Synthèse de sondes moléculaires pour l'imagerie multimodale et multi-échelle appliquée en science du vivant

Les travaux décrits dans ce mémoire ont pour objectif d'imager des cellules par des techniques d'imagerie complémentaires à la spectroscopie de fluorescence : la micro-spectroscopie Raman et l'imagerie NanoSIMS. Le présent manuscrit se divise en quatre chapitres et présente les différents aspects menant à la mise en place de ce projet. Le premier chapitre du manuscrit a pour but de positionner le projet d'un point de vue biologique. Ce chapitre présente l'organite que nous souhaitions marquer, le nucléole, et la façon dont nous avons procédé à sa localisation par l'intermédiaire de sondes moléculaires. Le marquage que nous avons choisi est un immuno-marquage indirect faisant intervenir deux anticorps : un anticorps primaire spécifique à la nucléophosmine, protéine majoritairement présente dans le nucléole et un anticorps secondaire, reconnaissant l'anticorps primaire, marqué par une sonde moléculaire d'intérêt. Ce chapitre a également permis de montrer par une analyse en immunofluorescence l'absence d'interférence des sondes moléculaires dans le cadre de ce marquage. Le second chapitre se focalise sur la synthèse des sondes moléculaires permettant le marquage des anticorps secondaires. Les sondes envisagées pour ce marquage possèdent un groupement ester de succinimide réactif capable de "s'accrocher" de manière covalente aux fonctions amines de l'anticorps secondaire et un ou plusieurs groupements fonctionnels visible par la technique d'imagerie voulue. Trois catégories de sondes ont été préparées et dépendent de la technique d'imagerie employée. Les sondes Raman comportant une triple liaison carbone-carbone visible par cette technique, les sondes NanoSIMS dont l'acquisition d'image sera possible par les halogènes présents dans la structure et les sondes bimodales comportant les deux éléments dans la même structure. Les sondes bimodales sont obtenues par le couplage pallado-catalysé de Sonogashira. Dans la dernière partie du chapitre, une nouvelle série de sonde a été envisagée dans le cadre d'une application future : le marquage de l'ATP- -SH grâce à une fonction mésylate. Les deux derniers chapitres ont pour but de mettre en application tout ce qui a été présenté dans les chapitres précédents. Les deux techniques employées permettent d'accéder à des types d'informations différentes : la spectroscopie Raman donne accès aux modes de vibrations d'une molécule tandis que l'imagerie NanoSIMS permet d'obtenir des informations élémentaires et isotopiques. Nous présenterons le cheminement suivi pour imager des cellules via leurs constituants par micro-spectroscopie Raman et imagerie NanoSIMS via les sondes moléculaires introduites.Life sciences imaging are widely used for different applications, they are interested in medical diagnosis as well as basic research. In cells biology, fluorescence microscopy is mainly used for organelles observation at sub-cellular scale. However, techniques based on fluorescence phenomena are limited by some drawbacks like technical resolution, fluorescent dye degradation and the number of channels, which can be visualized. In this context, the exploration and the development of new way for image acquisition are considered as an experimental and technical scientific challenge. Furthermore, it can lead to complementary technique to fluorescence microscopy.This PhD thesis is a life science imaging project development and application allowing image acquisition base on molecular vibrations phenomena and elementary analysis in cells. Two techniques have been chosen in relation to both specificity: micro Raman spectroscopy and NanoSIMS imaging. Micro Raman spectroscopy allows the observation of molecular vibration mode at micron scale and NanoSIMS leads to elementary and isotopic sample information at sub-micronic scale. Combination of both techniques will lead to multi-scale and multi-modal imaging of biological samples. Molecular probes designing and synthesis for both techniques were used to visualize an organelle inside the nucleus: the nucleolus. Nucleolus has a key role in ribosomal RNA transcription and researchers shows some interest in the study of this organelle for his multifunctional role like ribosome biogenesis and nuclear organization. An immuno-labelling method combine with the introduction of molecular probes will allow nucleolus imaging by micro-Raman spectroscopy and NanoSIMS spectrometry. This immuno-labelling is specific to a phosphoprotein mainly localized inside the nucleolus: the nucleophosmin (NPM). In this project, the introduction of molecular probes in an immuno-labelling will act as a Raman Tag or a NanoSIMS tag for NPM's nucleolus observation and studies.This work at the interface between different fields: chemistry, biology and physics shows all the aspect of this project starting from molecular probes synthesis, immuno-labelling methods uses to direct application of both Raman and NanoSIMS techniques.RENNES1-Bibl. électronique (352382106) / SudocSudocFranceF

(En)countering counterfeits in Bangkok: the urban spatial interlegalities of intellectual property law, enforcement and tolerance

In a Bangkok mall a fibreglass policeman warning against intellectual property (IP) piracy stands just metres away from vendors selling fake DVDs; a scene indicative of incomplete and unsuccessful attempts by foreign governments (the US and EU in particular) and corporate actors at enrolment towards ever-higher IP standards – the ‘IP ratchet’ that Drahos (2004 Intellectual property and pharmaceutical markets: a nodal governance approach Temple Law Review 77 401–24) describes. But the scene also reflects cultural resistance at the local level. Both readings exemplify the range of historical, cultural, and politico-legal factors at play that can only be understood through engagement with vendors and consumers in the markets and malls of Bangkok. IP laws may achieve partial ‘closure’ but are regularly changing, contested, variably enforced, and subject to existing social norms such as the ‘cult of imitation’, cultures of legal informality, and a lack of social contract. We found that this lack of legal closure was most pronounced in the day-to-day operation of the Pratunam Market. Whilst other sites host regular crack-downs by police, the IP-specific DSI force, and the Thai courts, markets like Pratunam are mostly immune despite being a transnational trade node for the production and export of counterfeit garments with other developing countries, and a non-conforming node in the IP enforcement context. In the face of persistent efforts to transpose Euro-American IP laws in countries like Thailand, alternative and resistant nodes representing ‘spaces of interlegalities’ are likely to persevere because of the historical context, and the socio-cultural norms of these places

Electrospun collagen-based nanofibres: A sustainable material for improved antibiotic utilisation in tissue engineering applications

For the creation of scaffolds in tissue engineering applications, it is essential to control the physical morphology of fibres and to choose compositions which do not disturb normal physiological function. Collagen, the most abundant protein in the human body, is a well-established biopolymer used in electrospinning compositions. It shows high in-vivo stability and is able to maintain a high biomechanical strength over time. In this study, the effects of collagen type I in polylactic acid-drug electrospun scaffolds for tissue engineering applications are examined. The samples produced were subsequently characterised using a range of techniques. Scanning electron microscopy analysis shows that the fibre morphologies varied across PLA-drug and PLA-collagen-drug samples − the addition of collagen caused a decrease in average fibre diameter by nearly half, and produced nanofibres. Atomic force microscopy imaging revealed collagen-banding patterns which show the successful integration of collagen with PLA. Solid-state characterisation suggested a chemical interaction between PLA and drug compounds, irgasan and levofloxacin, and the collagen increased the amorphous regions within the samples. Surface energy analysis of drug powders showed a higher dispersive surface energy of levofloxacin compared with irgasan, and contact angle goniometry showed an increase in hydrophobicity in PLA-collagen-drug samples. The antibacterial studies showed a high efficacy of resistance against the growth of both E. coli and S. Aureus, except with PLA-collagen-LEVO which showed a regrowth of bacteria after 48 h. This can be attributed to the low drug release percentage incorporated into the nanofibre during the in vitro release study. However, the studies did show that collagen helped shift both drugs into sustained release behaviour. These ideal modifications to electrospun scaffolds may prove useful in further research regarding the acceptance of human tissue by inhibiting the potential for bacterial infection

A Meeting of Masks : Status, Power and Hierarchy in Bangkok

Beneath the protest marches, rallies and sieges dividing Thailand in recent times are more subtle pressures that emerge from everyday encounters involving cultural notions of rank and hierarchy. These are the focus of this highly accessible ethnographic study, which ventures beyond the barricades to explore the connections between inequality, space and social life in modern-day Bangkok. The author argues that the notion of an urban–rural divide obscures a far more complex reality linking city and countryside in reciprocal relations within both urban and national systems of status and class. Global market forces have increased the emphasis on material wealth in contemporary status relations and exacerbated pre-existing inequalities informed by a premodern system of status ranking called sakdina. This has compounded the challenges facing the growing urban middle classes and further marginalised rural and economically disadvantaged Thais

A meeting of masks: Status, power and hierarchy in Bangkok

Beneath the protest marches, rallies and sieges dividing Thailand in recent times are more subtle pressures that emerge from everyday encounters involving cultural notions of rank and hierarchy. These are the focus of this highly accessible ethnographic study, which ventures beyond the barricades to explore the connections between inequality, space and social life in modern-day Bangkok. The author argues that the notion of an urban–rural divide obscures a far more complex reality linking city and countryside in reciprocal relations within both urban and national systems of status and class. Global market forces have increased the emphasis on material wealth in contemporary status relations and exacerbated pre-existing inequalities informed by a premodern system of status ranking called sakdina. This has compounded the challenges facing the growing urban middle classes and further marginalised rural and economically disadvantaged Thais