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Thematic Research Projects

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. Ten research projects led by junior investigators recruited to UW have been chosen. Career development plans and discrete mentorships have been developed for all project investigators.

CURRENT THEMATIC RESEARCH PROJECTS:

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 awareness.
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. Assitant Professor School of 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.

PAST THEMATIC INVESTIGATOR PROJECTS:
Krisztina Varga, Assistant Professor Chemistry. STRUCTURE AND FUNCTION OF TSPO, AN IMPORTANT DRUG TARGET.
John Oakey Ph.D., Chemical Engineering, High Throughput Screening of β Cell Encapsulation.
Machender Kandadi Ph.D., Role of Reactive oxygen species (ROS) in anthrax lethal toxin associated cardiac dysfunction.
Enette Larson-Meyer Ph.D., Family and Consumer Sciences, Role of Ghrelin and PYY in postpartum body weight regulation and presence in human milk.
Sreejayan Nair Ph.D., Cathepsin-K Mitigates Cardiac Dysfunction.
Baskaran Thyagarajan Ph.D., Analysis of Role of TRPV1 channels in the regulation of Metabolic Diseases.
Meijun Zhu Ph.D., Animal Sciences, Maternal Obesity, Stem Cell Factor/c-Kit Signaling and Mast Cells in the Development of Intestine and Incidence of Inflammatory Bowel Diseases in Progeny. 

Krisztina Varga, Assistant Professor Chemistry. STRUCTURE AND FUNCTION OF TSPO, AN IMPORTANT DRUG TARGET. The aim of this project is the structural and functional study of TSPO, an important drug and imaging target in cardiovascular disease and diabetic neuropathy and other diseases. We will elucidate the binding sites, determine its enzymatic activity, design high-affinity selective ligands, and test the effect of ligands on rat cardiac myocytes. TSPO is an 18-kDa transmembrane protein predominantly present in the outer membrane of mitochondria. TSPO has a role in regulating mitochondrial functions with implications in steroidogenesis and apoptosis. Recent studies have shown that TSPO inhibition by various ligands effectively prevented reperfusion injury after ischemia in rats. Activation of TSPO has been shown to exert neuroprotective action against diabetes induced peripheral nerve pathologies. However, we currently possess limited understanding about the interaction of TSPO with ligands. Recent evidence suggests that there are multiple ligand binding sites, and the binding pocket(s) are poorly characterized. As TSPO is widely distributed and has multiple roles, it is very important to identify the structural functional significance of TSPO ligands before their efficient application to treat specific diseases. It has been recently proposed that at least two of the bacterial TSPOs have enzymatic activity. As TSPO is widely distributed and has multiple roles, it is very important to identify the structural functional significance of ligands before their efficient application to treat specific diseases. We propose a structural functional and ligand binding study of TSPO. We have expressed and purified a well-characterized structural and functional homolog of TSPO from R. sphaeroides, and ligand binding, enzymatic studies, and NMR spectroscopy structural studies are currently underway. We are the first to show that RsTSPO has enzymatic activity (manuscript in preparation). The bacterial protein will serve as a structural and functional model to refine conditions for structure determination of the human TSPO. In order the explore the function of TSPO and test ligand binding in vivo, we will test the effect of ligands on rat myocytes. Results of this study will serve as a basis for an R01 proposal to NIH. The results of this study are expected to be transformative for drug design to combat reperfusion injury and diabetic neuropathy.

John Oakey Ph.D., Chemical Engineering, High Throughput Screening of β-Cell Response to Encapsulation
Pancreatic islet of Langerhans transplantation is an experimental, yet highly promising therapy for insulin-dependent diabetes mellitus (type I diabetes). However, a persistent obstacle to successful transplantation is the immunorejection of donor islets. One solution, islet encapsulation, protects islets from immunorejection by providing a physical barrier through which secreted insulin can diffuse while preventing contact with host cells and antibodies. This strategy promises to provide patients with functional, insulin-producing islets without need for immunosuppresants. Many biomaterials have been explored as candidate encapsulants, including a variety of natural and synthetic polymers. Photopolymerized poly(ethylene glycol) (PEG) hydrogels are particularly attractive cell encapsulants as a result of their excellent mechanical properties, diffusive properties, cytocompatibility and biopassivitiy within the body. The conditions that maximize β-cell efficacy and function, however, are a complex amalgam of PEG properties and the function of secondary extracellular matrix proteins that are formulated into the hydrogel. Given the number of components, formulation variables and degrees of freedom, a very small fraction of the overall composition space has been surveyed for the influence of cell-material interactions upon cell viability and function.

Our goal, therefore, is to create a platform that accelerates the process of design and discovery by screening candidate materials, conditions and cellular responses far more quickly and efficiently. This platform will utilize microfluidic cell encapsulation to rapidly generate micro-hydrogel particles containing individual cells and a broad range of material compositions. Hydrogel particles and indicators of cellular response will be evaluated by custom-built microfluidic flow cytometry. Success of this project will produce diabetes-specific outcomes in the form of high-throughput screening tools for guiding cellular therapies. More broadly, we will have created and demonstrated a platform by which we can screen any cell-matrix interaction, which represents a profound capability for the fields of tissue engineering regenerative medicine.

The initial phase of this project have been focused upon enabling technology and process development. We have successfully built and validated systems for producing hydrogel particles and screening particles. As a result, we have developed a hardware platform that is capable of producing large quantities of monodisperse polyethylene glycol (PEG)-based particles and a homebuilt flow cytometer that integrates with microfluidic flow devices. We have successfully made batches of particles with a broad range of PEG weight fraction and relative molecular weight that are barcoded to allow for the accurate optical discrimination of the particles’ polymer composition. We are currently producing batches of PEG particles that contain fluorescently-labeled proteins in order to study diffusion through hydrogel matrices in order to better understand the relationship of macromolecular composition to network structure and thereby molecular diffusivity. This study is important for three reasons: 1) It provides us with a simple, dynamic system with which we can validate our hardware, 2) it leverages the utility of high-throughput screening and multi-temporal screening to address an issue that has been data-limited and, 3) it leads us to the next phase of our project in which hydrogel particles and indicators of cellular viability and response will be evaluated in our flow cytometers. We are actively seeking funding for fundamental instrumentation development and disease-specific studies.

Machender Kandadi, Ph.D. School of Pharmacy, Role of Reactive oxygen species (ROS) in anthrax lethal toxin associated cardiac dysfunction
Bacillus anthracis infection is a highly lethal disease and a major bioterrorism health threat today. In US, the 2001 outbreak of B. anthracis resulted 22 cases of anthrax, including 5 deaths. Anthrax infections are frequently associated with severe and often irreversible cardiovascular complications, suggesting that the toxins generated from Bacillus anthracis, namely lethal toxin and edema toxin may possess pernicious cardiovascular effects. Lethal toxin depresses left ventricular ejection fraction and edema toxin produces pronounced tachycardia. In addition, lethal toxin has also been shown to trigger diastolic dysfunction, and suppress contractility in the hearts in in-vivo models. However, the underlying mechanisms were not explored. We hypothesis that anthrax lethal toxin can directly affect cardiac contractility and we plan to test this with the following: Aim #1 will examine whether anthrax lethal toxin directly affects cardiomyocyte contractile function and also does anthrax lethal toxin induces excessive ROS production in myocardium; Aim #2 will examine whether ROS inhibition confer protection against anthrax lethal toxin induces cardiomyocyte contractile anomalies; Aim #3 will evaluate whether TLR4 receptor knockout ameliorate anthrax lethal toxin induced cardiac contractile dysfunction. Cardiac contractile properties of individual myocytes from cardiac catalase overexpressing mice and Toll like Receptor 4 (TLR4) knockout mice will be assessed following lethal toxin exposure. Various signaling downstream signaling events that are involved in anthrax toxicity would be studied using standard molecular biology techniques. Given that autophagy plays an extremely important role in maintenance of normal heart function and morphology in stress conditions. We would elucidate whether autophagy has any role in anthrax lethal toxin induced cardiac contractile dysfunction. Autophagy would be studied for all the aims described above. The outcome of this study will help in understanding the pathology and molecular signaling involved anthrax toxicity which in turn may help in designing treatment strategies in clinics.

Enette Larson-Meyer, Ph.D., R.D. Family and Consumer Sciences and Brenda Alexander, Ph.D. and Ann Marie Hart, APRN, BC (FNP), Role of Ghrelin and PYY in postpartum body weight regulation and presence in human milk.
Introduction & Background: Epidemiological studies suggest that childbearing is an important contributor to the development of obesity in many women, and that breastfeeding protects against overweight and obesity in the mother and infant. It is possible that concentrations of the orexogenic peptide ghrelin, and the satiety hormones polypeptide YY (PYY), glucagon-like peptide (GLP-1) and leptin are altered following child birth and during lactation, and are associated with body weight/body fat retention during the postpartum period. It is also plausible these hormones are present in human milk where they may regulate appetite and/or energy homeostasis in the breastfed infant. Research Problem & Purpose: The purpose if this project is to determine whether ghrelin, PYY, GLP-1 and leptin are altered during fasting and following meal ingestion in postpartum women relative to never-pregnant controls, and whether these same hormones are present in human fore-and hind milk. Specific Aims and Rational: The specific aims of this project three fold:

Aim 1. Determine how pregnancy and lactation affect the fasting and meal-induced concentrations of the appetite-related hormones.
Aim 2. Determine the role of the appetite-related hormones in predicting body weight-retention/loss in the year following childbirth.
Aim 3. Identify specific appetite-related hormones in breast milk and determine whether concentrations of these hormones differ in fore- compared to hind milk throughout lactation.

The rational for this project is that when we understand how these hormones change following childbirth and during lactation and whether they are present in human milk (possibly in relation to maternal factors), we can then undertake targeted experimental and/or mechanistic studies to elucidate factors responsible for the obesity-protective effect of breastfeeding in mother and offspring. Biomedical Impact/Relevance to Field: Aberrations of ghrelin, PYY, GLP-1 and/or leptin may help explain why some women struggle to lose weight or are predisposed to body weight (or adiposity) gain following childbirth and during lactation. Concentrations of these hormones, if present in human milk, may also serve as short-term or long-term regulators of energy intake in the breastfed infant and offer a potential mechanism for the obesity-protective effects of breastfeeding. Current Project Status. As of March 3, 2012, we have enrolled and completed baseline data collection on 18 lactating postpartum women and 15 never-pregnant controls (for a total of 33 enrolled/baseline-completed volunteers) (Aim 1). We have enrolled 3 additional never-pregnant controls that are scheduled to complete baseline visits in April, and are waiting the delivery of several interested, currently pregnant participants. We have completed one- year follow-up visits on 11 postpartum lactating subjects and 8 never-pregnant controls (Aim 2). Additionally we submitted a manuscript on provocative findings from milk collected at baseline (4 weeks postpartum) in 13 postpartum women (Aim 3). In that publication we report the presence of PYY, GLP-1 and leptin in human milk, and that GLP-1 but not PYY or leptin concentrations change across a single feeding with concentrations of GLP-1 greater in hind- compared to fore-milk. Concentrations of milk leptin strongly correlated with maternal BMI. Action Plans for External Funding: Findings from our analysis of breast milk (Aim 3) will serve as preliminary data for a NIH grant application with a specific aim to determine whether the appetite regulation hormones in human milk—including ghrelin, PYY, GLP-1 and leptin-- are potential regulators of growth and body fat accretion in infants during the first year of life, and whether variations in these hormones among individual mothers are explained by differences in maternal nutrition and/or adiposity (October, 2012 submission).

Sreejayan Nair, Ph.D., School of Pharmacy,Cathepsin-K Mitigates Cardiac Dysfunction
Heart failure is the leading cause of morbidity and mortality in the United States. Cardiac hypertrophy, the adaptive response of the heart to pressure overload, has been recognized as a major independent risk factor for heart failure. Consequently, inhibition or regression of cardiac hypertrophy by pharmacologic interventions such as angiotensin converting enzyme inhibition has been shown to lower the risk of heart failure whereas persistence of hypertrophy has the opposite effect. However, despite the pivotal role of cardiac hypertrophy in heart failure, drugs that interrupt or reverse the progression of cardiac hypertrophy remain limited, warranting the development of novel therapeutic strategies to address this problem. Cathepsins are cysteine proteases that are ubiquitously expressed in various tissues and that play roles in cancer, autoimmune and cardiovascular diseases. Recent observations showing altered expression of cathepsins in the myocardium of humans with left-ventricular hypertrophy strongly suggest the involvement of cathepsins in heart failure. Our preliminary data show that cathepsin K (catK) the most potent proteolytic cathepsin is upregulated in the hypertrophic heart. However the causal role of catK in the development of cardiac hypertrophy and heart failure is largely unknown. The long-term goal of our research program is to understand how cathepsins can be manipulated for preventive and therapeutic purposes in heart failure. The objective of this research proposal, which is our next step in pursuit of that goal, is to determine the role of cat-K in the development of cardiac hypertrophy. Our central hypothesis is that cat-K is necessary for obesity- and pressure overload-mediated cardiac hypertrophic responses. Our hypothesis has been formulated on the basis of our preliminary data using cat-K-/- mice. The rationale for the proposed research is that, once it is known how cat-K regulates cardiac hypertrophy, pharmacological inhibitors of cat-K (which are currently used clinically to treat osteoporosis) can be assessed as new or innovative approaches for the prevention and treatment of heart failure. We plan to test our central hypothesis and, thereby, accomplish the objective of this application by pursuing the following two specific aims: 1. Determine the role of catK in the development of heart failure. Based on the preliminary data, the working hypothesis here is that catK knockout mitigates cardiac dysfunction and hypertrophy in a mouse model of pressure-overload-induced cardiac hypertrophy. 2. Identify intracellular signaling mechanisms modulated by cat-K leading to cardiac hypertrophy and dysfunction. We postulate, again on the basis of our preliminary data, that catK mediates cardiomyocyte apoptosis, autophagy and hypertrophy. The outcome of these studies will help identify molecular pathways that are mediated by cathepsins in the development of cardiac hypertrophy and dysfunctions. Such results are expected to have an important positive impact, because the identified mechanisms are highly likely to provide new targets for preventive and therapeutic interventions in addition to fundamentally advancing the growing knowledge of the mechanisms involved in the pathogenesis of heart failure. A NIHR15 grant proposal addressing the aforementioned aims was submitted in 2011, which received a priority score of 69. On resubmission the proposal receive a score of 26. The proposal will be submitted as a R01 for the June, 2012 deadline.

Baskaran Thyagarajan Ph.D., Analysis of Role of TRPV1 channels in the regulation of Metabolic Diseases.
Obesity is the hallmark of cardiovascular complications and a threat for current and future healthcare demands. Although life-style modification presents an effective way to manage obesity, the coexistence of comorbid conditions like hypertension, diabetes, or atherosclerosis often necessitates pharmacotherapy for management.

Recent research suggests the role of transient receptor potential vanilloid receptor 1 (TRPV1) in the pathophysiology of obesity, diabetes and cardiovascular complications. However, the precise role of TRPV1 in the regulation of these diseases remains unknown. This proposal stems from our hypothesis that TRPV1 is a potential target against adipogenesis and obesity. Our primary focus is to investigate how the molecular mechanisms of adipogenesis are regulated by TRPV1 via Ca2+ dependent protein kinases, phospholipids and phosphatases as these critically regulate the functions of TRPV1. Our work emphasizes the critical role of regulation of adipogenesis by TRPV1 agonists, which modulate lipid homeostasis in animals and humans. We propose to investigate the regulatory role of TRPV1 activation by capsaicin and SA13353 (a specific TRPV1 agonist) in weight gain leading to obesity in mouse model. We hypothesize that cellular mechanisms that facilitate TRPV1 desensitization inhibit adipogenesis.

Figure 1. Hypothetical scheme represents the catalysis of differentiation of preadipocytes to adipocytes (adipogenesis) by peroxisome proliferation activating receptor gamma (PPARγ). The effects of capsaicin, protein kinase C (PKC), Ca2+/calmodulin dependent protein kinase (CaM Kinase II), calcineurin, phospholipase C (PLC) and PPARγ agonists on adipogenesis and TRPV1 ion channel functions are described. +, –, and ? - denote activation, inhibition and undetermined effect, respectively. TM, transmembrane domain; N and C, N and C termini; [Ca2+]e, extracellular Ca2+.

To investigate this overarching hypothesis, we plan to study the modulations of TRPV1 activity by capsaicin/SA13353 using cultured preadipocyte 3T3-L1 and primary adipose cells and animal models (wild type and transgenic, TRPV1-/-) to analyze

1.  The effects of TRPV1 agonists on

i. Ca2+ influx induced calcineurin activation
ii. Ca2+ influx induced desensitization of TRPV1 (via calcineurin activation)
(i and ii down-regulate PPARγ)

2. The effects of long-term desensitization of TRPV1 via PLC pathway or loss of TRPV1 (TRPV1 knockout, TRPV1-/-) inhibits adipogenesis; and

3.  The effects of TRPV1 sensitization by

i. PKC activation
ii. CaM Kinase II activation
(i and ii promote adipogenesis)

The outcome of this proposal will provide new insight into the role of TRPV1 channels in the regulation of adipogenesis. Our experimental plans will investigate potentials of TRPV1 channel agonists as novel therapeutic agents to treat obesity and the related cardiovascular complications. The data obtained from this proposal will form the foundation for our future research work and for the submission of a compelling and fundable R01 grant application to NIH within 2 years. Besides serving our immediate goals, the data obtained will help developing new pharmacotherapy for the treatment of metabolic syndrome.

Meijun Zhu Ph.D., Animal Sciences, Maternal Obesity, Stem Cell Factor/c-Kit Signaling and Mast Cells in the Development of Intestine and Incidence of Inflammatory Bowel Diseases in Progeny.
The obesity rate increased more than 2 fold in recent decades. According to the latest NHANES survey (1999-2002), 29% of non-pregnant women 20-39 years of age are obese. At the same time, inflammatory bowel diseases (IBD) and related allergic diseases are also increasing. Is there a link between the increase in maternal obesity (MO) and the increase in IBD and related allergic diseases in offspring? Gut is the largest immune organ, and the majority of key developmental milestones of gut accomplishes during the fetal stage. Recent studies clearly show that gut epithelial integrity and barrier function is the central predisposing factor to IBD, food allergy as well as autoimmune diseases. The objective of this project is to explore the impact of MO on fetal and offspring gut development, inflammation, and barrier function, providing an explanation for the surge of IBD and related diseases in recent decades. We are employing the unique strength of the availability of transgenic mice including TLR4 knockout (TLR4-/-) mice, mast cell deficient (c-Kit-/-) mice and IBD-susceptible (IL10-/-) mice to exploring 1) role TLR4 signaling in maternal obesity and its associated inflammation in offspring gut development; and 2) role of mast cells in pre-disposing OB offspring mice to IBD.

Mice study is ongoing as planned. Currently, we have gotten sufficient TLR4 (-/+) mice which were treated with either control (Con) or obesogenic (OB) diet and given birth. The offspring born to both Con and OB mothers were fed with the same control diet after weaning till 2-month and 4-month of age, when offspring mice will be sacrificed and gut will be collected for analyzing the role of TLR4 signaling in maternal obesity and its associated inflammation in offspring gut through biochemical and histological analysis. Currently, we have collected 2-m-old TLR4 offspring mice and will collect samples from 4-month-old TLR4 knockout offspring mice in June.

Meanwhile, we are cross-breeding IL10 (-/-) mice with mast cell deficient mice (c-Kit-/-) to get mast cell heterozygous IL10 knockout mice (c-Kit-/+, IL10-/-). Currently, the colonies of c-Kit-/+, IL10-/- mice are increasing as planned and we have the first group of c-Kit-/+, IL10-/- mice under maternal obesity treatment at the end of 2011. We are expecting to have our first set of gut samples from offspring (c-Kit-/-, IL10-/- and c-Kit+/+, IL10-/-) mice born to C and OB mothers (c-Kit-/+, IL10-/-) collected in the following months.

In addition, we are also looking into the role of mast cells in IBD onset and incidence by cross-breeding c-Kit (-/-) and IL10 (-/-) mice that can spontaneously develop IBD. Currently, gut tissues from c-kit(-/-), IL10(-/-) and c-Kit (+/+), IL10 (-/-) have been harvested and we are undertaking related biochemical and histological analysis. Recently, we also looked into the protective roles of bioactive food components, especially grape seed extract on IBD onset and disease index.

Besides in vivo mice experiment, we have been conducting in vitro cell culture studies using murine mastocytoma cell line P815 and primary bone marrow derive mast cells derived from wild type and TLR4 knockout mice to test our hypothesis: palmitic acid (PA) induces mast cell activation and proliferation through TLR4 and SCF/c-Kit signaling. One abstract was submitted to 2012 Experimental Biology meeting and one manuscript is in preparation. Three papers in related topics were published in 2011.

We have been actively acquiring for external funds. Please see the following for details.

Mei-Jun Zhu (PI), Stephen P. Ford, and Claudio Fiocchi. NIH R01. Improving gastrointestinal immune response through fetal programming. $1,571,125 Not funded
Mei-Jun Zhu (PI), NIH R15. Maternal Obesity, AMPK and Neonatal Gut Development. $424,500 Impact score: 21
Mei-Jun Zhu (PI). NIH R01. Obesity, AMP-activated -protein kinase, and gut epithelial renewal and barrier function $1,809,875 Pending
Mei-Jun Zhu (PI). DOD. Grape seed extract, gut epithelium barrier function and inflammatory bowel diseases $750,000 Preparing

 


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