Matching Items (130)
Description
Microfluidic devices represent a growing technology in the world of analytical chemistry. Serial femtosecond crystallography (SFX) utilizes microfluidic devices to generate droplets of an aqueous buffer containing protein crystals, which are then fired out as a jet in the beam of an X-ray free electron laser (XFEL). A crucial part of the device is its method of droplet detection. This project presents a design for a capacitive sensor that uses a unique electrode configuration to detect the difference in capacitance between the aqueous and oil phases. This design was developed using MATLAB and COMSOL Multiphysics simulations and printed using high-resolution 3D printing. Results show that this design can successfully distinguish between the two immiscible liquids, confirming it as a possible detection method in future SFX experiments.
ContributorsCorder, Cameron Dean (Author) / Ros, Alexandra (Thesis director) / Williams, Peter (Committee member) / Hayes, Mark (Committee member) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Fluoroquinolone antibiotics have been known to cause severe, multisystem adverse side effects, termed fluoroquinolone toxicity (FQT). This toxicity syndrome can present with adverse effects that vary from individual to individual, including effects on the musculoskeletal and nervous systems, among others. The mechanism behind FQT in mammals is not known, although various possibilities have been investigated. Among the hypothesized FQT mechanisms, those that could potentially explain multisystem toxicity include off-target mammalian topoisomerase interactions, increased production of reactive oxygen species, oxidative stress, and oxidative damage, as well as metal chelating properties of FQs. This review presents relevant information on fluoroquinolone antibiotics and FQT and explores the mechanisms that have been proposed. A fluoroquinolone-induced increase in reactive oxygen species and subsequent oxidative stress and damage presents the strongest evidence to explain this multisystem toxicity syndrome. Understanding the mechanism of FQT in mammals is important to aid in the prevention and treatment of this condition.
ContributorsHall, Brooke Ashlyn (Author) / Redding, Kevin (Thesis director) / Wideman, Jeremy (Committee member) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Bioanalytes such as protein, cells, and viruses provide vital information but are inherently challenging to measure with selective and sensitive detection. Gradient separation technologies can provide solutions to these challenges by enabling the selective isolation and pre-concentration of bioanalytes for improved detection and monitoring. Some fundamental aspects of two of these techniques, isoelectric focusing and dielectrophoresis, are examined and novel developments are presented. A reproducible and automatable method for coupling capillary isoelectric focusing (cIEF) and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) based on syringe pump mobilization is found. Results show high resolution is maintained during mobilization and &beta-lactoglobulin; protein isoforms differing by two amino acids are resolved. Subsequently, the instrumental advantages of this approach are utilized to clarify the microheterogeneity of serum amyloid P component. Comprehensive, quantitative results support a relatively uniform glycoprotein model, contrary to inconsistent and equivocal observations in several gel isoelectric focusing studies. Fundamental studies of MALDI-MS on novel superhydrophobic substrates yield unique insights towards an optimal interface between cIEF and MALDI-MS. Finally, the fundamentals of isoelectric focusing in an open drop are explored. Findings suggest this could be a robust sample preparation technique for droplet-based microfluidic systems. Fundamental advancements in dielectrophoresis are also presented. Microfluidic channels for dielectrophoretic mobility characterization are designed which enable particle standardization, new insights to be deduced, and future devices to be intelligently designed. Dielectrophoretic mobilities are obtained for 1 µm polystyrene particles and red blood cells under select conditions. Employing velocimetry techniques allows models of particle motion to be improved which in turn improves the experimental methodology. Together this work contributes a quantitative framework which improves dielectrophoretic particle separation and analysis.
ContributorsWeiss, Noah Graham (Author) / Hayes, Mark A. (Thesis advisor) / Garcia, Antonio (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2011
Description
In an effort to begin validating the large number of discovered candidate biomarkers, proteomics is beginning to shift from shotgun proteomic experiments towards targeted proteomic approaches that provide solutions to automation and economic concerns. Such approaches to validate biomarkers necessitate the mass spectrometric analysis of hundreds to thousands of human samples. As this takes place, a serendipitous opportunity has become evident. By the virtue that as one narrows the focus towards "single" protein targets (instead of entire proteomes) using pan-antibody-based enrichment techniques, a discovery science has emerged, so to speak. This is due to the largely unknown context in which "single" proteins exist in blood (i.e. polymorphisms, transcript variants, and posttranslational modifications) and hence, targeted proteomics has applications for established biomarkers. Furthermore, besides protein heterogeneity accounting for interferences with conventional immunometric platforms, it is becoming evident that this formerly hidden dimension of structural information also contains rich-pathobiological information. Consequently, targeted proteomics studies that aim to ascertain a protein's genuine presentation within disease- stratified populations and serve as a stepping-stone within a biomarker translational pipeline are of clinical interest. Roughly 128 million Americans are pre-diabetic, diabetic, and/or have kidney disease and public and private spending for treating these diseases is in the hundreds of billions of dollars. In an effort to create new solutions for the early detection and management of these conditions, described herein is the design, development, and translation of mass spectrometric immunoassays targeted towards diabetes and kidney disease. Population proteomics experiments were performed for the following clinically relevant proteins: insulin, C-peptide, RANTES, and parathyroid hormone. At least thirty-eight protein isoforms were detected. Besides the numerous disease correlations confronted within the disease-stratified cohorts, certain isoforms also appeared to be causally related to the underlying pathophysiology and/or have therapeutic implications. Technical advancements include multiplexed isoform quantification as well a "dual- extraction" methodology for eliminating non-specific proteins while simultaneously validating isoforms. Industrial efforts towards widespread clinical adoption are also described. Consequently, this work lays a foundation for the translation of mass spectrometric immunoassays into the clinical arena and simultaneously presents the most recent advancements concerning the mass spectrometric immunoassay approach.
ContributorsOran, Paul (Author) / Nelson, Randall (Thesis advisor) / Hayes, Mark (Thesis advisor) / Ros, Alexandra (Committee member) / Williams, Peter (Committee member) / Arizona State University (Publisher)
Created2011
Description
Water-soluble, adenosine triphosphate (ATP)-stabilized palladium nanoparticles have been synthesized by reduction of palladium salt in the presence of excess ATP. They have been characterized by electron microscopy, energy dispersive X-ray spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, and X-ray diffraction in order to determine particle size, shape, composition and crystal structure. The particles were then subsequently attached to a glassy carbon electrode (GCE) in order to explore their electrochemical properties with regard to hydrogen insertion in 1 M sodium hydroxide. The particles were found to be in the size range 2.5 to 4 nm with good size dispersion. The ATP capping ligand allowed the particles to be air-stable and re-dissolved without agglomeration. It was found that the NPs could be firmly attached to the working electrode via cycling the voltage repeatedly in a NP/phosphate solution. Further electrochemical experiments were conducted to investigate the adsorption and absorption of hydrogen in the NPs in 1 M sodium hydroxide. Results for cyclic voltammetry experiments were consistent with those for nanostructured and thin-film palladium in basic solution. Absorbed hydrogen content was analyzed as a function of potential. The maximum hydrogen:Pd ratio was found to be ~0.7, close the theoretical maximum value for β phase palladium hydride.
ContributorsLamb, Timothy (Author) / Buttry, Daniel A (Thesis advisor) / Yarger, Jeffery (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2013
Description
Pathogenic Gram-negative bacteria employ a variety of molecular mechanisms to combat host defenses. Two-component regulatory systems (TCR systems) are the most ubiquitous signal transduction systems which regulate many genes required for virulence and survival of bacteria. In this study, I analyzed different TCR systems in two clinically-relevant Gram-negative bacteria, i.e., oral pathogen Porphyromonas gingivalis and enterobacterial Escherichia coli. P. gingivalis is a major causative agent of periodontal disease as well as systemic illnesses, like cardiovascular disease. A microarray study found that the putative PorY-PorX TCR system controls the secretion and maturation of virulence factors, as well as loci involved in the PorSS secretion system, which secretes proteinases, i.e., gingipains, responsible for periodontal disease. Proteomic analysis (SILAC) was used to improve the microarray data, reverse-transcription PCR to verify the proteomic data, and primer extension assay to determine the promoter regions of specific PorX regulated loci. I was able to characterize multiple genetic loci regulated by this TCR system, many of which play an essential role in hemagglutination and host-cell adhesion, and likely contribute to virulence in this bacterium. Enteric Gram-negative bacteria must withstand many host defenses such as digestive enzymes, low pH, and antimicrobial peptides (AMPs). The CpxR-CpxA TCR system of E. coli has been extensively characterized and shown to be required for protection against AMPs. Most recently, this TCR system has been shown to up-regulate the rfe-rff operon which encodes genes involved in the production of enterobacterial common antigen (ECA), and confers protection against a variety of AMPs. In this study, I utilized primer extension and DNase I footprinting to determine how CpxR regulates the ECA operon. My findings suggest that CpxR modulates transcription by directly binding to the rfe promoter. Multiple genetic and biochemical approaches were used to demonstrate that specific TCR systems contribute to regulation of virulence factors and resistance to host defenses in P. gingivalis and E. coli, respectively. Understanding these genetic circuits provides insight into strategies for pathogenesis and resistance to host defenses in Gram negative bacterial pathogens. Finally, these data provide compelling potential molecular targets for therapeutics to treat P. gingivalis and E. coli infections.
ContributorsLeonetti, Cori (Author) / Shi, Yixin (Thesis advisor) / Stout, Valerie (Committee member) / Nickerson, Cheryl (Committee member) / Sandrin, Todd (Committee member) / Arizona State University (Publisher)
Created2013
Description
The study of bacterial resistance to antimicrobial peptides (AMPs) is a significant area of interest as these peptides have the potential to be developed into alternative drug therapies to combat microbial pathogens. AMPs represent a class of host-mediated factors that function to prevent microbial infection of their host and serve as a first line of defense. To date, over 1,000 AMPs of various natures have been predicted or experimentally characterized. Their potent bactericidal activities and broad-based target repertoire make them a promising next-generation pharmaceutical therapy to combat bacterial pathogens. It is important to understand the molecular mechanisms, both genetic and physiological, that bacteria employ to circumvent the bactericidal activities of AMPs. These understandings will allow researchers to overcome challenges posed with the development of new drug therapies; as well as identify, at a fundamental level, how bacteria are able to adapt and survive within varied host environments. Here, results are presented from the first reported large scale, systematic screen in which the Keio collection of ~4,000 Escherichia coli deletion mutants were challenged against physiologically significant AMPs to identify genes required for resistance. Less than 3% of the total number of genes on the E. coli chromosome was determined to contribute to bacterial resistance to at least one AMP analyzed in the screen. Further, the screen implicated a single cellular component (enterobacterial common antigen, ECA) and a single transporter system (twin-arginine transporter, Tat) as being required for resistance to each AMP class. Using antimicrobial resistance as a tool to identify novel genetic mechanisms, subsequent analyses were able to identify a two-component system, CpxR/CpxA, as a global regulator in bacterial resistance to AMPs. Multiple previously characterized CpxR/A members, as well as members found in this study, were identified in the screen. Notably, CpxR/A was found to transcriptionally regulate the gene cluster responsible for the biosynthesis of the ECA. Thus, a novel genetic mechanism was uncovered that directly correlates with a physiologically significant cellular component that appears to globally contribute to bacterial resistance to AMPs.
ContributorsWeatherspoon-Griffin, Natasha (Author) / Shi, Yixin (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Misra, Rajeev (Committee member) / Nickerson, Cheryl (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2013
Description
Complex samples, such as those from biological sources, contain valuable information indicative of the state of human health. These samples, though incredibly valuable, are difficult to analyze. Separation science is often used as the first step when studying these samples. Electrophoretic exclusion is a novel separations technique that differentiates species in bulk solution. Due to its ability to isolate species in bulk solution, it is uniquely suited to array-based separations for complex sample analysis. This work provides proof of principle experimental results and resolving capabilities of the novel technique. Electrophoretic exclusion is demonstrated at a single interface on both benchtop and microscale device designs. The benchtop instrument recorded absorbance measurements in a 365 μL reservoir near a channel entrance. Results demonstrated the successful exclusion of a positively-charged dye, methyl violet, with various durations of applied potential (30 - 60 s). This was the first example of measuring absorbance at the exclusion location. A planar, hybrid glass/PDMS microscale device was also constructed. One set of experiments employed electrophoretic exclusion to isolate small dye molecules (rhodamine 123) in a 250 nL reservoir, while another set isolated particles (modified polystyrene microspheres). Separation of rhodamine 123 from carboxylate-modified polystyrene spheres was also shown. These microscale results demonstrated the first example of the direct observation of exclusion behavior. Furthermore, these results showed that electrophoretic exclusion can be applicable to a wide range of analytes. The theoretical resolving capabilities of electrophoretic exclusion were also developed. Theory indicates that species with electrophoretic mobilities as similar as 10-9 cm2/Vs can be separated using electrophoretic exclusion. These results are comparable to those of capillary electrophoresis, but on a very different format. This format, capable of isolating species in bulk solution, coupled with the resolving capabilities, makes the technique ideal for use in a separations-based array.
ContributorsKenyon, Stacy Marie (Author) / Hayes, Mark A. (Thesis advisor) / Ros, Alexandra (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2012
Description
The need for a renewable and sustainable light-driven energy source is the motivation for this work, which utilizes a challenging, yet practical and attainable bio-inspired approach to develop an artificial oxygen evolving complex, which builds upon the principles of the natural water splitting mechanism in oxygenic photosynthesis. In this work, a stable framework consisting of a three-dimensional DNA tetrahedron has been used for the design of a bio-mimic of the Oxygen-Evolving Complex (OEC) found in natural Photosystem II (PSII). PSII is a large protein complex that evolves all the oxygen in the atmosphere, but it cannot be used directly in artificial systems, as the light reactions lead to damage of one of Photosystem II's core proteins, D1, which has to be replaced every half hour in the presence of sunlight. The final goal of the project aims to build the catalytic center of the OEC, including the Mn4CaCl metal cluster and its protein environment in the stable DNA framework of a tetrahedron, which can subsequently be connected to a photo-stable artificial reaction center that performs light-induced charge separation. Regions of the peptide sequences containing Mn4CaCl ligation sites are implemented in the design of the aOEC (artificial oxygen-evolving complex) and are attached to sites within the tetrahedron to facilitate assembly. Crystals of the tetrahedron have been obtained, and X-ray crystallography has been used for characterization. As a proof of concept, metal-binding peptides have been coupled to the DNA tetrahedron which allowed metal-containing porphyrins, specifically Fe(III) meso-Tetra(4-sulfonatophenyl) porphyrin chloride, to be encapsulated inside the DNA-tetrahedron. The porphyrins were successfully assembled inside the tetrahedron through coordination of two terminal histidines from the orthogonally oriented peptides covalently attached to the DNA. The assembly has been characterized using Electron Paramagnetic Resonance (EPR), optical spectroscopy, Dynamic Light Scattering (DLS), and x-ray crystallography. The results reveal that the spin state of the metal, iron (III), switches during assembly from the high-spin state to low-spin state.
ContributorsRendek, Kimberly Nicole (Author) / Fromme, Petra (Thesis advisor) / Chen, Julian (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2012
Description
This work demonstrated a novel microfluidic device based on direct current (DC) insulator based dielectrophoresis (iDEP) for trapping individual mammalian cells in a microfluidic device. The novel device is also applicable for selective trapping of weakly metastatic mammalian breast cancer cells (MCF-7) from mixtures with mammalian Peripheral Blood Mononuclear Cells (PBMC) and highly metastatic mammalian breast cancer cells, MDA-MB-231. The advantage of this approach is the ease of integration of iDEP structures in microfliudic channels using soft lithography, the use of DC electric fields, the addressability of the single cell traps for downstream analysis and the straightforward multiplexing for single cell trapping. These microfluidic devices are targeted for capturing of single cells based on their DEP behavior. The numerical simulations point out the trapping regions in which single cell DEP trapping occurs. This work also demonstrates the cell conductivity values of different cell types, calculated using the single-shell model. Low conductivity buffers are used for trapping experiments. These low conductivity buffers help reduce the Joule heating. Viability of the cells in the buffer system was studied in detail with a population size of approximately 100 cells for each study. The work also demonstrates the development of the parallelized single cell trap device with optimized traps. This device is also capable of being coupled detection of target protein using MALDI-MS.
ContributorsBhattacharya, Sanchari (Author) / Ros, Alexandra (Committee member) / Ros, Robert (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2013