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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Structural and functional characterization of bitter taste receptors, T2R1 and T2R4

Pydi, Sai Prasad January 2014 (has links)
In humans, taste is one of the five senses, and helps in the recognition of nutritionally important and potentially harmful substances. It triggers innate behaviour to accept or reject food. Humans can sense five basic tastes, which are sweet, umami, bitter, salt and sour. The receptors that mediate bitter, sweet and umami tastes belong to the G protein-coupled receptor (GPCR) superfamily. A group of three receptors sense sweet and umami tastes, whereas bitter taste is sensed by 25 bitter taste receptors (referred as T2Rs). T2Rs are activated by structurally diverse natural and synthetic bitter compounds. Many common pharmaceutical compounds are bitter in taste and these are effective ligands for T2Rs. Recent finding of T2Rs in extra-oral tissues suggests these receptors are also involved in various physiological and pathophysiological processes. To understand the structure and function of these receptors, studies directed at elucidating their mechanisms of activation, and identification of novel ligands including bitter blockers (antagonists and inverse agonists), are required. To obtain mechanistic insights into the role of the highly conserved, and receptor specific residues, two bitter taste receptors (T2R1 and T2R4) were targeted. In this study, a combination of molecular, biochemical and pharmacological approaches were used to identify the amino acids and motifs, important for T2Rs to switch from inactive to active state. A hydrogen-bonding network between transmembrane (TM) helices 1-2-7 was identified as important for T2R activation. Alanine-scan mutagenesis of intracellular loops (ICLs) 2 and 3 identified T2R regions important for G protein binding, and receptor activation. A pharmacological method was developed, to screen potential bitter blockers for T2Rs. Using this method, three novel bitter blockers, which include two natural antagonists and one synthetic inverse agonist for T2R4, were discovered. The role of expression tags in enhancing T2R4 expression was also pursued. T2R4 expression on the cell surface was increased 2.5 fold, when its N-terminus was tagged with rhodopsin N-terminal 33 residues (Rho33- T2R4 chimera). In conclusion, work carried out provides novel insights into the mechanisms of T2R activation, and in the discovery of bitter blockers for T2R4.
2

Erzeugung und Charakterisierung von Mausmodellen mit lichtsensitivem Geschmackssystem zur Aufklärung der neuronalen Geschmackskodierung / Generation and characterization of transgenic lines of mice to elucidate neuralnetworks engaged in processing of gustatory information

Loßow, Kristina January 2011 (has links)
Die Wahrnehmung von Geschmacksempfindungen beruht auf dem Zusammenspiel verschiedener Sinneseindrücke wie Schmecken, Riechen und Tasten. Diese Komplexität der gustatorischen Wahrnehmung erschwert die Beantwortung der Frage wie Geschmacksinformationen vom Mund ins Gehirn weitergeleitet, prozessiert und kodiert werden. Die Analysen zur neuronalen Prozessierung von Geschmacksinformationen erfolgten zumeist mit Bitterstimuli am Mausmodell. Zwar ist bekannt, dass das Genom der Maus für 35 funktionelle Bitterrezeptoren kodiert, jedoch war nur für zwei unter ihnen ein Ligand ermittelt worden. Um eine bessere Grundlage für tierexperimentelle Arbeiten zu schaffen, wurden 16 der 35 Bitterrezeptoren der Maus heterolog in HEK293T-Zellen exprimiert und in Calcium-Imaging-Experimenten funktionell charakterisiert. Die Daten belegen, dass das Funktionsspektrum der Bitterrezeptoren der Maus im Vergleich zum Menschen enger ist und widerlegen damit die Aussage, dass humane und murine orthologe Rezeptoren durch das gleiche Ligandenspektrum angesprochen werden. Die Interpretation von tierexperimentellen Daten und die Übertragbarkeit auf den Menschen werden folglich nicht nur durch die Komplexität des Geschmacks, sondern auch durch Speziesunterschiede verkompliziert. Die Komplexität des Geschmacks beruht u. a. auf der Tatsache, dass Geschmacksstoffe selten isoliert auftreten und daher eine Vielzahl an Informationen kodiert werden muss. Um solche geschmacksstoffassoziierten Stimuli in der Analyse der gustatorischen Kommunikationsbahnen auszuschließen, sollten Opsine, die durch Licht spezifischer Wellenlänge angeregt werden können, für die selektive Ersetzung von Geschmacksrezeptoren genutzt werden. Um die Funktionalität dieser angestrebten Knockout-Knockin-Modelle zu evaluieren, die eine Kopplung von Opsinen mit dem geschmacksspezifischen G-Protein Gustducin voraussetzte, wurden Oozyten vom Krallenfrosch Xenopus laevis mit dem Zwei-Elektroden-Spannungsklemm-Verfahren hinsichtlich dieser Interaktion analysiert. Der positiven Bewertung dieser Kopplung folgte die Erzeugung von drei Mauslinien, die in der kodierenden Region eines spezifischen Geschmacksrezeptors (Tas1r1, Tas1r2, Tas2r114) Photorezeptoren exprimierten. Durch RT-PCR-, In-situ-Hybridisierungs- und immunhistochemische Experimente konnte der erfolgreiche Knockout der Rezeptorgene und der Knockin der Opsine belegt werden. Der Nachweis der Funktionalität der Opsine im gustatorischen System wird Gegenstand zukünftiger Analysen sein. Bei erfolgreichem Beleg der Lichtempfindlichkeit von Geschmacksrezeptorzellen dieser Mausmodelle wäre ein System geschaffen, dass es ermöglichen würde, gustatorische neuronale Netzwerke und Hirnareale zu identifizieren, die auf einen reinen geschmacks- und qualitätsspezifischen Stimulus zurückzuführen wären. / Taste impression is based on the interaction of taste, smell and touch. To evaluate the nutritious content of food mammals possess five distinct taste qualities: sweet, bitter, umami (taste of amino acids), sour and salty. For bitter, sweet, and umami compounds taste signaling is initiated by binding of tastants to G protein-coupled receptors. The interactions of taste stimuli, usually watersoluble chemicals, with their cognate receptors lead to the activation of the G protein gustducin, which, in turn, initiates a signal resulting in the activation of gustatory afferents. However, details of gustatory signal transmission and processing as well as neural coding are only incompletely understood. This is partly due to the property of some tastants to elicit several sensations simultaneously, unspecific effects caused by the temperature, viscosity, osmolarity, and pH of the solvents, as well as by mechanical stimulation of the tongue during stimulus application. The analysis of gustatory processing of taste information are mainly based on mouse models after stimulation with bitter taste stimuli. Even though it is known that the mouse genome codes for 35 bitter taste receptor genes only few of them had been analysed so far. For better understanding and interpretation of animal experiments 16 mouse bitter receptors had been analysed by Calcium Imaging experiments with HEK293T cells. The data reveal that mouse bitter taste receptors are more narrow tuned than human bitter taste receptors, proving that the ligand spectra of murine and human orthologous receptors are not complient. In order to avoid the disturbing effects of solvents and stimulus application on the analysis of gustatory information transfer and processing, I employ an optogenetical approach to address this problem. For this purpose I generated three strains of gene-targeted mice in which the coding regions of the genes for the umami receptor subunit Tas1r1, the sweet receptor subunit Tas1r2 or the bitter taste receptor Tas2r114 have been replaced by the coding sequences of different opsins (photoreceptors of visual transduction) that are sensitive to light of various wavelengths. In these animals I should be able to activate sweet, bitter, or umami signalling by light avoiding any solvent effects. In initial experiments of this project I demonstrated that the various visual opsins indeed functionally couple to taste signal transduction pathway in oocyte expression system, generating basic knowledge and foundation for the generation of the gene-targeted animals. The knockout-knockin strategies have been successfully realized in the case of all three mouse models, revealed by RT-PCR, in situ hybridization and immunohistochemical analysis of taste papillae. All data confirm that the particular taste receptors have been replaced by the different opsins in taste cells. Further analysis concerning the functional consequences of opsin knockin and taste receptor knockout are part of prospective work.

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