High Mass X-Ray Binary Stars (HMXBs) are stars that contain one early-type main sequence or giant star and one of a black hole, neutron star or white dwarf. HMXBs in the Large Magellanic Cloud (LMC) and the Small Magellanic Cloud (SMC) are instructive to study because both galaxies are metal poor in compari- son to the Milky Way and they are fairly transparent to both optical and X-ray radiation. This allows a more complete study of the whole population, without the biasing effects of gas and dust that occur in our own Galaxy. The objective of this study was to find the periods of HMXBs in the LMC and SMC with known optical counterparts in the dataset acquired by the Robotic Optical Transient Search Ex- periment telescope. Two possible orbital periods were found for the objects XTE J0055-724 and RX J0101.3-7211 of 1724 days and 478 days, respectively. Continued observations are recommended to conrm the two periods.

Graduation date: 1993

CD16 is a physiologically essential Fc and #947; receptor III as either a single- pass transmembrane protein (CD16A) or as a glycosylated phosphatidylinositol (GPI) anchored protein (CD16B) on the surface of immune cells that have been implicated in many autoimmune and immune complex-mediated diseases. Its functions include binding and clearing antibody (IgG) coated foreign pathogens, receptor-mediated phagocytosis, and triggering antibody dependent cellular cytotoxicity. It is well established that these functions depend on protein-protein interaction between CD16 and the Fc domain of IgG. However, the molecular details of CD16-IgG interactions are less well defined, but are essential to developing therapeutic compounds to treat many autoimmune and IC diseases. Stable mammalian cell lines expressing wild-type CD16 isoforms and site-specific mutants, including extracellular soluble fragments of CD16 have been established. Soluble forms of wild type CD16A and these CD16 mutants were expressed in a bacterial pathway in order to amass sufficient quantities for x-ray crystallographic studies. The soluble portions of wild-type CD16A and several site-specific CD16A and CD16B mutants were constructed by PCR amplification and ligation with a pET vector. The proteins were expressed in a prokaryotic pathway, BL21 AI, for 8-10 hours and lysed to obtain inclusion bodies. A hand-held sonicator was used to wash the inclusion bodies, while a Urea solution separated and dissolved the proteins. The target proteins were then refolded by rapid dilution, concentrated with a stir cell, and purified. Wild type sCD16A and four site specific mutants were constructed with good sequencing, while wild type sCD16A, sCD16A F176V, and sCD16A G147D were expressed and refolded to optimal levels. X-ray crystallographic data has been collected from sCD16A F176V as a result of these studies and crystals are currently being grown from wild type sCD16A and sCD16A G147D.

Nitrochemicals are widely used as explosives, biocides and drugs. In addition, 3-nitro-tyrosine and other nitrated protein residues are important markers for many cardiovascular, neurodegenerative, and malignant conditions. Because of the wide presence of the nitrocompounds as toxins, potential nitrogen/carbon sources, and metabolic intermediates, different organisms have evolved to produce enzymes that can biodegrade nitrocompounds. The structural studies of the enzymes, which catalyze the removal of nitro group from nitrochemicals, are of considerable interest for both applied and fundamental reasons. The insights into the reaction mechanism of these enzymes can be used for designing efficient biocatalysts for bioremediation and for developing antibiotics for disease resistant microbes. Nitroalkane oxidase (NAO) produced by

An x-ray fluorescence detection system has been designed by our research group for quantifying the amount of gold nanoparticles presented within the phantom and animals during gold nanoparticle-aided cancer detection and therapy procedures. The primary components of the system consist of a microfocus x-ray source, a Pb beam collimator, and a CdTe photodiode detector. In order to optimize and facilitate future experimental tasks, a Monte Carlo model of the detection system has been created by using the MCNP5 code. Specifically, the model included an x-ray source, a Pb collimator, a CdTe detector, and an acrylic plastic phantom with four cylindrical columns where various materials such as gold nanoparticles, aluminum, etc. can be inserted during the experiments. In this model, 110 kVp x-rays emitted into a 60o cone from the focal spot of the x-ray source were collimated to a circular beam with a diameter of 5 mm. The collimated beam was then delivered to the plastic phantom with and without a gold nanoparticle-containing column. The fluence of scattered and gold fluorescence x-rays from the phantom was scored within the detector's sensitive volume resulting in various photon spectra and compared with the spectra acquired experimentally under the same geometry. The results show that the current Monte Carlo model can produce the results comparable to those from actual experiments and therefore it would serve as a useful tool to optimize and troubleshoot experimental tasks necessary for the development of gold nanoparticle-aided cancer detection and therapy procedures.

Nitrochemicals are currently widely used as solvents, drugs, biocides, fuels and explosives and are consequently widely distributed in the environment. The reductive nitrite elimination from explosive compounds is catalyzed by two FMN-dependent, xenobiotic reductases (XenA or XenB). These genes for these regiospecific enzymes were cloned from Pseudomonas putida and P. fluorescens I-C respectively and isolated from the soil of a contaminated World War II munitions manufacturing plant. These enzymes enable the microbes to fulfill their nitrogen requirements from nitroglycerin by catalyzing the regiospecific, NADPH dependent, reductive denitration of nitroglycerin with differing selectivities. The two enzymes also transform a number of additional nitrocompounds in vitro, e.g. TNT and metronidazole, a leading drug in the treatment of Helicobacter pylori, a causative agent of human ulcers. Single crystals were obtained for XenA and XenB and complete X-ray diffraction datasets have been collected and analyzed to better understand these characteristics. The 1.6 Å resolution structure of XenA reveals a dimer of β/α)₈-TIM barrels, but the 2.3 Å resolution structure for XenB is a monomer. The (β/α)₈-TIM barrel protein fold is the most common fold in the PDB. However, the XenA structure exhibits a unique, C-terminal domain-swapped topology. Thus a portion of each active site is comprised of residues from the neighboring monomer. To probe the reaction cycle, crystal structures of ligand complexes and the reduced enzyme have been refined. For example, our structure of the XenA-metronidazole complex shows that ligands bind parallel to the FMN si-face. Our 1.5 Å resolution structure for reduced XenA reveals an FMN isoalloxazine ring with an angle of ~165° along the N5-N10 axis. We have also generated models of the reduced enzyme-nitroglycerin complexes by molecular dynamics. The results with both XenA and XenB reveal differences in enzyme-ligand hydrogen bonding. These differences correlate remarkably well with the regiospecific differences observed for nitrite elimination from nitroglycerin and reduction of TNT by the two enzymes.

This research developed an on-line measurement systemfor determining the amount of fluorochemicals on carpet fibers using energy-dispersive X-ray fluorescence (EDXRF).This system is designed as a complementary tool to an existingchemical burn test certified by the American Association ofTextile Chemists and Colorists (AATCC), which is performed off-line on randomly selected carpet samples and time consuming.This research reviewed XRF principles and determined parameters that affect XRF spectra such as measurement time, measurement number, X-ray tube voltage, X-ray tube current, primary beam filter, and carpet characteristics. For this application, XRF calibrations must be performed for carpets of all styles and types. However, preparing actual carpet calibration samples is expensive. This research introduced a methodology to synthetically generate reference spectra using XRF spectra from standard fluorochemical stock solution samples and from base carpet samples for each carpet type to be tested. Thus, actual, physical standards are not required for each carpet type or style. This study showed that the synthetically generated XRF spectra alone were not always sufficient to guarantee the confidence interval required by the certified AATCC test. Thus, it is recommended that for on-line implementation, burn test results should be used to create a historical data base for each carpet type to reduce margin of error for calibrations generated from the synthetic spectra.

Bi-annual report on the activities at the ROssendorf BeamLine (ROBL) at the ESRF in Grenoble. The report contains selected contributions on actual research topics, a list of all scheduled experiments, and short experimental reports.

X-ray crystallography is a form of microscopy that allows the three-dimensional arrangement of atoms belonging to molecules within crystals to be determined. In this method, a crystal is illuminated with a beam of X-rays and the diffracted amplitudes resulting from the illumination are measured and computationally processed to enable the electron density of the unit molecule, or the unit cell, constituting the crystal to be calculated. The recent development of the X-ray free-electron laser (XFEL) provides new routes for determining molecular structures via its ability to generate intense but brief X-ray pulses. These new instruments enable diffraction measurements to be obtained from crystals that have a small number of unit cells, referred to as nanocrystals, and molecular structure determination via this technique is known as serial femtosecond nanocrystallography (SFX). This thesis is concerned with the characterisation of diffraction data obtained from SFX experiments and the techniques for reconstructing the electron density of the molecule from such data. The noise characteristics of diffraction measurements from nanocrystals is developed. Methods for directly inverting nanocrystal diffraction to obtain the electron density of the molecule are analysed and an approach to ameliorate the effect of noise is proposed and evaluated by simulation. A model for diffraction by nanocrystals that include the effects of different unit cell arrangements and incomplete unit cells on the crystal surface is also developed and explored by simulation. The diffraction by finite crystals is shown to be equal to the incoherent average over a set of unit cells that contain different molecular arrangements related to the symmetry of the crystal at hand. The problem of image reconstruction under this circumstance is investigated. The more general problem of reconstructing multiple, unrelated, objects from their averaged diffraction is also explored and uniqueness properties along with reconstruction algorithms developed. The problem of reconstructing multiple, related, unit cells is studied and preliminary results are obtained. These results show that iterative phase retrieval algorithms can in principle be adapted to reconstruct the electron density of a crystalline specimen from the data obtained in SFX and the retrieval of phases from the diffracted intensity averaged over multiple objects is feasible.

This work describes the synthesis of dendrimer-encapsulated nanoparticles (DENs) and the expansion of the characterization ability for these materials. The dendrimer-template method for the synthesis of nanoparticles allows precise control over the size, composition and structure of nanoparticles in the 40-250 atom range. In this size regime, the surface structure of the nanoparticles dominates their catalytic properties. The long term goal of this research is to correlate the structure of these nanoparticles to their catalytic activity, improving the ability to predict superior catalysts a priori. As a prerequisite for this analysis, the precise structure of the catalytically active nanoparticle must be determined. Characterization of nanoparticles in the 1-2 nm region is significantly more difficult than more commonly used nanoparticles of 3-5 nm diameter or larger. Typical characterization of these nanoparticles involves UV-vis spectroscopy for Mie absorbance and transmission electron microscopy for size analysis. This work involves the use of extended X-ray absorption-fine structure (EXAFS) to determine the local structure of the nanoparticles. For monometallic Pt DENs, EXAFS was combined with UV-vis, TEM, X-ray photoelectron spectroscopy (XPS) and electrochemistry to determine that the Pt system is not simply nanoparticles but a more complex, bimodal state. EXAFS has also been used to differentiate between different bimetallic structures. For PdAu DENs, there are two synthetic methods used. When both metals are reduced simultaneously, the resulting nanoparticles have a quasi-random alloy structure. These nanoparticles were then extracted from the dendrimer into an organic solvent by use of alkanethiols. The extraction process changed the alloy structure into Au-core/Pd-shell. When Pd and Au were reduced in sequence, the DENs were formed as a Au-core/Pd-shell material, regardless of the order of the reduction of the metals. The Au-core/Pd-shell structure was also present after extraction. In addition to structural analysis to determine the result of different synthetic methods, EXAFS was also used in situ to measure the structure of Pt DENs during the oxidation of absorbed CO. These in situ measurements are important for determining the structure of the actual catalyst rather than the precursor nanoparticle. In this case, the Pt DENs changed from a bimodal distribution into fully reduced nanoparticles by the application of a reducing potential. The binding of CO to the Pt DENs and subsequent oxidation did not cause measurable agglomeration of the nanoparticles. This reduction of the Pt system by electrochemical means was also explored as a synthetic method. The Pt-dendrimer complex was placed on a TEM grid for electrochemical treatment. A potential step was shown to reduce some of the Pt-dendrimer complexes into Pt nanoparticles of the expected size. However, most of the complexes were not reduced. Therefore, only the standard chemical reduction followed by electrochemical treatment is sufficient to fully reduce the nanoparticle samples. This work has explored additional synthetic methods for the synthesis of monometallic and bimetallic DENs. The use of EXAFS, as well as other advanced characterization techniques, has advanced knowledge of the structure of various DENs. Both the characterization toolset and the synthetic methods will provide a basis for investigations of catalytically active materials. / text