Autofluorescence spectroscopy can improve the early detections of oral cancer. Biochemical and structural changes associated with dysplastic progression alter the optical properties of oral mucosa and cause diagnostically significant differences in spectra from normal and neoplastic sites. This dissertation describes experimental and modeling studies aimed at revealing biological reasons for the diagnostically significant differences observed in depth-resolved fluorescence spectra from normal and neoplastic oral mucosa. An experimental approach, based on high-resolution fluorescence imaging, is used to study the autofluorescence patterns of oral tissue. At UV excitation, most of the epithelial autofluorescence originates from cells occupying the basal and intermediate layers, while stromal signal originates from collagen and elastin crosslinks. With dysplasia, epithelial autofluorescence increases, while autofluorescence from subepithelial stroma drops significantly. Benign lesions also display a drop in autofluorescence from subepithelial stroma, but have different epithelium fluorescence patterns compared to dysplasia. Optical probes that measure mostly stromal fluorescence, may reveal a similar loss of fluorescence intensity and thus fail to distinguish benign inflammation from dysplasia. These results emphasize the importance of using probes with enhanced detection of epithelial fluorescence for improved diagnosis of different types of oral lesions. The second part of this work presents a Monte Carlo model that predicts fluorescence spectra of oral mucosa obtained using a depth-selective probe as a function of tissue optical properties. A model sensitivity analysis determines how variations in optical parameters associated with neoplastic development influence the intensity and shape of spectra, and elucidates the biological basis for differences in spectra from normal and premalignant oral mucosa. Spectra of oral mucosa collected with the depthselective probe, are affected by variations in epithelial optical properties and to a lesser extent by changes in superficial stromal parameters, but not by changes in the optical properties of deeper stroma. Changes in parameters associated with dysplastic progression lead to a decreased fluorescence intensity and a shift of the spectra to longer emission wavelengths. Decreased fluorescence is due to a drop in detected stromal photons, whereas the shift of spectral shape is attributed to an increased fraction of detected photons arising in the epithelium.

A novel approach of using near infrared region (NIR) dyes is applied to elucidate the binding interaction between human serum albumin (HSA) and mangiferin (MGF). HSA is a blood carrier protein used for drug delivery, while mangiferin is a natural polyphenol found in mangoes that possesses numerous beneficial health properties. The NIR dyes are used as a probe to investigate MGF binding interaction with HSA via monitoring fluorescence of Trp-214 residue. Molecular modeling is used for docking and semi-empirical analysis. The investigation of the binding interaction between Trp-214 and MGF is significant, for it may offer broader pharmacological insight and applications for the polyphenol. Mangiferin in proposed to bind with a 2:1 stoichiometric ratio with HSA to the Trp-214 residue in subdomain IIA and another possible binding site to be determined in future studies. Spectral changes suggest a stabilized protein conformation upon mangiferin binding with the addition of NIR dye E-06 and MHI-06.

Mitochondrial gene expression in trypanosomatid pathogens requires extensive post transcriptional modification called RNA editing. This unique molecular mechanism, catalyzed by a multiprotein complex (the editosome), generates translatable transcripts for essential components of parasite respiratory complex. How editosome proteins are assembled and perform RNA editing is not fully understood. Moreover, previous studies have shown that editosome proteins are essential for parasite survival, which makes editosome as a suitable target for drug discovery. Currently, researchers use radio-labeled based assays to monitor RNA editing process. However, these assays are not suitable for high throughput screening of editosome inhibitors, have low detection limits, and cannot monitor RNA editing in real time. / Therefore, to develop a sensitive high throughput RNA editing assay, we have designed a sensitive hammerhead ribozyme-based fluorescence assay. Ribozyme structure was remodeled by adding or removing uridylate in its conserved catalytic core to make an inactive ribozyme. In the presence of the editosome, inactive ribozyme is edited to an active ribozyme. Consequently, hammerhead ribozyme activity can be measured by cleaving its fluorescently labeled substrate. We have shown that higher sensitivity is achieved using fluorescent based assay than conventional radio-labeled assay. Moreover, we can use this assay for rapid identification and characterization of the editosome inhibitors against RNA editing activities in trypanosomatids.

Characterizing dissolved organic matter (DOM) composition remains a major unresolved problem in aquatic ecology. "Tryptophan-like" dissolved organic matter fluorescence (FDOM) was found to be a much better predictor of heterotrophic bacterial metabolism in 28 Quebec lakes than dissolved organic carbon (DOC), describing 52, 44, 51 and 55% of the variability in bacterial production (BP), bacterioplankton respiration (BR), total bacterial carbon consumption (TBCC), and total plankton community respiration (CR), respectively. In addition, the study provides indirect support for the view that FDOM represents a product of bacterial activity, rather than a bioavailable substrate. This is the first field study to show that fluorescence spectroscopy can be used to characterize an aspect of DOM composition that is related to bacterial metabolism, and provides results that encourage further exploration of the potential uses of DOM fluorescence spectroscopy as a predictive tool.

Radicals are interesting to study because of importance in so many processes such as semiconductor growth or stellar evolution. Laser induced fluorescence (LIF) and wavelength resolved emission spectra of jet cooled HPS, HAsO, AsD2, H2PS, and F2BO have been measured using the pulsed discharge jet technique. Several bands in the à 1A′′ − X̃ 1A′ transition of HPS were observed and assigned with the help of ab initio calculations. The ab initio geometries showed that HPS does not follow Walsh’s predictions for the angle change upon electronic excitation; Walsh predicts an increase in HPS upon excitation while a decrease is calculated. Ab initio Walsh-style orbital angular correlation diagrams for both electronic states show a change in correlation for some orbitals upon electronic excitation, an effect that Walsh did not predict. The à 1A′′ − X̃ 1A′ transitions were measured in HAsO and DAsO for the first time. A molecular geometry was derived for each electronic state from experimental rotational constants. The experimental geometries prove that HAsO also violates Walsh’s rules for the same reason shown in HPS. The à 2A1 – X̃ 2B1 electronic transition of AsD2 and AsHD were measured. Vibrational levels observed in emission were fit to a local mode vibrational Hamiltonian. Using the previously reported rotational constants for AsH2 and those determined for AsD2 in this work, an improved estimate of the excited state geometry was obtained. The discovery of the B̃ 2A′ − X̃ 2A′ band system of H2PS is the first report of this molecule. Both D2PS and HDPS were also observed. Ab initio calculations helped assign the transition. H2PS is one of the few tetra-atomic or larger molecules that violates Kasha’s empirical rule due to the large separation between the B̃ and à states. Finally, laser induced fluorescence spectra of the F2BO radical was observed for the first time. Previous work showed two band systems with only a tentative assignment. The measured LIF spectra confirm the identity of the two band systems as the B̃ 2A1 – X̃ 2B2 and the B̃ 2A1 – à 2B1 transitions showing F2BO also violates Kasha’s rule.

No description available.

We applied single-molecule fluorescence (SMF) methods to probe the properties of individual fluorescent probes, and to characterize the proteins of interest to which these probes were attached. One remarkable advantage of SMF spectroscopy is the ability to investigate heterogeneous subpopulations of the ensemble, which are buried in ensemble averaging in other measurements. Other advantages include the ability to probe the entire dynamic sequences of a single molecule transitioning between different conformational states. For the purpose of having an extended observation of single molecules, while maintaining the native nanoscale surroundings, we developed an improved vesicle preparation method for encapsulating scarce biological samples. SMF investigations revealed that molecules trapped in vesicles exhibit nearly ideal single-emitter behavior, which therefore recommends the vesicle encapsulation for reproducible and reliable SMF studies. Hyperactive Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein contributes significantly to human cancers, such as leukemia and lymphoma. We have proposed a novel therapeutic strategy by designing a cholesterol-based protein membrane anchor (PMA), to tether STAT3 to the cell membrane and thus inhibit unwanted transcription at the cell nucleus. We designed in vitro proof-of-concept experiments by encapsulating STAT3 and PMAs in phospholipid vesicles. The efficiency and the stability of STAT3 anchoring in the lipid membrane were interrogated via quantitative fluorescence imaging and multiparameter SMF spectroscopy. Our in vitro data paved the way for the in vivo demonstration of STAT3 inhibition in live cells, thus demonstrating that PMA-induced protein localization is a conceptually viable therapeutic strategy. The recent discovery of intrinsically disordered proteins (IDPs) highlights important exceptions to the traditional structure-function paradigm. SMF methods are very suited for probing the properties of such highly heterogeneous systems. We studied in detail the effects of electrostatics on the conformational disorder of an IDP protein, Sic1 from yeast, and found that the electrostatic repulsion is a major factor controlling the dimensions of Sic1. Based on our data we also conclude that a rod-like shape seems a better candidate than a random Gaussian chain to describe and predict the behavior of Sic1.

We applied single-molecule fluorescence (SMF) methods to probe the properties of individual fluorescent probes, and to characterize the proteins of interest to which these probes were attached. One remarkable advantage of SMF spectroscopy is the ability to investigate heterogeneous subpopulations of the ensemble, which are buried in ensemble averaging in other measurements. Other advantages include the ability to probe the entire dynamic sequences of a single molecule transitioning between different conformational states. For the purpose of having an extended observation of single molecules, while maintaining the native nanoscale surroundings, we developed an improved vesicle preparation method for encapsulating scarce biological samples. SMF investigations revealed that molecules trapped in vesicles exhibit nearly ideal single-emitter behavior, which therefore recommends the vesicle encapsulation for reproducible and reliable SMF studies. Hyperactive Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein contributes significantly to human cancers, such as leukemia and lymphoma. We have proposed a novel therapeutic strategy by designing a cholesterol-based protein membrane anchor (PMA), to tether STAT3 to the cell membrane and thus inhibit unwanted transcription at the cell nucleus. We designed in vitro proof-of-concept experiments by encapsulating STAT3 and PMAs in phospholipid vesicles. The efficiency and the stability of STAT3 anchoring in the lipid membrane were interrogated via quantitative fluorescence imaging and multiparameter SMF spectroscopy. Our in vitro data paved the way for the in vivo demonstration of STAT3 inhibition in live cells, thus demonstrating that PMA-induced protein localization is a conceptually viable therapeutic strategy. The recent discovery of intrinsically disordered proteins (IDPs) highlights important exceptions to the traditional structure-function paradigm. SMF methods are very suited for probing the properties of such highly heterogeneous systems. We studied in detail the effects of electrostatics on the conformational disorder of an IDP protein, Sic1 from yeast, and found that the electrostatic repulsion is a major factor controlling the dimensions of Sic1. Based on our data we also conclude that a rod-like shape seems a better candidate than a random Gaussian chain to describe and predict the behavior of Sic1.

No description available.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 143-151). Also available for download via the World Wide Web; free to University of Oregon users.