Doctor of Philosophy (PhD), Wright State University, 2020, Environmental Sciences PhD

When people allow fear to drive their decision making, they often make decisions that do more harm than good. Examples of this include stocking up on ciprofloxacin, flooding doctors' offices and buying black market antibiotics after the anthrax attacks of 2001. Therefore, it is important to be able to address what people are saying when another anthrax attack occurs. Supervised and unsupervised machine learning methodologies can be utilized to detect an event, classify the tweets by event, and to determine the main topics of discussion. Over the period of data collection, twenty events were detected. Three of these events concerned North Korean Threats, six discussed The Mueller Investigation, and three concerned Anthrax Scares. Other events included natural outbreaks in hippos and cattle, a conspiracy theory about Matt DeHart, an article on how long anthrax remains in the soil, wishing someone had anthrax, and tweets from those affected by the anthrax attacks. Parts of speech, unigrams, hashtags, URL's and at-mentions were all important for classifying tweets. These methods can be used on other social media sources and can detect other terrorism events. The Mueller Investigation demonstrated that people do not forget past failings of the Federal Bureau of Investigation (FBI) and continue to distrust them because of these failings. Anthrax scares indicate people use past scares to determine how to react to current scares. North Korean threats showed that people are fearful of new threats but stopped talking about them quickly after the story broke.

Committee: William Romine Ph.D. (Advisor); Terry Oroszi Ed.D. (Committee Member); Nancy Bigley Ph.D. (Committee Member); Cassie Barlow Ph.D. (Committee Member); Dawn Wooley Ph.D. (Committee Member) Subjects: Biology

Doctor of Philosophy, Case Western Reserve University, 2018, Pharmacology

The knowledge of a protein's structure is foundational to understanding how it accomplishes its function. Protein interactions with other proteins and DNA govern critical cellular events. However, protein complexes are difficult to study using traditional methods in structural biology. Electron microscopy (EM) has provided a way to study protein complexes at a level that is unachievable by other structural methods. Here we have used EM combined with various image processing methods to determine the structure of two protein complexes that have been either stabilized by crosslinking, or destabilized by pH. First, we have used negative-stain EM with random conical tilt procedures to determine a low resolution structure of the crosslinked Ccq1-Tpz1-Poz1 complex in fission yeast. This complex is intimately involved in events associated with telomere homeostasis, specifically regulating whether the telomere is in an extendable or a non-extendable state. Our results to the EM study were corroborated by Mapping Interfaces via Crosslinking Mass Spectrometry (MICro-MS), both of which indicated a C-terminal dimerization event in the Ccq1 component. Second, we used Cryo-EM and computational modeling to study the cascade of events that are involved in pore formation associated with the anthrax protective antigen (PA) heptamer. Using a D425A mutant which does not form the pore, we exposed the mutant heptamer to low pH, which initiates pore formation in the WT protein. Analysis of two dimensional (2D) class averages and fluorescence profiles across a pH range indicated a conformational change in the mutant at low pH, which suggested the presence of an intermediate state in pore formation. Analysis of the mutant three dimensional (3D) structure using molecular dynamics with flexible fitting showed that protonation of histidines is likely the pH-dependent step in anthrax pore formation and that D425 and K397 likely form an electrostatic interaction which is required for pore formati (open full item for complete abstract)

Committee: Derek Taylor (Advisor) Subjects: Biology; Pharmacology

PhD, University of Cincinnati, 2007, Medicine : Molecular Genetics, Biochemistry, and Microbiology

Bacillus anthracis is a high priority biothreat agent because of two virulence factors, an anti-phagocytic capsule and an exotoxin, consisting of protective antigen (PA), lethal factor (LF), and edema factor (EF). These virulence factors inhibit the host immune responses, allowing the bacilli to rapidly multiply and overwhelm the host. While the capsule is nonimmunogenic, the PA component of anthrax toxin has been shown to induce a protective antibody response and is the main component of human anthrax vaccines, including the United States' anthrax vaccine adsorbed (AVA). This dissertation aimed to further characterize anthrax toxin activity, examine human antibody responses to AVA vaccination, and investigate the influence of toxin receptor type on susceptibility to anthrax toxin. Initial studies characterized the activity of LF and EF using the mouse J774A.1 cell line and examined if human antibody-mediated neutralization of toxin could be more efficiently assessed using human cells. A variety of human and animal cells lines, and human primary cells were examined for susceptibility to anthrax toxin. J774A.1 cells were the only cells found to be susceptible to LF-induced lysis, but all cells tested were susceptible to EF-induced increases in cAMP. The activity of LF and EF antibodies was also assessed utilizing the LF- and EF-susceptible J774A.1 cells. The results revealed that LF and EF antibodies do not significantly contribute to anthrax toxin neutralization in humans, and that antibodies to PA are sufficient to neutralize toxin activity. Lastly, the influence of receptor type on susceptibility to anthrax toxin components using Chinese hamster ovary cells (CHO), CHO cells lacking functional toxin receptors (CHO-R1.1), and CHO-R1.1 cells expressing the human forms of two PA receptors, TEM8 (CHO-TEM8) or CMG2 (CHO-CMG2) was examined. Unexpectedly, PA alone, previously believed to only mediate entry of LF or EF, was found to be toxic to CHO-TEM8 cells. CHO-TEM8 cel (open full item for complete abstract)

Committee: Dr. Alison Weiss (Advisor) Subjects: Biology, Microbiology

MS, University of Cincinnati, 2004, Medicine : Molecular Genetics, Biochemistry, and Microbiology

The ability of an organism to cause infection or disease is related to a variety of factors, including the general health of the host. Bacillus anthracis is the organism responsible for anthrax, a disease most commonly associated with people who work with animals, but recently has been designated a bioterrorism agent. The organism uses a polysaccharide capsule and secretes three toxins in order to avoid and disable the host's immune response. I characterized the protective nature of the immune response of recipients of the anthrax vaccine. Escherichia coli O157:H7 is the causative agent of enterohemorrhagic colitis, a bowel disease associated with watery and bloody diarrhea. The organism's genome is lysogenized by a bacteriophage, which encodes Shiga toxin. Shiga toxin is produced when the phage enters the lytic cycle. I characterized the physical properties of a toxin-deleted derivative from bacteriophage isolated from clinical samples.

Committee: Dr. Alison Weiss (Advisor) Subjects: Biology, Microbiology

PhD, University of Cincinnati, 2004, Engineering : Industrial Engineering

In October 2001, the mail distribution system of the United States Postal Service was used to distribute envelopes containing Bacillus anthracis spores by unknown terrorist(s), resulting in 22 cases of anthrax, as well as the closing of the Brentwood Mail Facility in Washington, DC and the Hamilton Township Facility in New Jersey. The development and installation of local exhaust ventilation onto mail and/or package processing machinery is intended to reduce future operator and customer exposure to potentially hazardous contaminants, like anthrax, emitted from letter mail. This document presents the development of a model to be used as a basis for testing local exhaust ventilation for any organization that processes large quantities of mail; the recommended model focuses on a combination of tracer gas testing, smoke release observations and air velocity measurements. Additionally, the model is validated by results from field testing.

Committee: Dr. Richard Shell (Advisor) Subjects: Engineering, Industrial

Doctor of Philosophy, The Ohio State University, 2007, Microbiology

Bacillus anthracis, the causative agent of anthrax, produces two bipartite toxins (lethal and edema toxin). These toxins contribute a large portion of the pathogenesis of this organism; therefore, the common component of both toxins (protective antigen) is the primary antigen in vaccination regimens. Previous research has indicated that immunization with both protein components of lethal toxin (lethal factor and protective antigen) increases antibody response to each component. In this work, the functional activities of lethal toxin were investigated in order to identify the toxin actions producing the enhanced immune response. Each step of the cellular intoxication process was examined, as was the catalytic activity of lethal toxin. Single amino acid changes were introduced into each lethal toxin component in order to remove specific functional activities. The immune responses generated by toxin combinations lacking a single functional activity were then compared to the response stimulated by wild type lethal toxin. The enhanced antibody response to lethal toxin depended on its catalytic activity. Although the inactive forms of lethal toxin did not produce an increase in antibody titer, altering the intoxication ability of lethal toxin revealed an underlying competition between the immune responses to protective antigen and lethal factor. The antibody response to lethal factor depended on the production of IFN-γ and the cytosolic localization of this antigen. In contrast, the antibody response to protective antigen produced large amounts of IL-4. The resulting antagonism between these two cytokines led to competition under all circumstances tested. Antibody production was also enhanced by combining the edema toxin components (protective antigen and edema factor). Despite structural similarities to lethal toxin, edema toxin had a different interaction with the immune system. The combination of edema toxin components acted as an adjuvant and stimulated an increase in (open full item for complete abstract)

Committee: Darrell Galloway (Advisor) Subjects: Biology, Microbiology

Doctor of Philosophy, The Ohio State University, 2007, Veterinary Biosciences

Bacillus anthracis is a gram-positive, spore forming facultative anaerobic bacteria and is the etiologic agent for anthrax. Pulmonary anthrax is the form associated with the highest mortality in humans and is considered a significant threat due to its potential as a bioterrorist weapon. We examined the functionality and the expression of mRNA transcripts for anthrax toxin receptors in primary macrophages and several cell lines. We assessed receptor functionality by comparing the ability of macrophage cell lines and primary macrophages to translocate edema factor to the cytosol. We also examined the expression of TEM8 and CMG2 mRNA transcripts in human and mouse macrophages and mouse tissues by standard and real-time RT-PCR. Our results indicate that most cell lines and primary macrophages express functional anthrax toxin receptors and that CMG2 transcripts are preferentially expressed over TEM8 transcripts in primary macrophages. In order to determine if immunoglobulin and complement are involved in the phagocytosis of B. anthracis spores, we characterized the binding of human IgG and complement protein C3b to the surface of spores in nonimmune human serum. We showed that spores activate the classical complement pathway and that C3b, but not immunoglobulin, serves as an opsonin for spores resulting in enhanced phagocytosis by human macrophages. Thus, C3b opsonization of B. anthracis spores provides a mechanism for enhanced phagocytosis of spores by human macrophages. These findings led us to hypothesize that antibody against spores of other Bacillus species would be cross reactive with B. anthracis spores. We showed antibody and C3b deposition takes place when spores are incubated in antiserum against B. cereus and B. anthracis Sterne. In addition, we showed a complement and immunoglobulin dependent increase in spore phagocytosis by mouse macrophages in the presence of spore antiserum as compared to unvaccinated mouse serum or recombinant B. anthracis protective ant (open full item for complete abstract)

Committee: Michael Lairmore (Advisor) Subjects: Health Sciences, Immunology

Doctor of Philosophy, Miami University, 2012, Chemistry and Biochemistry

This dissertation contains six chapters demonstrating the use of Terahertz Time-Domain spectroscopy and imaging in a variety of applications, from the principle analysis of observed absorption features to the quantitation of threat agents. Chapter 1 focuses on the background of Terahertz, starting with its roots in Microwave and Infrared Spectroscopies and continuing on to modern time-domain techniques that dominate the field at present. Terahertz's interaction with different types of matter, various instrumentation setups, and several types of common time-domain measurements are also discussed. Chapter 2 discusses two separate studies attempting to further the understanding of collective mode absorption peaks observed in the THz spectral region. Absorption peaks found in the THz region of crystalline solids are typically described generically as collective modes or computationally analyzed with no supporting experimental data. These two studies demonstrate an experimental method that can be used concurrently with computational techniques to elucidate a more complete understanding of observed collective modes. Chapter 3 probes the feasibility of detecting a possible threat agent, dipicolinic acid, which is a major component in bacterial spores, such as Anthrax. It focuses on qualitative discovery and the ability to quantify its presence with Terahertz Spectroscopy and imaging. Chapter 4 presents a library of quality cryogenic and room temperature spectra for the 20 standard amino acids to be used as a reference for future research. In addition, trends observed by the groups of amino acids were assessed. Chapter 5 examines the spectral properties of a large biomolecule, heparin, in the terahertz spectral region. Several sample configurations are investigated, from heparin as-is to crystallized nitric acid digestion remnants. A novel trace metal analysis method of heparin utilizing Inductively Coupled Plasma Optical Emission Spectroscopy is also presented. Chapter 6 d (open full item for complete abstract)

Committee: Gilbert E. Pacey PhD (Advisor); Shouzhong Zou PhD (Committee Chair); Richard T. Taylor PhD (Advisor); C. Scott Hartley PhD (Committee Member); James R. Gord PhD (Committee Member) Subjects: Analytical Chemistry

PHD, Kent State University, 2007, College of Arts and Sciences / Department of Biological Sciences

The deliberate dissemination of anthrax spores through the US Postal System in 2001 resulted in mail decontamination using electron beam (EB) accelerators, and the realization for the need for faster, more reliable, and inexpensive detection systems. There is paucity of scientific literature addressing the questions of (1) how to successfully detect Bacillus spores in an efficient and cost-effective manner, and (2) how accelerated electrons effectively control Bacillus spores. To address these questions Bacillus atrophaeus (a traditional anthrax surrogate) was used to study a lyotropic chromonic liquid crystal (LCLC) biosensing technology and to evaluate EB irradiation as a sporicide. For the biosensor, B. atrophaeus spores and anti-B. atrophaeus antibodies were mixed into a LCLC and added to specially created glass slides. LCLC distortions in the slides (caused by the spores and antibodies clumping together within the liquid crystal) were viewed with polarizing microscopy as white spots against a black background and quantified. To answer the second question of how spores are controlled with EB irradiation, the D 10value (dose required to reduce a population by 90%) for B. atrophaeus spores was determined. Spores were dried onto filter papers and placed within small paper coin envelopes and irradiated over a range of doses to determine the dose required to kill 10 8spores. Surviving spores were counted using the standard plate count method. To more thoroughly address the effectiveness of spore control using EB radiation, DNA breakage as a function of increasing EB dose was measured using the agarose gel electrophoresis technique on extracted spore DNA. Also, EB induced damage of the spore outer structures was evaluated using flow cytometry to measure uptake of two fluorescent dyes. These experiments resulted in the following findings: (1) the LCLC biosensor was able to detect Bacillus spores with a detection sensitivity at 105 spores ml-1, (2) EB irradiation dose-d (open full item for complete abstract)

Committee: Christopher Woolverton (Advisor) Subjects: Biology, Microbiology