Sensitive and specific visualisation of biomolecules in living models is highly challenging because of the complexity of cellular systems. Raman spectroscopy provides chemical contrast based upon molecular vibrations within a sample. It is therefore a powerful approach in biomedicine for disease diagnosis, owing to its potential to provide a spectroscopic fingerprint of biological species. However, Raman scattering is a weak process, and therefore novel approaches are required in order to improve the detection sensitivity for biomolecular imaging in living systems. Here, novel approaches for biomolecular visualisation based upon stimulated Raman scattering (SRS) microscopy are reported. Spectroscopically bioorthogonal functional groups, those which produce spectrally isolated Raman peaks distinct from endogenous cellular Raman peaks, are utilised as a general strategy for small-molecule visualisation. A key issue in the pharmaceutical industry is the lack of available techniques which can probe drug uptake and retention in living cells. Spontaneous Raman spectroscopy and SRS imaging are used for the selective intracellular visualisation of small-molecule inhibitors using a spectroscopically bioorthogonal approach in Chapter 2. Ponatinib and erlotinib are tyrosine kinase inhibitors used in clinical cancer treatment. The spectroscopically bioorthogonal alkyne group present within each drug is used as a specific marker to visualise the uptake and distribution of these two compounds in relevant cellular models. However, only a limited number (~2%) of regulatory approved drugs contain spectroscopically bioorthogonal Raman functional groups. Hence, a general strategy for the design of novel bioorthogonal Raman labels would allow SRS microscopy to be fully exploited in the drug discovery process. Therefore, density functional theory (DFT) is utilised as an in silico screening technique for the design of novel Raman labels, which is presented in Chapter 3. A library of bioorthogonal Raman-labelled analogues of the natural product, anisomycin, have been synthesised in order to validate the DFT screening approach. The effects of labelling upon the biological activity of anisomycin is also assessed. Spectroscopically bioorthogonal Raman imaging of several biomolecules was performed using SRS imaging and the results of which are reported in Chapter 4. The intracellular detection of de novo DNA synthesis was investigated using SRS imaging through metabolic incorporation of alkyne-containing nucleosides. Secondly, the intracellular detection of the Raman-labelled anisomycin analogues is reported using both spontaneous Raman spectroscopy and SRS imaging. This approach has enabled the rate of drug uptake to be assessed in real-time. Furthermore, combining multi-colour SRS imaging and fluorescence imaging in a dual-modality approach, enabled the analysis of drug uptake to be visualised across intracellular structures, and to be correlated with markers of cell-cycle status. These studies represent novel approaches for the direct intracellular visualisation of the uptake and retention of small-molecule inhibitors in live cells.

Significant attention from the medicinal and pharmaceutical communities has been pushed towards the design and development of natural products for defence against many forms of illnesses. The Amaryllidaceae plant family has shown their prevalence over time aiding towards our needs and becoming viable sources of alkaloids due to their wide variety of bioactivities presented. The low availability towards these often-complex structures with at times comprising up to six contiguous chiral centers have made practical testing scarce. More dominantly the isocarbostyrils are well recognized, being hydroxylated phenanthridones providing increased activities making them model targets to test and develop new synthetic strategies towards. These compounds represent a subset of the Amaryllidaceae alkaloids that lack a basic nitrogen center. This thesis describes the total synthesis of four derivatives of the antiviral natural product (+)-trans-dihydronarciclasine from α-azidoacetone and m-anisaldehyde. Herein we demonstrate constructive routes towards ring-A modified, fully functionalized rings-B/C derivatives synthesized via asymmetric chemical syntheses providing further insight into SAR studies. This thesis expands on the organocatalytic [3+3]-cycloaddition sequence to produce aminocyclitol cores providing effective routes towards the development of five stereogenic centers in all targeted ring-C structures. Such studies were attributed to the enal adducts isolated from the Wittig reaction towards four natural product derivatives gaining knowledge related to the targeted molecules mode of action. One additional (+)-transdihydrolycoricidine analogue will be communicated, that enables the imaging while inside live cells with use of alkyne-tag Raman imaging. Limitations of the alkaloids include the toxicity that accompanies these agents and the poor aqueous solubilities they provide, eliciting an increased need for new antiviral agents. The syntheses communicated provide effective routes towards unnatural alkaloids and can be pushed towards alternative chiral aminocyclitol targets for future studies. All compounds have been sent away for screening including against coronavirus at Johns Hopkins. / Thesis / Bachelor of Science (BSc) / This thesis is primarily driven towards the development of four antiviral lycorane structural type alkaloids, and an analogue synthesized via a copper-cocatalyzed Sonogashira reaction, utilizing a labile phenol-derived sulfonated hydroxyl group in its coupling towards an alkyne tagged structure. This method provides easy access for a variety of compounds without a fluorescent tag, taking steps forward in elucidating how the Amaryllidaceae alkaloids are delivering their biological effects. The densely substituted ring-C was obtained via an asymmetric organocatalytic [3+3] sequence for the assembly of the aminocyclitol core and is described. This sequence has provided effective regio, diastereo, and enantioselective access to five unnatural products. Preparation of the precursors were prepared using a Wittig methodology previous reported by the McNulty group that has been used in many syntheses for various Amaryllidaceae alkaloids

Lors de certains scénarios accidentels, percement de cuve de réacteur avec entrée d’air, dénoyage de piscinesde manutention, de stockage ou d’entreposage de combustibles usés, ou encore certains accidents de transport,les assemblages combustibles des Réacteurs à Eau Pressurisée (REP), peuvent se retrouver privés de leurrefroidissement et exposés à l’air. Dans ces conditions, la température des assemblages s’élève et les gaines descrayons combustibles, en alliage à base de zirconium, s’oxydent. Par rapport à une oxydation sous vapeur, lavitesse de dégradation des gaines est beaucoup plus élevée, d’une part à cause de l’enthalpie élevée de laréaction zirconium-oxygène, comparée à la réaction zirconium-vapeur d’eau, et d’autre part parce que l’azotecontribue à la dégradation. Des phénomènes d’emballement sont attendus qui peuvent rapidement conduire à laperte de la fonction de confinement assurée par les gaines.L’objectif de cette thèse a été d’affiner la compréhension des mécanismes d’oxydation sous air à hautetempérature des deux alliages de zirconium les plus utilisés dans les REP français, le Zircaloy-4 et le M5®, et depréciser le rôle de l’azote.Des tronçons de gaines vierges de Zircaloy-4 et M5® ont été oxydés dans une thermobalance sous air enconditions isothermes à des températures comprises entre 800°C et 1000°C. Plusieurs techniquesexpérimentales (spectroscopie micro-Raman, EPMA, DRX, microscopies optique et électronique à balayage…)ont été mises en oeuvre pour caractériser les couches d’oxyde. La formation et l’évolution des différentes phasesqui composent ces couches (zircone monoclinique, quadratique et cubique, oxynitrure de zirconium et ZrN) ontainsi pu être mises en évidence et analysées en fonction de l’avancement de l’oxydation. Des mécanismesd’oxydation ont été proposés, et la résistance à l’oxydation plus importante de l’alliage M5® par rapport auZircaloy-4 a été expliquée.Les informations recueillies permettront d’alimenter des modèles visant à prédire le comportement des gaineslors de l’oxydation en situation accidentelle avec entrée d’air (transitoire de température, composition évolutive dela phase gazeuse…). / In nuclear plants, some accidental situations can result in air exposure of Pressurized Water Reactor (PWR) fuelassemblies: air ingress following a breach in the reactor vessel, deflooding during handling, spent fuel storagepool deflooding. Deprived of cooling source, the assemblies temperature raises and the fuel cladding, made out ofzirconium based alloys, oxidize. Compared to a steam oxidation, the degradation kinetic of the cladding is higher,on the one hand because of the high enthalpy of the zirconium-oxygen reaction (compared to zirconium-steamreaction), on the other hand because of the nitrogen contribution to the degradation. Temperature escalation andreaction runaway are expected and can rapidly lead to the loss of integrity of the cladding tubes.The objective of this PhD thesis was to affine the understanding of the high temperature air oxidation mechanismsof the two mostly used zirconium alloys in French PWR, Zircaloy-4 and M5®. Special attention has been paid toclarify the role of nitrogen.As-received Zircaloy-4 and M5® claddings segments have been oxidized in a thermobalance in air in isothermalconditions at temperatures between 800°C and 1000°C. Several characterization techniques (micro-Ramanspectroscopy, EPMA, XRD, optical and scanning electron microcopies...) have been used to analyze the oxidelayers. Identification and evolution of the different phases (monoclinic, tetragonal and cubic zirconia, zirconiumoxynitride and ZrN) has been evidenced and analyzed at several step of the oxidation process. Oxidationmechanisms have been proposed and the better oxidation resistance of the M5® alloy, compared to Zircaloy-4alloy, has been explained.The collected information will allow improvement of modeling aiming to predict the behavior of the claddings invarious accidental situations with air ingress (temperature transients, evolution of the gas phase composition…).

The diagnosis of cancer in early stages is an unmet clinical need, especially in view that current treatments for cancer cannot address metastatic disease. Cancer aberration processes are associated to an increase in the production of reactive oxygen species (ROS). Chemical probes that can specifically detect these species are potentially useful as medical diagnostics and research tools for cancer imaging. One of the aims of my thesis was the design and synthesis of the activatable fluorescent probes based on small molecule fluorophores modified with chemically reactive moieties. The activation of these moieties by defined targets (e.g. ROS) results in the activation of the fluorophore and subsequent emission of a fluorescent signal. Two libraries of fluorescence probes for the detection of ROS have been designed and synthesised: 1) hydrocyanine-based probes as silent fluorophores that can be activated with superoxide ions, 2) coumarin-based hydrogen peroxide probes with red-shifted fluorescent properties and different boronate activatable groups for hydrogen peroxide sensing. We have performed in vitro assays to evaluate the fluorescence response of our probes as well as experiments in relevant live cells to assess their application for detection of ROS in live cells with molecular resolution. Moreover, cancer cells also overexpress Epidermal Growth Factor Receptors (EGFR). Surface-enhanced Raman scattering (SERS) nanotags that can recognize specifically EGFR receptors in cells are promising tools for the enhanced diagnosis of cancer. Two near-infrared cyanine Raman reporters were synthesized with a carboxylic group that was conjugated to cysteamine for derivatization of gold nanoparticles (AuNPs). This work was performed in the CSIR-NIIST (Kerala, India), where I did a 3-month PhD placement. I conjugated the cyanine reporters to spherical AuNPs of 40 nm diameter, and measured their Raman intensity and stability. The best SERS nanotags were selected for encapsulation with PEG and subsequently derivatization with anti-EGFR-EP22 antibodies. In vitro characterization of the SERS nanotags was performed: SERS and absorbance spectra, electron microscopy images as well as SERS imaging experiments in A549 lung cancer cells.

Cette thèse s’inscrit dans le domaine de la catalyse hétérogène pour des applications en raffinage et en pétrochimie. Plus particulièrement, nous avons choisi d’étudier le cas des catalyseurs d’hydrotraitement largement utilisés dans les procédés de conversion du pétrole en carburant. Ces catalyseurs sont constitués d’une phase métallique sulfurée supporté sur un support poreux inorganique.Dans ce travail, nous nous sommes particulièrement intéressés à la compréhension et la modélisation de la première étape de préparation d’un catalyseur d’hydrotraitement : l’étape d’imprégnation. Une méthodologie innovante basée sur la technique d’imagerie RMN qui permet d’observer operando le transport des précurseurs métalliques dans le support et sur la technique d’Imagerie Raman a été développée. Les résolutions spatiales et temporelles obtenues ont permis de valider cette méthodologie pour caractériser l’étape d’imprégnation en termes de phénomènes physiques (capillarité et diffusion) et chimiques (interaction de surface) et pour identifier les descripteurs clés de cette étape.En conclusion, ces travaux permettront un meilleur contrôle de l'étape d'imprégnation et donc du profil de distribution de la phase active, ce qui aura une forte répercussion sur la performance catalytique / The objective of this work is to characterize the impregnation step in terms of physical phenomena (capillarity and diffusion) and chemical phenomena (surface interaction) in order to identify the key descriptors of this step in order to improve the preparation hydrotreating catalysts. This involves monitor spatial and temporally the transport of the species in the impregnation solution within the porosity, more particularly in the case of Ni(Co)MoP/?-Al2O3 catalysts preparation.An original MRI approach has been developed to follow in-situ the impregnation step from a solution of mixed nature with more than one metal precursor in the presence of an additive. MRI provides access to the spatial distribution of all species within the catalyst with a spatial resolution of 39 × 39 µm. Raman imaging allows an identification of the chemical nature of the species in solution with a spatial resolution of 16.2 × 16.2 µm.First, the phenomena involved in the impregnation of model solutions composed of Ni (or Co), were studied by modifying the parameters of the solution. These experiments were then used as the basis for the development of a mathematical model to rationalize the impregnation step. In a second part, this study was extended to the preparation of hydrotreatment catalysts. Several phenomena have been evaluated here, notably the impact of the presence of phosphorus on the transport of Mo species, possible competition of adsorption between P, Co and Mo and the effect of the presence of citric acid.This study allows a better control of the impregnation step and thus of the distribution profile of the active phase, which has an impact on the catalytic performance

L’objet de cette thèse consiste en des développements de méthodes en spectroscopie et imagerie Raman. Après une revue des outils statistiques permettant de traiter de gros volumes de données (analyse multivariée, classification), l’étude est appliquée à deux familles de matériaux déjà bien connus, utilisés comme modèles pour tester les limites des développements mis en oeuvre. La première famille est une série de matériaux carbonés, traités à différentes températures, avec des inhomogénéités à l’échelle du nm, dont la taille est déduite d’une approche conjointe Raman-diffraction des rayons X. Un autre résultat concerne l’effet du polissage, qui induit des artefacts Raman conduisant à surestimer le désordre structural local, et une méthode basée sur la largeur de la bande G est proposée. L’autre classe de matériaux présente cette fois des inhomogénéités à des échelles supérieures au micromètre, il s’agit de céramiques d’oxyde d’uranium appauvri. L’imagerie Raman, particulièrement bien adaptée en termes d’échelle spatiale, est utilisée pour sonder les surfaces obtenues, Le traitement des données est effectué par un approche mêlant analyse multivariée (analyse en composantes principales), et ajustement classique par Lorentziennes. L’interprétation des données obtenues est soutenue par une analyse par electron backscattering diffraction (EBSD), permettant de séparer l’effet d’orientation des grains de céramiques, d’autres effets sous-jacents. Ceux-ci, principalement localisés aux joints de grains, avec l’existence d’un mode Raman caractéristique, paraissent liés à des variations de stoechiométrie en oxygène ou d’impuretés, ou à des inhomogénéités de contrainte. Les perspectives de ce travail portent notamment sur la mise en oeuvre d’autres méthodes mathématiques, et pour l’aspect matériaux, sur l’analyse fine d’endommagements par irradiation des UO2, (effets d’anisotropie, rôle des joints de grains. / This thesis is aimed at developing methodologies in Raman spectroscopy and imaging. After reviewing the statistical instruments which allow treating giant amount of data (multivariate analysis and classification), the study is applied to two families of well-known materials which are used as models for testing the limits of the implemented developments. The first family is a series of carbon materials pyrolyzed at various temperatures and exhibiting inhomogeneities at a nm scale which is suitable for Raman--X-ray diffraction combination. Another results concern the polishing effect on carbon structure. Since it is found to induce Raman artifacts leading to the overestimation of the local structural disorder, a method based on the use of the G band width is therefore proposed in order to evaluate the crystallite size in both unpolished and polished nanographites. The second class of materials presents inhomogeneities at higher (micrometric) scales by the example of uranium dioxide ceramics. Being well adapted in terms of spatial scale, Raman imaging is thus used for probing their surfaces. Data processing is implemented via an approach combining the multivariate (principal component) analysis and the classical fitting procedure with Lorentzian profiles. The interpretation of results is supported via electron backscattering diffraction (EBSD) analysis which enables us to distinguish the orientation effects of ceramic grains from other underlying contributions. The last ones are mainly localized at the grain boundaries, that is testified by the appearance of a specific Raman mode. Their origin seems to be caused by stoichiometric oxygen variations or impurities, as well as strain inhomogeneities. The perspectives of this work include both the implementation of other mathematical methods and in-depth analysis of UO2 structure damaged by irradiation (anisotropic effects, role of grain boundaries).

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No description available.

Compte tenu de leur bioactivité et de leur ostéoconduction, les revêtements d’hydroxyapatite (HA) favorisent le développement de tissu osseux et contribuent à l’adhérence et à la prolifération des cellules osseuses tout en préservant l’intégrité mécanique du dispositif métallique de l’implant. Toutefois, des signes de résorption de cette couche ont été constatés et l’introduction du procédé de projection plasma pour la réalisation de dépôts d’HA a donné lieu à plusieurs controverses. Certaines études ont montré que les dépôts d’HA par projection plasma présentent une mauvaise adhérence sur le titane, une dégradation et écaillage avec le temps. Ce phénomène est attribuable à la décomposition de l'HA au cours de la projection et à l'existence de phases secondaires cristallisées ou amorphes qui fragilisent le dépôt. A l’heure actuelle, les recherches de nouvelles apatites thermiquement plus stables se développent. Ainsi des dépôts à base de fluorapatite ou de fluorhydroxyapatite fournissent des dépôts plus stables et plus adhérents. Il existe d’autres formes d’apatites comme la chlorapatite (ClA) qui fond sans se décomposer ce qui permettrait d’éviter la formation des phases étrangères qui provoquent l’écaillage du dépôt. Ce travail porte sur la synthèse et la caractérisation de phosphates de calcium apatitiques destinés au recouvrement de prothèses ostéoarticulaires et/ou d’implants dentaires métalliques. L’élaboration des poudres d’apatite a été réalisée par réaction solide-solide, en solution aqueuse ou en sel fondu. Plusieurs apatites ont été étudiées : la fluorapatite, la fluorhydroxyapatite, la chlorapatite et l’apatite calcostrontique. On obtient des poudres pures, bien cristallisées et stoechiométriques. Nous avons ensuite étudié la stabilité thermique des poudres d’apatite par analyse thermogravimétrique. La stabilité thermique de la ClA et sa fusion sans décomposition permettent d’obtenir des dépôts sans phases secondaires avec un taux de cristallinité élevé. Parmi toutes les poudres étudiées, nous avons choisi de sélectionner la chlorapatite pour la suite de l’étude ainsi que l’hydroxyapatite comme référence. La poudre de ClA est déposée sur un substrat en titane via une mini-torche plasma mobile à basse énergie, conçue pour les implants dentaires ou orthopédiques de petites tailles et de géométries complexes. La projection par la mini-torche plasma permet de réaliser des dépôts fins et adhérents. Les propriétés structurales et microstructurales des dépôts de ClA et d’HA effectués dans les mêmes conditions ont été évaluées par diffraction des rayons X, spectroscopies FTIR et Raman. L’analyse des revêtements de ClA et d’HA par diffraction des rayons X atteste de la présence d’une seule phase cristalline. Néanmoins, le rapport de cristallinité des dépôts de ClA est largement supérieur à celui des dépôts d’HA, pour lesquels le rapport de cristallinité est supérieur à la norme requise. Ce résultat est du à la stabilité thermique de la ClA par rapport à l’HA. Les spectroscopies FTIR et Raman montrent la présence d’un faible taux de phase amorphe et d’oxyapatite. Nous avons également développé une nouvelle méthode semi-quantitative par cartographie Raman permettant de déterminer l’homogéneité de composition du dépôt. Enfin, nous avons montré que l’adhérence des dépôts de ClA au substrat déterminée suivant la norme ASTM C633 est équivalente à celle de l’HA. La réponse biologique des dépôts a été étudiée in vitro avec des cellules pré-ostéoblastes humaines sur les deux compositions ClA et HA. Les tests montrent que quelle que soit la composition du dépôt, la prolifération cellulaire augmente au cours du temps d’incubation. Une étude in vivo a été menée sur un modèle animal ovin en site fémur et humérus pour des durées de 2 et 6 mois afin d’évaluer le potentiel ostéoconducteur des dépôts d’HA et de ClA. Aucune réaction inflammatoire n’a été observée et l’interprétation des résultats d’analyses histologiques et physico-chimiques est en cours. / Plasma-sprayed hydroxyapatite (HA) has been shown to enhance bone apposition as compared with uncoated metal implants. In spite of their good clinical performances, implant bio-integration is still limited due to the poor adhesion of HA-coating on the titanium surface and the decomposition of HA into several foreign phases during plasma spray. The plasmaspray process was thus considered not to be optimum for HA coatings. To circumvent these disadvantages, other types of calcium phosphates have been proposed to replace HA, such as tricalcium phosphate or biphasic calcium phosphate with little improvement. Chlorapatite (ClA) however has never been tested despite its ability to melt without decomposition which could be a decisive advantage to avoid apatite decomposition and could increase coating crystallinity. This work concern the synthesis and the characterization of apatitic calcium phosphate as coating for endoprostheses and dental implants. The syntheses of the different apatite powders were performed by solid-solid reaction, in aqueous solution or by molten salt reaction. Several apatites were studied: fluorapatite, fluorhydroxyapatite, chlorapatite and strontium substituted apatite. The as-synthesized powders are pure, well crystallized and stoechiometric. The thermal properties of apatite powders were compared using thermogravimetric analysis. The thermal stability of ClA and its melting without decomposition allows to obtain deposits with no secondary phases and with a degree of crystallinity close to 100 %. We have therefore chosen ClA and HA as feedstock powders to achieve coatings. The as-synthesized ClA powder was deposited on a titanium substrate using a novel low energy plasma mini-gun characterized by a low power range (<13 kW) and portability allowing in-situ coating deposition designed for dental and orthopedic small implants with complex geometry. The plasma spray with the mini-gun gives thin and adherent coatings. It also provides economic benefits such as low energy and a higher yield of feedstock powder than conventional gun. The structural and microstructural properties of ClA and HA coatings performed under the same conditions were evaluated by X-ray diffraction, Raman and infrared spectroscopy. X-ray diffraction analyses on the ClA and HA coatings indicate the presence of crystalline apatite as the only crystalline phase. However, the crystallinity ratio of ClA coatings was much higher than that of HA coatings but superior to the required standard in both cases. This difference is due to the thermal stability of ClA powder. IR and Raman spectroscopy shows the presence of low levels of amorphous phase and oxyapatite. We have also developed a new semiquantitative method by Raman imaging in order to determine coating homogeneity. The mechanical properties of coatings were determined according to the standard test measurements ASTM C633. The adhesion of ClA coatings to the substrate is equivalent to HA coatings. The biological response of the coatings was studied in vitro with human preosteoblast cells on both ClA and HA coatings. Tests show that wathever the composition of coating, cell proliferation increases with time of incubation. The in vivo osteoconductive properties of coatings were studied in femur and humerus sites of an ovine animal model during 2 and 6 months. No inflammatory reaction has been observed and the interpretation of histological and physico-chemical results is in progress.

No description available.