A Pipeline Analytical Program and Dredging Knowledge{Base Expert{System (DKBES) determines a pipeline dredge's production and resulting cost and schedule. Pipeline dredge engineering presents a complex and dynamic process necessary to maintain navigable waterways. Dredge engineers use pipeline engineering and slurry transport principles to determine the production rate of a pipeline dredge system. Engineers then use cost engineering factors to determine the expense of the dredge project. Previous work in engineering incorporated an object{oriented expert{system to determine cost and scheduling of mid{rise building construction where data objects represent the fundamental elements of the construction process within the program execution. A previously developed dredge cost estimating spreadsheet program which uses hydraulic engineering and slurry transport principles determines the performance metrics of a dredge pump and pipeline system. This study focuses on combining hydraulic analysis with the functionality of an expert{system to determine the performance metrics of a dredge pump and pipeline system and its resulting schedule. Field data from the U.S. Army Corps of Engineers pipeline dredge, Goetz, and several contract daily dredge reports show how accurately the DKBES can predict pipeline dredge production. Real{time dredge instrumentation data from the Goetz compares the accuracy of the Pipeline Analytical Program to actual dredge operation. Comparison of the Pipeline Analytical Program to pipeline daily dredge reports shows how accurately the Pipeline Analytical Program can predict a dredge project's schedule over several months. Both of these comparisons determine the accuracy and validity of the Pipeline Analytical Program and DKBES as they calculate the performance metrics of the pipeline dredge project. The results of the study determined that the Pipeline Analytical Program compared closely to the Goetz eld data where only pump and pipeline hydraulics a ected the dredge production. Results from the dredge projects determined the Pipeline Analytical Program underestimated actual long{term dredge production. Study results identi ed key similarities and di erences between the DKBES and spreadsheet program in terms of cost and scheduling. The study then draws conclusions based on these ndings and o ers recommendations for further use.

Funktionalisierte organische Perylenbisimidfarbstoffe (PBI) und aus Cadmiumselenid bestehende Halbleiternanokristalle werden hinsichtlich physikalischer sowie chemischer Wechselwirkungsprozesse miteinander und mit ihrer Umgebung mittels zeitaufgelöster optischer Spektroskopie untersucht. Im Mittelpunkt der Studien an diesem organisch/anorganischen Modellsystem nanoskopischer Größe steht die Aggregatbildungskinetik und die Identifikation und Quantifizierung von Transferpozessen. Die Anbindung der gut löslichen PBI-Farbstoffe an die Oberfläche solcher Halbleiternanokristalle mittels spezieller Ankergruppen wird durch Selbstorganisation in Lösung realisiert. Die Kombination von Absorptions- und zeitaufgelöster Fluoreszenzspektroskopie zeigt einen unterschiedlich starken Einfluss von Liganden und Farbstoffen auf die Fluoreszenzlöschung der Nanokristalle und belegt, dass Resonanzenergietransfer zum Farbstoff nur in sehr geringem Maße die physikalische Ursache der Fluoreszenzlöschung ist. Die Anzahl adsorbierter Farbstoffe und die Stärke der Fluoreszenzlöschung eines einzelnen Farbstoffmoleküls werden aus zeitaufgelösten Einzelmolekülexperimenten an immobilisierten Emittern gewonnen, welche den direkten spektroskopischen Zugang zur Verteilung gebundener und freier Farbstoffe/Nanokristalle erlaubt. Darüber hinaus werden ankergruppen- und umgebungsspezifische Einflüsse auf die Konformations- und Orientierungsdynamik von Perylenbisimidmolekülen dargestellt. Abschließend werden photo-physikalische Gemeinsamkeiten chemisch unterschiedlich hervorgerufener Fluoreszenzlöschungsprozesse herausgearbeitet und im Kontext von Einzelkristall-Blinkprozessen diskutiert.

The homo and hetero dimerisation of two spectroscopically different chromophores were studied, namely: 4,4-difluoro-4-bora-3a,4a-diazas-indacene (g-BODIPY) and its 5-styryl-derivative (r-BODIPY). Various spectroscopic properties of the r-BODIPY in different common solvents were determined. It was shown that g- and r-BODIPY in the ground state can form homo- as well as hetero dimers. We demonstrate that the ganglioside GM1 in lipid bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exhibits a non-uniform lateral distribution, which is an argument in favour of self-aggregation of GM1 being an intrinsic property of the GM1. This was concluded from energy transfer/migration studies of BODIPY-labelled gangliosides. An algorithm is presented that quantitatively accounts for donor–donor energy migration (DDEM) among fluorophore-labelled proteins forming regular non-covalent polymers. The DDEM algorithm is based on Monte Carlo (MC) and Brownian dynamics (BD) simulations and applies to the calculation of fluorescence depolarisation data, such as the fluorescence anisotropy. Thereby local orientations, as well as reorienting motions of the fluorescent groups are considered in the absence and presence of DDEM among them. A new method, in which a genetic algorithm (GA) was combined with BD and MC simulations, was developed to analyse fluorescence depolarisation data collected by the time-correlated single photon counting technique. It was applied to study g-BODIPY-labelled filamentous actin (F-actin). The technique registered the local order and reorienting motions of the fluorophores, which were covalently coupled to cysteine 374 (C374) in actin and interacted by means of electronic energy migration within the polymer. Analyses of F-actin samples composed of different fractions of labelled actin molecules revealed the known helical organiszation of F-actin, and demonstrated the usefulness of this technique for structure determination of complex protein polymers. The distance from the filament axis to the fluorophore was found to be considerably less than expected from the proposed position of C374 at a high filament radius. In addition, polymerisation experiments with BODIPY-actin suggest a 25-fold more efficient signal for filament formation than pyrene-actin.

No description available.

No description available.

Les récepteurs couplés aux protéines G (RCPGs) constituent la plus grande classe de récepteurs membranaires impliqués dans la transmission des signaux extracellulaires. Traditionnellement, la transmission de la signalisation par les RCPGs implique l’activation d’une protéine G hétéro-trimérique qui pourra à son tour moduler l’activité de divers effecteurs intracellulaires. Ce schéma classique de signalisation s’est complexifié au fils des années et l’on sait maintenant qu’en plus d’interagir avec les protéines G, les RCPGs s’associent avec une panoplie d’autres protéines afin de transmettre adéquatement les signaux extracellulaires. En particulier, la découverte d’une famille de protéines transmembranaires modulant la fonction des RCPGs, baptisées protéines modifiant l’activité des récepteurs (« receptor activity-modifying proteins » ; RAMPs), a changé la façon de concevoir la signalisation par certains RCPGs. Dans le cas du récepteur similaire au récepteur de la calcitonine (« calcitonin-like receptor » ; CLR), l’association avec les RAMPs permet l’acheminement à la surface cellulaire du récepteur tout en modulant ses propriétés pharmacologiques. Lorsqu’il est associé avec RAMP1, le CLR fonctionne comme un récepteur du peptide relié au gène de la calcitonine (« calcitonin gene-related peptide » ; CGRP), alors qu’il devient un récepteur de l’adrénomedulline lorsqu’il interagit avec RAMP2 ou RAMP3. D’autre part, en plus d’interagir avec des protéines accessoires transmembranaires telles les RAMPs, les RCPGs peuvent aussi s’associer entre eux pour former des oligomères de récepteurs. Dans cette thèse, nous nous sommes penchés sur les interactions entre les RCPGs et les RAMPs, et plus particulièrement sur l’interrelation entre ce type d’association RCPG/RAMP et l’assemblage en oligomères de récepteurs, en utilisant le récepteur du CGRP comme modèle d’étude. Une première étude nous a tout d’abord permis de confirmer l’interaction entre le récepteur CLR et RAMP1, dans un contexte de cellules vivantes. Nous avons démontré que ce complexe CLR/RAMP1 active la protéine G et recrute la protéine de signalisation -arrestine suite à une stimulation par le CGRP. Ensuite, nous avons déterminé que même s’il doit obligatoirement former un hétéro-oligomère avec les RAMPs pour être actif, le CLR conserve malgré tout sa capacité à interagir avec d’autres RCPGs. En plus d’observer la présence d’homo-oligomère de CLR, nous avons constaté que tout comme les RCPGs, les RAMPs peuvent eux-aussi s’associer entre eux pour former des complexes oligomériques pouvant comprendre différents sous-types (RAMP1/RAMP2 et RAMP1/RAMP3). Cette observation de la présence d’homo-oligomères de CLR et de RAMP1, nous a amené à nous questionner sur la stœchiométrie d’interaction du complexe CLR/RAMP1. Dans une deuxième étude ayant pour but d’établir la composition moléculaire du récepteur CGRP1 in vivo, nous avons développé une nouvelle approche permettant l’étude de l’interaction entre trois protéines dans un contexte de cellules vivantes. Cette technique baptisée BRET/BiFC, est basée sur le transfert d’énergie de résonance de bioluminescence entre un donneur luminescent, la Renilla luciférase, et un accepteur fluorescent, la protéine fluorescente jaune (YFP), reconstituée suite au ré-assemblage de ces deux fragments. En utilisant cette approche, nous avons pu déterminer que le récepteur CGRP1 est constitué d’un homo-oligomère de CLR interagissant avec un monomère de RAMP1. En démontrant un assemblage oligomérique asymétrique pour le récepteur CGRP1 à partir d’une nouvelle approche biophysique, nous croyons que les travaux présentés dans cette thèse ont contribué à élargir nos connaissances sur le fonctionnement de la grande famille des RCPGs, et seront utile à la poursuite des recherches sur les complexes protéiques impliqués dans la signalisation. / G protein coupled receptors (GPCRs) constitute the largest family of membrane receptors involved in signal transduction. Traditionally, signal transduction by GPCRs involves the activation of a hetero-trimeric G protein which will then modulate the activity of several intracellular effectors. We can now appreciate the fact that in addition to their interaction with G proteins, GPCRs also associate with several other proteins, in order to allow proper signal transduction. In particular, the discovery of a family of proteins called receptor activity-modifying proteins (RAMPs) has challenged the traditional views of signal transduction by some GPCRs. In the case of the calcitonin-like receptor (CLR), the association with RAMPs allows the proper cell surface targeting of the receptor in addition to modulate it’s pharmacological properties. Co-expression of CLR with RAMP1 leads to a calcitonin gene-related peptide (CGRP) receptor, whereas CLR association with RAMP2 or RAMP3 promotes the formation of an adrenomedullin receptor. In addition to their interaction with transmembrane accessory proteins such as RAMPs, GPCRs can also interact with other receptors to form receptors oligomers. In this thesis, we were interested in the interactions between GPCRs and RAMPs, and particularly, in the link between these GPCR/RAMP interactions and the assembly of receptor oligomers, using CGRP1 receptor as a model. We first confirmed the interaction between CLR and RAMP1 in living cells. We showed that this CLR/RAMP1 complex activates G proteins and recruits the signalling protein -arrestin upon CGRP stimulation. Next, we demonstrated that even if the CLR requires hetero-oligomeric assembly with RAMPs in order to be active, this receptor can still interact with other GPCRs. In addition to CLR homo-oligomers, we observed that RAMPs can also self-associate to form oligomeric complexes which can involve different subtypes (RAMP1/RAMP2 and RAMP1/RAMP3). This observation of the presence of CLR and RAMP1 homo-oligomers raised the question of the stoiechiometry of interaction of the CLR/RAMP1 complex. In order to establish the molecular composition of the CGRP1 receptor in vivo, we developed a novel approach allowing the detection of the interaction between three proteins in living cells. This method called BRET/BiFC is based on the bioluminescence resonance energy transfer between a luminescent energy donor, Renilla luciferase, and a fluorescent energy acceptor, the yellow fluorescent protein (YFP), reconstituted after the re-association of its two fragments. Using this approach, we showed that the CGRP1 receptor consist of a homo-oligomer of CLR interacting with a monomer of RAMP1. By demonstrating the asymmetrical organization of the CGRP1 receptor complex using a novel biophysical approach, we believe that the results presented herein have contributed to increase our knowledge of the mechanisms of function of the large family of GPCRs and will be useful for the pursuit of research on protein complexes involved in signalling pathways.

No description available.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The SNABP [40SiO2.30Na2CO3.1Al2O3.25B2O3.PbO2 (mol%)] glass system, nanostructured with PbS Quantum Dots (QDs) and/or doped with Er3+ ions, was successfully synthesized by the Fusion Method, when it was subjected to appropriate thermal annealing. The glass transition temperatures (Tg) were obtained by Differential Thermal Analyze (DTA), in which it was possible to define a suitable temperature to be used in the thermal annealing of the synthesized samples. As results of these thermal annealing, the formation and growth of PbS QDs have occurred in the glass environment. The optical properties of samples were investigated by Optical Absorption (OA), Photoluminescence (PL), and Photoluminescence with Temporal Resolution (PLTR). Moreover, the Atomic Force Microscopy (AFM) and X-Ray Diffractometry were employed in study of morphological and structural properties of samples, respectively. The size dispersions of PbS QDs were determined from OA spectra. Once using both the Method and OA data, it was also possible to estimate the average diameters of these nanoparticles, which grow with the increase in annealing time. The characteristic transitions of Er3+ ions were clearly identified in OA spectra. In addition, it was observed that the increase in annealing time of samples had provoked amplification in the overlapping between PL bands of both the PbS QDs and Er3+ ions, as well as with the absorption 4I15/2 -> 4I13/2 of these ions. Thus, the PLTR measurements have confirmed the decrease in lifetime of the 4I13/2 level (of Er3+ ions), with the amplification in the overlapping of PL emissions. Finally, as a main result of this work, it was proved that the SNABP glass system, nanostructured with PbS QDs and doped with Er3+ ions, displays to be quite favorable to the radiative energy transfer process (from PbS QDs to Er3+ ions), as well as the occurrence of stimulated emission of 4I13/2 level. / O sistema vítreo SNABP [40SiO2.30Na2CO3.1Al2O3.25B2O3.PbO2 (mol%)] nanoestruturado com pontos quânticos (PQs) de PbS e/ou dopados com íons Er3+ foi sintetizado com sucesso pelo Método de Fusão, quando submetido a tratamentos térmicos apropriados. As temperaturas de transição vítrea (Tg) foram obtidas por Análise Térmica Diferencial (DTA), em que foi possível definir uma temperatura adequada para ser utilizada nos tratamentos térmicos das amostras sintetizadas. Como resultados desses tratamentos térmicos, a formação e crescimento dos PQs de PbS ocorreram no ambiente vítreo. As propriedades ópticas das amostras foram investigadas por Absorção Óptica (AO), Fotoluminescência (PL) e Fotoluminescência com Resolução Temporal (PLRT). Além disso, a Microscopia de Força Atômica (AFM) e Difratometria de Raios-X (DRX) foram empregadas no estudo das propriedades morfológicas e estruturais das amostras, respectivamente. As dispersões de tamanho dos PQs de PbS foram determinadas a partir dos espectros de AO. Com a utilização do Método e os dados de AO, foi também possível estimar os diâmetros médios dessas nanopartículas, que aumentaram com o aumento no tempo de tratamento térmico. As transições características dos íons Er3+ foram claramente identificadas nos espectros de AO. Em adição, foi observado que o aumento no tempo de tratamento das amostras provocou um aumento na sobreposição entre as bandas de PL dos PQs de PbS e dos íons Er3+, bem como com a absorção 4I15/2 -> 4I13/2 desses íons. Assim, as medidas de PLRT confirmaram o decréscimo no tempo de vida do nível 4I13/2 (dos íons Er3+) com o aumento dessa sobreposição das emissões de PL. Finalmente, como principal resultado deste trabalho, foi comprovado que o sistema vítreo SNABP nanoestruturado com PQs de PbS dopados com íons Er3+ mostrou-se bastante favorável ao processo de transferência de energia radiativa (dos PQs de PbS para os íons Er3+), bem como a ocorrência da emissão estimulada do nível 4I13/2. / Mestre em Física

Time resolved spectroscopy is one of the crucial methods used to study processes on molecular level in biological systems. It is useful especially for monitoring fast processes that take a place in photosynthetic apparatus of photosynthetic organisms, such as electron and energy transfer. The integral parts of photosynthetic apparatus are carotenoids, whose role in the photosynthetic apparatus is not as well explored as it is for chlorophylls. It was proved that carotenoids actively participate in energy transfer processes in photosynthetic antennas. They have a crucial role in protection against excess energy damage. They are also electron donors in both antennas and reaction centers. The fact that photo-physical properties of carotenoids are much different from properties of others organic pigments, complicates studies of their functions in photosynthesis as well as in other biological systems. This thesis employs advanced methods of femtosecond spectroscopy to obtain more information about carotenoid functions in some biological systems and in solution with special focus on carotenoids containing carbonyl group.

Manipulating light by controlling surface plasmons on metals is being discussed as a means for bridging the size gap between micrometer-sized photonic circuits and nanometer-sized integrated electronics. Plasmonic waveguides based on metal nanoparticles are of particular interest for circumventing the diffraction limit, thereby enabling high-speed communication over short-range distances in miniaturized micro-components. However, scalable, inexpensive fine-tuning of particle assemblies remains a challenge and near-field probing is required to reveal plasmonic interactions. In this thesis, self-assembled waveguides should be produced on DNA scaffolds. DNA origami is an extremely versatile and robust self-assembly method which allows scalable production of nanostructures with a fine control of assemblies at the nanoscale. To form the plasmonic waveguides, six-helix bundle DNA origami nanotubes are used as templates for attachment of highly monodisperse and monocrystalline gold nanoparticles with an inter-particle distance of 1-2 nm. In the first part of this thesis, the effects of parameters which are involved in assembly reactions are systematically investigated. The assembly yield and binding occupancy of the gold nanoparticles are determined by an automated, high-throughput image analysis of electron micrographs of the formed complexes. As a result, unprecedented binding site occupancy and assembly yield are achieved with the optimized synthesis protocol. In addition, waveguides with different sizes of gold nanoparticles and different inter-particle distances, quantum dots attachments to the waveguides and multimerization of the waveguides are successfully realized. In the second part of this thesis, direct observation of energy transport through a self-assembled waveguide towards a fluorescent nanodiamond is demonstrated. High-resolution, near-field mapping of the waveguides are studied by electron energy loss spectroscopy and cathodoluminescence imaging spectroscopy. The experimental and simulation results reveal that energy propagation through the waveguides is enabled by coupled surface plasmon modes. These surface plasmon modes are probed at high spatial and spectral resolutions. The scalable self-assembly approach presented here will enable the construction of complex, sub diffraction plasmonic devices for applications in high-speed optical data transmission, quantum information technology, and sensing. / Die Manipulation des Lichts durch die Kontrolle von Oberflächenplasmonen auf metallischen Oberflächen und Nanopartikeln gilt als vielversprechende Methode zur Überbrückung der Größen-Lücke zwischen Mikrometer-großen photonischen und nanometer-großen elektronischen Schaltkreisen. Plasmonische Wellenleiter basierend auf metallischen Nanopartikeln sind vom besonderen Interesse, da sie die Umgehung des Beugungslimits und somit eine Hochgeschwindigkeitskommunikation über kurze Distanzen in immer kleiner werdenden Schaltkreisen ermöglichen könnten. Allerdings ist die skalierbare und kostengünstige Anordnung von Partikeln eine große Herausforderung und es werden Nahfelduntersuchungen benötigt um plasmonische Interaktionen detektieren zu können. Das Ziel dieser Arbeit ist die Selbstassemblierung von multi-partikel Wellenleitern auf DNA Gerüsten. Die Verwendung von DNA-Origami bietet eine äußerst vielseitige Plattform zur skalierbaren Herstellung von Nanostrukturen mittels Selbstassemblierung und ermöglicht eine präzise Kontrolle der Anordnungen im Nanobereich. Für den Aufbau der plasmonischen Wellenleiter werden DNA-Origami Nanoröhren, bestehend aus sechs Helices als Templat für die Anbindung von monodispersen und monokristallinen Goldnanopartikeln mit einem interpartikulären Abstand von 1-2 nm verwendet. Im ersten Abschnitt dieser Arbeit werden die beeinflussenden Faktoren dieser Assemblierungsreaktion systematisch untersucht. Die Ausbeute der assemblierten Strukturen und die Besetzung der Bindungsstellen werden durch eine automatisierte und effiziente Bildanalyse von Elektronenmikroskopieaufnahmen ausgewertet. Durch die Entwicklung eines optimierten Syntheseprotokolls werden bisher unerreichte Assemblierungsausbeuten ermöglicht. Zusätzlich erfolgen die experimentelle Realisierung von Strukturen mit verschieden großen Goldnanopartikeln und unterschiedlichen interpartikulären Abständen, sowie die Anbindung von Quantenpunkten an die Wellenleiter und eine Verknüpfung der assemblierten Strukturen. Der zweite Abschnitt dieser Dissertation befasst sich mit der Untersuchung des Energietransports in selbstassemblierten Wellenleitern über einen fluoreszierenden Nanodiamanten. Dazu erfolgen hochaufgelöste Nahfeldmessungen der Wellenleiter mittels Elektronenenergieverlustspektroskopie und Kathodolumineszenz-mikroskopie. Die experimentellen Ergebnisse und zusätzlich durchgeführte Simulationen bestätigen eine durch gekoppelte Oberflächenplasmonenmoden induzierte Weitergabe der Energie innerhalb des Wellenleiters. Diese Oberflächenplasmonenmoden werden bei hoher räumlicher und spektraler Auflösung untersucht. Das hier umgesetzte Konzept der Selbstassemblierung wird den Aufbau komplexer plasmonischer Geräte für Anwendungen im Bereich der optischen Hochgeschwindigkeitsdatenübertragung, der Quanteninformations-technolgie und der Sensorik ermöglichen.