3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DHS) catalyzes the first step of the shikimate pathway - a pathway involved in Tyrosine (Tyr), Tryptophan (Trp) and Phenylalanine (Phe) biosynthesis - by condensation of phosphoenolpyruvate and erythrose-4-phosphate to DAHP. Our lab previously demonstrated that Arabidopsis thaliana shikimate pathway flux is regulated by Tyr and Trp. This project suggests that A. thaliana DHS1 overexpressor lines have increased Trp accumulation with Tyr treatment, and that an A. thaliana DHS2 overexpressor line treated with Tyr has unchanged Trp accumulation, indicating that AtDHS2 is Tyr-sensitive. Confocal microscopy of all 3 AtDHS isoforms fused to yellow fluorescent protein demonstrates chloroplast localization. Bimolecular fluorescence complementation indicates that protein-protein interactions occur in the cytoplasm, and not in the chloroplast, for AtDHS1 and AtDHS2 with the metabolic regulator At14-3-3ω. These findings suggest that protein-protein interactions could regulate accumulation of AtDHS2 in the chloroplast, and are perhaps modulated by Tyr.

This diploma thesis deals with the analysis of structural and dynamic organization of thyrotropin releasing hormone receptor (TRH-R) and δ-opioid receptor (DOR) within plasma membrane (PM) in relation to the specific sub-compartments of PM denominated as domains or membrane rafts. Modern fluorescence microscopy techniques FLIM, FRAP and RICS were used for this purpose. The experiments were performed on the live cells derived from HEK293 cell line. To reach the main goal of this work, the integrity of PM structure was altered by depletion of cholesterol which was performed by incubation of cells with β cyclodextrin. Results clearly support our previously suggested idea that the vast majority of TRH-R is localized in non-raft regions of plasma membrane. This work also compared different modes of performance of FRAP and results obtained by FRAP and RICS because these methods are to some extent analogous. This is one of the first works that used the RICS approach to characterize the G protein-coupled receptors. In the second part of this work, the setup of transient transfection of the HEK293 cells with DOR-ECFP and DOR EYFP constructs was established. Simultaneously, the functionality of these constructs, i.e. the ability of DOR to activate the cognate G protein was determined. Powered by TCPDF (www.tcpdf.org)

No description available.

No description available.

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DHS) catalyzes the first step of the shikimate pathway - a pathway involved in Tyrosine (Tyr), Tryptophan (Trp) and Phenylalanine (Phe) biosynthesis - by condensation of phosphoenolpyruvate and erythrose-4-phosphate to DAHP. Our lab previously demonstrated that Arabidopsis thaliana shikimate pathway flux is regulated by Tyr and Trp. This project suggests that A. thaliana DHS1 overexpressor lines have increased Trp accumulation with Tyr treatment, and that an A. thaliana DHS2 overexpressor line treated with Tyr has unchanged Trp accumulation, indicating that AtDHS2 is Tyr-sensitive. Confocal microscopy of all 3 AtDHS isoforms fused to yellow fluorescent protein demonstrates chloroplast localization. Bimolecular fluorescence complementation indicates that protein-protein interactions occur in the cytoplasm, and not in the chloroplast, for AtDHS1 and AtDHS2 with the metabolic regulator At14-3-3ω. These findings suggest that protein-protein interactions could regulate accumulation of AtDHS2 in the chloroplast, and are perhaps modulated by Tyr.

Abstract In mice, gene targeting has become a useful tool for resolving the functions of proteins and for creating new animal models. Cre/loxP technology has been used broadly for generation of genetically modified mice. The Cre recombinase recognizes a specific DNA sequence, called loxP, and removes any DNA fragment between two loxP-sites. The activity of the Cre recombinase can be controlled spatially and temporally with cell- or tissue-specific promoters and synthetic inducing agents, such as tamoxifen or tetracycline. In this thesis, we employed tamoxifen-induced Cre recombination in vitro. Cre-ERTM mice were crossed to ROSA26LacZ reporters and Cre-recombination induced by 4OH-TM was monitored by LacZ staining. 0.5 μM 4OH-TM treatment was competent for tamoxifen-induced Cre-mediated activation of LacZ both in kidney cultures and in experimentally induced kidney mesenchymes. Wnt4 is a secreted signaling molecule, which is necessary for the development of several organs including kidney, ovary, adrenal gland, mammary and pituitary glands. Wnt4 is crucial for kidney development and conventional Wnt4-/- mice die soon after birth, likely due to renal failure. In this thesis, two different Wnt4 alleles, Wnt4EGFPCre and floxed Wnt4, were generated and analyzed to learn more about the Wnt4 functions and to apply these mouse lines to renal functional genomics. In the Wnt4EGFPCre, the EGFPCre fusion cDNA was targeted into exon I of the Wnt4 locus. EGFP-derived fluorescence was observed in the pretubular aggregates from E12.5 embryonic kidney onwards. Further characterization by crossing with the floxed ROSA26LacZ and yellow fluorescent protein (YFP) reporter lines demonstrated that in addition to the kidney, reporter expression was observed in the gonad, spinal cord, lung and the adrenal gland. The expression pattern of the Cre activity recapitulates well the known pattern of the Wnt4 gene. Time-lapse analysis in organ culture settings showed that the Wnt4 expressing cells contributed to the nephrons, some cells near the stalk of the developing ureter, as well as a number of positive supposed medullary stromal cells. In the conditional Wnt4 knock-out, loxP sites were placed to flank exons 3 to 5. The Wnt4 gene was specifically inactivated with CAGCre and Wnt4EGFPCre lines. In both of these crosses deletion of Wnt4 gene function led to impaired kidney development. In conclusion, we identified the culture conditions that can be used for the tamoxifen-induced conditional mutagenesis in tissue cultures. In addition, the created Wnt4 mouse lines serve as new useful tools for addressing the roles of Wnt4 function in diverse tissues and in different stages of development. / Tiivistelmä Hiirillä geenikohdennuksesta on muodostunut hyödyllinen väline proteiinien tehtävien selvittämisessä ja uusien eläinmallien luomisessa. Cre/loxP -tekniikkaa on käytetty laajasti muuntogeenisten hiirien tuottamisessa. Cre-rekombinaasi tunnistaa spesifisen DNA-jakson, niin kutsutun loxP:n, ja poistaa kaikki DNA-jaksot kahden loxP-sekvenssin väliltä. Cre-rekombinaasin aktiivisuutta voidaan säädellä paikallisesti ja ajallisesti solu- tai kudosspesifisillä promoottoreilla ja synteettisillä indusoivilla kemikaaleilla, kuten tamoksifeenillä tai tetrasykliinillä. Tässä väitöskirjassa hyödynsimme tamoksifeenin aiheuttamaa Cre-rekombinaatiota in vitro -kudosviljelmissä. Cre-ERTM-hiirilinja risteytettiin ROSA26LacZ-reportterilinjan kanssa, ja 4-hydroksitamoksifeenin indusoima Cre-rekombinaasin aktiivisuutta monitoroitiin LacZ–värjäyksellä. 0.5 µM:n 4OH-TM konsentraatiolla LacZ-reportterigeeni saatiin aktivoitua tehokkaasti Cre-rekombinaasin avulla sekä munuaisviljelmissä että munuaismesenkyymiviljelmissä. Wnt4 on erittyvä signalointimolekyyli, jolla on keskeinen rooli useiden elinten, kuten munuaisen, munasarjan, lisämunuaisen, rintarauhasen ja aivolisäkkeen kehittymisessä. Wnt4-geenillä on ratkaisevan tärkeä rooli munuaisen kehityksessä, ja poistogeeninen Wnt4-/-hiiri kuolee pian syntymän jälkeen, todennäköisesti munuaisen vajaatoimintaan. Tässä väitöskirjatyössä tuotettiin kaksi eri Wnt4 alleelia, Wnt4EGFPCre ja konditionaalinen Wnt4. Nämä hiirilinjat analysoitiin, jotta saisimme lisää tietoa Wnt4-geenin toiminnasta ja pystyisimme soveltamaan kyseisiä hiirikantoja munuaisten toiminnan selvittämisessä. Wnt4EGFPCre-alleelissa EGFPCre-fuusio -cDNA kohdennettiin osaksi endogeenisen Wnt4-geenin ykköseksonia. Vihreän fluoresoivan proteiinin (EGFP) aktiivisuus havaittiin varhaisen munuaisen kehityksen aikana. Wnt4EGFPCre-alleelin lisäkarakterisointi reportterilinjoilla (Rosa26LacZ ja Rosa26YFP) osoitti, että Wnt4-geenin ilmentyminen havaittiin munuaisen lisäksi sukurauhasissa, selkäytimessä, keuhkoissa sekä lisämunuaisessa. Wnt4EGFPCre-alleeli ilmentyi niissä kudoksissa, joissa endogeenisen Wnt4-geenin tiedetään olevan aktiivinen. Time-lapse -analyysin avulla osoitettiin, että Wnt4-geeniä ilmentävät solut muodostavat tiettyjä rakenteita munuaisen kehityksen aikana. Wnt4-geeni ilmentyi nefroneissa, kehittyvän virtsajohtimen soluissa sekä useissa medullaarisissa stroomasoluissa. Konditionaalisessa (ehdollisessa) Wnt4 knock-out-hiirilinjassa loxP-sekvenssit sijoitettiin eksonien kolme sekä viisi ympärille. Wnt4-geenin toiminta inaktivoitiin CAGCre- ja Wnt4EGFPCre-hiirilinjojen avulla. Näissä molemmissa tapauksissa Wnt4-geenin toiminnan poistaminen johti munuaisen kehityshäiriöön. Yhteenvetona voimme todeta, että olemme tunnistaneet ne kasvatusolosuhteet, joita voidaan hyödyntää, kun halutaan aktivoida reportterigeenejä tai kehityksen kannalta tärkeitä geenejä tamoksifeenin aiheuttamaa Cre/loxP -rekombinaatiota hyväksikäyttäen kudosviljelmissä. Samoja olosuhteita ja menetelmää käyttäen voidaan myös poistaa jonkun kehityksen kannalta tärkeän geenin toiminta ja tutkia sitä kudosviljelmässä. Tuotetut Wnt4-hiirikannat ovat lisäksi uusia hyödyllisiä työkaluja, kun halutaan tutkia Wnt4-geenin toimintaa erilaisissa kudoksissa ja eri kehitysvaiheiden aikana.

No description available.

Les protéines fluorescentes, comme la GFP (green fluorescent protein), sont des protéines naturellement fluorescentes qui sont utilisées pour leur rôle de marqueur, permettant de localiser des protéines dans les cellules et d'en suivre les déplacements. De nombreuses études expérimentales et théoriques ont été menées ces dix dernières années sur les protéines fluorescentes. De là, se forge une compréhension essentiellement qualitative du rôle de la protéine vis-à-vis de l’obtention ou non d’une émission radiative : il apparaît que la protéine permet la fluorescence en bloquant les processus qui la désactivent ; ces processus de désactivation sont très rapides et efficaces (à l'échelle de la picoseconde) dans le cas du chromophore seul, et ils sont bien identifiés comme étant des torsions autour des liaisons intercycles (tau et phi). Dans la protéine, la sensibilité des temps de vie de fluorescence à des mutations proches ou non du chromophore, à des modifications de pH ou de température laisse supposer un contrôle de la dynamique du chromophore par différents paramètres, sans qu’ils soient pour autant identifiés et mis en relation.Une étude de la dynamique de la protéine permettrait de faire la lumière sur les mécanismes responsables de ces phénomènes photophysiques pour lesquels une analyse structurale ne suffit pas. Cependant l'étude de la dynamique est limitée par la taille du système (>30 000 atomes), par l'échelle de temps des phénomènes photophysiques considérés (dizaine de nanosecondes) et par le fait que les deux torsions tau et phi sont fortement couplées dans l'état excité du chromophore. Ces trois facteurs excluent les méthodes de dynamique existantes aujourd'hui ; dynamique quantique (AIMD), dynamique mixte classique-quantique (QM/MD) et dynamique moléculaire classique (MD).Nous avons surmonté le problème par la modélisation de la surface d’énergie potentielle de torsion du chromophore à l’état excité basée sur des calculs quantiques de haute précision, par une interpolation des valeurs obtenues par une expression analytique appropriée en fonction des angles de torsion tau et phi et avec une précision suffisante pour reproduire des barrières de l’ordre de la kcal/mol, et enfin, par l’implémentation de cette expression analytique dans le programme parallèle AMBER. Une deuxième difficulté théorique concerne la simulation et l’analyse statistique d’événements peu fréquents à l’échelle de la nanoseconde, et dont on ne connait pas le chemin de réaction, ici les déformations de la protéine et du chromophore conduisant aux géométries favorables à la conversion interne. Grâce à ces développements et aux simulations qu'ils ont permises, nous avons réalisé la première modélisation de la désactivation non-radiative par conversion interne à l’échelle de la nanoseconde dans trois protéines fluorescentes différentes. L’analyse des dynamiques moléculaires classiques nous donne une évaluation quantitative des temps de vie de l’extinction de fluorescence, en accord avec les données expérimentales. Par ailleurs elle nous a permis d'identifier les mouvements moléculaires concertés de la protéine et du chromophore conduisant à cette extinction. De ces résultats, émerge une représentation plus complète du mécanisme qui libère la torsion du chromophore ou qui la déclenche : il peut venir d’un mouvement spécifique de la protéine, qui se produit à l’échelle de la nanoseconde, ou bien de plusieurs mouvements spécifiques, plus fréquents (rupture de liaisons hydrogène, rotation de chaînes latérales, dynamique d'agrégats d’eau), mais qui coïncident seulement à l’échelle de la nanoseconde. Ces mouvements spécifiques n’ont pas un coût énergétique important mais la nécessité de leur coïncidence crée un délai de l’ordre de quelques nanosecondes alors que dans le vide la torsion se produit en quelques picosecondes. Dans le cas des protéines étudiées, on a identifié en grande partie les mécanismes et les acides aminés qui sont impliqués. / Fluorescent proteins, like GFP (green fluorescent protein), are efficient sensors for a variety of physical-chemical properties and they are extensively used as markers in living cells imaging. These proteins have been widely studied both experimentally and theoretically the last decade. The comprehension of the protein's role in the regulation of the radiative emission is today essentially qualitative: it appears that the protein enables the fluorescence by blocking the processes that deactivates it; the deactivating processes are very quick and efficient (on the picosecond time scale) when the chromophore is isolated, and they are identified as being the torsions around the central bonds of the chromophore (tau and phi). The fluorescence lifetimes of a protein is very sensitive to mutations in the vicinity of the chromophore, to modifications in pH or in temperature. This seems to indicate a control of the dynamics of the chromophore by different parameters, that are not necessarily identified.A study of the dynamics of the protein would allow a deeper understanding of the mechanisms that are responsible for the fluorescence quenching. From a theoretical point of view, one is faced with three difficulties in this type of study: the size of the system (>30 000 atoms including a water box), the required time scale (tens of nanoseconds) and the fact that the torsions tau and phi are strongly coupled in the excited state of the chromophore. We must thus rule out the already existing dynamics methods: quantum dynamics (AIMD), mixed classical-quantum dynamics (QM/MD) and classical molecular dynamics (MD).We have overcome this problem by modeling the torsional potential energy surface of the chromophore in the first excited state trough high precision quantum calculations, by interpolating the energy values with an analytical fitting expression depending on the torsions tau and phi and with a precision high enough to reproduce barriers of the order of 1 kcal/mol, and lastly, by implementing this fitting expression in a parallelized version of the MD program AMBER. Another theoretical difficulty concerns the simulation and the statistical analysis of rare events on the nanosecond time scale without knowing the reaction path in advance, i.e. the deformations of the protein and of the chromophore leading to geometries where the internal conversion is favored. As a result of these developments and of the simulations they have enabled, we have been able to model, for the first time, the non-radiative deactivation by internal conversion at the nanosecond time scale in three different fluorescent proteins. The analysis of the classical molecular dynamics gives us a quantitative evaluation of the lifetime of the fluorescence extinction, in agreement with experimental results. In addition, it has allowed us to identify the concerted molecular movements between the protein and the chromophore leading to this extinction. A more complete representation of the mechanism that liberates or provokes the chromophore torsion emerges from these results: it could be a specific movement of the protein, that occurs on the nanosecond timescale, or several specific movements that occur more frequently (breakage of a hydrogen bond, rotation of side chains, dynamics of a water cluster), but that coincide only on the nanosecond time scale. These specific movements do not have a high energy cost but the need for them to coincide creates a delay of several nanoseconds compared to the chromophore torsion in vacuo which occurs after a few picoseconds. In the proteins we have studied (GFP, YFP and Padron), we have identified the principle components of the mechanisms and the amino acids that are implicated in this chromophore-protein interplay.

No description available.

No description available.