Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [202]-225).

Thesis (Ph. D.)--University of Nevada, Reno, 2008. / "December of 2008." Includes bibliographical references (leaves 195-197). Online version available on the World Wide Web.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Rebecca Richards-Kortum. Vita. Includes bibliographical references. Also available from UMI.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 188-199). Available also in a digital version from Dissertation Abstracts.

Thesis (M.S.)--Brigham Young University. Dept. of Chemistry and Biochemistry, 2008. / Includes bibliographical references.

Thesis (M.E.E.)--University of Delaware, 2005. / Principal faculty advisor: Balaji Panchapakesan, Dept. of Electrical & Computer Engineering. Includes bibliographical references.

The advent and establishment of systems biology has cemented the idea that real understanding of biological systems requires quantitative models, that can be integrated to provide a complete description of the cell and its complexities. At the same time, synthetic biology attempts to leverage such quantitative models to efficiently engineer novel, predictable behaviour in biological systems. Together, these advances indicate that the future understanding and application of biology will require the ability to create, parameterise and discriminate between quantitative models of cellular processes in a rigorous and statistically sound manner. In this thesis we take the regulation of GAL1 expression in Saccharomyces cerevisiae as a test case and look at all aspects of this process: from reporter selection to data acquisition and statistical analysis. In chapter B we will discuss optimal fluorescent reporter selection and construction for investigating transcriptional dynamics, as well as procedures for quantifying and correcting the various sources of error in our microscope system. In chapter 3 we will describe software developed to analyse fluorescent microscopy images and convert them to gene expression data. A number of iterations of the software are tested against manually curated data sets, and the measurement error produced by its imperfections is quantified and discussed. In chapter 4 a method, based on fluctuations in photobleaching, is developed for quantifying both measurement error and the relationship between protein concentration and measured fluorescence. The method is refined and its efficacy discussed. In the last section I make a preliminary application of these methods to investigating the regulatory effect of the GAL10-lncRNA. Interesting phenomena are observed and further investigated using two new strains: genetic variants expressing a fluorescent reporter from the GAL1 promoter, one harbouring a wild type GAL1 promoter and one in which the binding site for the Gal10 noncoding RNA has been removed. The methods developed throughout the thesis are applied and the data analysed. A heterogeneous response, distinguishable between the two strains, is observed and related to cell-to-cell variations in growth rate.

The development of facile and reliable methods to image and detect important biomolecules has drawn considerable attention owing to their potential applications in clinical, bioanalytical and forensic analysis. One-photon microscopy (OPM) has traditionally been used in cell biology research. However, probes based on OPM are associated with shortcomings including photobleaching, cell damage, and intracellular autofluorescence interference. Many researchers are seeking better tools to overcome these obstacles. Two-photon microscopy (TPM) is a convenient and powerful tool to explore the intracellular environment and provides the opportunity to overcome the abovementioned obstacles. Probes based on TPM have become important for bioimaging and sensing because of their low photodamage, reduced fluorescence interference, and better tissue penetration depth. With the development of fluorescence molecules in recent decades, a wide range of organic fluorescence probes based on TPM has been rapidly developed and used in biomedicine and bioimaging. Cyanine dye, one of the classic synthetic dyes, continues to be used in many fields, especially in bio-related applications, owing to its ability to interact with biomolecules through non-covalent and electrostatic bonds. Based on cyanine models, we designed a series of structural modifications of cyanine fluorophores used as two-photon (TP) probes to detect and image the intracellular environment in which new cyanine compounds, namely SLSO3, SLCOOH-Pr, F-SLOH, SLOH, Me-SLM, SLE, SAM, SAOH, SLG, F-SPG, SLOH-Pr, SLAD, F-SLAD, Me-SLG, SLNA, SLAD-Pr, SLCOOH, SLAce, SLM, SPC, SIOH, PSIOH, DMA-SLOH, DBA-SLOH, DPA-SLM, GBPM, HBBM, HBLM, SBM, SIBM, SIM, PLOH, and PTM, was successfully synthesized. All of these newly designed compounds were characterized with 1H NMR, 13C NMR, and HRMS and found to show good agreement with the desired structures. To our surprise, some of the novel cyanine molecules were also able to detect and image amyloid-β (Aβ) peptide species and showed excellent biological properties including neuroprotective effects against the cytotoxicity induced by different forms of Aβ species, blood-brain barrier permeability, and high in vivo stability. The photophysical and biological properties of these newly synthesized compounds included optical properties such as UV-vis absorption, emission, fluorescence quantum yield in different solvents, dissociation constant determined by fluorescence titration, and circular dichroism spectroscopy, cytotoxicity assay, neuroprotection, and inhibition of Aβ aggregation were investigated.

Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / This dissertation describes the application of fluorescence microscopy techniques to investigations of mass transport phenomena in self-assembled nanomaterials. The microscopic morphologies of the materials and the mass-transport dynamics of probe molecules dispersed within them were assessed with high temporal and spatial resolution by single molecule imaging and spectroscopic methods. Three distinct sets of experiments were performed in completing the work for this dissertation. In the first study, single molecule imaging was employed to explore the interactions and field-induced migration of double-stranded DNA (ds-DNA) molecules with nanostructured Pluronic F127 gels. While DNA interactions with nanostructured gels have been explored in the past, none had apparently looked at these interactions in gels comprising hexagonally ordered arrays of cylindrical micelles. Therefore, these studies focused on materials DNA dispersed in flow aligned hexagonal F127. DNA molecules were found to be strongly confined in the hexagonal mesophase structures from their elongation, alignment, and exclusively occurred electrophoretic migration in the direction parallel to the cylinder long axis. These observations will lead to a better understanding of macromolecular interactions with nanostructured gels like those now being investigated for use in drug delivery and chemical separations. In the second study, imaging-fluorescence correlation spectroscopy (imaging-FCS) was used to study the rate and mechanism of sulforhodamine B (SRB) dye within novel bolaamphiphile-based self-assembled nanotubes. These nanotubes were only recently developed and their mass transport properties remain largely unexplored. The nanotubes employed here are unique because they incorporate amine groups and glucose groups on their inner and outer surfaces, respectively. Wide-field fluorescence video microscopy was first applied to locate and image dye-doped nanotubes dispersed on a glass surface. Imaging-FCS was employed as it allows for the dynamics to be recorded simultaneously from a large sample region, thus the SRB mass transport within nanotubes can be spatially resolved. The coulombic interactions between cationic ammonium ions on the inner nanotube surface and the anionic SRB molecules was shown to play a critical role in governing dye dynamics under varied pH and ionic strength conditions. Mass transport of SRB within the nanotubes is concluded to occur by a desorption-mediated Fickian diffusion mechanism. In the third set of experiments, solvatochromic dye molecules were employed in novel imaging-FCS studies of the role played by partitioning in governing mass transport phenomena within the same organic nanotubes used above. Two forms of the solvatochromic dye Nile Red (NR) were employed: the commercial hydrophobic form of NR, and a more polar derivative 2-hydroxybenzophenoxazinone (named NR-OH). The partitioning of dye molecules within the nanotubes was investigated assessing the diffusion rate for each dye. The preliminary results suggested NR and NR-OH preferentially partitioned into the tube walls and the ethanol phase filling the tubes, respectively. The diffusion coefficient data indicated NR-OH diffused faster than NR, consistent with the presence of NR-OH in a relatively less viscous environment (e.g., the ethanol phase filling the tubes). The results of these studies afford information essential to the use of organic nanotubes in controlled drug release and possibly in catalysis applications.

The diffraction limit restricts conventional light microscopes to approximately 250 nm laterally and 500 nm axially, these limits being first proposed by Abbe in 1873. Despite this, optical microscopes have found many applications in biological research and single cells that are 10 - 100 um in size. Furthermore by coupling the non-invasive nature of a light microscope with highly sensitive fluorescent probes, fluorescence microscopy has also become a standard imaging technique. Recent advances in fluorescence microscopy now provide a number of methods to circumvent the Abbe diffraction limit, with many techniques becoming prevalent over the last 10 years including direct Stochastic Optical Reconstruction Microscopy (dSTORM). A dSTORM system has been constructed and calibrated using a commercially available inverted florescence microscope and total internal reflection florescence (TIRF) imaging. dSTORM relies on the ability to switch sparse subsets of fluorophores and temporally separate them. Provided the spatial separation is sufficient between any member of a subset, the average error with which the emission can be localized is much less than size of the emission profile itself. The underlying mechanism for this switching is detailed based on the principle of photoinduced electron transfer (PET). The switching characteristics of the common florescent dye Alexa Fluor 568 are investigated and shown to be controlled by a number of factors including the excitation intensity and concentration of the primary thiol cysteamine beta-MEA. A number of parameters are defined, including the dye switching rate, for a given set of physical parameters. U2OS cells are labelled for the microtubule protein Tubulin using immunofluorescent labelling strategies. A direct comparison is made between diffraction limited TIRF images and dSTORM reconstructed images, with an average width for microtubules determined to (58.2 ± 8.1) nm. Further measurements are made by labelling the Rab5 effector Early Endosome Antigen 1 (EEA1). From this the aspect ratio for early endosomes is determined to be 1.68 ± 0.7 with an average radius of (45.8 ± 18.8) nm. The point spatial distribution of EEA1 is investigated by using the linearised form of Ripley's K-function H(r) and the null hypothesis of complete spatial randomness tested. EEA1 is shown to cluster at radius of 58.7 nm on individual endosomes, thought to be due to the well defined binding domains present on early endosomes for EEA1. Further evidence suggests that clustering is also exhibited at another maximum of approximately 500 nm when looking at an ensemble of EEA1 and early endosomes.