The unique spectroscopic properties of quantum dots (QDs) are of interest for application in intracellular studies of gene expression. QDs derivatized with single-stranded probe oligonucleotides were used to detect complementary target sequences via hybridization and fluorescence resonance energy transfer (FRET). As nucleic acid targets are not labeled within cells, a displacement assay for nucleic acid detection featuring QDs as FRET donors was developed. QDs conjugated with oligonucleotide probes and then pre-hybridized with labeled target yielded efficient FRET in vitro. Studies in vitro confirmed that displacement kinetics of pre-hybridized target was a function of the stability of the initial hybridized complex. Displacement was observed as reduction in FRET intensity coupled with regeneration of QD fluorescence. By engineering the sequence of the labeled target, faster displacement was possible. The QDprobe+target system was successfully delivered into cells via transfection. Although QDs with their cargo remained sequestered in endosomal vesicles, fluorescent properties were retained.

Assay technologies for GPCRs and their associated G-proteins are in demand for drug screening and other biotechnology applications such as biosensors for diagnostic purposes or odorant/flavour assessment as well as for elucidating the remaining controversial mechanisms in G-protein mediated signalling. This study aims to make progress towards developing a TR-FRET assay for G-protein interactions that could be used as a generic assay platform for GPCR signalling that would be fluorescent, homogeneous and amenable to miniaturization. The first chapter of this study investigates the use of small molecule labels, CS124-DTPA-EMCH:Tb and Alexa546 in a TR-FRET assay. This TR-FRET pair had previously been applied to Gα, Gβγ and RGS4 proteins and during the characterization of this assay, the protein CrV2 was observed to interact with the G-protein. Using TR-FRET, it was demonstrated that a high affinity interaction appears to occur between Gαi1 and CrV2 (Apparent Kd 6.2 nM). CrV2 is encoded by a polydnavirus from endoparasitoid wasps, which is thought to mediate immune suppression, and the interaction with Gα could have important implications in the regulation of the immune system of invertebrates. Improvements to the labelling strategy used in this assay are then attempted through the creation of various G-protein subunit fusions with small, genetically encoded lanthanide binding tags (LBTs) or tetracysteine motifs (TCMs) for site-specific labelling with terbium or FlAsH, respectively. The consequence of the fusions on maintaining G-protein subunit integrity and on the affinity of the tags for their labels is characterized, and then the utility of these constructs as TR-FRET partners is demonstrated. TCM:FlAsH complexes could successfully be used as TR-FRET acceptors for CS124-DTPA-EMCH:Tb labelled binding partners. The interaction between Gβγ2-TCM:FlAsH and Gα:Tb could be measured using TR-FRET and generated an apparent Kd of 3.6 nM. Likewise, LBT:Tb complexes could be used as TR-FRET donors to Alexa546 labelled binding partners which was demonstrated using the chimeric, promiscuous Gα subunit, LBT2:Tb-GαS25 and Gβγ:Alexa. Furthermore, the two site-specific labelling strategies can be used together in TR-FRET studies and an interaction between LBT2:Tb-Gα[subscript]S25 and Gβγ₂-TCM:FlAsH was shown to have an apparent Kd of 2.3 nM. The TRFRET assays were further validated using protease treatments and the addition of unlabelled binding partners reduced the TR-FRET signal. Finally, the feasibility of fusing lanthanide binding tags to GPCRs for alternate assay platforms or other applications was investigated. The β₂- adrenergic and M₂-muscarinic receptors were fused to LBTs and the integrity of the receptors determined using ligand binding and [[superscript]35 S]GTPγS signalling assays. Terbium binding to the LBT was then demonstrated. The utility of these constructs in alternative TR-FRET platforms with Gproteins was then explored. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1363937 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20530号 / 生博第372号 / 新制||生||49(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 井垣 達吏, 教授 HEJNA,James / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

En se basant sur l’approche des corridors de transport de fret, cette thèse se propose d’étudier les impacts différenciés des politiques d’infrastructures de transport et de télécommunication sur la spécialisation régionale et, par conséquent, sur le développement polycentrique et durable de l’espace européen. Ce faisant, un premier modèle théorique mixte de la NouvelleEconomie Géographique et de l’économie urbaine a été développé. Il montre d’un côté que le commerce interrégional dépend de la taille et des structures internes des zones urbaines ; d’un autre côté, il souligne le fait que l’intégration régionale influence la taille, les structures de ces zones et, par conséquent, leur spécialisation régionale. Ensuite, un deuxième modèle théorique de la Nouvelle Economie Géographique est développé pour étudier les impacts des politiques d’infrastructures de transport et de télécommunication sur la spécialisation régionale. On montre cependant que le secteur technologique joue un rôle essentiel dans la répartition spatiale des activités industrielles et, par conséquent, dans le développement polycentrique de l’espace européen. Enfin, un modèle économétrique basé sur la problématique de couplage est mobilisé pour tester la relation entre la demande de transport maritime international de marchandises et la croissance économique. On souligne ainsi le fait qu’une approche par corridors peut être un outil de découplage, en permettant un système de transport efficace et durable dans la région euro-méditerranéenne. / Based on the freight corridors approach, in this thesis we explore differentiated impacts of transport and telecommunication infrastructure policy on regional specialization and consequently on polycentric and sustainable development of the European area. Within this framework the first mixed theoretical model of New Economic Geography and urban economics was developed. On the one hand it shows that interregional trade depends on the size and internal structure of urban zones; on the other hand it emphasizes that regional integration influences the size and structure of these zones and, as a result, their regionalspecialization. Then the second theoretical model of the New Economic Geography is developed to study the impact of transport and telecommunication infrastructure policy on regional specialization. We demonstrate, however, that technological sector plays an essential role in the spatial distribution of industrial activities, and consequently in the polycentric development of the European space. Finally, an econometric model based on the problem of coupling is applied to test the relation between the demand for international maritime transport and economic growth. Thus it is emphasized that the approach of corridors may beused as an instrument of decoupling, allowing to develop an efficient and sustainable transport system in the Euro-Mediterranean region.

The Transforming Growth Factor (TGF) superfamily of cytokines and their serine/threonine kinase receptors play an important role in the regulation of cell division, differentiation, adhesion, migration, organization, and death. Smad proteins are the major intracellular signal transducers for the TGF receptor superfamily that mediate the signal from the membrane into the nucleus. Bone Morphogenetic Protein-4 (BMP-4) is a representative of the TGF superfamily, which regulates the formation of teeth, limbs and bone, and also plays a role in fracture repair. Binding of BMP-4 to its receptor stimulates phosphorylation of Smad1, which subsequently recruits Smad4. A hetero-oligomeric complex consisting of Smad1 and Smad4 then translocates into the nucleus and regulates transcription of target genes by interacting with transcription factors. Although the individual steps of the signaling cascade from the receptor to the nucleus have been identified, the exact kinetics and the rate limiting step(s) have remained elusive. Standard biochemical techniques are not suitable for resolving these issues, as they do not offer sufficiently high sensitivity and temporal resolution. In this study, advanced optical techniques were used for direct visualization of Smad signaling in live mammalian cells. Novel fluorescent biosensors were developed by fusing cyan and yellow fluorescent proteins to the signaling molecules Smad1 and Smad4. By measuring Fluorescence Resonance Energy Transfer (FRET) between the two fluorescent proteins, the kinetics of BMP/Smad signaling was unraveled. A rate-limiting delay of 2 - 5 minutes occurred between BMP receptor stimulation and Smad1 activation. A similar delay was observed in the complex formation between Smad1 and Smad4. Further experimentation indicated that the delay is dependent on the Mad homology 1 (MH1) domain of Smad1. These results give new insights into the dynamics of the BMP receptor – Smad1/4 signaling process and provide a new tool for studying Smads and for testing inhibitory drugs. / Die Transforming Growth Factor" (TGF)-Superfamilie der Cytokine und ihrer Serin/Threonin-Kinase-Rezeptoren spielt eine bedeutende Rolle bei der Regulierung der Zellteilung, -differenzierung, -adhäsion, -migration, -organisation, und beim Zelltod. Die Smad-Proteine sind die wichtigsten intrazellulären Signalüberträger für die TGF-Rezeptor-Familie, da sie das Signal von der Zellmembran zum Kern übermitteln. Das ,,Bone Morphogenetic Protein4" (BMP-4) ist ein Vertreter der TGF-Familie, der die Bildung von Zähnen, Gliedmaßen und Knochen reguliert und darüber hinaus eine Rolle bei der Frakturheilung spielt. Das Binden von BMP-4 an seinen Rezeptor stimuliert die Phosphorylierung von Smad1, welches in der Folge Smad4 rekrutiert. Ein hetero-oligomerer Komplex bestehend aus Smad1 und Smad4 verlagert sich dann in den Zellkern, wo er durch Interaktion mit Transkriptionsfaktoren die Transkription von Zielgenen reguliert. Obwohl die einzelnen Schritte der Signalkaskade vom Rezeptor bis in den Zellkern bereits identifiziert wurden, blieben die Kinetik und die geschwindigkeitsbegrenzenden Schritte bisher unbekannt. Gängige biochemische Methoden eignen sich nicht um diese Fragen zu lösen, da sie nicht über ausreichende Empfindlichkeit und zeitliches Auflösungsvermögen verfügen. In der vorliegenden Arbeit wurden hochentwickelte optische Techniken angewandt, um die Smad-vermittelte Signaltransduktion direkt in lebenden Zellen sichtbar zu machen. Neue fluoreszierende Biosensoren wurden konstruiert, indem gelb- und cyan-fluoreszierende Proteine mit den Signalmoleküle Smad1 und Smad4 fusioniert wurden. Durch Messung des "Fluorescent Resonance Energy Transfer" (FRET) zwischen den zwei fluoreszierenden Proteinen konnte die Kinetik der BMP-Smad-Signalkaskade bestimmt werden. Zwischen der Stimulation des Rezeptors und der Aktivierung von Smad1 trat eine geschwindigkeitsbegrenzende Verzögerung von 2-5 Minuten auf. Eine ähnliche Verzögerung wurde bei der Bildung des Komplexes aus Smad1 und Smad4 beobachtet. Weitere Experimente zeigten, dass die Verzögerung von der Mad-Homologie-Domäne 1 (MH1) von Smad1 abhängt. Die Ergebnisse dieser Arbeit geben neue Einblicke in die Dynamik der BMP-Rezeptor-Smad1/4 Signaltransduktion und stellen neue Werkzeuge zur Untersuchung von Smads und zur Austestung inhibitorischer Wirkstoffe zur Verfügung.

No description available.

J E. coli On-Off &ldquo, &rdquo, °, &ndash The specific objectives of the work presented in this dissertation are to design novel molecular sensors based on fluorescence resonance energy transfer (FRET) between fluorophore (donor) and polydiacetylene (PDA, acceptor) for selective detection of biomolecules in solution. The work described in this dissertation is divided into three sections. In the first section, we report here two novel systems where the rate of energy transfer is based on changes in the spectral overlap between the emission of the donor and the absorption of the acceptor (J) as well as changes in the quantum yield of the acceptor. In the second section, we discuss modified these high sensitive molecular sensors based on FRET by using different receptors for selective detection of biomolecules such as proteins or bacteria in solution. The third section develops reversibility studies on FRET based sensors in solution or solid state. In the Chapters two and three, conjugated polydiacetylene (PDA) possessing stimuli-responsive properties have been intensively investigated for developing efficient sensors. Sensors based on FRET between conjugated polymers and fluorophores can be more sensitive than colorimetric based sensors. We use the fluorophore dansyl as the donor and polydiacetylene (PDA) as the acceptor to demonstrate the modulation of FRET efficiency through conformationally induced changes in the PDA absorption spectrum following thermal treatment that converts the PDA backbone of the liposome from the blue form to the red form. We have used steady-state electronic absorption, emission and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). Energy transfer was found to be significantly more efficient from dansyl to the red-form PDA. This is due to large increase in the J values between dansyl emission and absorption red-form of PDA. We also have found that the monomer ratio of acceptor to donor (Rad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. A decrease in Rad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. Increase in Rad led to increase probability of FRET from donor to acceptor as larger number of acceptors are present around a given donor. The competition between donor for energy transfer increases with decrease in Rad that contributed to lower FRET efficiency between donors and acceptors. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to the insertion of dansyl in the bilayer of the liposomes which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. Much larger emission amplification for FRET was observed as compared to direct-excitation of PDA. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. These findings support the basis of a new sensing platform that utilizes J-modulated FRET as an actuating mechanism. A FRET based protein sensor by using sulforhodamine 101 as donor and PDA as acceptors was developed. This novel FRET based system primarily utilizes changes in J values (the spectral overlap between the emission of the donor and absorption of the acceptor) for the modulation of FRET efficiency between donors and acceptors. These FRET based sensors can be modified by tagged receptors (for proteins, viruses, and bactria) onto PDA liposomes which can interact with ligands present on proteins or bacteria. The biotin-streptavidin interactions were used as a sensing model system to test our FRET sensor response. In chapter 4, four different biotin-tagged lipids were used as receptors to investigate the effect of interactions between ligand-receptors on the FRET efficiency. The biotin was covalently linked to the liposome surface when using biotin-tagged diacetylene; whereas the biotin-tagged lipids with hydrophobic chains but without diacetylene functionalities provided non-covalently inserted lipids in liposomes. These studies were used to elucidate the effect of molecular interactions on FRET sensor response. The conjugated polymerized liposomes consisted of sulforhodamine-tagged-diacetylene and receptors linked lipids in different molar ratios. The characterization of the liposomes and sensing mechanism was investigated using UV-Vis and steady-state emission spectroscopy. The liposome solution yielded a weak donor emission (sulforhodamine 101) from after photo-polymerization of diacetylene monomers. This is due to energy transfer from the donor to PDA backbone chains (acceptors). The addition of streptavidin which interacted with biotin receptors resulted in increase in the sulforhodamine 101 emissions. The stress, due to interactions between biotin and streptavidin, induced the chromatic shift in the absorption spectrum of PDA which led to a decrease in the spectral overlap (J) between the emission spectra of donor and the absorption spectra of acceptor, leading to a decrease in the FRET efficiency from sulforhodamine 101 to PDA. These sensors, thus, show an "On-Off" type optical mechanism based on FRET between fluorophores and PDA where the donor emission was highly quenched in the "Off" state but was turned "On" due to receptor-ligand interactions. Large electronic absorbance and emission intensity differences between covalently and non-covalently bound biotin liposome systems were observed which indicated that the molecular interactions between biotin and PDA backbone play a crucial role in the FRET sensor response. In Chapter 5, we also developed FRET sensor for the detection of E. coli in aqueous media. Two glucose-based receptors were used in this study: (1) glucose-tagged lipid which can be inserted non-covalently in the bilayer of liposome, and (2) glucose-tagged diacetylene monomer in which the receptors were covalently bound to the backbone of the PDA liposome. The steady-state UV/Vis absorbance and fluorescence emission spectroscopy, and the fluorescence microscopy analysis of the receptors-containing liposomes were investigated for the detection of E. coli. The blue shift in the absorption spectrum of the conjugated PDA backbone induced through the interactions between receptors and bacteria resulted in decrease in the spectral overlap between the emission of SR-101 (donor) and the absorption of PDA (acceptor). This, ultimately, led to change in FRET efficiency between SR-101 and PDA after glucose - E. coli binding and caused increase in the emission intensity of SR-101. Polydiacetylenes have been exploited because of their sensitivity to external stimuli, such as temperature, pH, ions, and ligands. Unfortunately, the majorities of the sensors developed are not reversible but used as a one-time use. Here we report our preliminary results of a benzoic acid monomer of polydiacetylene (PDA-mBzA) to investigate reversible FRET characteristics between fluorophore and PDA. The LS films containing dansyl-tagged-diacetylene monomers and m-aminobenzoic acid derivatized- diacetylene monomers in different molar ratios were self-assembled and polymerized. The UV/Vis and steady-state fluorescence emission analysis of these LS films were investigated. These systems have shown partial reversible FRET over many "on-off" cycles. We believe that this incomplete FRET reversibility is due to liposomes preparation conditions used for liposomes which decreased PDA-mBzA amount in liposomes. We also reported reversible FRET studies on the liposome solutions, made from the monomer of m-aminobenzoic acid derivatized-10,12-pentacosadiynoic acid (PDA-mBzA) monomers and 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA) or dansyl-tagged diacetylene. After photo-polymerization, the solution appeared blue in color at room temperature. Heating and cooling cycles (between 25 ºC and 95 ºC temperature range), illustrated a visible color change from blue to red and a complete return to blue over many thermal cycles. Our preliminary reversible absorption and emission measurements showed that there exist opportunities for reversibility in FRET response. We are now performing more experiments to increase the FRET reversibility in these experiments. Although our system does not display full reversibility, the preliminary absorption and emission measurements strongly suggest that there exist opportunities for fully reversible selective and sensitive FRET-based sensors after further optimization of the system.

The main aim of work presented here is to design, develop and characterize a colorimetric model membrane (liposome) systems, which can bind with proteins, enzymes, bacteria, virus and other biomolecules. PDA molecules are utilized as a scaffold for the bilayer membrane, and a colorimetric assay is carried out. The holy grail of present work contributes towards the better understanding of protein interactions with the cell bilayer surface. Chapter 1 introduces a brief history on the advent of bilayer systems for cellular research exploration. We presented a literature survey about how liposome systems are used as a complementary technique to understand the fundamental principles of cellular membrane functions. Furthermore, we describe about membrane protein functions and recent findings on how proteins interact with the cell membrane. Finally, we explain conjugated systems and their exploration in bilayer membrane as a colorimetric scaffold. We also touch bases with major fluorescence techniques used in our experiments. Chapter 2 provides details on the preparation protocols of liposome and liposome-protein complexes. We confirmed protein-bilayer interactions by monitoring FRET between PDA and rhodamine molecules. Furthermore, we performed streptavidin-biotin binding studies on the PDA bilayer. Protein binding changed the spectral overlap (J) between PDA and rhodamine, which ultimately increased the fluorescence emission of rhodamine. The goal of performing these studies was to present a complete protocol for the preparation of liposome and protein-liposome complex. In chapter 3, we investigate how proteins bind on the cell membrane. Additionally, we propose a model of protein-bilayer complex. We reported that, by harnessing cell bilayer with specific bio-molecules, we monitored protein--bilayer, protein--protein and enzyme--substrate signal transduction. We have developed a colorimetric system for monitoring vital stimulations occur on the liposomal membrane surface. Bilayer was modified to covalently bind the amino group of lysine residues present on protein molecules. These bio-molecular interactions on bilayer surface provide differential stimulus, which turned out to be the major cause of differential spectroscopic signals depending upon size and shape of the protein bounded to the bilayer. Polydiacetylene (PDA) liposomes are the core of our color based system. These liposomes are used to monitor subtle interactions on the bilayer surface. We have also developed a semi-quantitative method based on the colorimetric response of PDA liposomes; we were able to detect protein molecules at sub-nanomolar concentrations in the solution. It's capability of distinguishing protein molecules based on their chemical and physical interactions to bilayer contributes towards the identity of our system. Interestingly, our mass spectroscopic data suggested non-specific enzymatic cleavage of membrane-bound proteins. These fragments were not present in bulk protein cleavage. We also proposed a model that depicts the covalent binding of protein at the bilayer of liposomes. These studies are intended to investigate protein-bilayer and enzyme-protein interaction occurring on the cell surface. In chapter 4, we focus on the kinetics of protein interaction on bilayer surface and we also attempt to visualize these interactions by exploring fluorescence microscopy. A self-assembled cell membrane is consisted of various lipids, which cluster themselves in their preferred phase separated regions. Lipid clusters are very important for lipid specific protein interactions. We investigated protein binding on such phase separated regions under a fluorescence microscope. Furthermore, we enzymatically catalyzed proteins, which were covalently bonded on the bilayer surface. This catalytic reaction was monitored both spectroscopically and under a fluorescence microscope. These studies were performed to help us in the better understanding of biological interactions at cell surface. Chapter 5, describes the encapsulation and controlled delivery of antimicrobial compounds from liposomes. Use of antimicrobial coatings on food packaging is one of the important technologies of active packaging for improving food safety. There is growing demand for natural antimicrobials because of fear of adverse health effects of synthetic preservatives. The main objective of this study is to compare antimicrobial activity of free versus encapsulated curcumin. Glass surfaces coated with nano-encapsulated curcumin may be used as an active packaging material in preserving liquid foods; however, further study is required to improve antimicrobial activities of polylactic acid PLA surfaces. In chapter 6, we investigate interactions between receptors and ligands at bilayer surface of polydiacetylene (PDA) liposomal nanoparticles using changes in electronic absorption spectroscopy and fluorescence resonance energy transfer (FRET). We study the effect of mode of linkage (covalent versus noncovalent) between the receptor and liposome bilayer. We also examine the effect of size-dependent interactions between liposome and analyte through electronic absorption and FRET responses. Glucose (receptor) molecules were either covalently or noncovalently attached at the bilayer of nanoparticles, and they provided selectivity for molecular interactions between glucose and glycoprotein ligands of E. coli. These interactions induced stress on conjugated PDA chain which resulted in changes (blue to red) in the absorption spectrum of PDA. The changes in electronic absorbance also led to changes in FRET efficiency between conjugated PDA chains (acceptor) and fluorophores (Sulphorhodamine-101) (donor) attached to the bilayer surface. Interestingly, we did not find significant differences in UV−Vis and FRET responses for covalently and noncovalently bound glucose to liposomes following their interactions with E. coli. We attributed these results to close proximity of glucose receptor molecules to the liposome bilayer surface such that induced stress were similar in both the cases. We also found that PDA emission from direct excitation mechanism was ∼2−10 times larger than that of the FRET-based response. These differences in emission signals were attributed to three major reasons: nonspecific interactions between E. coli and liposomes, size differences between analyte and liposomes, and a much higher PDA concentration with respect to sulforhodamine (SR-101). We have proposed a model to explain our experimental observations. Our fundamental studies reported here will help in enhancing our knowledge regarding interactions involved between soft particles at molecular levels. In chapter 7, we conclude the summary of all work carried out in previous chapters.

abstract: The primary carbon fixing enzyme Rubisco maintains its activity through release of trapped inhibitors by Rubisco activase (Rca). Very little is known about the interaction, but binding has been proposed to be weak and transient. Extensive effort was made to develop Förster resonance energy transfer (FRET) based assays to understand the physical interaction between Rubisco and Rca, as well as understand subunit exchange in Rca. Preparations of labeled Rubisco and Rca were utilized in a FRET-based binding assay. Although initial data looked promising, this approach was not fruitful, as no true FRET signal was observed. One possibility is that under the conditions tested, Rca is not able to undergo the structural reorganizations necessary to achieve binding-competent conformations. Rca may also be asymmetric, leading to less stable binding of an already weak interaction. To better understand the structural adjustments of Rca, subunit exchange between different oligomeric species was examined. It was discovered that subunit exchange is nucleotide dependent, with ADP giving the fastest exchange, ATP giving slower exchange and ATPS inhibiting exchange. Manganese, like ADP, destabilizes subunit-subunit interactions for rapid and facile exchange between oligomers. Three different types of assemblies were deduced from the rates of subunit exchange: rigid types with extremely slow dissociation of individual protomers, tight assemblies with the physiological substrate ATP, and loose assemblies that provide fast exchange due to high ADP. Information gained about Rca subunit exchange can be used to reexamine the physical interaction between Rubisco and Rca using the FRET-binding assay. These binding assays will provide insight into Rca states able to interact with Rubisco, as well as define conditions to generate bound states for structural analysis. In combination with assembly assays, subunit exchange assays and reactivation studies will provide critical information about the structure/function relationship of Rca in the presence of different nucleotides. Together, these FRET-based assays will help to characterize the Rca regulation mechanism and provide valuable insight into the Rubisco reactivation mechanism. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2017

Mon travail de thèse aborde différents développements physico-chimiques qui reposent sur le principe de transfert d'énergie par résonance de Foster (FRET). Le but est de parvenir à étudier et caractériser des assemblages moléculaires ainsi que des changements structurels de biomo-lécules (ou macro-ions) en phase gazeuse. Le transfert d'énergie par résonance de type Förster est un procédé par lequel de l'énergie s'échange de manière non radiative entre un chromo-phore dit donneur dans un état excité et un second chromophore accepteur en proximité di-recte. Conventionnellement, cette technique permet de localiser et déterminer l'écart entre deux molécules (de l'ordre de 10 à 100nm). Principalement utilisée pour étudier des systèmes biologiques, des résultats marquants ont été obtenus sur l'étude de système tel que l'appareil de Golgi, le cytosquelette ou les membranes cellulaires. Elle n'est cependant appliquée qu'à des systèmes en phase liquide. Il nous a paru intéressant de transposer cette technique en phase gazeuse, en utilisant la capacité des spectromètres de masse à sélectionner, isoler et activer des espèces moléculaires, nous permettant d'obtenir de nouvelles informations structurelles. Une grande partie de ma thèse a consisté à premièrement, valider le concept de FRET en phase gaz puis à développer et optimiser, la technique FRET dit ‘d'action'. L'Action-FRET est une tech-nique d'analyse par couplage de spectrométrie de masse et spectroscopie LASER mise au point par l'équipe Spectrobio afin d'étudier les molecules isolées en phase gazeuse. A travers ce dis-positif, je me suis particulièrement investi à contrôler, étudier et caractériser l'évolution des conformations de biomacromolécules d'intérêt biochimique et biologique. Dans une première partie je ferai une courte introduction générale sur les fondamentaux des protéines, de leur composition et élaboration en entités structurelles complexes, diverses et fonctionnelles. La manière dont les protéines s'arrangent successivement en niveaux structural quaternaire est aussi décrite. La deuxième partie est consacrée à une présentation des chromo-phores utilisés. Je présente ensuite leurs utilisations et détaille la synthèse des édifices molécu-laires produits pour réaliser les expériences de FRET. Ceux-ci sont constitués de composés bio-logiques (peptides ou protéines), couplés aux chromophores, (donneur-accepteur). Dans le contexte de ce chapitre se trouve également une discussion sur les mécanismes et produits uti-lisés lors de l'étape de conjugaison qui permet d'obtenir les composants désirés. En troisième partie vient un chapitre qui relate le fonctionnement des appareils utilisés dans le montage expérimental; le LASER et le spectromètre de masse. La méthode de couplage est dé-crite et spécifiée, détaillant comment les appareils commerciaux ont été modifiés pour interagir avec l'un avec l'autre. Avec ce nouveau montage, un suivi de la signature optique de FRET ap-partenant aux protéines entières greffées et à différents états de charge a été possible. Le quatrième chapitre est dédié dans les premières sections à la théorie et l'état de l'art en ce qui concerne le FRET. Les éléments emblématiques et leurs applications en solution de ces der-nières années et les travaux plus récents en phase gazeuse y sont présentés. Par ailleurs, nous avons voulu démontrer dans ce chapitre que nos diverses manipulations ont l'avantage critique de ne pas dépendre d'une mesure de l'émission de lumière suite au transfert résonant d'éner-gie. A la place, on dispose de la fragmentation spécifique de l'ion piégé du chromophore à tra-vers l'analyse de masse conventionnelle du spectromètre de masse pour détecter et quantifier une manifestation de FRET. Nous démontrerons aussi la possibilité cette méthode appliquée à la biologie moléculaire... [etc] / In this thesis, I discuss the application and development of mass spectrometry (MS) - LASER coupled techniques for the characterization and measurement of trapped biomolecules in the gas phase. In broad terms, this thesis demonstrates the potential and perspectives of action-FRET a novel structural biology tool amenable to the gas phase. The fundamentals rely on a well attested resonance quantic process known as Förster resonance energy transfer (FRET). As of yet it has been a widely utilized method to scrutinize molecular structure in solution. The mo-tivation has been to transpose this occurrence to the instrumental settings of a mass spectrom-eter, its gas confinement and, in doing so, overcome the earlier limitations of the technique and stride into the theoretical and experimental study of well determined systems as well as those whose structure were presently undetermined - all without the influence of the environment of a solvated medium. The first chapter of this thesis offers a general overview on peptides and proteins plus how they can be studied. Subsequent chapters include how the work carried out herein ads towards their study, moving the technique towards a gold standard of native mass spectrometry (native MS). In the second chapter, a treatment of the synthetic steps and preparations is given detailing the mechanistics of the reactions at play and above all outlining the experimental procedures and providing any information on any observations made. The third chapter describes, and is devoted to an introduction of the instrumental setup outlaid as it stands giving an account on the LASER, optical pieces and the mass spectrometer employed throughout the course of this thesis, effectively setting the premises of thought and understand-ing for subsequent chapters and methodology. Chapter four presents energy transfer, in particular to the Foster Resonance Energy Transfer and furthermore outlines the developed technique central to the mass spectrometry method to look at donor-acceptor chromophores espoused to biomolecular systems and their photofrag-ments — what is nicknamed as action-FRET. Chapter five reviews and discusses the study of a macromolecule: Ubiquitin, by action-FRET. The first gas phase experiment on the protein to have ever been realised. The information and content gathered from this adapted experiment is compared to work found elsewhere giving an appraisal on the potential of action-FRET and providing an idea to what future insights the technique could bring. In chapter six the reader is introduced to the project of establishing action-FRET in the negative mode of the mass spectrometer’s ionization source as opposed to its positive mode. Suitable pairs of donor-acceptor chromophores to validate the energy transfer under a negative regime were explored. These where profiled and characterized before being adapted to a biomolecular system. The results provide a different flavor of complimentary structural and conformation information, the first of its kind for negative mode action-FRET. The seventh and final chapter is devoted to future developments. The conclusive work tends to grant a further understanding of neurodegenerative diseases that afflict our societies. Chiefly that of the likes of Alzheimer’s: it’s the mechanism of action pertinent to other neurodegenera-tive pathologies; Parkinson’s, Huntington’s but also prion diseases or amyloid neuropathy. In doing so a contribution is presented on a way to trace a strategy in how to tackle and treat such diseases