Reverse transcription of HIV-1 genome involves multiple nucleic acids structural rearrangements chaperoned by nucleocapsid protein (NC). One such critical step is that the DNA transactivation response element (TAR) anneals to it complementary RNA region on the genome. It has been challenging to investigate mechanistic details on the annealing process because of the involvement of heterogeneous sets of protein/nucleic acid complexes. Here, we use single-molecule spectroscopy to study the NC induced melting of nucleic acid structure and the annealing activity of different regions along TAR structure. We find that NC induced secondary fluctuations are limited to the terminal stems, and the mechanism for the fluctuations is complex. By employing complementary targeted oligomers, we kinetically trap and investigate stable states of the putative nucleation complexes for the annealing process. This single molecule spectroscopy method directly probes kinetic reversibility and the chaperone role of NC at various stages along the reaction sequence. The new results lead to detailed understanding of NC chaperoned reverse-annealing and the partially annealed conformational sub-states. Argininamide, because of its specific binding to the loop regions of TAR, was studied on its specific inhibition to strand transfer. The loopmediated annealing is found to be more potentially inhibited than stem-mediated one.

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The purpose of this dissertation was two-fold: 1) explore the micelle structure and microenvironment of monorhamnolipids (mRL), produced by Pseudomonas aeruginosa ATCC 9027, and their mixtures with synthetic surfactants in order to postulate possible applications of these materials in industrial products and 2) examine the effects of mRL on commercial metal oxide nanoparticle (NP) aqueous dispersion behavior to reveal the potential impact of microbial secondary metabolites on NP fate and transport in the environment. The mixing behavior of mRL with cetylpyridinium chloride (CPC) was measured using surface tensiometry. Electrostatics resulted in cooperative enhancement in mixture properties, but were not significant until α(CPC) ≥ 0.25. Steady-state and time resolved fluorescence quenching measurements in mRL micelles revealed that quenching proceeded via a combined static and dynamic mechanism. Static quenching was preferred in mRL illustrating the reactants form a globular micelle. Changing the structure of the reactants displayed changes in the degree and mechanism of quenching further supporting this aggregate model. Fluorescence measurements on mRL-Tween 20 micelles supported that a geometrically-driven shape transition occurs as mRL decreases. The corresponding decrease in probe lifetime indicated the polarity of the micelle was decreasing. Tween "sealed" the mRL micelles making them less susceptible to water penetration. The effect of mRL on metal oxide NP dispersions was evaluated on adsorption strength, NP aggregate size and stability, and zeta potential under different conditions. Silica NPs showed little adsorption of mRL and was impervious to all variables in altering the solution stable aggregate size. NP aggregate size decreased at very high mRL concentrations due to osmotic and electrosteric repulsions of mRL micelles in solution. Titania, despite expectations, indicated fairly low adsorption of mRL and displayed similar aggregate dispersion stability as that of silica. Spectroscopic investigations exposed that the commercial titania NPs were contaminated with silica altering NP surface properties. Zinc oxide (ZnO) dispersions were substantially affected by the adsorption of mRL. Without mRL, ZnO NPs were unstable independent of pH. The addition of mRL stabilized the ZnO dispersions and lowered the zeta potentials. Furthermore, mRL coating prevented the dissolution of ZnO, the major factor implicated in ZnO toxicity.

The application and use of non-destructive portable x-ray fluorescence (XRF) analysis is a critical tool in the preservation and interpretation of cultural material. Portable XRF instrumentation produce elemental compositional data that is used to reconstruct current artifact composition, which can be related to materials and methods of manufacture, technological practice, as well as object condition and presence of corrosion surfaces. Portable XRF analysis is used to assess a variety of material classes utilized in artifact manufacture. The dissertation research is based on a series of three case studies that represent typical groups of material culture commonly encountered in conservation and conservation science research.Conservators and conservation scientists frequently undertake analysis and interpretation of disparate groups of materials. Often, these objects are tied together by research questions or themes directed by outside influences including preservation issues requiring action; curatorial research interests; museum exhibition programs; as well as many other cultural heritage stakeholders. To this end, both non-destructive and destructive tools that provide measurements of interest play critical roles in analysis. The case studies have been designed to answer common compositional questions relating to (a) bulk analysis of Chinese coins, (b) characterization of Southwestern ceramic colorants, and, (c) chemical examination of post-depositional manganese dioxide accretions occurring on archaeological ceramic materials. They evaluate the value of data produced using effectiveness of non-destructive portable XRF analysis for the interpretation of archaeological materials. The case studies provide a template for the development of conservation science research, predicated on object preservation, which produce meaningful data for the interpretation and conservation of the analyzed archaeological artifacts. Portable XRF provides useful data that is used to successfully interpret archaeological materials through (a) classification of metal alloys that can be related to published coin data, (b) identification of ceramic colorants and production technologies, and, (c) characterization of post-depositional product composition when used with established visual typologies.

The interaction of different formulations of the antifungal drug amphotericin B (AmB) with model cell membranes was studied and new techniques of measuring this interaction using electrochemical and/or spectroscopic methods were developed. Two model cell membrane systems were used: sterol-free lipid monolayers adsorbed to a Hg electrode and sterol-free or sterol-containing floating lipid monolayers on a Langmuir trough. Electrochemical control over the adsorbed monolayer allowed the defectiveness of the layer to be varied and the interaction of AmB with both well-ordered and defective monolayers characterized. Measurements of monolayer capacitance and permeability were used to indicate the nature of the interaction. Capacitance provides a measure of the lipid organization, while permeability was measured via electro reduction of thallium (I)cation. The three AmB formulations and two control samples were examined and showed different interaction behaviour. The disruption of lipid order and permeabilization induced by the two commercial formulations correlated generally with in vivo studies of their toxicity. An experimental and possibly less toxic AmB formulation made monolayer significantly more permeable. In situ fluorescence microscopy of the monolayer on Hg was carried out after introducing a low concentration of fluorophore into the layer. Fluorescence intensity as a function of electrode potential was measured and was used to characterize the lipid on Hg model membrane system before we attempted to measure AmB's influence on the fluorescence. The fluorescence excitation and emission spectra of AmB itself were measured ex situ for two of the formulations. Using added surfactant to control AmB aggregation state, the relationship between AmB aggregation and its fluorescence properties was examined. We discovered AmB to have unusual dual fluorescence properties, the extent of which differed between formulations. We measured AmB's fluorescence in situ as the drug interacted with floating lipid monolayers on the Langmuir trough. Both the variation in fluorescence during compression of a mixed AmB/lipid monolayer and penetration of AmB into a phospholipid monolayer were measured. This experimental setup was configured to collect fluorescence only from AmB at the monolayer, and not from AmB in bulk solution. Fluorescence excitation was made using a laser diode extracted from a consumer electronics device.

Le projet de recherche de cette thèse consiste à investiguer les mécanismes d'inhibition du transport d'électrons photosynthétiques induite par les xénobiotiques qui affectent les réactions photochimiques du photosystème II (PSII). Des méthodes fluorimétriques et biochimiques ont été utilisées pour étudier les effets de xénobiotiques sur les réactions photochimiques et le transport des électrons photosynthétiques. Le changement des différents paramètres de fluorescence chlorophyllienne, indiquant la dissipation de l'énergie via le transport d'électrons ou via les voies non photochimiques, a été utilisé comme indicateur de toxicité de xénobiotiques. L'induction du stress oxydatif a été analysée par l'investigation de la teneur en espèces réactives de l'oxygène. Nous avons utilisé les plantes Lemna gibba et Lactuca sativa ainsi que l'algue Chlamydomonas reinhhardtii comme matériel pour les expériences. Dans la partie concernant l'étude des mécanismes de toxicité du dichromate chez L. gibba, nous avons démontré que ce xénobiotique inhibe le transport d'électrons du PSII et induit la dissipation d'énergie sous forme non photochimique. Sous ces conditions, une inhibition de l'activité photochimique du PSII et une diminution de la proportion des centres réactionnels en état fonctionnel ont été obtenues. Le changement du rendement et de la cinétique rapide de fluorescence montre que le dichromate induit une inhibition du complexe de la photolyse de l'eau et de la réduction de l'accepteur primaire d'électrons (QA). Ces effets ont été associés à l'altération de la protéine Dl du centre réactionnel du PSII, ainsi que la protéine 33 kDa constituant le complexe de photolyse de l'eau. L'induction d'un stress oxydatif a été démontrée par l'augmentation de la teneur en espèces réactives de l'oxygène chez les plantes exposées au dichromate. La diminution de la proportion des PSII en état fonctionnel et la formation des espèces réactives de l'oxygène induites par le dichromate ont été corrélées avec le changement des polypeptides Dl. On a pu établir que l'altération fonctionnelle de l'activité du PSII par le dichromate est étroitement associée au changement structurel se produisant dans le complexe PSII. Nous avons également investigué les effets toxiques du dichromate sur l'activité du PSII chez des algues ayant une faible capacité de dissipation de l'énergie sous forme non photochimique. On a utilisé deux algues mutantes de C. reinhardtii qui ont une altération de la biosynthèse de pigments xanthophylles: le premier mutant, npq1, est déficient en zéaxanthine et le deuxième, npq2, est déficient en violaxanthine et accumule la zéaxanthine. On a étudié l'effet du dichromate sur l'activité du PSII sous des conditions lumineuses favorisant la photoinhibition. Après exposition au dichromate, ces algues ont été illuminées par différentes intensités lumineuses pour mesurer le changement du rendement photochimique opérationnel du PSII et la dissipation de l'énergie sous forme non photochimique. L'exposition aux dichromate des mutants npq1 et npq2 a induit une inhibition de transport d'électrons de PSII à faible intensité lumineuse, indiquant une accélération de la photoinhibition par le dichromate. Lorsque ces mutants d'algues ont été exposés aux dichromate et à une intensité lumineuse élevée, la dissipation d'énergie sous forme non photochimique, impliquant des processus non régulateurs, est devenue majoritaire, atteignant la valeur de 80 %. Par conséquent, les mutants d'algues npq1 et npq2 ayant le cycle de xanthophylles non fonctionnel étaient plus sensibles aux effets toxiques du dichromate. Ces résultats ont permis l'élaboration d'un modèle des effets du dichromate sur l'activité du PSII chez les algues ayant un cycle de xanthophylles non fonctionnel. Dans la troisième partie, on a analysé les effets toxiques du trinitrotoluène sur le changement des paramètres de fluorescence utilisés comme indicateurs de toxicité sur la photosynthèse et l'état physiologique de Lactuca sativa. L'activité photosynthétique a été mesurée en utilisant la cinétique rapide de la fluorescence chlorophyllienne et la fluorimétrie modulée. Nos résultats ont montré une diminution de la fluorescence variable provoquée par la désactivation des centres réactionnels PSII. En plus de cet effet, les paramètres de fluorescence indiquant l'état de réduction du PSII et la dissipation d'énergie sous forme non photochimique ont été les indicateurs les plus sensibles de la toxicité du trinitrotoluène. Ces paramètres ont montré une dépendance entre l'inhibition de l'activité du PSII et la concentration du trinitrotoluène. Le changement des paramètres photosynthétiques de fluorescence était une indication fiable de l'état physiologique des plantes exposées à ce polluant. Nous avons proposé que l'état de réduction du PSII et la dissipation non photochimique de l'énergie représentent des indicateurs utiles dans les bioessais. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Xénobiotiques, Bioessais, Fluorescence chlorophyllienne, Réactions photochimiques, Photosystème II, Chrome hexavalent, Trinitrotoluène, Fluorométrie PAM et PEA.

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The carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a human receptor that facilitates adhesion with neighbouring cells, as well as with certain pathogens. CEACAM1 at the cell surface exists as a mixture of monomers and dimers in a heterogeneous distribution that is thought to regulate the balance of its functions, including those associated with pathogen binding. We used live cell fluorescence and homogeneous Förster resonance energy transfer (homo-FRET) microscopy on a combined total internal reflection fluorescence polarization (TIRFPM) confocal microscopy platform to investigate the distribution, dynamics, and monomer-dimer equilibrium of CEACAM1-4L-EYFP on live cells that were parachuted onto surfaces coated with CEACAM1-binding Neisseria gonorrhoea. Both CEACAM1-4L-EYFP and a monomeric mutant form of the receptor are rapidly recruited to bacteria and lead to downstream effector recruitment. Homo-FRET data indicate that wild-type CEACAM1-4L-EYFP was predominantly monomeric at bacterial contact sites. Preferential monomeric binding during bacterial adhesion controls the infection process.

Nanoplasmonics has opened up the gates for numerous innovations. Recent studies showed that metal nanoparticles, when introduced into the solar cells and organic light emitting diodes, would greatly enhance their efficiencies. Though these advances are promising, they require a tool for investigating the interactions occuring at the microscopic level to further optimize their performance. In that context, we are interested in understanding the energy transfer mechanism between molecules in the vicinity of metal nanoparticle. Time-resolved fluorescence intensity and anisotropy experiments on single and clusters of Silver-Silica core-shell nanoparticles coated with Rhodamine B(RB) dye molecules, (Ag-SiO2-RB) were performed. We witnessed the signature of the interaction between RB molecules and metal nanoclusters in the form of the enhanced fluorescence intensity decay rates. The fluorescence lifetime of RB in the vicinity of the nanoparticles was (600 +/- 100) ps, as compared to (2.4+/-0.3)ns in the absence of nanoparticle. While the anisotropy of RB molecules in the absence of nanoparticle has remained almost constant(0.075+/-0.029) over long times; anisotropy in the presence of particles showed wide range of values immediately after excitation. Surprisingly high anisotropy values, at about 10 ns after excitation, were observed with a mean of about (0.145+/-0.025). We interpret the high and low initial anisotropies of the clusters, relative to the case of RB alone, to be due to the interaction of dye molecules with collective plasmon modes of the clusters. / Thesis (Master, Physics, Engineering Physics and Astronomy) -- Queen's University, 2011-06-30 23:29:38.658