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2016 Research Showcase
CADB Abstracts
Abstract Title : Enhanced wound repair and regeneration in heparan sulfate biosynthetic mutant mice
Abstract : Mammals have limited ability to repair wounds and regenerate from traumatic physical injury. Studies have shown that heparan sulfate is critical for regulating blood clotting and regeneration. Since the EXT1 and EXT2 genes encode for the copolymerase complex that synthesizes heparan sulfate, we hypothesized that heparan sulfate biosynthetic mutant mice, Ext1+/-Ext2+/- may have altered wound repair and regeneration. To investigate the possible effects on wound repair, full-thickness dorsal skin excisional wounds were performed on mice. Interestingly, these Ext1+/-Ext2+/- mice closed their dorsal skin wounds 25% faster (2 days) than the wild type mice. Also, when we compared the wounded skin of heparan sulfate mutant with wild type mice, we observed that mutant had a thicker dermis layer with signs of unorganized angiogenesis. This suggests the role of heparan sulfate affects the angiogenesis, which can contribute to the mutant phenotype of enhanced wound repair. We also investigated the signs of regeneration in these mutant mice. Wildtype mice, like human, cannot regenerate when the digit amputations were performed at regions that are more proximal to the 3rd phalangeal element. However, we found that mice deficient in Ext1 can regenerate the joint between the 2nd and 3rd phalangeal element, the 3rd phalangeal element, and the nail organ. These results suggest the importance of heparan sulfate in wound repair and regeneration in mammals. ADDITIONAL CONTRIBUTOR: Erica Trinh
Advisor : DR. AMY KIGER
Abstract Title : The Role of Rab35 and Endosomal Trafficking in T-tubule Remodeling
Abstract : T-tubules are specialized muscle cell plasma membrane invaginations critical for contraction, yet T-tubule formation and remodeling is poorly understood. We are studying these processes in Drosophila pupal abdominal muscles that disassemble and reassemble T-tubules during metamorphosis. We identified genes important to T-tubule remodeling, including several known regulators of endosomal trafficking and recycling. Specifically, Rab35 GTPase knockdown or dominant negative in muscle accelerated T-tubule disassembly but blocked reassembly with an accumulation of membrane vacuoles. I showed that the Rab35 loss of function phenotype requires dynamin large GTPase activity, pointing to a shared and regulated endosomal pathway involved in T-tubule disassembly. Knockdown of other known Rab35 pathway components, such as CenB1A Arf6 GAP, or of the myopathy-relevant gene, mtm PI3-phosphatase, resulted in phenocopy of Rab35 depletion. My findings point to a role for regulated Rab35 endosomal recycling and its possible novel connection to an endosomal Mtm phosphoinositide pathway in regulating T-tubule remodeling.
Abstract Title : The synergistic effect of sustained release of Daunorubicin and Ketorolac in the treatment of proliferative vitreoretinopathy utilizing a porous silicon particle delivery system
Abstract : Proliferative vitreoretinopathy (PVR) is a potentially blinding eye disease that occurs in people who have undergone invasive eye surgery, recent retinal repair surgery, or have experienced an ocular trauma that results in retinal detachment (RD). Daunorubicin and ketorolac, usually administered individually, exhibit anti-proliferative and anti-inflammatory effects in the treatment of PVR, respectively. In this study, a pretreatment strategy was adopted in which drug loaded porous silicon particles were intravitreally injected two weeks before an experimental retinal detachment. We aimed to assess the potentially synergistic effect of the Daunorubicin and Ketorolac dual-loaded particles at treating the PVR model in comparison to particles loaded with either drug alone. Three days after RD and four hours prior to sacrifice, animal eyes were injected with BRDU to assess cellular proliferation. In order to compare the effectiveness of the different drug treatments, immunohistochemical stains of BRDU, and Vimentin and GFAP, which are intermediate filament markers shown to upregulate their expression following retinal detachment, were conducted and analyzed. Photos were taken using an EVOS FL Auto Fluorescent microscope, and BRDU cell counts per mm of retina were recorded. For Vimentin and GFAP, measurements of the length of filament protrusion were graded and compared between all groups. In addition, stain intensity over predetermined retinal areas were statistically compared across groups using the ImageJ image analysis program.
Abstract Title : The Exportin Crm1 Not Only Mediates Nuclear Export, But Also Pore Assembly
Abstract : The nuclear pore complex (NPC) is a multiprotein structure that controls communication between the nucleus and the cytoplasm. Transport through the NPC is mediated by import receptors (Importins) or export receptors (Exportins). Interestingly, Importins have also been shown to separately act in mitosis, specifically by regulating key mitotic events, such as spindle assembly and NPC assembly. Here we show that the Exportin Crm1 has a role in pore assembly. A novel way to test for such a role is to study the process in annulate lamellae (AL). AL are composed of stacked flattened cytoplasmic membranes containing regularly spaced pore structures that are identical in size, shape, and protein content to nuclear pores. We found that Crm1 negatively regulates AL formation in an in vitro system derived from Xenopus egg extracts. These results can now be extended to investigate the molecular mechanism by which Crm1 regulates AL and NPC pore formation.
Abstract Title : The role of transmembrane protein Tmem2 in heart development
Abstract : During embryonic development, migration of cardiac muscle cells, or cardiomyocytes, play a crucial role in early heart formation. Movement of these cells toward the midline is required for assembly of the heart tube. However, the factors driving this process, known as cardiac fusion, are not well understood. We have identified the transmembrane protein, Tmem2, as a novel regulator of cardiac fusion.  In tmem2 mutants, cardiomyocyte migration fails to occur completely, leaving bilateral populations of cardiomyocytes instead of a midline heart tube. The biochemical function of Tmem2, however, is not yet understood. In order to gain insight into its function, we are examining the molecular characteristics of Tmem2.Tmem2 has a short cytoplasmic tail, two Pander-like regions, a series ofPbH1 repeats, and a G8 domain. Our initial domain analysis indicated that all four extracellular domains in Tmem2 are required for its function. We next wanted to test whether these required domains were sufficient for Tmem2 function. To begin this work, I have created a variant of Tmem2 in which a single Pander-like domain is fused to a C-terminus GFP tag. I will overexpress this construct in tmem2 mutant embryos and assess rescue as compared to the rescue seen with full-length Tmem2. In parallel, we aim to identify Tmem2 protein interactors that will yield insight into its function. To do this, I am immunoprecipitating GFP-tagged Tmem2 and blotting for potential protein interactors found through a literature-based candidate approach. By analyzing the structure and function of Tmem2, as well as its interactions, we hope to elucidate the mechanisms of its action and to gain a better understanding of heart development.
Abstract Title : NF-kB and Sp3 Interactions Regulate Key Developmental Genes in the Lung
Abstract : In preterm infants and animal models, inflammation and subsequent NF-kB activation inhibit normal lung development. The Prince lab has shown that NF-kB reduces expression of key developmental genes in the lung. In the case of fibroblast growth factor 10, NF-kB -Sp3 interactions inhibit promoter activity and gene transcription. We therefore hypothesize that Sp3 is required for inflammation-induced loss of developmental gene expression. RNA sequencing showed that the bacterial endotoxin and NF-kB activator lipopolysaccharide (LPS) inhibited expression of meningioma1 (Mn1) and C/EBPα, two transcription factors important for cellular proliferation and differentiation. However, LPS had no effect on Mn1 and C/EBPα in Sp3 mutant cells. We will next determine how these changes might regulate lung development. Real-time PCR will measure expression of Mn1 and C/EBPα in Sp3 wild-type, Sp3 heterozygous, and Sp3 mutant fetal lung explants treated with LPS. Immunostaining will confirm the cellular sites of Mn1 and C/EBPα by protein expression. Control experiments will measure expression of C/EBPβ, which is not influenced by LPS or loss of Sp3. These findings will determine how interactions between NF-kB and Sp3 regulate changes in the developing lung exposed to inflammation.
Abstract Title : Transcription Factor Rhox10 Involvement in Establishment of Spermatogonial Stem Cells in Early Germ Cell Development
Abstract : Spermatogonial stem cells (SSCs) are critical for maintaining spermatogenesis throughout adult life. Little is known about how SSCs are first generated. Here, we report the identification of a transcription factor 'RHOX10' that promotes the initial establishment of SSCs. It has been found that Rhox10 is essential for efficient SSC establishment through its ability to promote the differentiation of Pro-Spermatogonia into SSCs. We provide evidence that Rhox10 acts by promoting the migration of ProSG into a specialized niche located in the periphery of seminiferous tubules. The discovery of a transcription factor important for both ProSG differentiation and SSC establishment provides an opportunity to gain insights into these critical steps of gametogenesis.
Advisor : DR. KANG ZHANG
Abstract Title : The effects of vitamin C on senescence and proliferation in human fibroblasts
Abstract : The effects of vitamin C treatment on human cells have been linked to both cell proliferation and cell death based on variations in cell type. While vitamin C is known to be an antioxidant, it can be spontaneously oxidized to dehydroascorbic acid (DHAA). Studies have shown that the oxidation of vitamin C to DHAA within the cell results in oxidative stress. Therefore, it is unclear what determines when vitamin C acts as a beneficial or harmful agent. In this paper, we examine the phenotypic and genotypic effects of vitamin C and DHAA on a mortal fibroblast cell line. Our results indicate that appropriate concentration of vitamin C treatment induces the proliferation of fibroblast cells as shown through cell count and reduced senescence levels. On the other hand, excessive concentrations of vitamin C cause cell death in human fibroblasts. Furthermore, qRT-PCR results show that appropriate amount of vitamin C and its derivatives upregulate the expression of an established age-related gene, a marker for cell proliferation. Our results demonstrate that vitamin C does indeed induce proliferation of fibroblast cells which could be reaffirmed by further results in vivo.
Abstract Title : The Effects of Hypoxia on the Proliferation and Differentiation of Trophoblast Stem Cells
Abstract : Trophoblast stem (TS) cells, derived from the polar trophectoderm, differentiate into all trophoblast lineage of the placenta and contribute to healthy placental development. Dysfunctional differentiation can be characteristic of placental insufficiency. Studies show that a prolonged hypoxic environment can adversely alter placental development, affecting cell proliferation and differentiation. Thus, we hypothesized that hypoxia affects proliferative trophoblast cells. To investigate the effects of hypoxia, mouse TS cells and two distinct TS cell subpopulations, Sca1+ and Epcam+, were independently cultured in hypoxic (1% O2) and normoxic chambers in proliferative and differentiating conditions. Histological and qRTPCR analyses of cultures showed that hypoxia increased cell growth in in proliferative conditions. In differentiating conditions, there was increased growth accompanied by prolonged expression of progenitor genes, particularly promoting differentiation to glycogen cells, shown by increased glycogen accumulation and elevated Aldh1a3 and Pcdh12 gene expression. These results show that hypoxia alters both trophoblast cell proliferation and differentiation.