Master of Science (MS), Bowling Green State University, 2024, Biological Sciences

Muco-obstructive illness is caused by any number of defects in the respiratory tract that result in viscous or static mucus. This creates an environment conducive for pathogen colonization with subsequent synergistic interactions that drive selection of a smaller set of apex pathogens, resistant to immune and antibiotic intervention. Successful control and potential resolution of such infections will require the development of new therapeutic strategies. One potential approach involves identification of bacterial viruses that target the dominant bacterial strains that manifest in patients with compromised respiratory functions. This study considers a set of bacteriophage specific for, and toxic to Burkholderia species that often dominate in such patient populations. I isolated a set of bacteriophage from environmental samples (including soil, human sewage, and agricultural waste for consideration. Some environmental isolates were capable of infecting pathogenic Burkholderia and a suite of lab adapted and pathogenic Burkholderia strains. As a result of this bacteriophage hunt, 15 potential bacteriophage isolates have been maintained, four of which have been sequenced and analyzed further. To do so, DNA was extracted from the purified and amplified isolates and sequenced. Thus, it was postulated that the novel bacteriophages “Clear “and “Cloudy” may be related to the P2-family of bacteriophage: whereas novel bacteriophage “P1P” contains elements similar to mu-like phage. Further research is necessary to detail the genome structure, genetic relationships, and gene function; however, these novel isolates reiterate the notion that pathogen-infecting bacteriophage are potentially isolated from the environment using non-pathogenic or pathogenic strains.

Committee: Raymond Larsen Ph.D. (Committee Chair); Hans Wildschutte Ph.D. (Committee Member); Simon Morgan-Russell Ph.D. (Committee Member) Subjects: Microbiology

Master of Science (MS), Bowling Green State University, 2020, Biological Sciences

Multidrug resistant (MDR) pathogens are predicted to cause more than ten million annual deaths worldwide by 2050, making bacterial infections the leading cause of death. Although bacteria are evolving resistance to all known antibiotics, no large pharmaceutical companies are involved in drug discovery due to high cost and low profitability; thus, no new antibiotics are available, and the current ones are increasingly becoming ineffective at treating MDR infections. Moreover, recent studies suggest that there are few remaining new antibiotics in the environment left for discovery. Since MDR bacterial infections are predicted to be a major crisis, a new or augmented therapy to treat infections is imperative. Bacteriophage therapy is an alternative solution and has been internationally used for over 100 years. In bacteriophage therapy, phage bind to bacteria through specific protein-protein interactions that result in narrow host range infectivity. Although this specificity is beneficial because the interaction precisely targets a single pathogen, it is also problematic because a single phage usually cannot infect multiple strains of the same bacterial species. The phage discovery process to treat a specific pathogen is both time consuming and phage can fall short of the ability to antagonize multiple infections. Thus, phage with broad host range killing phenotypes are more beneficial when using phage therapy to treat infections caused by a particular pathogen. Therefore, this study set out to isolate phage with broad host range killing phenotypes such that phage that could inhibit more than one MDR pathogen could be found. In this study, 29 phage that antagonize cystic fibrosis (CF) derived MDR Pseudomonas aeruginosa were isolated from equine fecal water, purified, characterized through host range assays, and shown to kill two to eight CF derived MDR P. aeruginosa strains. Since phage have been shown to drive bacterial evolution toward increased antibiotic susce (open full item for complete abstract)

Committee: Hans Wildschutte (Advisor); George Bullerjahn (Committee Member); Raymond Larsen (Committee Member) Subjects: Biology; Biomedical Research; Health; Microbiology; Therapy

Master of Science, University of Toledo, 2004, Chemistry

In the Mueser laboratory, we study how DNA replication and repair proteins recognize DNA in a structure-specific manner. Bacteriophage T4 is used as a model system to study DNA replication as it encodes all ten proteins required for DNA replication. Much is known about how the individual proteins function in replication but not much is known about the structural aspects of the protein-protein or protein-DNA interactions at the replication fork. The goal of our research is to study how these replication proteins interact with each other and with DNA. We work towards achieving this goal by crystallizing the protein-protein and protein-DNA complexes and then solving their structures, using macromolecular crystallography techniques. We then use the structural information gathered to analyze the interactions. The overall goal of this master's thesis project was to learn many of the techniques involved in protein chemistry and protein crystallization. My research was tailored to protein expression, purification and crystallization so I could learn an array of techniques and become familiar with various pieces of instrumentation. I wanted to be able to use this knowledge in future research positions. My work was focused on two of the replication proteins from Bacteriophage T4: T4 gene 59 helicase assembly protein and T4 gene 32 single-stranded binding protein. These two proteins interact in the absence of DNA and form a complex at the replication fork. I was responsible for expressing mutated and truncated forms of the native proteins on a large scale and developing purification protocols in order to prepare pure protein for crystal screening. After my research with the T4 helicase assembly protein began, I also started working on a similar helicase assembly protein from a related system – bacteriophage KVP40 59 protein. I was also responsible for developing a purification protocol for single-stranded DNA substrates that were used to prepare forked substrates for the cryst (open full item for complete abstract)

Committee: Timothy Mueser (Advisor) Subjects: Chemistry, Biochemistry

Doctor of Philosophy, Miami University, 2025, Chemistry and Biochemistry

The continuing rise of antibiotic resistance (AR) is an emerging public health problem. This problem is further compounded by a reduction in the development of novel antibiotics to combat these persistent pathogens. This is due to the cost to bring the drug to market and the low likelihood of developing a successful drug. However, one alternative and promising approach is the application of bacteriophage (phage) viruses to selectively infect and kill pathogenic bacteria. These viruses are ubiquitous and innumerable, making them a compelling therapeutic alternative. However, this innumerable nature has a downside for therapy development. Current methods are limited in their throughput, generation of quantitative data regarding phage-host interactions, and are not amenable to multiplexing or parallel screening (i.e. scalability). Because phage therapy is personalized and requires screening collections of phages to identify efficacious phages to formulate into a therapeutic cocktail, new methods are required to address these gaps. Traditional screening methods include plaque and kinetic growth assays, but these assays are not representative of native conditions and are limited in multiplexing and scalability. Phage-layer Interferometry (PLI), engineered multiplex compatible reporter phages (MultiPhlex), and an indirect phage susceptibility assay called Phage-Assisted Droplet Sorting (PhADS) are aimed at addressing these current challenges to the field of phage therapy. PLI was developed to provide a standardized method for generating quantitative data regarding phage-host interactions. The development of MultiPhlex is ongoing but seeks to address the current inability to multiplex phage screening by developing fluorescently barcoded reporter phages with fluorogenic RNA aptamers. PhADS addresses the main short coming of MultiPhlex, not all phages are amenable to engineering, through indirect detection and provides a high throughput massively parallel phage screening sys (open full item for complete abstract)

Committee: Kevin Yehl (Advisor); Rick Page (Committee Chair); Jason Berberich (Committee Member); Neil Danielson (Committee Member); Gary Lorigan (Committee Member) Subjects: Biochemistry; Biology; Chemistry

Doctor of Philosophy (Ph.D.), University of Dayton, 2023, Biology

Antibiotic resistance has been declared a global concern by the World Health Organization and is increasing the rate of mortality of once-treatable, common infections. Antibiotic resistance is conferred by multiple mechanisms both intrinsic (horizontal gene transfer) and extrinsic (production of biofilms). The eradication of biofilms produced by bacterial colonization remains a serious threat to human infections. Bacterial biofilms produce an extracellular matrix composed of proteins, polysaccharides, and extracellular DNA (eDNA). This matrix acts as a scaffold for growth and imparts a form of protection against predators, harsh conditions, and chemicals (e.g., bacteriophage, pH, and antibiotics). The biofilm-associated cells of Pseudomonas aeruginosa (PsA) are up to 1000-fold more resistant to antibiotics than planktonic cells. Additionally, PsA has been linked to many infections that can be mortally dangerous for individuals with compromised immune systems such as Cystic Fibrosis (CF). PsA colonization in individuals with CF causes a decreased quality of life. Thus, there is a search for alternative strategies for antimicrobial management. Our lab has produced a patented zinc-containing porphyrin, Zn(II)meso-5,10,15-triyl-tris(1-methylpyridin-1-ium)-20-(pentafluorophenyl) porphine tritosylate (ZnPor), which exhibits broad antibacterial activity against planktonic and biofilm-associated cells of PsA. ZnPor presents itself as a unique possible surrogate for traditional antibiotics by its interaction with eDNA of biofilms. ZnPor intercalates between base pairs and binds to the outside of the helix, resulting in a more porous biofilm that dissembles and detaches from substrata. Furthermore, ZnPor has potent photoactivity that increases both its bactericidal and viricidal properties when exposed to light. The ability to disrupt the inherent matrix structure makes biofilm-associated cells more accessible to other treatments such as antibiotics and bacteriophage (open full item for complete abstract)

Committee: Jayne Robinson (Committee Chair); Karolyn Hansen (Committee Member); Shawn Swavey (Committee Member); Madhuri Kango-Singh (Committee Chair); Kristen Krupa (Committee Chair) Subjects: Biology; Molecular Biology; Virology

Master of Science (MS), Bowling Green State University, 2021, Biological Sciences

Pharmaceutical companies have slowed the discovery and development of antibiotics due to low-profit margins. Therefore, antibiotic discovery is at an all-time low, and pathogens have evolved resistance to all currently available drugs. As a result, multi-drug resistant (MDR) bacterial infections are becoming more difficult to treat, especially in individuals at a high risk for infection such as cystic fibrosis (CF) patients. CF is a genetically inherited disease that inhibits or decreases chloride ion transport across epithelial cell membranes, resulting increased mucus viscosity, impairing normal clearance in the lungs. This environment is ideal for bacterial colonization and leads to a chronic lung infection. A major pathogen that colonizes the CF lung over time is Pseudomonas aeruginosa. A promising alternative treatment against MDR P. aeruginosa is bacteriophage therapy which has several advantages compared to antibiotics. First, phage therapy exhibits minimal side effects because phage are highly host-specific and do not inhibit other bacteria that are part of the human microbiome. Second, phage replicate itself exponentially when killing its host; and third, phage can be applied directly to the site of infection. However, like antibiotics, bacteria can evolve resistance to phage. To circumvent the problem of phage and drug resistance, trade-off effects may promote opportunities against both entities that may be exploited to treat MDR infections. I hypothesize that the effectiveness of antibiotics can be restored after selective pressure from bacteriophage. To test this hypothesis, MDR P. aeruginosa strains were exposed to phage in trade-off experiments, and results showed that the evolved phage resistant P. aeruginosa strain became antibiotic susceptible. In one trade-off experiment, a temperate phage recombined in the P. aeruginosa pathogen at a location downstream of a multidrug resistance efflux pump that may directly affect antibiotic susceptibility. In an (open full item for complete abstract)

Committee: Hans Wildschutte Ph.D (Advisor); George Bullerjahn Ph.D (Committee Member); Ray Larsen Ph.D (Committee Member) Subjects: Bioinformatics; Biology; Biomedical Research; Microbiology; Molecular Biology

Doctor of Philosophy, Miami University, 2021, Chemistry and Biochemistry

Holins are a family of lytic membrane proteins responsible for the lysis of the cytosolic membrane in host cells of double stranded DNA bacteriophages. Recently a new family of holins have been discovered, the pinholin, which have been shown to depolarize the cytosolic membrane leading to the release of membrane-bound signal-anchor-released (SAR) endolysin from the bilayer. Despite the biological importance of pinholin the structure and dynamic properties have not been well characterized. The work in this dissertation will use a variety of powerful biophysical techniques to study these structure and dynamic properties of pinholin. First, we report the first in vitro synthesis of both the active S2168 and inactive S21IRS pinholin using solid phase peptide synthesis (SPPS) and reveals the first experimental data indicating the global α-helical structure of pinholin using circular dichroism (CD) spectroscopy. After sample optimization, electron spin echo envelope modulation (ESEEM) spectroscopy was used to determine the local α-helical secondary structure of the inhibitory and functional helices of pinholin. Following that, SS-NMR spectroscopy was utilized to probe the differences in the way the active and inactive forms of pinholin interact with the membrane and gives the first piece of direct quantitative evidence indicating TMD1 interacts with the lipid headgroups of the bilayer. Finally, DEER spectroscopy was used to probe the structural models of the active and inactive form of pinholin the membrane. This work led to a newly proposed model of active S2168 pinholin with TMD1 partially externalized from the membrane. This study expanded the application of SS-NMR and EPR spectroscopic techniques and provided a deeper understanding of the structure and dynamic properties of the complex pinholin membrane protein system.

Committee: Gary Lorigan Ph.D (Advisor); Rick Page Ph.D (Committee Chair); David Tierney Ph.D (Committee Member); Paul Urayama Ph.D (Committee Member); Ellen Yezierski Ph.D (Committee Member) Subjects: Chemistry

Doctor of Philosophy, Miami University, 2020, Chemistry and Biochemistry

Bacteriophages have evolved an efficient, protein-mediated host cell lysis mechanism that terminates the infection cycle and facilitates the release of progeny virions at an optimal time. Among the lytic proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time and concentration. Recently, a prototype holin called pinholin has been reported which makes nanoscale holes that are too small for the passage of endolysin. However, the holes formed by pinholin dissipate the cytoplasmic membrane potential leading to the release and activation of membrane-tethered signal anchor release endolysins which are already exported to the periplasm. Pinholin is the evolutionary ancestor of holin, but was discovered more recently and, as such, has not been fully studied in the literature. Of all the pinholin systems, S21 from lambdoid phage 21 is one of the most well-known. Pinholin S21 consists of two holin proteins: the active pinholin S2168 and the inactive antipinholin S2168IRS. Each of these proteins have a short N-terminal domain followed by two transmembrane domains connected by a short loop and terminate in a long, positively charged C-terminal region. However, the precise structural details of the proteins in this system is not well understood. The works presented in this dissertation were carried out to structurally characterize the proteins of pinholin S21 using biophysical techniques including state-of-the-art electron paramagnetic resonance (EPR) spectroscopic methods. Continuous Wave (CW) EPR line shape analysis and power saturation (PS) experiments showed that both transmembrane domains (TMDs) of S2168IRS had restricted mobility meaning they were incorporated in the lipid bilayer while the termini regions had higher mobility as they were solvent exposed. However, TMD1 of S2168 was found to be partially externalized from and interacting with surface of the lipid bilayer while TMD2 remaine (open full item for complete abstract)

Committee: Gary Lorigan Ph.D. (Advisor); Carole Dabney-Smith Ph.D. (Committee Chair); Rick Page Ph.D. (Committee Member); David Tierney Ph.D. (Committee Member); Paul Urayama Ph.D. (Committee Member) Subjects: Biochemistry; Biophysics; Chemistry

Master of Science (MS), Bowling Green State University, 2020, Biological Sciences

The CDC predicts that 10 million deaths will occur worldwide by 2050 due to multi-drug resistant (MDR) bacterial infections. As microbial resistance increases, the number of new antibiotics coming to market, and the number of pharmaceutical companies that discover them, continues to decrease. Furthermore, recent data from the Wildschutte Lab suggests that there are few natural remaining antibiotics to be discovered from environmental bacteria that are effective against MDR pathogens, necessitating the use of alternative treatments. Bacteriophage therapy has been successfully used to treat infections for over 100 years in countries other than the United States and represents an alternative strategy for treating MDR pathogens. While phage therapy is a promising approach, like antibiotic regimes, it comes with drawbacks. For instance, phage are generally host specific, infecting only one or a few highly similar strains, and bacteria can rapidly evolve resistance to specific phage. Here, we identify a set of phage capable of infecting a broad host range (BHR) of MDR pathogens isolated from cystic fibrosis patients, with the potential for use in phage therapy. We isolated multiple phage and characterized them using restriction digests, host ranges assays, and imaging; as a prelude to whole genome sequencing. To further support our broad host range data, we sequenced both the16S rRNA and gyrB genes of the MDR Pseudomonas aeruginosa and Burkholderia spp. hosts to verify the diversity of the pathogens susceptible to these phage. Altogether, these results support that the isolation of at least 14 unique BHR phage capable of inhibiting multiple MDR pathogens. We have also begun to investigate the synergistic relationships between phage and antibiotics that may be exploited and used in tandem to treat pathogenic infections. While this work is still in its infancy, the results support the contention that phage therapy represents a valuable tool to combat the global antibiotic (open full item for complete abstract)

Committee: Hans Wildschutte Ph.D (Advisor); George Bullerjahn Ph.D (Committee Member); Raymond Larsen Ph.D (Committee Member) Subjects: Biology; Microbiology; Molecular Biology

Bachelor of Science, Ashland University, 2020, Biology/Toxicology

Bacteriophages are viruses that infect bacteria. They are found in different environments, including soil, which was the source utilized for this research on bacteriophage host range. Host range is a characteristic of viruses that describes which cells the virus is capable of infecting. Knowing which bacteria are infected by which bacteriophages facilitates a greater understanding of the microbial ecology of soil. My hypothesis asked whether adding a specific host or multiple hosts to the isolation sample would produce bacteriophages with a broader host range than isolation relying on bacteria already in the same soil sample to initially grow the bacteriophages. Bacteriophages were isolated from several soil samples found in a variety of locations in Ohio. Some samples were inoculated with Bacillus cereus 6A3 bacteria, some with multiple strains of Bacillus, while others were left uninoculated. Bacteriophages were isolated from these samples, and, through procedures of passaging and amplification, stocks of phage were obtained. From these stocks, host range was determined. There did not seem to be a distinct difference in the host range pattern between the samples with bacteria and the samples without. Throughout, there was a mixture of broader and narrower host ranges, indicating that the number of hosts added to the initial sample does not have an effect on the host range of isolated phages.

Committee: Paul Hyman (Advisor); Tawse Merrill (Other) Subjects: Biology; Microbiology; Virology

Doctor of Philosophy (Ph.D.), University of Dayton, 2017, Biology

It is now generally recognized that the dominant state of microorganisms in nature is that of the biofilm, a community of microorganisms that grows, not as a free-swimming community, but in close association with an interface between two phases, be it a liquid and solid surface, liquid and air, or even two non-miscible liquids (such as water and oil). This growth form possesses many unique attributes when compared to its planktonic, free-swimming counter part. Biofilms are composed of cells with diverse metabolic states, are protected from their environment by the extracellular matrix, can differ greatly in conditions (e.g. pH, diffusion of nutrients) within the biofilm compared to the bulk medium. These factors result in biofilms being more resistant to biocide and antibiotic treatment than planktonic cells. This resistance has resulted in the pursuit for new ways to both deter the formation of biofilms and eradicate those that have already been established. This current work takes two approaches, in two very different environments, to accomplish these goals. The first chapters address the fouling of aviation fuels by Pseudomonas aeruginosa biofilms and introduces the use of bacteriophage as a method preventing such fouling. The latter portion of this work introduces a cationic porphyrin with the ability to prevent and remediate Pseudomonas aeruginosa and Staphylococcus aureus biofilms in the presence and absence of photoactivation and begins to suggest mechanisms for this activity. This porphyrin has the potential to be applied across a many fields including medicine and industry. Together these approaches begin to address the challenges posed by biofilms.

Committee: Jayne Robinson (Advisor); Mark Nielsen (Committee Member); Karolyn Hansen (Committee Member); Wendy Goodson (Committee Member); Amit Singh (Committee Member) Subjects: Biology; Microbiology

Doctor of Philosophy, The Ohio State University, 2017, Food Science and Technology

Fresh produce is low in calories and high in the fibers, essential vitamins and minerals. Consumption of fresh fruits and vegetables helps prevention of cardiovascular diseases, cancer, diabetes and improves gastrointestinal health. Thus, fruit and vegetables have been gaining popularity among consumers and the increased consumption has been endorsed by many health organizations. On the contrary, fresh produce has continuously been associated with foodborne diseases outbreaks. It is technically challenging to decontaminate fresh produce due to the lack of microbial kill-step that effectively eliminate pathogens without causing product quality deterioration. Currently, chlorine wash is the most-used antimicrobial treatment of postharvest fresh produce. Gaseous ozone and bacteriophages have become popular as natural and environmental-friendly alternative decontamination technologies. Bacteriophages can be applied as alternative to chlorine spray or used as a final rinse before packing. Gaseous ozone was previously found very effective when combined with vacuum cooling to inactivate Escherichia coli O157:H7 on baby spinach. The objectives of this research were: (i) to evaluate the suitability of a single lytic bacteriophage, Escherichia phage OSYSP, as a fresh produce decontaminant; (ii) to assess the efficacy of bacteriophage OSYSP and gaseous ozone against E. coli O157:H7 on spinach leaves; and (iii) to develop a combination treatment involving bacteriophage and gaseous ozone to eliminate E. coli O157:H7 on spinach leaves. To justify using Escherichia phage OSYSP in fresh produce decontamination and other applications, the phage characteristics and genomic makeup were investigated (Chapters 2-4). The method to determine phage titer was optimized for maximum phage recovery and plaque clarity during enumeration. Stability of the phage at different incubation temperatures was investigated. The titer of a phage preparation did not change considerably during storage (open full item for complete abstract)

Committee: Ahmed E. Yousef (Advisor); V.M. Balasubramaniam (Committee Member); Farnaz Maleky (Committee Member); Jiyoung Lee (Committee Member) Subjects: Food Science

Master of Science (M.S.), University of Dayton, 2016, Biology

Antibiotic resistance is a major threat to human health. Because of the rise of antibiotic resistant Pseudomonas aeruginosa bacterial infections in cystic fibrosis and other immunocompromised patients, many individuals are left with untreatable and potentially lethal lung infections. Novel methods of treating infections, such as the use of bacteriophages, may provide new means to attenuate these dangerous and oftentimes antibiotic resistant bacteria. The goal of this project was to determine the degree of innate resistance of lung cells to two P. aeruginosa bacteriophages in an in vitro model. The model system used in this project was a human lung tissue co-culture comprised of A549 type II alveolar cells and U937-derived macrophages. In both monoculture and co-culture, it was discovered that after 24 hours, bacteriophage PEV2 induced significant production of IL-8. DMS3 did not yield innate resistance from our panel of four cytokines: IL-6, IL-8, IL-10, and TNF-alpha. Simulated lung infections were carried out with two strains of P. aeruginosa, PAO1 and PAO1-NP, in which the selected bacteriophages demonstrated their ability to protect the A549 monoculture 12 and 24 hours after bacterial exposure.

Committee: Jayne Robinson Ph.D. (Advisor); Robert Kearns Ph.D. (Committee Member); Kristen Comfort Ph.D. (Committee Member); Amit Singh Ph.D. (Committee Member) Subjects: Biology; Immunology; Microbiology

Master of Science (MS), Bowling Green State University, 2014, Biological Sciences

Ray Larsen, Advisor JK5 is a novel environmental isolate bacteriophage that was isolated from horse feces. JK5 utilizes the TonB system coupled with the siderophore transporter FhuA for successful infection Escherichia coli cells. This study focused on the characterization of this novel phage and the importance of the J gene that is thought to be responsible for host range specificity. The hypothesis of this study is that the C-terminal region of the J gene is responsibly for both host range specificity and TonB dependence. Plaque assays resulted into a similarity of T1, large clear plaques when plating on laboratory K12 E. coli strain. Similar to T1, JK5 has a genome size of roughly 40,000 base pairs. After genomic comparison, JK5 is similar to RTP as well as T1. After full genomic sequencing, I found that another gene (herein gene38) in addition to the J gene is responsible for host range specificity.

Committee: Ray Larsen Dr. (Advisor); Vipa Phuntumart Dr. (Committee Member); George Bullerjahn Dr. (Committee Member) Subjects: Biology; Microbiology; Molecular Biology

Master of Science (MS), Wright State University, 2007, Biochemistry and Molecular Biology

The ability to detect chemical and biological agents is arguably one of the highest priority technical challenges today. The capability to obtain specific information at and near single-molecule resolution is the ultimate goal in chemical and biological agent detection. Metallic nanostructures, nanoshells and nanorods in particular, are attractive substrates because of their plasmonic properties. Combining the specificity of biomolecular recognition with these nanostructures might lead to increased sensitivity and selectivity. Localization of biological recognition motifs to the surface of these nanostructures could provide a mechanism for highly specific and directed energy transfer when bound to its target. This study utilizes nanoshells functionalized with antibodies specific for Escherichia coli, and investigates at both the microscopic and macroscopic scales the ability of these biofunctionalized nanoshells to bind and destroy their target micro-organism when excited using 808 nm near infrared laser radiation. Extension of the technique to Bacillus subtilis spores as well as bacteriophage specific to Escherichia coli are also explored. The bacteriophage is a viral surrogate, and provides a means to explore proof of principle of the interactions between nanoshells and viruses. It is demonstrated that appropriately biofunctionalized nanoshells recognize and bind to their target species, and that the nanoshell successfully couples the energy transfer from an IR laser to the target species. A ratio of nanoshells to Escherichia coli of ~104 for a 50% bacterial cell survival rate is determined, and a possible mechanism for this is discussed. Finally, this ratio is found to decrease by 4-5 orders of magnitude for the case of two Escherichia coli bacteriophage considered, and the significance of this is described.

Committee: Madhavi Kadakia (Advisor) Subjects: Biology, Molecular

PhD, University of Cincinnati, 2012, Engineering and Applied Science: Biomedical Engineering

RNA nanotechnology is to extract defined RNA structure motifs and tertiary interactions, apply them as the building blocks to self-assemble nano-scaled scaffolds with rational designs, and incorporate functional molecules such as siRNA, ribozyme, aptamer and therapeutical compounds to form functionalized RNA nanoparticles. Bacteriophage phi29 packaging RNA (pRNA) has two defined domains: the 5'/3'-end helical domain and the interlocking loop region which is located at the central part of the pRNA sequence. pRNA dimer is formed by hand-in-hand interaction via 4-bp interlocking base pairing. The dimeric pRNA nanoparticle has been shown to be an efficient vector for the specific delivery of small interfering RNA (siRNA) into specific cancer or viral infected cells. However, there are several problems hindering the therapeutic applications of pRNA nanoparticles. In this thesis, I will try to address: 1) The problem of large-scale synthesis of longer RNA molecules. Industrial scale production of RNA by chemical synthesis is limited to ~ 80nt. In order to chemically synthesize pRNA and its functionalized chimeric constructs (generally > 120 nt) in large scales, pRNA nanoparticles were constructed using two synthetic RNA fragments within the size limit for chemical synthesis. The resulting bipartite pRNAs were competent to form dimers, package DNA via the nanomotor, and assemble phi29 phage in vitro. The pRNA subunit assembled from bipartite fragments harboring siRNA or receptor-binding ligands were equally competent in binding cancer cells specifically, entering the cell, and silencing specific genes of interest as the intact constructs. 2) The problem of RNA degradation. 2'-fluorine (2'-F) modification was introduced into the RNA sugar ring and the modified RNAs were resistant to RNase degradation and suitable for in vivo delivery. 3) The dissociation problem of pRNA nanoparticles. The lack of covalent linkage or crosslinking in nanoparticles causes dissociation of pRNA (open full item for complete abstract)

Committee: Jing-Huei Lee PhD (Committee Chair); Peixuan Guo PhD (Committee Member); Andrew Herr PhD (Committee Member); Malak Kotb PhD (Committee Member) Subjects: Biomedical Research

PhD, University of Cincinnati, 2012, Engineering and Applied Science: Biomedical Engineering

Linear double-stranded DNA (dsDNA) viruses package its genome into a preformed procapsid fueled by the energy from ATP hydrolysis. The bacteriophage phi29 motor has a truncated cone shaped protein component, named connector, with a central channel of 3.6 nm at its narrowest part. The connector protein has been successfully inserted into an artificial lipid bilayer membrane, and the channel exhibited robust capability under various salt and pH conditions as revealed by single channel studies. This channel is suitable for extremely precise assessment of the transportation of small molecules, such as ions, DNA and RNA. There is an urgent need to development a highly sensitive detection system, for the applications in the area of pathogen detection, disease diagnosis, environmental monitoring, etc. The current challenges and limitations of these technologies are the sensitivity and accuracy issues arising from background noise and nonspecific reactions. The property of phi29 motor channel has been studied at various conditions, and was incorporated into lipid membrane. The motor channel exercised a one-way traffic property during the process of dsDNA translocation with a valve mechanism. In addition, the opening and closure of the channel also exhibit reversible and controllable. A modified version of the connector channel is founded to have a smaller channel size, which is able to detect the ssDNA and ssRNA. These findings have important implications since this artificial membrane-embedded channel would allow detailed investigations into the mechanisms of viral motor operation, as well as future applications for therapeutic molecule packaging, delivery, single molecule sensing and drug screening.

Committee: Jing-Huei Lee PhD (Committee Chair); Chong Ahn PhD (Committee Member); Peixuan Guo PhD (Committee Member); Jaroslaw Meller PhD (Committee Member); Marepalli Rao PhD (Committee Member) Subjects: Biomedical Research

PhD, University of Cincinnati, 2012, Arts and Sciences: Chemistry

The advantages of high sensitivity, low detection, large dynamic range and multi-element/isotope capability for most elements in the periodic table have enabled inductively coupled plasma mass spectrometry (ICPMS) to become a popular technique for elemental analysis. However, there are challenges from sample loss, matrix effects, interferences, or contamination when applying this technique in various analysis. To explore methods for reducing these difficulties in ICPMS-based analysis, the first part of this thesis is focused on removing inorganic phosphate interferences for the determination of organophosphate nerve agent degradation products based on the detection of 31P+. Calcium chloride and ammonium hydroxide were chosen as the coagulant and for pH adjustment to remove inorganic phosphate interferences. Applications to apple juice and cola drink indicate that this method is suitable for enhancing the determination of organophosphate species in high inorganic phosphate matrices. The growing interest in metals and their biological functions have promoted the utilization of ICPMS in the biological area for trace element determination and metal-containing speciation studies. The remaining focus of this dissertation is to investigate and characterize the metal-protein associations in various biological systems, which is referred as metallomics. To achieve overall screening and sufficient confidence in protein identifications, state-of-the-art metallomics techniques via two-dimensional chromatographic separations in combination with elemental and molecular mass spectrometries were utilized. Two biological systems were studied with different purposes and possible applications. Human cerebrospinal fluid (CSF) from healthy control patients and patients who suffered subarachnoid hemorrhage and its complication, cerebral vasospasm was screened at different metal detection points (Fe, Ni, Cu, Zn and Pb) for molecular distribution patterns and was investigated for possible (open full item for complete abstract)

Committee: Joseph Caruso PhD (Committee Chair); Bruce Ault PhD (Committee Member); Patrick Limbach PhD (Committee Member); Richard Thompson PhD (Committee Member) Subjects: Analytical Chemistry

PhD, University of Cincinnati, 2009, Medicine : Molecular Genetics, Biochemistry, and Microbiology

Escherichia coli O157:H7 causes an estimated 73,000 of food borne illness annually. Varying levels of disease severity exist and include diarrhea, bloody diarrhea, and hemolytic uremic syndrome which can result in kidney damage or death. E. coli O157:H7 produces Shiga toxins 1 and/or 2, although it is Shiga toxin 2 that is linked to severe disease. Currently, a Shiga toxin-producing isolate will yield a positive diagnostic result, regardless of which Shiga toxin variant is produced. We have developed an ELISA that can differentiate between Shiga toxin 1 and Shiga toxin 2 in the presence of fecal material, and if used in the clinical setting, can increase the accuracy of prognosis for a patient infected with E. coli O157:H7. Epidemiological studies have suggested that antibiotics may be linked to HUS development. As such, we have completed an extensive study to determine which, if any, antibiotics are safe for the treatment of E. coli O157:H7. The data suggest that antibiotic-mediated Shiga toxin induction or reduction is based on the mechanism. Antibiotics that target DNA increase Shiga toxin production while protein synthesis inhibitors decrease Shiga toxin production. Human intestinal E. coli have been shown to amplify Shiga toxin. Our data suggest that protein synthesis inhibitors may be effective, even if the patient harbors a Shiga toxin-amplifying isolate, provided that the commensal isolate is susceptible to the antibiotic. Conversely, antibiotics would likely be of no benefit if the Shiga toxin-amplifier is resistant to the antibiotic. Future studies should focus on characterizing the flora of HUS patients, and examining antibiotic treatments within the context of these strains.

Committee: Alison Weiss PhD (Advisor); Gary Dean PhD (Committee Member); George Deepe MD (Committee Member); Michael Lieberman PhD (Committee Member); Suri Iyer PhD (Committee Member) Subjects: Microbiology

MS, University of Cincinnati, 2008, Engineering : Electrical Engineering

In recent years technology has made possible more complex and CPU intensive simulations in the area of biological modeling and biochemical kinetic modeling. Biological systems are stochastic and function in highly noisy surroundings. For a simple system, with a low number of molecules, fluctuations can be large and can play an enormous role in system decisions. Many regulatory proteins and RNA molecules are present in very small numbers, typically on the order of 10 to 100 molecules. But almost all simulation systems are ultimately based on solving either ordinary differential equations (ODEs), partial differential equations (PDEs) or stochastic differential equations (SDEs). Using an ODE approach to model a biological pathway is unrealistic, because spatial phenomena must be considered, discrete events (binding, switching) and non-continuous variables (low copy numbers) must be modeled, and important parameters may be found to be indeterminate. In addition, since proteins and RNA are often present in the cell in small quantities, certain reactions are subject to large statistical fluctuation. Differential equation (DE) methods don't easily capture stochasticity or noise (common in biology). The reality is that cellular behavior is not deterministic and the DE approach is not accurate. This thesis discusses an approach involving the chemical kinetics of spatially homogenous systems that is complete and attentive to detail such as the stochastic kinetics formulation (e.g. Gillespie algorithm) applied to a series of synthetic systems.Our knowledge of how synthetic biological systems can be designed would benefit from biophysically realistic models that can make accurate predictions on the time-evolution of molecular events given arbitrary arrangements of genetic regulatory elements. Building blocks for constructing intracellular logic circuits are useful in achieving circuits of significant complexity. Modularization of a GRN (genetic regulatory network) can help us u (open full item for complete abstract)

Committee: Carla Purdy PhD (Committee Chair); Robert Ewing PhD (Committee Member); George Purdy PhD (Committee Member) Subjects: Electrical Engineering