Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 137-152). Also available in electronic version.

In the current work, the photophysics and photochemistry of the phototoxic red fluorescent protein (RFP) KillerRed was investigated. KillerRed's phototoxicity makes it useful for studying oxidative stress on cell physiology and for cell killing in photodynamic therapy. Spectroscopic probes were used to show that the phototoxicity of KillerRed stems primarily from a type I photosensitization mechanism producing radicals. The production of radicals was supported by electron paramagnetic resonance (EPR) studies, where a long-lived radical was observed in KillerRed and two other RFPs (mRFP and DsRed) following excitation. Transient absorption spectroscopy, various other spectroscopic techniques, and the published crystal structure of KillerRed indicate that the long-filled water channel is likely responsible for the increased phototoxicity of KillerRed. In the blue fluorescent protein (BFP) mKalama1, some of the same techniques were applied to understand the photophysics and photochemistry on the timescale ranging from femtoseconds to seconds. Transient absorption spectroscopy and previously published results demonstrate that two-photon excitation of mKalama1 likely results in the formation of a radical cation and solvated electrons. This may explain the blinking behavior which has been observed on the single molecule level for many fluorescent proteins, the identity of which has remained elusive. It was also shown that the chromophore, while neutral in the ground state, does not exhibit excited-state proton transfer (ESPT) during its nanosecond excited-state lifetime; however, the chromophore undergoes a deprotonation in the ground state after electronic relaxation. This work plays a key role in our understanding of fluorescent proteins and will help pave the way to developing new ones. The research on the BFPs was extended to improve them for cellular imaging. This was accomplished by identification of dark states in the BFPs which are longer in wavelength than the collected fluorescence. Using dual lasers, it was shown that these dark states could be optically depleted, thereby increasing the overall fluorescence without enhancing the background fluorescence. Rational site-directed mutagenesis was carried out on the BFPs and the mutants were screened for fluorescence enhancement. These proteins were then analyzed using transient absorption spectroscopy to elucidate the identity of the dark state(s) used for fluorescence enhancement.

Fluorescence resonance energy transfer (FRET)-based biosensors have been designed to fluorometrically detect everything from proteolytic activity to receptor-ligand interactions and structural changes in proteins. While a wide variety of fluorophores have demonstrated effectiveness in FRET probes, several potential sensor components are particularly notable. Semiconductor quantum dots (QDs) are attractive FRET donors because they are rather bright, exhibit high quantum yields, and their nanoparticulate structure enables the attachment of multiple acceptor molecules. Fluorescent proteins (FPs) are also of particular interest for fluorescent biosensors because design elements necessary for signal transduction, probe assembly, and device delivery and localization for intracellular applications can all be genetically incorporated into the FP polypeptide. The studies described in this thesis elucidate the important parameters for concerted QD-FP FRET probe design. Experimental results clarify issues of FRET pair selection, probe assembly, and donor-acceptor distance for the multivalent systems. Various analysis approaches are compared and guidelines asserted based on the results. To demonstrate the effectiveness of the QD-FP FRET probe platform, a ratiometric pH sensor is presented. The sensor, which uses the intrinsic pH-sensitivity of the FP mOrange to modulate the FP/QD emission ratio, exhibits a 20-fold change in its ratiometric measurement over a physiologically interesting pH range, making it a prime candidate for intracellular imaging applications.

Deux différents types de vecteurs, les nanocapsules lipidiques (LNC) et les systèmes multimodulaires (MMS) ont été développés pour l'administration par voie systémique de deux types d'acides nucléiques, l'ADN et les petits ARN à interférence (siRNA). Ces vecteurs sont formulés à base de complexes d'acides nucléiques et de lipides cationiques (lipoplexes) qui ont été soit encapsulés au coeur des LNC, soit recouverts de stabilisateurs stériques afin de former des MMS. Une partie du travail a consisté à développer des vecteurs de siRNA et à les caractériser par des méthodes physico-chimiques. En fonction du lipide cationique utilisé, jusqu'à 65% de siRNA ont pu être encapsulés dans les LNC, en présentant des caractéristiques appropriées pour une administration par voie systémique. La seconde partie a consisté à approfondir la caractérisation des vecteurs d'ADN et à analyser leur profil de distribution en utilisant de l'imagerie par fluorescence in vivo. Chez la souris saine, les vecteurs d'ADN ont présenté des profils de biodistribution spécifiques à leur composition. Sur deux modèles tumoraux (gliome sous-cutané et mélanome), les vecteurs ayant un temps de circulation prolongé ont également montré une co-localisation intéressante avec les cellules tumorales. Afin de mettre en évidence l'efficacité de ces vecteurs, un plasmide codant pour la tymidine kinase du virus herpes simplex (HSV-tk) a été encapsulé et administré. Puis un traitement par le ganciclovir (GCV) basé sur l'approche par gène suicide a été effectué. Les premiers résultats sont concluants, montrant une baisse de croissance tumorale après quelques jours de traitement aussi bien dans le modèle de gliome que dans celui du mélanome. Ces résultats indiquent que ces outils sont prometteurs pour une variété d'applications en thérapie génique.