DOCSLIB.ORG

Alsaai Udel 0060D 14351.Pdf

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Alsaai Udel 0060D 14351.Pdf INVESTIGATION OF TDRD7 FUNCTION IN OCULAR LENS DEVELOPMENT by Salma Mohammed Al Saai A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Sciences Summer 2020 © 2020 Salma Mohammed Al Saai All Rights Reserved . INVESTIGATION OF TDRD7 FUNCTION IN OCULAR LENS DEVELOPMENT by Salma Mohammed Al Saai Approved: __________________________________________________________ Velia M. Fowler, Ph.D. Chair of the Department of Biological Sciences Approved: __________________________________________________________ John A. Pelesko, Ph.D. Dean of the College of Arts & Sciences Approved: __________________________________________________________ Douglas J. Doren, Ph.D. Interim Vice Provost for Graduate and Professional Education and Dean of the Graduate College I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Salil A. Lachke, Ph.D. Professor in charge of dissertation I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Melinda K. Duncan, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Donna Woulfe, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Shawn Polson, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Jeffrey Caplan, Ph.D. Member of dissertation committee ACKNOWLEDGMENTS To my father, who has instilled in me the passion for knowledge and science. My father always believed in me, and whenever I come across hard tasks he says: “Salma I know that if you want something you can do it! No matter how challenging it is!” To my husband who has been my best supporter/ friend/ mentor/ harshest critique of my work, best colleague to discuss science with……and the list goes on! Thank you! To my mother and all my family who have accompanied me throughout this joyful journey and have been relentlessly encouraging and praying for me, despite being miles away... Thank you all! To Dr. Lachke, my advisor, who accepted me to his lab and gave me the privilege to work on all the amazing cutting-edge projects. I learned A LOT!!! and I will continue to seek knowledge and learn. While being in your lab, you have supported me all the time. You gave me a great freedom in conducting research which I always appreciated. The freedom you provided helped me build on more skills and become an independent thinker. Under your mentorship I became a scientist who sets up the bar so high, cares about the big question and picture but yet never dismisses any minute details because ultimately everything contributes to a well-constructed image of the whole story. Many, Many thanks to you! v To all my committee members who, despite their other commitments, have provided me with advice, critiqued my work and asked me hard question because they wanted me to become a better scientist. I appreciate all the time you have spent to support me during this journey. Many thanks to you all. To all the past and current lab members, to all my students whom I have mentored, to all my friends and colleagues from other labs: you made the PhD journey a special experience! Together we shared moments of joy and laughter… moments of stress and frustration which would sporadically be healed with an invaluable moment of success. Thank you! To the animal facility, who has always helped us in maintaining our experimental animals, so that we can focus on producing great science. Thank you! To Betty, thanks for your smile which you greet me with every time I walk to your desk. Thanks for providing me with an answer/help for everything I ever asked you for. Thank you! To all my friends who stayed by my side, kept checking on me, encouraged me, helped me, and always spent quality time with me. Thank you! vi TABLE OF CONTENTS LIST OF TABLES ......................................................................................................... x LIST OF FIGURES ....................................................................................................... xi ABSTRACT ................................................................................................................ xiv Chapter 1 INTRODUCTION .............................................................................................. 1 1.1 The ocular lens .......................................................................................... 1 1.2 Lens fiber cell architecture ........................................................................ 3 1.3 Lens cytoskeleton ...................................................................................... 7 1.3.1 F-actin dynamics ......................................................................... 11 1.4 Cataract .................................................................................................... 12 1.5 Tdrd7 gene and protein ............................................................................ 14 1.6 Tdrd7 function in development ............................................................... 17 1.7 Loss of Tdrd7 causes fully penetrant cataracts in mouse ........................ 20 1.8 Tdrd7-/- mice exhibit severe lens fiber cell defects prior to overt cataract formation .................................................................................... 21 2 MATERIALS AND METHODS ..................................................................... 24 2.1 Animals studies ....................................................................................... 24 2.2 DNA isolation from mouse tails .............................................................. 25 2.3 Mouse genotyping ................................................................................... 26 2.4 Western Blot Analysis ............................................................................. 27 2.5 Cryopreservation of eye tissue and cryosectioning ................................. 28 2.6 Immunofluorescence ............................................................................... 28 2.7 Fluorescence in situ hybridization coupled with Immunostaining .......... 30 2.8 RNA sequencing for Postnatal stage 15 in Tdrd7-/- mouse lens ............. 31 2.9 Analysis of RNA sequencing data ........................................................... 31 2.10 Tandem Mass Tag (TMT) Mass spectrometry for Postnatal stage 15 in Tdrd7-/- mouse lens ................................................................................. 32 2.11 Cluster heatmap for normal expression pattern of misregulated proteins and genes in P15 Tdrd7-/- lens .................................................. 32 vii 2.12 Gene Set Enrichment Analysis (GESA) .................................................. 33 2.13 Immunoprecipitation for Tdrd7 protein coupled with mass spectrometry ............................................................................................ 33 2.14 RNA immunoprecipitation assay for Tdrd7 protein ................................ 35 2.15 RNA Isolation .......................................................................................... 36 2.16 Reverse Transcriptase for Quantitative PCR ........................................... 36 2.17 Scanning electron microscopy for Cap2-/- mouse lens ........................... 37 2.18 Correlation analysis for Tdrd7 and downstream misexpressed genes ..... 38 2.19 Immunofluorescence Quantification analysis ......................................... 39 3 TDRD7 CONTROLS THE LEVEL OF HEAT SHOCK PROTEIN HSPB1 (HSP27) AND LENS FIBER CELL MORPHOLOGY ................................... 40 3.1 Introduction ............................................................................................. 40 3.2 Results ..................................................................................................... 44 3.2.1 Tdrd7-/- mice exhibit morphological defects in lens fiber cells that have undergone nuclear degradation .................................... 44 3.2.2 Hspb1 is a top priority candidate down-regulated gene in Tdrd7-/- lens ............................................................................... 48 3.2.3 Hspb1 is expressed in normal lens development and is reduced in Tdrd7-/- lens ............................................................................ 50 3.2.4 Tdrd7 protein associates with Hspb1 mRNA .............................. 52 3.2.5 Single molecule fluorescence in situ hybridization shows that Tdrd7 protein co-localizes
Load more

Recommended publications
  • Recherche De Nouvelles Mutations Génétiques À Effet Majeur Dans La Maladie De Crohn Sara Frade Proud’Hon-Clerc
    Recherche de nouvelles mutations génétiques à effet majeur dans la maladie de Crohn Sara Frade Proud’Hon-Clerc To cite this version: Sara Frade Proud’Hon-Clerc. Recherche de nouvelles mutations génétiques à effet majeur dans la maladie de Crohn. Médecine humaine et pathologie. Université de Lille, 2019. Français. NNT : 2019LILUS016. tel-02465045 HAL Id: tel-02465045 https://tel.archives-ouvertes.fr/tel-02465045 Submitted on 3 Feb 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. École doctorale Biologie Santé de Lille THÈSE de DOCTORAT pour obtenir le grade de docteur délivré par l’Université de Lille - Communauté d’Universités et d’Etablissements Lille Nord de France Discipline doctorale “Recherche Clinique, Innovation Technologique, Santé Publique” Spécialité doctorale “Génétique” présentée et soutenue publiquement par Sara FRADE PROUD’HON-CLERC le 12 septembre 2019 Recherche de nouvelles mutations génétiques à effet majeur dans la maladie de Crohn Directeur de thèse : Francis VASSEUR Jury Mme Mouna BARAT-HOUARI, Maitre de conférence T M. Jean-Pierre HUGOT, Professeur M. Edouard LOUIS, Professeur H M. Xavier TRETON, Professeur E EA 2694, 1 Place Verdun, 59045 Lille S E ii iii iv Résumé Résumé : Le gène NOD2, impliqué dans les réponses immunitaires innées contre le peptidogly- cane bactérien, est étroitement associé à la maladie de Crohn (MC) avec des Odd Ratio (OR) allant de 3 à 36 selon le génotype et a été initialement identifié par des analyses de liaisons génétiques.
    [Show full text]
  • MARK4 with an Alzheimer's Disease-Related Mutation Promotes
    bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107284; this version posted May 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. MARK4 with an Alzheimer’s disease-related mutation promotes tau hyperphosphorylation directly and indirectly and exacerbates neurodegeneration Toshiya Obaa, Taro Saitoa,b, Akiko Asadaa,b, Sawako Shimizua, Koichi M. Iijimac,d and Kanae Andoa,b, * aGraduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan, bDepartment of Biological Sciences, School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan, cDepartment of Alzheimer’s Disease Research, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511, Japan, dDepartment of Experimental Gerontology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi 467-8603, Japan *Corresponding author Kanae Ando, Ph.D. E-mail: [email protected] Running title: Mutant MARK4 enhances phospho-tau accumulation Abstract Accumulation of the microtubule-associated protein tau is associated with Alzheimer’s disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser262 and Ser356 play critical roles in tau accumulation and toxicity. Microtubule-affinity regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a double de novo mutation in the linker region of MARK4, ΔG316E317InsD, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration. Here, we report that MARK4ΔG316E317D increases the abundance of highly phosphorylated, insoluble tau species and exacerbates neurodegeneration via Ser262/356-dependent and -independent mechanisms.
    [Show full text]
  • Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model
    Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 T + is online at: average * The Journal of Immunology , 34 of which you can access for free at: 2016; 197:1477-1488; Prepublished online 1 July from submission to initial decision 4 weeks from acceptance to publication 2016; doi: 10.4049/jimmunol.1600589 http://www.jimmunol.org/content/197/4/1477 Molecular Profile of Tumor-Specific CD8 Cell Hypofunction in a Transplantable Murine Cancer Model Katherine A. Waugh, Sonia M. Leach, Brandon L. Moore, Tullia C. Bruno, Jonathan D. Buhrman and Jill E. Slansky J Immunol cites 95 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html http://www.jimmunol.org/content/suppl/2016/07/01/jimmunol.160058 9.DCSupplemental This article http://www.jimmunol.org/content/197/4/1477.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 25, 2021. The Journal of Immunology Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model Katherine A.
    [Show full text]
  • A Feed-Forward Cycle
    GMJ.2020;9:e1681 www.gmj.ir Received 2019-08-08 Revised 2019-11-11 Accepted 2019-11-24 Tau Abnormalities and Autophagic Defects in Neurodegenerative Disorders; A Feed-forward Cycle Nastaran Samimi 1, 2, Akiko Asada 3, 4, Kanae Ando 3, 4 1 Noncommunicable Diseases Research Center, Fasa University of Medical Sciences, Fasa, Iran 2 Department of Brain and Cognitive Sciences, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 3 Department of Biological Sciences, School of Science, Tokyo Metropolitan University, Tokyo, Japan 4 Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan Abstract Abnormal deposition of misfolded proteins is a neuropathological characteristic shared by many neurodegenerative disorders including Alzheimer’s disease (AD). Generation of excessive amounts of aggregated proteins and impairment of degradation systems for misfolded proteins such as autophagy can lead to accumulation of proteins in diseased neurons. Molecules that contribute to both these effects are emerging as critical players in disease pathogenesis. Furthermore, impairment of autophagy under disease conditions can be both a cause and a consequence of abnormal protein accumulation. Specifically, disease-causing proteins can impair autophagy, which further enhances the accumulation of abnormal proteins. In this short review, we focus on the relationship between the microtubule-associated protein tau and autophagy to highlight a feed-forward mechanism in disease pathogenesis. [GMJ.2020;9:e1681] DOI:10.31661/gmj.v9i0.1681 Keywords: Neurodegenerative Diseases; Tauopathy; Autophagy; Microtubule Binding Pro- tein; Tau; Phosphorylation; Vesicle Trafficking Tau phosphorylation in physiology and dis- However, tau detaches from microtubules and ease misfolds to form insoluble filaments in neuro- isfolded tau protein is deposited in a fibrillary tangles in the brains of patients with Mgroup of neurodegenerative diseases tauopathies [4-9].
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Genome-Wide Identification and Analysis of Prognostic Features in Human Cancers
    bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446243; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Genome-wide identification and analysis of prognostic features in human cancers Joan C. Smith1,2 and Jason M. Sheltzer1* 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724. 2. Google, Inc., New York, NY 10011. * Lead contact; to whom correspondence may be addressed. E-mail: [email protected]. bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446243; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Abstract Clinical decisions in cancer rely on precisely assessing patient risk. To improve our ability to accurately identify the most aggressive malignancies, we constructed genome-wide survival models using gene expression, copy number, methylation, and mutation data from 10,884 patients with known clinical outcomes. We identified more than 100,000 significant prognostic biomarkers and demonstrate that these genomic features can predict patient outcomes in clinically-ambiguous situations. While adverse biomarkers are commonly believed to represent cancer driver genes and promising therapeutic targets, we show that cancer features associated with shorter survival times are not enriched for either oncogenes or for successful drug targets.
    [Show full text]
  • 1 Metabolic Dysfunction Is Restricted to the Sciatic Nerve in Experimental
    Page 1 of 255 Diabetes Metabolic dysfunction is restricted to the sciatic nerve in experimental diabetic neuropathy Oliver J. Freeman1,2, Richard D. Unwin2,3, Andrew W. Dowsey2,3, Paul Begley2,3, Sumia Ali1, Katherine A. Hollywood2,3, Nitin Rustogi2,3, Rasmus S. Petersen1, Warwick B. Dunn2,3†, Garth J.S. Cooper2,3,4,5* & Natalie J. Gardiner1* 1 Faculty of Life Sciences, University of Manchester, UK 2 Centre for Advanced Discovery and Experimental Therapeutics (CADET), Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Sciences Centre, Manchester, UK 3 Centre for Endocrinology and Diabetes, Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester, UK 4 School of Biological Sciences, University of Auckland, New Zealand 5 Department of Pharmacology, Medical Sciences Division, University of Oxford, UK † Present address: School of Biosciences, University of Birmingham, UK *Joint corresponding authors: Natalie J. Gardiner and Garth J.S. Cooper Email: [email protected]; [email protected] Address: University of Manchester, AV Hill Building, Oxford Road, Manchester, M13 9PT, United Kingdom Telephone: +44 161 275 5768; +44 161 701 0240 Word count: 4,490 Number of tables: 1, Number of figures: 6 Running title: Metabolic dysfunction in diabetic neuropathy 1 Diabetes Publish Ahead of Print, published online October 15, 2015 Diabetes Page 2 of 255 Abstract High glucose levels in the peripheral nervous system (PNS) have been implicated in the pathogenesis of diabetic neuropathy (DN). However our understanding of the molecular mechanisms which cause the marked distal pathology is incomplete. Here we performed a comprehensive, system-wide analysis of the PNS of a rodent model of DN.
    [Show full text]
  • Modulating Hallmarks of Cholangiocarcinoma
    University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Fall 12-14-2018 Modulating Hallmarks of Cholangiocarcinoma Cody Wehrkamp University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Molecular Biology Commons Recommended Citation Wehrkamp, Cody, "Modulating Hallmarks of Cholangiocarcinoma" (2018). Theses & Dissertations. 337. https://digitalcommons.unmc.edu/etd/337 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. MODULATING HALLMARKS OF CHOLANGIOCARCINOMA by Cody J. Wehrkamp A DISSERTATION Presented to the Faculty of the University of Nebraska Graduate College in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Biochemistry and Molecular Biology Graduate Program Under the Supervision of Professor Justin L. Mott University of Nebraska Medical Center Omaha, Nebraska November 2018 Supervisory Committee: Kaustubh Datta, Ph.D. Melissa Teoh‐Fitzgerald, Ph.D. Richard G. MacDonald, Ph.D. Acknowledgements This endeavor has led to scientific as well as personal growth for me. I am indebted to many for their knowledge, influence, and support along the way. To my mentor, Dr. Justin L. Mott, you have been an incomparable teacher and invaluable guide. You upheld for me the concept that science is intrepid, even when the experience is trying. Through my training, and now here at the end, I can say that it has been an honor to be your protégé. When you have shaped your future graduates to be and do great, I will be privileged to say that I was your first one.
    [Show full text]
  • Genes in Eyecare Geneseyedoc 3 W.M
    Genes in Eyecare geneseyedoc 3 W.M. Lyle and T.D. Williams 15 Mar 04 This information has been gathered from several sources; however, the principal source is V. A. McKusick’s Mendelian Inheritance in Man on CD-ROM. Baltimore, Johns Hopkins University Press, 1998. Other sources include McKusick’s, Mendelian Inheritance in Man. Catalogs of Human Genes and Genetic Disorders. Baltimore. Johns Hopkins University Press 1998 (12th edition). http://www.ncbi.nlm.nih.gov/Omim See also S.P.Daiger, L.S. Sullivan, and B.J.F. Rossiter Ret Net http://www.sph.uth.tmc.edu/Retnet disease.htm/. Also E.I. Traboulsi’s, Genetic Diseases of the Eye, New York, Oxford University Press, 1998. And Genetics in Primary Eyecare and Clinical Medicine by M.R. Seashore and R.S.Wappner, Appleton and Lange 1996. M. Ridley’s book Genome published in 2000 by Perennial provides additional information. Ridley estimates that we have 60,000 to 80,000 genes. See also R.M. Henig’s book The Monk in the Garden: The Lost and Found Genius of Gregor Mendel, published by Houghton Mifflin in 2001 which tells about the Father of Genetics. The 3rd edition of F. H. Roy’s book Ocular Syndromes and Systemic Diseases published by Lippincott Williams & Wilkins in 2002 facilitates differential diagnosis. Additional information is provided in D. Pavan-Langston’s Manual of Ocular Diagnosis and Therapy (5th edition) published by Lippincott Williams & Wilkins in 2002. M.A. Foote wrote Basic Human Genetics for Medical Writers in the AMWA Journal 2002;17:7-17. A compilation such as this might suggest that one gene = one disease.
    [Show full text]
  • Methods in and Applications of the Sequencing of Short Non-Coding Rnas" (2013)
    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2013 Methods in and Applications of the Sequencing of Short Non- Coding RNAs Paul Ryvkin University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Bioinformatics Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Ryvkin, Paul, "Methods in and Applications of the Sequencing of Short Non-Coding RNAs" (2013). Publicly Accessible Penn Dissertations. 922. https://repository.upenn.edu/edissertations/922 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/922 For more information, please contact [email protected]. Methods in and Applications of the Sequencing of Short Non-Coding RNAs Abstract Short non-coding RNAs are important for all domains of life. With the advent of modern molecular biology their applicability to medicine has become apparent in settings ranging from diagonistic biomarkers to therapeutics and fields angingr from oncology to neurology. In addition, a critical, recent technological development is high-throughput sequencing of nucleic acids. The convergence of modern biotechnology with developments in RNA biology presents opportunities in both basic research and medical settings. Here I present two novel methods for leveraging high-throughput sequencing in the study of short non- coding RNAs, as well as a study in which they are applied to Alzheimer's Disease (AD). The computational methods presented here include High-throughput Annotation of Modified Ribonucleotides (HAMR), which enables researchers to detect post-transcriptional covalent modifications ot RNAs in a high-throughput manner. In addition, I describe Classification of RNAs by Analysis of Length (CoRAL), a computational method that allows researchers to characterize the pathways responsible for short non-coding RNA biogenesis.
    [Show full text]
  • Characterization of a Monoclonal Antibody Panel Shows That the Myotonic Dystrophy Protein Kinase, DMPK, Is Expressed Almost Exclusively in Muscle and Heart
    © 2000 Oxford University Press Human Molecular Genetics, 2000, Vol. 9, No. 14 2167–2173 Characterization of a monoclonal antibody panel shows that the myotonic dystrophy protein kinase, DMPK, is expressed almost exclusively in muscle and heart L.T. Lam, Y.C.N. Pham, Nguyen thi Man and G.E. Morris+ MRIC Biochemistry Group, PP18, North East Wales Institute, Mold Road, Wrexham LL11 2AW, UK Received 11 May 2000; Accepted 28 June 2000 DDBJ/EMBL/GenBank accession no. AF250871 Myotonic dystrophy (DM) is a multisystemic disorder ness), brain (mental retardation), eyes (cataracts), testis caused by an inherited CTG repeat expansion which (atrophy) and heart (conduction defects) (10). It is important, affects three genes encoding the DM protein kinase therefore, to establish the normal tissue distribution of all three (DMPK), a homeobox protein Six5 and a protein proteins produced from the DM locus. containing WD repeats. Using a panel of 16 mono- The human DMPK cDNA has 15 exons and predicts a ∼ clonal antibodies against several different DMPK protein of 70 kDa, although there may be variation in the size epitopes we detected DMPK, as a single protein of and sequence of the first exon (1–3,11,12). DMPK is a serine/ threonine protein kinase in which the catalytic domain ∼80 kDa, only in skeletal muscle, cardiac muscle and, (∼43 kDa) is followed by a coiled-coil domain (∼12 kDa) and to a lesser extent, smooth muscle. Many earlier a hydrophobic C-terminal domain. Alternative splicing has reports of DMPK with different sizes and tissue distri- been observed in a short VSGGG sequence between domains butions appear to be due to antibody cross-reactions and in the C-terminal domain (13).
    [Show full text]
  • Regulation of Hematopoietic Activity Involving New Interacting Partners (RRAGC & PSMC2, CKAP4 & MANF and CTR9 & CNTNAP2)
    CellBio, 2020, 9, 123-141 https://www.scirp.org/journal/cellbio ISSN Online: 2325-7792 ISSN Print: 2325-7776 Regulation of Hematopoietic Activity Involving New Interacting Partners (RRAGC & PSMC2, CKAP4 & MANF and CTR9 & CNTNAP2) Swati Sharma1, Gurudutta U. Gangenahalli2*, Upma Singh3 1Department of Pharmacology, All India Institute of Medical Science (AIIMS), New Delhi, India 2Stem Cell & Gene Therapy Research Group, Institute of Nuclear Medicine & Allied Sciences, New Delhi, India 3Department of Applied Chemistry, School of Vocational Studies & Applied Sciences, Gautam Buddha University, Greater Noida, India How to cite this paper: Sharma, S., Gan- Abstract genahalli, G.U. and Singh, U. (2020) Regu- lation of Hematopoietic Activity Involving Hematopoietic stem cells (HSCs) are tissue-specific cells giving rise to all New Interacting Partners (RRAGC & PSMC2, mature blood cell types regulated by a diverse group of hematopoietic cyto- CKAP4 & MANF and CTR9 & CNTNAP2). kines and growth factors that influences the survival & proliferation of early CellBio, 9, 123-141. https://doi.org/10.4236/cellbio.2020.93007 progenitors and differentiation mechanisms by modulating the functional ac- tivities of HSCs. In this study, the functional yet distinctive role of three novel Received: August 13, 2020 combinations of gene pairs RRAGC & PSMC2; CKAP4 & MANF; and CTR9 Accepted: September 27, 2020 & CNTNAP2 have been newly identified. These novel combinations of genes Published: September 30, 2020 were confirmed and expressed in K562 human leukemic cell line in the pres- Copyright © 2020 by author(s) and ence of cytokine combination (IL-3, FLT-3 and SCF) using RT-PCR and Scientific Research Publishing Inc. siRNA-mediated gene knock down strategy.
    [Show full text]