Chemical Oxygen Iodine Laser(COIL) is an electronic transition high energy chemical laser having a wavelength of 1.315 /mi. This is the first chemical laser to operate on an electronic rather than a rotational or vibrational transition. In principle the COIL can be operated either in pulsed or cw mode. Its interest lies in high chemical efficiency, high power and wavelength which is shortest among all the chemical lasers. COIL finds a wide range of applications as its output wavelength at 1.315/zm couples well with the surface of most metals. The applications include surface hardening and modification of metals, welding, drilling and cutting of metals, cutting of ceramics, micro machining, laser deposition of non metallic coatings on metallic surfaces, monitoring of atmospheric pollutants and solar hazardous waste detoxification. Moreover, its wavelength is suitable for fiber optic transmission. In COIL the laser output at 1.315 /an is achieved by stimulated emission on the f (2-PL/2) -* -f (2-p3/2) magnetic dipole transition in atomic iodine. The population inversion on this transition is obtained by resonant collisions! energy transfer from metastable excited Oj^A) molecules produced by a chemical reaction of KOH, H2O? and Cl2. The chemical reaction of H2O2 and Cl2 that produces oxygen molecules is highly exothermic, and because of spin conservation considerations, channels its energy directly into the metastable electronically excited singlet delta state of oxygen molecule. Since the O2(1A) has a 45 mins lifetime and hence an extremely low small signal gain coefficient, it cannot be lased directly. Lasing can be achieved, however, if this energy is transferred to an atom or molecule which has a reasonable transition moment between its excited and ground states. The iodine 52P^2 -> 52P3/2 magnetic dipole transition has an acceptable transition moment and is nearly resonant with the 02{lA) state in oxygen. Excited iodine atoms are obtained by mixing O2(l A) and l2 molecules resulting in their dissociation and subsequent excitation. Power levels in excess of 25 kW have been reported in COIL. Due to wide range of applications and mainly for its use as a laser weapon, efforts are being made to enhance the power to higher levels. The dissociation of I2 controls the gain of the coil and hence power. In the pure COIL scheme some of the I2 remains undissociated due to the recombination reactions. Hence if we add a mechanism to dissociate the residual I2 molecules, we can enhance the performance of the COIL. So we propose to add a solar pumping to conventional COIL, which by photo exciting the undissociated I2lead to increase in efficiency. The thesis contains six chapters in which chapter 1 contains a general introduction and the definition of the research problem. The basic theory and the chemical reactions are discussed in chapter 2, The proposed model is discussed and the rate equations are solved in chapter 3. The numerical scheme and the computer code along-with the validation of the code are presented in chapter 4. The numerical results for the species concentrations, population inversion density and the output power for the proposed solar pumped COIL are presented in chapter 5, Final conclusions and future scope of the proposed research are presented in the final chapter 6. (Pl refer the original document for formulas)

L’apparition ces dernières années de supraconducteurs réalisés industriellement utilisant des composés à haute température de transition offre la possibilité de nouveaux développements en magnétisme supraconducteur. En effet ils permettent d’augmenter le champ magnétique généré en conservant une cryogénie classique à 4,2K d’une part, et ils ouvrent la voie à des développements d’aimants supraconducteurs fonctionnant entre 10 et 30K d’autre part. Les matériaux supraconducteurs à haute température critique sont alors indispensables pour dépasser les inductions magnétiques de 16 T (cas de l’insert dipolaire HTc pour le Large Hadron Collider du CERN) ou augmenter la densité spécifique d’énergie stockée dans un SMES (Superconducting Magnetic Energy Storage, cas du projet ANR SuperSMES).Les atouts incontestables (température critique, champ magnétique critique, résistance mécanique) apportés par l’utilisation des matériaux supraconducteurs à haute température critique tels que l’YBaCuO dans les aimants supraconducteurs demandent de relever quelques défis. Leur comportement est encore mal compris, surtout lors des transitions résistives. Arriver à protéger ces conducteurs requiert une réflexion nouvelle sur les systèmes de protection destinés à éviter les dégradations thermiques et mécaniques. La réponse à la question « peut-on utiliser ces matériaux de manière pérenne dans les aimants supraconducteurs ? » est incontournable.Des éléments de réponse sont donnés ici. L’utilisation des conducteurs est abordée à travers différentes études expérimentales permettant de mieux connaître le conducteur (caractérisation électrique et modélisation de la surface critique) d’une part et de définir les étapes clés de la fabrication des aimants supraconducteurs à haute température de transition (étude des jonctions entre conducteurs ou entre galettes) d’autre part. Cette étude a abouti à la réalisation de deux prototypes d’aimants ayant permis d’identifier les difficultés liées à l’utilisation des rubans d’YBaCuO. Un modèle thermoélectrique des supraconducteurs à haute température de transition est développé et un code numérique basé sur le logiciel de calcul par Eléments Finis CASTEM permet d’étudier le phénomène de transition résistive, ou quench, dans un conducteur et dans un aimant. Le code a été validé sur des essais réalisés au Laboratoire National des Champs Magnétiques Intenses de Grenoble. Les résultats obtenus ont permis la définition des conducteurs pour les deux projets liés à la thèse et la validation de la protection. / The new industrial superconductors using high critical temperature compounds offer new possibilities for superconducting magnetism. Indeed they allow higher magnetic field with the same classical cryogenics at 4.2 K on one hand, and on the other hand they also pave the way for superconducting magnets working between 10 K and 30 K. The high temperature superconductors are then needed in order to produce magnetic fields higher than 16 T (case of HTS dipole insert for Large Hadron Collider at CERN) or to increase the specific density stored in one SMES (Superconducting Magnetic Energy Storage, in the case of the SuperSMES ANR Project).Nevertheless the indisputable assets (critical temperature, critical magnetic field, mechanical stresses) brought by the use of High critical temperature superconductors like YBCO, used in superconducting magnets, require to solve some challenges. Their behavior is still badly understood, especially during the resistive transitions. To succeed in protecting these conductors we need a new reflection on protection schemes designed to avoid the thermal and mechanical damages. The answer to the question: “Can we use those materials in the long run inside superconducting magnets?” is now inescapable.Some answers are given here. The use of the conductors is approached through various experimental studies to understand the material (electrical characterization and modeling of the critical surface) and to define the key stages of high critical temperature superconducting magnets manufacturing (work on the junctions between conductors and pancakes). This study led to the creation of two coils in order to identify the issues related to the use of YBCO tapes. A numerical thermo-electrical model of the high critical temperature superconductor has been developed and a numerical code based on the CEA software CASTEM (Finish Elements Model) allowed to study the resistive transition (or quench) behavior of those conductor and coil. The code has been confirmed by comparison with some experimental data obtained by the Laboratoire National des Champs Magnétiques Intenses in Grenoble. The results have allowed to define the conductors for the two projects of this thesis and to validate the feasibility of the protection of those conductors.

Produktkoncept för en dubbelhaspel för plåt – För effektivisering inom stålindustrin vid hantering av plåt, är ett projekt som genomförts på Karlstads universitet i samarbete med Camatec Industriteknik AB och är ett examensarbete i högskoleingenjörsutbildningen ’Innovationsteknik och Design’. Projektet handlar om att ta fram ett produktkoncept för en dubbelhaspel. Camatec Industriteknik AB är ett konsultföretag och arbetar i en nära relation med företaget Camcoil Systems AB. Camcoil Systems AB tillverkar idag enkelhasplar, maskiner som används inom stålindustrin vid hantering av tunn plåt, bandplåt. Camcoil Systems AB har uttryckt en önskan att på sikt även tillverka dubbelhasplar. Genom detta projekt läggs en grund för Camcoil Systems AB att ta vid och vidareutveckla det framtagna konceptet. För att ta fram ett hållbart produktkoncept har fyra huvudkategorier undersökts; marknad, teknik, design samt budget. Säkerhet är en kategori som också tas hänsyn till i projektet. I projekt tillämpas designprocessen för att nå målet. En förstudie ligger till grund för hela projektet, där de fyra nämnda huvudkategorierna samt vetenskapliga teorier vad gäller perception, färglära, produktutveckling samt gestaltningslagar behandlas. Det framtagna konceptet består av enkla former uttryckt i runda former där dess utseende och funktion går hand i hand och på så sätt förstärker varandra. Konceptet besitter samtidigt en hållbar teknik och en god säkerhet som ryms inom Camcoil Systems AB’s kostnadsram. Med det framtagna konceptet skapas ett hållbart underlag och möjliggör en vidareutveckling för Camcoil Systems AB att på sikt vara en potentiell aktör på marknaden vad gäller dubbelhasplar. / Product concept for a double-reel for sheet metal – For a more effective process in steel-industry in handling sheet metal, is a project as implemented at Karlstad university in cooperation with Camatec Industriteknik AB and is an examination of the engineering education ’Innovationsteknik och Design’. The project is about to develop a product concept for a double-reel. Camatec Industriteknik AB is a consulting company and works in a close relationship with the company Camcoil Systems AB. Camcoil Systems AB manufactures single-reel’s, machines as used in the steel industry in handling thin sheet, strip steel. Camcoil Systems AB has expressed the desire to in the future be a double-reel manufacture. By this project Camcoil Systems AB wants to build a base to develop the produced concept. In order to develop a sustainable product concept there are four major categories in this project which are investigated; Market, technology, design and budget. Security is something that is also taken into consideration in the project. The design-process is applied in order to achieve the goal. A feasibility study is the basis for the entire project, where all these four main categories, scientific theories regarding to perception, colour theory, product development and design laws are treated. The produced concept is built upon a clean design in which the functions and the design are synchronized towards each other. In that way, they reinforce each other. There are also a sustainable technology and a good security. At the same time the costs are not too high. With the produced concept, there is a foundation which enables a further development for Camcoil Systems AB to be a potential entrant for the market in producing double-reels.

The coiled-coil motif is present in proteins from all kingdoms of life. Its structure is based on a repeating sequence of 7 amino acids with hydrophobic residues at positions 1 and 4, which folds into an alpha-helix. Two, or more, alpha-helices wind around each other based on hydrophobic interactions forming the coiled-coil. Structural variations include length, deviations from the canonical form based on the heptad repeat, as well as the orientation and number of alpha-helices. They are involved in a wide variety of cellular processes including vesicle tethering and signal transmission along their length. In order to transmit signal, the protein must be able to dynamically rearrange its structure. An outstanding example of a coiled-coil that needs to rearrange its structure to perform its function is the early endosomal tether EEA1, which has been shown to increase its flexibility upon binding to the active form of the small GTPase Rab5. That conformational change generates an entropic collapse that brings the ends of the protein closer to each other. Nevertheless, the recycling from the more flexible state to its original extended conformation was not addressed. Herein, the entropic collapse mechanism was further studied and the full EEA1 cycle between extended and flexible states described. In addition to these studies, other coiled-coil proteins were assessed to determine if they also experience a binding-induced entropic collapse. One of the strategies to investigate the entropic collapse mechanism was to compare the adhesive forces along the two alpha-helices of the EEA1 dimer in its extended and flexible conformations. To this end, an experiment was designed to unwind the dimer using optical tweezers, a force-spectroscopy method that uses a highly focused laser beam to manipulate microscopic objects. Each EEA1 monomer was attached to a distinct DNA piece using a site-specific enzymatic reaction. The DNA pieces were linked to two optically trapped micron-sized beads. And the distance between the optical traps increased to unwind the EEA1. A second strategy to investigate the entropic collapse was to evaluate EEA1 dynamics in solution using dual color fluorescence cross-correlation spectroscopy (dcFCCS). EEA1 C-termini was labeled with two different fluorophores. Fluctuations on fluorescent intensities caused by the dyes crossing a confocal volume were recorded over time. Based on an analysis of these fluctuations, a conformational change in EEA1 from semi-flexible to flexible upon addition of active Rab5 was described. This is in agreement with the previously reported entropic collapse. More importantly, EEA1 was shown to cycle between semi-flexible and flexible states by adding Rab5:GTP and waiting for the GTP to hydrolyse. To determine whether other proteins experience a binding-induced entropic collapse, coiled-coil proteins that share structural and functional similarities with EEA1 were evaluated. Rotary shadowing EM images of the target protein alone and binding with its suspected allosteric effector were compared. It was found that ELKS, a coiled-coil protein involved in vesicle trafficking, undergoes an increase in flexibility upon binding with the active form of Rab6. Thus, hinting that the entropic collapse may indeed be a general mode of action for at least a sub-group of long coiled-coil proteins. Overall, the major contributions of this thesis are to describe the full entropic collapse cycle on EEA1 and to show a second example of a coiled-coil protein experiencing a binding induced flexibility increase.:List of Figures List of Tables List of Equations List of Abbreviations 1 Introduction 1.1 EEA1 as an endosomal tether 2 Materials and Methods 2.1 Materials 2.2 Methods 2.2.1 Sub-cloning 2.2.2 Protein expression and purification 2.2.3 Protein-protein binding assays 2.2.4 Electron microscopy 2.2.5 Analysis of electron microscopy 2.2.6 Generation of DNA handles for protein-DNA conjugates 2.2.7 Adding SortaseA recognition site to EEA1 2.2.8 Protein-DNA conjugation3 2.2.9 Sample preparation for optical tweezers 2.2.10 Dual color labeling of EEA1 2.2.11 Fluorescence cross-correlation spectroscopy 2.2.12 Generation of dsDNA for dcFCCS calibration 2.2.13 RabGTPase nucleotide loading 2.2.14 Liposome preparation 2.2.15 MCBs preparation 3 Unwinding EEA1 coiled-coil domain 3.1 Introduction 3.1.1 Optical tweezers for EEA1 unwinding 3.1.2 SortaseA-catalysed ligation 3.2 Aims 3.3 Results 3.3.1 Optimization of SortaseA-catalysed ligation 3.3.2 Formation of EEA1-DNA handle conjugate 3.3.3 EEA1 unwinding experiments 3.4 Discussion 4 EEA1 entropic collapse is recyclable 4.1 Introduction 4.1.1 Advantages of dcFCCS vs FCS 4.1.2 Requirements for dcFCCS measurements 4.1.3 dcFCCS for end polymer dynamics analysis 4.2 Aims 4.3 Results 4.3.1 System preparation and dcFCCS calibration 4.3.2 Labelling of EEA1 4.3.3 Comparing FCS vs dcFCCS 4.3.4 EEA1 entropic collapse shown by dcFCCS 4.3.5 EEA1 flexibility change is recyclable 4.4 Discussion 5 Entropic collapse as a general mechanism 5.1 Introduction 5.2 Aims 5.3 Results 5.3.1 ELKS increases its flexibility upon binding active Rab6 5.3.2 p115-GM130 complex observed by rotary shadowing EM 5.4 Discussion 6 Conclusions and outlook References

With advancements in educational digitalisation, new opportunities for international collaboration on education have been created. Through the KTH Global Development Hub collaboration, students from universities in Botswana, Kenya, Sweden and Tanzania participate in modules with Challenge Driven Education (CDE). These modules allows students to gain intercultural and interdisciplinary experiences while working with real societal challenges. In this master’s degree project, Batswana, Swedish and Kenyan students’ and teachers’ experiences with CDE in blended campus-based and remote international education have been investigated. This was done through qualitative interviews, and through observations of a CDE module at Botho University in Botswana. The data was then analysed using thematic analysis with an inductive approach and discussed through the lens of previous research on CDE, e-learning and Collaborative Online International Learning (COIL), as well as contextualised through discussion of a student project on hydroponics. Based on the data and analysis, guidelines for working with Online-CDE were created. The guidelines are intended to aid teachers to bridge the gap between their previous knowledge and experience in CDE and the difficulties prevalent when planning an Online-CDE module. The modules allowed students to develop core competencies. The intercultural exchange of Online-CDE is appreciated by students and inspires them to work harder, although it may sometimes lead to conflicts. Issues with internet access and a limited knowledge of how to use digital tools provided created difficulties for both students and teachers. Although teachers aimed for students to work interdisciplinarily, project groups often divided work internally, therefore only achieving multi-disciplinarity. / I takt med att digitala lösningar för utbildning utvecklas, uppkommer nya möjligheter för internationella samarbeten kring utbildning. Genom samarbetet i KTH Global Development Hub (GDH) kan studenter från universitet i Botswana, Kenya, Sverige och Tanzania läsa kurser med utmaningsdriven utbildning (CDE). Kurserna ger studenter en möjlighet att få interkulturella och interdisciplinära erfarenheter samtidigt som de arbetar med riktiga samhällsutmaningar. I det här examensarbetet undersöks botswanska, svenska och kenyanska studenter och lärares erfarenheter av CDE i hybridundervisning där vissa studenter deltar digitalt från andra länder än arrangerande universitet. Data samlades in genom kvalitativa intervjuer och observationer av en CDE-kurs på Botho University i Botswana. Insamlad data analyserades genom tematisk analys och diskuterades utifrån tidigare forskning inom CDE, e-lärande och internationella virtuella samarbeten inom lärande (COIL). Resultaten kontextualiserades genom en diskussion av ett studentprojekt om hydroponisk odling. Baserat på resultat och analys togs riktlinjer för Online-CDE fram. Riktlinjerna är tänkta som ett komplement till lärares tidigare erfarenheter av CDE och behandlar de unika svårigheter som uppkommer med Online-CDE. Kurserna gav studenterna möjlighet att utveckla mjuk kompetens. Den interkulturella aspekten av Online-CDE uppskattas av studenterna och får dem att anstränga sig mer, även om det ibland leder till konflikter. Otillräcklig och dyr internetuppkoppling samt begränsad kunskap om de digtala verktyg som används under kurserna skapar problem för både lärare och studenter. Även om lärare eftersträvar interdisciplinära elevsamarbeten delar projektgrupper ofta upp arbetet på ett sätt som leder till multidisciplinärt arbete.

In dieser Arbeit wurde eine neue Templat-gesteuerte fluorogene Peptidverknüpfung vorgestellt. Speziell modifizierte Peptidfragmente wurden durch die Bindung an ein Templatmolekül zu einer chemischen Reaktion befähigt, wodurch ein Fluoreszenzsignal erzeugt werden konnte. Das erlaubte eine Reaktionskontrolle in Echtzeit. Die fluorogene Peptidverknüpfung konnte erfolgreich mit einem Coiled-coil Peptid-Model etabliert werden. Dabei wurden Peptidthioester derart modifiziert, dass in räumlicher Nähe zur Thioestergruppe ein Fluorophor platziert wurde und die acetylierte Mercaptogruppe als Fluoreszenzlöscher agierte. Die modifizierten Thioester können nach dem Reaktionsmechanismus der nativen chemischen Peptidverknüpfung (NCL) unter der Bildung einer Amidbindung mit N-terminalen Cysteinylpeptiden reagieren. Die fluoreszenzlöschende Mercaptogruppe verlässt dabei den Peptidthioester als Nukleofug, wodurch ein fluoreszierendes Reaktionsprodukt entsteht. Die Templat-gesteuerte Durchführung dieser fluorogenen nativen chemischen Peptidverknüpfung (fNCL) erlaubte die Reaktionsdurchführung bei sehr geringen Peptidkonzentrationen. Die Synthese der benötigten fluorogenen Thioester gelang zum einem durch die Anwendung der selbstreinigenden Thioestersynthese mit einer Tandem-Entschützungs-Kupplungs-Strategie und zum Anderen mit Hilfe eines synthetisierten fluorogenen Azid-Thioesterbausteins, welches mit einem Alkin-modifizierten Peptid zur Reaktion gebracht wurde. Neben Coiled-coil Peptiden, wurden auch doppelsträngige DNA und Antikörper als Template für die fNCL eingesetzt. Die fNCL konnte zur Durchführung eines Abstandsscreenings angewendet werden. Es wurde eine Abhängigkeit zwischen der Reaktionsgeschwindigkeit und dem Abstand der Bindungsstellen im Templat für zwei reaktive Peptidbindungspartner gezeigt. Durch diese Untersuchung konnte die räumliche Abstandsgrenze zwischen zwei Bindungsstellen in einem Templatmolekül bestimmt werden, die keinen Templat-Effekt mehr beobachten lässt. / In this thesis a new template-controlled fluorogenic peptide linkage was presented. Specially modified peptide fragments were enabled to undergo a chemical reaction by binding to a template molecule, which resulted in a fluorescent signal. This allowed a reaction control in real time. The fluorogenic peptide linkage was successfully established using a coiled coil peptide model. Peptide thioesters were modified in such a way that a fluorophore was placed in close proximity to the thioester group and the acetylated mercapto group acted as fluorescence quencher. These modified thioesters can react with N-terminal cysteinyl peptides according to the reaction mechanism of the native chemical ligation (NCL) under the formation of an petide bond. The fluorescence-quenching mercapto group leaves the peptide thioester as a leaving group, resulting in a fluorescent reaction product. Template-controlled execution of this fluorogenic native chemical ligation (fNCL) allowed the reaction to be performed at very low peptide concentrations. The synthesis of the required fluorogenic thioesters was achieved on the one hand by applying a self-purifying thioester synthesis with a tandem-protective coupling strategy and on the other hand by using a synthesized fluorogenic azide thioester building block which was reacted with an alkine-modified peptide. In addition to the coiled coil peptides, double-stranded DNA and antibodies were used as templates for fNCL. Finally, the fNCL could be used to perform a distance screening. A dependence between the reaction rate and the distance between the binding sites in the template for two reactive peptide binding partners was shown. By this investigation a distance between two binding sites in a template molecule could be determined, which does not show a template effect anymore.

Das Verständnis natürlicher biologischen Materialien für die Entwicklung neuer biomimetischer Materialien ist von großem Interesse in der Chemie und den Materialwissenschaften. In vielen komplexen biomolekularen Materialien ist die Etablierung der Struktur-Funktionsbeziehungen von Proteinbausteine notwendig, um die Eigenschaften der daraus aufgebauten weichen, biologischen Materialien zu verstehen, wie z. B. die extrazelluläre Matrix. Inspiriert durch bekannte Faltungsmotive von ECM-Proteinen, wurden vereinfachte Modellpeptide entwickelt, um deren Funktion zu untersuchen oder diese als biomimetische Bausteine für synthetische Biomaterialien zu verwenden. Ziel dieser Arbeit war die Synthese von hybriden Hydrogelen, die aus einem synthetischen Polymer und ECM-inspirierten Modellpeptiden zusammengesetzt sind. Insbesondere Kollagen-mimetische Peptide und Coiled-Coil-formende Peptide wurden benutzt, um das biokompatible und hydrophile Polymer Polyethyleneglykol zu vernetzen. Dabei wurde von der Fähigkeit dieser Peptide zur dynamischen Selbstassemblierung Gebrauch gemacht. Unter Verwendung von Kollagen-mimetischen Peptiden mit langsamer Dissoziationskinetik wurden Hydrogele synthetisiert, die weichen, glasartigen Materialien mit einem gestauchten exponentiellen Relaxationsverhalten entsprechen und auch einen Alterungsprozess zeigen. Darüber hinaus wurde gezeigt, dass Netzwerkkonnektivität ein bis dato selten gebrauchter Designparameter ist, um die rheologischen Eigenschaften von Hydrogelen nach Wunsch zu kontrollieren. Die Kombination molekular einstellbarer Vernetzer mit einem Fluoreszenz-Reportersystem, welches deren Zustand auslesen kann, kann detaillierte Einblicke in das Reaktionsvermögen solcher Netzwerke auf mechanische Stimuli ermöglichen. Das Verständnis molekularer Prozesse erlaubt langfristig die Synthese von ECM-inspirierten Biomaterialien, deren Eigenschaften nach Wunsch einstellbar sind und die selbst ihren mikroskopischen und mesoskopischen Zustand anzeigen. / Understanding natural biological materials for the development of novel biomimetic materials has drawn enormous attention from the areas of chemistry and material science. In many complex biomolecular materials, establishing molecular structure-function relationships of proteins forms the basis for understanding the emerging properties of various biological soft materials, such as the extracellular matrix (ECM). Inspired by common association motifs of ECM proteins, simplified model peptides have been developed for functional studies and as biomimetic building blocks for synthetic biomaterials. The aim of this thesis was to utilize ECM-inspired and molecularly controlled model peptides for the synthesis of peptide-polymer hybrid hydrogels. Specifically, collagen-mimetic peptides (CMPs) and coiled coil (CC)-forming peptides were utilized to crosslink the biocompatible and hydrophilic polymer poly(ethylene glycol) (PEG), making use of the ability of these peptides to dynamically self-assemble. Employing CMPs with slow dissociation kinetics, hydrogels have been synthesized that resemble soft glassy materials with compressed exponential relaxation and aging. Furthermore, network connectivity has been shown to be an underutilized design parameter for tuning the rheological properties of hydrogels. Combining molecularly controlled crosslinks with a fluorescence reporter system that allows to read out crosslink status will ultimately allow for more detailed insights into the response of such networks to mechanical perturbation and thus aid the synthesis of ECM-inspired biomaterials with tunable and self-reporting properties.

Fluoreszenzmarkierungstechniken für lebende Zellen ermöglichen es Biologen, einen Blick in eine komplexe biologische Umgebung zu werfen und Informationen über ein bestimmtes Ziel in einer nahezu natürlichen Umgebung zu erhalten. Dank der konzertierten Bemühungen der wissenschaftlichen Gemeinschaft gibt es eine Fülle von kommerziell erhältlichen, genetisch kodierbaren Markern und Reportern für die Fluoreszenzmikroskopie. Allerdings gibt es nur wenige Lebendzellmethoden, die eine direkte Konjugation von Nukleinsäuren mit Proteinen erlauben, obwohl es robuste DNA-Technologien gibt, die mit Oligo-Antikörper-Konjugaten auf Zelloberflächen durchgeführt werden. Ein weiterer, oft einschränkender Aspekt der Markierung ist die Fähigkeit, Ziele selektiv zu multiplexen. In dieser Studie wurde eine Methode der Tag-Probe-Markierung entwickelt, die eine selektive, gleichzeitige Markierung von zwei verschiedenen Zielen mit zwei Peptid-Nukleinsäure-Strängen (PNA) ermöglicht. Diese Methode verwendet ein Paar von Coiled-Coil-Peptiden, um die Konjugation einer PNA-Gruppe an ein Zielprotein zu steuern, das ein Peptid-Tag exprimiert. Die Verwendung orthogonaler Coiled-Coils ermöglicht Multiplexing. Die Markierung von synthetischen Tag-Peptiden, die mittels Flüssigchromatographie analysiert wurden, hat gezeigt, dass der orthogonale duale Transfer von PNA selektiv, quantitativ und schnell ist. Die PNA-Konjugation von exemplarischen Membranrezeptoren, gefolgt von der Hybridisierung mit komplementären Fluorophor-DNAs, ermöglichte eine unkomplizierte Visualisierung von dualen Rezeptoren in lebenden Zellen. Durch den Einsatz einfacher molekularer Hilfsmittel, die die Grundlage der DNA-Nanotechnologie bilden, konnte durch die Rekrutierung mehrerer DNAs eine zunehmend hellere Markierung erreicht werden und die löschbare Oberflächenmarkierung ermöglichte eine quantitative Untersuchung der Rezeptorinternalisierung. / Live-cell fluorescent labelling techniques allow biologists to glimpse into a complex biological environment and derive information about a specific target in a near-native environment. Thanks to a concerted effort from the scientific community, a plethora of commercially available, genetically encodable tags and reporters for fluorescence microscopy exist. However, few live-cell methods allow direct conjugation of nucleic acids with proteins despite the robust DNA technologies carried out on cell surfaces using oligo-antibody conjugates. Another aspect of labelling which is often limiting is the ability to selectively multiplex targets. In this study, a method of tag–probe labelling was developed that accomplishes selective, simultaneous labelling of two distinct targets with two peptide nucleic acid (PNA) strands. The technique uses a pair of coiled-coil peptides to guide conjugation of a PNA group to a target protein expressing a peptide tag and using orthogonal coiled-coil enables multiplexing. Initially, the labelling of synthetic tag-peptides analysed by liquid chromatography revealed the orthogonal dual transfer of PNA to be selective, quantitative, and rapid. PNA conjugation of exemplar membrane receptors followed by hybridization with complementary fluorophore-DNAs achieved straightforward live-cell dual receptor visualization. Finally, using simple molecular tools that form the basis of DNA nanotechnology, recruitment of multiple DNAs facilitated progressively brighter labelling, and erasable surface labelling allowed quantitative study of receptor internalisation.

Entailing of phosphorus exchanges in most bio-chemicals as a key factor in disease, increases researcher’s interest to develop the technologies capable of detecting this metabolite. Phosphorus magnetic resonance spectroscopy is able to detect key metabolites in a non-invasive manner. Particularly, it offers the ability to measure the dynamic rate of phosphocreatine(PCr) degeneration through the exercise and recovery. This metric as a valid indication of mitochondrial oxidative metabolism in muscle, differentiate between normal and pathological state. To do magnetic resonance imaging and spectroscopy, clinical research tools provide a wide variety of anatomical and functional contrasts, however they are typically restricted to the tissues containing water or hydrogen atoms and they are still blind to the biochemicals of other atoms of interests. Through this project we intended to obtain the phosphorus spectrum in human body – specificadenerativelly in muscle – using 31P spectroscopy. To do so a double loop RF surface coil, tuned to phosphorus frequency, is designed and fabricated using bench work facilities and then validated through in vitro spectroscopy using 3 Tesla Siemens scanner. We acquired in vitro as well as in vivo phosphorus spectrum in a 100 mM potassium phosphate phantom and human calf muscle in rest-exercise-recovery phase in a 3T MR scanner. The spectrum demonstrates the main constituent in high-energy phosphate metabolism. We also observed the dynamic variation of PCr for five young healthy subjects who performed planter flexions using resistance band during exercise and recovery. The took steps in this project pave the way for future application of spectroscopic quantification of phosphate metabolism in patients affected by carotid artery disease as well as in age-matched control subjects. / L’importance des échanges de phosphore liés à l’apparition de troubles et de maladies neurodégénèratives a suscité l’intérêt des chercheurs en ce qui concerne le développement de technologies pouvant détecter ces composés métaboliques. Á l’aide de la spectroscopie en résonance magnétique du phosphore, il est possible de détecter ces métabolites d’une manière non-invasive. Cette technique permet plus particulièrement de mesurer le taux de degénération de la phosphocréatine (PCr) lors de périodes d’exercice et de récupération post-activité. Cette dernière métrique agit en tant qu’indicateur valide du métabolisme oxydatif mitochondrial dans le muscle et permet de différentier un muscle sain d’un muscle pathologique. Pour effectuer l’imagerie ou la spectroscopie par résonance magnétique, de nombreux outils cliniques peuvent être utilisés afin de générer des images comprenant une variété de contrastes anatomiques et fonctionnels. Par contre, ces contrastes ne peuvent être habituellement produits qu’à partir des molécules d’eau ou des atomes d’hydrogène renfermés dans les tissus biologiques et les outils permettant de générer les images correspondantes demeurent, pour la plupart, insensibles à la présence d’autres atomes d’intérêt. Au cours de ce projet, il a été question d’obtenir un spectre du phosphore à partir de l’activité musculaire chez l’humain in vivo en se servant de la technique de spectroscopie de type 31P. Pour ce faire, une antenne radiofréquence à deux canaux, devant être syntonisée à la fréquence de résonance du phosphore, a d’abord été conçue et fabriquée en laboratoire pour ensuite être validée lors d’expériences sur une plateforme IRM 3T de marque Siemens. Les spectres du phosphore furent acquis sur un fantôme, une solution de phosphate de potassium à une concentration de 100 mM, ainsi que sur le mollet d’un sujet humain au cours d’une période de repos, d’exercice et de récupération. Le spectre obtenu à la suite de cette dernière expérience démontre la présence accrue du phosphore lors de procédés métaboliques à haute teneur énergétique impliquant la dégradation de phosphates. Par la suite, il a été question d’observer la variation des niveaux de PCr durant des périodes d’exercice et de récupération chez 5 jeunes adultes effectuant une série iv de flexions plantaires ayant un élément résistif attaché à leurs pieds. La démarche entreprise au cours de ce projet permettra l’utilisation future des techniques quantitatives en spectroscopie pour l’évaluation du métabolisme des phosphates chez des patients souffrant de maladies coronariennes ainsi que chez des sujets contrôles sains provenant du même ensemble démographique.

Este trabalho descreve o desenvolvimento de um sistema integrado transdutor/bobinas de gradientes de alto desempenho para Imagens por Ressonância Magnética. Este sistema é composto por um transdutor de radiofreqüência tipo sela e um conjunto de 3 bobinas locais assimétricas. No desenho do transdutor foram otimizados os parâmetros: relação sinal ruído e uniformidade do campo magnético por ele gerado. A densidade de corrente de cada bobinas local foi otimizada mediante técnicas numéricas estocásticas para gerar um gradiente de campo magnético uniforme em cada uma das 3 direções do espaço numa região das dimensões do crânio. O conjunto de bobinas de gradientes construído possui um diâmetro livre de 31.5 cm e gera em média 25 mT/m/A por bobina, com indutâncias inferiores a 310 mH. São mostradas as aplicações clínicas desenvolvidas nas áreas de imagens tridimensionais e angiografia, a partir das seqüências de pulsos implementadas e aproveitando o uso do sistema integrado, em um tomógrafo de ressonância magnética de 0.5 Tesla. Imagens de phantom foram adquiridas em menos de 500 ms usando o conjunto integrado e técnicas do tipo Echo Planar Imaging. Aspectos referentes à caracterização e correção de campos magnéticos estáticos e homogêneos são também comentados. As soluções descritas nesta tese têm um amplo conteúdo tecnológico e beiram nas fronteiras da Física Aplicada e a Engenharia Biomédica. / Here we describe the development of a high performance radio-frequency/gradient coil integrated system for Magnetic Resonance Imaging. A saddle radio-frequency coil and a three-axis asymmetric local gradient coil composed this system. Two parameters were optimized in the RF coil design: signal-to-noise ratio and magnetic field uniformity. The current density of each local coil was optimized using stochastic numerical techniques, in order to generate a uniform magnetic field gradient by axis in a region representing a human head. The build gradient coil set has an inner diameter of 31 cm. The average gradient efficient of the three-axis is 25 mT/m/A and the maximum inductance is less than 310 mH. We show the clinical applications performed in three-dimensional and angiography imaging areas in a 0.5 Tesla magnetic resonance tomograph. These applications were optimized to taking advantage from the integrated system. Phantom images were acquired in less than 500 millisecond using echo planar techniques and the integrated set. Some aspects about static and homogeneous magnetic field characterization and correction are also commented. In this work we described solutions with wide technologic content close to the boundaries of the Applied Physics and Biomedical Engineering.