Objective: To assess biofilm proliferation within the sealer-dentin interfaces of methacrylate resin-based sealers, self-etch (SE) and total-etch (TE), and an epoxy resin-based sealer (EP). Methods: Standardized human root specimens were filled with the test materials and were aged for 1 week, 1, 3 or 6 months in saline (n=3/group). Monoclonal biofilms of Enterococcus faecalis were grown on the specimens for 7 days in continuous media reactor. The extent of biofilm proliferation of E. faecalis within the sealer-dentin interface for each material at each incubation period was assessed using fluorescence microscopy of dihydroethidium-stained specimens. Results: TE had less biofilm proliferation than EP and SE (p<0.01). Deeper biofilm proliferation was detected in SE and EP specimens aged for 1 and 3 months than those aged for 1 week or 6 months (p<0.05). Conclusion: Self-etch and epoxy resin-based sealers were more susceptible to interfacial biofilm proliferation than total-etch system at shorter incubation periods.

With the emergence of new imaging techniques, researchers are always eager to push the boundaries by examining objects either smaller or further away than what was previously possible. The development of image analysis techniques has greatly helped to introduce objectivity and coherence in measurements and decision making. It has become an essential tool for facilitating both large-scale quantitative studies and qualitative research. In this Thesis, methods were developed for analysis of low-resolution (in respect to the size of the imaged objects) three-dimensional (3D) images with low signal-to-noise ratios (SNR) applied to images from cryo-electron tomography (cryo-ET) and fluorescence microscopy (FM). The main focus is on methods of low complexity, that take into account both grey-level and shape information, to facilitate large-scale studies. Methods were developed to localise and represent complex macromolecules in images from cryo-ET. The methods were applied to Immunoglobulin G (IgG) antibodies and MET proteins. The low resolution and low SNR required that grey-level information was utilised to create fuzzy representations of the macromolecules. To extract structural properties, a method was developed to use grey-level-based distance measures to facilitate decomposition of the fuzzy representations into sub-domains. The structural properties of the MET protein were analysed by developing a analytical curve representation of its stalk. To facilitate large-scale analysis of structural properties of nerve cells, a method for tracing neurites in FM images using local path-finding was developed. Both theoretical and implementational details of computationally heavy approaches were examined to keep the time complexity low in the developed methods. Grey-weighted distance definitions and various aspects of their implementations were examined in detail to form guidelines on which definition to use in which setting and which implementation is the fastest. Heuristics were developed to speed up computations when calculating grey-weighted distances between two points. The methods were evaluated on both real and synthetic data and the results show that the methods provide a step towards facilitating large-scale studies of images from both cryo-ET and FM.

The pericellular matrix (PCM) is a voluminous polymer network adhered to and surrounding many different types of mammalian cells, and which extends out into the environment outside the cell for distances ranging up to twenty microns. It is comprised of very long flexible polymers (hyaluronan) which are tethered to the cell surface and which have binding sites for large, highly charged bottle brush proteoglycans (aggrecan). The PCM plays an important role in many cell functions such as cell proliferation, cell adhesion, cell migration, and cancer development, however the precise way it influences these processes remains unclear. Three original biophysical tools are developed in this thesis in order to study the PCM: the quantitative particle exclusion assay (qPEA), optical force probe assay (OFPA), and exogenous fluorescent aggrecan mapping assays. These tools are used to measure the polymeric and biophysical properties of the matrix in order to make further advancements in the understanding the PCMs role in adhesion, transport to and from the cell surface, its purported function as a chemical micro-reservoir, as well as basic studies on the kinetics of its formation, turnover and maintenance. The qPEAs measure the penetration and distribution of sub-micron particles after they diffuse into the cell coat, where their distribution maps the interior structure of the PCM. The qPEA assays reveal that the PCM acts a sieve, separating incoming particles by their size, preventing micron sized particles from entering the PCM while allowing sub 100 nm particles to pass to the cell surface. The OFPA uses an optically-trapped bead to study the force response of the matrix as it encounters the probe. The assay not only reveals new details about the PCM such as the fact that it is larger than initially thought, having a two layer structure, but when combined with a polymer physics model which relates the observed equilibrium forces to an existing osmotic pressure gradient within the PCM, the OFPA studies produce the first discovery and measurement of the correlation length distribution in the cell coat. The OFPA and qPEA assays are also performed on cells modified with exogenous aggrecan, resulting in a model for possible proteoglycan mediated cell coat transformations. The fluorescent exogenous aggrecan assays measure the dynamics of the exogenous aggrecan binding to and releasing from the coat, revealing that the PCM can be rapidly modified by a changing environment, and quantitatively measure how the exogenous aggrecan modifies the existing PCM. Together, these assays provide an unprecedented look into the interior structure of the PCM, and the mechanisms responsible both for this structure and its modification.

The objective of this thesis is to present the fabrication of a multiphoton microscope and the underlying theory responsible for its proper functioning. A basic introduction to nonlinear optics will give the necessary knowledge to the reader to understand the optical effects involved. Femtosecond laser pulses will be presented and characterized. Each part of the microscope, their integration and the design of the microscope will be discussed. The basic concepts of laser scanning microscopy are also required to explain the design of the scanning optics. Fast scanning problems and their solutions are also briefly viewed. As a working proof, the first images taken with the microscope will be presented. Fluorescent beads, rat tail tendon, gold nanoparticles and pollen grain images using various nonlinear effects will be shown and discussed.

Toxic elements are present in polluted water from mines, industrial outlets, storm water etc. Wetland plants take up toxic elements and increase the pH of the medium. In this thesis was investigated how the shoots of submerged plants and roots of emergent plants affected the pH of the surrounding water in the presence of free toxic ions. The aim was to clarify the mechanisms by which these plants change the surrounding water pH in the presence of toxic ions. The influence of Elodea canadensis shoots on the pH of the surrounding water was studied in the presence of cadmium (Cd) at low initial pH (4-5). The involvement of photosynthetic activity in the pH changes was investigated in the presence and absence of Cd. The cytosolic, vacuolar and apoplasmic pH changes as well as cytosolic Cd changes in E. canadensis were monitored. The influence of Eriophorum angustifolium roots on the pH of the surrounding water was investigated in the presence of a combination of Cd, copper, lead, zinc and arsenic at low initial pH (3.5). Eriophorum angustifolium root exudates were analyzed for organic acids. Elodea canadensis shoots increased the pH of the surrounding water, an effect more pronounced with increasing Cd levels and/or increasing plant biomass and increased plant Cd uptake. The pH increase in the presence of free Cd ions was not due to photosynthesis or proton uptake across the plasmalemma or tonoplast. Cadmium was initially sequestered in the apoplasm of E. canadensis and caused its acidosis. Eriophorum angustifolium roots increased the surrounding water pH and this effect was enhanced in the presence of arsenic and metals. This pH increase was found to depend partly on the release of oxalic acid, formic acid and succinic acid by the plants. In conclusion, E. canadensis shoots and E. angustifolium roots were found to increase the low initial pH of the surrounding water. The pH modulation by these species was enhanced by low levels of free toxic ions in the surrounding water. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 3: Submitted. Paper 4: Manuscript.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [96]-100).

Thesis (Ph. D.)--University of Western Australia, 2007.

Thesis (Ph. D. in Physics)--Vanderbilt University, May 2006. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Metal ions play essential roles in biological functions including catalysis, protein stability, DNA-protein interactions and cell signaling. It is estimated that 30% of proteins utilize metals in some fashion. Additionally, methods by which metal ions can be visualized have been utilized to study metal concentrations and localizations in relation to disease. Understanding the roles metals play in biological systems has great potential in medicine and technology. Chapters 1 and 2 of this dissertation analyzes the structure and function of the Mn-dependent enzyme oxalate decarboxylase (OxDc) and Chapter 2 presents a bioinformatic analysis of the cupin superfamily that provides the structural scaffold of the decarboxylase. The X-ray crystal structure of the W132F variant was determined and utilized together with EPR data to develop a computational approach to determining EPR spectra of the enzyme’s two metal-binding centers. Furthermore, a variant in which the catalytic Glu162 was deleted revealed the binding mode of oxalate, the first substrate-bound structure of OxDc. OxDc is a member of the cupin superfamily, which comprises a wide variety of proteins and enzymes with great sequence and functional diversity. A bioinformatics analysis of the superfamily was performed to analyze how sequence variation determines function and metal utilization. Chapters 3 and 4 discuss the expansion of lanthanide-binding tags (LBTs) to in cellulo studies. Lanthanide-binding tags are short sequences of amino acids that have high affinity and selectivity for lanthanide ions. An EGF-LBT construct used to quantify EGF receptors on the surface of A431 and HeLa cells. The results from the LBT quantification are consistent with previous studies of EGFR receptors in these cell types, validating the use of this method for future studies. The potential of using LBTs for X-ray fluorescence microscopy (XFM) was also investigated. LBT-labeled constructs were utilized to investigate if membrane bound as well as cytosolic LBT-containing proteins could be visualized and localized to their cell compartments via XFM; both membrane-localized and cytosolic proteins were successfully visualized. With the high resolution (< 150 Å) obtainable with new synchrotron beamline configurations LBTs could be used to study nanoscale biological structures in their near-native state.