Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 156-160).

Bacteria are implicated in endodontic infections. They not only infect the root canal lumen but also invade the dentinal tubules where they may remain untouched by contemporary chemomechanical preparation during root canal therapy. The contentious issue is whether the bacteria within these tubules contribute to secondary infections. Many studies have shown that clinicians fail to completely eradicate them during root canal therapy. At present there are no techniques available to detect the effectiveness of the current chemomechanical treatment regime within dentinal tubules. It is difficult to detect bacteria within the dentinal tubules. Culturing techniques have been used routinely as they are versatile and easy to use. However, they are unable to show the distribution of the bacteria within the dentinal tubules. Scanning electron microscopy, on the other hand, shows detailed surface structure in association with bacteria. Histological examination of root dentine specimens under the light microscope also shows the distribution of bacteria within the specimen but not viability. The dilemma posed by these existing techniques is that the results offer limited information; either demonstrating bacterial viability or bacterial distribution within specimens. No techniques able to show both the viability and the distribution of bacteria within the dentinal tubules have been reported to date. Fluorescent stains, in particular SYTO�9 and propidium iodide (LIVE/DEAD� Baclight[TM] viability kit, Molecular Probes Inc., Eugene, Oregon), have made it possible not only to stain bacteria but to differentiate live and dead bacteria. The combination of these two stains has yet to be applied to dental hard tissue in situ and they provide the basis for this investigation. The aim of this study was to evaluate the potential of the LIVE/DEAD� Baclight[TM] stains in conjuction with confocal laser scanning microscopy in the development of a technique to evaluate the viability and distribution of bacteria within dentinal tubules. This was extended to demonstrate the application of this technique by examining three different means of root canal disinfection both qualitatively and quantitatively. An important aspect of this study was to maintain bacterial viability, as well as to get maximum bacterial invasion into dentinal tubules. Results indicated that when the root canals were instrumented with Protaper� files and then irrigated with sodium hypochlorite (NaOCl) and ethylene diaminetetraacetic acid with cetrimide (EDTAC), there was more bacterial invasion into the dentinal tubules than when the root canals were only irrigated with NaOCl and EDTAC. Daily replenishments of nutrients resulted in deeper bacterial invasion into the dentinal tubules. Bacteria colonized the dentinal tubules up to a distance of 594 � 133 [mu]m from the canal. In the untreated tubules, 96 � 4 % of bacteria remained viable (green-fluorescent), whereas the Amoxicillin-treated tubules contained 94 � 6 % dead (red-fluorescent) bacteria. The calcium hydroxide-treated tubules resulted in 92 � 7 % bacterial death while the laser-treated tubules contained 81 � 12 % dead cells, frequently displaying an inner zone of dead cells surrounded by an outer zone of viable cells. The application of the fluorescent stains combined with confocal microscopy offers a new method for assessing the in vitro efficacy of root canal disinfection regimens.

Thesis (Ph. D.)--Missouri University of Science and Technology, 2009. / Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed April 29, 2009) Includes bibliographical references.

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 (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.