Nih short-term research training for minority students

Cholecystokinin Expression and Localization in Human Placenta Yamilette Borja, Robert Boudreaux, M.S., and Kirk P. Conrad, M.D. Departments of Physiology and Functional Genomics, and of Ob/Gyn, University of Florida College of Medicine Background: Cholecystokinin (CCK) is a gastrointestinal hormone that primarily regulates digestion through neural and hormonal pathways. In response to ingested free fatty acids and amino acids, CCK is released into the bloodstream from I-cells located in the duodenal mucosa. It is also secreted through the stimulation of I-cells by two factors, CCK-releasing peptide or monitor peptide. CCK-releasing peptide is secreted into the small intestinal lumen by paracrine cells within the epithelium in response to products of fat or protein digestion. Monitor peptide is released by pancreatic acinar cells and is found in pancreatic juice. Both factors can also be released by neural input, which is significant for initiating pancreatic secretion, preparing the system for digestion of a meal as soon as it enters the small intestine. Presence of these factors stimulates the release of CCK which then travels in the bloodstream to the brain, stomach, gallbladder and pancreas, where CCK receptors, CCK-AR or CCK-BR are expressed. CCK actions include neurotransmission, regulation of gastric emptying, gallbladder contraction, relaxation of the sphincter of Oddi and pancreatic secretion, all of which can contribute to satiety [1]. The placenta is an organ that forms in the uterus during pregnancy and connects to the fetus via the umbilical cord. It functions as a pulmonary, alimentary and excretory system for the fetus. Additionally, it produces a myriad of steroid and protein hormones including progesterone, estrogen and human chorionic gonadotropin. These hormones are vital for implantation and maintenance of pregnancy [2]. Preeclampsia is a condition during pregnancy that is known to cause hypertension, intrauterine growth restriction and proteinuria. It is a leading cause of maternal and fetal mortality and morbidity [3]. A previous microarray experiment from our laboratory on first trimester chorionic villous samples (CVS) revealed expression of CCK, which was markedly up regulated in CVS from women who developed preeclampsia compared to women who experienced normal pregnancy. Subsequent RT-PCR corroborated the expression of CCK and its receptors in first trimester and term placental tissues, as well as in choriocarcinoma (trophoblast-derived) cell lines. We hypothesize that CCK protein will be localized to trophoblast in first and third trimester placental tissues. Methods: Paraffin-embedded blocks of first trimester placentae were kindly provided by Dr. Post Uiterweer (Utrecht Netherlands). The placentae were acquired from elective terminations under approval of The Medical Ethical Review Board of the University Medical Center Utrecht. These tissues contain both fetal chorionic villous and maternal decidual tissue. A third trimester placenta was acquired under approval of University of Florida IRB #147-2009, and tissue samples of the decidual basal plate and chorionic villous were obtained and fixed in 4% formaldehyde overnight at 4°C. They were then embedded in paraffin, 4m sections cut and mounted on slides that were used for immunohistochemical localization of CCK. Two CCK antibodies were used, one directed against a Cand another against a Nterminal peptide. The tissues sections on the slides were permeabilized for 10 minutes with 0.3% Triton X-100 in PBS, treated with 1% hydrogen peroxide for 3 minutes in methanol to quench endogenous peroxidase activity, and then blocked for 20 minutes at RT with 1% normal horse serum in PBS. The sections were then immunostained overnight at 4°C with the primary antibodies goat anti-CCK C-20 (2g/mL), goat anti-CCK58 N-19 (2g/mL), mouse anti-cytokeratin (1g/mL) or control isotype normal goat IgG (2g/mL). This was followed by addition of the corresponding biotinylated secondary antibodies horse anti-goat (1g/mL) or biotinylated horse anti-mouse (3g/mL) for 30 minutes at RT, and then ABC reagent for 1 hour at RT. Sections were finally developed using DAB substrate. Subsequently the slides were mounted with Cytoseal and observed with a Nikon bright field microscope. Images were then obtained using an Olympus BX41 system microscope and an Olympus DP71 digital imaging system. Results: CCK was localized to syncytiotrophoblast and extravillous trophoblast cells in both first trimester and term placenta. This was concluded based on anatomical location or parallel sections of CCK and cytokeratin antibody, the latter staining trophoblast cells. When using blocking peptides with the corresponding CCK antibody a diminished signal was observed, and when the blocking peptides were used with the opposite CCK antibody there was little or no signal reduction. Conclusions: These findings suggest that CCK is expressed by trophoblast, but its role in normal pregnancy or preeclampsia is presently unknown, and requires further study. Trophoblast CCK may be secreted serving an endocrine, paracrine, or autocrine function. We will investigate the possibility that CCK is secreted into conditioned medium by cultured trophoblasts and villous explants, and attempt to manipulate its secretion with somatostatin and CCK-releasing peptide. Other future goals include examination of additional placenta, explorations of CCK receptors via in situ hybridization (antibodies that work appear not to be available), investigations of cell signaling, as well as of potential roles for CCK in trophoblast migration and syncytialization using pharmacologic and molecular strategies. The Role of Estrogen Receptors in Epithelial to Mesenchymal Transition (EMT) in Idiopathic Pulmonary Fibrosis Kristal L. Gant, BS1, Cody Smith, MS2, and Tara Sabo-Attwood, PhD2 1Elizabeth City State University, Elizabeth City, NC; 2Department of Environmental and Global Health and Department of Physiological Sciences, University of Florida, Gainesville, FL Idiopathic Pulmonary Fibrosis (IPF) is a fatal lung disease in which an unknown injury to alveolar epithelial cells initiates various mechanisms that yield an excess deposition of extracellular matrix components. This leads to a build-up of scar tissue and causes lung dysfunction. Epithelial to mesenchymal transition (EMT) is one such mechanism that involves a change in phenotype of alveolar epithelial cells to fibroblastic cells, the effector cells of fibrosis. Estrogen's role in the development of lung fibrosis and in particular EMT isn't well-known, but gender-based differences in the prevalence and incidence of IPF suggest estrogen and estrogen receptors may be involved in pathogenesis. Understanding estrogen's role in EMT is important in further understanding the cause of lung fibrosis and in possibly controlling its progression. The aim of this research was to develop a model of EMT in which to test the effect of estrogen receptor modulation. In this model, exposure of lung epithelial cells (BEAS-2B) to transforming growth factor beta 1 (TGF-β1), a key modulator of fibrosis, causes a decrease in expression of epithelial marker genes (Ecadherin, CDH1) and an increase in expression of fibroblast marker genes (α-smooth muscle actin, ACTA2, and vimentin, VIM) thus indicating EMT and a change in cellular phenotype. Our hypothesis was that cells exposed to TGF-β1 would adopt the mesenchymal/fibroblastic phenotype and that the expression of estrogen receptors (ESR1, ESR2, and GPER) and EMT markers would increase suggesting a feed-forward mechanism where increased ER expression further increases EMT. For these experiments, BEAS-2B cells were exposed to variable concentrations of TGF-β1 (0.1 ng/μl, 1 ng/μl, and 5 ng/μl), and cells were harvested after 0, 24, 48, 72, 96, and 120 hours of exposure. Next, mRNA was purified, reverse transcribed, and qPCR was performed with validated primers specific to the EMT markers and estrogen receptors. Data analysis indicated a general trend at each dose tested supporting the original hypothesis. The expression of ACTA2 and VIM and ESR2 increased while the expression of ESR1 and EMT marker CDH1 decreased indicating that EMT occurred and that ESR activity may have changed. It is yet to be determined whether the change in expression of ESRs affected EMT. Expression changes in estrogen receptor GPER were unclear but also trended toward increased expression at the highest dose tested. The data obtained is preliminary and further testing and exposures must take place before prominent conclusions can be reached. Gene Therapy for Friedreich's Ataxia Michael Hamm, Thiago Love-Lenoir, GA The lab that I participated in was Dr. Barry Byrne's lab. He has been involved in many gene therapy projects. In the project I was involved in, we hypothesized that introducing a functional FXN gene via gene therapy would increase frataxin production. My specific project was to aid in the cloning of the expression and viral plasmid. Friedriech's Ataxia (FRDA) is a neuromuscular genetic disorder named after Nicolaus Friedreich, which has many symptoms affecting coordination, vision, hearing, etc. It is caused by inheriting two recessive alleles for the FXN gene and it makes a GAA expansion in the FXN gene expand greatly. Frataxin is found in the mitochondria, so lack of frataxin leads to lack of energy production and possibly buildup of oxidative stress and iron in the mitochondria. After putting the pcDNA 3.1 expression backbone into the viral pTR-UF12 plasmid, we transformed the plasmid, picked 11 colonies, and grew them up in 5ml LB buffer. After confirming the insert with NsiI/SpeI and SmaI, we grew with LB overnight for a maxi prep. The last step I was involved in was confirming the single and doublestranded clone. The effects of maternal hypercortisolemia during late gestation on the fetal ovine circulatory system Innah Lachica, Andrew Antollic and M