L'oxygène singulet, premier état électronique excité du dioxygène, est considéré comme l'agent cytotoxique principal de la thérapie photodynamique (PDT) dans le traitement du cancer.En PDT, l'oxygène singulet est créé via l'activation par la lumière d'une molécule photo-sensible qui transfère son énergie au dioxygène pour conduire à l'excitation de ce dernier dans l'état singulet.Notre étude a permis de montrer qu'il est possible par activation optique directe à 1270 nm, sans agent photosensibilisant, de l'oxygène singulet d'obtenir un stress oxydant conduisant à la mort de cellules in vitro. Ceci a pu être réalisé grâce au développement d'une expérience de vidéo-microscopie, de lasers de puissance accordables autour de 1270 nm et d'une mesure in situ de l'échauffement induit par le laser. Ces trois points seront exposés avant de présenter les preuves de l'implication de l'oxygène singulet dans la mort cellulaire par irradiation laser à 1270 nm.Dans une seconde partie du travail, nous nous sommes intéressés à des méthodes optiques de détection de l'oxygène singulet, excité directement par absorption à 1270 nm, pouvant être exportées en microscopie. / Singlet oxygen is the first electronically excited state of molecular oxygen. This specie is considered as the main cytotoxic agent in photodynamic therapy (PDT) of cancer. In PDT, singlet oxygen is produced via the activation by light of a photosensitizer molecule which will transfer its energy to molecular oxygen. This process leads to the excitation of molecular oxygen into the excited singlet state.In our study we have shown that it is possible by direct optical activation at 1270 nm (without a photosensitizer) of singlet oxygen to obtain oxidative stress that leads to cell death in vitro. This was done throught the developpement of a time-lapse experiment and the creation of high power tunable laser around 1270 nm and the in situ measurement of laser-induced temperature increase. These three points will be described. Then the prooves of the implication of singlet oxygen in cell death induced by 1270 nm irradiation will be exposed.In a second part of this work we studied optical methods for the detection of singlet oxygen directly, created by light absorption at 1270 nm, that could be exported to microscopy.

No description available.

Gelsolin was labelled with the sulphydryl-specific fluorescent reagent 6-acryloyl-2-dimethylaminonaphthalene (acrylodan). The degree of labelling using non-denaturing conditions was 1.9 ± 0.5 acrylodans per gelsolin molecule. Circular dichroism and viscosity studies showed no significant effect on gelsolin structure and function on incorporation of the label. Circular dichroism studies did not detect Ca²⁺ effects on aerylodan-labelled gelsolin, but fluorescence studies detected subtle changes in the protein. The presence of Ca²⁺ causes a decrease and red-shift in fluorescence emission, an increase in sensitivity to quenching by I⁻ and a decrease in fluorescence polarisation of the acrylodan-labelled gelsolin. These indicate an increased degree of exposure of the fluorescent label to the solvent environment on interaction of gelsolin with Ca²⁺. Actin binding to gelsolin was evident from a decrease in fluorescence intensity, an increase in sensitivity to quenching and an increase in fluorescence polarisation. Actin binding increases the exposure of the acrylodan label to solvent, as does Ca²⁺ binding. / Science, Faculty of / Chemistry, Department of / Graduate

Fluorescence spectroscopy can be used to probe protein conformation and is recognized as a technique that provides very specific information. It has been applied/ in recent years/ to the study of tropomyosin (TM) and its role in regulation of contractile processes. In this thesis, two different approaches were used to further the understanding of the structure/function relationship in the two chain coiled coil of tropomyosin. The first involves a comparative study on TM and non-polymerizable TM (NPTM) (Mak, A.S., and Smillie, L.B. (1981) Biochim. Biophys. Res. Commun., 101, 208-214). Fluorescence involving pyrene (Py) and acrylodan (AD) bound at the only cysteine residue in the molecule (Cys-190), and circular dichroism (CD) studies led to the main conclusion that, while the two species, are very similar in stability, the COOH-terminus is required to hold the Cys-190 region in a specific conformation. This long-range structural effect may play a role in regulation of contraction. A species having one intact COOH-terminus, made by hybridizing TM and NPTM, was found to be non-polymerizable suggesting that one intact COOH-terminus is insufficient to permit overlap with the NH₂-terminus of a neighbouring TM under polymerizing conditions. Unlike the TM/NPTM hybrid, the hybrid of TM and platelet TM (P-TM) was difficult to make due to the sequence mismatches in the terminal regions, but small quantities could be detected by loss of excimer fluorescence from Py-P-TM on rapid cooling of a heated mixture of Py-P-TM and cardiac TM (C-TM). The second approach was to investigate the effect of actin-binding proteins on the structure and function of tropomyosin. DNase I depolymerizes F-actin and is known to interfere with the end-to-end polymerizability of tropomyosin (Payne, M.R., Baydoyannis, H., and Rudnick, S.E. (1986) Biochim. Biophys. Acta 883, 454-459). Results presented here from fluorescence studies suggest that this effect is caused by a localized loss of structure in the tropomyosin at the sites of labelling upon binding of DNase I. This result is supported by CD studies on labelled and unlabelled tropomyosins. Gelsolin is another actin-binding protein found in many cell types and in extracellular fluids. It is shown here to be able to depolymerize tropomyosin, but its mechanism of action is not the same as that of DNase I. The effect of interaction of gelsolin on the structure of tropomyosin, as determined from fluorescence studies, is negligible. / Science, Faculty of / Chemistry, Department of / Graduate

Les oxo-anions tels que le sélénite et le sélénate (SeO32-/ SeO42-) et les oxo-cations tels que l’uranyle (UO22+) sont des polluants que l’on trouve dans les déchets nucléaires ultimes destinés à être stocké en couches géologiques profondes. L’adsorption des oxo-anions a été étudiée sur une surface d’oxyde modèle : le dioxyde de titane TiO2. Le but étant de comparer les comportements en fonction des différentes faces cristallographiques, les travaux ont été réalisés sur poudres et monocristaux. Une étape préliminaire importante s’est attachée à la caractérisation de l’hydroxylation de la surface des monocristaux. Des travaux d’adsorption en batch ont ensuite été réalisés suivis d’une caractérisation spectroscopique des interfaces eau/oxyde au moyens de diverse méthodologies telles que la spectroscopie de photoélectrons X (XPS), la spectroscopie par diffusion Raman en champ lointain et en champ proche, la spectroscopie de génération de seconde harmonique (SGSH), la microscopie à force atomique (AFM). Les poudres de rutile ont été caractérisées par mesures électrocinétiques et des courbes de cinétique de sorption ainsi que des sauts de sorption en fonction du pH ont été réalisés. L’influence du sel de fond (NaCl, NaClO4) ainsi que l’influence de la lumière sur l’adsorption ont été étudiés. Ceci a permis de mettre en évidence un processus de photoréduction du sélénium(6) en sélénium(4). La seconde partie de ces travaux de thèses sont des études exploratoires basées sur des méthodes spectroscopiques innovantes telles que la spectroscopie de fluorescence en champ proche. Elles concernent le suivi et l’analyse du type d’interactions entre le cation Europium(III), cation modèle pour les actinides et lanthanides trivalents, et de deux matrices disctinctes : un verre aluminate de type verre de stockage de déchets nucléaires et une argile de la famille des kaolins : la dickite. / Oxo-anions such as selenite/selenate (SeO32-/ SeO42-) and oxo-cationss such as uranyl (UO22+) are contaminants found in ultimate nuclear wastes (79Se) which could be stored in deep geological media. Furthermore, selenium mobile species can be found in high amount in some polluted soils. Adsorptions of selenite and selenate were studied on a model oxide surface, namely rutile TiO2. The final goal was to find out possible differences in reactivity among crystallographic faces and try to extrapolate the results to the powder behaviour. The goal of these studies was to use spectroscopic characterizations and batch experiments in order to propose surface complexes. As our interest is focused on the chemistry of the water/oxide interface, surface specific methods are thus required. Techniques used in this work are Angle Resolved X-Ray Photoelectron Spectroscopy, Near-Field Raman Spectroscopy (NFRS), Surface Second Harmonic Generation (SSHG), and Atomic Force Microscopy (AFM). Batch experiments were performed on powders, electrokinetics measurements, sorption edge curves and sorption kinetics curves were also realized. The influence of the salt used for adjusting the ionic strength (NaClO4 and NaCl) and, as titanium dioxide exhibits strong photocatalytic properties, the influence of sunlight were also studied. A photoreduction processus from Se(VI) to Se(IV) was evidenced. The second part of this manuscript presents some exploratory works dealing with innovative spectroscopies like Near-Field Luminescence applied to the study of Eu3+ interaction with an aluminate glass and a model clay : dickite.

The use of fluorescent molecules as probes of protein conformation is recognized as a technique which provides very specific information and has been applied, in recent years, to the study of the role of tropomyosin (TM) in the regulation of contractile processes. The isolation and sequencing of TM from horse blood platelets (P-TM) has shown it to be different from muscle TM, especially near the NH₂-and COOH-termini. These differences have been suggested to weaken end-to-end interaction of P-TM molecules. TM's are two chain coiled coils and P-TM has cysteine residues at the penultimate COOH-terminus position of adjacent chains. These can be labelled with sulfhydryl-specific fluorescent probes that reflect conformational changes in that region of the molecule via changes in their emission characteristics. The results of experiments on both pyrene (Py) (40) and acrylodan (AD) labelled P-TM show that there is a preferred interaction of the COOH-terminus of P-TM with the NH₂-terminus of cardiac TM over that with the NH₂-terminus of P-TM. This indicates that the altered NH₂-terminus of P-TM, with respect to muscle TM, is responsible for the relative loss of polymerizability of P-TM at low salt concentration. Addition of actin to the Py-P-TM (40) and AD-P-TM species showed changes in emission characteristics indicative of binding to the F-actin filaments, suggesting that the presence of the probes had not affected the function of the P-TM adversely. However, the presence of pyrenes at the COOH-terminus seemed to reduce further the ability of P-TM to self-polymerize. Thermal denaturation of AD-P-TM, AD-C-TM and AD-labelled truncated P-TM followed by fluorescence polarization suggested that, contrary to the theory of Skolnick and Holtzer on the stability of two chain coiled coils, the region towards the COOH-terminus is among the last to lose its helical character. / Science, Faculty of / Chemistry, Department of / Graduate

No description available.

Les derniers évènements planétaires et plus particulièrement l'avènement sans précédent du nouveau coronavirus augmente la demande pour des appareils de test à proximité du patient. Ceux-ci fonctionnent avec une batterie et peuvent identifier rapidement des biomarqueurs cibles. Pareils systèmes permettent aux utilisateurs, disposant de connaissances limitées en la matière, de réagir rapidement, par exemple dans la détection d'un cas positif de COVID-19. La mise en œuvre de l'élaboration d'un tel instrument est un projet multidisciplinaire impliquant notamment la conception de circuits intégrés, la programmation, la conception optique et la biologie, demandant tous une maîtrise pointue des détails. De plus, l'établissement des spécifications et des exigences pour mesurer avec précision les interactions lumière-échantillon s'additionnent au besoin d'expérience dans la conception et la fabrication de tels systèmes microélectriques personnalisés et nécessitent en elles-mêmes, une connaissance approfondie de la physique et des mathématiques. Ce projet vise donc à concevoir et à mettre en œuvre un appareil sans fil pour détecter rapidement des biomarqueurs impliqués dans des maladies infectieuses telles que le COVID-19 ou des types de cancers en milieu ambulatoire. Cette détection se fait grâce à des méthodes basées sur la fluorescence. La spectrophotométrie de fluorescence permet aux médecins d'identifier la présence de matériel génétique viral ou bactérien tel que l'ADN ou l'ARN et de les caractériser. Les appareils de paillasse sont énormes et gourmand énergétiquement tandis que les spectrophotomètres à fluorescence miniatuarisés disponibles dans le commerce sont confrontés à de nombreux défis. Ces appareils miniaturisés ont été découverts en tirant parti des diodes électroluminescentes (DEL) à semi-conducteurs peu coûteuses et de la technologie des circuits intégrés. Ces avantages aident les scientifiques à réduire les erreurs possibles, la consommation d'énergie et le coût du produit final utilisé par la population. Cependant, comme leurs homologues de paillasse, ces appareils POC doivent quantifier les concentrations en micro-volume d'analytes sur une large gamme de longueurs d'onde suivant le cadre d'une économie en ressources. Le microsystème envisagé bénéficie d'une approche de haute précision pour fabriquer une puce microélectronique CMOS. Ce procédé se fait de concert avec un boîtier personnalisé imprimé en 3D pour réaliser le spectrophotomètre à la fluorescence nécessaire à la détection quantitative d'analytes en microvolume. En ce qui a trait à la conception de circuits, une nouvelle technique de mise à auto-zeroing est appliquée à l'amplificateur central, celui-ci étant linéarisé avec des techniques de recyclage et de polarisation adaptative. Cet amplificateur central est entièrement différentiel et est utilisé dans un amplificateur à verrouillage pour récupérer le signal d'intérêt éclipsé par le bruit. De plus, l'augmentation de la sensibilité de l'appareil permet des mesures quantitatives avec des concentrations en micro-volume d'analytes ayant moins d'erreurs de prédiction de concentration. Cet avantage cumulé à une faible consommation d'énergie, un faible coût, de petites dimensions et un poids léger font de notre appareil une solution POC prometteuse dans le domaine de la spectrophotométrie de fluorescence. La validation de ce projet s'est fait en concevant, fabriquant et testant un prototype discret et sans fil. Son article de référence a été publié dans IEEE LSC 2018. Quant à la caractérisation et l'interprétation du prototype d'expériences in vitro à l'aide d'une interface MATLAB personnalisée, cet article a été publié dans IEEE Sensors journal (2021). Les circuits intégrés et les photodétecteurs ont été fabriqués ont été conçus et fabriqués par Cadence en 2019. Relativement aux solutions de circuit proposées, elles ont été fabriquées avec la technologie CMOS 180 nm et publiées lors de la conférence IEEE MWSCAS 2020. Tout comme cette dernière contribution, les expériences in vitro avec le dispositif proposé incluant la puce personnalisée et le boîtier imprimé en 3D ont été réalisés et les résultats électriques et optiques ont été soumis au IEEE Journal of Solid-State Circuits (JSSC 2022). / The most recent and unprecedented experience of the novel coronavirus increases the demand for battery-operated near-patient testing devices that can rapidly identify the target biomarkers. Such systems enable end-users with limited resources to quickly get feedback on various medical tests, such as detecting positive COVID-19 cases. Implementing such a device is a multidisciplinary project dealing with multiple areas of expertise, including integrated circuit design, programming, optical design, and biology, each of which needs a firm grasp of details. Alongside the need for experience in designing and manufacturing custom microelectronic systems, establishing the specifications and requirements to precisely measure the light-sample interactions requires an in-depth knowledge of physics and mathematics. This project aims to design and implement a wireless point-of-care (POC) device to rapidly detect biomarkers involved in infectious diseases such as COVID-19 or different types of cancers in an ambulatory setting using fluorescence-based methods. Fluorescence spectrophotometry allows physicians to identify and characterize viral or bacterial genetic materials such as DNAs or RNAs. The benchtop devices that are currently available are bulky and power-hungry, whereas the commercially available miniaturized fluorescence spectrophotometers are facing many challenges. Many of these difficulties have been resolved in literature thanks to inexpensive semiconductor light-emitting diodes (LEDs) and integrated circuits technology. Such advantages aid scientists in decreasing the size, power consumption, and cost of the final product for end-users. However, like the benchtop counterparts, such POC devices must quantify micro-volume concentrations of analytes across a wide wave length range under an economy of resources. The envisioned microsystem benefits from a high-precision approach to fabricating a CMOS microelectronic chip combined with a custom 3D-printed housing. This implementation results in a fluorescence spectrophotometer for qualitative and quantitative detection of micro-volume analytes. In terms of circuit design, a novel switched-biasing ping-pong auto-zeroed technique is applied to the core amplifier, linearized with recycling and adaptive biasing techniques. The fully differential core amplifier is utilized within a lock-in amplifier to retrieve the signal of interest overshadowed by noise. Increasing the device's sensitivity allows quantitative measurements down to micro-volume concentrations of analytes with less concentration prediction error. Such an advantage, along with low-power consumption, low cost, low weight, and small dimensions, make our device a promising POC solution in the fluorescence spectrophotometry area. The approach of this project was validated by designing, fabricating, and testing a discrete and wireless prototype. Its conference paper was published in IEEE LSC 2018, and the prototype characterization and interpretation of in vitro experiments using a custom MATLAB interface were published in IEEE Sensors Journal (2021). The integrated circuits and photodetectors were designed and fabricated by the Cadence circuit design toolbox (2019). The proposed circuit solutions were fabricated with 180-nm CMOS technology and published at IEEE MWSCAS 2020 conference. As the last contribution, the in vitro experiments with the proposed device, including the custom chip and 3D-printed housing, were performed, and the electrical and optical results were submitted to the IEEE Journal of Solid-State Circuits (JSSC 2022).

Photodynamic therapy (PDT) has been considered a favorable approach in certain oncology applications for its little invasiveness and better targeting specificity compared to conventional therapies. In PDT, localized photosensitizers can be activated by light to produce cytotoxic oxygen species. However, the prescribed drug and light doses do not provide satisfying outcomes as the PDT efficacy relies strongly on the interplay between localized dose factors. Therefore, the fluorescence emission from active photosensitizers has been investigated extensively for real-time PDT dosimetry. This dissertation focuses on characterizing fluorescence properties of two photosensitizers, Photofrin® and PpIX, in cellular models and discusses about their potential clinical applications. First, we introduc time-resolve fluorescence (TRF) of photosensitizers as a potential tool in PDT dosimetry. TRF acquires fluorescence decay profiles and it is sensitive to drug-microenvironment interactions that occur frequently in PDT. Therefore, it provides complementary information in addition to fluorescence spectra that could be subject to intensity artifacts. In this dissertation, we review TRF studies on PDT photosensitizers, and quantify TRF parameters of Photofrin® at various subcellular locations. Moreover, analytical solutions are developed to correct distorted TRF measurements from commonly used time-domain data acquisition. Second, we report a new concept – integrated detection and treatment of Barrett’s Esophagus (BE). BE is a pre-cancerous lesion considered as a major risk factor in developing esophageal cancers. However, early intervention of BE has remained a challenging issue as tissue biopsy introduces significant sampling errors and the separate procedures between diagnosis and treatment add relocation errors. We proposed to use PpIX fluorescence to highlight morphological features at the cellular level for quantitative classification, followed by well-characterized treatment. Current proof-of-concept studies were performed separately, whilst the detection and treatment can be integrated using confocal endomicroscopy technology. Overall, these studies examine the potential benefits provided by fluorescence of photosensitizers for cancer diagnosis and treatment monitoring. / Thesis / Doctor of Philosophy (PhD)

For this dissertation, I investigated the laser-induced fluorescence (LIF) of solid uranyl compounds to develop an optical screening technique for the presence of uranyl compounds and I applied Raman spectroscopy to tissue-engineered myocardial scaffolds. Uranyl (UO2+2) compounds yield an easily detectable, characteristic emission. The fluorescence emission is in the 450–600 nm (22200 cm-1 to 16700 cm-1) spectral region. Typically five emission bands are observable, regardless of the excitation wavelength and the emission spectrum can be used as a fingerprint for uranyl compounds. In order to develop an optical screening technique for the presence of uranyl compounds, I investigated the dependence of uranyl LIF emission intensity on laser excitation wavelength and on laser power, and compared the advantages of continuous wave lasers with pulsed lasers. Based on our studies, we successfully designed a field-deployable Fluorescence Spectral Imaging (FSI) system for in situ detection of uranyl compounds. Tissue-engineered myocardial scaffolds are used to study cell recellularization into a three-dimensional (3-D) scaffold for true multilayered 3-D cellular growth and organization. Decellularization and recellularization processes necessarily are accompanied by specific molecular composition changes in the tissues and Raman spectroscopy is highly sensitive to these molecular composition changes. Raman spectroscopy is suitable for accurate molecular-level elucidation of reconstruction mechanisms. In this study, I recorded characteristic Raman spectra of fresh native porcine myocardium, decellularized porcine myocardium, fresh native rat myocardium, and decellularized porcine myocardium recellularized with rat stem cells. The results show that for fresh porcine myocardium and fresh rat myocardium have characteristic Raman peaks at 1004, 1448 and 1661 cm-1, which reflects cell compositions such as lipids. The Raman spectra of decellularized samples exhibit seven peaks at Raman shifts of 857, 938, 1063, 1253, 1300, 1448 and 1661 cm-1, which reflects that the extracellular matrix (ECM) after removal of cells. We also found that long time exposure to continuous laser irradiation produced the Raman spectra with increased background and blackened tissue in fresh heart tissue due to thermal denaturation. Our results demonstrate the feasibility of using Raman spectroscopy to analyze the recellulization process in tissue engineering myocardium.