Theses and Dissertations
This collection includes both ASU Theses and Dissertations, submitted by graduate students, and the Barrett, Honors College theses submitted by undergraduate students.
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Description
Osteocalcin (Oc) is the most abundant non-collagen protein found in the bone, but its precise function is still not completely understood. Three glutamic acid (Glu) residues within its sequence are sites for vitamin K-dependent post-translational modification, replacing a hydrogen with a carboxylate located at the γ-carbon position, converting these to γ-carboxyglutamic acid (Gla) residues. This modification confers increased binding of Oc to Ca2+ and hydroxyapatite matrix. Presented here, novel metal binding partners Mn2+, Fe3+, and Cr3+ of human Oc were determined, while the previously identified binders to (generally) non-human Oc, Ca2+, Mg2+, Pb2+ and Al3+ were validated as binders to human Oc by direct infusion mass spectrometry with all metals binding with higher affinity to the post-translationally modified form (Gla-Oc) compared to the unmodified form (Glu-Oc). Oc was also found to form pentamer (Gla-Oc) and pentamer and tetramer (Glu-Oc) homomeric self-assemblies in the absence of NaCl, which disassembled to monomers in the presence of near physiological Na+ concentrations. Additionally, Oc was found to form filamentous structures in vitro by negative stain TEM in the presence of increased Ca2+ titrations in a Gla- and pH-dependent manner. Finally, by combining circular dichroism spectroscopy to determine the fraction of Gla-Oc bound, and inductively-coupled plasma mass spectrometry to quantify total Al concentrations, the data were fit to a single-site binding model and the equilibrium dissociation constant for Al3+ binding to human Gla-Oc was determined (Kd = 1.0 ± 0.12 nM). Including citrate, a known competitive binder of Al3+, maintained Al in solution and enabled calculation of free Al3+ concentrations using a Matlab script to solve the complex set of linear equations. To further improve Al solubility limits, the pH of the system was lowered to 4.5, the pH during bone resorption. Complementary binding experiments with Glu-Oc were not possible due to the observed precipitation of Glu-Oc at pH 4.5, although qualitatively if Glu-Oc binds Al3+, it is with much lower affinity compared to Gla-Oc. Taken together, the results presented here further support the importance of post-translational modification, and thus adequate nutritional intake of vitamin K, on the binding and self-assembly properties of human Oc.
ContributorsThibert, Stephanie (Author) / Borges, Chad R (Thesis advisor) / LaBaer, Joshua (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021
Description
Without a doubt, protein is the most crucial biomolecule performing life and biological functions of any living cell. Profiling various protein expression in individual cells has raised a great interest for scientist and researchers over decades in attempts to reveal cell-to-cell variation, which used to be masked in many previous population average measurement methods. Immunofluorescence (IF) has been a well-established single cell protein analysis technique as for its fast and high-resolution detection and localization, simple and adaptable workflows, and affordable instrumentation. However, inadequate detection sensitivity and multiplexing capability are the two limitation of this platform that remain incompletely addressed in many decades. In this work, several improvements have been proposed and demonstrated to improve existing drawbacks of conventional immunofluorescence. An azide-based linker featured in the novel fluorescent probes synthesis has enable iterative protein staining on the same tissue sample, which subsequently increase the multiplex capacity of IF. Additionally, the multiple fluorophore introduction to the proteins target via either layer by layer biotin-cleavable fluorescent streptavidin or tyramide signal amplification (TSA) have significantly increase the detection sensitivity of the platform. With these advances, IF has the potential to detect, image and quantify up to 100 protein targets in single cell in the tissue sample. In addition of desirable features of IF, these improvements have further turned the technique into a powerful proteomic study platform for not only research setting but also clinical study setting. It is anticipated this highly sensitive and multiplexed, renovated IF method will soon be translated into biomedical studies.
ContributorsPham, Thai Huy (Author) / Guo, Jia (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2023
Description
Global industrialization and urbanization have led to increased levels of air pollution. The costs to society have come in the form of environmental damage, healthcare expenses, lost productivity, and premature mortality. Measuring pollutants is an important task for identifying its sources, warning individuals about dangerous exposure levels, and providing epidemiologists with data to link pollutants with diseases. Current methods for monitoring air pollution are inadequate though. They rely on expensive, complex instrumentation at limited fixed monitoring sites that do not capture the true spatial and temporal variation. Furthermore, the fixed outdoor monitoring sites cannot warn individuals about indoor air quality or exposure to chemicals at worksites. Recent advances in manufacturing and computing technology have allowed new classes of low-cost miniature gas sensor to emerge as possible alternatives. For these to be successful however, there must be innovations in the sensors themselves that improve reliability, operation, and their stability and selectivity in real environments. Three novel gas sensor solutions are presented. The first is the development of a wearable personal exposure monitor using all commercially available components, including two metal oxide semiconductor gas sensors. The device monitors known asthma triggers: ozone, total volatile organic compounds, temperature, humidity, and activity level. Primary focus is placed on the ozone sensor, which requires special circuits, heating algorithm, and calibration to remove temperature and humidity interferences. Eight devices are tested in multiple field tests. The second is the creation of a new compact optoelectronic gas sensing platform using colorimetric microdroplets printed on the surface of a complementary-metal-oxide-semiconductor (CMOS) imager. The nonvolatile liquid microdroplets provide a homogeneous, uniform environment that is ideal for colorimetric reactions and lensless optical measurements. To demonstrate one type of possible indicating system gaseous ammonia is detected by complexation with Cu(II). The third project continues work on the CMOS imager optoelectronic platform and develops a more robust sensing system utilizing hydrophobic aerogel particles. Ammonia is detected colorimetrically by its reaction with a molecular dye, with additives and surface treatments enhancing uniformity of the printed films. Future work presented at the end describes a new biological particle sensing system using the CMOS imager.
ContributorsMallires, Kyle Reed (Author) / Tao, Nongjian (Thesis advisor) / Forzani, Erica (Thesis advisor) / Wiktor, Peter (Committee member) / Wang, Di (Committee member) / Alford, Terry (Committee member) / Xian, Xiaojun (Committee member) / Arizona State University (Publisher)
Created2020
Description
This work demonstrates a capable reverse pulse deposition methodology to influence gap fill behavior inside microvia along with a uniform deposit in the fine line patterned regions for substrate packaging applications. Interconnect circuitry in IC substrate packages comprises of stacked microvia that varies in depth from 20µm to 100µm with an aspect ratio of 0.5 to 1.5 and fine line patterns defined by photolithography. Photolithography defined pattern regions incorporate a wide variety of feature sizes including large circular pad structures with diameter of 20µm - 200µm, fine traces with varying widths of 3µm - 30µm and additional planar regions to define a IC substrate package. Electrodeposition of copper is performed to establish the desired circuit. Electrodeposition of copper in IC substrate applications holds certain unique challenges in that they require a low cost manufacturing process that enables a void-free gap fill inside the microvia along with uniform deposition of copper on exposed patterned regions. Deposition time scales to establish the desired metal thickness for such packages could range from several minutes to few hours. This work showcases a reverse pulse electrodeposition methodology that achieves void-free gap fill inside the microvia and uniform plating in FLS (Fine Lines and Spaces) regions with significantly higher deposition rates than traditional approaches. In order to achieve this capability, systematic experimental and simulation studies were performed. A strong correlation of independent parameters that govern the electrodeposition process such as bath temperature, reverse pulse plating parameters and the ratio of electrolyte concentrations is shown to the deposition kinetics and deposition uniformity in fine patterned regions and gap fill rate inside the microvia. Additionally, insight into the physics of via fill process is presented with secondary and tertiary current simulation efforts. Such efforts lead to show “smart” control of deposition rate at the top and bottom of via to avoid void formation. Finally, a parametric effect on grain size and the ensuing copper metallurgical characteristics of bulk copper is also shown to enable high reliability substrate packages for the IC packaging industry.
ContributorsGanesan, Kousik (Author) / Tasooji, Amaneh (Thesis advisor) / Manepalli, Rahul (Committee member) / Alford, Terry (Committee member) / Chan, Candace (Committee member) / Arizona State University (Publisher)
Created2018