Protein-protein interactions are important for biological processes. Therefore, many small molecules target a specific protein or interaction in the cell to have biological consequence. While we can measure some protein-protein interactions in a test tube, many proteins cannot be purified making it difficult to properly test that a drug is “on target”. An alternative is to measure these interactions in live cells. We express the proteins of interest fused to fluorophores allowing the use of fluorescence techniques. Förster Resonance Energy Transfer (FRET) provides a molecular level ruler to measure the distance, within a few nanometers, between two proteins. FRET indicates binding. The gold standard for measuring FRET in live cells is by quantifying changes in fluorescence lifetime using Fluorescence lifetime imaging microscopy (FLIM). The change in fluorescence lifetime is inversely proportional to the ratio of bound to non-bound proteins. Tradition FLIM-FRET microscopy is too slow for screening applications. Our aim was to develop a highly multiplexed confocal system for rapid FLIM-FRET acquisition. We present the development of multiple prototypes for confocal multiplexing. In this work, our final design includes 32×32 multiplexed excitation points which scan the sample using refractive window scanners. We coupled this excitation scheme to a 64×32 time-gated single-photon avalanche photodiode (SPAD) sparse array detector. This multiplexed setup allows the use of the sparse array with high frame rate and sub-nanosecond time-gating to achieve high throughput FLIM acquisition. Using our multiplexed FLIM prototype we measured Bcl-2 family protein-protein interactions in live cells (310×310 μm FOV) with two-channel confocal FLIM in 1.5 s. Protein binding affinities were estimated by measuring the changes in FRET as a function of acceptor to donor ratio. The resulting speed of this system meets requirements for implementation in screening applications. / Thesis / Candidate in Philosophy / Inside a cell, proteins are the “workers” and they interact with each other, doing that work. Many of these interactions are important for the cell to live. Pharmaceutical companies may design drugs that can interfere with a specific interaction in order to cause an effect in the cell. Scientists are interested in measuring these interactions and we can do this by “taking a picture” of the interaction using a specialized microscope. One of the major issues with these microscopes is that it takes scientists a long time to collect pictures of these interactions. This means only a few drugs can be tested in a day. To speed up the drug discovery and testing we want to design faster microscopes that can test hundreds of drugs in a day. In my thesis I contributed to building a state-of-the-art super fast microscope. We made progress in steps, and by the third attempt we successfully measured interactions in cells in seconds! Our new microscope is ~400x faster than current technologies. We hope that this research will be useful to speed up drug discovery in the future.

Metallic nanoparticles (NPs) are exploited for their ability to interact with organic compounds and to increase significantly the fluorescence intensity and the photostability of many fluorescent dye molecules. Metal enhanced fluorescence (MEF) is therefore widely investigated for biosensing applications. When used in immunoassays, silver island films (SIFs) could augment the fluorescence intensity of fluorescein by a factor of seventeen; SIFs were also able to double or triple the emission intensity of cyanine dyes which are commonly used in (deoxyribonucleic acid) DNA microarrays. The emission intensity of indocyanine green — widely used as a contrast agent in medical imaging — was about twenty times higher in the proximity of SIFs. This enhancement phenomenon — due to the surface plasmon polaritons associated with the metallic NPs — can be explained by energy transfer from the metal NPs to the fluorescent dye molecules or by a modified local electromagnetic field experienced by the fluorophores in the vicinity of metal surfaces. Our research focused on the optical characterization of colloidal gold-silver alloy NPs containing different ratios of gold and silver (Au_1.00-Ag_0.00, Au_0.75-Ag_0.25, Au_0.50-Ag_0.50, and Au_0.25-Ag_0.75), as well as their interaction with three fluorophores: rose bengal, rhodamine B, and fluorescein sodium. Depending upon the dye quantum yield and its concentration in solution, enhancement or quenching of fluorescence was obtained. Thus, a three to five times increase in fluorescence intensity was observed in a 2.0 mM solution of rose bengal with all nanoalloys, a slight enhancement of fluorescence (1.2 – 1.6 times) was noticed in a 0.13 mM solution of rhodamine B with all four types of NPs, and fluorescence quenching occurred in all the fluorescein-NP solutions regardless of the dye concentration. / Master of Science

Call number: LD2668 .T4 1958 W49

Le syndrome de détresse respiratoire aigue (SDRA) est une maladie très grave du poumon. Malgré les nombreuses études entreprises au cours des ans, le taux de mortalité est encore élevé et aux alentours de 35% à 40%. Comme il n'existe aucun traitement spécifique, le praticien doit s'appuyer sur des outils d'évaluations fiables, qui vont lui permettre d'évaluer la réparation (ou la dégradation) du poumon et se contenter d'un traitement de support. Les outils d'imagerie dont dispose le praticien sont nombreux et variés (biopsie, tomographie, résonance magnétique,...). Ces modalités fournissent certes une image globale de la structure du poumon, et permettent de détecter facilement de nombreuses pathologies. Cependant, malgré les dernières avancées en matière de résolution et de qualité d'image, les systèmes d'imageries utilisés actuellement ne donnent pas d'information au niveau cellulaire. Or, c'est à ce niveau que l'on peut vraiment voir les mécanismes d'évolution de cette maladie. Il serait donc intéressant de trouver un moyen de visualiser le poumon à l'échelle cellulaire, sans pour autant être trop invasif, comme l'est la biopsie pulmonaire. La microscopie in vivo peut être une solution à ce problème. A l'aide d'une sonde, placée directement sur le poumon, le praticien pourra évaluer l'état du poumon de son patient en temps réel. C'est ici le but de l'étude, qui a été réalisée sur des rats. Après injection de marqueurs spécifiques (reconnaissance de certaines cellules par exemple), il devient possible d'évaluer l'état du poumon, par des considérations structurelles, cellulaires, et fonctionnelles notamment. Outre l'étape où il a fallu choisir les bons produits de marquage selon ce qu'il était visé de visualiser, il a été nécessaire de développer des outils de stabilisation de la sonde. En effet, les mouvements du coeur et des poumons, apportent sur les images un flou cinétique qui corrompt les résultats, ce qui peut amener une erreur de diagnostic. Il a donc été nécessaire de supprimer ce flou en développant notamment un système de succion pour maintenir un contact entre la sonde et le poumon.

A large variety of post-translational modifications can exist on the N-terminal tails of histone proteins H2A, H2B, H3 and H4. These have been of great interest as they have increasingly been shown to influence fundamental biological processes and human disease. Studying these modifications provides insight into their physiological functions and enables the search for potent small molecule inhibitors. In this thesis, new fluorescence-based supramolecular tools were developed and used to a) measure the binding of covalently modified peptide tails to a collection of synthetic receptors in neutral aqueous solution and b) monitor an enzyme that installs a post-translational modification (PTM) in real-time. Two different approaches were used to detect binding in these systems. The first was the optimization of a competitive dye-displacement method that relies on the ability of the cationic dye lucigenin. The second was the synthesis of novel conjugates that consist of calixarenes covalently appended with multiple different fluorescent dyes. / Graduate / 0487 / 0490 / 0491

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