Transfert d'énergie par résonance de type Förster entre des complexes de terbium et des boîtes quantiques pour des immunodosages et des reglettes moléculaires multiplexés

Förster resonance energy transfer (FRET) is a non-radiative energy transfer from a donor to an acceptor in close proximity. Due to its extremely sensitive distance dependence in the 1 – 20 nm range, FRET plays an important role in nanobiotechnology. Thereby FRET can be used as signal transduction system but also for the distance estimation between donor and acceptor. The selected FRET acceptors in this work were semiconductor nanocrystals (quantum dots, QDs). This type of luminophore is well known for its superior photophysical properties. Their strong and broad absorption and their bright, narrow-band, and size-tunable photoluminescence (PL) emission make QDs ideally suited for FRET application. Combing QDs as FRET acceptors with luminescent terbium complexes (LTC) as FRET donors offers exceptionally large Förster distances of more than 10 nm. The Förster distance is characteristic of a FRET pair and is the distance at which the FRET efficiency equals 50 %. A large Förster distance is desirable as it offers the detection of biological interactions over large distances. LTC are suitable FRET donors for QDs because they provide long excited-state lifetimes in the millisecond range. This long PL decay time enables time-gated measurements for the suppression of autofluorescence and PL of directly excited QDs, which strongly increases the detection sensitivity. Additionally, the structured PL emission bands of LTCs together with the size-tunable PL emission bands of QDs make this FRET pair ideal for the application in multiplexed diagnostics, which is the measurement of multiple biomarkers in a single sample.The PhD thesis consists of two parts. In the first part the LTC-QD FRET pair was used within homogeneous FRET immunoassays for the detection of the biomarkers prostate specific antigen (TPSA), neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), and epidermal growth factor receptor (EGFR). The immunoassay sensitivity was optimized using different types of antibodies IgG, F(ab’)2,F(ab), and for EGFR single heavy chain antibodies, which differ largely in their size. The use of small-volume serum samples and measurements on clinical as well customized fluorescence plate readers result in picomolar detection limits for all measured biomarkers. In addition to these QD-based in vitro diagnostic tests, a detailed study of the different FRET-systems using time-resolved spectroscopy was performed. The investigation revealed the influence of the different antibodies on distance, functionality, and sensitivity of the FRET immunoassays. The study was completed by the measurement of NSE and CEA in a duplexed format and real patient samples were investigated.The second part was to use FRET for nanometric distance measurements as molecular or spectroscopic ruler. Time-resolved FRET measurements enabled the calculation of the distance between donor and acceptor. Therefore two different binding strategies were investigated to establish a close proximity between the LTC-donor to the QD-acceptor, namely biotin-streptavidin recognition and polyhistidine mediated self-assembly. A detailed time-resolved study was performed of QDs with different sizes, shapes, and surface coatings in combination with LTC bound to three different host biomolecules, which also possessed different sizes, shapes, orientations, and binding conditions. The analysis of the multi-exponential decay curves of donor and acceptor allowed to obtain information about the size, shape, and biofunctionality of the investigated QD bioconjugates. The results were in agreement with other structural analysis methods, such as transmission electron microscopy (TEM) or dynamic light scattering (DLS), but with the advantage of a homogeneous measurement with three-dimensional resolution (not possible for TEM), without the inclusion of a hydration shell (drawback for DLS), and at low concentration in the same environment as used for the biological application.Le transfert d'énergie par résonance de type Förster (FRET) est un transfert d'énergie non radiatif d'un donneur à un accepteur à proximité. En raison de sa dépendance de la distance extrêmement sensible entre env. 1 et 20 nm, FRET joue un rôle important dans la nanobiotechnologie. Ainsi FRET peut être utilisé comme système de transduction du signal, mais aussi pour l'estimation de la distance entre le donneur et l'accepteur.Les accepteurs de FRET utilisés dans ce travail étaient des nanocristaux semi-conducteurs (quantum dots, QD). Ce type de luminophore est bien connu pour ses propriétés photophysiques supérieures. Leur absorption forte et spectralement large, et leur photoluminescence (PL) brillante et spectralement fine et de l'accordabilité spectrale de la PL sont idéalement adaptés aux applications de FRET. La combinaison des QDs comme accepteurs de FRET avec des complexes luminescents de terbium (CLT) comme donneurs permet des grandes distances de FRET (> 10 nm). La distance de FRET est caractéristique d'une paire FRET et décrit la distance à laquelle l'efficacité de FRET est égale à 50%. CLT sont idéal comme donneurs de FRET parce qu'ils fournissent des longues durées de vie des états excités à l'ordre de la milliseconde. Cette longue période de décroissance de PL permet de mesurer en temps décalé pour une répression d’autofluorescence et la PL des QDs directement excités, ce qui augmente fortement la sensibilité de détection. Les bandes d'émission de PL structurés de CLT et la PL accordable de QDs sont idéales pour l'application dans le diagnostic multiplexé.La thèse se compose de deux parties. Dans la première le couple FRET CLT-QD a été utilisé dans des immunodosages de FRET homogènes pour la détection de marqueurs biologiques antigène prostatique spécifique (TPSA), énolase specifique des neurones (NSE), antigène carcino-embryonnaire (CEA), et le récepteur du facteur de croissance épidermique (EGFR). La sensibilité du dosage immunologique a été optimisé en utilisant des différents types d'anticorps IgG, F (ab')2, F (ab), et pour EGFR un anticorps de chaîne lourde unique. Des limites de détection picomolaires étaient réalisées en utilisant des échantillons de sérum de petit volume et des mesures sur des lecteurs de microplaques cliniques. Une étude détaillée des différents systèmes de FRET en utilisant la spectroscopie résolue en temps a été réalisée pour étudier l'influence des différents anticorps sur la distance, la fonctionnalité, et la sensibilité des immunodosages. L'étude a été complétée par la mesure de NSE et CEA dans un format duplexé et des échantillons réels de patients.Dans la deuxième partie le FRET pour des mesures de distance nanométriques (réglette moléculaire ou spectroscopique) étaient étudiés. FRET en résolution temporelle a permis de calculer la distance entre le donneur et l’accepteur. Par conséquent, deux stratégies de liaisons différentes ont été étudiées pour établir une proximité entre le CLT et le QD : la reconnaissance biotine-streptavidine et l’auto-assemblage médié par polyhistidine. Une étude en résolution temporelle détaillée a été effectuée avec des QDs de différentes tailles, formes et revêtements de surface combiné avec des CLT liés à trois différentes biomolécules. L'analyse des courbes de décroissance multiexponentielle des donneurs et accepteurs permettait à obtenir des informations sur la taille, la forme et la biofonctionnalité des bioconjugués CLT-QD. Les résultats étaient en accord avec d'autres méthodes d'analyse de structure, telles que la microscopie électronique à transmission (MET) ou la diffusion de lumière dynamique (DLS), mais avec l'avantage d'une mesure homogène à la résolution 3-dimensionelle (impossible pour le MET), sans l'inclusion d'une couche d'hydratation (l’inconvénient de DLS) et en faible concentration dans le même environnement que celui utilisé pour l'application biologique

Snake River Dam Breaching: River & Salmon Politics in the George W. Bush Administration

It is the objective of this paper to outline some of the administrative and legislative history that has led to the present state of salmon affairs in the Snake River basin. In addition, it is the intent to outline some of the compounding reasons that have led to the decline of the salmon and finally to outline some of the actions that are necessary to move beyond the bureaucratic stalemate that the salmon find themselves in today

Snake River Dam Breaching: River & Salmon Politics in the George W. Bush Administration

It is the objective of this paper to outline some of the administrative and legislative history that has led to the present state of salmon affairs in the Snake River basin. In addition, it is the intent to outline some of the compounding reasons that have led to the decline of the salmon and finally to outline some of the actions that are necessary to move beyond the bureaucratic stalemate that the salmon find themselves in today

GEOMETRICAL STRING and DUAL SPIN SYSTEMS

We are able to perform the duality transformation of the spin system which was found before as a lattice realization of the string with linear action. In four and higher dimensions this spin system can be described in terms of a two-plaquette gauge Hamiltonian. The duality transformation is constructed in geometrical and algebraic language. The dual Hamiltonian represents a new type of spin system with local gauge invariance. At each vertex $\xi$ there are $d(d-1)/2$ Ising spins $\Lambda_{\mu,\nu}= \Lambda_{\nu,\mu}$, $\mu \neq \nu = 1,..,d$ and one Ising spin $\Gamma$ on every link $(\xi,\xi +e_{\mu})$. For the frozen spin $\Gamma \equiv 1$ the dual Hamiltonian factorizes into $d(d-1)/2$ two-dimensional Ising ferromagnets and into antiferromagnets in the case $\Gamma \equiv -1$. For fluctuating $\Gamma$ it is a sort of spin glass system with local gauge invariance. The generalization to $p$-branes is given.Comment: 16 pages,Late

Renormalization Group for Matrix Models with Branching Interactions

We develop a method to obtain the large N renormalization group flows for matrix models of 2 dimensional gravity plus branched polymers. This method gives precise results for the critical points and exponents for one matrix models. We show that it can be generalized to two matrices models and we recover the Ising critical points.Comment: 19 pages, 1 latex2e + 7 eps files, revised version (misprints corrected and a few points made more precise

Self-Avoiding Gonihedric Srting and Spin Systems

We classify different theories of self-intersecting random surfaces assigning special weights to intersections. When self-intersection coupling constant $\kappa$ tends to zero, then the surface can freely inetrsect and it is completely self-avoiding when $\kappa$ tends to infinity. Equivalent spin systems for this general case were constructed. In two-dimension the system with $\kappa = 0$ is in complete disorder as it is in the case of 2D gauge Ising system.Comment: Preprint CRETE-TH-21, October 1993,8 pages,Late

The Peculiar Motions of Early-Type Galaxies in Two Distant Regions VI: The Maximum Likelihood Gaussian Algorithm

The EFAR project is designed to measure the properties and peculiar motions of early-type galaxies in two distant regions. Here we describe the maximum likelihood algorithm we developed to investigate the correlations between the parameters of the EFAR database. One-, two-, and three-dimensional gaussian models are constructed to determine the mean value and intrinsic spread of the parameters, and the slopes and intrinsic parallel and orthogonal spread of the Mgb'-Mg2, Mg2-sigma, Mgb'-sigma relations, and the Fundamental Plane. In the latter case, the cluster peculiar velocities are also determined. We show that this method is superior to ``canonical'' approaches of least-squares type, which give biased slopes and biased peculiar velocities. We test the algorithm with Monte Carlo simulations of mock EFAR catalogues and derive the systematic and random errors on the estimated parameters. We find that random errors are always dominant. We estimate the influence of systematic errors due to the way clusters were selected and the hard limits and uncertainties in the selection function parameters for the galaxies. We explore the influence of uniform distributions in the Fundamental Plane parameters and the errors. We conclude that the mean peculiar motions of the EFAR clusters can be determined reliably. In particular, the placement of the two EFAR sample regions relative to the Lauer and Postman dipole allows us to strongly constrain the amplitude of the bulk motion in this direction.Comment: 43 pages, 19 figures, accepted for publication in MNRA

RETAIL HEALTH & SAFETY DIGITAL MANAGEMENT SYSTEM

Health and Safety procedures and documentations are one of the many tasks that need to be done by a small retail shop, they are also one of the most difficult things to manage daily as all those can build up during the day/week. Inside Health and Safety there are many subcategories that need to be administered (maintenances, trainings and checklists for example) which can be very overwhelming for any management team notably in small retail shops as they do not have the permanent personnel to be able to maintain an organised system. This project aims to create a management software to assist a business to conduct all those procedures in an easier and more efficient manner