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Design and synthesis of mechanical systems with coupled units / Conception et synthèse des systèmes mécaniques aux unités coupléesZhang, Yang 19 April 2019 (has links)
Ce mémoire traite de nouveaux principes de conception qui sont inspirés par le couplage de deux unités représentant les différentes structures mécaniques. Les critères de conception optimale et les types d'unités combinées sont différents. Cependant, toutes les tâches sont considérées dans le couplage de ces unités. L'examen critique présenté dans le premier chapitre est divisé en trois sections en raison de la nature des problèmes traités: les robots marcheurs, les compensateurs de gravité et les robots collaboratifs. Le deuxième chapitre traite du développement de robots marcheurs à actionneur unique, conçus par couplage de deux mécanismes ayant les fonctionnants de jambe. Basée sur l'algorithme génétique, la synthèse proposée permet d'assurer la reproduction de la trajectoire obtenue à partir de la marche humaine. Par l'ajustement des paramètres géométriques des unités conçues, il devient possible non seulement d'assurer une marche du robot à des pas variables, mais également de monter les escaliers. Ensuite la conception et la synthèse des équilibreurs pour les robots sont considérés. Un costume robotisé type exosquelette permettant d'aider aux personnes transportant des charges lourdes est examiné dans le chapitre suivant La conception proposée présente une symbiose d'un support rigide et léger et d'un système de câbles monté sur ce support. L'étude et l'optimisation statique et dynamique ont conduit aux tests sur un mannequin. Le dernier chapitre propose l'étude et 'optimisation d'un système couplé, comprenant un manipulateur équilibré à commande manuelle et un robot collaboratif. Le but d'une telle coopération est de manipuler de lourdes charges avec un cobot. / This thesis deals with the design principles, which arc based on the coupling of two mechanical structures. The criteria for optimal design and the types of combined units are different. However, all the tasks are considered in coupling of given mechanical units. The critical review given in the first chapter is divided into three sections due to the nature of the examined problems: legged walking robots, gravity compensators used in robots and collaborative robots. Chapter two deals with the development of single actuator walking robots designed by coupling of two mechanisms. Based on the Genetic Algorithm, the synthesis allows one to ensure the reproduction of prescribed points of the given trajectory obtained from the walking gait. By adjusting the geometric parameters of the designed units, it becomes possible not only to operate the robot at variable steps, but also to climb the stairs. The next chapter deals with the design and synthesis of gravity balancers. A robotic exosuit that can help people carrying heavy load is the subject of chapter four. The proposed exosuit presents a symbiosis of two systems: rigid lightweight support and cable system. Static and dynamic studies and optimization are considered. Experiments are also carried out on a mannequin test bench. The last chapter presents a coupled system including a hand-operated balanced manipulator and a collaborative robot. The aim of such a cooperation is to manipulate heavy payloads with less powerful robots. Dynamic analysis of the coupled system is perfonned and methods for reducing the oscillation of the HOBM at the final phase of the prescribed trajectories are proposed.
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A Coupled Human and Natural Systems Approach to Understanding an Invasive Frog, Eleutherodactylus Coqui, in HawaiiKalnicky, Emily A. 01 May 2012 (has links)
Human activities worldwide have altered nature in ways that create new combinations of species and environmental processes. To understand so-called "novel ecosystems" it is important to consider both the natural and the societal factors that shape them, and how those factors are interconnected or "coupled." We used such an approach to explore options for managing a non-native invasive frog, the coqui, which has become established on the island of Hawaii and threatens to spread to other parts of the state.
The nighttime calls of the coqui create a nuisance for property owners when populations become dense enough, as often occurs in Hawaii where the frogs have no natural enemies. Humans have tried various ways to eliminate coqui on the island of Hawaii with little success. Therefore we studied how property owners cope with their presence, both through management practices and psychological coping strategies. We also examined results of those efforts. People whose properties had more frogs were more likely to take action to reduce their numbers, but also attitudes toward the coqui were less negative when people had grown used to having to share their properties with the frogs. For those who cannot cope psychologically, we found it would be possible to manage properties to reduce densities but only when leaf litter and low shrubs were completely removed from near a home. Information campaigns about managing coqui should be different when targeting people that already host frogs and those that do not.
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Investigation on Pre- and Postsynaptic Ca<sup>2+ </sup>Signaling in Neuronal Model SystemsKrjukova, Jelena January 2004 (has links)
<p>Communication between neuronal and non-neuronal is called volume transmission when the released neurotransmitter (NT) acts via diffusion and affects several target cells. Both the neurosecretory and postsynaptic cell responses are linked to [Ca<sup>2+</sup>]<sub>i</sub> elevations. </p><p>In the present thesis the role of pre-and postsynaptic Ca<sup>2+</sup> elevations has been investigated in the reconstituted "synapse" model comprised of NGF-differentiated PC12 and HEL cells as well as in SH-SY5Y neuroblastoma cells. In PC12 cells, both 70mM K<sup>+</sup> and nicotine triggered NT release, which could be detected as a secondary [Ca<sup>2+</sup>]<sub>i</sub> increase in surrounding HEL cells. Both secretagogues shared the same voltage-dependent Ca<sup>2+</sup> influx pathway as judged from the pharmacological profile blockers of voltage-gated Ca<sup>2+</sup> channels. The coupling of electrical responses to the activation of Ca<sup>2+</sup> signaling via muscarinic receptors in SH-SY5Y cells was also studied. These data revealed that depolarization caused a considerable potentiation of the muscarinic Ca<sup>2+</sup> response. The potentiated Ca<sup>2+</sup> increase was mainly dependent on the enhanced Ca<sup>2+</sup> influx and to a lesser extent on [Ca<sup>2+</sup>]<sub>i</sub> release from intracellular stores. A phospholipase C (PLC) activator, m-3M3FBS was used to further study the role of G-protein coupled receptor (GPCR)-coupled Ca<sup>2+</sup> signaling. However, it was found that m-3M3FBS instead triggered [Ca<sup>2+</sup>]<sub>i</sub> elevations independently of PLC activation. </p><p>In conclusion, the results indicate that the magnitude of NT release from PC12 cells is sufficient to cause a robust activation of neighboring target cells. Postsynaptic muscarinic signaling is amplified due to integration of electrical excitation and GPCR signaling. The PLC activator, m-3M3FBS is not suitable for studies of PLC-mediated signals in intact cells.</p>
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GaN Nanopore Arrays: Fabrication and CharacterizationWang, Yadong, Peng, Chen, Sander, Melissa, Chua, Soo-Jin, Fonstad, Clifton G. Jr. 01 1900 (has links)
GaN nanopore arrays with pore diameters of approximately 75 nm were fabricated by inductively coupled plasma etching (ICP) using anodic aluminum oxide (AAO) films as etch masks. Nanoporous AAO films were formed on the GaN surface by evaporating an Al film onto a GaN epilayer and subsequently anodizing the aluminum. To minimize plasma-induced damage, the template was exposed to CF4-based plasma conditions. Scanning electron microscopy (SEM) analysis shows that the diameter and the periodicity of the nanopores in the GaN were directly transferred from the original anodic alumina template. The pore diameter in the AAO film can be easily controlled by tuning the anodization conditions. Atomic force microscopy (AFM), photoluminescence (PL) and micro-Raman techniques were employed to assess the quality of the etched GaN nanopore surface. Such a cost-effective method to produce nano-patterned GaN template would be useful for growth and fabrication of III-Nitrides based nanostructures and photonic band gap materials. / Singapore-MIT Alliance (SMA)
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A true virtual windowRadikovic, Adrijan Silvester 17 February 2005 (has links)
Previous research from environmental psychology shows that human well-being suffers in windowless environments in many ways and a window view of nature is psychologically and physiologically beneficial to humans. Current window substitutes, still images and video, lack three dimensional properties necessary for a realistic viewing experience primarily motion parallax. We present a new system using a head-coupled display and image-based rendering to simulate a photorealistic artificial window view of nature with motion parallax. Evaluation data obtained from human subjects suggest that the system prototype is a better window substitute than a static image and has significantly more positive effects on observers moods. The test subjects judged the system prototype as a good simulation of, and acceptable replacement for, a real window, and accorded it much higher ratings for realism and preference than a static image.
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Model error space and data assimilation in the Mediterranean Sea and nested grids / Espace d'erreur et assimilation de données dans un modèle de la Mer Mediterranée et des grilles gigognes.Vandenbulcke, Luc 11 June 2007 (has links)
In this work, we implemented the GHER hydrodynamic model in the Gulf of
Lions (resolution 1/100°). This model is nested interactively in another model
covering the North-Western basin of the Mediterranean Sea (resolution 1/20°),
itself nested in a model covering the whole basin (1/4°). A data assimilation
filter, called the SEEK filter, is used to test in which of those grids observations taken in the Gulf of Lions are best assimilated. Therefore, twin experiments are used: a reference run is considered as the truth, and another run, starting from different initial conditions, assimilates pseudo-observations coming from
the reference run. It appeared that, in order to best constrain the coastal
model, available data should be assimilated in that model. The most efficient setup, however, is to group all the state vectors from the 3 grids into a single vector, and hence coherently modify the 3 domains at once during assimilation cycles.
Operational forecasting with nested models often only uses so-called passive
nesting: no data feedback happens from the regional models to the global model.
We propose a new idea: to use data assimilation as a substitute for the feedback.
Using again twin experiments, we show that when assimilating outputs
from the regional model in the global model, this has benecial impacts for the
subsequent forecasts in the regional model.
The data assimilation method used in those experiments corrects errors in the
models using only some privileged directions in the state space. Furthermore, these directions are selected from a previous model run. This is a weakness of the method when real observations are available. We tried to build new directions of the state space using an ensemble run, this time covering only the Mediterranean basin (without grid nesting). This led to a quantitative characterization of the forecast errors we might expect when various parameters and external forcings are affected by uncertainties.
Finally, using these new directions, we tried to build a statistical model supposed to simulate the hydrodynamical model using only a fraction of the computer resources needed by the latter. To achieve this goal, we tried out artifficial neural networks, nearest-neighbor and regression trees. This study constitutes only the first step toward an innovative statistical model, as in its present form, only a few degrees of freedom are considered and the primitive equation model is still required to build the AL method. We tried forecasting at 2 different time
horizons: one day and one week.
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Untersuchung und Modellierung von Wasserhaushalt und Stofftransportprozessen in grundwassergeprägten Landschaften am Beispiel der Unteren Havel / Experimental and model based investigations of water balance and nutrient dynamics of groundwater influenced floodplains - the example of the Lower Havel RiverKrause, Stefan January 2005 (has links)
Das
Ziel dieser Arbeit ist die Untersuchung der Wasserhaushaltsprozesse und Stofftransportvorgänge
innerhalb der grundwassergeprägten Talauenlandschaften von Tieflandeinzugsgebieten am Beispiel
der im Nordostdeutschen Tiefland gelegenen Havel. Die Arbeiten in verschieden skaligen
Teileinzugsgebieten der Havel beschäftigen sich dabei zum einen mit der experimentellen Untersuchung
und vorrangig qualitativen Beschreibung der Wasserhaushaltsdynamik, zum anderen
mit der Entwicklung eines zur quantitativen Analyse von Wasserhaushalts- und Stofftransportprozessen
geeigneten Modells und der anschließenden Modellsimulation von Wasserhaushalt
und Stickstoffmetabolik im Grundwasser sowie der Simulation von Landnutzungs- und
Gewässerstrukturszenarien.<br><br>
Für die experimentelle Untersuchung der
Abflussbildung und der Wasserhaushaltsprozesse in den Talauenlandschaften des Haveleinzugsgebiets
wurde Einzugsgebiet der
”Unteren Havel Niederung“ ein umfangreiches Messnetz
installiert. Dabei wurden an mehreren Messstationen und Pegeln meteorologische
Parameter, Bodenfeuchte sowie Grundwasserstände und Abflüsse beobachtet. Die Analyse
der Messergebnisse führte zu einem verbesserten Verständnis von Wasserhaushaltsprozessen
in der durch das oberflächennahe Grundwasser und die Oberflächengewässerdynamik beeinflussten Talauenzone. Darüber hinaus konnten durch die Implementierung der Messergebnisse
konsistente Anfangs- und Randbedingungen für die Wasserhaushalts- und Grundwassermodellierung
im Modellkonzept IWAN realisiert werden.
Mit dem Modell IWAN (Integrated Modelling of Water Balance and Nutrient
Dynamics) wurde ein Werkzeug geschaffen, welches die Berücksichtigung spezifischer
hydrologischer Eigenschaften von Tieflandauen, wie z. B. den Einfluss des oberflächennahen
Grundwassers bzw. der Dynamik von Oberflächenwasserständen auf den Wasserhaushalt, ermöglicht. Es basiert auf der Kopplung des deterministischen distribuierten hydrologischen Modells
WASIM-ETH mit dem dreidimensionalen Finite-Differenzen-basierten Grundwassermodel
MODFLOW. Die Modellierung der Stickstoffmetabolik im Grundwasser erfolgt durch das mit Grundwassermodell
gekoppelte Stofftransportmodel MT3D.
Zur modellbasierten Simulation des Wasserhaushalts der Tieflandauenlandschaften wurde das Modellkonzept IWAN für verschieden skalige Teileinzugsgebiete
an der Havel für Simulationszeiträume von 2 Wochen
bis zu 13 Jahren angewandt. Dabei wurden die Teilmodelle für Wasserhaushalts- und Grundwassermodellierung
in zwei unterschiedlichen Teileinzugsgebieten der
”Unteren Havel Niederung“
kalibriert. Die anschließende Validierung erfolgte für das gesamte Einzugsgebiet der
”Unteren
Havel“. Die Unsicherheiten des Modellansatzes sowie die Anwendbarkeit des Modells im Untersuchungsraum
wurden geprüft und die Limitierung der Übertragbarkeit auf andere grundwasserbeeinflusste
Tieflandeinzugsgebiete analysiert. Die Ergebnisse der Wasserhaushaltssimulationen
führen einerseits zum erweiterten Prozessverständnis des Wasserhaushalts in Flachlandeinzugsgebieten,
andererseits ermöglichten sie durch die Quantifizierung einzelner Prozessgrößen die
Beurteilung der Steuerungsfunktion einzelner Wasserhaushaltsprozesse. Auf der Basis lokaler
Simulationsergebnisse sowie geomorphologischer und gewässermorphologischer Analysen
wurde ein Algorithmus entwickelt, welcher die Abgrenzung des direkten Eigeneinzugsgebiets
der Havel als Raum der direkten Interaktion zwischen Oberflächengewässer und umgebendem
Einzugsgebiet beschreibt. Durch Simulation des Wasserhaushalts im Eigeneinzugsgebiet mit
dem Modell IWAN konnten die Interaktionsprozesse zwischen Fluss und Talauenlandschaft
quantitativ beschrieben werden. Dies ermöglichte eine Bewertung der Abflussanteile aus dem
Eigeneinzugsgebiet sowie eine Quantifizierung der zeitlich variablen Retentionskapazität der
Auenlandschaft während Hochwasserereignissen.
Zur Abschätzung des Einflusses veränderter Landnutzung und angepassten
Managements auf den Wasserhaushalt der Talaue wurden Szenarien entwickelt, welche
Änderungen der Landnutzung sowie der Gewässergeometrie implizieren. Die Simulation
des Wasserhaushalts unter jeweiligen Szenariobedingungen ermöglichte die detaillierte Analyse
sich ändernder Randbedingungen auf den Gebietswasserhaushalt und auf die Austauschprozesse
zwischen Grundwasser und Oberflächengewässer.
Zur Untersuchung der Stickstoffmetabolik
im Grundwasser der Talauenlandschaft wurde das im Modellkonzept IWAN integrierte
Stofftransportmodell MT3D für das Eigeneinzugsgebiet der Havel angewandt. Dies ermöglichte eine Bilanzierung der aus dem Grundwasser des Eigeneinzugsgebiets stammenden
Nitratfrachtanteile der Havel sowie von Nitratkonzentrationen im Grundwasser. Szenariensimulationen, welche verminderte
Nitrateinträge aus der durchwurzelten Bodenzone annehmen, ermöglichten die Quantifizierung der Effizienz von Managementmaßnahmen
und Landnutzungsänderungen in Hinblick auf die Minimierung von Einträgen
in Grundwasser und Oberflächengewässer. / For a sustainable management of lowland river basins, a comprehensive knowledge about floodplain
water balance and nutrient transport processes is required. This investigation aims to
analyse water balance processes and nutrient dynamics and transport within the groundwater
influenced floodplains of lowland areas. Thus, the investigation was focused on the Havel river
catchment at the Northeast German Lowlands, which is a typical example of a lowland
floodplain. Experimental investigations were performed at different spatial scales for qualitative
analysis of water balance. The complex model IWAN was developed which enables the
simulation and quantitative analysis of water balance and nutrient dynamics. Furthermore it allows
the scenario based simulation and analysis of changing landuse management and boundary
conditions.<br><br>
For experimental investigation of runoff generation
and water balance processes within the floodplains of the Havel river an extensive measurement
campaign was installed at several testsites of the 198 km2 catchment of the “Lower Havel river
basin“. These testsites include stations and gauges for the observation of meteorological parameters,
soil moisture, groundwater depths and river runoff. Analysis of the observed data led to
an improved understanding of water balance processes within the specific part of the floodplain
which is influenced by the dynamics of the uppermost groundwater and by the surface water
dynamics. Furthermore the implementation of the observed data within the model concept realised
the consistent definition of time variable, spatial distributed initial and boundary conditions.
The IWAN model was developed as a tool which implements the speci-
fic hydrological characteristics of floodplains within the concept of modelling water balance
and nutrient dynamics. It is based on the coupling of the distributed deterministic hydrological
model WASIM-ETH with the three-dimensional finite difference based numerical groundwater
model MODLFOW. Simulation of the metabolism of nitrogen within the groundwater
passage was realised by the nutrient transport model MT3D which was coupled with the
groundwater model.
For model based simulation of the water balance within lowland
river floodplains the IWAN model was applied for different scaled subcatchments of the Havel
river with varying sizes from 2 to 1000 km2 and simulation periods from 2 weeks up to 13 years.
Calibration of the model was performed for two different sized subcatchments of the “Lower Havel river basin“. The subsequent validation of the model focused on the entire “Lower Havel
river basin“. Uncertainties of the model approach and the limited applicability and transferability
for further groundwater influenced floodplain landscapes were analysed. The results of
the water balance simulations led to an improved understanding of the processes and dynamics
within floodplains. It furthermore enabled the quantification and impact analysis of certain processes
and interactions. Based on local simulation results and on the analysis of surface and
groundwater morphology an algorithm was developed which was used for delineation of the direct
catchment of the Havel river. This direct catchment is specified as the part of the floodplain
which is characterised by the direct interaction between river and adjacent catchment. Water
balance simulations with the IWAN model in the direct catchment led to the quantification of
interaction processes between river and floodplain. This enabled the assessment of the runoff
fraction from the direct catchment during the ecologically sensitive low flow periods in summer
and of the retention capacity of the floodplain during flood events.
For the evaluation of the influences of alternative landuse management
on the water balance within floodplains, complex scenarios were developed which implemented
alterations of landuse or changes of surface water geometries and drainage structures. Simulation
of water balances for each scenario allowed the detailed analysis of changing boundary
conditions on the floodplain water balance and on the interaction processes between groundwater
and river.
Modelling nitrogen metabolism within the groundwater: For investigation of the nitrogen
metabolism within the groundwater of floodplains the nutrient transport and dynamics model
MT3D, which considers also interactions between groundwater and surface waters, was integrated
in the IWAN concept. The model was applied for the simulation of nitrate dynamics within
the direct catchment of the Havel river.With this approach, the nitrate loads between groundwater
and river could be quantified. In addition, nitrate concentrations within the groundwater were
analised in dependence of surface water dynamics. Scenario simulations, assuming a decrease
of incoming nitrate loads from the root zone, caused by landuse techniques, led to the quantification
of the efficiency of landuse changes and advanced management strategies to inhibit
pollution of groundwater and surface waters.
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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 informationLoßow, 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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Functional Studies of the Neuropeptide Y System : Receptor-Ligand Interaction and Regulation of Food IntakeÅkerberg, Helena January 2009 (has links)
The members of the mammalian neuropeptide Y family, i.e. the peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), are all involved in regulation of food intake. In human and most other mammals they act via receptors Y1, Y2, Y4 and Y5. NPY is released in the hypothalamus and is one of the strongest appetite-stimulating neurotransmitters whereas PP and PYY are secreted from gut endocrine cells after meals and function as appetite-reducing hormones. This thesis describes studies of the NPY system at both the molecular and the physiological level. The first part describes two investigations of receptor-ligand interactions with the human Y1 and Y2 receptors. The results clarify the importance of several amino-acid residues of the human Y1 receptor. Three amino acids previously suggested by others to form a binding pocket for the carboxy-terminus of the peptide were confirmed to be crucial for interaction with peptide ligands. However, they were found to be too distantly located from each other to be able to form a binding pocket. Further investigation of the three corresponding positions in the human Y2 receptor showed that only one of the positions was important for interaction with full-length peptides. The results indicate overlapping but, surprisingly, non-identical binding of the different peptides to human Y1 and Y2 receptors, despite the fact that the two receptors share a common ancestor. The second part of the thesis describes an investigation of the effect of PP on food intake in six beagle dogs and a test for personality characteristics in dogs (TFPC). Treatment with physiological doses of PP decreased both the appetitive and the consummatory drive but had no effect on the amount food consumed. The TFPC protocol was used to map individual behavioral differences in a population of sixteen beagle dogs. The test, which included several situations that may appear in an experimental study, revealed considerable inter-individual differences in behavioral responses despite the fact that the dogs were born and housed in the same animal facility in constant controlled conditions. These results demonstrate that PP can influence food intake in distantly related mammals and emphasize the importance of considering differences in personality in experimental animals.
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Investigation on Pre- and Postsynaptic Ca2+ Signaling in Neuronal Model SystemsKrjukova, Jelena January 2004 (has links)
Communication between neuronal and non-neuronal is called volume transmission when the released neurotransmitter (NT) acts via diffusion and affects several target cells. Both the neurosecretory and postsynaptic cell responses are linked to [Ca2+]i elevations. In the present thesis the role of pre-and postsynaptic Ca2+ elevations has been investigated in the reconstituted "synapse" model comprised of NGF-differentiated PC12 and HEL cells as well as in SH-SY5Y neuroblastoma cells. In PC12 cells, both 70mM K+ and nicotine triggered NT release, which could be detected as a secondary [Ca2+]i increase in surrounding HEL cells. Both secretagogues shared the same voltage-dependent Ca2+ influx pathway as judged from the pharmacological profile blockers of voltage-gated Ca2+ channels. The coupling of electrical responses to the activation of Ca2+ signaling via muscarinic receptors in SH-SY5Y cells was also studied. These data revealed that depolarization caused a considerable potentiation of the muscarinic Ca2+ response. The potentiated Ca2+ increase was mainly dependent on the enhanced Ca2+ influx and to a lesser extent on [Ca2+]i release from intracellular stores. A phospholipase C (PLC) activator, m-3M3FBS was used to further study the role of G-protein coupled receptor (GPCR)-coupled Ca2+ signaling. However, it was found that m-3M3FBS instead triggered [Ca2+]i elevations independently of PLC activation. In conclusion, the results indicate that the magnitude of NT release from PC12 cells is sufficient to cause a robust activation of neighboring target cells. Postsynaptic muscarinic signaling is amplified due to integration of electrical excitation and GPCR signaling. The PLC activator, m-3M3FBS is not suitable for studies of PLC-mediated signals in intact cells.
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