survive drying, are sufficient to increase the desiccation tolerance in heterologous systems (e.g., yeast and bacteria) by up to 100X, and preserve sensitive purified biomaterials (e.g., enzymes) up to an order of magnitude more efficiently than current FDA approved protectants. We will achieve our goal of developing technology for the dry preservation of Drosophila embryos through the execution of three specific aims. Aim 1: we will carry out a series of quantitative assays coupled with advanced fluorescence microscopy to understand what perturbations arise in cultured, desiccation-sensitive, Drosophila S2 cells as well as one cell-stage embryos. Aim 2: we will develop strategies using our existing knowledge of desiccation tolerance to prevent these perturbations in S2 cells, for example by the introduction of tardigrade IDPs and other protectants. Aim 3: we will apply the lessons learn in Aim 2 to develop technology allowing us to preserve one-cell stage Drosophila embryos in a dry state. Completion of these three aims is in line Wyoming INBRE-4’s thematic goals. Aim 1 will define the cellular and molecular perturbations induced by a pertinent stress (desiccation), while Aims 2 and 3 will develop technology to enhance the value and utility of Drosophila, a key model for the study of chronic human disease. INBRE support of this project will be essential for developing a future grant that is competitive in NIH’s PAR-19-176 funding announcement or similar solicitations.
Danielle Bruns, Assistant Professor Kinesiology and Health. Identification of juvenile factors to treat age-related declines in cardiac adrenergic reserve. Heart failure (HF) is an enormous public health problem which impacts patients across the entire human life-course. Traditionally, HF has been treated with a one-size-fits-all approach, a strategy which has largely been unsuccessful, as evidenced by stagnant 5-year mortality over the last few decades. We believe this is in part due to significant differences biological variables such as age, which influences disease pathogenesis between the young and old patients. We recently tested this hypothesis in pediatric, adult, and geriatric mice treated with the same stimulus- adrenergic activation by isoproterenol (ISO). While pediatric mice robustly responded to ISO to increase cardiac contractility, the aged mice did not. Our preliminary data also demonstrate significantly different pathway activation by age, with pediatric hearts robustly upregulating genes implicated in cardiac and sarcomere contractility, a molecular signature completely absent in the aged heart. Thus, our overall hypothesis is that factors present in the pediatric heart must be responsible for its responsiveness to ISO, and that these factors change with increased age. We propose that identifying these putative protective factor(s) will improve therapeutic options for older patients with HF who are well-characterized by diminished responsiveness to ISO. The purpose of this INBRE Thematic Proposal is to identify these factors by mechanistic dissection of the adrenergic signaling cascade in pediatric and geriatric mice. We will specifically test whether differences in adrenergic receptor activity or downstream sarcomere protein and mechanics are responsible for the different responses to ISO between pediatric and geriatric mice. This Thematic Research Project proposal comes from a junior faculty currently supported by a Career Development Award (K) from NIH. This K award provides Dr. Bruns with the scientific and professional training as well as mentorship team necessary to build a successful independent career in the field of cardiac aging. While this proposal does not scientifically or budgetarily overlap with the K, it allows Dr. Bruns to take advantage of the training and mentorship she’s received during the K, using similar techniques to answer similarly significant biomedical research questions. Further, this proposal integrates WY community college collaboration in Dr. Bud Chew’s group to comprehensively assess the mechanisms by which the pediatric and geriatric hearts respond differently to the same stress. Together, this Thematic Research Project will support the generation of preliminary data to continue Dr. Bruns’ lab efforts to treat HF in the aging population and accelerate her trajectory and promise for future R-level funding in this significant area of biomedical research.
Todd Schoborg, Assistant Professor Molecular Biology. Role of glial cells and the inflammatory response in brain growth control. Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by reduced brain size and life span resulting from mutations in MCPH genes. While the clinical aspects of the disorder are well characterized, the underlying molecular mechanisms remain poorly understood. This has limited our ability to fully understand how genetic & cellular defects contribute to altered tissue function in MCPH patients. This proposal seeks to uncover the mechanisms by which MCPH genes promote proper brain size, leading to a better understanding of brain development, growth and evolution. Specifically, it focuses on the role of abnormal spindle (asp) and WD repeat containing protein (wdr62), the two most commonly mutated genes in human MCPH patients, exclusively in glial cells. The role of glial cells in brain growth control has been largely ignored in the context of MCPH, despite their involvement in other neurodegenerative disorders. Recent work has shown that wdr62 activity in glial cells is important for proper brain size in Drosophila, and my preliminary data on asp mutant brains suggests activation of an inflammatory response, likely mediated by glia. This proposal is designed to dissect the relationship between asp & wdr62, glial cells, the immune response, and other cellular pathways in the etiology of MCPH. To do so, we will utilize Drosophila genetic tools, reporter assays and high resolution imaging to test a series of hypotheses for how glial cells influence brain growth. Completion of this project will not only identify and characterize a novel cellular pathway for MCPH, but also lay the foundation for a future R01 grant aimed at dissecting the molecular mechanism of glial cell function in brain growth control.
Pejman Tamasebi, Assistant Professor Petroleum and Civil Engineereing. Cell-Based Blood Flow Simulation using a Coupled Blood-Cell Modeling. Several challenges avoid modeling the behavior of living tissue, such as flowing blood, from a biomechanical viewpoint. This problem has been investigated as a fluid that contains solid particles; e.g. cell transport in blood flow, and platelet deposition on blood vessel walls. Due to complex interactions between the particles and blood, their dynamic behavior is a very complicated problem. The existing cells and elements in blood adapt their functions and configurations to their environment and they continuously change the morphology and behavior. This characteristic must be taken into account in the modeling process. More typical examples are blood coking, drug carrier design, cell separation, blood clotting, flow-induced blood clotting and thrombus formation in cardiovascular pathologies, and in cardiovascular devices. Furthermore, the presence of highly deformable particles makes such problems very challenging. The available methods treat all the constituents in the blood flow field by a unified method and adopting a particle-based method such that the plasma flow, as well as the motion of the cellular components, is modeled via particle-based representations. For instance, the solid components are modeled as connected particles whose connection is fixed and can be deformed unrealistically. Although various numerical methods have been proposed, there is a growing interest in the particle-based methods and a crucial need for a method that can take the real morphology of cells and particles into account. In this research, we plan to study such an important phenomenon by considering the particles as deformable cells that are similar in morphology in the vessel, thus requiring explicit consideration of the particle mechanics. We aim to conduct such simulations for the first time and provide very realistic results of the interactions between deformable particles and blood. It should be noted that other than the morphology, we also plan to consider the softness degree (i.e. plasticity) for the utilized particles such that they can experience a plastic and elastic deformation. The results of this research will provide an accurate calculation of the lubrication forces between particles as well as long-range hydrodynamic interactions among many interacting cells. Furthermore, this method will be a type of molecular dynamics modeling and can be used to phenomena in the nanoscales where biochemistry plays a major role.
Evan Johnson, Assistant Professor Exercise Physiology, UW. Genetic and Hematological Risk for Acute Kidney Injury during High Intensity Exercise. The proposed work is designed to be the first in a series of studies investigating the health benefits and risks related to high intensity training (HIT) exercise. Our specific aims are to determine, 1) if participation in a single bout of HIT induces hematological markers consistent with acute kidney injury (AKI), and 2) if risk is predicted by the number of risk alleles present in the participant and/or by the pre-exercise concentration of plasma proenkephalin-A. This investigation is an observational case control study. In year one, data collection procedures will be refined with ~50 participants local to UW and training will occur for collaborators from Wyoming community and tribal colleges. In year two, data collection will expand to some of the 12 CrossFit® gyms in Wyoming with assistance from the community and tribal colleges. Blood and urine samples will be collected before and up to 48 h after a standardized bout of HIT exercise on ~150 participants. Baseline blood samples will be, genetically typed in order to quantify the number of single nucleotide polymorphisms (SNPs) related to kidney function, and analyzed for proenkephalin-A. All blood samples will be analyzed for markers of muscle damage (e.g., creatine kinase and myoglobin), and markers of kidney function (e.g., serum creatinine and blood urea nitrogen). Urine will be analyzed for markers of filtration function (e.g., albumin, creatinine, neutrophil gelatinase-associated lipocalin [NGAL], and kidney injury molecule 1 [KIM-1]). Lastly, the severity of kidney damage will be compared with the number of risk alleles and proenkaphalin-A concentration. We envision that the bout of HIT exercise will induce markers consistent with skeletal muscle damage in most participants and, based on literature from other styles of intense exercise, that acute kidney injury will be diagnosable in between 50-75% of participants. Secondarily, we predict that both, the number of risk alleles, and the concentration of proenkaphalin-A will be inversely related to the change in kidney function from before to after the HIT exercise bout.
Jill Keith, Assistant Professor of Family and Consumer Sciences, UW. Reclaiming indigenous food and health: a pilot RCT on health impacts of sovereign nation diets. Native Americans suffer disproportionately from chronic health conditions influenced by dietary patterns such as type 2 diabetes and obesity. A transition from healthier traditional foods to a "westernized" diet (which includes more processed, energy-dense convenience foods) has contributed to a pattern of higher fat and saturated fat intake and lower fruit and vegetable intake among Native Americans. These contemporary eating patterns are a contributing factor to health disparities among Native Americans. Previous research regarding value, use, accessibility, and impact of indigenous plants among tribal communities is limited. Research that has been conducted has shown the superior value of indigenous foods compared to commonly consumed contemporary reference foods. Research focused on indigenous plants and their role in nutrition and health is especially limited within Wyoming and the Mountain West region. The overall purpose of this proposed action-research project is to measure the feasibility of consuming and the impact of indigenous plants and foods on health outcomes among Native American participants on the Wind River Indian Reservation (WRIR) in Wyoming. Participants from the WRIR will be recruited to pilot a randomized control trial (RCT) with a delayed intervention to measure the impacts of consuming a diet that is 50% indigenous foods (based on participant total energy needs and recommended dietary pattern). Specific objectives of the pilot RCT will be to: (1) evaluate the feasibility of access, collection, and consumption of indigenous plants/animals, (2) define precisely what constitutes a "50% indigenous food diet" that will be supplied as the intervention in this trial, (3) quantify the time commitment and logistical challenges to access and collect indigenous foods, (4) evaluate the consumption of indigenous foods on health outcomes including waist circumference, body mass index, blood pressure, blood glucose control, and blood lipid levels, (5) evaluate the impact of consuming indigenous foods on cultural identity, (6) build potential for culturally relevant education focused on indigenous foods and cultural identity in the community through a traditional foods class offered at the Central Wyoming College, and (7) use information gathered from objectives 1-6 to design a feasible, significant, and innovative proposal for external funding.
Breanna Krueger, Assistant Professor Communication Disorders. Age-related correlates of treatment efficacy and efficiency for late-acquired sounds. Children with speech sound disorders often struggle to resolve their late-acquired errors because speechlanguage pathologists wait until the expected age of acquisition for these speech sounds. This practice puts children at risk for persistent speech sound errors that may never be resolved. My previous work suggests that there are age-related differences in terms of treating speech sound disorders. Younger children (age 4- 5) progressed through treatment more accurately earlier than older children (age 7-8). Therefore, the proposed study intends to examine whether these age-related differences can be mitigated by targeting treatment for each age groups’ age-related advantages. A single-subjects 2x2 treatment design will explore the following aims: Aim 1 Measure treatment efficacy of motor-based and phonology-based approaches in younger and later age groups for late-acquired sounds. Aim 2 Measure treatment efficiency of motor-based and phonology-based approaches in younger and later age groups for late-acquired sounds. In achieving these aims, the proposed study will improve current clinical practice and improve treatment for children with speech sound disorders.
Katie (Dongmei) Li-Oakey. Assistant Professor Chemical Engineering. Tunable Biodegradable Multimodal Hydrogel Nanoparticles for Targeted Therapeutics. Many classes of nanoparticles have been designed with cancer-specific therapeutic applications in mind. The function of these particles can include, for instance, localized delivery of an imaging dye or contrast agent, targeted delivery of a chemotherapeutic molecule, or a metallic nanoparticle for hyperthermic treatment. Synthetic polymer-based hydrogels such as polyethylene glycol (PEG) are ideal candidates for therapeutic drug delivery because they provide biopassivitiy, programmed drug release, and ease of functionalization with antibodies, nucleic acids, and peptide sequences. Despite the broad diversity of techniques by which polymer hydrogels may be customized, there exist no reliable methods to prepare PEG particles on nanometer length scales with narrow size distributions and well-defined loading. Aqueous hydrogels also struggle to retain and deliver hydrophyilic small chemotherapeutic molecules. Recently, we have successfully developed a platform to address the above challenges by fabricating structured, nano-scale, biodegradable polyethylene glycol (PEG) particles with narrow size distribution. Here we propose to apply this platform to produce nanoparticle carriers for applications in cancer therapeutics. The proposed microfluidic processing approach is superior to existing techniques due to its exquisite control over particle size and uniformity and facile tunability of the macromolecular network. The Specific Aims of this two year INBRE Thematic Project are: Specific Aim #1: To validate the function and efficacy of micron and sub-micron PEG-based MMNPs against tumor cells in culture, and in tumor-bearing rodent models. These particles will be functionalized with tumor-localizing antibodies and will contain antibody-based therapeutics and magnetic nanoparticles for hyperthermia therapy. Specific Aim #2: To provide nanoporous hydrophilic PEG-hydrogels with the ability to retain and release, over tunable and long time periods, hydrophobic small molecule therapeutics. This aim will combine conventional PLGA carriers with secondary and tertiary encapsulation within hydrogel nanoparticles to extend, stage, and tailor release profiles of individual therapeutics.
Alison Looby, Assistant Professor Psychology. Examining Expectancy Challenges to Prevent Nonmedical Prescription Stimulant Use. Nonmedical prescription stimulant use (NPS) is commonly reported among college students for cognitive enhancement purposes, though it is associated with numerous negative psychological and physical consequences. Despite increasingly high prevalence rates and widespread acknowledgement of the need for efficacious interventions, little is known regarding how to prevent or treat this behavior. An intervention that targets cognitive enhancement motives and expectancy effects related to NPS may be particularly effective in light of recent research purporting limited evidence for meaningful NPS-related cognitive improvements among individuals without legitimate attention deficits. The primary objective of this proposal is to examine the efficacy of an intervention that successfully prevents NPS among college students by modifying expectations for NPS-related effects, while at the same time providing alternative means of enhancing cognition and arousal. Participants will be 126 stimulant-naïve college students who report a combination of risk factors for NPS. They will be randomized to one of three treatment conditions: a placebo- based expectancy challenge intervention that solely aims to modify expectancies related to NPS, a caffeine- based expectancy challenge intervention that includes expectancy modification combined with a safer alternative for cognitive enhancement, or a control group. Multilevel mixed modeling and survival analyses will be used to 1) examine changes in NPS-related expectancy effects across a 6-month follow-up period, and 2) assess incidence of NPS over the follow-up period, respectively, across the three groups. It is hypothesized that both expectancy challenge interventions will successfully modify expectancies compared to the control group and that they will be maintained over the follow-up period. It is also expected that the caffeine-based intervention will most successfully prevent NPS through a combination of expectancy modification and encouraging safe use of caffeine rather than prescription stimulants to achieve desired outcomes. Mediational analyses will also be employed to assess whether changes in expectancy effects via the interventions are responsible for differences in initiation rates between groups. The results of this project will facilitate the development of larger-scale prevention efforts to target the high rate of NPS on college campuses.
Maysam Mousaviraad, Assistant Professor Mechanical Engineering. Computational FSI Modeling for Heart Failure Treatment with Titin Manipulation. Heart failure with preserved ejection but abnormal diastolic function is a major public health issue that is currently not very well understood and has no effective therapeutic options available. It is hypothesized that altered myocardial passive stiffness is the main cause for the diastolic disfunction, and that manipulation of RBM20 levels may improve the diastolic function by restoring the wall stiffness. However, the mechanistic behavior of heart failure due to increased compliance, and the amount of stiffness restoration needed to treat the condition are not yet clear. The previous in vivo experiments have not isolated the effects of wall stiffness, and are unable/expensive to visualize/quantify the modifications in the interactive fluid-structure patterns due to stiffness alterations. Aim 1 of the proposed research will attempt to design an in vitro experiment to study the deformation of the arterial walls of the healthy and unhealthy animals in response to pulsating flows. These results will also provide the structural properties to be used in the computational models (Chew lab, year 1). Aim 2 will develop a high-performance physics-based computational fluidstructure interaction (FSI) model that can simulate the arterial and the left ventricle fluid and structure dynamics and interactions (Mousaviraad lab, years 1 and 2). Aim 3 will perform verification and validation (V&V) studies of the computational methods against the in vivo pressure-volume loop measurements as well as the in vitro arterial structural dynamics measurements (Chew and Mousaviraad labs, year 2). Aim 4 will perform systematic studies to understand and characterize/quantify the modifications in the fluid-structure interactions, and therefore the heart functionality, due to stiffness alterations. The high-resolution computational results will also be used to study/explain the physical phenomena behind the complex interactions between the fluid and the solid with varying wall stiffness (Mousaviraad lab, year 2).
Rebecca Carron, Assistant Professor School of Nursing. POLYCYSTIC OVARIAN SYNDROME IN AMERICAN INDIAN WOMEN: AN EXPLORATORY STUDY. The NIH mission is, in part, to discover new knowledge about the behavior of living systems and to use that knowledge to improve health. Women with polycystic ovary syndrome (PCOS) are at risk for many significant health care problems including cardiometabolic syndrome (CMS), type 2 diabetes, obesity, infertility, psychosocial stress, suicide, and decreased health-realted quality of life (HRQL). No specific knowledge about the effects of PCOS in American Indian women exists. The long-term goal of the project is to improve the HRQL of Al women with PCOS. The project goal will be accomplished with the specific aims designed to fill PCOS knowledge gaps:
Aim #1 : Determine Al ethnic specific PCOS symptoms, problems, and psychosocial stress
in a sample of Al women with confirmed PCOS, and increase health care provider PCOS
Aim #2: Estimate PCOS population prevalence and cardiometabolic profile, including risk for diabetes, in a sample of Al women with PCOS.
Aim #3: Determine cultural and societal practices of Al women with PCOS for beginning development of patient-centered, culturally competent PCOS self-care interventions.
Aim #4. Develop a conceptual model of the Al experience of PCOS and initial development of an instrument to measure the Al experience of PCOS based on results from Aims 1-3.
The research design uses a mixed methods approach with both qualitative (interviews, group meetings, conceptual model) and quantitative (survey results, cardimetabolic profile, instrument development) methods to answer the specific aims.The evidence-based, translatable knowledge gained will significantly increase the ability of health care providers in a variety of settings to measure Al ethnic specific symptoms/problems and provide beginning evidence of effects of patient-centered, culturally competent interventions to reduce the risk for cardiometabolic diseases, psychosocial stress, and improve HRQL in Al women with PCOS.
Brian Cherrington, Assistant Professor Zoology and Physiology. The Effect of Obesity Induced Hyperinsulinemia on Lactation. Obesity negatively affects lactation, yet a mechanistic understanding of how this occurs is lacking. This gap in knowledge is an important medical problem because breastfeeding protects both the mother and infant against obesity, diabetes, and metabolic disorders later in life. At parturition, a dramatic rise in prolactin produced by anterior pituitary gland lactotrope cells is indispensable for initiating breast milk production by mammary epithelial cells – a process termed secretory activation. In obese mothers, however, secretory activation is disrupted potentially due to the negative effects of chronic hyperinsulinemia on prolactin production by lactotrope cells. Our long term goal is to understand the defects in the lactation mechanism in obese mothers which results in lactation problems. The overall objective of this proposal, which advances our long-term goal, is to determine if delayed secretory activation is due to obesity induced hyperinsulinemia and test if treatment with an insulin sensitizing agent can restore normal lactation. Our central hypothesis is that obesity induced hyperinsulinemia alters prolactin production by lactotrope cells delaying secretory activation. We will test our central hypothesis by the following aims: (1) determine the molecular mechanisms through which hyperinsulinemia alters prolactin production in lactotrope GH3 cells; (2) establish if changes in lactotrope proliferation or insulin signaling activity alter prolactin production delaying secretory activation in obese mice. In aim 1, we will use GH3 cells to investigate the effects of hyperinsulinemia on activity of insulin signaling and prolactin mRNA and protein levels. In aim 2, we will use a mouse obesity model to investigate the effects of hyperinsulinemia on lactotrope cell number, insulin signaling activity, and prolactin production in vivo. Aim 2 will also test if treating obese mice with metformin restores normal prolactin production and secretory activation. The proposed research is innovative because it investigates a side effect of chronic hyperinsulinemia on lactation. The work is significant because it is an initial step in a line of research to understand the mechanisms causing lactation problems in obese mothers and test novel treatment options.
Wei Guo, Assistant Professor Animal Sciences. ROLE OF RBM20 IN THE REGULATION OF CARDIAC GENE SPLICING IN HEART FAILURE. Heart failure (HF) is a serious, chronic condition that gradually deteriorates over time. The onset and development of HF are unpredictable and present individual variation possibly due to unclear etiology for HF progression. With development of HF, cardiac remodeling will occur, leading to impaired cardiac structure and function. Sarcomeric proteins are essential determinants of sarcomeric structure and function. The abnormal expression of sarcomeric proteins will result in impaired sarcomeric structure and function, enroute to impaired cardiac structure and function. Therefore, aberrant expression of sarcomeric proteins is one of the major causes for HF progression. Sarcomeric proteins such as myosin, actin and titin take up -60 to 80% of myocyte mass, so abnormal alterations of these protein will significantly affect cardiac structure and function. All of these proteins undergo isoform switch with development and under disease condition. The abnormal isoform switch of these proteins has been identified in failing heart. However, the mechanisms of isoform switch of these proteins remain elusive. Previous studies have shown that metabolic and hemodynamic switch under stress environment can cause isoform switch of these proteins, but it isunknown how these metabolic changes link to protein isoform switch. Recently, our group identified a muscle-specific splicing factor-RNA binding motif 20 (RBM20) that is a master regulator of cardiac protein isoform switches. RBM20 has been found to regulate over 30 gene splicing in cardiac muscle to date. Among these genes, titin and myosin are the major targets of RBM20, but the detailed mechanisms of how RBM20 regulates isoform switch of these proteins remain elusive. Hence, this project is proposed to use titin, the major target of RBM20 as a molecular model for investigation of the mechanisms of RBM20- mediated protein isoform switch. The increased understanding of the regulatory mechanisms regarding RBM20-mediated protein isoform switch may partially address metabolic change-induced gene expression in the heart and the progression of HF at the molecular levels.
Guanglong He, Associate Professor School of Pharmacy. CARD9 Signaling and Childhood Obesity-Associated Cardiac Dysfunction. While obesity has emerged as an epidemic, strategies to curb this disease are limited. Childhood obesity and its persistance into adulthood is associated with insulin resistance and glucose intolerance with dire consequences on cardiovascular system. Therefore, research strategies that focus on the underlying mechanisms are much needed. Obesity is accompanied with a low-grade chronic inflammation with infiltration of macrophages in target organs such as heart and vessels. As activated macrophages release pro-inflammatory cytokines with detrimental effects on the target cells/organs, our approach is to dissect the obesity-induced inflammatory response pathways and their potential impact on cardiovascular diseases. Recently, it has been reported that the pro-inflammatory protein CARD9, which is exclusively expressed in macrophages, associates with BCL10 and MALT1 as a signalosome, robustly activates NFkappaB signaling, and eventually eradicates the invaded pathogens. However, whether or not CARD9 plays a role in obesityinduced chronic inflammation is not known. Our preliminary data indicated that CARD9 knockout reconciled obesity-induced insulin resistance and glucose intolerance. We also observed that CARD9 knockout ameliorated obesity-induced cardiac dysfunction. Therefore, we hypothesize that obesity activates CARD9 signaling in macrophages and this pro-inflammatory signaling is responsible for obesity-induced insulin resistance and myocardial dysfunction. To test this hypothesis, we will utilize an obese mouse model and the CARD9 knockout mouse strain to determine whether or not: 1) obesity induces systemic insulin resistance via activation of CARD9-BCL10-MALT1 signalosome and related TNFalpha/IL6/IL1beta signaling in macrophages; 2) obesity suppresses myocardial mitogenesis and cardiac function via activation of CARD9- BCL10-MALT1 sinalosome and related NFkappaB/TNFalpha signaling in macrophages in a paracrine manner. We believe that successful completion of the proposed studies will provide potential novel targets for the treatment of obesity-associated metabolic syndrome and cardiovascular abnormalities.
Anya Lyuksyutova, Assistant Researcher, Molecular Biology. OPTOGENETIC CONTROL OF GCS VIA MICRORNAS AS TREATMENT FOR LIVER STEATOSIS. Sphingolipid accumulation contributes to cardiometabolic syndrome. Inhibition of the first enzyme in sphingolipid biogenesis, glucosylceramide synthase (GCS), emerged as a powerful new approach for treating cardiometabolic syndrome and type II diabetes. Current GCS inhibitors have a long list of side effects. Inhibitory small RNAs, siRNAs and microRNA, represent an alternative approach for GCS inhibition. Recently, we showed that overexpression of microRNA-190 and microRNA-200 significantly reduces GCS mRNA levels in cell culture. However, inhibiting GCS in the whole animal is deleterious. To inhibit GCS specifically in the liver and to control the extent of GCS expression over time, we propose to use an optogenetic expression system developed in the Gomelsky lab. This system relies on light in the near infrared window (NIRW), which penetrates to the depth of several centimeters in mammalian tissues. It has been validated in mammalian cell culture. First, we will test and optimize the NIRW light expression system in mice, using hydrodynamic transfection of microRNA-190 and 200 to achieve their strong expression in the liver. Next, we will characterize effects of microRNA-190 and 200 on reducing GCS and reversing cardiometabolic symptoms in vivo. The effect of the microRNAs will be quantified by measuring changes in insulin resistance, liver lipid levels and blood free triglyceride levels over time. Precise temporal control, made possible by the NIRW light expression of the microRNAs, will allow us to fine-tune microRNA-190 and 200 expression for optimal GCS reduction. If successful, this project will provide a novel and innovative approach for cardiometabolic symptom reversal. In a broader sense, it will open up opportunities for temporal and spatial control of therapeutically important genes (i.e. cell reprogramming) in live animals, and subsequently in humans. This multidisciplinary project relies on the synergy in expertise of Dr. Lyuksyutova in eukaryotic gene regulation and mouse genetics and Dr. Gomelsky’s expertise in NIRW optogenetics. The collaboration between Drs. Gomelsky and Lyuksyutova has been ongoing for the last several years and it resulted in joint funding, high-profile publications, and jointly supervised Ph.D. students.
Amy Navratil, Assistant Professor Zoology and Physiology. MOLECULAR MECHANISMS OF LUTEINIZING HORMONE DYSREGULATION IN PCOS. Polycystic ovary syndrome (PCOS) is complex reproductive disorder with unclear pathophysiology. One of the hallmarks of PCOS includes elevated plasma luteinizing hormone (LH) levels that in combination with metabolic dysfunction and excess androgen lead to reproductive abnormalities. Despite recent advances, the precise mechanisms that are involved in PCOS LH hyper-secretion directly at the level of the gonadotrope are largely unknown. We propose to utilize a prenatal androgenized mouse model to test the central hypothesis that metabolic and steroid hormone dysregulation in PCOS alters gonadotrope function at both the population and cellular level to disproportionately increase LH levels. We propose three comprehensive aims to examine the hypotheses 1. the gonadotrope network increases its size and cell-tocell contacts in PCOS to more effective synchronize increased pulstile LH 2. gonadotropes will have hightened calcium activity in repsonse to GnRH to not only enhance LH secretory events but also increase ERK activation necessary for LH synthesis.3. histone citrullination of the LHβ gene leads to increased transcription and expression, contributing to the pathogenesis of PCOS. Taken together, this proposal seeks to advance our understanding of the relationship between PCOS, the gonadotrope, and LH secretion. We are hopeful that the experiments outlined will help in understanding the underlying mechanisms of increased LH secretion in the pituitary and provide critical insight into the pathophysiology of PCOS and impaired reproductive function.
John Oakey, Assistant Professor Chemical Engineering. CIRCULATING TUMOR CELL CAPTURE AND RELEASE FROM DEGRADABLE HYDROGEL SURFACES. This proposal proposes the development of a microfluidic rare cell capture and release device that can be applied to the isolation of viable circulating tumor cells (CTCs) from clinical whole blood samples. This work is organized into two interacting components: 1) cell capture and release device platform development, 2) viable CTC isolation and recovery and genomic analysis. Central to the proposed work is the development of an immunoaffinity cell capture and release device based upon moldable photodegradable hydrogels. Antibodies lend tremendous recognition specificity and have been successfully used in surface-capture microchips to immobilize and isolate rare (1 in 109) circulating tumor cells (CTCs) from whole blood. Many devices with different geometries and surface capture motifs have already been tested to optimize for either rare population enrichment or for higher volume cell capture. Regardless of the capture target, it is of tremendous interest to subsequently release captured cells from devices in as viable a state as possible. We have found that trypsin or other enzymatic digestion approaches provide poor viable cell yields and, therefore, we have developed hydrogel-based sacrificial capture surfaces. These surfaces are fabricated by mold-polymerizing a photo-degradable hydrogel capture surface with the footprint of a microfluidic capture chamber. Capture antibodies are bound to the hydrogel by either post-polymerization conjugation or by copolymerization. Following capture and device flushing, cells can be released from the device by a short exposure to low-intensity UV light, which degrades the gel’s photocleavable crosslinks. Once fluidized, the gel’s degradation products and cells may be gently eluted from the device and captured. As capture pads are molded in place, multiple capture zones, each with a unique antibody, may be created within a single device.
Christine Porter, Assistant Professor Kinesiology and Health. GROWING RESILIENCE PHASE II: ALBANY COUNTY REDESIGN AND WIND RIVER EXPANSIONS. The four facets of this proposed project expand on the one-year pilot Growing Resilience program (“Phase I”) that Porter led in 2013 with partners in Albany County and in Wind River Indian Reservation (WRIR). In Phase I, each team co-designed a randomized controlled trial (RCT) of the impacts of home gardens on multiple health outcomes and we trialed the design with a few families in each place. This Phase II proposed project will, in decreasing order of duration and cost:
1. Redesign and conduct a larger pilot trial of the RCT with Albany County partners,
including establishing recruitment partnerships with new organizations and determining
what we should select as the primary health outcome for the study design (2015-2018).
2. Expand the Native American young adult internship/mentorship program with Dr. Tarissa Spoonhunter at Central Wyoming College (CWC) for student involvement in this study (2015-2018).
3. Support the collection of health outcome data with WRIR families who participated in Phase I, and other work to sustain WRIR Growing Resilience partnerships while an R01 proposal to NIH is under review for a 2016 start (2015 only).
4. Seed-fund the nursing leadership in Native American health care action research that emerged as a priority during Phase I (2015 only).
Finally, each of the four arenas above will serve as a foundation for four different externally-funded projects.
Baskaran Thyagarajan, Assistant Professor Pharmacy. TRPV1 ACTIVATION PREVENTS FROM HIGH FAT DIET-INDUCED NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD) IN OBESITY VIA SIRT-1. High fat diet-induced obesity is associated with abnormal fat accumulation in the liver, This affects the metabolic functions of the liver leading to non-alcoholic fatty liver disease (NAFLD). The staggering statistics of increase in NAFLD in the USA necessitates the development of strategies to prevent and treat NAFLD. In our efforts to analyze the role of transient receptor potential vaniloid 1 (TRPV1) channel protein in the regulation of metabolic syndrome, we discovered that capsaicin (CAP; a TRPV1 agonist) significantly prevented high fat diet (HFD)-induced obesity and NAFLD by increasing the expression and activity of sirtuin-1 (SiRT-1) protein. SiRT-1 activates and deacetylates metabolically important cellular proteins (PPARα, PGC-1α) to trigger lipolysis and to regulate the self-digestion of lipid cells (lipoautophagy). Forkhead box O1 (FOXO1) is a nuclear transcription factor and is a substrate for deacetylation by SiRT-1. FOXO1 regulates lipophagy and is a potential target for treating NAFLD. We hypothesize that “HFD inhibits SiRT-1 and causes NAFLD by 1) Down-regulating TRPV1 and suppressing TRPV1/CaMKII/AMPKdependent SiRT-1 activation; 2) Disrupting SiRT-1/PPARα interaction to decrease lipolysis; and 3). Inhibiting FOXO1 deacetylation by SiRT-1 to impair lipophagy. CAP stimulates TRPV1 and increases CaMKIIα/AMPKα-dependent SiRT-1 phosphorylation to facilitate 1). SiRT-1/PPARα interaction to promote lipolysis; 2). PGC-1α deacetylation by SiRT-1 leads to activation of UCP-1 and promotes lipophagy; and 3). Deacetylation of FOXO1 by SiRT-1 promotes lipophagy to prevent NAFLD”. We propose three specific aims Specific Aim 1: Determine the effect of dietary CAP on SiRT-1/PPARα interaction, which stimulates lipolysis. Specific Aim 2: Evaluate the effect TRPV1 activation on deacetylation of PGC-1α by SiRT-1 to activate UCP- 1. Specific Aim 3: Analyze the effect of SiRT-1 activation on FOXO1 deacetylation and induction of lipophagy to prevent NAFLD. Our research will provide a new insight in the use of CAP to combat obesity and NAFLD.