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2015 Research Showcase
CADB Abstracts
Abstract Title : Understanding the Role of Wnt9a Proteins in Hematpoiesis
Abstract : Blood is composed of many different cell types that are all formed in the biological process known as hematopoiesis. The origin of these blood cells can be traced back to self-renewing multipotent stem cells, called hematopoietic stem cells (HSCs), which have the unique ability to give rise to all the different blood cell lineages (erythrocytes, leukocytes, platelets, etc). In regenerative medicine, HSCs are utilized to treat hematologic disorders such as different types of anemias, leukemias, and myelomas via hematopoietic stem cell transplantation. This effective but risky therapeutic procedure has met challenges, such as the inability to find immunologically compatible donors to minimize rejection complications, and finding a reliable source of functional self-renewing HSCs as the current demand greatly outweighs the supply. Therefore, the goal for many researchers within the field is to induce the formation of HSCs from hPS cells, which have the potential to differentiate to all mature cell types, including HSCs. In addition, hPS cells can be derived from any individual, thereby overcoming risks of immunological rejection after transplant. HSCs arise early in the developing embryo through a poorly understood process before eventually migrating to the bone marrow in the adult system. Wnt signaling is an important signaling pathway that has been shown to play a vital role in regulating HSC development. In this pathway, Wnt proteins, which are secreted lipid modified growth factors, bind to receptors encoded by Frizzled (Fzd) gene family, thereby triggering a downstream signal cascade. In the absence of a Wnt signal, a destruction complex comprised of multiple proteins, targets β-catenin for degradation by proteasomes. The binding of Wnt to Fzd disrupts the destruction complex allowing the accumulation of β-catenin in the cytoplasm from where it translocates to the nucleus to activate target gene expression. Using a zebrafish model, whose genome contains 23 Wnt ligands, current research in the Willert and Traver labs have demonstrated that Wnt9a was found to be highly expressed in tissues important for HSC development. Building on these observations on the critical role of Wnt9a in zebrafish hematopoiesis, we hypothesize that manipulation of Wnt9a signaling will significantly improve the differentiation of hPS cells to HSCs. Therefore our goal is to to isolate the human and mouse Wnt9a and paralog Wnt9b genes and overexpress them in mammalian cell culture systems to test the ability of the recombinant Wnt9 proteins to influence the differentiation of hPSCs to HSCs.
Abstract Title : The Role of MAP4K3 in Autophagy Induction
Abstract : Autophagy is a cellular recycling pathway that is critical in the normal aging process and in neurodegenerative disorders such as Parkinson's, Huntington's, and Alzheimer's diseases. Misfolded proteins are removed from the cell by the ubiquitin-proteasome system, but many aggregate-prone proteins, such as polyglutamine-containing proteins, are inefficiently degraded by the proteasome. These aggregate-prone proteins require alternative protein turnover pathways such as autophagy, which removes misfolded proteins and damaged organelles by sequestration into double membrane autophagosomes, followed by lysosome fusion and degradation. We have found that MAP4K3, a Ste20 kinase that regulates the amino acid sensing pathway upstream of mTORC1, regulates autophagy induction in a mechanism independent of the mTOR pathway. Our studies indicate that knock-down of MAP4K3 in N2a cells or in primary neurons is sufficient to induce autophagy, based upon LC3-II immunoblotting and analysis of autophagic vesicle formation. These findings highlight MAP4K3 as a pivotal regulatory factor in cellular metabolism.
Abstract Title : Role of Heparan Sulfate in Adipocyte Biology
Abstract : Obesity is a causal factor for insulin resistance and its parallel epidemic is driving the incidence of Type-2 Diabetes (T2D). Adipose tissue plays a central role in insulin resistance. The quality of adipose tissue rather than its quantity is important; therefore understanding the mechanisms that regulate adipocyte number and size are urgent research goals for the development of treatments for T2D. Adipose-derived glypican-4, a heparan sulfate proteoglycan (HSPG), serves as a marker for body-mass index and insulin sensitivity. We propose that variations in heparan sulfate (HS) composition on adipocytes serve as a rheostat for adipocyte differentiation and function and can predict susceptibility to develop insulin resistance and T2D. To address the hypothesis we used N-deacetylase/N-sulfotransferase 1 (Ndst1)-deficient mouse embryonic fibroblasts (MEFs) and evaluated adipocyte differentiation. Ndst1 is an enzyme responsible for catalyzing N-sulfation of glycosaminoglycans heparan sulfate. Ndst1 inactivation had impaired adipocyte differentiation and failed to accumulate lipids. This lack of lipid accumulation was associated with decrease glucose uptake as Ndst1-deficient MEFs consumed less glucose and produced less lactate when compared with wild type MEFs. The reduced glucose uptake could not be explained by reduced expression of GLUT4 or differences in FGF reception and signaling. Mice deficient for Ndst1 in adipose tissue (Ndst1fl/flAdipoqCre+) had impaired glucose tolerance and increased insulin resistance compared to wild-type mice. Our findings suggest that variation in HSPG can affect glucose metabolism and support a new mechanism by which proteoglycans can affect adaptation of adipose tissue in the context of obesity and T2D.
Advisor : HEMAL H. PATEL
Abstract Title : Severity in the Progression of Cardiomyopathy Dependent on Dietary Content in a Murine Type 2 Diabetes Model
Abstract : The causes of diabetes-associated heart disease are multifactorial. Several studies have emphasized the importance of diabetic cardiomyopathy, or ventricular dysfunction independent of coronary artery disease or hypertension. However, these works did not focus on understanding the pathogenesis of diabetic cardiomyopathy. In our long-term observational study, we assessed how dietary content affects the manifestation and progression of cardiomyopathy in a murine type 2 diabetes mellitus (T2DM) model. 3 months into a high fat diet (HFD), T2DM mice exhibited significant hyperglycemia, hyperinsulinemia, and altered glucose-tolerance response alongside significantly increased body weights. After 6 months, T2DM mice exhibited eccentric cardiac hypertrophy and mildly reduced cardiac contractility by echocardiography. After 8 months, T2DM mice exhibited no significant difference in systolic blood pressure or heart rate, indicating that cardiac hypertrophy was not caused by hypertension. Future studies are underway to investigate kidney function and ultrastructural changes in the cardiac muscle of T2DM mice.
Abstract Title : Plg-RKT has an important role in mouse mammary gland development
Abstract : Plasmin is the major enzyme responsible for degrading fibrin within the circulation. Plasmin is formed when the circulating zymogen, plasminogen (Plg), is cleaved by urokinase-type (uPA) and tissue-type Plg activators. In addition to its role in fibrin degradation, when plasminogen is bound to cells its activation is markedly promoted. Cell associated plasmin plays a key role in processes in which cells must degrade an extracellular matrix in order to migrate. Plasminogen Receptor KT (Plg-RKT) is a recently discovered transmembrane receptor for Plg that promotes Plg activation to plasmin. Plasmin remains bound to Plg-RKT on cell surfaces where it is protected from inactivation by plasmin inhibitors. Here we explore the role of Plg-RKT in lactation using Plg-RKT knockout mice because breast development in lactation requires degradation of the extracellular matrix when the epithelial component of the mammary glands proliferates into the fat pad, forming hollow alveoli with secretory epithelial cells surrounded by contractible myoepithelial cells and a basement membrane. Here we show, using histological analysis, that mice deficient in Plg-RKT have reduced alveolar diameter and epithelial content when compared to the Plg-RKT Wild-Type (WT). Furthermore, we show that Plg-RKT-KO/fibrinogen heterozygous mice have reduced alveolar diameter and epithelial content when compared to Plg-RKT-WT/fibrinogen heterozygotes. Thus, the inability to proteolytically remodel the extracellular matrix may present a barrier to lobular/alveolar development during pregnancy.