Abdominal Aortic Aneurysm (AAA) is a complex disease that leads to a localized dilation of the infrarenal aorta that develops over years. Longitudinal information in humans has been difficult to obtain for this disease, therefore mouse models have become increasingly used to study the development of AAAs. The objective of this study was to determine any changes that occur in the biomechanical response and fiber microstructure in the apolipoprotein E difficient (ApoE-/-) angiotensin II (AngII) infused mouse model of aneurysm during disease progression, as well as determine some of the underlying changes in biochemistry, and demonstrate a novel method of reducing any pathogenic protease activity. Using a Microbiaxial Opto-Mechanical Device (MOD), ex vivo studies included adult aortas of ApoE-/- AngII infused mice excised and tested for mechanical response simultaneously imaged using two-photon microscopy to assess the microstructure at multiple time points. In vitro and ex vivo studies have shown changes in protease concentrations with the use of FRET based proteolytic beacons able to provide a non-destructive method to quantify protease activity measured against mechanical and microstructural changes. In vitro studies have demonstrated protease activity can be reduced using a molecule providing a positive feedback mechanism for protease inhibition and possibly provide a reduction in aneurysm progression.

Les travaux présentés dans ce manuscrit abordent l’étude de deux familles distinctes de molécules photoactivables induisant des processus biologiques différents sous excitation 2-photon : les triphénylamines (TPAs) et les Nanotriggers de deuxième génération (NTs). Les TPAs présentent un grand intérêt pour la thérapie photodynamique (PDT) 2-photon, grâce à leur section efficace d’absorption 2-photon élevées (325-764 GM) et leur capacité à induire la mort cellulaire, médiée par la production d’espèces réactives de l’oxygène (ROS) sous excitation 1- ou 2-photon. De plus, leur forte fluorescence permet d’imager la mort cellulaire grâce à un comportement singulier dans les cellules : les TPAs sont initialement localisés dans le cytoplasme des cellules vivantes, principalement au niveau mitochondrial, mais se re-localisent dans le noyau des cellules en apoptose. Les NTs de deuxième génération ont été conçus suivant le modèle de NT1, le premier Nanotrigger synthétisé. Leur structure générale a été conceptualisée pour se fixer à la protéine NO synthase (NOS) et générer un potentiel redox élevé uniquement sous excitation lumineuse, initiant ainsi un transfert d’électron à travers la NOS aboutissant à une production de NO. Les structures chimiques des sept molécules nouvellement synthétisées diffèrent à plusieurs niveaux. Cette étude a permis d’identifier, parmi les sept NT de deuxième génération, deux molécules ayant la capacité de pénétrer dans les cellules endothéliales, de cibler la eNOS et d’initier le transfert d’électron à travers la eNOS sous excitation 1- ou 2-photon. Les expériences réalisées au cours de cette thèse sur les deux familles de composés, ont également permis de mettre en évidence les relations entre la structure des molécules et les différents mécanismes induits par leur photo-activation. / The presented works in this manuscript approach the study of two distinct families of photoactivatable molecules that induce different biological processes under 2-photon excitation : triphenylamines (TPAs) and second generation Nanotriggers (NTs). TPAs present a great interest for 2-photon photodynamic therapy (PDT), thanks to their high 2-photon absorption cross-sections (325-764 GM) and their ability to induce cell death mediated by reactive oxygen species (ROS) production under 1- or 2-photon excitation. Moreover, their high fluorescence allows the imaging of cell death due to their particular behaviour in cells: TPAs initially localize in the cytoplasm of living cells, but translocate to the nucleus of apoptotic cells. Second generation NTs were designed based on the model of NT1, the first synthesized Nanotrigger. Their general structure has been designed to bind to the NO Synthase protein (NOS) and generate a high redox potential under excitation only, leading to the initiation of the electron flow through the protein and resulting in NO production. The chemical structures of the seven newly synthesized molecules differ on many levels. This study has identified, among the seven second generation NTs, two molecules which are able to penetrate in endothelial cells, target eNOS and initiate the electron transfer at the eNOS level under 1- or 2-photon excitation. The experiments presented in this thesis allowed to establish structure-activity relationships for each compound family regarding the different mechanisms induced by their photoactivation.

Die Relaxation der Styrylfarbstoffe DCM und LDS-750 nach Photoanregung in flüssiger Phase wurde mittels zeitaufgelöster optischer Spektroskopie untersucht. Dabei wurde die Breitband-Pump-Probe-Technik angewandt. Zur Charakterisierung der Relaxation von DCM im elektronischen Grundzustand wurde außerdem die Breitband-Dump-Probe-Technik (stimuliertes Emissionspumpen) eingesetzt. Für die beobachtete schnelle Relaxation von DCM wurde eine annähernd lösungsmittelunabhängige Zeitkonstante von 0.23 (( 0.04) ps im elektronisch angeregten und von 0.28 (( 0.07 ps) im elektronischen Grundzustand gefunden. Sie wurde als Konforma tionsänderung mit nur geringer Ladungsverschiebung charakterisiert. Die weitere spektrale Entwicklung wird in polarer Lösungsmittelumgebung vorwiegend von der Solvatation bestimmt. Für das ionische Polymethin LDS-750 wurden nach der Anregung solvensabhängige Kinetiken beobachtet, die sich durch die Annahme dreier möglicher Konformationen im S1 erklären lassen. / Relaxation of the stryryl dyes DCM and LDS-750 after photoexcitation in the liquid phase has been investigated by means of time-resolved optical spectroscopy. For this purpose, the broadband pump-probe technique was used. To characterize the relaxation of DCM in the electronic ground state, additionally the broadband dump-probe technique (stimulated emission pumping) was applied. An approximately solvent-independent time constant was found typical for the observed fast relaxation of DCM, with values of 0.23 ((0.04) ps in the excited and 0.28 (( 0.07 ps) in the electronic ground state. The relaxation was characterized as conformational change with only a small amount of charge transferred. The further spectral evolution in polar solvents was dominated by solvation dynamics.For the ionic polymethine species LDS-750 solvent-dependent kinetics were found after photoexcitation. They could be accounted for by assuming the existence of three different conformers within the S1 state.

This PhD project has explored the use of novel nitroxide annulated porphyrinic macrocycles as photosensitisers (PSs) for photodynamic therapy (PDT). The PSs have been synthesised, structurally, photophysically and photochemically characterised and investigated biologically through cell assays to determine their potential as photoactivated anticancer drugs. Tetra-nitroxide annulated phthalocyanines (Pcs) were initially investigated. Each of these compounds exhibit four annulated nitroxide-containing rings in a rigid, planar arrangement with fixed distance and geometry with respect to the macrocycle core, which is a novel structural motif in Pc chemistry. The presence of the nitroxides purportedly has two effects: to quench fluorescence and to increase photochemical singlet oxygen production by the compound. While nitroxides, as paramagnetic compounds, are non-fluorescent, their reduction to diamagnetic hydroxylamines results in an increased fluorescence yield. In this way, the nitroxide Pcs can potentially be used as probes for metabolic activity in biological systems, where the primary fate of the nitroxide moiety is reduction. Here, the fluorescence properties of the nitroxide-annulated Pcs were investigated through reduction of the nitroxide moieties by biologically significant reductants and calculation of fluorescence quantum yields. The singlet oxygen quantum yields of the Pcs were determined by two methods, the chemical trapping of singlet oxygen and the direct observation of singlet oxygen luminescence. While the quantum yields of the Pcs were promising when compared to clinically relevant PSs, the tetra-nitroxide Pcs did not exhibit increased quantum yields with respect to their nitroxide-free analogues. Additionally, there was minimal photodynamic action in cell assays. Subsequent fluorescence microscopy confirmed that this was most probably due to the fact that the Pcs were not localising within the cells. To improve the photodynamic action of the Pcs, two polymer-based delivery strategies were employed to enhance their delivery in biological environments. Firstly, the nitroxide Pcs were coupled to linear polymers to create polymer Pc hybrids. The second method involved the encapsulation of the nitroxide Pcs within polymer-based micelles. The synthesis of the polymer Pc hybrids was via a fast and efficient Atom Transfer Nitroxide Radical Coupling (ATNRC) reaction. The hybrids were synthesised as Mg, Zn or free-base (2H) Pc complexes, with either hydrophobic or hydrophilic polymer arms. The hybrids displayed high fluorescence quantum yields and reasonable singlet oxygen quantum yields. Again, these attributes this did not extend to any cell growth inhibition, even for the hydrophilic derivatives. Micellisation of the Pcs with a poly(styrene)-poly(acrylic acid) (PSty-PAA) star diblock copolymer afforded micelles with a range of concentrations of Pcs encapsulated within the glassy PSty core. Fluorescence studies showed that the micelles protected the nitroxide moieties from reduction by ascorbate, a result that could find application in EPR imaging and oximetry. During singlet oxygen experiments, the micelles were found to perform as nanoreactors, supramolecular assemblies which provide a reaction volume for other reagents. Pcs in the micelle cores effectively generated singlet oxygen and while this failed to escape the micelles, it was very effective in the oxidation of a hydrophobic model compound. Although there are potential applications for the micelle systems in waste-water remediation, these systems were ineffective in the PDT assays. With the difficulties associated with the biological delivery of the nitroxide Pcs in mind, hydrophilic mono-nitroxide annulated porphyrazine (Pz) macrocycles were designed and synthesised. Pzs exhibit many of the favourable optical properties of the Pcs. Synthesis of these compounds proceeded via Linstead macrocyclisation to give the target MgPz with A3B type substitution. A nitroxide-free A4 Pz was also isolated as a side-product. Transmetallation and/or hydrolysis gave access to Mg and Zn A3B and A4 carboxylate Pzs. These compounds were structurally characterised and their fluorescence characteristics investigated. The 1O2 quantum yields of the Pzs were also studied. Significantly, nitroxide annulation was found to enhance 1O2 generation of the Pzs relative to nitroxide-free analogues. The Zn carboxylate Pzs were shown to effectively inhibit the growth of tumour cell lines in PDT assay and the MgA4 carboxylate displayed strong 2-photon fluorescence within the cytoplasm of NFF cells. Experiments on freshly excised skin showed the carboxylate Pzs to be highly effective 2-photon PSs for PDT. In conclusion, we have identified several carboxylate Pzs which should be tested further in in vivo PDT experiments.

Early experimental work suggested that the retina’s main role was to detect changes in brightness and contrast, namely working as a light detector, and that most of the complex computations in the visual system happened upstream in the brain. In reality, there is a growing wealth of literature indicating that the retina itself processes multiple channels of visual information (contrast, motion, orientation, etc.), making it much more complex than it originally appeared. For instance, there now appear to be over 20 types of retinal ganglion cells. To this end, the work in this thesis will focus on the identification and characterization of a single type of retinal ganglion cell in the mouse retina. In the first section of my results, I will show that this cell type, identified as the only GFP+ ganglion cell in the transgenic Hb9::eGFP retina, is a directionally selective ganglion cell (DSGC), that preferentially responds to objects moving upward through the visual field. This cell has a pronounced morphological asymmetry that helps it to synergistically (along with asymmetric inhibition) generate directionally selective responses. In the second results section, I will describe a novel phenomenon exhibited by Hb9+ DSGCs: Thanks to gap junction mediated signals, Hb9+ cells are able to anticipate moving stimuli and correct for lags that are inherent in visual signals generated by photoreceptors. In the third results section I will elucidate the mechanisms for the gap junction mediated anticipatory signals outlined in the second results section. Together, these results provide a significant advancement in our understanding of how the retina processes moving stimuli and provide a compelling example of how chemical and electrical synapses interact to allow for exquisite signal multiplexing.

abstract: Sample delivery is an essential component in biological imaging using serial diffraction from X-ray Free Electron Lasers (XFEL) and synchrotrons. Recent developments have made possible the near-atomic resolution structure determination of several important proteins, including one G protein-coupled receptor (GPCR) drug target, whose structure could not easily have been determined otherwise (Appendix A). In this thesis I describe new sample delivery developments that are paramount to advancing this field beyond what has been accomplished to date. Soft Lithography was used to implement sample conservation in the Gas Dynamic Virtual Nozzle (GDVN). A PDMS/glass composite microfluidic injector was created and given the capability of millisecond fluidic switching of a GDVN liquid jet within the divergent section of a 2D Laval-like GDVN nozzle, providing a means of collecting sample between the pulses of current XFELs. An oil/water droplet immersion jet was prototyped that suspends small sample droplets within an oil jet such that the sample droplet frequency may match the XFEL pulse repetition rate. A similar device was designed to use gas bubbles for synchronized “on/off” jet behavior and for active micromixing. 3D printing based on 2-Photon Polymerization (2PP) was used to directly fabricate reproducible GDVN injectors at high resolution, introducing the possibility of systematic nozzle research and highly complex GDVN injectors. Viscous sample delivery using the “LCP injector” was improved with a method for dealing with poorly extruding sample mediums when using full beam transmission from the Linac Coherent Light Source (LCLS), and a new viscous crystal-carrying medium was characterized for use in both vacuum and atmospheric environments: high molecular weight Polyethylene Glycol. / Dissertation/Thesis / Doctoral Dissertation Physics 2015

Le projet de thèse a pour but de synthétiser de nouveaux matériaux siliciques à microstructure contrôlée en utilisant des procédés de polycondensation à basse température, et de développer de nouvelles approches de microfabrication 3D à haute résolution par excitation bi-photonique d’une formulation sol-gel. La synthèse de matériaux microstructurés est ici centrée sur le procédé sol-gel, impliquant des réactions d’hydrolyse-polycondensation. Les mtériaux les plus étudiés par ces approches en particulier dans le cadre d’un contrôle de la porosité sont préparés à partir de tetraéthoxide de silicium (TEOS)/ Le matériau est structuré généralement par des agent moléculaires structurants (« templates ») tels que les composés tensioactifs. Ces derniers sont en général éliminés, après condensation des silanols, par traitement thermique ou lavage afin de libérer la porosité et générer la structuration. Cette étape d’élimination des agents structurants est souvent problématique, en particulier au niveau industriel, car elle utilise soit des hautes températures, soit des quantités importantes de solvants. Il y a un enjeu important à développer des approches de microstructuration s’affranchissant de ces agents moléculaires structurants, cela représentant le principal objectif de cette thèse.Deux stratégies sont abordées aux cours de ce travail de recherche. La première implique l’utilisation de précurseurs organosilylés donc la structure moléculaire permet une microstructuration du matériau synthétisé. Cette partie explore l’utilisation de précurseurs de la famille des silsesquioxanes et leur transformation vers les matériaux. La deuxième approche propose d’évaluer pour la première fois l’utilisation de l’excitation biphotonique pour contrôler l’étape de condensation sol-gel par modification de pH au point focal d’un laser et ainsi apporter une résolution micrométrique à la structuration du matériau / The main goal of this research was the synthesis of new siliceous materials with controlled microstructures, using low-temperature polycondensation process, and develop the new approach towards 3D microfabrication under two-photon excitation of a sol-gel formulation. Synthesis of microstructured materials is based on the sol-gel process, i.e. hydrolytic polycondensation. Most known siliceous materials with controlled microstructure are synthesized from tetraethoxysilane (TEOS). The materials are generally structured by structuring molecular agents ("templates") such as surfactant compounds, which are then removed by heat treatment or washing with appropriate solvents. This stage of removal of structuring agents is often problematic, particularly at the industrial level, because it uses either high temperatures or large amounts of solvents. Therefore, new methods to obtain materials with controlled porosity without the need to use templates are still being developed. Two strategies are discussed during this research work. First one involved the use of organosilicon precursors with a define structure that at molecular level modify the structure of the final materials. The use of well-defined precursors (silsesquioxanes), in appropriate proportions, allows the control of the porosity of the obtained silica materials. The second approach investigated in this work is the local photo-induced structuration of silicon based pre-hydrolyzed precursors under two-photon excitation allowing high 3D resolution

Mit dem Ziel stabile und photomodulierbare Fluorophore für ihre direkte Anwendung in der hochaufgelösten Mikroskopie, wurde die Synthese und Charakterisierung von neuen fern Rot emittierender photoschaltbarer Fluorophore erreicht. Hierbei wurde die elektronische Kopplung zwischen den Komponenten tiefgreifend untersucht, und die Struktur Eigenschafts Beziehung etabliert. Durch die Wechselwirkung der photochromen Einheit mit dem Fluorophor, konnte die Fluoreszenzemission ein- und ausgeschaltet werden. Zusätzlich wurden Einkapselungs Experimente in Micellen durchgeführt, um die Wasserlöslichkeit der synthetisierten Verbindungen zu untersuchen. Letzlich, wurden mögliche Anwendungen mittels Fluoreszenzmikroskopie geprüft. Mit dem Ziel hochaufgelöste Bilder unter biologischen Bedingungen zu erhalten, wurde die Verkapselung der Dyaden in riesige unilamellare Vesikel erprobt. Unter Ausnutzung der Vorteile nichtlinearer Prozesse wurde die Synthese und Charakterisierung von photochromen Verbindungen, die in der Lage indirekt durch einen anfänglichen sensibilisierten Prozess geschaltet werden, untersucht. Zu diesem Zweck wurde ein Triarylamin Chromophor als Zwei-Photonenabsorber kovalent an einen Azobenzol verbunden. Die Charakterisierung der angeregten Zustandsdynamik wurde ausgeführt und die zwei Photonen induzierte Isomerisierung der Dyade bestätigt. Eine detaillierte Untersuchung der elektrochemischen Eigenschaften wurde durchgeführt und Richtlinien zur Verbesserung des Systems wurden kurz genannt. / With the purpose of designing stable and photomodulable fluorophores for their direct application in subdiffraction microscopy techniques, the synthesis and characterization of new far-red emitting photoswitchable fluorophores was accomplished. Fluorescence emission was efficiently modulated or switched On and Off by the interaction of the photochromic unit with the fluorescent-unit. Additionally, encapsulation experiments in micelles were performed to investigate the water solubility of the synthesized compounds. Finally, potential applications were examined with fluorescence microscopy, encapsulating the dyads in giant uni-lamellar vesicles under biological conditions to explore the feasibility to obtain highly resolved subdiffraction images. Exploiting the advantages of nonlinear processes, the synthesis and characterization of photochromes which are able to be switched indirectly through an initial sensitized event were studied. With this determination a two-photon absorbing triarylamine chromophore was covalently linked to an electron poor azobenzene. In-depth characterization of the excited state dynamics was performed and two photon induced isomerization of the dyad was confirmed. A detailed study of the electrochemical properties was set and guidelines towards the improvement of the system were succinctly mentioned.

Melanoma is a pigmented tumor that originates from the melanocytes; pigmented cells present throughout the body, including skin and iris. The cutaneous form is the most common type, and it represents about 5% of the skin tumors diagnosed in Brazil. Although it does not have a high incidence, it represents about 80% to 85% of all skin tumor deaths. The second most frequent type of melanoma is ocular. It represents 5% of all melanoma cases and is a potentially lethal disease, especially when it causes metastasis. The main therapeutic approach for melanomas, in general, is surgery, with resection of the cutaneous lesion or enucleation in the case of ocular melanoma. Other techniques such as adjuvant immunotherapy, palliative chemotherapy, and radiotherapy are also used. However, they have low efficacy and several side effects. Photodynamic therapy is a therapeutic modality based on the interaction of light at specific wavelength and photosensitizer, in the presence of molecular oxygen, leading the cell to death. As melanoma is a pigmented cancer, it usually does not respond well to photodynamic therapy due to the high absorption of light on the surface of the tumor, making volumetric eradication impossible. This project investigated optical strategies for the diagnosis and treatment of melanoma. For the diagnosis, it was evaluated the fluorescence lifetime technique to differentiate melanoma and normal skin. A sensitivity of 99.4%, specificity of 97.4% and accuracy of 98.4% were achieved using linear discrimination analysis. For the cutaneous melanoma treatment, PDT combined to optical clearing agents (OCAs) was investigated. Vascular and cell-target photosensitizers were evaluated combined or not to OCAs. OCA improved PDT response in all pigmented tumors treated, but the best results were achieved when a dual-photosensitizer treatment combined to OCA was performed. The treatment of conjunctival melanoma was conducted using 2-photon excitation photodynamic therapy. The advantage of this technique is the use of infrared light, in a wavelength that melanin has a low absorption, improving the light penetration into the tumor. The tumor histology shows that apoptosis was induced only at the treatment site, with no damage to the surrounding tissue. Additionally, a single TPE-PDT session could treat the entire tumor. / O melanoma é um tumor pigmentado que surge dos melanócitos, células pigmentadas presentes em todo o corpo, incluindo a pele e a íris. A forma cutânea é a mais comum e representa cerca de 5% dos tumores cutâneos diagnosticados no Brasil. Embora não tenha uma alta incidência, representa cerca de 80% a 85% de todas as mortes por tumor de pele. O segundo tipo de melanoma mais frequente é o ocular. Representa 5% de todos os casos de melanoma e é uma doença potencialmente letal, especialmente em casos de metástase. A principal abordagem terapêutica para melanomas, em geral, é a cirurgia, com ressecção da lesão cutânea ou enucleação no caso do melanoma ocular. Outras técnicas, como imunoterapia adjuvante, quimioterapia paliativa e radioterapia também são usadas, porém, apresentam baixa eficiência e muitos efeitos colaterais. A terapia fotodinâmica é uma modalidade terapêutica baseada na interação da luz em um comprimento de onda específico e um fotossensibilizador, na presença de oxigênio molecular, levando a célula à morte. Como o melanoma é um câncer pigmentado, geralmente não responde bem à terapia fotodinâmica devido à alta absorção de luz na superfície do tumor, impossibilitando a erradicação volumétrica. Este projeto investigou estratégias ópticas para o diagnóstico e tratamento do melanoma. Para o diagnóstico, foi avaliada a técnica de tempo de vida de fluorescência para distinguir melanoma de pele normal. Utilizando análise de discriminação linear, obteve-se uma sensibilidade de 99,4%, especificidade de 97,4% e precisão de 98,4%. Para o tratamento de melanoma cutâneo, a PDT combinada com clareadores ópticos (OCAs) foi investigada. Um fotossensibilizador que tem como alvo vaso sanguíneo e um fotossensibilizador de alvo celular foram avaliados combinados ou não com OCAs. OCAs são soluções hiperosmóticas que desidratam o tecido, diminuindo o espalhamento da luz e melhorando a penetração de luz em profundidade. OCA melhorou a resposta de PDT em todos os tumores melanóticos tratados, mas os melhores resultados foram obtidos quando a PDT foi realizada com a combinação dos fotossensibilizadores e clareador óptico em uma única sessão. O tratamento do melanoma conjuntival foi realizado utilizando a terapia fotodinâmica por excitação de 2 fótons (TPE-PDT). A vantagem desta técnica é o uso de luz na região do infravermelho, em um comprimento de onda que melanina tem baixa absorção, melhorando a penetração de luz no tumor. A histologia do tumor mostrou que a apoptose foi induzida apenas no local do tratamento, sem danos no tecido adjacente. Além disso, uma única sessão de TPE-PDT foi capaz de tratar todo o tumor.

Cette thèse est consacrée à l’étude des mécanismes d’interaction laser-matière en régime femtoseconde pour l’analyse de circuits intégrés par stimulation photoélectrique non-linéaire. Cette technique permet d’accroitre la résolution pour répondre à la miniaturisation des composants électroniques. Les milieux étudiés dans ce travail sont plus particulièrement le silicium, matériau constitutif des circuits intégrés, et la silice pour le transport des impulsions laser dans une fibre optique. En effet, l’émergence de cette technique d’analyse en milieu industriel requiert l’utilisation de systèmes compacts, fiables et sécuritaires. Les simulations réalisées montrent la génération de charges dans le silicium et la propagation des impulsions dans des fibres photoniques à cœur creux identifiées pour limiter les effets non-linéaires. Des expérimentations sur composants permettent de les confronter aux simulations et de valider l’utilisation de ce type de fibres.Enfin, ce travail a permis de déterminer les paramètres optiques et laser essentiels ainsi que les technologies compatibles avec les contraintes industrielles en analyse de circuits intégrés. / This thesis is dedicated to the study of laser-matter interaction mechanisms in femtosecond regime for the analysis of integrated circuits by nonlinear photoelectric laser stimulation. This technique improves the resolution in order to deal with the miniaturization of electronic components. The materials studied in this work are silicon, a major chemical component in semiconductor electronics, and silica used for transporting laser pulses in optical fibers. Indeed, the emergence of this analysis technique in industrial environments needs the use of compact, reliable and safe systems. The performed simulations show charge generation in silicon and propagation of pulses in photonic fibers identified for limiting nonlinear effects. Some experiments on components allow a comparison with simulations and validation of the use of this type of fibers. Finally, this work was able to identify key optical and laser parameters along with technologies compatible with industrial constraints in integrated circuit analysis.