Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 109-127).

A novel optical calorimetry approach is proposed for the dosimetry of therapeutic radiation, based on the optical technique of Digital Holographic Interferometry (DHI). This detector determines the radiation absorbed dose to water by measurement of the refractive index variations arising from radiation induced temperature increases. The output consists of a time series of high resolution, two dimensional images of the spatial distribution of the projected dose map across the water sample. This absorbed dose to water is measured directly, independently of radiation type, dose rate and energy, and without perturbation of the beam. These are key features which make DHI a promising technique for radiation dosimetry. A prototype DHI detector was developed, with the aim of providing proof-of-principle of the approach. The detector consists of an optical laser interferometer based on a lensless Fourier transform digital holography (LFTDH) system, and the associated mathematical reconstruction of the absorbed dose. The conceptual basis was introduced, and a full framework was established for the measurement and analysis of the results. Methods were developed for mathematical correction of the distortions introduced by heat di usion within the system. Pilot studies of the dosimetry of a high dose rate Ir-192 brachytherapy source and a small eld proton beam were conducted in order to investigate the dosimetric potential of the technique. Results were validated against independent models of the expected radiation dose distributions. Initial measurements of absorbed dose demonstrated the ability of the DHI detector to resolve the minuscule temperature changes produced by radiation in water to within experimental uncertainty. Spatial resolution of approximately 0.03 mm/pixel was achieved, and the dose distribution around the brachytherapy source was accurately measured for short irradiation times, to within the experimental uncertainty. The experimental noise for the prototype detector was relatively large and combined with the occurrence of heat di usion, means that the method is predominantly suitable for high dose rate applications. The initial proof-of-principle results con rm that DHI dosimetry is a promising technique, with a range of potential bene ts. Further development of the technique is warranted, to improve on the limitations of the current prototype. A comprehensive analysis of the system was conducted to determine key requirements for future development of the DHI detector to be a useful contribution to the dosimetric toolbox of a range of current and emerging applications. The sources of measurement uncertainty are considered, and methods suggested to mitigate these. Improvement of the signal-to-noise ratio, and further development of the heat transport corrections for high dose gradient regions are key areas of focus highlighted for future development.

Nanoparticles are promising platforms for biomedical applications ranging from diagnostic tools to therapeutic delivery agents. During the course of these applications, nanoparticles are exposed to a complex mixture of extracellular serum proteins that nonspecifically adsorb onto the surface. The resulting protein layer, or protein "corona," creates an interface between nanoparticles and the biological environment. Protecting the nanoparticle surface can reduce protein adsorption, but complete inhibition remains a challenge. As a result, the corona, rather than the nanoparticle itself, mediates the cellular response to the nanoparticle. The following dissertation describes the fundamental characterization of the cellular binding of charged nanoparticles, interactions of protein-nanoparticle complexes with cellular receptors, and the structural and thermodynamic properties of adsorbed corona proteins.

With the emergence of Matrix Assisted Laser Desorption/Ionization-Time-of-flight as a tool for diagnosis of diseases via proteomics, there is an increasing need for greater sensitivity. Analysis of peptides by MALDI-TOF-MS is affected by sample formulation and spotting onto a MALDI target. This dissertation investigates a novel MALDI sample preparation technique, Induction Based Fluidics (IBF), for depositing precise volumes (pL to nL) of samples onto the target. We have seen that while using IBF, the induced electric field accompanying deposition enhances matrix crystallization yielding smaller crystals with more homogeneity, as compared to conventional manual micropipette (MP) depositions. An investigation of the signal-to-noise (S/N) for IBF deposition of tryptic digested Bovine Serum Albumin (BSA) showed a significant improvement in the signal-to-noise ratio for 0.5 and 0.25 pmol/µL BSA sample compared to equivalent MP depositions. The S/N enhancement for IBF and MP depositions of BSA were studied using à-cyano-4-hydroycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) matrices, and CHCA showed better results than DHB . The exciting results obtained by IBF prompted us to probe sample morphology more fully and to relate morphology to the detections level and hopefully, to increase the utility of MALDI-TOF-MS for detection of a larger range of peptides. Morphology results were correlated to sensitivity limits using both dispensing techniques. Because of dissimilar rates of evaporation, different or uneven deposition thickness, or crystal lattice morphology, discontinuous crystallization patterns were observed for MP depositions. However, IBF deposited samples occupied less planar area with uniform distribution of crystals, thereby reducing sample crystal heterogeneity and laborious hunt for a "sweet" or "hot" spot to produce high quality spectra. The application of IBF was extended to the tryptic digested BSA protein using peptide mass fingerprinting. IBF deposition resulted in a larger number of detectable peptides as well as higher sequence coverage as compared to equivalent MP depositions. In last few decades, advanced research and potential applications in the field of microelectronics have spurred interest in the development of reticulated doped polymer films. Bis (ethylenedioxy) tetrathiafulvalene (BEDO-TTF)/Polycarbonate (PC) films were synthesized and characterized for use in hand-held real time explosives sensors, capable of detecting nitro-based compounds (nitroaromatics, nitoamines and nitroesters), which are the main components of Improvised Explosive Devices or IEDs. Reticulated doped polymer films were prepared by exposing solid solutions of BEDO-TTF in PC to iodine to form conductive charge transfer complexes. The resulting films exhibited room temperature conductivities ranging from 6.33-90.4*10-5 S cm-1. The colored iodine complexes in the film were reduced by cyclic voltammetry yielding conductive, colorless, transparent films. Dielectric analysis (DEA) was used to probe relaxations in neat PC and BEDO-TTF/PC showed that BEDO-TTF plasticized the PC and decreased the glass transition temperature. Two secondary relaxations appeared in PC films, whereas the transitions merged in the doped film. DEA also revealed conductivity relaxations above 180°C, which were characterized by the electric modulus formalism and showed that BEDO-TTF increased the alternating current, (AC) conductivity in PC.

Due to strict placement time and strength constraints during the construction of concrete pavement repair slabs, accelerators must be incorporated into the mixture design. Since the most common accelerator, calcium chloride, promotes corrosion of concrete reinforcement, a calcium nitrate-based accelerator was studied as an alternative. To replicate mixtures used in the field, commercial accelerators commonly used in concrete pavement repair slabs were used in the current study. Crack risk of different mixtures was assessed using modeling and cracking frame testing. HIPERPAV modeling was conducted using several measured mixture properties; namely, concrete mechanical properties, strength-based and heat of hydration-based activation energies, hydration parameters using calorimetric studies, and adiabatic temperature rise profiles. Autogenous shrinkage was also measured to assess the effect of moisture consumption on concrete volume contraction. The findings of the current study indicate that the cracking risk associated with calcium nitrate-based accelerator matches the performance of a calcium-chloride based accelerator when placement is conducted during nighttime hours.

The tumor suppressor protein, p53, is mutated or dysregulated in nearly all human cancers(1). The amino terminal domains are essential for transcriptional activation in stressed cells and play a vital role in cell cycle regulation, apoptosis and senescence. The transactivation (TAD) and proline rich domains in this region are dynamic and intrinsically disordered; lacking stable secondary or tertiary structure. This region contains multiple binding sites; arguably, the most significant of these is for p53's negative regulator, the E3 ligase, MDM2. An important, but less understood interaction involving the single stranded DNA binding protein, RPA70A, is hypothesized to be involved in maintaining genome integrity(2-4). Additionally, the amino terminus contains an important single nucleotide polymorphism that has demonstrated different affinity for MDM2 and is of significant biological importance in the induction of apoptosis (5). Isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy were employed to investigate how the thermodynamics and the inherent flexibility of the amino terminus of p53 play a role in complex formation with the MDM2 or RPA70 proteins. Understanding the structure, dynamics, and function of p53 is paramount in the fight against cancer.

Different optical and calorimetric techniques were utilized to gain a better understanding of aggregated macromolecules. This research looked at two different macromolecules: poly(9,9'-dioctylfluorene), a conjugated polymer that forms aggregates in organic solvents; and bovine insulin, which forms amyloid fibrils. Conjugated polymers are of increasing interest due to their thermal stability and ease of solution processing for use in devices. A member of the polyfluorene family, poly(9,9'-dioctylfluorene) (PFO), has been studied due to its blue-emitting spectral properties. However, PFO has been found to form aggregates in solution, which is detected by the presence of a red-shifted absorption peak. This peak is caused when a section of the backbone planarizes forming the [beta]-phase. The [beta]-phase can be removed from the solution upon heating and will not return until the solution is cooled, making it a non-equilibrium process. The dissolution and reformation of the -phase were monitored using absorption spectroscopy and differential scanning calorimetry. Atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) were able to probe the aggregates in films. It is important to understand polymer properties in solution in order to understand film morphology. Amyloid fibrils contribute to over 20 different neurodegenerative diseases, in which cures have yet to be found. The fibrils form when a soluble protein misfolds and self-assembles to form insoluble protein aggregates, and the cause of the fibril formation in vivo has still yet to be determined. Spectroscopy studies have been made possible with the use of fluorescent dyes: thioflavin T (ThT), BTA-2, and Congo red (CR). These dyes bind to amyloid fibrils and exhibit changes in their spectral properties. However, the exact mechanism for the binding of these dyes has only recently been studied. Through the use of calorimetry, the forces involved with binding of ThT and CR to amyloid fibrils can be determined. Absorption and fluorescence spectroscopy techniques were employed to study the spectral properties of these dyes. Polarized NSOM was used to determine the ThT or BTA-2's orientation with an individual fibril. Understanding how these dyes bind to fibrils will enable researchers to use spectroscopy to study the early stages of fibril formation. / text

Apoptosis is a crucial process in multicellular organisms in sculpting them, especially during embryogenesis. In addition, apoptosis is responsible for the clearance of harmful or damaged cells which can otherwise be detrimental to the organism. The Bcl-2 family proteins are key players in the regulation of the intrinsic pathway of the apoptotic machinery. This family consists of three subfamilies with B-cell CLL/lymphoma 2 (Bcl-2) protein itself representing anti-apoptotic members, the Bcl-2-associated X protein (Bax), and pro-apoptotic BH3-only signaling proteins. The interplay between pro- and anti-apoptotic proteins on the mitochondrial membranes is central to the balance between the life and death decision of whether the membrane should be permeabilized or not. The cytosolic Bax protein can upon cellular stress translocate to the mitochondrial membrane where it can either carry out its action of forming homo-oligomers that cause outer membrane permeabilization or be inhibited there by the anti-apoptotic membrane protein Bcl-2. Upon mitochondrial outer membrane permeabilization (MOMP) apoptogenic factors leak out from the intermembrane space (IMS) of the mitochondria, leading to caspase activation and ultimately cell death. A common stress signal initiating apoptosis is an increased formation of reactive oxygen species (ROS in the mitochondria, who can cause oxidative damage to lipid membranes. This membrane damage presumably influences the lipid landscape and the membrane features and hence the interactions of the Bcl-2 family proteins with each other and the mitochondrial outer membrane (MOM). To investigate the significance of membrane oxidation on the behavior of the Bcl-2 family proteins, especially Bax, synthetically produced oxidized phospholipids (OxPls) were incorporated in MOM-mimicking vesicles. Differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD) spectroscopy revealed a major perturbation in membrane organization in the presence of OxPls. These changes in membrane properties increase the affinity of Bax to its target membrane and enable its partial penetration and formation of pores, as fluorescence leakage assays confirmed. However, in the absence of BH3-only proteins these pores are not sufficiently large for the release of apopototic factors such as cytochrome C (CytC). To understand the inhibition of Bax by the full-length Bcl-2 protein, suitable detergent solubilizing conditions were carefully chosen to enable the measurement of their direct binding to each other outside the membrane, by an antimycin A2 fluorescence assay. The observed protein-protein interaction was confirmed by surface plasmon resonance (SPR). An established protocol for the reconstitution of Bcl-2 into stable proteoliposomes now paves the way for structural studies of this key protein, in its membrane environment near physiological conditions; information essential for understanding its function, on a molecular level, and its potential as a cancer drug target.

Polyethylene (PE) is produced in a huge scale globally and has plenty of desirable properties. It is used in coating, packaging, and artificial joint replacements. The growing need for high performance polyethylene led to the development of new catalysts, monomers and polymerizations. The synthesis of polymethylene (equivalent to polyethylene) by living polyhomologation opened the way to well-defined polymethylenes-based polymeric materials with controlled structure, molecular weight and narrow polydispersity. Such model polymers are substantial to study the structure-properties relationships. This research presents a new strategy based on the in situ formation of B-thexyl-silaboracyclic serving as initiating sites for the polyhomologation of dimethylsulfoxonium methylide. Combination with metal-free ring-opening polymerization (ROP) of ɛ-caprolactone (CL) and atom transfer radical polymerization (ATRP) of styrene led to three polymethylene-based 3-miktoarm stars copolymers PCL(PM-OH)2, Br-PCL(PM-OH)2 and PS(PM-OH)2.

For a number of years the NRC group has been working on a laboratory protocol for the analysis of gas hydrate that has been recovered from various natural sites. The expectation was that a comprehensive set of techniques would become available for the general use of hydrate researchers around the world. With the current set of available techniques a good picture of natural gas hydrates can be obtained, although the emerging complexity of the hydrate-mineral system still demands additional work. Here we present a suite of techniques that will take a researcher from preservation techniques to hydrate occurrence, gas/water/sediment ratios, gas and isotope analysis, P-T behaviour, structure, composition, degree of water conversion to hydrate, hydrate homogeneity and decomposition behaviour. As more detailed studies become possible a variety of more subtle features are revealed, for instance the role of minor gas components in hydrate stability, decomposition behaviour and heterogeneity in structure and composition.