Thèses sur le sujet « MFS proteins »
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Debbiche, Rim. « Influence des lipides sur la dynamique du transport du fer médié par la ferroportine-1 et sa modulation par des composés amphiphiles ». Electronic Thesis or Diss., Brest, 2024. http://www.theses.fr/2024BRES0006.
Texte intégralFerroportin-1(FPN1), the only known mammalian iron exporter, is expressed on the surface of various specialized cells involved in iron metabolism. This protein belongs to the Major Facilitator Superfamily (MFS) and releases intracellular iron through conformational changes oscillating between an open structure towards the cytoplasm (Inward-Facing) and an open structure towards the bloodstream (Outward-Facing; OF). It has been reported that FPN1 is preferentially localized in lipid-rafts, microdomains of the plasma membrane particularly enriched in cholesterol (CHOL). Early in the thesis, we hypothesized that direct interactions between FPN1 and surrounding lipids, notably CHOL, are necessary to stabilize FPN1 in the OF conformation and/or promote certain conformational changes. I confirmed the preferential colocalization of FPN1 in the lipid rafts of human embryonic kidney cells. The dependence of FPN1's iron export function on CHOL was examined by depletion/repletion (CHOL/epicholesterol). Mutational screening experiments supported by structural analyses of the experimental 3D structure of FPN1 in an OF state have identified three possible CHOL-binding sites (of the CARC/CRAC type). Based on molecular dynamics simulations in a simplified POPC-type lipid environment, we identify certain interactions between charged residues of the human FPN1 3D structure and the polar heads of the surrounding phospholipids, which could facilitate conformational changes of the transporter. Besides, I show, for the first time, that FPN1 function is modulated by synthetic amphiphilic compounds, ohmline and its derivatives. Through the development of a novel in vitro approach (PLA: Proximity Ligation Assay), I show that ohmline delocalizes FPN1 from lipid-rafts, thereby decreasing its interaction with its functional partner, ceruloplasmin (CP), a ferroxidase that catalyzes the oxidation of ferrous iron, and consequently its iron export function
Yousefian, Narek. « The three-component multidrug MFS-type efflux pump EmrAB-TolC from Escherichia coli : from cloning to structural analysis ». Thesis, Bordeaux, 2020. http://www.theses.fr/2020BORD0065.
Texte intégralCurrently, due to the misuse of antibiotics, we are facing a major public health problem. The resistance to antibiotics of certain bacterial strains makes the treatment of infections very complex. In this context, the present thesis project concerns the study of a bacterial efflux complex capable of transporting antibiotics from the cytoplasm to the outside of the cell. This complex is composed of an inner-membrane Major Facilitator Superfamily (MFS) transporter (EmrB, E. coli multidrug resistance), a channel of the outer membrane TolC (Tolerance to Colicin E1) and a periplasmic adapter (EmrA, E. coli multidrug resistance). Unlike RND-type efflux systems (such as AcrAB-TolC), little is known about the MFS-type EmrAB-TolC system. It is therefore important to study the entire complex on a structural and functional level, to analyse the marked differences between these two types of transport systems. The goal of my thesis project was to study at least one EmrAB-TolC complex from a structural point of view. For my studies the aim was to isolate the complex directly from bacteria overexpressing the three protein partners. In a first step, 15 homologous EmrAB-TolC systems were identified and their corresponding genes amplified from genomic DNA of different Gram-negative bacteria. Among the genes of the 15 systems, the genes coding for the E. coli and V. cholerae systems were further studied. The expression vectors encoded fluorescent markers for the monitoring of the expression levels of different proteins and for studying the formation of complexes. In a first step, the different protein expression levels (EmrB-mRFP1 and EmrA-sfGFP) were studied for several expression strains of E. coli by measuring the red and green fluorescence levels and by Western blot (anti-His, Myc, and Strep for EmrB, EmrA, and TolC). The E. coli strain C41(DE3) was best suited for co-expression of EmrAB-TolC. In a second step, the FSEC (Fluorescence detection Size Exclusion Chromatography) methodology was used to identify a complex suitable for structural study. Thus this method enabled the observation that the EmrAB-TolC complex of E. coli was produced in higher amount than that of V. cholerae. The final co-purification protocol consists in perfoming a gentle lysis of the bacteria using lysozyme, then after solubilization with DDM, the purification is started by a Ni2+-NTA affinity chromatography step followed by a size exclusion chromatography step. Finally, the fractions containing the three protein partners are used for the detergent-exchange by amphipol A8-35 before the structural study by electron microscopy. Negative stain EM-micrographs displayed elongated objects with a length of 33 nm in side view. An average image of EmrAB-TolC shows similarities to that of the AcrAB-TolC complex observed under similar conditions. Similarities included the characteristic densities of TolC. Whereas differences were found in the lower part of EmrAB which is thinner than the lower part of AcrAB. The densities visible above the amphipol-ring correspond to EmrA, which displays a channel-like structure as in AcrA. The channel however seems to extend further towards the amphipol belt. Since EmrB does not have an extended periplasmic domain as the RND proteins have, these densities are therefore solely assigned to EmrA. EmrA, on the other side, contacts TolC akin to the interaction of AcrA/MexA to their cognate outer membrane channels (TolC/OprM) in a ‘tip-to-tip’ fashion
Aufgrund des Missbrauchs von Antibiotika stehen wir derzeit vor einem großen Problem deröffentlichen Gesundheit. Die Antibiotikaresistenz bestimmter Bakterienstämme macht die Behandlungvon Infektionen sehr komplex.In diesem Zusammenhang befasst sich diese Arbeit mit der Untersuchung eines bakteriellenEffluxkomplexes, der Antibiotika vom Zytoplasma zur Außenseite der Zelle transportieren kann. DieserKomplex besteht aus einem Major Facilitator Superfamily (MFS) Transporter der inneren Membran(EmrB, E. coli multidrug resistance), einem Kanal der äußeren Membran TolC (Tolerance to Colicin E1)und einem periplasmatischen Adapter (EmrA, E. coli multidrug resistance).Im Gegensatz zu Effluxsystemen vom RND-Typ (wie AcrAB-TolC) ist über das EmrAB-TolCSystemvom MFS-Typ wenig bekannt. Es ist daher wichtig, den gesamten Komplex auf struktureller undfunktioneller Sicht zu untersuchen, um die deutlichen Unterschiede zwischen diesen beiden Arten vonEffluxsystemen zu analysieren.Ziel meiner Doktorarbeit war es, mindestens einen EmrAB-TolC-Komplex aus struktureller Sichtzu untersuchen. Ziel meiner Studien war es, den Komplex direkt aus Bakterien, die die dreiProteinpartner überexprimieren, zu isolieren. In einem ersten Schritt wurden 15 homologe EmrAB-TolCSystemeidentifiziert und ihre entsprechenden Gene aus der genomischen DNA verschiedenergramnegativer Bakterien amplifiziert. Unter den Genen der 15 Systeme wurden die Gene, die für die E.coli und V. cholerae Systeme kodieren, weiter untersucht. Die Expressionsvektoren codiertenfluoreszierende Marker zur Untersuchung der Expression verschiedener Proteine und zur Untersuchungder Komplexbildung. In einem ersten Schritt wurden die verschiedenen Niveaus der Proteinexpression(EmrB-mRFP1 und EmrA-sfGFP) für mehrere E. coli Expressionsstämme untersucht durch Messen derroten und grünen Fluoreszenzniveaus und durch Western Blot (Anti-His, Myc und Strep für EmrB, EmrAund TolC). Der Stamm von E. coli C41(DE3) war am besten für die Koexpression von EmrAB-TolC14 geeignet. In einem zweiten Schritt wurde die FSEC-Methode (Fluorescence Detection Size ExclusionChromatography) verwendet, um einen für Strukturuntersuchungen geeigneten Komplex zuidentifizieren. Somit konnte mit dieser Methode festgestellt werden, dass der EmrAB-TolC-Komplex vonE. coli in größerer Menge als der von V. cholerae produziert wurde.Das endgültige Ko-Reinigungsprotokoll besteht darin, eine sanfte Lyse der Bakterien unterVerwendung von Lysozym durchzuführen. Nach der Solubilisierung mit DDM wird die Reinigung durcheinen Ni2+-NTA Affinitätschromatographieschritt gefolgt von einemGrößenausschlusschromatographieschritt gestartet. Schließlich werden die Fraktionen, die die dreiProteinpartner enthalten, für den Detergensaustausch durch Amphipol A8-35 vor derStrukturuntersuchung durch Elektronenmikroskopie verwendet.EM-Aufnahmen mit negativer Kontrastierung zeigten längliche Objekte mit einer Länge von 33nm in Seitenansicht. Ein durch Mittlung der Partikel erhaltenes Bild von EmrAB-TolC zeigt Ähnlichkeitenmit dem des AcrAB-TolC-Komplexes, der unter ähnlichen Bedingungen beobachtet wurde.Ähnlichkeiten schlossen die charakteristischen Dichten von TolC ein. Während im unteren Teil vonEmrAB Unterschiede festgestellt wurden, der dünner ist als der untere Teil von AcrAB. Die über demAmphipolring sichtbaren Dichten entsprechen EmrA, das wie bei AcrA eine kanalartige Strukturaufweist. Der Kanal scheint sich jedoch weiter in Richtung des Amphipolgürtels zu erstrecken. Da EmrBkeine erweiterte periplasmatische Domäne aufweist wie die RND-Proteine, werden diese Dichten daherausschließlich EmrA zugeordnet. Auf der anderen Seite kontaktiert EmrA TolC, ähnlich der Interaktionvon AcrA/MexA mit ihren jeweiligen Außenmembrankanälen (TolC/OprM), von “tip-to-tip”
Symington, Vicki F. « Structure and function of nitrate and nitrite transporters, NrtA and NitA, from Aspergillus nidulans ». Thesis, University of St Andrews, 2009. http://hdl.handle.net/10023/748.
Texte intégralBayro, Marvin J. « Protein MAS NMR methodology and structural analysis of protein assemblies ». Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/57800.
Texte intégralVita. Cataloged from PDF version of thesis.
Includes bibliographical references.
Methodological developments and applications of solid-state magic-angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy, with particular emphasis on the analysis of protein structure, are described in this thesis. MAS NMR studies of biomolecules ranging from model peptides and proteins in crystalline form to amyloid fibrils and whole bacterial organelles are reported. The methods presented include novel pulse sequences and optimized pulse sequence elements, experimental approaches designed for multiple-spin systems, a protocol for efficient sequential resonance assignment of proteins in the solid state, and techniques to determine the inter-molecular organization of amyloid fibrils formed by moderately sized proteins. Notably, an efficient dipolar recoupling technique, bandselective radio frequency-driven recoupling (BASE RFDR), is introduced and combined with alternating 13C-12C labeling to yield highly sensitive 13C-13C correlation spectra between distant nuclei in proteins. Various applications of the BASE RFDR scheme are presented, including protein resonance assignment, determination of tertiary structure of amyloid fibrils, and variable-temperature studies of protein dynamics. The main biological systems analyzed are amyloid fibrils formed by the SH3 domain of P13 kinase (P13-SH3) and intact gas vesicles from anabaena flos-aquae, for which atomic-level structural information was previously unavailable. P13-SH3 (86 residues) is a system thoroughly studied as a model of protein misfolding and amyloid formation by a natively globular protein. Gas vesicles are bacterial buoyancy organelles, with walls composed almost entirely by a single protein (GvpA, 70 residues), whose formation and structure constitute a highly intriguing biophysical problem. Nearly complete 13C and 'IN resonance assignments and the molecular conformations of the polypeptide backbones of both P13-SH3 and GvpA have been obtained via MAS NMR spectroscopy, enabling the proposal of models for the structure of these two protein assembly systems. In addition, the tertiary structure of P13-SH3 amyloid fibrils has been elucidated by the application of novel methodology introduced in this thesis. Finally, investigations regarding the effects of temperature and protein dynamics on MAS NMR experiments and biomolecular dynamic nuclear polarization studies are presented.
by Marvin J. Bayro.
Ph.D.
Hunnewell, Mary E. « Probing for Conformational Changes in the Repair Enzyme Mfd Using Mutant Protein Constructs ». Connect to this title, 2008. https://scholarworks.umass.edu/theses/154.
Texte intégralAgarwal, Vipin. « Development and application of MAS solid state NMR methodologies to biomolecules ». Berlin mbv, 2009. http://d-nb.info/998718602/04.
Texte intégralVahl, Martin [Verfasser]. « Identifizierung von Liganden des G-Protein gekoppelten Rezeptors Mas und Mas-Sequenz-ähnlicher Rezeptoren / Martin Vahl ». Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2010. http://d-nb.info/102410494X/34.
Texte intégralRaynor, James E. Jr. « Characterization of the mas protein as an angiotensin ii receptor ». DigitalCommons@Robert W. Woodruff Library, Atlanta University Center, 1994. http://digitalcommons.auctr.edu/dissertations/2831.
Texte intégralJordan, Katherine Jo. « Guanidine-stable chymoelastase : a comparative study of its hydrolytic specificity in the presence and absence of denaturant ». Virtual Press, 1987. http://liblink.bsu.edu/uhtbin/catkey/481688.
Texte intégralShevelkov, Veniamin. « Development of MAS solid state NMR methods for structural and dynamical characterization of biomolecules ». Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16260.
Texte intégralUnderstanding the mechanisms how biological systems work is an important objective of current structural biology. Nuclear magnetic resonance (NMR) spectroscopy is a well suited technique to approach these goals and to study structure and dynamics of biomolecules in order to obtain complimentary information for understanding functionality of proteins. Recently, rapid progress has been made in the field of biological solid state NMR (ssNMR), which resulted in complete structure elucidation of several peptides and small proteins, the characterization of protein complex formation and the characterization of dynamic properties of small proteins. Solid state NMR is the method of choice for structural and dynamic characterization of membrane proteins and aggregated amyloidogenic systems, which are poorly soluble and can not be easily studied by solution state NMR and X-ray spectroscopy. Modern solid state NMR is still limited in resolution and sensitivity, and requires developments in sample preparation and pulse sequence design. In my thesis, I study the potential use of deuteration in protein solid state NMR for sensitivity, as well as for resolution enhancement in 15N-1H correlation experiments. Achieved progress in these fields allows to monitor backbone motion with high accuracy, which has not been available before. We show for the first time that TROSY type experiments can be beneficial for solid state NMR. In addition, a pulse sequence for 13C-13C J decoupling was developed to increase resolution in the carbon dimension.
Friedrich, Daniel [Verfasser]. « Establishing Advanced MAS NMR Methods to Investigate Protonation Dynamics in Proteins / Daniel Friedrich ». Berlin : Freie Universität Berlin, 2018. http://d-nb.info/1176640615/34.
Texte intégralCisneros, Delgadillo Fiorella Melina. « Maize fine streak virus (MFSV) gene expression and protein interaction ». The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1366204177.
Texte intégralWilson, Theodore James. « Taking shape : regulating mitochondria morphology through alternative splicing and phosphorylation of fission factor proteins ». Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/4796.
Texte intégralWeldai, Lydia. « Do Major Facilitator Superfamily Domain Containing Proteins Respond to Glucose Starvation ? » Thesis, Uppsala universitet, Institutionen för farmaceutisk biovetenskap, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-348673.
Texte intégralHiller, Matthias. « Sample preparation of membrane proteins suitable for solid-state MAS NMR and development of assignment strategies ». Phd thesis, Universität Potsdam, 2009. http://opus.kobv.de/ubp/volltexte/2009/3724/.
Texte intégralBiologische Membranen bestehen hauptsächlich aus Lipiden, ihre Funktion wird jedoch vor allem durch die eingebetteten Membranproteine (z.B. Kanäle, Ionenpumpen und Rezeptoren) bestimmt. Mutationen in dieser Proteinklasse können zum Auftreten verschiedener Krankheitsbilder führen, weshalb die Untersuchung der dreidimensionalen Struktur von Membranproteinen nicht nur von strukturbiologischem, sondern auch von pharmakologischem Interesse ist. In den letzten Jahren wurde eine Methode, die Festkörper NMR Spektroskopie, für Strukturuntersuchungen an Proteinproben im festen Aggregatzustand entwickelt. Diese Arbeit beschäftigt sich mit drei verschiedenen Präparationsarten von Membranproteinen, die eine Aufnahme von hochaufgelösten Festkörper NMR Spektren erlauben. Als Modelsystem wurde das Protein G der äußeren Membrane (outer membrane protein G, OmpG) von Escherichia coli gewählt. Eine wichtige Vorraussetzung zur Berechnung der Proteinstruktur aus den NMR-Spektren, ist die Zuordnung der einzelnen Signale zur jeweiligen Aminosäure in der Proteinsequenz. In dieser Arbeit wurde eine Methode entwickelt, die das Auffinden von Startpunkten für die sequentielle Zuordnung in großen Membranproteinen, wie zum Bsp. OmpG (281 Aminosäuren), erlaubt. Multidimensionale NMR Experimente mit verschieden spezifisch markierten Proben wurden durchgeführt und ermöglichten die Zuordnung von 50 % der NMR Signale der OmpG Proteinsequenz. Zur Überprüfung der gewonnenen Daten wurden diese zur Vorhersage von Sekundärstrukturelementen genutzt. Es konnte gezeigt werden, dass die berechneten Strukturmotive in guter Übereinstimmung zu den bisher veröffentlichten OmpG Strukturen liegen. Die in dieser Arbeit angewendeten Methoden sollten auf eine Vielzahl anderer Membranprotein anwendbar und somit einen neuen Weg zur Strukturbiologischen Untersuchung von Membranproteinen eröffnen.
Ma, Qijun [Verfasser]. « Protein interactions in living cells studied by multiparameter fluorescence imaging spectroscopy (MFIS) / Qijun Ma ». Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2016. http://d-nb.info/108283713X/34.
Texte intégralThomas, Lars, Julian Kahr, Peter Schmidt, Ulrike Krug, Holger A. Scheidt et Daniel Huster. « The dynamics of the G protein-coupled neuropeptide Y2 receptor in monounsaturated membranes investigated by solid-state NMR spectroscopy ». Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-193569.
Texte intégralThebault, Sabine. « Caractérisation d'une nouvelle protéine humaine contenant un domaine homologue à l'I-mfa : implication dans la régulation de l'expression de deux rétrovirus humains, HTLV-I et HIV-1 ». Montpellier 1, 2000. http://www.theses.fr/2000MON1T013.
Texte intégralPelé, Julien. « ANALYSE EVOLUTIVE DES RECEPTEURS COUPLES AUX PROTEINES G (RCPG) ». Phd thesis, Université d'Angers, 2010. http://tel.archives-ouvertes.fr/tel-00858597.
Texte intégralShahid, Shakeel Ahmad [Verfasser]. « Structure and function of the autotransporter protein YadA : a solid-state MAS NMR study / Shakeel Ahmad Shahid ». Berlin : Freie Universität Berlin, 2012. http://d-nb.info/1030383162/34.
Texte intégralThomas, Lars, Julian Kahr, Peter Schmidt, Ulrike Krug, Holger A. Scheidt et Daniel Huster. « The dynamics of the G protein-coupled neuropeptide Y2 receptor in monounsaturated membranes investigated by solid-state NMR spectroscopy ». Journal of biomolecular NMR (2015) Apr, 61 (3-4) : S. 347-359, 2015. https://ul.qucosa.de/id/qucosa%3A14207.
Texte intégralLange, Adam. « Three-dimensional protein structure determination by high-resolution solid-state NMR spectroscopy ». Doctoral thesis, [S.l.] : [s.n.], 2006. http://webdoc.sub.gwdg.de/diss/2006/lange.
Texte intégralAsami, Sam. « Method development for biomolecular solid-state NMR spectroscopy ». Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2014. http://dx.doi.org/10.18452/17044.
Texte intégralIn this thesis, a novel labeling scheme for solid-state NMR spectroscopy, the Reduced Adjoining Protonation (RAP) scheme, is introduced, which allows proton detection of all aliphatic sites, as shown for the microcrystalline SH3 domain of alpha-spectrin. These samples yield high-resolution, 1H-detected 1H,13C correlation spectra. In addition, the benefit of high MAS frequencies was investigated. 1H- and 13C-detected 3D assignment experiments are implemented, which allowed us to assign 90% of all aliphatic resonances of alpha-spectrin SH3. As the chemical shift is dependent on the structural motif, it can be employed to derive secondary structure information. Furthermore, a 1H-detected H(H)CH 3D experiment is introduced, to obtain long-range 1H,1H contacts, which can be used for the determination of the tertiary structure. To obtain artifact-free relaxation data, the RAP labeling scheme was modified to obtain sparsely proton labeled, 13C dilute samples, in which spin diffusion is suppressed. To probe sub-microsecond dynamics, we report experiments to determine 13C T1 relaxation times and 1H,13C dipolar coupling tensors for backbone and side chain resonances, respectively. Furthermore, we show, that the RAP labeling scheme can be applied to non-crystalline systems, such as amyloid fibrils of the Alzheimer’s disease peptide Abeta1-40. Using 1H-detection, we obtained high-resolution 1H,13C correlation spectra. Finally, we applied the perdeuteration approach to the L7Ae-box C/D protein-RNA complex from P. furiosus. We obtained high-resolution, 1H-detected 1H,15N, as well as 13C,13C correlation spectra of the protein-RNA complex. In addition, we established a methodology to determine accurate distance and angular restraints for the protein-RNA interface and propose approaches for the chemical shift assignment of RNA resonances.
Karhu, T. (Toni). « Isolation of novel ligands for MAS-related G protein-coupled receptors X1 and X2, and their effect on mast cell degranulation ». Doctoral thesis, Oulun yliopisto, 2017. http://urn.fi/urn:isbn:9789526216331.
Texte intégralTiivistelmä Syöttösolut on tärkeä osa ihmisen immuunijärjestelmää. Ne ovat tärkeitä tulehdus- ja fysiologistenprosessien säätelijöitä. Syöttösolujen vaikutus välittyy degranulaation ja siinä vapautuvien tulehdusvälittäjäaineiden kautta. Vapautuviin aineisiin lukeutuu esim. histamiini ja lukuisia sytokiinejä, sekä proteaaseja. Syöttösolujen aktivaatio voi tapahtua immunoglobuliineista riippuvaa tai immunoglobuliineista riippumatonta reittiä pitkin. Monet ei-immunologiset tekijät voivat laukaista jälkimmäisen reitin ja kaksi uutta tähän vaikuttavaa G-proteiinikytkentäistä reseptoria on löydetty, MAS-related G protein-coupled receptor X1 (MRGPRX1) ja X2. MRGPRX1:llä ja MRGPRX2:lla on kaksi tunnettua tehtävää: i) ne laukaisevat syöttösolujen degranulaation ja ii) ne osallistuvat kivun ja kutinan aistimiseen tietyissä tuntohermoissa. Näitä reseptoreita ei ilmennetä kaikissa syöttösoluissa, vaan ainoastaa tryptaasia ja kymaasia sisältävissä syöttösoluissa, ja täten osaltaan selittävät syöttösolujen monimuotoisuutta. Useimmista G-proteiinikytkentäisistä reseptoreista poiketen MRGPRX1 ja MRGPRX2 ovat laajakirjoisia, sitoen monia erilaisia ligandeja. Ligandeihin kuuluu endogeenisia neuropeptidejä, antimikrobiaalisia peptidejä ja proteiinin fragmentteja, sekä synteettisiä yhdisteitä kuten erilaisia antibiootteja. Reseptoreiden endogeeniset ligandit voivat toimia laukaisijana jossain syöttösoluihin liittyvissä sairauksissa, degranuloidessaan syöttösoluja ja aiheuttaen paikallisen tulehdustilan. Reseptoreiden laajakirjoisuudesta johtuen niillä on oletettavasti monia vielä tuntemattomia ligandeja. Tämän tutkimuksen tarkoitus oli eristää uusia endogeenisiä ligandeja MRGPRX1:lle ja MRGPRX2:lle ihmisen kudoksista ”kääteisfarmakologista lähestymistapaa” hyödyntäen ja selvittää ligandien kyky syöttösolujen degranulaatioon. Lähtömateriaalina käytetyt ihmisen verihiutaleet ja plasma sisälsivät MRGPRX1:ta ja MRGPRX2:ta aktivoivia yhdisteitä. Plasmasta eristettiin ja sekvensoitiin kolme albumiinin fragmenttia, jotka aktivoivat MRGPRX2:ta. Nämä fragmentit aktivoivat MRGPRX2:ta ja degranuloivat syöttösoluja annosriippuvaisesti. Kaksi MRGPRX1:tä aktivoivaa hemoglobiinin β-ketjun fragmenttia eristettiin ihmisen verihiutaleista. Nämä fragmentit tunnistettiin hemorfiineiksi ja ne aktivoivat MRGPRX1:tä annosriippuvaisesti, mutta eivät vaikuttaneet syöttösolujen degranulaatioon
Hakizimana, Pierre. « Phosphatidylethanolamine regulates the function and the structure of LmrP, a bacterial multidrug transporter protein associated to antibiotic resistance ». Doctoral thesis, Universite Libre de Bruxelles, 2008. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210486.
Texte intégral
Doctorat en Sciences
info:eu-repo/semantics/nonPublished
Tureli, Akif Emre. « Antimicrobial Spectrum Determination Of The K5 Type Yeast Killer Protein And Its Kinetics Of Cell Killing ». Master's thesis, METU, 2005. http://etd.lib.metu.edu.tr/upload/12606847/index.pdf.
Texte intégral#946
-1,3- glucans. As mammalian cells lack cell walls research and development of novel highly selective antifungals are mostly focused on the agents which target the components of the fungal cell wall. We have previously characterized the K5 type killer protein. This protein is an exo &
#946
-1,3-glucanase which is stable at pH&rsquo
s and temperatures appropriate for its medical usage. &
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-1,3- glucan hydrolyzing activity of the K5 type killer protein highlighted the potential use of this protein as a selective antimycotic agent. Antifungal activity of the K5 type yeast killer protein was tested against 26 human pathogenic yeast and 9 dermathophyte strains and found to be affective on all of the tested strains. Toxin MIC50, MIC100 and MFC values were found to be between 0.25-4, 0.5-8, 1-8 µ
g/ml respectively except Candida krusei isolates. Cell killing analysis revealed that toxin activity starts within first 2 hours and complete cell death time differs due to the susceptibility of strains to the K5 type yeast killer protein. K5 type yeast killer protein would be used as a novel and selective agents with the results obtained from this study.
Bertarello, Andrea. « Magic-angle Spinning NMR of paramagnetic metalloproteins ». Thesis, Lyon, 2018. http://www.theses.fr/2018LYSEN004/document.
Texte intégralMost of our understanding of metalloproteins derives from atomic or molecular structures obtained from diffraction methods on single crystal samples. However, not all proteins are amenable for diffraction studies, and even when a highly-resolved structure is available, often the nature of the metal ion, its coordination geometry or its oxidation state are not determined. The aim of the present thesis is the investigation of structural properties of metal sites in paramagnetic metalloproteins by Magic-Angle Spinning Nuclear Magnetic Resonance (MAS NMR). MAS NMR is a powerful technique for the investigation of biological systems, and may represent a direct probe of the structure at the active site of paramagnetic metalloproteins. However, it suffers from limited sensitivity and resolution when applied to nuclei close to a paramagnetic center.In this thesis, we address these limitations by developing NMR methods based on ultra-fast (60-111 kHz) MAS rates. A “toolkit” of suitably designed pulse sequences is built for the detection and the assignment of nuclei in close proximity of a paramagnetic center. State-of-the-art computational techniques are also employed to convert the experimental data into structural restraints for obtaining atomic-resolution geometries of active sites. We benchmark this approach with the study of Fe, Cu and Co sites in two microcrystalline proteins, and we also provide preliminary data on a non-diffracting divalent metal ion transporter in lipid membranes. We anticipate that the techniques described here are an essential tool to elucidate many currently unanswered questions about structure and function of metal sites in structural biology
Marchetti, Alessandro. « Sviluppi metodologici per la cristallizzazione e l’analisi strutturale di proteine tramite Risonanza Magnetica Nucleare allo stato solido ». Doctoral thesis, Scuola Normale Superiore, 2012. http://hdl.handle.net/11384/85789.
Texte intégralVaidilaite-Pretorius, Agita. « Mechanistic approaches towards understanding particle formation in biopharmaceutical formations : the role of sufactant type and level on protein conformational stability, as assessed by calorimetry, and on protein size stability as assessed by dynamic light scattering, micro flow imaging and HIAC ». Thesis, University of Bradford, 2013. http://hdl.handle.net/10454/13482.
Texte intégralIsobe, Yuu. « Direct evidence for the age-dependent demise of GNAS-mutated cells in oral fibrous dysplasia ». Kyoto University, 2019. http://hdl.handle.net/2433/242351.
Texte intégralPASQUA, IRENE. « Significato clinico dell'espressione della proteina ZAP-70 nelle leucemie linfatiche croniche ». Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/1024.
Texte intégralDysfunctional apoptosis and cell cycle are the main reasons for the clinical enigma, that CLL can not yet be cured with conventional chemotherapy. In B-CLL, malignant cells seem to be arrested in the G0/early G1 phase of the cell cycle, and inhibition of spontaneous apoptosis and upregulation of the anti-apoptotic protein bcl-2 define clinical prognosis. However, increasing evidence exists that disease progression relies upon cycling B-CLL cells: a proliferating pool of cells has been described in lymph nodes and bone marrow and might feed the accumulating pool in the blood. Moreover, the lack of immunoglobulin (Ig) VH gene mutation also has been shown to predict a rapid disease progression (DP) and an inferior overall survival (OS) (Damle, Hamblin, 1999). B-CLL cells that use non-mutated IgVH genes express ZAP-70 RNA, which encodes ZAP-70, a 70-kDa protein tyrosine kinase, associated both with an enhanced B cell receptor signaling and with an early DP risk in B-CLL (Del Principe, 2006). Moreover, the today availability of rapamycin or proteasome inhibitors effective against proliferating B-CLL cells and bcl-2 antisense oligonucleotides prompted us to evaluate the real impact of proliferation and apoptosis pathways on B-CLL prognosis. The primary aims of our study were: 1) to determine progression-free survival (PFS) upon apoptosis/proliferation subgroups and ZAP-70 expression; 2) whether apoptosis/proliferation could predict varied outcome within ZAP-70 subgroups; and finally 3) whether ZAP-70 and apoptosis/proliferation groups were independent prognostic factors. Therefore we investigated 265 pts, median age 64 years (range 37-84), 136 males and 129 females. With regard to modified Rai stages, 87 patients had a low stage, 170 an intermediate stage and 8 a high stage. ZAP-70 was quantified by a multicolor flow cytometric method fixing a cut-off value of 20%. Bcl-2 was determined by flow cytometry, dividing mean fluorescence intensity (MFI) of CD19+B-CLL cells / MFI of T-cells (Bcl-2B/T). The threshold was set at the median value >1.6. Transferrin receptor (CD71) was used as a measure of the proliferation and the threshold was set at the median value >8%. Combining Bcl-2B/T with CD71 (Bcl2CD71) we enucleated three subgroups: 1) Bcl2CD71- [106 pts] with low proliferation (CD71 <8%) and high apoptosis (Bcl-2B/T <1.6); 2) Bcl2CD71+ [49 pts] with high proliferation (CD71>8%) and low apoptosis (Bcl-2B/T >1.6); and 3) Bcl2CD71+/- [110 pts] with low proliferation and low apoptosis or with high proliferation and high apoptosis. ZAP-70+ B-CLL patients were 95/265 (36%). In 111 studied pts ZAP-70 expression and Ig V gene mutational status were significantly correlated (p<0.00001). Furthermore, we found significant associations either between lower ZAP-70 and lower Bcl-2B/T index (p=0.001) or lower ZAP-70 and Bcl2CD71- (p=0.002), confirming that low levels of ZAP-70 were characterized by high apoptosis and low proliferation. With regard to clinical outcome, a significant shorter progression-free survival (PFS) was observed in ZAP-70+ pts vs ZAP-70 negative pts (0% vs 58% at 13 years; p<0.00001) and in Bcl2CD71+ pts vs Bcl2CD71- pts (10% vs 56% at 12 years; p<0.00001). The Bcl2CD71+/- subgroup showed an intermediate outcome (30% at 12 years). To further explore the prognostic impact of Bcl2CD71 index, we investigated its expression within ZAP70+ (95 pts) and ZAP70- (170 pts) subsets. As a matter of fact, Bcl2CD71 was not able to identify prognostic subsets within ZAP-70+ pts, because all these cases presented a shorter PFS without significant differences. On the other hand, this index identified subsets at different PFS within the ZAP-70 negative subgroup (73% for Bcl2CD71- pts vs 29% for Bcl2CD71+ at 12 years, p=0.00009). In multivariate analysis of PFS, in which age, Rai modified stages, CD38, soluble CD23 (sCD23), lymphocyte doubling time (LDT), Bcl-2CD71 and ZAP-70 entered, ZAP-70 (p=0.00005), LDT (p=0.006), Rai modified stages (p=0.03) and sCD23 (p=0.01) resulted to be independent prognostic factors. Therefore, ZAP-70 was confirmed as the most important indipendent prognostic factor with regard to PFS. However, our apoptotic/proliferative index (Bcl2CD71), performed by flow cytometry, was very useful to identify pts at different progression rate within the ZAP-70 negative subgroup. Since the ZAP-70 negative subset represents a large and heterogeneous B-CLL population with a variable progression, other biological factors, such as the amount of apoptosis and the proliferative rate, have to be added in order both to identify early progressive pts and to take timely accurate therapeutic decisions.
Hizbai, Biniam T. « Comparative Mapping of QTLs Affecting Oil Content, Oil Composition, and other Agronomically Important Traits in Oat (Avena sativa L.) ». Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23481.
Texte intégralMoss, Éric. « Etude in situ par RMN HRMAS sur des épidermes reconstruits du métabolisme et de la réactivité de xénobiotiques allergisants ». Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAF003/document.
Texte intégralContact dermatitis is a skin pathology particularly prevalent in industrialized countries. No therapy currently exists and only complete avoidance of the particular allergen can prevent an allergic reaction. Historically, the assessment of skin sensitisation potential of molecules placed on the market was always carried out by animal testing. However, the scope of this testing method is now limited by the new European cosmetics legislation. In this way, the development of alternative methods, not based on animal experimentation, become an important issue. Contact dermatitis results of a chemical key step: the formation of an antigenic complex allergen-protein complexe able to activate the cutaneous immune system. The aim of this PhD work was to study the in situ behaviour of allergens in reconstructed human epidermis (SkinEthic® model). By using an appropriate non-invasive analysis technique, HR-MAS NMR spectroscopy, it has been possible to study the mode of action of different allergens, from their possible activation through the metabolic pathway to the binding with epidermal proteins
Pavoni, Serena. « Mise au point d’un nouveau modèle d’organoïde cérébral humain pour l’étude des mécanismes d’interaction de la protéine prion et de l’amyloïde β ». Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS427.
Texte intégralPrion-like mechanisms are known to underlie most of human neurodegenerative diseases including Alzheimer’s disease (AD), which is characterized by two important pathological markers, β amyloid (or Aβ at the origin of the etiopathogenic amyloid cascade hypothesis) and phosphorylated tau protein. Furthermore, the prion protein (PrPC) interacts at multiple levels with the metabolism of Aβ, by mechanisms which are not well understood. To overcome the current limits in the development of efficient strategies to treat AD, the pharmaceutical industry requires innovative experimental models. However, even if a lot of progress has been achieved by using transgenic mouse models, to date no in vivo model can reflect the complexity of human brain or reproduce a clinical context. 2D in vitro cell culture models are unable to allow the aggregation and accumulation of pathological proteins as observed in vivo. The aim of this study consists in taking advantage of the research prospects offered by induced pluripotent stem cell (iPSCs) in the field of neurosciences. iPSCs can be used to generate 3D models of differentiation also called human cerebral organoids or mini-brains (MBs). Their ability to self-organise in 3D neuroectodermic tissue leds to a complex system that mimics different human cerebral structures in which we were able to characterize the expected markers. The study of the two proteins of interest (APP and PrPC) during neural differentiation has allowed us to follow the modulation of protein expression level occurring during the in vitro development of the human MBs. In order to use this model to reproduce the protein accumulation mechanisms seen in AD, we have tested chemical inductors such as Aftin-5 in order to modulate the APP post-transcriptional pathway towards a pathological outcome. Many strategies of treatment are adopted to lead APP cleavage and Aβ generation. The production of soluble fragments Aβ38, Aβ40, Aβ42 in the supernatant of organoids has been showed using ELISA technique. The levels generated are reproducible and the increase of Aβ42/Aβ40 ratio is consistent with extrapolated data from mouse and human models thus validating our model. Analysis at the gene and protein level has been assessed in order to understand the interaction between PrPC and APP after treatment. The long-term goal consists in improving this model which is notably hampered by the absence of vascularization and the low level of maturation of the neural tissue. The main challenge in MB culture thus consists in the integration of the vascular system, and also in increasing the speed of ageing process in vitro for the study of neurodegenerative diseases. In the long term, the prospect of automating the culture of MBs would allow the use of the system for cytotoxicity testing and/or high throughput screening for the discovery of new drugs for AD
Sousa, Ana Rita Lopes Nogal Lemos de. « Tool generation to characterize DTR1, a member of the poorly characterized DHA1 transporter family of proteins in yeast ». Master's thesis, 2021. http://hdl.handle.net/10400.1/16851.
Texte intégralO azoto é um nutriente mineral crítico em todos os organismos vivos, pois é necessário para a síntese de um grande número de compostos, incluindo hormonas, nucleotídeos e aminoácidos. Os aminoácidos são muito importantes para várias dinâmicas da levedura, como síntese de proteínas, metabolismo de hormonas, transmissão nervosa, crescimento celular, geração de energia, metabolismo do azoto e síntese de bases azotadas. Os aminoácidos também são importantes para fins industriais, como aplicações em alimentos como o aminoácidos glutamato (intensificador de sabor) ou aspartato, fenilalanina (adoçantes); para alimentação, tais como os aminoácidos lisina, metionina, treonina; e para aplicações farmacêuticas, como soluções de infusão e blocos de construção, triptofano (indutor do sono) e fenilalanina (antidepressivo). Este projeto visa a identificação e caracterização de novos transportadores envolvidos na excreção de aminoácidos por meio do estudo de um gene específico da superfamília MFS (Major Facilitator Superfamily) de compostos azotados em eucariontes, o gene DTR1. De acordo com, Sá-Correia et al., (2009) vários transportadores MDR foram identificados e estudados em diferentes organismos, particularmente aquele pertencentes à superfamília ATP-binding cassette (ABC). Um grupo de transportadores envolvidos na resistência a multidrogas menos caracterizado pertence à família MFS-MDR. Proteínas deste grupo têm vindo a receber mais atenção, maioritariamente em bactéria. Em Saccharomyces cerevisiae, a maioria dos membros desta família foi apenas descobertos aquando revelada a sequencia genómica desta levedura e caracterizados em alguns aspetos nos últimos 12 anos. A família MDR-MFS (Multidrug Resistance-Major Facilitator Superfamily) de proteínas tem aproximadamente 300 proteínas transportadoras de membrana, com até 100 sendo desconhecidas. 24 proteínas são MDR da superfamília MFS, algumas delas podem estar envolvidas na excreção de aminoácidos, como DTR1. Dentro da DHA1 (Drug: H+ antiporter family 1) que é composta por 46 proteínas, sete genes são individualmente necessários para conferir resistência à quinidina, um fármaco antiarrítmico e anti malária, entre esses genes encontram-se os genes AQR1, TPO1 e DTR1. Estes sete transportadores podem, também, proteger a célula contra outros compostos que estão normalmente ausentes no ambiente natural de células de levedura e poderão ter substratos fisiológicos específicos, onde fármacos são transportados fortuitamente ou oportunisticamente. Ainda na família DHA1, 9 proteínas mostraram ser bombas de multirresistência, 15 são provavelmente bombas de efluxo específicas para drogas e 22 são proteínas hipotéticas ou não caracterizadas. Os genes de resistência a múltiplas drogas, isto é, a aquisição simultânea de resistência a uma variedade de químicos citotóxicos, é encontrada numa grande variedade de organismos, desde bactérias a mamíferos, e isto poderá causa um severo problema clínico, principalmente no tratamento de cancros humanos e infeções de origem bacteriana e fúngica, tendo atingido proporções alarmantes nos últimos anos. DTR1 é a primeira proteína de resistência a múltiplas drogas da superfamília de facilitadores principais com um papel fisiológico atribuído na célula de levedura, mas o transporte por DTR1p pode não ser restrito ao seu substrato natural, bisformil ditirosina. As camadas de quitosana e ditirosina da parede externa dos esporos conferem maior resistência a estresses ambientais no esporo, incluindo a capacidade de passar pelo trato digestivo dos insetos, permitindo a dispersão para o meio ambiente. No presente estudo é realizada a caraterização do gene DTR1 através da sua localização e expressão com recurso a variadas ferramentas, tais como a proteína Green Fluorescent Protein, produzida pelo cnidário Aequorea victoria que emite fluorescência na zona verde do espectro visível, observando quando esta está a ser expressa no controle de seu próprio promoter. Uma fusão com o promotor Gal1, um operão procariótico que codifica as enzimas necessárias para o metabolismo da galactose, para verificar a sobrexpressão da proteína ou se a expressão é feita normalmente como no seu estado natural. Com essas fusões podemos analisar se a proteína está bem expressa, superexpressa e onde ela ocorre na célula e, usando plasmídeos identificados como GAP1 e UGA4 ambos contendo o promotor GAP1, ativado pela falta de azoto, para observação do comportamento do gene em estudo. Essas ferramentas contribuem para a caracterização a nível genómico e plasmídico da proteína de interesse, DTR1, para compreender sua função e localização no genoma da levedura. O objetivo principal deste trabalho foi construir uma cassete combinando primers para o plasmídeo, primeiro e para localização do DTR1 foi utilizado o plasmídeo PKT140, que contém a proteína GFP e resistência à canamicina. Os primers construídos são homólogos à proteína GFP, no caso do F5, e à resistência à canamicina, no caso do primer R3. A estratégia adotada foi a utilização de um fragmento do plasmídeo contendo os primers, GFP e a resistência à canamicina, que foi amplificado com um tamanho esperado de 2515 pares de bases e transformado em S. cerevisiae. A tag GFP foi aplicada a jusante do gene de interesse, DTR1. A canamicina foi usada como marca de seleção no momento da transformação e a integração no genoma da levedura foi concluída por recombinação homóloga. Para compreender se a proteína de interesse, DTR1, está a ser superexpressa, um fragmento do plasmídeo PGAL1 foi usado, novamente os primers foram construídos contendo parte de DTR1 e o promoter Gal1 no caso do iniciador R2 e parte de DTR1 e resistência à nourseotricina em o caso do primer F4. Para análise deste estudo recorreu-se a técnicas como transformação em E. coli e em S. cerevisiae, digestão com recurso a enzimas de restrição, PCR, RT-PCR (Reverse Transcriptase PCR), análise de expressão por qPCR (Quantitative Real Time PCR), Cross-feeding e ainda Western Blotting. O fragmento do plasmídeo mais primers foi amplificado por PCR com 1850 pares de bases e a integração no genoma da levedura é feita por recombinação homóloga. A nível funcional concluímos que não foi possível a construção da cassete PGAL1-DTR1-GFP tendo sido efetuadas várias tentativas da mesma não tendo sido possível a observação da sobrexpressão do gene DTR1 e a sua localização. A nível plasmídico foi possível uma observação de analise de expressão de diferentes estudos de comportamento da construção GAP1-DTR1 com alteração de meio. Foi ainda feito um estudo de influência do glicerol e temperatura em cultura de S. cerevisiae em meio mínimo. Tendo em conta que o gene DTR1 faz parte da reprodução de S. cerevisiae através esporulação em condições de stress, verificou-se que uma temperatura de 30ºC e glicerol 10% poderão representar condições de stress suficientes para que este gene seja expresso.
« Characterization of an orphan G protein-coupled receptor mas-induced tumor formation ». Thesis, 2005. http://library.cuhk.edu.hk/record=b6074087.
Texte intégralIn order to identify the cellular mechanism of mas-induced tumor formation, a full-length mas cDNA was cloned into a mammalian expression vector pFRSV with dihydrofolate reductase gene as a selection marker. Detailed analyses of mas-transfected cell lines by Southern blot, Northern blot and tumorigenicity assay indicated that tumorigenicity of mas-transfected cells depended on the sites of chromosomal integration and the levels of mas expression. These results suggest that overexpression of mas is not sufficient to induce tumor formation. In line with the ability of mas-transfected cells Mc0M80 to form solid tumor in nude mice, MTT cell proliferation assay indicated that the mas-transfected cells Mc0M80 proliferated faster than vector-transfected cells. Moreover, mas-transfected cells Mc0M80 exhibited significantly increased anchorage-independent growth. Furthermore, mas-transfected cells Mc0M80 showed higher percentage cells in G2/M phase but lower in S-phase in comparison with vector-transfected cells.
Interestingly, Southern blot analysis of individual xenografted tumor tissue indicated that tumor was composed of cells not only derived from injected mas-transfected CHO cells but also cells from mouse tissues. The presence of mouse stromal cells in the tumor was confirmed by immunohistochemistry and in situ hybridization. Previously our laboratory had identified some up- and down-regulated genes in mas-transfected cells by fluorescent differential display (FluoroDD). Northern blot showed that these differential expressed genes were up- or down-regulated in mas-transfected cells and tumor samples, which might play an important role in cancerous growth.
Taken together, these results suggest that over-expression of GPCR mas up-regulated tumor-related genes, resulting in promoting excessive cell growth and tumorigenic transformation. In addition, when the tumor mass formed they secreted some growth factor(s) which altered the migration of mouse stromal cells into tumor mass. The interactions of transformed cells and stromal cells further aggravate the tumorigenicity process.
To complement our fluorescent differential display study and to compare changes of gene expression when transformed cells were exposed to the microenvironment in nude mice, protein expression profiles of mas over-expressing cells as well as tumor tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. The 2D-PAGE analysis showed that a similar but distinct protein expression profiles in mas-transfected cells and in mas-induced tumor. Mass spectrometry analysis identified several cancerous growth-related proteins and they are involved in processes such as cell signaling, energy metabolism, transcription and translation and cytoskeleton organization.
Lin Wenzhen.
"December 2005."
Adviser: Cheung Wing Tai.
Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6381.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references (p. 222-240).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.
« Modulations of receptor activity of orphan G protein-coupled receptor mas by C-terminal GFP tagging and experssion level ». Thesis, 2009. http://library.cuhk.edu.hk/record=b6074750.
Texte intégralMBP7-like motif was identified in human facilitative GLUT1 and GLUT7 indicating that mas might interact with glucose transporter (GLUT) and regulate cellular glucose uptake. GLUT4 was found to be expressed endogenously in the CHO cell by RT-PCR, but expression of insulin receptor was not detectable. Although no statistical difference was detected in basal glucose uptake among control cells Vc0M80 and cells with different levels of mas expression, cells expressing mas-(Gly10Ser5)-GFP showed a high glucose uptake in response to insulin. Furthermore, basal 2-DOG uptake in Mc0M80 cells was not affected by pretreatment with various kinase inhibitors or transient expression of Rho variants. By contrast, MBP7 was found to induce a significant elevation of glucose uptake specifically in Mc0M80 cells transiently transfected with GLUT1.
Orphan G protein-coupled receptor (GPCR) mas was initially isolated from a human epidermal carcinoma. Previous study from our lab identified a surrogate ligand---MBP7 (mas binding peptide 7) for mas, and suggested that GFP tagging might affect the receptor activity of mas. In this project, three stable CHO cell lines expressing native mas, mas-GFP and mas-(Gly10Ser 5)-GFP were used to characterize receptor activity of mas.
To summarize, direct GFP tagging at the C-terminus of mas decreased its interactions with ligand and downstream signaling molecules. Partial recovery of mas receptor activity by adding a peptide linker was confirmed by phage binding, membrane fusion protein translocation and calcium response. In addition, mas was possibily coupled with GLUT1 to affect cellular glucose uptake via signaling pathways yet to be fully characterized.
Sun, Jingxin.
Adviser: Cheung Wing Tai.
Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0104.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 150-170).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
« Biological roles of mas oncogene ». 2002. http://library.cuhk.edu.hk/record=b5895980.
Texte intégralThesis (M.Phil.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (leaves 176-185).
Abstracts in English and Chinese.
Acknowledgments --- p.1
Abstract --- p.2
摘要 --- p.4
List of Abbreviation --- p.6
Chapter Chapter 1 --- General Introduction
Chapter 1.1 --- Isolation and activation of mas oncogene --- p.11
Chapter 1.2 --- Amino acid sequence of mas oncogene --- p.14
Chapter 1.3 --- Expression of mas oncogene --- p.18
Chapter 1.4 --- Possible physiological role of mas oncogene --- p.20
Chapter 1.5 --- Gene related to mas family --- p.23
Chapter 1.6 --- Aims of study --- p.26
Chapter Chapter 2 --- Over-expression of mas oncogene
Chapter 2.1 --- Introduction --- p.28
Chapter 2.2 --- Materials and Methods --- p.29
Chapter 2.2.1 --- Materials --- p.30
Chapter 2.2.1.1 --- Chemicals --- p.30
Chapter 2.2.1.2 --- Enzyme --- p.30
Chapter 2.2.1.3 --- DNA Purification Kit --- p.31
Chapter 2.2.1.4 --- Others --- p.31
Chapter 2.2.2 --- Methods --- p.31
Chapter 2.2.2.1 --- Strategy of construct preparation --- p.31
Chapter 2.2.2.2 --- "Preparation of linearized vector, pFRSV" --- p.32
Chapter 2.2.2.2.1 --- Cloning of vectors --- p.32
Chapter 2.2.2.2.2 --- Restriction enzyme digestion and DNA dephosphorylation --- p.34
Chapter 2.2.2.2.3 --- DNA purification by agarose gel electro-elution --- p.34
Chapter 2.2.2.3 --- Preparation of pFRSV/mas construct --- p.35
Chapter 2.2.2.3.1 --- PCR amplification --- p.35
Chapter 2.2.2.3.2 --- Restriction enzyme digestion --- p.36
Chapter 2.2.2.4 --- Ligation and analysis --- p.37
Chapter 2.2.2.5 --- Purification of DNA by cesium chloride --- p.38
Chapter 2.2.2.5.1 --- Large-scale bacterial culturing --- p.38
Chapter 2.2.2.5.2 --- Ethanol precipitation --- p.39
Chapter 2.2.2.5.3 --- Cesium chloride purification --- p.39
Chapter 2.2.2.5.4 --- Removal of DNA dye by dialysis and ethanol precipitation --- p.40
Chapter 2.2.2.6 --- Transfection by electroporation --- p.41
Chapter 2.2.2.7 --- Screening for the stably transfected cells --- p.42
Chapter 2.2.2.8 --- RT-PCR analysis of the mas transfectant --- p.43
Chapter 2.2.2.8.1 --- Isolation of the total RNA from the mas transfectants by TRIzol ® Reagent --- p.43
Chapter 2.2.2.8.2 --- Reverse transcription of the total RNA into cDNA --- p.44
Chapter 2.2.2.8.3 --- Analysis of the transfected mas expression by PCR --- p.44
Chapter 2.2.2.8.4 --- Analysis of the transfected DHFR expression by PCR --- p.45
Chapter 2.2.2.8.5 --- Analysis of endogenous GAPDH expression by PCR --- p.46
Chapter 2.2.2.9 --- Amplification of mas transgene by using methotrexate --- p.47
Chapter 2.2.2.9.1 --- Amplification by low dosage MTX treatment --- p.47
Chapter 2.2.2.9.2 --- Amplification by high dosage MTX treatment --- p.49
Chapter 2.2.2.10 --- Southern blot analysis --- p.50
Chapter 2.2.2.10.1 --- Preparation of DIG-labelled mas probe --- p.51
Chapter 2.2.2.10.2 --- Preparation of DIG-labelled DHFR probe --- p.51
Chapter 2.2.2.10.3 --- Preparation of DIG-labelled GAPDH probe --- p.52
Chapter 2.2.2.10.4 --- Isolation of Genomic DNA from the mas transfectants by DNAzol® Reagent
Chapter 2.2.2.10.5 --- Enzymatic restriction of genomic DNA and Gel electrophoresis --- p.54
Chapter 2.2.2.10.6 --- DNA transferring to positive charged Nylon membrane --- p.54
Chapter 2.2.2.10.7 --- Pre-hybridization and hybridization --- p.56
Chapter 2.2.2.10.8 --- Post-hybridization washing and blocking --- p.56
Chapter 2.2.2.10.9 --- Detection --- p.57
Chapter 2.2.2.11 --- Northern blot analysis --- p.57
Chapter 2.2.2.11.1 --- Preparation of the agarose gel containing formaldehyde --- p.58
Chapter 2.2.2.11.2 --- Preparation of the RNA sample --- p.58
Chapter 2.2.2.11.3 --- Gel electrophoresis and transferring --- p.59
Chapter 2.2.2.11.5 --- Pre-hybridization and hybridization --- p.60
Chapter 2.2.2.11.4 --- Post-hybridization washing and blocking --- p.60
Chapter 2.2.2.11.6 --- Detection --- p.61
Chapter 2.2.2.11.7 --- Stripping and rehybridization --- p.61
Chapter 2.3 --- Results --- p.62
Chapter 2.3.1 --- RT-PCR analysis of gene expression in the stably transfectant --- p.62
Chapter 2.3.2 --- Morphology of the mas trasnfectant --- p.64
Chapter 2.3.3 --- Determination of mas gene copy number by Southern blot analysis in the mas transfectants --- p.66
Chapter 2.3.4 --- Northern blot analysis of the transcriptional level of mas transcriptsin mas transfectants --- p.76
Chapter 2.4 --- Discussion --- p.87
Chapter Chapter 3 --- In vivo study of physiological effect of over-expression of mas
Chapter 3.1 --- Introduction --- p.92
Chapter 3.2 --- Materials and Methods --- p.93
Chapter 3.2.1 --- Materials --- p.93
Chapter 3.2.2 --- Methods --- p.93
Chapter 3.2.2.1 --- Cell culture --- p.93
Chapter 3.2.2.2 --- Subcutaneous injection of nude mice --- p.94
Chapter 3.2.2.3 --- Isolation of the total RNA from the tumor tissues --- p.95
Chapter 3.2.2.4 --- Northern blot analysis --- p.96
Chapter 3.3 --- Results --- p.96
Chapter 3.3.1 --- Tumorgenicity assay of mas oncogene in nude mice --- p.96
Chapter 3.3.2 --- Northern blot analysis of mas expression in the tumor tissues --- p.103
Chapter 3.4 --- Discussion --- p.109
Chapter Chapter 4 --- Fluorescent differential display analysis of mas transfectants
Chapter 4.1 --- Introduction --- p.111
Chapter 4.2 --- Materials and Methods --- p.112
Chapter 4.2.1 --- Materials --- p.112
Chapter 4.2.1.1 --- Chemicals --- p.112
Chapter 4.2.1.2 --- Enzyme --- p.113
Chapter 4.2.1.3 --- Kits --- p.113
Chapter 4.2.1.4 --- Others --- p.114
Chapter 4.2.2 --- Methods --- p.114
Chapter 4.2.2.1 --- Isolation of the total RNA from the mas transfectants by TRIzol ® Reagent --- p.114
Chapter 4.2.2.2 --- DNase I treatment --- p.115
Chapter 4.2.2.3 --- Reverse transcription (RT) and non-fluorescent PCR --- p.116
Chapter 4.2.2.4 --- Reverse transcription and fluorescent differential display-PCR --- p.118
Chapter 4.2.2.5 --- High resolution fluorescent differential display (Fluoro DD) gel --- p.118
Chapter 4.2.2.6 --- Gel band excision of differentially expressed cDNA fragments --- p.120
Chapter 4.2.2.7 --- Gel band reamplification --- p.120
Chapter 4.2.2.8 --- Subcloning of reamplified cDNA fragments --- p.121
Chapter 4.2.2.9 --- Purification of plasmid DNA from recombinant clones for reverse dot blot analysis --- p.122
Chapter 4.2.2.10 --- Reverse dot blot analysis --- p.123
Chapter 4.2.2.10.1 --- Preparation of cDNA dot blot --- p.123
Chapter 4.2.2.10.2 --- Preparation of DIG-labeled cDNA library probes --- p.124
Chapter 4.2.2.10.3 --- Hybridization --- p.126
Chapter 4.2.2.11 --- Northern blot analysis --- p.127
Chapter 4.3 --- Results --- p.128
Chapter 4.3.1 --- Fluorescent differential display (FluoroDD) --- p.128
Chapter 4.3.2 --- Reverse dot blot analysis --- p.135
Chapter 4.3.3 --- DNA sequencing analysis of the clones --- p.141
Chapter 4.3.4 --- Confirmation of differential display pattern of the subclones by Northern blot analysis --- p.160
Chapter 4.4 --- Discussion --- p.166
Chapter Chapter 5 --- General Discussion
Chapter 5.1 --- General model for mos-induced tumor formation --- p.169
Chapter 5.2 --- Future aspect --- p.174
References --- p.176
Appendix I Buffer composition --- p.186
Appendix II Sequences of fluoroDD TMR-Anchored primers and arbitrary primers --- p.190
« Identification and characterization of surrogate peptide ligands for mas, an orphan G protein-coupled receptor using phage-displayed random peptide library ». 2004. http://library.cuhk.edu.hk/record=b6073722.
Texte intégral"August 2004."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (p. 212-223)
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.
Lai, Yan-Ren, et 賴彥任. « Characterization of genotoxin MMS-induced Cebpd protein in MEFs ». Thesis, 2008. http://ndltd.ncl.edu.tw/handle/97813600132310174435.
Texte intégral高雄醫學大學
醫學研究所
96
The transcription factors C/EBP (CCAAT/Enhancer Binding Protein) family is a highly conserved family of leucine zipper composed of six members: Cebpa, Cebpb, Cebpg, Cebpd, Cebpe and Cebpz. Cebpd originally was identified as an inflammatory response gene. Cebpd is expressed in a variety of tissues and cell types and involved in the control of cellular proliferation, differentiation, metabolism, inflammation and maintenance of genomic stability. In our previous studies, we had demonstrated that Cebpd knockout (KO) mouse embryonic fibroblasts (MEFs) exhibited significant higher resistance to cisplatin through altering the p53 and Erk signaling pathways. This suggests that Cebpd could serve as a mediator in response to genotoxic agents. Methyl methanesulophonate (MMS) is an alkylating agent. The removal of MMS-induced DNA adducts is mainly through base excision repair. In this study, we found that Cebpd KO MEFs (NKO) showed higher resistance to MMS when compared to wild type. In addition, Cebpd protein and p53 protein activation (phospho-Ser-15) can be induced by MMS within five hours in NWT. p21 protein, a well-known p53 target gene, was also induced at the same kinetics. However, Cebpd and p53 mRNA level were not induced after MMS treatment. The phosphorylation of p38 can also be induced by MMS, but there was no difference whether Cebpd existence or not. Phosphorylated p38 can directly activate p53. These data indicate that Cebpd may play an important role in regulating of apoptosis and DNA repair in MEFs. The molecular mechanism by which Cebpd might involve in MMS-induced DNA damage response needs to be explored. This study reveals a potential role of Cebpd in MMS-induced DNA damage response in MEFs.
« Role of Mas oncogene on angiotensin receptor expression ». 1999. http://library.cuhk.edu.hk/record=b5896324.
Texte intégralThesis (M.Phil.)--Chinese University of Hong Kong, 1999.
Includes bibliographical references (leaves 142-147).
Abstract also in Chinese.
Abstract --- p.i
摘要 --- p.iii
Acknowledgement --- p.v
Lists of Abbreviations --- p.vi
Table of Contents --- p.vii
Chapter Chapter 1: --- Introduction
Chapter 1.1 --- Isolation of Mas Oncogene --- p.1
Chapter 1.2 --- Distribution of Mas Oncogene..........…… --- p.3
Chapter 1.3 --- Developmental Expression of Mas Oncogene --- p.5
Chapter 1.4 --- Study of Mas-deficient Mice --- p.7
Chapter 1.5 --- Signal Transduction of Mas Oncogene --- p.8
Chapter 1.6 --- Other Family Member of Mas Oncogene --- p.9
Chapter 1.7 --- Mas and Angiotensin Receptor --- p.11
Chapter 1.8 --- Angiotensin Receptors
Chapter 1.8.1 --- Classification of Angiotensin AT1 Receptor --- p.14
Chapter 1.8.2 --- Cloning of Angiotensin Receptor --- p.15
Chapter 1.9 --- Expression of Angiotensin Receptor
Chapter 1.9.1 --- Physiological Factors --- p.17
Chapter 1.9.2 --- Cis-regulatory Elements
Chapter 1.9.2.1 --- Organization and Regulatory Elements of AT1 Receptor --- p.19
Chapter 1.9.2.2 --- Expression of AT1a Receptor Promoter was Induced by AP-1 and GATA-4 in Pressure Overload Model --- p.20
Chapter 1.9.2.3 --- AT1a Receptor Reveals Three Glucocorticoid Responsive Elements --- p.22
Chapter 1.10 --- Signal Transduction of Angiotensin Receptor --- p.22
Chapter 1.11 --- Aim of Project --- p.25
Chapter Chapter 2: --- Mas Oncogene in AR4-2J cells
Chapter 2.1 --- Introduction --- p.26
Chapter 2.2 --- Materials and Methods
Chapter 2.2.1 --- Materials
Chapter 2.2.1.1 --- Reagents --- p.27
Chapter 2.2.1.2 --- Enzymes --- p.27
Chapter 2.2.1.3 --- DNA Purification Kits --- p.28
Chapter 2.2.1.4 --- Materials and Antibodies for Western Blot --- p.28
Chapter 2.2.1.5 --- Others --- p.28
Chapter 2.2.2 --- Restriction Enzyme Digestion --- p.29
Chapter 2.2.3 --- Agarose Gel Electrophoresis --- p.29
Chapter 2.2.4 --- DNA Extraction and Purification --- p.29
Chapter 2.2.5 --- Plasmid Vector Modification and DNA Ligation --- p.30
Chapter 2.2.6 --- Bacterial Transformation --- p.31
Chapter 2.2.7 --- Preparation of Plasmid DNA
Chapter 2.2.7.1 --- Minipreps --- p.32
Chapter 2.2.7.2 --- Midipreps and Maxipreps --- p.33
Chapter 2.2.8 --- Genomic DNA Extraction From Tissue and Cell Culture --- p.34
Chapter 2.2.9 --- RT-PCR Cloning of Mas Oncogene --- p.35
Chapter 2.2.10 --- Construction of Full Length Mas cDNA into pBluescript® II SK Vector --- p.38
Chapter 2.2.11 --- Southern Blot Analysis
Chapter 2.2.11.1 --- Preparation of DIG-labeled Mas Probe --- p.38
Chapter 2.2.11.2 --- Enzyme Restriction of Genomic DNA --- p.39
Chapter 2.2.11.3 --- Transferring DNA to Nylon Membrane --- p.40
Chapter 2.2.11.4 --- Prehybridization and Hybridization --- p.40
Chapter 2.2.11.5 --- Post-hybridization Washes and Blocking --- p.41
Chapter 2.2.11.6 --- Detection --- p.41
Chapter 2.2.12 --- DNA Sequencing
Chapter 2.2.12.1 --- Manual Sequencing --- p.42
Chapter 2.2.12.2 --- Autosequencing --- p.43
Chapter 2.2.12.3 --- Sequencing Primers --- p.44
Chapter 2.2.13 --- Cell Culture --- p.45
Chapter 2.2.14 --- Protein Assay by Modified Lowery --- p.46
Chapter 2.2.15 --- SDS-PAGE and Western Blot Analysis --- p.47
Chapter 2.3 --- Results --- p.49
Chapter 2.4 --- Discussion --- p.60
Chapter Chapter 3: --- Analysis of Transfected Mas Cell Lines
Chapter 3.1 --- Introduction --- p.61
Chapter 3.2 --- Materials and Methods
Chapter 3.2.1 --- Materials --- p.62
Chapter 3.2.2 --- Cell Culture and Transfection
Chapter 3.2.2.1 --- Cell Culture --- p.62
Chapter 3.2.2.2 --- Transfection Optimization --- p.62
Chapter 3.2.2.3 --- Fluorescent SEAP Assay --- p.63
Chapter 3.2.2.4 --- Transient Transfection --- p.64
Chapter 3.2.2.5 --- Stable Cell Line Construction --- p.64
Chapter 3.2.3 --- Protein Assay ESL --- p.65
Chapter 3.2.4 --- SDS-PAGE and Western Blot Analysis --- p.65
Chapter 3.2.5 --- Preparation of an AT1a Receptor Internal Standard for Quantitative RT-PCR Analysis
Chapter 3.2.5.1 --- Preparation of an AT1a Receptor cDNA by RT-PC --- p.66
Chapter 3.2.5.2 --- Cloning of AT1A Receptor cDNA into pBluescript® II SK Vector --- p.67
Chapter 3.2.5.3 --- Autosequence of pBluescript® II SK Vector/AT1AR --- p.68
Chapter 3.2.5.4 --- Preparation of 100 bp Deleted AT1a Receptor cDNA by RT- PCR --- p.68
Chapter 3.2.5.5 --- Cloning of Deleted AT1a R cDNA into pCAPs Vector --- p.71
Chapter 3.2.6 --- Construction of Full Length Mas cDNA into pOPRSVI/MCS Operator Vector --- p.71
Chapter 3.2.7 --- Preparation of an Mas Internal Standard for Quantitative RT-PCR Analysis
Chapter 3.2.7.1 --- Preparation of 100 bp Deleted Mas cDNA by RT- PCR --- p.72
Chapter 3.2.7.2 --- Cloning of 100 bp Deleted Mas cDNA into pCAPs Vector (Mas/pCAPs) --- p.73
Chapter 3.2.8 --- Quantitative RT-PCR Analysis of AT1A R Expression --- p.74
Chapter 3.2.9 --- Quantitative RT-PCR Analysis for the Expression of Mas --- p.74
Chapter 3.3 --- Results --- p.76
Chapter 3.4 --- Discussions --- p.100
Chapter Chapter 4: --- Cloning of AT1A Receptor Promoter
Chapter 4.1 --- Introduction --- p.104
Chapter 4.2 --- Materials and Methods
Chapter 4.2.1 --- Materials --- p.105
Chapter 4.2.2 --- Genomic DNA Extraction From Rat Pancreas --- p.105
Chapter 4.2.3 --- "Nest PCR Amplification of 3.2, 2.8 and 1.4kb AT1a Receptor Promoter" --- p.105
Chapter 4.2.4 --- PCR Amplification of 2.2 kb Aproximal Portion of AT1a Receptor Promoter --- p.107
Chapter 4.2.5 --- Construction of PCR Fragment of Angiotensin Receptor Promoter into Various Vector --- p.108
Chapter 4.2.5.1 --- pSEAP2-Basic --- p.108
Chapter 4.2.5.2 --- pBluescript® II SK Vector --- p.109
Chapter 4.2.5.3 --- PCR Cloning Kit (pCAPs vector) --- p.109
Chapter 4.2.5.4 --- PCR-TRAP Cloning System --- p.109
Chapter 4.2.6 --- Direct PCR Analysis --- p.110
Chapter 4.2.7 --- Autosequencing of PCR Fragment of AT1A Receptor Promoter --- p.111
Chapter 4.3 --- Results --- p.114
Chapter 4.4 --- Discussions --- p.130
Chapter Chapter 5: --- General Discussion --- p.131
Chapter Appendix 1 --- Composition of Solutions --- p.133
Chapter Appendix 2 --- Published Abstract --- p.141
References --- p.142
Etzkorn, Manuel. « Protein precipitates, aggregation kinetics and membrane protein receptors characterized by solid-state NMR ». Doctoral thesis, 2008. http://hdl.handle.net/11858/00-1735-0000-0006-B647-3.
Texte intégralTiemann, Johanna Katarina Sofie. « Interactive Analysis of Protein Structure and Dynamics ». 2019. https://ul.qucosa.de/id/qucosa%3A72400.
Texte intégralMurphy, Michael N. « Structure of MfdN and its influence on the stability and activity of the MFD protein ». 2009. https://scholarworks.umass.edu/dissertations/AAI3349734.
Texte intégralChou, Chi-Hsien, et 周季賢. « Roles of ACE2-Ang-(1-7)-Mas pathway and vitamin D-binding protein in diabetic nephropathy ». Thesis, 2015. http://ndltd.ncl.edu.tw/handle/a47zkt.
Texte intégral高雄醫學大學
醫學研究所博士班
103
TGF-β1 and high glucose are pivotal factors in diabetic nephropathy (DN) and they induce irreversible renal fibrosis. ACE2-Ang-(1-7)-Mas pathway is also a key mediator in DN. In previous studies, ACE2 and Mas were decreased in DN, meanwhile, high glucose inhibited ACE2 and Mas mRNA in kidney cells. However, the influence of TGF-β1 on ACE2-Ang-(1-7)-Mas pathway remains unclear. Moverover, we also investigated that the interaction between TGF-β1, high glucose and ACE2-Ang-(1–7)-Mas in NRK-52E cells. In our research, the results showed that high glucose inhibited ACE2, Mas and Ang-(1-7) production through TGF-β pathway. TGF-β1 also reduced ACE2-Ang-(1-7)-Mas pathway. Further, Ang-(1-7) inhibited TGF-β1 mRNA expression through Mas receptor、JAK2-STAT3 and PI3K-AKT pathways. However, Ang-(1–7) alone did not attenuate TGF-β1 and high glucose-induced fibronectin and collagen IV protein expression. In contrast, Ang-(1–7) in combination with Mas overexpression attenuated TGF-β1-induced fibronectin. We suggested that there is a negative feedback function between TGF-β1 and ACE2-Ang-(1-7)-Mas pathway. In animal experiment, we detected serum protein of rats with DN by using proteomic techniques. The results showed that the level of serum DBP was changed in diabetic rats. Therefore, our research focus on the role of DBP in DN. First, we confirmed that serum DBP was phosphorylated in diabetic rats. Meanwhile, DBP levels were increased in kidney and liver of diabetic rats, and the pI of DBP was shifted to the lower values. Next, we confirmed that high glucose increased DBP expression in NRK-49F renal fibroblasts and Clone-9 hepatocytes. Finally, we found that vitamin D attenuated of HG-induced renin, collagen IV and fibronectin protein expression in NRK-49F cells, however, DBP silencing only attenuated vitamin D-induced attenuation of HG-induced renin protein expression. In conclusion, both of TGF-β1 and high glucose inhibited the renoprotective effects of ACE2-Ang-(1-7)-Mas pathway, resulting in high level of Ang II. High level of Ang II induced renal fibrosis. Renin is a important enzyme for RAS activation, and DBP is required for vitamin D-induced attenuation of HG-induced renin. Therefore, the efficient inhibitory effect of vitamin D on RAS activation is very important.
Caeiro, Sara Inês de Matos. « Search for antagonists of the orphan G protein-coupled R Mas-related gene receptor (Mrg) X2 ». Master's thesis, 2013. http://hdl.handle.net/10451/29294.
Texte intégralThe large majority of receptors in human organism belongs to the superfamily of G protein-coupled receptors (GPCR). This superfamily is constituted of eleven families with distinct ligands and physiological roles, meaning they are a good drug target. Many GPCRs do not have any known ligand being denominated as orphan receptors. Over the past years several compounds were found as ligands for these receptors using reverse pharmacology. One of the families belonging to the GPCR superfamily is the Mas-related gene X (MrgX) receptor family. The MrgX2 receptor is a member of this family and is implicated in nociception and in inflammatory process. Cortistatin-14 has been reported as a natural ligand, but other agonists have also been identified. Despite this, no MrgX2 antagonist has yet been described. In this study two compound libraries were screened using β-arrestin assay in order to identify antagonists for the MrgX2 receptor. From the 1560 screened compounds one demonstrated a possible antagonistic activity: menadione. This is an analogue of vitamin K. Further studies must be performed in order to assess the mechanism of the antagonistic effect and the specificity of menadione. This molecule may serve as a lead structure for the development of more potent MrgX2 antagonists.
A grande maioria dos recetores no organismo humano pertence à superfamília dos recetores acoplados à proteína G (GPCR). Esta superfamília é constituída por onze famílias com ligandos e funções fisiológicas distintas, conferindo-lhes especial interesse como alvos terapêuticos. Muitos GPCRs não têm ligando conhecido e são denominados recetores órfãos. Nos últimos anos muitos compostos foram classificados como ligandos destes recetores, usando farmacologia reversa. Uma das famílias que pertence à superfamília GPCR é a dos recetores Masrelated gene X (MrgX). O recetor MrgX2 é um membro desta família que participa na nocicepção e no processo inflamatório. A cortistatina-14 foi identificada como o seu ligando natural, mas outros agonistas também têm sido reportados. No entanto nenhum antagonista foi descrito até à data. Neste estudo duas bibliotecas de compostos foram testadas usando o ensaio da β-arrestina com o objetivo de encontrar um antagonista para o recetor MrgX2. Dos 1560 compostos testados um mostrou possível atividade antagonista: a menadiona. Esta é um análogo da vitamina K. É ainda necessário o desenvolvimento de estudos posteriores que permitam verificar o mecanismo do efeito antagonista e a especificidade da menadiona. Esta molécula poderá servir como estrutura modelo para o desenvolvimento de antagonistas mais potentes para o recetor MrgX2.
Rheinische Friedrich-Wilhelms Universität Bonn, Pharmazeutisches Institut
Seidel, Karsten. « Structural characterization of membrane proteins by solid-state NMR spectroscopy ». Doctoral thesis, 2008. http://hdl.handle.net/11858/00-1735-0000-0006-B46E-1.
Texte intégralGouveia, Ana Maria da Silva. « Functional analysis of the peroxisomal and mitochondrial proteins MFF and FIS1 in HCMV evasion of the cellular antiviral response ». Doctoral thesis, 2021. http://hdl.handle.net/10773/31441.
Texte intégralOs peroxissomas são organelos celulares de membrana simples que desempenham funções metabólicas cruciais. Eles foram descritos pela primeira vez nos anos 60 e, ao longo dos anos, sua importância para a homeostasia celular tem sido cada vez mais destacada. Ao nível celular, os peroxissomas e as mitocôndrias cooperam em vários mecanismos metabólicos e recentemente foi descoberto que também têm funções complementares ao nível da resposta imune antiviral. Estes organelos também partilham várias proteínas, incluindo as proteínas chave das suas maquinarias de divisão MFF, FIS1 e DLP1 bem como a proteína antiviral MAVS. Como a correta função destes organelos depende em grande parte da capacidade de adequar a sua morfologia e localização celular de acordo com as necessidades das células e, por isso, da correta regulação dos eventos de fissão membranar, neste trabalho decidimos avaliar o papel da maquinaria de divisão peroxissomal e mitocondrial, especificamente dos adaptadores chave da DLP1, MFF e FIS1 na resposta antiviral contra um dos vírus mais disseminados na comunidade: o citomegalovírus humano (HCMV). Assim, iniciámos este trabalho com a caracterização da maquinaria de divisão peroxissomal a com a distinção de diferentes funções das proteínas de fissão partilhadas com as mitocôndrias (MFF e FIS1). Seguidamente, analisámos em detalhe a função destas proteínas na sinalização antiviral peroxissomal na defesa contra o HCMV. Os nossos resultados indicam que a MFF desempenha um papel crucial na regulação da fissão peroxissomal, enquanto que a FIS1 parece ter maior impacto na divisão mitocondrial. Além disso, foi descoberto que a MFF interage com a proteína viral vMIA, e assim parece desempenhar um papel crucial na infeção pelo HCMV. No seu conjunto, estes resultados enfatizam a importância da maquinaria de divisão peroxissomal na defesa antiviral mediada pelos RLR e poderá levar, em última instância, à descoberta de novos mecanismos peroxissomais, que poderão ser usados com alvos terapêuticos na infeção viral.
Programa Doutoral em Biomedicina
Shih, Yu-Hung, et 施昱宏. « Molecular cloning, protein expression and characterization of milk-fat globule EGF factor 8 (MFG-E8) variant forms ». Thesis, 2010. http://ndltd.ncl.edu.tw/handle/27903166987867815310.
Texte intégral國立臺灣大學
醫學檢驗暨生物技術學研究所
98
Background: Milk-fat globule EGF factor 8 (MFG-E8) is a secreted glycoprotein found in milk-fat globule derived from lactating mammary. MFG-E8 proteins express mainly in the mammary cells and immuno-modulating cells such as tingible-body macrophages in spleen and lymph node. MFG-E8 contains EGF domains (Epidermal growth factor-like) and C1/C2 domains (Coagulation factor V and VIII type C-like) for integrin αvβ3/5 on (MΦ) binding and phosphatidylserine (PS)(on apoptotic cell) recognition, respectively. The importance of MFG-E8 function as a bridging molecule during apoptotic cell clearance has been emphasized in recent years. Decreased phagocytic clearance and accumulation of apoptotic cells was noted in MFG-E8-deficient mice. Intriguingly, the 40-week-old female MFG-E8-deficient mice present higher titer of autoantibodies and suffered splenomegaly/glomerulonephritis more prevalently than the male mice, which were characterized in human systemic lupus erythematosus (SLE). These results indicate the correlation between MFG-E8 deficiency-related apoptotic cell clearance defect and SLE pathogenesis. Our previous studies revealed that the variant on MFG-E8-76th residue (76Met) was a predisposing factor for human SLE. Interestingly, a concensus Kozac sequence (A/G-3NNATGG+4) resides around residue 76Met, which may potentially act as an alternative translation initiation site and subsequently produce short form MFG-E8-Δ1-75 in cells of MFG-E8-76Met variant type. Methods: Several approaches were applied to test the hypothesis whether there is alternative translation - 1. Molecular cloning of MFG-E8 cDNA and express recombinant MFG-E8 protein in E. coli. 2. Examine whether MFG-E8-76Met variant form perform alternative translation in different MFG-E8-76 variant forms-transfected 293T cell lines and PBMC-derived human macrophages with different MFG-E8-76 variant forms. 3. Detect serum MFG-E8 expression level in SLE cohort to realize the correlation between MFG-E8-76 variations and serum MFG-E8 level. Results: 1. MFG-E8 cDNA was successfully cloned and then expressed in E. coli. However, the protein expression levels are considered low even after optimizing the protein induction conditions. Further protein expression was hampered due to low expression level. 2. Short form MFG-E8 protein was produced in MFG-E8 (Δ1-75)-transfected 293T cell lines but the expression efficiency were considerably lower than that found in 293T cell transfected with full length MFG-E8. 3. No short form MFG-E8 protein was observed in MFG-E8-76Met-transfected 293T cell lines. 4. No short form MFG-E8 protein were observed in peripheral blood monocytic cells-derived macrophage of MFG-E8-76Met variant form. Interestingly, the MFG-E8 detected in human macrophage was of smaller size than the expected. The protein expression level differed independently of MFG-E8-76 variant forms. 5. Serum MFG-E8 expression levels in SLE cohort were significantly higher than that of non-lupus controls but the elevated MFG-E8 level did not show significant correlation with MFG-E8-76 variant forms. Conclusions: 1. MFG-E8-76th codon ATG does not show to be an appropriate alternative translational start site. 2. Serum MFG-E8 levels were higher in part of SLE patients than in healthy controls but the elevated MFG-E8 level did not correlation with the MFG-E8-76 variant forms. 3. MFG-E8 expression levels had no correlation with the MFG-E8-76 variant forms in human macrophage. The proteins determined in human macrophage were smaller than estimated size suggested that there have distinct processes in human macrophage. The differential processing and expression of MFG-E8 in the phagocytes await further investigation.
Hiller, Matthias [Verfasser]. « Sample preparation of membrane proteins suitable for solid state MAS NMR and development of assignment strategies / vorgelegt von Matthias Hiller ». 2009. http://d-nb.info/999193430/34.
Texte intégral