Matching Items (233)
Description
Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet would bring fully hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses ( 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. At the initial experiments at the AMO beamline using 6.9- Å wavelength, Bragg peaks were recorded to 8.5- Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage. Recently, femtosecond X-ray protein nanocrystallography experiments were done at the CXI beamline of the LCLS using 1.3- Å wavelength, and Bragg reflections were recorded to 3- Å resolution; the data are currently being processed. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.
ContributorsHunter, Mark (Author) / Fromme, Petra (Thesis advisor) / Wolf, George (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2011
Description
With the increasing focus on developing environmentally benign electronic packages, lead-free solder alloys have received a great deal of attention. Mishandling of packages, during manufacture, assembly, or by the user may cause failure of solder joint. A fundamental understanding of the behavior of lead-free solders under mechanical shock conditions is lacking. Reliable experimental and numerical analysis of lead-free solder joints in the intermediate strain rate regime need to be investigated. This dissertation mainly focuses on exploring the mechanical shock behavior of lead-free tin-rich solder alloys via multiscale modeling and numerical simulations. First, the macroscopic stress/strain behaviors of three bulk lead-free tin-rich solders were tested over a range of strain rates from 0.001/s to 30/s. Finite element analysis was conducted to determine appropriate specimen geometry that could reach a homogeneous stress/strain field and a relatively high strain rate. A novel self-consistent true stress correction method is developed to compensate the inaccuracy caused by the triaxial stress state at the post-necking stage. Then the material property of micron-scale intermetallic was examined by micro-compression test. The accuracy of this measure is systematically validated by finite element analysis, and empirical adjustments are provided. Moreover, the interfacial property of the solder/intermetallic interface is investigated, and a continuum traction-separation law of this interface is developed from an atomistic-based cohesive element method. The macroscopic stress/strain relation and microstructural properties are combined together to form a multiscale material behavior via a stochastic approach for both solder and intermetallic. As a result, solder is modeled by porous plasticity with random voids, and intermetallic is characterized as brittle material with random vulnerable region. Thereafter, the porous plasticity fracture of the solders and the brittle fracture of the intermetallics are coupled together in one finite element model. Finally, this study yields a multiscale model to understand and predict the mechanical shock behavior of lead-free tin-rich solder joints. Different fracture patterns are observed for various strain rates and/or intermetallic thicknesses. The predictions have a good agreement with the theory and experiments.
ContributorsFei, Huiyang (Author) / Jiang, Hanqing (Thesis advisor) / Chawla, Nikhilesh (Thesis advisor) / Tasooji, Amaneh (Committee member) / Mobasher, Barzin (Committee member) / Rajan, Subramaniam D. (Committee member) / Arizona State University (Publisher)
Created2011
Description
Molybdenum (Mo) is a key trace nutrient for biological assimilation of nitrogen, either as nitrogen gas (N2) or nitrate (NO3-). Although Mo is the most abundant metal in seawater (105 nM), its concentration is low (<5 nM) in most freshwaters today, and it was scarce in the ocean before 600 million years ago. The use of Mo for nitrogen assimilation can be understood in terms of the changing Mo availability through time; for instance, the higher Mo content of eukaryotic vs. prokaryotic nitrate reductase may have stalled proliferation of eukaryotes in low-Mo Proterozoic oceans. Field and laboratory experiments were performed to study Mo requirements for NO3- assimilation and N2 fixation, respectively. Molybdenum-nitrate addition experiments at Castle Lake, California revealed interannual and depth variability in plankton community response, perhaps resulting from differences in species composition and/or ammonium availability. Furthermore, lake sediments were elevated in Mo compared to soils and bedrock in the watershed. Box modeling suggested that the largest source of Mo to the lake was particulate matter from the watershed. Month-long laboratory experiments with heterocystous cyanobacteria (HC) showed that <1 nM Mo led to low N2 fixation rates, while 10 nM Mo was sufficient for optimal rates. At 1500 nM Mo, freshwater HC hyperaccumulated Mo intercellularly, whereas coastal HC did not. These differences in storage capacity were likely due to the presence in freshwater HC of the small molybdate-binding protein, Mop, and its absence in coastal and marine cyanobacterial species. Expression of the mop gene was regulated by Mo availability in the freshwater HC species Nostoc sp. PCC 7120. Under low Mo (<1 nM) conditions, mop gene expression was up-regulated compared to higher Mo (150 and 3000 nM) treatments, but the subunit composition of the Mop protein changed, suggesting that Mop does not bind Mo in the same manner at <1 nM Mo that it can at higher Mo concentrations. These findings support a role for Mop as a Mo storage protein in HC and suggest that freshwater HC control Mo cellular homeostasis at the post-translational level. Mop's widespread distribution in prokaryotes lends support to the theory that it may be an ancient protein inherited from low-Mo Precambrian oceans.
ContributorsGlass, Jennifer (Author) / Anbar, Ariel D (Thesis advisor) / Shock, Everett L (Committee member) / Jones, Anne K (Committee member) / Hartnett, Hilairy E (Committee member) / Elser, James J (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Biological and immunological characterization of plant-produced HIV-1 Gag/dgp41 virus-like particles
Description
Anti-retroviral drugs and AIDS prevention programs have helped to decrease the rate of new HIV-1 infections in some communities, however, a prophylactic vaccine is still needed to control the epidemic world-wide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although recent clinical trials have shown promising results. Recent successes have focused on highly conserved, mucosally-targeted antigens within HIV-1 such as the membrane proximal external region (MPER) of the envelope protein, gp41. MPER has been shown to play critical roles in the viral mucosal transmission, though this peptide is not immunogenic on its own. Gag is a structural protein configuring the enveloped virus particles, and has been suggested to constitute a target of the cellular immunity potentially controlling the viral load. It was hypothesized that HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (dgp41) could be expressed in plants. Plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a tobacco mosaic virus-based expression system or a combination of both. Results of biophysical, biochemical and electron microscopy characterization demonstrated that plant cells could support not only the formation of HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These particles were purified and utilized in mice immunization experiments. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR - a fusion of MPER and the B-subunit of cholera toxin) were administered to BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens could be elicited in mice systemically primed with VLPs and these responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a robust boosting response against Gag and gp41 when boosted with either candidate. Functional assays of these antibodies are in progress to test the antibodies' effectiveness in neutralizing and preventing mucosal transmission of HIV-1. This immunogenicity of plant-based Gag/dgp41 VLPs represents an important milestone on the road towards a broadly-efficacious and inexpensive subunit vaccine against HIV-1.
ContributorsKessans, Sarah (Author) / Mor, Tsafrir S (Thesis advisor) / Matoba, Nobuyuki (Committee member) / Mason, Hugh (Committee member) / Hogue, Brenda (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Description
The cyanobacterium Synechocystis sp. PCC 6803 performs oxygenic photosynthesis. Light energy conversion in photosynthesis takes place in photosystem I (PSI) and photosystem II (PSII) that contain chlorophyll, which absorbs light energy that is utilized as a driving force for photosynthesis. However, excess light energy may lead to formation of reactive oxygen species that cause damage to photosynthetic complexes, which subsequently need repair or replacement. To gain insight in the degradation/biogenesis dynamics of the photosystems, the lifetimes of photosynthetic proteins and chlorophyll were determined by a combined stable-isotope (15N) and mass spectrometry method. The lifetimes of PSII and PSI proteins ranged from 1-33 and 30-75 hours, respectively. Interestingly, chlorophyll had longer lifetimes than the chlorophyll-binding proteins in these photosystems. Therefore, photosynthetic proteins turn over and are replaced independently from each other, and chlorophyll is recycled from the damaged chlorophyll-binding proteins. In Synechocystis, there are five small Cab-like proteins (SCPs: ScpA-E) that share chlorophyll a/b-binding motifs with LHC proteins in plants. SCPs appear to transiently bind chlorophyll and to regulate chlorophyll biosynthesis. In this study, the association of ScpB, ScpC, and ScpD with damaged and repaired PSII was demonstrated. Moreover, in a mutant lacking SCPs, most PSII protein lifetimes were unaffected but the lifetime of chlorophyll was decreased, and one of the nascent PSII complexes was missing. SCPs appear to bind PSII chlorophyll while PSII is repaired, and SCPs stabilize nascent PSII complexes. Furthermore, aminolevulinic acid biosynthesis, an early step of chlorophyll biosynthesis, was impaired in the absence of SCPs, so that the amount of chlorophyll in the cells was reduced. Finally, a deletion mutation was introduced into the sll1906 gene, encoding a member of the putative bacteriochlorophyll delivery (BCD) protein family. The Sll1906 sequence contains possible chlorophyll-binding sites, and its homolog in purple bacteria functions in proper assembly of light-harvesting complexes. However, the sll1906 deletion did not affect chlorophyll degradation/biosynthesis and photosystem assembly. Other (parallel) pathways may exist that may fully compensate for the lack of Sll1906. This study has highlighted the dynamics of photosynthetic complexes in their biogenesis and turnover and the coordination between synthesis of chlorophyll and photosynthetic proteins.
ContributorsYao, Cheng I Daniel (Author) / Vermaas, Wim (Thesis advisor) / Fromme, Petra (Committee member) / Roberson, Robert (Committee member) / Webber, Andrew (Committee member) / Arizona State University (Publisher)
Created2011
Description
ATP synthase is a large multimeric protein complex responsible for generating the energy molecule adenosine triphosphate (ATP) in most organisms. The catalysis involves the rotation of a ring of c-subunits, which is driven by the transmembrane electrochemical gradient. This dissertation reports how the eukaryotic c-subunit from spinach chloroplast ATP synthase has successfully been expressed in Escherichia coli and purified in mg quantities by incorporating a unique combination of methods. Expression was accomplished using a codon optimized gene for the c-subunit, and it was expressed as an attachment to the larger, more soluble, native maltose binding protein (MBP-c1). The fusion protein MBP-c1 was purified on an affinity column, and the c1 subunit was subsequently severed by protease cleavage in the presence of detergent. Final purification of the monomeric c1 subunit was accomplished using reversed phase column chromatography with ethanol as an eluent. Circular dichroism spectroscopy data showed clear evidence that the purified c-subunit is folded with the native alpha-helical secondary structure. Recent experiments appear to indicate that this monomeric recombinant c-subunit forms an oligomeric ring that is similar to its native tetradecameric form when reconstituted in liposomes. The F-type ATP synthase c-subunit stoichiometry is currently known to vary from 8 to 15 subunits among different organisms. This has a direct influence on the metabolic requirements of the corresponding organism because each c-subunit binds and transports one H+ across the membrane as the ring makes a complete rotation. The c-ring rotation drives rotation of the gamma-subunit, which in turn drives the synthesis of 3 ATP for every complete rotation. The availability of a recombinantly produced c-ring will lead to new experiments which can be designed to investigate the possible factors that determine the variable c-ring stoichiometry and structure.
ContributorsLawrence, Robert Michael (Author) / Fromme, Petra (Thesis advisor) / Chen, Julian J.L. (Committee member) / Woodbury, Neal W. (Committee member) / Arizona State University (Publisher)
Created2011
Description
ABSTRACT études; written for violin ensemble, which include violin duets, trios, and quartets, are less numerous than solo études.; These works rarely go by the title "étude;," and have not been the focus of much scholarly research. Ensemble études; have much to offer students, teachers and composers, however, because they add an extra dimension to the learning, teaching, and composing processes. This document establishes the value of ensemble études; in pedagogy and explores applications of the repertoire currently available. Rather than focus on violin duets, the most common form of ensemble étude;, it mainly considers works for three and four violins without accompaniment. Concentrating on the pedagogical possibilities of studying études; in a group, this document introduces creative ways that works for violin ensemble can be used as both études; and performance pieces. The first two chapters explore the history and philosophy of the violin étude; and multiple-violin works, the practice of arranging of solo études; for multiple instruments, and the benefits of group learning and cooperative learning that distinguish ensemble étude; study from solo étude; study. The third chapter is an annotated survey of works for three and four violins without accompaniment, and serves as a pedagogical guide to some of the available repertoire. Representing a wide variety of styles, techniques and levels, it illuminates an historical association between violin ensemble works and pedagogy. The fourth chapter presents an original composition by the author, titled Variations on a Scottish Folk Song: étude; for Four Violins, with an explanation of the process and techniques used to create this ensemble étude.; This work is an example of the musical and technical integration essential to étude; study, and demonstrates various compositional traits that promote cooperative learning. Ensemble études; are valuable pedagogical tools that deserve wider exposure. It is my hope that the information and ideas about ensemble études; in this paper and the individual descriptions of the works presented will increase interest in and application of violin trios and quartets at the university level.
ContributorsLundell, Eva Rachel (Contributor) / Swartz, Jonathan (Thesis advisor) / Rockmaker, Jody (Committee member) / Buck, Nancy (Committee member) / Koonce, Frank (Committee member) / Norton, Kay (Committee member) / Arizona State University (Publisher)
Created2011
Description
The name of Geechie Wiley has surfaced only rarely since 1931, when she recorded her second session with the Paramount Company in Grafton, WI. A few scholars including Paul Oliver and Greil Marcus unearthed and promoted her music and called for further research on this enigmatic figure. In other publications, Wiley is frequently given only passing mention in long lists of talented female blues singer-guitarists, or briefly discussed in descriptions of songsters. Her music is lauded in the liner notes of the myriad compilation albums that have re-released her recordings. However, prior to this study, Marcus's three-page profile is the longest work written about Wiley; other contributions range between one sentence and two paragraphs in length. None really answers the question: who was Geechie Wiley? This thesis begins by documenting my attempt to piece together all information presently available on Geechie Wiley. A biographical chapter, supplemented with a discussion of the blues songster, follows. I then discuss my methodology and philosophy for transcription. This is followed by a critical and comparative analysis of the recordings, using the transcriptions as supplements. Finally, my fifth chapter presents conclusions about Wiley's life, career, and disappearance. My transcriptions of Wiley's six songs are found in the first appendix. Reproductions of Paramount Records advertisements are located in the final appendix. In these ways, this thesis argues that Wiley's work traces the transformation of African-American music from the general secular music of the songsters to the iconic blues genre.
ContributorsCordeiro, AnneMarie Youell (Author) / Norton, Kay (Thesis advisor) / Mook, Richard (Committee member) / Sunkett, Mark (Committee member) / Arizona State University (Publisher)
Created2011
Description
Finger motion and hand posture of six professional clarinetists (defined by entrance into or completion of a doctorate of musical arts degree in clarinet performance) were recorded using a pair of CyberGloves® in Arizona State University's Center for Cognitive Ubiquitous Computing Laboratory. Performance tasks included performing a slurred three-octave chromatic scale in sixteenth notes, at sixty quarter-note beats per minute, three times, with a metronome and a short pause between repetitions, and forming three pedagogical hand postures. Following the CyberGloves® tasks, each subject completed a questionnaire about equipment, playing history, practice routines, health practices, and hand usage during computer and sports activities. CyberGlove® data were analyzed to find average hand/finger postures and differences for each pitch across subjects, subject variance in the performance task and differences in ascending and descending postures of the chromatic scale. The data were also analyzed to describe generalized finger posture characteristics based on hand size, whether right hand thumb position affects finger flexion, and whether professional clarinetists use similar finger/hand postures when performing on clarinet, holding a tennis ball, allowing hands to hang freely by the sides, or form a "C" shape. The findings of this study suggest an individual approach based on hand size is necessary for teaching clarinet hand posture.
ContributorsHarger, Stefanie (Author) / Spring, Robert (Thesis advisor) / Hill, Gary (Committee member) / Koonce, Frank (Committee member) / Norton, Kay (Committee member) / Stauffer, Sandy (Committee member) / Arizona State University (Publisher)
Created2011
Description
Early-age cracks in fresh concrete occur mainly due to high rate of surface evaporation and restraint offered by the contracting solid phase. Available test methods that simulate severe drying conditions, however, were not originally designed to focus on evaporation and transport characteristics of the liquid-gas phases in a hydrating cementitious microstructure. Therefore, these tests lack accurate measurement of the drying rate and data interpretation based on the principles of transport properties is limited. A vacuum-based test method capable of simulating early-age cracks in 2-D cement paste is developed which continuously monitors the weight loss and changes to the surface characteristics. 2-D crack evolution is documented using time-lapse photography. Effects of sample size, w/c ratio, initial curing and fiber content are studied. In the subsequent analysis, the cement paste phase is considered as a porous medium and moisture transport is described based on surface mass transfer and internal moisture transport characteristics. Results indicate that drying occurs in two stages: constant drying rate period (stage I), followed by a falling drying rate period (stage II). Vapor diffusion in stage I and unsaturated flow within porous medium in stage II determine the overall rate of evaporation. The mass loss results are analyzed using diffusion-based models. Results show that moisture diffusivity in stage I is higher than its value in stage II by more than one order of magnitude. The drying model is used in conjunction with a shrinkage model to predict the development of capillary pressures. Similar approach is implemented in drying restrained ring specimens to predict 1-D crack width development. An analytical approach relates diffusion, shrinkage, creep, tensile and fracture properties to interpret the experimental data. Evaporation potential is introduced based on the boundary layer concept, mass transfer, and a driving force consisting of the concentration gradient. Effect of wind velocity is reflected on Reynolds number which affects the boundary layer on sample surface. This parameter along with Schmidt and Sherwood numbers are used for prediction of mass transfer coefficient. Concentration gradient is shown to be a strong function of temperature and relative humidity and used to predict the evaporation potential. Results of modeling efforts are compared with a variety of test results reported in the literature. Diffusivity data and results of 1-D and 2-D image analyses indicate significant effects of fibers on controlling early-age cracks. Presented models are capable of predicting evaporation rates and moisture flow through hydrating cement-based materials during early-age drying and shrinkage conditions.
ContributorsBakhshi, Mehdi (Author) / Mobasher, Barzin (Thesis advisor) / Rajan, Subramaniam D. (Committee member) / Zapata, Claudia E. (Committee member) / Arizona State University (Publisher)
Created2011