DOCSLIB.ORG

Chemoreception: a Consideration of Olfactory Signalling Proteins in Non-Olfactory Systems

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Chemoreception: a Consideration of Olfactory Signalling Proteins in Non-Olfactory Systems Chemoreception: A Consideration of Olfactory Signalling Proteins in Non-Olfactory Systems Yuliya Makeyeva A thesis in fulfillment of the requirements for the degree of Doctor of Philosophy School of Women’s and Children’s Health Faculty of Medicine November 2018 Thesis/Dissertation Sheet Australia's Global UNSWSYDNEY University Surname/Family Name Makeyeva Given Name/s Yuliya Abbreviation for degree as given in the University calendar PhD Faculty Faculty of Medicine School School of Women's and Children's Health Thesis Title Chemoreception: A Consideration of Olfactory Signalling Proteins in Non­ Olfactory Systems Abstract 350 words maximum: Chemoreception is a biological process whereby cells and/or organisms are stimulated by chemicals in their environment. The detectibn of chemicals triggers a response that can be the attraction or aversion by a single II cell or a highly integrative process that initiates a complex behaviour. Olfactory receptors (ORs) are a multigene family of molecules used to monitor extracellular chemical cues. They were originally described in the olfactory system as mediators of the sense of smell but have since been observed in a number of non-olfactory tissues. OR-mediated chemoreception in the olfactory system involves a G protein (Gait), adenylyl cyclase Ill (AC3), and OMP. ORs are difficultto study so little is known about their functions and expression profiles in these tissues. I, Olfactory marker protein (OMP) is a highly expressed protein found in mature olfactory sensory neurons of all vertebrates, co-labels with individual ORs, and is involved in intracellular signal transduction. OMP expression may indicate OR-mediated chemoreception in non-olfactory systems. Cells that are mobile are optimal candidates because they might use chemoreception to guide their movements. My thesis used OMP, Gait, and AC3 to identify chemoreception in non-olfactory cells: spermatozoa, mast cells, and Leydig cells. The distribution of these proteins was investigated using immunohistochemistry and microscopic image analysis. In rats, expression of these proteins was documented in spermatozoa, mast cells, and Leydig cells. In humans, expression was investigated in spermatozoa in three modes of activation: control, activated, and hyper-activated. The results showed that OMP, Gait, and AC3 were co-expressed in spermatozoa, mast cells, and Leydig cells. Due to the adverse reaction of tissues to a high-fat diet, the response of mast cells, the I olfactory epithelium, and olfactory bulb was tested and found to be rapidly altered. This presence of an OR-mediated signalling pathway suggests chemoreception and guidance in diverse cells of non-olfactory tissues. A high-fat diet, even in the short term, adversely affected immunity and chemoreception. My data reveal the widespread and complex interactions among diverse systems that are mediated by chemoreception. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). ....i.f ... !.{.49/.8 Witness Signature Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on us�. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. OR OFFICE USE ONLY Date of completion of requirements for Award: COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ……………………………………………........................... Date ……………………………………………........................... AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………………………........................... Date ……………………………………………........................... INCLUSIONOF PUBLI CATIONS STATEMENT UNSW is supportive of candidates publishing their research results during their candidature as detailed in the UNSW Thesis Examination Procedure. Publications can be used in their thesis in lieu of a Chapter if: • The student contributed greater than 50% of the content in the publication and is the "primary author'', ie. the student was responsible primarily for the planning, execution and preparation of the work for publication • The student has approval to include the publication in their thesis in lieu of a Chapter from their supervisor and Postgraduate Coordinator. • The publication is not subject to any obligations or contractual agreements with a third party that would constrain its inclusion in the thesis Please indicate whether this thesis contains published material or not. This thesis contains no publications, either published or submitted for publication (if this box is checked, you may delete all the material on page 2) Some of the oork described in this thesis has been published and it has been documented in the relevant Chapters Vvith acknowedgement (if this box is D checked, you may delete all the material on page 2) This thesis has publications (either published or submitted for publication) incorporated into it in lieu of a chapter and the details are presented below D l, CANDIDATE 'S DE CLARATION I declare that: • I have complied with the Thesis Examination Procedure • where I have used a publication in lieu of a Chapter, the listed publication(s) below meet(s) the requirements to be included in the thesis. Name Date (dd/mmfyy) \du)1_('.j Q_, N f\� '::!E V 14- l60Y.;l0(9 Postgraduate Coordinator's Declaration (to be filled in where publications are used in lieu of Chapters) I declare that: • the information below is accurate • where listed 'publication(s) have been used in lieu of Chapter(s), their use complies with the Thesis Examination Procedure • the minimum requirements for the format of the thesis have been met. PGC'sName PGC's Signature Date (dd/mmfyy) Originality Statement 'I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own wofk, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.' Signed: Date: iii Supervisors Supervisor: Professor William Ledger School of Women’s and Children’s Health UNSW Sydney Co-Supervisor: Professor David Ryugo Hearing Research Garvan Institute of Medical Research and UNSW Sydney Co-Supervisor: Dr Steven Leach School of Women’s and Children’s Health UNSW Sydney iv Dedication For my father Valentine, my mother Elmira, and my daughter Kseniya—for their unflagging love and support v Acknowledgements I wish to thank the following people for their assistance with my PhD. My sincere thanks go: to Professor Laura Poole-Warren for her support through my PhD despite considerable difficulties; to my primary supervisor, Professor William Ledger, for inspiring my interest in reproduction and for his support and guidance during the final stages of my PhD. He gave me the strength and motivation to complete what has been an arduous journey; to my co-supervisor, Professor David Ryugo, for allowing me to complete my PhD in his laboratory, for his encouragement and feedback. I am grateful for his gentle guidance and the freedom he gave me to pursue my aims and the confidence to keep going. I also thank him for helpful comments and discussions on my thesis; to Dr Steven Leach, my co-supervisor, for his helpful assistance in interpreting
Load more

Recommended publications
  • Atypical Functions of Leukocyte Chemoattractant Receptors
    EDITORIAL published: 09 October 2020 doi: 10.3389/fimmu.2020.596902 Editorial: Atypical Functions of Leukocyte Chemoattractant Receptors José Luis Rodríguez-Fernández 1*, Mario Mellado 2*, Marcus Thelen 3* and Philip M. Murphy 4* 1 Department of Molecular Cell Biology, Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas, Madrid, Spain, 2 Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain, 3 Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera Italiana, Bellinzona, Switzerland, 4 Laboratory of Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States Keywords: chemotaxis, chemokine, atypical chemokine receptor, G protein-coupled receptor, signaling, arrestin, GPCR Edited and reviewed by: Silvano Sozzani, Sapienza University of Rome, Italy Editorial on the Research Topic *Correspondence: José Luis Rodríguez-Fernández Atypical Functions of Leukocyte Chemoattractant Receptors [email protected] Mario Mellado Chemoattraction is a process that involves directed cell movement toward extracellular chemical [email protected] stimuli. It is a fundamental feature of all biological systems that was discovered in the late Marcus Thelen nineteenth century, when the word “chemotaxis” was coined by the German biologist Wilhelm [email protected] Pfeffer. Metchnikoff’s 1882 description of macrophages
    [Show full text]
  • Stromal Cell–Derived Factor-1/CXCL12 Stimulates Chemorepulsion of NOD/Ltj T-Cell Adhesion to Islet Microvascular Endothelium Christopher D
    ORIGINAL ARTICLE Stromal Cell–Derived Factor-1/CXCL12 Stimulates Chemorepulsion of NOD/LtJ T-Cell Adhesion to Islet Microvascular Endothelium Christopher D. Sharp,1 Meng Huang,1 John Glawe,1 D. Ross Patrick,1 Sible Pardue,1 Shayne C. Barlow,2 and Christopher G. Kevil1 OBJECTIVE—Diabetogenic T-cell recruitment into pancreatic islets faciltates ␤-cell destruction during autoimmune diabetes, yet specific mechanisms governing this process are poorly un- iabetogenic T-cell infiltration into pancreatic derstood. The chemokine stromal cell–derived factor-1 (SDF-1) islets is a key pathophysiological feature of controls T-cell recruitment, and genetic polymorphisms of SDF-1 autoimmune diabetes. Recruitment of autoreac- are associated with early development of type 1 diabetes. tive T-cells into islets, known as insulitis, ini- D ␤ tiates the process of -cell damage, which may occur over RESEARCH DESIGN AND METHODS—Here, we examined a protracted period of time, eventually leading to frank the role of SDF-1 regulation of diabetogenic T-cell adhesion to ␤ islet microvascular endothelium. Islet microvascular endothelial destruction of -cells and loss of insulin production (1,2). cell monolayers were activated with tumor necrosis factor-␣ Cellular and molecular mechanisms necessary for recruit- (TNF-␣), subsequently coated with varying concentrations of ment and homing of diabetogenic T-cells have been pur- SDF-1 (1–100 ng/ml), and assayed for T-cell/endothelial cell posed, with antigen presentation and changes in adhesion interactions under physiological flow conditions. molecule expression playing important roles in this pro- cess (3–6). However, T-cell recruitment is regulated by RESULTS—TNF-␣ significantly increased NOD/LtJ T-cell adhe- other factors besides antigen presentation and adhesion sion, which was completely blocked by SDF-1 in a dose-depen- molecule expression.
    [Show full text]
  • The Olfactory Receptor Associated Proteome
    INTERNATIONAL GRADUATE SCHOOL OF NEUROSCIENCES (IGSN) RUHR UNIVERSITÄT BOCHUM THE OLFACTORY RECEPTOR ASSOCIATED PROTEOME Doctoral Dissertation David Jonathan Barbour Department of Cell Physiology Thesis advisor: Prof. Dr. Dr. Dr. Hanns Hatt Bochum, Germany (30.12.05) ABSTRACT Olfactory receptors (OR) are G-protein-coupled membrane receptors (GPCRs) that comprise the largest vertebrate multigene family (~1,000 ORs in mouse and rat, ~350 in human); they are expressed individually in the sensory neurons of the nose and have also been identified in human testis and sperm. In order to gain further insight into the underlying molecular mechanisms of OR regulation, a bifurcate proteomic strategy was employed. Firstly, the question of stimulus induced plasticity of the olfactory sensory neuron was addressed. Juvenile mice were exposed to either a pulsed or continuous application of an aldehyde odorant, octanal, for 20 days. This was followed by behavioural, electrophysiological and proteomic investigations. Both treated groups displayed peripheral desensitization to octanal as determined by electro-olfactogram recordings. This was not due to anosmia as they were on average faster than the control group in a behavioural food discovery task. To elucidate differentially regulated proteins between the control and treated mice, fluorescent Difference Gel Electrophoresis (DIGE) was used. Seven significantly up-regulated and ten significantly down-regulated gel spots were identified in the continuously treated mice; four and twenty-four significantly up- and down-regulated spots were identified for the pulsed mice, respectively. The spots were excised and proteins were identified using mass spectrometry. Several promising candidate proteins were identified including potential transcription factors, cytoskeletal proteins as well as calcium binding and odorant binding proteins.
    [Show full text]
  • Spontaneous Neural Activity Is Required for the Establishment and Maintenance of the Olfactory Sensory Map
    Neuron, Vol. 42, 553–566, May 27, 2004, Copyright 2004 by Cell Press Spontaneous Neural Activity Is Required for the Establishment and Maintenance of the Olfactory Sensory Map C. Ron Yu,1,2 Jennifer Power,2 Gilad Barnea,2 mus and cortex, results from complementary gradients Sean O’Donnell,3 Hannah E.V. Brown,4 of ephrin receptors on retinal afferents and ephrins ex- Joseph Osborne,3 Richard Axel,2,3,4,* pressed by the multiple targets in the brain (Wilkinson, and Joseph A. Gogos2,5,* 2001). These retinotopic representations deconstruct 1Department of Anatomy and Cell Biology the visual scene into submaps that refine the image, 2 Center for Neurobiology and Behavior including ocular dominance columns and orientation 3 Department of Biochemistry columns, as well as cortical blobs that uniquely respond and Molecular Biophysics to color, motion, and spatial frequency (Van Essen et al., 4 Howard Hughes Medical Institute 1992). These refinements are sensitive to visual activity, 5 Department of Physiology and Cellular Biophysics and this has led to efforts to distinguish the relative College of Physicians and Surgeons contributions of innate determinants (timing of axon ar- Columbia University rival and molecular cues) and experiential or activity- New York, New York 10032 dependent mechanisms in the generation of the visual circuitry (Katz and Shatz, 1996). The problem is perhaps best illustrated by early recordings by Hubel and Wiesel Summary that demonstrated that cortical neurons respond prefer- entially to one or the other eye and segregate into ocular We have developed a genetic approach to examine dominance columns (LeVay et al., 1978; Wiesel and Hu- the role of spontaneous activity and synaptic release bel, 1963).
    [Show full text]
  • Proteomic Atlas of the Human Olfactory Bulb
    JOURNAL OF PROTEOMICS 75 (2012) 4005– 4016 Available online at www.sciencedirect.com www.elsevier.com/locate/jprot Proteomic atlas of the human olfactory bulb Joaquín Fernández-Irigoyena, Fernando J. Corralesb, Enrique Santamaríaa,⁎ aProteomics Unit, Biomedical Research Center (CIB), Navarra Health Service, 31008 Pamplona, Spain bProteomics Unit, Centre for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain ARTICLE INFO ABSTRACT Article history: The olfactory bulb (OB) is the first site for the processing of olfactory information in the Received 12 March 2012 brain and its deregulation is associated with neurodegenerative disorders. Although Accepted 7 May 2012 different efforts have been made to characterize the human brain proteome in depth, the Available online 15 May 2012 protein composition of the human OB remains largely unexplored. We have performed a comprehensive analysis of the human OB proteome employing protein and peptide Keywords: fractionation methods followed by LC-MS/MS, identifying 1529 protein species, correspond- Olfactory bulb ing to 1466 unique proteins, which represents a 7-fold increase in proteome coverage with Brain respect to previous OB proteome descriptions from translational models. Bioinformatic Proteomics analyses revealed that protein components of the OB participated in a plethora of biological Mass spectrometry process highlighting hydrolase and phosphatase activities and nucleotide and RNA binding Bioinformatics activities. Interestingly, 631 OB proteins identified were not previously described in protein datasets derived from large-scale Human Brain Proteome Project (HBPP) studies. In particular, a subset of these differential proteins was mainly involved in axon guidance, opioid signaling, neurotransmitter receptor binding, and synaptic plasticity. Taken together, these results increase our knowledge about the molecular composition of the human OB and may be useful to understand the molecular basis of the olfactory system and the etiology of its disorders.
    [Show full text]
  • Immunotherapy in the Treatment of Human Solid Tumors: Basic and Translational Aspects
    Journal of Immunology Research Immunotherapy in the Treatment of Human Solid Tumors: Basic and Translational Aspects Guest Editors: Roberta Castriconi, Barbara Savoldo, Daniel Olive, and Fabio Pastorino Immunotherapy in the Treatment of Human Solid Tumors: Basic and Translational Aspects Journal of Immunology Research Immunotherapy in the Treatment of Human Solid Tumors: Basic and Translational Aspects Guest Editors: Roberta Castriconi, Barbara Savoldo, Daniel Olive, and Fabio Pastorino Copyright © 2016 Hindawi Publishing Corporation. All rights reserved. This is a special issue published in “Journal of Immunology Research.” All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Editorial Board B. D. Akanmori, Congo Eung-Jun Im, USA G. Opdenakker, Belgium Stuart Berzins, Australia Hidetoshi Inoko, Japan Luigina Romani, Italy Kurt Blaser, Switzerland Peirong Jiao, China Aurelia Rughetti, Italy Federico Bussolino, Italy Taro Kawai, Japan Takami Sato, USA N. G. Chakraborty, USA Hiroshi Kiyono, Japan Senthamil Selvan, USA Robert B. Clark, USA Shigeo Koido, Japan Naohiro Seo, Japan Mario Clerici, Italy Herbert K. Lyerly, USA Ethan M. Shevach, USA Nathalie Cools, Belgium Enrico Maggi, Italy George B. Stefano, USA Mark J. Dobrzanski, USA Mahboobeh Mahdavinia, USA T. J. Stewart, Australia Nejat K. Egilmez, USA Eiji Matsuura, Japan J. Tabarkiewicz, Poland Eyad Elkord, UK C. J. M. Melief, The Netherlands Ban-Hock Toh, Australia S. E. Finkelstein, USA Chikao Morimoto, Japan Joseph F. Urban, USA Luca Gattinoni, USA Hiroshi Nakajima, Japan Xiao-Feng Yang, USA David E. Gilham, UK Toshinori Nakayama, Japan Qiang Zhang, USA Douglas C.
    [Show full text]
  • Expressing Exogenous Functional Odorant Receptors in Cultured Olfactory Sensory Neurons Huaiyang Chen1, Sepehr Dadsetan2, Alla F Fomina2 and Qizhi Gong*1
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Springer - Publisher Connector Neural Development BioMed Central Methodology Open Access Expressing exogenous functional odorant receptors in cultured olfactory sensory neurons Huaiyang Chen1, Sepehr Dadsetan2, Alla F Fomina2 and Qizhi Gong*1 Address: 1Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, California 95616, USA and 2Department of Physiology and Membrane Biology, School of Medicine, University of California, Davis, California 95616, USA Email: Huaiyang Chen - [email protected]; Sepehr Dadsetan - [email protected]; Alla F Fomina - [email protected]; Qizhi Gong* - [email protected] * Corresponding author Published: 11 September 2008 Received: 22 May 2008 Accepted: 11 September 2008 Neural Development 2008, 3:22 doi:10.1186/1749-8104-3-22 This article is available from: http://www.neuraldevelopment.com/content/3/1/22 © 2008 Chen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Olfactory discrimination depends on the large numbers of odorant receptor genes and differential ligand-receptor signaling among neurons expressing different receptors. In this study, we describe an in vitro system that enables the expression of exogenous odorant receptors in cultured olfactory sensory neurons. Olfactory sensory neurons in the culture express characteristic signaling molecules and, therefore, provide a system to study receptor function within its intrinsic cellular environment.
    [Show full text]
  • Genomics of Mature and Immature Olfactory Sensory Neurons Melissa D
    University of Kentucky UKnowledge Physiology Faculty Publications Physiology 8-15-2012 Genomics of Mature and Immature Olfactory Sensory Neurons Melissa D. Nickell University of Kentucky, [email protected] Patrick Breheny University of Kentucky, [email protected] Arnold J. Stromberg University of Kentucky, [email protected] Timothy S. McClintock University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits oy u. Follow this and additional works at: https://uknowledge.uky.edu/physiology_facpub Part of the Genomics Commons, and the Physiology Commons Repository Citation Nickell, Melissa D.; Breheny, Patrick; Stromberg, Arnold J.; and McClintock, Timothy S., "Genomics of Mature and Immature Olfactory Sensory Neurons" (2012). Physiology Faculty Publications. 66. https://uknowledge.uky.edu/physiology_facpub/66 This Article is brought to you for free and open access by the Physiology at UKnowledge. It has been accepted for inclusion in Physiology Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact [email protected]. Genomics of Mature and Immature Olfactory Sensory Neurons Notes/Citation Information Published in Journal of Comparative Neurology, v. 520, issue 12, p. 2608-2629. Copyright © 2012 Wiley Periodicals, Inc. This is the peer reviewed version of the following article: Nickell, M. D., Breheny, P., Stromberg, A. J., and McClintock, T. S. (2012). Genomics of mature and immature olfactory sensory neurons. Journal of Comparative Neurology, 520: 2608–2629, which has been published in final form at http://dx.doi.org/ 10.1002/cne.23052. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
    [Show full text]
  • Mechanisms of Ciliary Targeting of the Olfactory Cyclic Nucleotide- Gated Channel
    Mechanisms of Ciliary Targeting of the Olfactory Cyclic Nucleotide- Gated Channel by Paul Michael Jenkins A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Pharmacology) in The University of Michigan 2010 Doctoral Committee: Associate Professor Jeffrey Randall Martens, Chair Professor Lori L. Isom Professor Benjamin L. Margolis Associate Professor Kristen J. Verhey © Paul Michael Jenkins All Rights Reserved 2010 To my family ii ACKNOWLEDGEMENTS I would like to first extend my sincerest gratitude to my research mentor, Dr. Jeffrey R. Martens. The time I have spent in his laboratory has shaped me both professionally and personally. His drive and dedication to science serve as a model that I strive towards every day. The past few years have been extremely rewarding for me due to his friendship, patience, and continual mentoring. I would not be where I am today without his guidance. I would also like to acknowledge my thesis committee members, Dr. Lori Isom, Dr. Ben Margolis and Dr. Kristen Verhey. I have been extremely lucky to have a committee that not only acted as thesis advisors, but also as collaborators, mentors, and friends. I would like to thank the numerous members of the Martens laboratory, present and former: Kristin Arendt, Dave Dudek, Nikhil Iyer, Sajida Jackson, Qiuju Li, Dyke McEwen, Jeremy McIntyre, Sarah Schumacher, Laurie Svoboda, Kristin van Genderen, Eileen Vesely, Tiffney Widner, Liz Williams, Kendra Yum, and Lian Zhang. Your antics in lab have kept me alternating
    [Show full text]
  • A Haptotaxis Assay for Neutrophils Using Optical Patterning and a High-Content Approach Received: 5 September 2016 Joannie Roy 1,2, Javier Mazzaferri 1, János G
    www.nature.com/scientificreports OPEN A Haptotaxis Assay for Neutrophils using Optical Patterning and a High-content Approach Received: 5 September 2016 Joannie Roy 1,2, Javier Mazzaferri 1, János G. Filep1,3 & Santiago Costantino1,2,4 Accepted: 21 April 2017 Neutrophil recruitment guided by chemotactic cues is a central event in host defense against infection Published: xx xx xxxx and tissue injury. While the mechanisms underlying neutrophil chemotaxis have been extensively studied, these are just recently being addressed by using high-content approaches or surface-bound chemotactic gradients (haptotaxis) in vitro. Here, we report a haptotaxis assay, based on the classic under-agarose assay, which combines an optical patterning technique to generate surface-bound formyl peptide gradients as well as an automated imaging and analysis of a large number of migration trajectories. We show that human neutrophils migrate on covalently-bound formyl-peptide gradients, which influence the speed and frequency of neutrophil penetration under the agarose. Analysis revealed that neutrophils migrating on surface-bound patterns accumulate in the region of the highest peptide concentration, thereby mimicking in vivo events. We propose the use of a chemotactic precision index, gyration tensors and neutrophil penetration rate for characterizing haptotaxis. This high-content assay provides a simple approach that can be applied for studying molecular mechanisms underlying haptotaxis on user-defined gradient shape. Neutrophils are rapidly recruited into infected or injured tissues where they play a pivotal role in host defense against invading pathogens and in wound healing1. Neutrophil trafficking into inflamed tissues is governed by chemotactic molecules secreted by invading bacteria and tissue-resident sentinel cells2 and coordinated expres- sion of adhesion molecules on neutrophils and endothelial cells3.
    [Show full text]
  • Studio Delle Vie Di Trasduzione Del Segnale Inositide-Dipendente Nelle Sindromi Mielodisplastiche
    UNIVERSITA' DEGLI STUDI DI BOLOGNA Scuola di Dottorato in Scienze Mediche e Chirurgiche Cliniche Dottorato di Ricerca in Scienze Morfologiche Umane e Molecolari Settore Disciplinare BIO/16 Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell’Apparato Locomotore STUDIO DELLE VIE DI TRASDUZIONE DEL SEGNALE INOSITIDE-DIPENDENTE NELLE SINDROMI MIELODISPLASTICHE Tesi di Dottorato Tutore: Presentata da: CHIAR.MO PROF. LUCIO COCCO DOTT.SSA MATILDE YUNG FOLLO XIX Ciclo Anno Accademico 2005/2006 INDICE Introduzione 3 1. Sindromi Mielodisplastiche (MDS) 4 1.1.Trattamento delle MDS: 5’-azacitidina 8 2. Signalling Inositide-Dipendente: Fosfolipasi cβ1 (PI-PLCβ1) 10 2.1 Struttura del Gene della PI-PLCβ1 11 2.2 Struttura Proteica della PI-PLCβ1 12 3. Asse di Attivazione Fosfoinositide-3-Chinasi (PI3K)/Akt 14 3.1 Isoforme di Akt 16 3.2. Ruolo di Akt nei Disordini Ematopoietici 18 3.3. Ruolo di Akt nei Meccanismi Apoptotici 19 3.4. Ruolo di Akt nella Progressione attraverso il Ciclo Cellulare 20 4. Target Molecolari a Valle di Akt: mTOR, 4E-BP1e p70S6K 21 Scopo della Ricerca 23 Materiali e Metodi 25 1. Colture Cellulari in vitro 26 2. Caratteristiche dei Pazienti 26 3. Separazione delle Cellule Mononucleate 26 4. Ibridazione Fluorescente in Situ (FISH) 27 5. Estrazione del DNA ed Analisi Mutazionale 28 6. Estrazione dell’RNA e Sintesi del cDNA 28 7. Real-Time PCR 28 8. Analisi Immunocitochimica 29 9. Separazione delle Cellule CD33 31 10. Analisi Citofluorimetrica per la Quantificazione dell’Apoptosi 31 11. Analisi Citofluorimetrica per l’Analisi del Fenotipo 32 12. Separazione delle Cellule CD34 33 13.
    [Show full text]
  • Cephalic Sensory Cell Types Provides Insight Into Joint Photo
    RESEARCH ARTICLE Characterization of cephalic and non- cephalic sensory cell types provides insight into joint photo- and mechanoreceptor evolution Roger Revilla-i-Domingo1,2,3, Vinoth Babu Veedin Rajan1,2, Monika Waldherr1,2, Gu¨ nther Prohaczka1,2, Hugo Musset1,2, Lukas Orel1,2, Elliot Gerrard4, Moritz Smolka1,2,5, Alexander Stockinger1,2,3, Matthias Farlik6,7, Robert J Lucas4, Florian Raible1,2,3*, Kristin Tessmar-Raible1,2* 1Max Perutz Labs, University of Vienna, Vienna BioCenter, Vienna, Austria; 2Research Platform “Rhythms of Life”, University of Vienna, Vienna BioCenter, Vienna, Austria; 3Research Platform "Single-Cell Regulation of Stem Cells", University of Vienna, Vienna BioCenter, Vienna, Austria; 4Division of Neuroscience & Experimental Psychology, University of Manchester, Manchester, United Kingdom; 5Center for Integrative Bioinformatics Vienna, Max Perutz Labs, University of Vienna and Medical University of Vienna, Vienna, Austria; 6CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; 7Department of Dermatology, Medical University of Vienna, Vienna, Austria Abstract Rhabdomeric opsins (r-opsins) are light sensors in cephalic eye photoreceptors, but also function in additional sensory organs. This has prompted questions on the evolutionary relationship of these cell types, and if ancient r-opsins were non-photosensory. A molecular profiling approach in the marine bristleworm Platynereis dumerilii revealed shared and distinct *For correspondence: features of cephalic and non-cephalic r-opsin1-expressing cells. Non-cephalic cells possess a full set [email protected] (FR); of phototransduction components, but also a mechanosensory signature. Prompted by the latter, [email protected] (KT-R) we investigated Platynereis putative mechanotransducer and found that nompc and pkd2.1 co- Competing interest: See expressed with r-opsin1 in TRE cells by HCR RNA-FISH.
    [Show full text]