Master of Science, Miami University, 2025, Cell, Molecular and Structural Biology (CMSB)

We aimed to optimize the post-expression processing of the thermophilic cellulase FnCel5a by determining conditions that maximizes enzyme yield and activity. The gene encoding FnCel5a was cloned into a pET-15b vector, confirmed via genomic sequencing, and expressed in Escherichia coli BL21 cells. Large-scale cultures were initiated in Terrific Broth, based on small-scale expression trials, to obtain enough biomass for purification. The preparation of high-purity enzyme was accomplished by nickel-affinity chromatography followed by size-exclusion chromatography to yield high-quality preparation for additional assays. Hydrolysis of the substrate, carboxymethylcellulose, was assayed using a 3,5-dinitrosalicylic acid assay to calculate Michaelis-Menten parameters (Vmax, Km, kcat) and catalytic efficiency. To enable high-throughput production, parameters of sonication were optimized by modifying the duty cycle and duration to achieve effective cell lysis while maintaining enzyme activity and an additional heat precipitation step at 90 °C for 30 minutes to remove impurities without compromising maximum cellulase activity. Long-term stability testing at 4, 17, and 37 °C revealed that storage at 37 °C maintained the highest activity over 30 days. These findings enhance our understanding of thermophilic cellulases and demonstrate a practical strategy for efficient production, stabilization, and application of FnCel5a in biofuel production and other industrial biotechnology processes.

Committee: Richard Page (Advisor); Carole Dabney-Smith (Committee Member); Kevin Yehl (Committee Member) Subjects: Biochemistry; Molecular Biology

Master of Science, The Ohio State University, 2007, Graduate School

Committee: Not Provided (Other) Subjects:

Doctor of Philosophy, The Ohio State University, 2023, Chemical Engineering

The biopharmaceutical market has seen tremendous growth over the past few decades as new recombinant proteins, monoclonal antibody-based therapeutics and gene therapies have been commercialized. After production in different mammalian, yeast and microbial expression hosts, each product must be concentrated and highly purified before clinical use. Protein A affinity methods provide a convenient and widely used platform for capturing and purifying monoclonal antibodies (mAbs), Fc-fusion proteins, antibody drug conjugates (ADCs) and bispecific antibodies (BsAbs). In this work, an attempt has been made to develop novel Protein A affinity ligands having higher binding capacity than the ligands on commercial resins. There is currently no similar platform technology for purifying increasingly important non-mAb protein therapeutics. Protein therapeutics such as single domain antibodies, single chain variable fragments, Fab fragments, interferons, epoetins, clotting factors, growth factors, insulin and insulin like analogues, enzymes etc. have traditionally been purified using multiple column steps based on ion exchange, hydrophobic interaction, mixed mode, and ceramic hydroxyapatite chromatography. These multicolumn approaches require significant optimization and often result in low product yields and recoveries. Thus, scalable and cost-effective alternatives to these currently used approaches are needed. Furthermore, these alternative methods should be convenient to use and allow for easy technology transfer between clinical drug discovery, process development and manufacturing. In this work, we propose the use of pH-sensitive self-removing affinity tags as a potential solution. Purification strategies based on self-removing and self-precipitating tags have been developed previously for laboratory scale protein purification. However, these methods utilize pH sensitive contiguous inteins which suffer from premature cleavage, resulting in significant product loss during p (open full item for complete abstract)

Committee: David Wood (Advisor); Eduardo Reategui (Committee Member); Jeffrey Chalmers (Committee Member) Subjects: Biochemistry; Chemical Engineering

Master of Science, The Ohio State University, 2023, Animal Sciences

The finite nature of natural gas, along with environmental and health problems arising from combustion of fossil fuels, have spurred interest in development of clean and renewable alternative energy sources. Biohydrogen (H2) production through acetone-butanol-ethanol (ABE) fermentation by solventogenic Clostridium species, offers a promising way to achieve the goal of substituting fossil fuels with clean renewable energy sources. Low H2 yield and productivity with ABE fermentation by solventogenic Clostridium species is a major barrier to commercialization of biohydrogen. Consequently, metabolic engineering strategy is among the methods researchers have been exploring to develop industrially applicable H2 producing strains. This study, therefore, explored simultaneous deletion of negative transcription regulator (iscR) and overexpression of Fe-Fe hydrogenase genes (hydA) in C. beijerinckii NCIMB 8052 and C. pasteurianum ATCC 6013 to enhance H2 production. Specifically, chapter 3 (objective 1) explored metabolic engineering strategy to improve H2 production in C. beijerinckii. Using allele exchange construct for homologous recombination, simultaneous deletion of iscR from isc operon and overexpression of Fe-Fe hydA in the iscR locus in C. beijerinckii was conducted. The 2 copies of hydA in C. beijerinckii (hydAi and hydAii) were overexpressed separately in C. beijerinckii to generate recombinant strains: C. beijerinckii_hydAi and C. beijerinckii_hydAii. This strategy led to 1.2- and 1.3-fold increases in the growth of C. beijerinckii_hydAi and C. beijerinckii_hydAii, respectively, compared to C. beijerinckii_wildtype. Surprisingly, there was 2.9-, 1.5-, 1.4- and 1.7-fold decreases in acetone, butanol, ethanol, and total ABE produced, respectively, by C. beijerinckii_hydAi compared to C. beijerinckii_wildtype. Similarly, there was 3.7-, 1.7-, 1.9-, and 1.7-fold decreases in acetone, butanol, ethanol, and total ABE produced, respectively, by C. beijerinckii_hydAii (open full item for complete abstract)

Committee: Thaddeus Ezeji Dr. (Advisor) Subjects: Molecular Biology

Doctor of Philosophy, The Ohio State University, 2023, Biomedical Sciences

Gene therapy refers to the use of genetic material to treat disease through gene addition, gene correction/alteration, or gene knockdown. The goal of the technique is to address the root causes of disease rather than symptoms alone. Gene therapy holds promise for treating a wide range of diseases, including many genetic disorders and certain types of cancer. Gene therapy was first conceptualized in the 1970s and 1980s following advances in our knowledge of genetic material. The first clinical trials were rightly met with skepticism due to lack of concern for patient safety and regulatory bodies. Ultimately, the first attempts came too soon and resulted in various adverse events and widely publicized patient deaths. The field slowed in the 1990s and early 2000s as we began to understand the basic science of gene therapy vectors and expand a so-called “toolkit” of vectors to increase safety and efficacy. Subsequent clinical trials proved more promising and resulted in the approval of the first gene therapies. Currently, gene therapy remains a clinical reality, though more work is needed to expand the number of therapeutics and indications. Adeno-associated viral (AAV) vectors are one tool within the gene therapy toolkit. AAVs are thought to be relatively safe for gene transfer due to their largely non-integrative nature and tendency to trigger mild immunogenic responses. Certain challenges remain in the field to maximize the utility of AAVs as vectors, including the need for cell-specific targeting, appropriate levels of the therapeutic construct, and the ability to evade immunogenic responses. The development of recombinant AAVs (rAAVs) through engineering of the transgene cassette and viral capsid can address many of these concerns, as both can dictate cellular transduction, transgene expression levels, and immune responses. Other areas of optimization include the use of different administration routes and dosing schedules. The combination of all the above will be (open full item for complete abstract)

Committee: Lei Cao (Advisor); Dana McTigue (Committee Member); Takeshi Kurita (Committee Member); Allison Bradbury (Committee Member) Subjects: Endocrinology; Neurobiology; Neurosciences

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

Infectious hematopoietic necrosis virus (IHNV) is a deadly fish pathogen that poses a global threat to aquatic ecosystems and the aquaculture industry. For decades, research has focused on developing vaccine therapeutics utilizing a variety of techniques and strategies. While these studies have met with some success in identifying potential vaccine targets that provided protective immunity, a commercially viable IHNV vaccine is currently unavailable. Here we explore the relationship between the structure and function of the IHNV matrix (M) protein through the introduction of mutations that reduce anti-host effects, with the goal of developing a novel recombinant IHNV with reduced pathogenicity that provides protective immunity. Our data suggest that the N- and C-termini of IHNV M contribute to its antitranscriptional effects and protein stability and are therefore functionally important. IHNV M mutants that exhibited reduced anti-host effects, but retained wild type protein expression levels, are prime candidates for additional study and incorporation into a novel recombinant IHNV system established in these studies. Overall, our studies highlight the feasibility of developing novel IHNV antiviral candidates through a mutational strategy that will yield additional information about viral protein function.

Committee: Douglas W. Leaman Ph.D. (Advisor); Shulin Ju Ph.D. (Committee Member); Abimbola O. Kolawole Ph.D. (Committee Member) Subjects: Immunology; Microbiology; Molecular Biology; Virology

Doctor of Philosophy, The Ohio State University, 2021, Food Science and Technology

Salmonella and Campylobacter are leading bacterial agents both nationally and globally, making them high public health concerns. Both are significant important zoonotic pathogens commonly found in livestock. Poultry and poultry products are the most common sources of human infections. Infected poultry show little to no clinical signs, and risk the chance of entering the food system, potentially contaminating consumers. Direct links between the high load of bacteria in the chicken intestinal tract and the high contamination of poultry carcasses support the need for pre-harvest control. Previously, antibiotics were used to control bacterial infections and growth; but increasing occurrence of antibiotic resistant bacteria has caused laws and practices to shift. Targeting the control of foodborne pathogens in the pre-harvest stage can improve animal welfare and public health. Alternative treatment methods are needed to combat Salmonella and Campylobacter in production animals, improve antibiotic stewardship, and subsequently strengthen the economy. We discovered the antimicrobial efficacy of Lactobacillus acidophilus (LA), Lactobacillus rhamnosus GG (LGG), and Bifidobacterium animalis subsp. lactis (Bb12) in vitro. More importantly, we showed LGG significantly reduced Salmonella in the chicken cecum by 1.9 logs (P<0.001) at 10 days post infection. LGG was also able to inhibit the growth/ presence of other food safety significant Salmonella serovars in an agar well diffusion assay. Characterization of antibacterial activity of LGG revealed that although organic acids are present, the most crucial part of inhibition are the heat and protease stable peptides that were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). After which, we tested the efficacy of Lactobacillus rhamnosus GG (LGG) derived small peptides (P1-NPSRQERR, P2- PDENK, and P4-MLNERVK) against Salmonella Typhimurium (ST) in chickens and examined their antibacterial effects a (open full item for complete abstract)

Committee: Gireesh Rajashekara (Advisor); Anastasia Vlasova (Committee Member); Melvin Pascall (Committee Member); Scott Kenney (Committee Member) Subjects: Food Science; Microbiology

Master of Science in Biology, Cleveland State University, 2018, College of Sciences and Health Professions

Our laboratory has recently shown that strategic placement of rare and frequent synonymous codons in an mRNA ensuring that in the target host organism it would possess kinetics of translation similar to that of the natural host, increasing the yield of correctly folded soluble proteins. This observation is based on the idea that codon choice might have functional implications beyond amino acid coding and that synonymous codons may modulate in vivo/co-translational protein folding by tuning the kinetics of translation. To understand the link between synonymous codon usage, translation, and protein folding, our laboratory has utilized mammalian (bovine) eye lens gamma-B crystallin as a model protein and investigated its expression in vitro and in vivo in Escherichia coli. Two variants of the mRNA coding for gamma-B crystallin were designed and used, one with the codon usage that would be optimal for protein translation in E. coli (with an mRNA codon distribution similar to that found in Bos taurus, which was expected to result in more natural translation kinetics) and the other with unaltered codon composition un-optimal for translation in E. coli. Expression of the optimal variant yielded more soluble and protease-resistant protein than that of the variant with non-optimal codon distribution along the gamma-B crystallin mRNA. 2D NMR of the E. coli expressed and purified gamma-B crystallin variants detected considerable structural differences of the synonymous variants arising from different protein oxidation states and, particularly, of a cysteine pair (18-22) in the N-terminal domain of the protein. Disulfide bonds are known to stabilize protein structure and maintain correct folding, but it is not known when (co- or post- translationally) the disulfide bond forms in gamma-B crystallin. Previous data showed that structural differences between synonymous variants arise co-translationally. For the H variant this disulfide bond may form early during co-translational fo (open full item for complete abstract)

Committee: Anton Komar Dr. (Advisor); Roman Kondratov Dr. (Committee Member); Barsanjit Mazumder Dr. (Committee Member) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 1983, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Master of Science (MS), Wright State University, 2016, Biochemistry and Molecular Biology

The unique protein-based structure of Sucker Ring Teeth (SRT) of cephalopods have spurned research into the molecular design, physical characteristics, functionality and mechanical properties to explore biomimetic engineering and biochemical potential for eventual industrial production. Previous research has elucidated the potential for scientific and industrial exploitation. However, much of the previous research focused on the most abundant protein isoform of the sucker ring teeth, suckerin-19 (also known as suckerin-39) from the Jumbo or Humboldt Squid (Dosidicus Gigas). There is little known about the characteristics of the other 37 protein isoforms of Sucker Ring Teeth. Although the other isoforms have similar modular repeats in the primary and secondary structures, the other isoforms are smaller and may provide some additional clues into the biochemical characteristics of the suckerin genes. Of the 37 protein isoforms, the suckerin-12 isoform displayed some sequence and modular similarities to suckerin-19 that warranted further evaluation. The procedures and techniques used to study suckerin-12 focused on the expression and purification techniques, mechanical and structural analysis, and fine-tuning strategies for future functionalization. Experiments were performed to evaluate protein isoform suckerin-12 as a candidate to provide a suitable biopolymer for development of highly durable and strong biomaterials that rival other suckerin isoforms and may provide some insight into protein functionality in both dry and wet environments. By mimicking post-transcriptional cellular processes in an aqueous and dry environment, suckerin-12 displayed special physical and chemical characteristics to those seen in suckerin-19. Specifically, the procedures used to form testable suckerin-12 based materials via di-Tyrosine cross-linking required alternate methods than the ruthenium-based cross-linking observed in suckerin-19 studies. T (open full item for complete abstract)

Committee: Patrick Dennis Ph.D. (Advisor); Madhavi Kadakia Ph.D. (Committee Member); Yong-jie Xu Ph.D. (Committee Member) Subjects: Biochemistry; Biomedical Engineering; Materials Science; Microbiology; Molecular Biology; Nanotechnology; Polymers

Doctor of Philosophy, The Ohio State University, 2016, Chemical Engineering

Recent advances in upstream bioprocessing have largely increased the productivities of recombinant protein expression in various expression systems. This has led to an increasingly urgent need for rapid, cost-effective and large-scale downstream unit operations. Affinity tag-based purification provides an efficient platform to purify virtually any uncharacterized protein in a highly selective, high-throughput manner. With the combination of a self-cleaving intein, the purification and the tag removal processes can all be carried out in a single chromatography step, thus dramatically reducing the cost of downstream processing. Over the past decades, inteins have been genetically modified for better controllability and cleaving ability. However, unwanted in vivo premature intein cleaving remains a major bottleneck for applications in mammalian systems. In this dissertation, recent progresses in the control of intein cleaving will be discussed. A continuous delta I-CM intein and a zinc binding motif fusion allow the intein-tagged precursor proteins to be expressed in mammalian systems with less than 40-50% in vivo premature cleaving in the presence of 200 micro molar zinc chloride. An alternative, the Npu split intein system, has been shown to completely eliminate premature cleaving and has been altered into a pH sensitive cleaving intein. These efforts to control intein cleaving have led to the development of a robust and efficient platform technology for non-antibody recombinant protein downstream purification.

Committee: David Wood (Advisor); Andre Palmer (Committee Member); Shang-Tian Yang (Committee Member); Werner Tajrks (Committee Member) Subjects: Chemical Engineering

Doctor of Philosophy (PhD), Wright State University, 2015, Biomedical Sciences PhD

Organophosphates (OPs) are highly toxic insecticides and nerve agents that have been designed to inhibit the hydrolysis of acetylcholine by binding to the serine active site of acetylcholinesterase (AChE). They are one of the most common causes of human poisoning worldwide and are frequently intentionally used in suicides in agricultural areas. For this reason, there is a need for therapeutics to rescue those from intoxication. Obvious ethical concerns prevent humans from being subjected to OP exposure for therapeutic efficacy and safety testing. Therefore, animal surrogates for humans must be appropriately selected. A new paradigm, described herein, incorporating both in silico and in vitro techniques may be able to reduce the use of animals in biomedical research. Historically, the guinea pig (Cavia porcellus) has been believed to be the best non-primate model for OP toxicology and therapeutic development because, similarly to humans, guinea pigs have low amounts of OP metabolizing carboxylesterase (CaE) in blood and tissues. To explore the hypothesis that guinea pigs are the most appropriate human substitute for studying OP toxicology and therapeutic development, I cloned, purified and enzymatically compared a recombinant guinea pig acetylcholinesterase (gpAChE) with the human and mouse enzyme variants. The guinea pig, mouse and human apparent inhibition constants for diisopropyl fluorophosphate were found to be 8.4+/-0.6 uM, 4.9+/-0.6 uM and 0.42+/-0.01 uM, respectively, indicating that species differences exist for OP inhibition. Furthermore, I developed a mechanistic quantitative structure-property relationship (QSPR) to predict OP and therapeutic tissue: plasma partition coefficient (Kt:pl) parameters for each species. Differences in tissue lipid, water and protein content contributed to species specific Kt:pl. For example, guinea pig and human lung Kt:pl predictions for paraoxon were found to be 0.3 and 0.17, respectively. Biological and chemical (open full item for complete abstract)

Committee: Jeffery Gearhart Ph.D. (Advisor); Adrian Corbett Ph.D. (Committee Member); James Lucot Ph.D. (Committee Member); Mateen Rizki Ph.D. (Committee Member); Gerald Alter Ph.D. (Committee Member) Subjects: Biochemistry; Molecular Biology; Pharmacology; Toxicology

Master of Science, Northeast Ohio Medical University, 2014, Integrated Pharmaceutical Medicine

ABSTRACT Introduction: Healthy bone constantly remodels, in balance between osteoclast bone resorption and osteoblast bone formation. Osteoclasts differentiate in vitro from osteoclast progenitor cells in the presence of macrophage-colony stimulating factor and receptor activated nuclear factor κB-ligand, while osteoblasts differentiate in vitro from mesenchymal stem cells in the presence of ascorbic acid, β-glycerol phosphate, and dexamethasone. Imbalance between the function of osteoclasts and osteoblasts results in disease. In osteoporosis, bone resorption dominates, leading to decreased bone mass. Osteoporosis is of critical concern to an aging population, and new treatments are needed. Osteoactivin is an anabolic bone growth factor in vivo, causing increased bone mass when injected into mice. In vitro, osteoactivin has been shown to increase osteoblast differentiation and function. However, the effects of osteoactivin on osteoclasts are not as well defined. In this study, we hypothesized that recombinant osteoactivin inhibits osteoclastogenesis. In the future, osteoactivin could be a therapeutic agent for bone healing and preventing or even reversing osteoporosis. Material and Methods: Osteoclast progenitor cells were isolated from C57BL/6 murine tibias, femurs, and humeri. Several aspects of osteoclastogenesis were tested in the presence of dose-dependent recombinant osteoactivin. First, proliferation and survival assays, with and without macrophage-colony stimulating factor respectively, were conducted on the osteoclast progenitor cells in the presence of recombinant osteoactivin. Next, the osteoclast progenitor cells were differentiated into osteoclasts in the presence of recombinant osteoactivin. Osteoclast tartrate resistant acid phosphatase activity was measured and stained as a marker for terminally differentiated osteoclasts. Differential cell counting was performed for osteoclasts containing 3-20 and >20 nuclei. Two functiona (open full item for complete abstract)

Committee: Fayez Safadi Ph.D. (Advisor); Werner Geldenhuys Ph.D. (Committee Member); Moses Oyewumi Ph.D. (Committee Member) Subjects: Biology, Cell

PhD, University of Cincinnati, 2005, Medicine : Pathobiology and Molecular Medicine

Apolipoprotein (apo) A-IV is a protein synthesized by the small intestine in response to lipid absorption. It has been proposed to play a role in cholesterol efflux, lipoprotein metabolism and food intake. Unfortunately, little information on its structure/function relationship is known. Therefore, it was important to establish a recombinant expression system for apoA-IV so deletion mutagenesis could be performed to achieve the specific aims. The following aims address the hypothesis that specific regions of apoA-IV are involved in its ability to interact with lipid and inhibit food intake. Aim 1: Determine the region of apoA-IV that is responsible for its ability to bind lipid as well as to identify the generalities of its structure. Initially, it was found that removing the C-terminal 44 amino acids from human apoA-IV caused a significant increase in lipid binding ability as compared to WT. Eventually, a smaller region from amino acid 333-343 was established as the ‘inhibitory' region of apoA-IV at the C-terminus. Using the Δ333-343 as a template, the N-terminus was also mutated and it was found that the N-terminal amino acids from 11-20 were required for the mutants to be fast lipid binders. Therefore, we propose there is an interaction between the ‘inhibitory' region from 333-343 and the N-terminus of the protein that does not allow the proper conformation for accelerated lipid binding. Aim 2: Determine the region of apoA-IV that is responsible for its role in the inhibition of food intake. Originally, several C-terminal deletion mutants were made in rat apoA-IV. The mutants were injected into the 3rd ventricle of rat brains and their food intake was determined. It was found that the food intake region was located in the N-terminal 116 amino acids. Next a 3rd mutant was made that removed the N-terminal 61 amino acids. This mutant did not inhibit food intake as compared to WT suggesting the region involved in food intake is located in the first 61 amino acids. In (open full item for complete abstract)

Committee: Dr. William Davidson (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2006, Pathology

Respiratory syncytial virus (RSV) is the most common viral cause of lower respiratory tract disease in infants and children. The incidence of RSV infection worldwide is high among infants, children, immunosuppressed adults, and the elderly. Despite the fact that nearly all individuals are infected by the age of two, complete immunological protection is never attained and re-infections are common throughout life. Currently, there is no licensed vaccine or effective therapy against RSV, and vaccine development has been hampered by the legacy of enhanced lung disease observed during trials of a formalin-inactivated, alum precipitated whole virus RSV (FIRSV) formulation. Other obstacles in designing an effective vaccine against RSV include its weak immunogenicity and the ability of viral proteins to induce an allergic-type memory response. Since the upper respiratory tract is never effectively protected against RSV infection, of particular need are vaccines that induce a sterilizing mucosal immune response. The experiments detailed herein demonstrate poor stimulation of innate defenses following RSV infection and test the hypothesis that the inability to induce a robust innate response following RSV infection hinders the development of a protective adaptive immune response against this virus and may therefore contribute to the high frequency of re-infections throughout life.

Committee: Joan Durbin (Advisor) Subjects: Health Sciences, Immunology

Doctor of Philosophy, The Ohio State University, 2005, Veterinary Preventive Medicine

Noroviruses (NoV) and sapoviruses (SaV) are emerging pathogens that cause gastroenteritis in humans and animals. Whether NoVs and SaVs are zoonotic pathogens is unclear due to limited studies of animal NoVs and SaVs. We screened for porcine NoVs by reverse transcription-PCR (RT-PCR) on fecal samples from US pigs. Based on sequence analysis of the 3 kb of the genomes of 5 porcine NoVs, 3 genotypes within genogroup II (GII) and two potential recombinant strains were identified. One genotype of porcine NoVs was genetically and antigenically related to human NoVs and replicated in gnotobiotic pigs. By similar approaches, porcine SaVs were identified as genetically diverse viruses comprising at least 2 genogroups. Two porcine SaVs were potential recombinants. One porcine SaV strain (Po/SaV/MI-QW19/2002/US) was genetically most closely related to human SaVs based on the partial RdRp sequence (286 nt). Furthermore, several primer pairs were designed and evaluated for the detection of porcine NoVs and SaVs, respectively. An internal control RNA was developed and used in RT-PCR to monitor for RT-PCR inhibition. Microwell hybridization assays were developed to confirm the porcine NoV- and SaV-specific amplicons. Finally, a prevalence study of porcine NoVs and SaVs in US swine was performed by the newly developed RT-PCR-hybridization assays. The porcine NoVs were detected exclusively from finisher pigs in 4 of 7 farms and 1 slaughterhouse with an overall prevalence of 20% in finisher pigs. Porcine SaVs were detected from all ages of pigs. The prevalence of porcine SaVs was 62% overall, lowest in nursing pigs (21%) and highest in post-weaning pigs (83%). Mixed infections of NoVs and SaVs were common in finisher pigs with an overall prevalence of 27% among NoV- or SaV-positive pigs. These findings have improved our understanding of the genetic diversity of porcine NoVs and SaVs and their relationships to human strains. Certain porcine NoVs and SaVs are genetically or antigenical (open full item for complete abstract)

Committee: Linda Saif (Advisor) Subjects:

Doctor of Philosophy (PhD), University of Toledo, 2011, College of Medicine

Benzodiazpines (BZs) are clinically useful anxiolytics, sedatives, and anticonvulsants. Their mechanism of action is positive allosteric modulation of γ-aminobutyric acid type A (GABAA) receptors, the main inhibitory neurotransmitter receptors in the mammalian central nervous system. Long-term administration of BZs and other positive allosteric GABAA receptor modulators, neurosteroids, barbiturates, and ethanol can lead to physical dependence manifested by a characteristic withdrawal syndrome. A common mechanism proposed to contribute to this withdrawal syndrome is functional up-regulation of L-type voltage-gated calcium channels (L-VGCCs). Our lab models BZ dependence using a 1-week oral treatment of rats with flurazepam (FZP) followed by 1 or 2 days of withdrawal. This treatment paradigm resulted in a near doubling of voltage-gated Ca2+ currents in hippocampal CA1 neurons. Enhanced L-VGCC-mediated Ca2+ influx may activate Ca2+/calmodulin-dependent protein kinase II (CaMKII), which potentiated excitatory synaptic function in CA1 neurons correlating with expression of FZP withdrawal anxiety. The current studies tested three hypotheses: 1) GABAA receptor modulators directly inhibit recombinantly expressed L-VGCCs containing neuronal α1 subunits, Cav1.2 or Cav1.3; 2) L-VGCC subunit expression is increased in the rat hippocampal CA1 region; and 3) CaMKII enhances CA1 excitatory synaptic function via activation and autophosphorylation at Thr286 and/or enhanced localization to the postsynaptic density (PSD). The findings suggested that while the barbiturate pentobarbital and ethanol directly inhibit L-VGCCs at clinically relevant concentrations, the concentrations of BZs and neurosteroids required to inhibit recombinant L-VGCCs were likely too high to be clinically relevant. Interestingly, Cav1.2 channels were more sensitive to inhibition by pentobarbital and FZP and were less sensitive to inhibition by the L-VGCC benzothiazepine (BTZ) antagonist, diltiazem, than Cav1.3 (open full item for complete abstract)

Committee: Elizabeth Tietz Ph.D. (Committee Chair); Zi-Jian Xie Ph.D. (Committee Member); David Giovannucci Ph.D. (Committee Member); Scott Molitor Ph.D. (Committee Member); Bryan Yamamoto Ph.D. (Committee Member) Subjects: Neurosciences

Doctor of Philosophy in Medical Sciences (Ph.D.), University of Toledo, 2006, College of Graduate Studies

Evaluation of recombinant proteins of C. posadasii for their protective efficacy in a murine model of coccidioidomycosis has revealed several potential vaccine candidates. One of the most promising is a proline rich-antigen (Ag2/Pra). We have demonstrated that vaccination of C57BL/6 and BALB/c mice with the combination of rAg2/Pra plus rPra2 enhanced protection against a lethal intranasal challenge with Coccidioides, compared to vaccination with either of the single antigens. Enhanced protection was based both on higher percentage of surviving mice (as demonstrated by the BALB/c strain) or significantly better clearance of the fungal burden (C57BL/6). Despite the related primary structures of Ag2/Pra and Pra2, we have identified differences in their helper T cell (Th) and continuous B cell epitope profiles using immune CD4+ T cells and sera from both C57BL/6 and BALB/c mice. The mapping of the Th and continuous B cell epitopes of Ag2/Pra and Pra2 induced by the immunization of C57BL/6 (H-2b) mice with either rAg2/Pra or rPra2 facilitated the rational design of three divalent chimeric antigens composed of immunoreactive domains selected from both antigens. The three recombinantly expressed and purified divalent chimeric antigens were evaluated for their ability to protect C57BL/6 mice from both sub-lethal and lethal intranasal challenges with Coccidioides (47 and 90 arthroconidia, respectively). The results of these protection experiments provide evidence that the three divalent recombinant chimeric antigens we designed afford a level of protective efficacy that in some cases is statistically no different than the full-length rAg2/Pra plus rPra2 divalent vaccine. However, comparisons of pulmonary fungal burdens (sub-lethal challenge) and survival rates (lethal challenge) indicate that the chimeric antigens in all cases are noticeably (and in some cases statistically) less protective than the combination of rAg2/Pra plus rPra2. These observations suggest that, though (open full item for complete abstract)

Committee: Garry Cole, Ph.D. (Advisor) Subjects:

Doctor of Philosophy, Case Western Reserve University, 1992, Biochemistry

Recombinant mouse β-nerve growth factor was expressed in bacterial and baculovirus systems. Expression of the recombinant NGF in a bacterial gene fusion system reached a high level of over 4 mg from one liter of bacterial cell culture. The recombinant NGF had the same molecular weight and shared the same N-terminal amino acid sequences as that of the native mouse β-NGF (mNGF). The recombinant NGF was immunoreactive to anti-NGF antiserum and was biologically active. However, the protein did not have the same specific biological activity as that of the native mNGF, indicating that the recombinant NGF did not completely fold into a native conformation. The problems of folding of the recombinant NGF were at least partially attributed to interchain disulfide formation between NGF monomers. Recombinant NGF was also expressed in a baculovirus system to a level of 1 mg from one liter of insect cell culture. Isolation of the recombinant NGF (rNGF) indicated that it was present in three stable, yet distinct forms. The three forms of rNGF exhibited distinct biological activities and differed in their abilities to compete with mNGF for high affinity NGF receptors. However, they were chemically and structurally indistinguishable from each other. All three forms of rNGF differed from mature mNGF from mouse submaxillary gland in that the C-terminal Arg-Gly dipeptide had not been proteolytically removed. Removal of the C-terminal dipeptide by γ-NGF peptidase treatment converted the three forms into a single form identical to mature mNGF. The above results demonstrate that a single polypeptide of rNGF, due to the presence of a C-terminal dipeptide, exhibits three stable dimeric protein conformations with distinct biological activities. The apparent lack of γ-NGF peptidase in the nervous system raises the possibility that the biologically significant form of NGF may differ from mature mNGF; such a difference may be of physiological relevance. Several NGF mutants from bacterial and ba (open full item for complete abstract)

Committee: Kenneth Neet (Advisor) Subjects: Chemistry, Biochemistry

MS, University of Cincinnati, 2013, Arts and Sciences: Biological Sciences

Pneumocystis is a genus of opportunistic fungi responsible for a type of pneumonia in immunocompromised hosts. The pathways by which these organisms synthesize sterols have been found to be unique amongst fungi. While most fungi have ergosterol as their main sterol, its de novo synthesis does not occur in Pneumocystis. However, Pneumocystis does produce unique Δ7 C28 and C29 24-alkyl sterols, while using cholesterol scavenged from the mammalian host as its main sterol. This thesis presents several distinct lines of research, all of which address sterol biosynthesis in Pneumocystis. The first aim was to elucidate the sterol biosynthesis pathways utilized by the organism. For this, cryopreserved P. carinii were intratracheally administered to corticosteroid-immunosuppressed rats to initiate infection. When moribund, the rats were sacrificed and P. carinii organisms were isolated and purified from the infected lungs. These organisms were incubated with labeled sterol precursors, namely [2-13C] leucine to determine whether this amino acid is a precursor of the organism's sterols. Sterols were then isolated from the organisms and analyzed by Nuclear Magnetic Resonance (NMR) spectroscopy. The second aim was to continue studies initiated in our laboratory on a critical P. carinii enzyme S-adenosylmethionine: sterol C24-methyltransferase (SAM:SMT) expressed in the budding yeast Saccharomyces cerevisiae. This protein, coded by the erg6 gene, has been previously expressed in the bacterium Escherichia coli and the ciliate Tetrahymena thermophila, but these systems were shown to be of limited utility. In addition to S. cerevisiae, fission yeast Schizosaccharomyces pombe was studied as a potential expression system. Work previously done in the laboratory initiated studies on the expression of the P. carinii SAM:SMT in the S. cerevisiae erg6 null mutant. My contribution to this project was to provide gas-liquid chromatography (GLC) analyses of the sterol profil (open full item for complete abstract)

Committee: Edna Sayomi Kaneshiro Ph.D. (Committee Chair); Charlotte Paquin Ph.D. (Committee Member); Jodi Shann Ph.D. (Committee Member) Subjects: Biology