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Agu, Chidozie VictorUse of process design and metabolic engineering to enhance bioconversion of lignocellulosic biomass and glycerol to biofuels
Doctor of Philosophy, The Ohio State University, 2016, Animal Sciences
Recent efforts to reduce dependency on food-based substrates for industrial applications aim towards the use of inexpensive and readily available non-food based substrates such as lignocellulosic biomass (LB) and biodiesel-derived glycerol. Interestingly, the utilization of lignocellulosic sugars for biofuel production is contingent on the disruption of recalcitrant LB cell wall structure prior to enzyme hydrolysis. Disruption and hydrolysis processes generate lignocellulose-derived microbial inhibitory compounds (LDMIC) including acids, aldehydes and phenolics. Additionally, fermentation of glycerol to butanol, a next-generation biofuel, is hampered by the inability of Clostridium beijerinckii NCIMB 8052, a butanol fermentation workhorse, to efficiently metabolize glycerol. Therefore, this study investigated novel strategies for enhancing butanol and ethanol production through process design and metabolic engineering. Towards process design, the bacterium Cupriavidus basilensis ATCC®BAA-699 was used to detoxify 98% of the LDMIC present in acid-pretreated Miscanthus giganteus (MG) lignocellulosic biomass hydrolysates. Fermentation of the detoxified MG hydrolysates by C. beijerinckii resulted in 70%, 50%, and 73% improvement in acetone-butanol-ethanol (ABE) concentration, yield and productivity, respectively, when compared to the fermentation of undetoxified MG hydrolysates. The second objective was to explore metabolic engineering strategies to enhance glycerol utilization by C. beijerinckii and improve butanol production in the presence of LDMIC. To realize this objective, genes that encode glycerol dehydrogenases (Gldh) and dihydroxyacetone kinase (Dhak) in a hyper-glycerol utilizing bacterium (Clostridium pasteurianum ATCC 6013) were systematically cloned into C. beijerinckii. By over-expressing two C. pasteurianum Gldh genes (dhaD1+gldA1) as a fusion protein in C. beijerinckii, we achieved 50% increase in cell growth, ABE production (up to 40%), and enhanced rate of furfural detoxification (up to 68%) during the fermentation of furfural-challenged (4 to 6 g/L) glucose+glycerol medium. Further, co-expression of dhaD1+gldA1 resulted in significant payoff in cell growth (57%), glycerol consumption (14%), and ABE productivity (27.3%) compared to over-expression of a single Gldh. In parallel, while co-expression of dhak and gldA1 in C. beijerinckii improved glycerol consumption by 37% relative to the plasmid control, over-expression of all three genes (dhaD1+gldA1+dhak) improved butanol production by >50% in the presence of 5 and 6 g/L furfural relative to the plasmid control. Objective 3 aimed to develop a high-throughput alcohol dehydrogenase (ADH)-dependent assay for screening hyper- or hypo- butanol producing C. beijerinckii mutant libraries. Screening of the activities of ADHs from different microorganisms showed that Thermotoga hypogea derived ADH has ~7-fold activity towards butanol than ethanol. It was rationalized that T. hypogea ADH can be used to selectively quantify butanol in the presence ofethanol (e.g., in ABE broth). Objective 4 aimed to use allopurinol to inhibit xanthine dehydrogenase/oxidase and improve ethanol fermentation of LB hydrolysates by Saccharomyces cerevisiae. Allopurinol increased S. cerevisiae growth (19%), ethanol titer (21%), ethanol productivity (20%), ethanol yield (24%), and the chronological lifespan of S. cerevisiae (>16 h) during the fermentation of 100% corn stover hydrolysate. Taken together, this study encompasses novel strategies to enhance LB and glycerol utilization and potentially improve the economics of biobutanol and bioethanol production.

Committee:

Thaddeus Ezeji (Advisor); Jeff Firkins (Committee Member); Zhongtang Yu (Committee Member); Macdonald Wick (Committee Member); Gireesh Rajashekara (Committee Member)

Subjects:

Agriculture; Animal Sciences; Biochemistry; Biology; Microbiology; Molecular Biology

Mojica, Maria FernandaACTIVE SITE LOOPS AND SUBSTRATE PROFILE IN CLASS B METALLO-ß-LACTAMASES: FROM MECHANISTIC STUDIES TO THERAPY DEVELOPMENT
Doctor of Philosophy, Case Western Reserve University, 2017, Biochemistry
ß-lactams are the most important class of antibiotics used to treat infections caused by both Gram-negative and Gram-positive bacteria. ß-lactams are characterized by their four membered cyclic amide ring, and act by inhibiting transpeptidases involved in cell wall synthesis. In particular, carbapenems, the “last resort” ß-lactam antibiotics, are used to treat the most resistant Gram-negative pathogens. Unsurprisingly, bacteria developed several mechanisms to resist the action of ß-lactams, including efflux, reduced permeability, altered transpeptidases, and most importantly, inactivation by ß-lactamases, enzymes that selectively hydrolyse the ß-lactam ring, rendering the antibiotics ineffective against their natural targets. Metallo-ß-Lactamases (MßLs) are metal-dependent enzymes of particular interest and concern given i) their ability to hydrolyse and provide resistance to virtually all ß-lactams including carbapenems; and ii) unavailability of clinically useful MßL inhibitors. Structurally, these enzymes feature a aß/ßa fold, in which the Zn-containing active site is positioned within a shallow groove formed by the interface of the a and ß domains, and is surrounded by active site loops (ASL). Herein, we study the effects that amino acid substitutions in the ASLs of two clinically relevant MßLs, VIM-2 and NDM-1, have on substrate specificity, enzyme catalysis and stability. We show that the substitution Arg228Leu at the ASL-10 of VIM-like enzymes expands the substrate spectrum to include 3rd and 4th cephalosporins, while changes at the ASL-3 in NDM-1 not only affect substrate catalysis but also alter the metal coordination center. We also introduce a novel class of mechanism-based MßL inhibitors, the bisthiazolidines, and demonstrate their in vitro effectiveness at restoring the susceptibility toward carbapenems in MßL-producing gram negative bacteria. We then investigate the sequence diversity of the L1 MßL of Stenotrophomonas maltophilia, an emerging multi-drug resistant bacterium for which antibiotic treatment options are limited. Based upon our knowledge of the mechanism and biochemistry of MßL and other ß-lactamases, we show that a novel antibiotic combination, ceftazidime-avibactam and aztreonam, is effective against S. maltophilia and other MßL-producers. Lessons learned during the development of this thesis provide valuable information concerning the catalytic mechanism of these enzymes, towards the development of effective MßL inhibitors and to the testing of novel combination therapies.

Committee:

BONOMO ROBERT (Advisor); MERRICK WILLIAM (Committee Chair); van den AKKER FOCCO (Committee Member); CAREY PAUL (Committee Member); VILA ALEJANDRO (Committee Member)

Subjects:

Biochemistry; Microbiology

Guzzo, Marissa BlaireMolecular Mechanisms Underlying the Intrinsic Sulfonamide Resistance in Bacteria
Doctor of Philosophy, Case Western Reserve University, Molecular Biology and Microbiology
The increasing frequency of MDR- and XDR-bacterial infections continues to pose a serious threat to global public health. This concern has not only intensified with the reduced efficacy of currently available treatments but with the significant decline in drug development and discovery. Through knowledge and understanding of bacterial intrinsic molecular resistance mechanisms, an emerging concept of “targeting resistance," or potentiation, has gained much interest as a means of re(sensitizing) pathogenic bacteria to available antibiotics. With a renewed interest in reviving the use of antifolates, such as sulfonamides, in the treatment of Mycobacterium tuberculosis (Mtb) infections, our lab developed a chemo-genomic screening approach for determining antifolate resistance determinants in mycobacterial species. We found that disruption of 5,10-methenyltetrahydrofolate (5,10-CH+-H4PteGlun) synthetase (MTHFS), an enzyme responsible for the conversion of 5-formyltetrahydrofolate (5-CHO-H4PteGlun, folinic acid) to 5,10-CH+-H4PteGlun in the folate-dependent one-carbon (1C) metabolic network led to severe defects in cellular folate homeostasis thus weakening the intrinsic antifolate resistance in both Mycobacterium smegmatis and Escherichia coli resulting in enhanced sensitivity to sulfonamides. Further investigation into folate mediated 1C metabolism led to the discovery of a novel mechanism of intrinsic sulfonamide resistance referred to as the methylfolate (MF) trap. Until now, this phenomenon has only been characterized in mammals as a means of explaining the relationship of Vitamin B12 (VB12), folate, and homocysteine (Hcy) homeostasis in many medical conditions such as anemia, neural tube defects, and Alzheimer’s dementia. This trap occurs as a result of defects in the multi-cycling reaction catalyzed by the B12-dependent methionine synthase (MetH). We demonstrate that in the presence of sulfonamide, MF trap induction, either through VB12 deficiency and/or mutations affecting MetH enzymatic activity, causes impaired homeostasis of folate and related metabolites as well as the cytotoxic accumulation of Hcy-thiolactone. This metabolic blockage ultimately leads to thymineless death rendering pathogenic bacteria, such as M. tuberculosis, E. coli, Pseudomonas aeruginosa, and Salmonella typhimurium, more susceptible to existing sulfonamides. We also provide proof-of-concept demonstrating that chemical induction of the MF trap by inhibiting VB12 uptake represents a novel method of potentiation by enhancing the antimicrobial activity of available, clinically approved sulfonamides.

Committee:

Arne Rietsch (Committee Chair); Liem Nguyen (Advisor); W. Henry Boom (Committee Member); Robert Bonomo (Committee Member)

Subjects:

Microbiology

Fyffe, Deanna LynneMethods to Monitor Lake Erie's Harmful Algal Blooms: A Fellowship with the Cooperative Institute for Great Lakes Research
Master of Environmental Science, Miami University, 2017, Environmental Sciences
To fulfill the professional experience requirement for a Master of Environmental Science degree at Miami University, I completed a fellowship with the Cooperative Institute for Great Lakes Research (CIGLR). My work involved investigating data trends of recent Lake Erie harmful algal blooms and potential monitoring methods. Data trends revealed monitoring stations closest to the mouth of the Maumee River had the highest average cyanobacteria concentrations. Bloom biomass distribution tended to favor the surface of the water column but was likely influenced by wind speed in 2016 and 2017. I also compared chlorophyll-a data from a CIGLR-owned bbe FluoroProbe to laboratory extraction data. The bbe FluoroProbe provided consistent results when used in the field and in the laboratory, but generally identified less chlorophyll-a than pigment extraction methods. Additionally, I performed field, laboratory, and analytical work to evaluate commercial in situ fluorometers. Due to proprietary reasons, the individual fluorometer data could not be presented in this report. I provided an example field deployment evaluation for the YSI EXO2 Multiparameter Sonde, a CIGLR-owned fluorometer that was used during the field tests for ancillary data. The EXO2 ultimately had low accuracy when compared to traditional laboratory methods, but both methods produced similar data trends.

Committee:

Suzanne Zazycki, JD (Advisor); Bartosz Grudzinski, PhD (Committee Member); Vanni Michael, PhD (Committee Member)

Subjects:

Aquatic Sciences; Environmental Health; Environmental Science; Environmental Studies; Freshwater Ecology; Microbiology; Water Resource Management

Keywords:

Lake Erie, Great Lakes, harmful algal bloom, HAB, water quality, in situ, fluorometer, cyanobacteria, biomass, microcystis, chlorophyll, phycocyanin, pigment, research, laboratory, EXO2, FluoroProbe, NOAA, GLERL, CIGLR, monitoring

Wu, XiaojunIdentification of two novel in vivo-upregulated Francisella tularensis proteins involved in metal acquisition and virulence
Doctor of Philosophy (PhD), University of Toledo, 2016, Biomedical Sciences (Infection, Immunity, and Transplantation)
Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs) are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal binding and uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. We have identified two new proteins, FTT0267 (named fmvA, for Francisella metal and virulence) and FTT0602c (fmvB), which are homologs of those iron acquisition genes and demonstrated that both are upregulated during mouse infections. Based on sequence homology and in vivo upregulation, we hypothesized that FmvA and FmvB are OMPs involved in metal acquisition and virulence. Despite sequence similarity to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither fmvA nor fmvB mutants exhibited growth defects in iron limitation. However, among other metals examined in this study, magnesium limitation significantly induced fmvB expression, fmvB mutant was found to express significantly higher levels of lipopolysaccharide (LPS) in magnesium-limiting medium, and increased numbers of surface protrusions were observed on fmvB mutant in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of fmvB mutant revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in fmvB mutant, compared with wild-type F. tularensis. To provide further evidence for the potential role of FmvB in magnesium uptake, we demonstrated that FmvB was outer membrane-localized. Finally, both fmvA and fmvB mutants were found to be significantly attenuated in mice and cytokine analyses revealed that fmvB mutant-infected mice produced lower levels of pro-inflammatory cytokines, including GM-CSF, IL-3, and IL-10, compared with mice infected with wild-type F. tularensis. Taken together, these studies have characterized two previously-unstudied F. tularensis proteins, have shown that both play roles in F. tularensis virulence, and provide new insights into the importance of magnesium for intracellular pathogens.

Committee:

Jason Huntley (Committee Chair); Robert Blumenthal (Committee Member); William Maltese (Committee Member); Kevin Pan (Committee Member); R.Mark Wooten (Committee Member)

Subjects:

Biology; Biomedical Research; Health Sciences; Immunology; Microbiology

Keywords:

Francisella tularensis; outer membrane proteins; virulence; metal acquisition; hypothetical proteins

McDonald, Kristina MarieMycoplasma bovigenitalium qPCR Detection and Multilocus Sequence Typing Strain Differentiation
Doctor of Philosophy, The Ohio State University, 2017, Comparative and Veterinary Medicine
Mycoplasma bovigenitalium (Mbvg) is a common inhabitant of the reproductive tract of diseased and apparently normal cattle. The bovine mycoplasma has been associated with multiple reproductive tract disorders in both male and female cattle. Additionally, Mbvg can colonize the prepuce and distal urethra of bulls, often without producing disease, resulting in Mbvg contaminated semen. The use of Mbvg-contaminated semen for AI has been reported as a potential source of infection for the bovine female genital tract. Although the pathogenic nature of Mbvg has been reproduced experimentally, it is not evident what triggers certain animals to exhibit disease while others remain asymptomatic carriers. Therefore, it is critical to increase the sensitivity of Mbvg detection as well as improve our knowledge regarding the pathogenicity and epidemiology of Mbvg. The first objective of this study was to develop and validate a TaqMan qPCR for the rapid detection of Mbvg in bovine semen. The assay exclusively amplified 81 field and laboratory strains of Mbvg when tested against a panel 15 bovine mycoplasma species. Fresh bovine semen samples (n=77) were assessed for Mbvg by qPCR and culture. M. bovigenitalium was recovered in 57 (74%) semen cultures and detected by qPCR in 62 (80.5%) samples. The rapid nature of the assay (results obtained in 5 h) may provide an alternative diagnostic and research tool for the detection and quantification of Mbvg in bovine semen samples as opposed to traditional culture, which requires up to 10 days. The second objective of this study was to develop a Multilocus Sequence Typing (MLST) assay for Mbvg strain differentiation. The sequences of three targeted loci (ppa, tpiA, rpoB) were analyzed from a collection of 86 Mbvg isolates from around the world. A high degree of nucleotide variability was observed and the isolates were organized into 61 strain types (ST). A phylogenetic tree constructed from the concatenated sequences displayed coherent relationships between branching and the geographic and species origin of the isolates. The third objective of the study was to assess Mbvg in bovine semen using MLST to identify potentially pathogenic strains. Semen samples from 41 bulls were cultured for mycoplasma. A total of 33 Mbvg isolates were recovered (80.5%) and subjected to MLST. Despite isolates originating from the same location, a high degree of nucleotide variability was present resulting in 24 STs. Phylogenetic analysis of the concatenated sequences revealed four distinct clusters (strain family A-D). Fertility potential (SCR) and six months (± 3 months from collection date) semen quantity/quality data for each bull were compared to strain family. A statistically significant association was observed between strain family and SCR (P = 0.002). Further analysis revealed that bulls carrying strain family C had significantly lower SCR than bulls negative for Mbvg (P = 0.006). These findings may allow the development and refinement of additional hypotheses for future research studies to identify additional carriers of Mbvg strain family C, assess the impact of Mbvg on bull fertility, and intervention strategies to control and prevent its propagation in cattle.

Committee:

Thomas Wittum (Advisor); Gustavo Schuenemann (Advisor); Gregory Habing (Committee Member); Christopher Premanandan (Committee Member); James M. DeJarnette (Committee Member)

Subjects:

Animal Sciences; Epidemiology; Microbiology; Molecular Biology

Keywords:

Mycoplasma bovigenitalium; qPCR; MLST; bovine semen; genotyping; fertility

Agans, Richard ThomasModeling Effects of Diet on Human Gut Microbiota
Doctor of Philosophy (PhD), Wright State University, 2016, Biomedical Sciences PhD
The human gut microbiota is integrally involved in the metabolism of nutrients contained within the human diet. Studies into human nutrition have primarily been carried out using human and animal models. These studies are extremely important in our understanding of human nutrition, however, suffer from inherent limitations including unique microbial compositions between individuals, compliance in human studies, inability to carry out mechanistic studies, and inability to interrogate proximal regions of the gut without applying invasive techniques. In vitro gut simulator systems circumvent many of these limitations in animal and human models by allowing control of gut environmental conditions, decreasing variability observed between subjects, and enabling mechanistic investigations and interrogations of inaccessible regions of the gut. In this work a custom biofermentation system, the human gut simulator, was designed, validated, utilizing previously reported gut conditions, capable of temperature, pH, and atmosphere regulation, nutrient transit, and it allows real-time sampling of vessel contents or addition of exogenous agents. The human gut simulator was further employed to the study of gut microbiota response to dietary long chain fatty acids as a sole nutrient source, following stabilization on a rich `western’ medium. Microbiota showed rapid responses to the transition from western to fat medium; where a lack of carbohydrates and proteins resulted in decreased community density. Specific members of the microbiota were capable of utilizing long chain fatty acids, including Bilophila, Alistipes, and Escherichia/Shigella. Interestingly, members of the microbiota incapable of metabolizing long chain fatty acids included beneficial microbes Roseburia, Bifidobacterium, and Akkermansia. Ordination and principal response curves analyses highlighted a significant effect of medium change on shifts in microbial composition over time. In conjunction with in vitro studies, human volunteers were enrolled to assess responses of microbiota to diets high in proteins, carbohydrates, or fats. Microarray analysis revealed specific individual host responses to test diets with smaller community wide effects. Increasing the amount of protein in the diet had a positive impact on relative abundance of Akkermansia, Alistipes, Enterococcus, and Lactococcus, while higher carbohydrates and fats resulted in higher abundances of Bifidobacterium, and Alistipes and Escherichia/Shigella, respectively. Together these results indicate that the Human Gut Simulator allows for robust studies of the human gut microbiota, and offers a foundation for conducting nutritional interventions in human subjects.

Committee:

Oleg Paliy, Ph.D. (Advisor); Volker Bahn, Ph.D. (Committee Member); Nancy Bigley, Ph.D. (Committee Member); Mill Miller, Ph.D. (Committee Member); Lawrence Prochaska, Ph.D. (Committee Member)

Subjects:

Biomedical Research; Ecology; Microbiology; Nutrition

Keywords:

Microbiota, in vitro gut simulator, bioreactor, nutrition, fatty acid

Gandhi, RomaThe Natural Acquisition of the Oral Microbiome in Childhood: A Cross-Sectional Analysis
Master of Science, The Ohio State University, 2016, Dentistry
This cross-sectional study explored the development of the oral microbiome throughout childhood. Our previous studies of infants up to 1 year of age have shown early presence of exogenous species not commonly found in the oral cavity followed by rapid replacement with a small, shared core set of oral bacterial species. Following this initial colonization, we hypothesize that the complexity of the microbial community will steadily increase with advancing age as the oral cavity develops more intricate environmental niches for bacterial growth, and as children are exposed to new strains of bacteria and novel foods. We sampled 116 children and adolescents ranging from age 1 to 14 years and collected salivary, supragingival and subgingival samples. Bacterial community composition was analyzed at the level of species using rRNA gene amplicon sequencing. This data allowed us to determine commonality among core species and the relationship of age to microbial complexity and community composition. Understanding when the establishment of bacterial communities will occur will help us determine if species are acquired in a specific order and will provide clues as to whether some species require the presence of others to colonize. Taken together, insight will be provided into the reconstruction of the natural acquisition of the human oral microbiome from birth through the establishment of the permanent dentition. Changes in species complexity and the establishment of shared order of the oral microbiota have been examined in relation to age and site-specific samples. More specifically, our analyses suggest that the overall oral microbiome remains fairly stable after the first year of life, with very little influence from age, particular oral niches, and caries status.

Committee:

Ann Griffen, DDS (Advisor)

Subjects:

Dentistry; Microbiology

Hariharan, JananiPredictive Functional Profiling of Soil Microbes under Different Tillages and Crop Rotations in Ohio
Master of Science, The Ohio State University, 2015, Environmental Science
Food production and security is dependent on maintaining soil health and quality. Thus, the emphasis on sustainable and healthy soil function is a top priority for scientists and land managers. One of the most important factors that influences soil function is the microbial community. Recent advances have allowed us to quantify more accurately the composition of such communities, but there is still a knowledge gap with regard to the contribution of microorganisms to various processes occurring in the soil. Understanding this will facilitate the development of healthier agroecosystems. In this thesis, a predictive functional approach is used to elucidate bacterial species–function relationships. Bacterial community profiles were compared across two tillage systems and two crop rotations in Northern Ohio (Wooster and Hoytville). 16S rRNA gene-targeted sequencing was performed and the raw data obtained were filtered, denoised and processed using QIIME. Open-reference OTU picking and taxonomic assignment was performed using the Greengenes database. I then used a computational approach called PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) to predict metagenomes and the most likely functions performed by individual species of bacteria. Sequence analysis reveals a large number of unidentified OTUs, which is consistent with our expectations of the soil ecosystem. Comparison of sequencing data from different platforms indicates that the dataset generated using Illumina sequencing provided better hits with the reference database than pyrosequencing, and was associated with a greater number of putative soil bacterial functions. PICRUSt allows an estimation of the level of involvement each OTU has with a specific gene function, which enables comparisons to be made across bacterial species and treatment conditions. Predicted functions of the bacterial community revealed a large number of proteins connected with metabolism and maintenance of natural organic molecules in soil as well as enzymes related to degradation of xenobiotics. Using this approach, I was also able to map specific OTUs to their functional potential. Bacterial enzymes implicated in the cycling of nitrogen, sulfur, carbon and methane through the soil were examined, as were enzymes that catalyzed the oxidative degradation of hydrocarbon compounds that are considered soil pollutants. Specialized groups of bacteria were linked to functions like nitrogen fixation and degradation of compounds like atrazine and chlorohydrocarbons. A broader range of OTUs was found to contain genes for carbon utilization and sulfur metabolism. These predictions are supported by previous ecological studies. There were other OTU-function relationships predicted in these studies that are novel and could be valuable in identifying commercially important microorganisms. These leads will require experimental validation. A clear difference was seen between the no-till and plow-till treatments, with no-till being functionally enriched for most major nutrient cycles. No such differences were observed between the different crop rotations. Proteobacteria, Actinobacteria and Acidobacteria were some of the most abundant phyla found in these soil samples, along with Nitrospirae, and Bacteroidetes. I concluded that long-term and continuous application of different tillage systems, and to a lesser extent crop rotation, result in unique bacterial communities that affect the overall functioning of the soil.

Committee:

Warren Dick (Advisor); Parwinder Grewal (Advisor); Margaret Staton (Committee Member)

Subjects:

Agriculture; Biogeochemistry; Bioinformatics; Ecology; Environmental Science; Microbiology; Soil Sciences

Keywords:

PICRUSt; soil metagenomics; soil bacteria; soil function; nutrient cycling

Ellis, David HaroldSilver nanoparticles: the immediate benefits of low bacterial resistance and the long-term risk of persistent stress in mammalian cells
Doctor of Philosophy (PhD), Wright State University, 2015, Biomedical Sciences PhD
Silver nanoparticles (AgNPs) are the fastest growing sector of nanotechnology, due mostly to their antibacterial properties. The antibacterial effectiveness of AgNPs is well known and derives from the shedding of silver ions which have multiple antibacterial targets in the bacterial cell. Due to their continuous release of ions and demonstrated antibacterial potency, some predict that AgNPs have a low potential for resistance development, which would make them a valuable asset in wound management. The ability for AgNPs to cause oxidative imbalance in mammalian cells is also well known, but the potential long-term impact of such a stress has not been studied despite its implication for negative outcomes in wound management. In this thesis, I demonstrate by using a stepwise increasing exposure protocol that Pseudomonas aeruginosa, but not Acinetobacter baumannii or Staphylococcus aureus could develop resistance to 10 nm, citrate-coated AgNPs. The potential for resistance development was lower than the antibacterial drug, ciprofloxacin, but not as low as silver nitrate to which none of the bacteria developed resistance. The resistance mechanism is not yet clear but appeared to involve the phenazine pigments produced by P. aeruginosa which can bind and reduce silver ions. In mammalian cells, I demonstrated the persistence and time-dependent oxidative stress of AgNPs in the A549, epithelial cell model, by using specialized imaging techniques and a common probe for oxidative stress. In addition, I showed that AgNPs can induce a senescent-like phenotype in A549 cells after an exposure that appears non-toxic in the typical viability assays used for assessing cytotoxicity. I confirmed that senescence was induced by showing an increase in senescence-associated, beta-galactosidase activity and the hypertrophic morphology of exposed cells, as well as a decrease in proliferation. The implication of this research for wound management is that AgNPs can be properly applied to wounds in order to inhibit bacterial colonization with little potential for resistant strains to emerge; however, the nanoparticles may persist in wound-associated, mammalian cells. There, the AgNPs will cause persistent oxidative stress with the potential to induce cellular senescence and reduce the long-term health and function of the surrounding tissue.

Committee:

Courtney Sulentic, PhD (Advisor); Saber Hussain, PhD (Advisor); Mill Miller, PhD (Committee Member); David Cool, PhD (Committee Member); Dan Halm, PhD (Committee Member)

Subjects:

Biology; Biomedical Research; Microbiology; Toxicology

Keywords:

silver nanoparticle; nanotechnology; toxicology; nanotoxicology; senescence; antibacterial; antibiotic resistance

Belyaevskaya, Anna VCharacterization of T box riboswitch gene regulation in the phylum Actinobacteria
Doctor of Philosophy, The Ohio State University, 2015, Molecular, Cellular and Developmental Biology
Riboswitches are cis-acting RNA regulatory elements located in the 5' untranslated region of a gene. These elements modulate gene expression by structural rearrangements in response to an array of physiological signals. T box riboswitches regulate expression of amino acid-related genes by responding to the aminoacylation status of a specific tRNA that matches the amino acid identity of the regulated gene. Most T box RNAs function at the level of transcription attenuation. A terminator that prevents transcription of the downstream gene forms when aminoacylation of the cognate tRNA is high, whereas uncharged tRNA promotes stabilization of an antiterminator that prevents termination and therefore increases gene expression. T box riboswitches are typically composed of three conserved helical domains, designated Stem I, II, III, the Stem IIA/B pseudoknot, and the competing terminator and antiterminator elements; these domains include conserved primary sequence and several secondary structure elements. However, the predicted structure of many T box riboswitches from the phylum Actinobacteria differ from those found in other phyla. A major goal of this research was to characterize the unusual T box RNAs found in Actinobacteria. These riboswitches are present in ileS genes, and were divided into three groups based on the arrangement of the Stem I domain: canonical Stem I; Ultrashort Stem I (US); and Unusually Structured Stem I Region (USSR). The US and USSR domains lack conserved elements in the canonical Stem I that were previously thought to be essential for T box riboswitch regulation. In addition, most of T box riboswitches in Actinobacteria are predicted to regulate gene expression at the level of translation initiation instead of transcription attenuation. In the current study, we demonstrated that several T box RNAs from Actinobacteria are functional in vitro and undergo structural rearrangements and changes in ribosomal binding in response to uncharged tRNAIle that are consistent with regulation at the level of translation initiation. Results from fluorescence assays demonstrated that the US domain binds the cognate tRNAIle in the absence of other riboswitch sequences, and nuclear magnetic resonance spectroscopy revealed structural rearrangements in this unusual domain in response to interaction with the anticodon stem-loop of the cognate tRNAIle. The US riboswitch variant also was used to identify a novel tRNA interaction site between the conserved nts of the Loop E motif in the Stem II domain and the T arm of the tRNA. This work has investigated the molecular details of T box riboswitch gene regulation, explored the codon-anticodon interaction in the context of unusual RNA structures, and identified tRNA elements important for tRNA noncanonical role in gene regulation. Overall, the obtained information increased our understanding of RNA as a regulatory element.

Committee:

Tina Henkin (Advisor); Irina Artsimovitch (Committee Member); Venkat Gopalan (Committee Member); Anita Hopper (Committee Member)

Subjects:

Microbiology

Keywords:

Riboswitch, Gene Regulation, Actinobacteria

Kulkarni, Aditya S.Metabolic Studies of Albomycin Biosynthesis
Doctor of Philosophy (PhD), Ohio University, 2015, Biological Sciences (Arts and Sciences)
Albomycin is a sulfur containing metabolite produced by Streptomcyes sp. ATCC 700974. It is a structurally unique molecule with potent antibiotic activity. Unfortunately, it is produced in low amounts and this is thought to be the stumbling block for its commercial development. Cytosine is thought to be a precursor for albomycin production and experiments were done to increase intracellular cytosine to possibly increase albomycin production. Isolation of a codA mutant was attempted. Feeding cytosine in the growth medium and overexpressing a blsM gene (which generates cytosine) were found to have no effect on albomycin production. Experiments were also done to gain insights into the sulfur source utilized for albomycin biosynthesis by Streptomyces sp. ATCC 700974. Addition of iron and propargylglycine altered intracellular homocysteine and cysteine levels and albomycin production suggesting homocysteine as a precursor. Based on these results, sulfur amino acid metabolism of the albomycin producing strain was manipulated by overexpressing select pathways to observe effects on albomycin production. Albomycin production increased with overexpression of pathways producing homocysteine, namely the transsulfuration and direct sulfhydrylation pathways. No effect was observed on expression of the reverse transsulfuration pathway. Overexpression of the genes in the active methyl cycle leading to homocysteine was also found to increase albomycin production. AbmD is proposed to be the first enzyme of the albomycin biosynthetic pathway in Streptomyces sp. ATCC 700974 and to utilize cysteine or homocysteine as one of its substrates. Enzyme assays using purified AbmD demonstrated that homocysteine was utilized along with a co-substrate. Five pathway genes directing homocysteine biosynthesis along with the abmD gene were integrated into the Streptomcyes sp. ATCC 700974 genome to produce the strain SAK9. Albomycin production by SAK9 was found to be four times the wild type strain in glycerol containing media. These results suggest homocysteine is the precursor for albomycin biosynthesis. 13C labeling experiments conducted by feeding labeled cysteine and methionine were inconclusive. The seleno-amino acids selenomethionine and selenocystine were not incorporated into albomycin. Future directions for albomycin biosynthesis research are discussed.

Committee:

Donald Holzschu, Ph.D (Advisor); Erin Murphy, Ph.D (Committee Member); Tomohiko Sugiyama, Ph.D (Committee Member); Martin Tuck, Ph.D (Committee Member); Mark McMills, Ph.D (Committee Member)

Subjects:

Biochemistry; Microbiology; Molecular Biology

Keywords:

Albomycin; sulfur metabolism; secondary metabolite biosynthesis; metabolic engineering; Streptomyces

Richardson, Edward ThompsonRegulation of Macrophages by Mycobacterium tuberculosis and the ERK MAP Kinase Signaling Pathway
Doctor of Philosophy, Case Western Reserve University, 2015, Pathology
Mycobacterium tuberculosis, the cause of tuberculosis, survives for long periods in a latent state in infected individuals, and the immune system is typically able to control but not eliminate the bacteria. Latency is a complex phenomenon but involves, in part, interactions of the bacteria and its unique lipoproteins and lipoglycans with macrophages, the main cells that become infected. The purpose of this dissertation was to expand understanding of how M. tuberculosis engages with macrophages. In the first part, we characterized the lipoglycan binding function of M. tuberculosis lipoprotein LprG. We determined the binding properties of these M. tuberculosis lipoglycans to LprG using surface plasmon resonance. We also verified the presence of a non-acyl chain dependent binding mode to LprG, and determined that LprG also binds mannan. Finally, we determined that one function of LprG is to facilitate exposure of LAM on the bacterial cell surface for interaction with macrophages. LprG-deficient M. tuberculosis had reduced surface-exposed lipoarabinomannan, and had reduced ability to block phagolysosome maturation, a known immune evasion mechanism that requires lipoarabinomannan. These studies contribute to understanding of LprG, and develop increased knowledge of how M. tuberculosis lipoarabinomannan is exposed to macrophages to block phagolysosome fusion, a process involved in bacterial persistence and intracellular survival. In the second part, we studied the TLR2 signaling response of macrophages to M. tuberculosis. We determined that TLR2 was required for M. tuberculosis to trigger NF-κB and ERK, and that TLR2 signaling results in balanced downstream effects. NF-κB is required for expression of pro-inflammatory IL-12, and M. tuberculosis-stimulated Tpl2-ERK signaling suppressed IL-12 while inducing anti-inflammatory IL-10. These effects reduced CD4+ T cell responses against M. tuberculosis. Tpl2-deficient macrophages expressed IL-12 in response to M. tuberculosis, and were more potent at stimulating antigen-specific T cells, upon initial stimulation and recall. These findings contribute to understanding of the signaling triggered by M. tuberculosis, and the role of the macrophage-intrinsic ERK cascade in inhibiting T cell-mediated host defense. Together, these studies expand understanding of the regulation of macrophages by M. tuberculosis in ways that promote long-term survival of the bacteria, and may potentiate latent infection.

Committee:

Clifford Harding (Advisor); W Henry Boom (Advisor); Alex Huang (Committee Chair); Clive Hamlin (Committee Member); Liem Nguyen (Committee Member); Roxana Rojas (Committee Member); Pamela Wearsch (Committee Member)

Subjects:

Immunology; Microbiology

Keywords:

Tuberculosis; Mycobacterium tuberculosis; lipoprotein; LprG; lipoarabinomannan; Tpl2; MAP3K8; ERK; macrophages

Ohtola, Jennifer APneumococcal Vaccination in Aging HIV-Infected Individuals
Doctor of Philosophy (PhD), University of Toledo, 2015, Biomedical Sciences (Infection, Immunity, and Transplantation)
Advanced age and human immunodeficiency virus (HIV) infection are both risk factors for Streptococcus pneumoniae infections due to immunological dysfunction. The aging HIV-infected (HIV+) population may be at higher risk for pneumococcal disease due to the combination of these factors on humoral immunity. Current recommendations for pneumococcal vaccination in HIV+ adults include a priming dose of the 13-valent pneumococcal conjugate vaccine followed by one dose of the 23-valent pneumococcal polysaccharide vaccine 8 weeks later (PCV/PPV). We compared quantitative and qualitative antibody responses to PCV/PPV versus a single dose of PPV in HIV+ adults aged 50-65 years with CD4+ T cells/µl (CD4) >200 on antiretroviral therapy ≥1 year. We found that PCV/PPV did not demonstrate a clear immunological advantage to PPV alone, as serotype-specific IgG levels and functional titers postvaccination were similar between groups. In addition, these antibody responses were significantly reduced in HIV+ subjects vaccinated with PCV/PPV compared to age-matched, uninfected (HIV–) controls who received PCV/PPV. We also characterized the phenotype and surface expression of several receptors on serotype-specific B cells that may influence vaccine responses. HIV+ subjects vaccinated with PCV/PPV generated significantly reduced frequencies of circulating serotype-specific B cells postvaccination compared to those who received PPV only. However, phenotypic distributions of serotype-specific memory B cell subsets were similar between groups. Transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI)+ serotype-specific B cell percentages were significantly decreased in HIV+ PCV/PPV compared to PPV groups, indicating that prior PCV altered TACI expression. It remains unclear if this impact provides any benefit to vaccine responses. CD21+ serotype-specific B cells were also significantly reduced in HIV+ compared to HIV– PCV/PPV groups which may contribute to diminished antibody responses. Collectively, our findings suggest that continued efforts aimed at developing more effective vaccination strategies in susceptible adult populations are warranted, and further investigation into the immunological mechanisms that increase the risk of pneumococcal disease and induce potent vaccine responses are necessary.

Committee:

M. A. Julie Westerink, MD (Advisor); Deepak Malhotra, MD, PhD (Committee Member); Z. Kevin Pan, MD, PhD (Committee Member); Stanislaw Stepkowski, DVM, PhD, DSc (Committee Member); R. Mark Wooten, PhD (Committee Member)

Subjects:

Immunology; Microbiology

Keywords:

Streptococcus pneumoniae; pneumococcal conjugate vaccine; pneumococcal polysaccharide vaccine; HIV infection; aging; B cells; antibody

Njus, Kelsey AnneMolecular Techniques for the Identification of Commensal Fungal Populations on Cave Roosting Bats
Master of Science, University of Akron, 2014, Biology
Since the emergence of White-nose Syndrome (WNS) in 2006, North American bat populations have experienced devastating population declines. WNS is caused by the psychrophilic fungus Pseudogymnoascus destructans (Pd), which invades the wing tissues of bats during torpor, resulting in up to a 97% mortality rate. Yet some species, including the Indiana bat (Myotis sodalis) and the Virginia big-eared bat (Corynorhinus townsendii virginianus), appear to have reduced mortality from WNS, despite close proximity to infected bats. In an attempt to determine whether bats possess commensal fungal populations that confer resistance to WNS, we analyzed the culturable and non-culturable fungal communities of bat fur. To generate a library of the non-culturable fungal species, DNA was isolated from bat fur clippings, amplified by PCR of the internal transcribed spacer (ITS) region, sequenced, and identified using BLAST. Culturable fungal populations were obtained by swabbing and isolation in pure culture, and also identified using the ITS sequence. By comparing the communities of bats belonging to five species for four locations, we were able to determine that the species of the bat is the most important contributor to the composition of a bat’s fungal community, and this was best analyzed using molecular methods. While the majority of shared fungal species consisted of saprotrophic fungi, a small portion of these shared species consisted of yeast, the most abundant of which was Debaryomyces udenii; Virginia big-eared bats, which have a 0% mortality rate, have a fungal community that consists of 55% D. udenii. This data supports the hypothesis that bats harbor commensal fungal communities, and that these communities may confer resistance to WNS. Identifying whether commensal populations provide a mechanism of WNS resistance in bats could help in our understanding of the emergence of this pathogen, the future of the disease, and potential biological controls.

Committee:

Hazel Barton, Dr. (Advisor); James Holda, Dr. (Committee Member); Greg Smith, Dr. (Committee Member)

Subjects:

Biology; Ecology; Microbiology; Molecular Biology

Keywords:

White-nose Syndrome; commensal; fungi; bats; Pseudogymnoascus; PCR inhibition; Corynorhinus townsendii

Vadia, Stephen EListeriolysin O activates Listeria monocytogenes internalization into human hepatocytes through a novel pore-dependent mechanism
Doctor of Philosophy, The Ohio State University, 2014, Microbiology

The causative agent of listeriosis, Listeria monocytogenes, is a frequent contaminant of fruits, vegetables, cheeses, and processed foods. In certain high risk groups, L. monocytogenes can cause potentially fatal infections. The bacterium can traverse the intestinal epithelial barrier and spread throughout the body via the bloodstream and lymphatic system to infect the liver, cross the blood-brain barrier, and in pregnant women, the placental barrier. L. monocytogenes is able to cross these biological barriers due to its ability to proliferate within certain phagocytic and nonphagocytic cells. Bacterial invasins, most notably internalin (InlA) and InlB, bind to receptors on the surface of nonphagocytic cells to induce endocytosis of the bacterium through a zipper-like mechanism. L. monocytogenes can then escape from the endocytic vacuole and enter the cytosol through the activity of the pore-forming toxin listeriolysin O (LLO). In the cytosol, the bacterium can replicate and disseminate to adjacent cells by cell-to-cell spreading.

LLO belongs to the cholesterol-dependent cytolysin (CDC) family of bacterial pore-forming toxins. These toxins are secreted as monomers, oligomerize on cholesterol-rich membranes, and form large transmembrane pores. In addition to mediating vacuolar escape, LLO stimulates signaling pathways in host cells. We hypothesized that the signaling activity of extracellular LLO affects the intracellular lifecycle of L. monocytogenes. Using quantitative immunofluorescence microscopy to assess the importance of LLO for bacterial association to and internalization into epithelial cells, we found that LLO is required for efficient L. monocytogenes internalization into hepatocytes, and is sufficient to induce internalization of noninvasive bacteria and polystyrene beads. Using novel LLO variants that bind to host cells but are unable to form the pore complex, we demonstrated that pore formation was required for LLO-mediated internalization.

The CDC streptolysin O is known to stimulate a Ca2+-dependent membrane repair response that involves endocytosis of the toxin from the perforated plasma membrane. LLO stimulates a Ca2+-dependent membrane repair response, but this response is insufficient to account for L. monocytogenes internalization. LLO-mediated internalization and membrane repair both require the influx of extracellular Ca2+, but differ in their requirement for F-actin and K+ efflux. Surprisingly, using ionophores to stimulate Ca2+ and K+ fluxes, as happens when the plasma membrane is perforated by LLO, was sufficient to induce internalization of large particles. LLO mediates bacterial internalization in all human hepatocytes we tested and in additional epithelial cell lines. However, LLO does not induce bacterial internalization in all epithelial cells that it perforates.

In light of this data, LLO emerges as a significant invasion factor expressed by L. monocytogenes, which is coexpressed with InlA and InlB during infection. With the same approaches used to assess the role of LLO, we determined the contribution of InlA and InlB to bacterial association, internalization, and intracellular viability in nonphagocytic cells that express the InlA and InlB receptors. We found significant variability in the involvement of each of these invasins in bacterial association, internalization, and intracellular viability, demonstrating that the activity of invasins is not governed solely by the presence or absence of their receptors.

Committee:

Stephanie Seveau, PhD (Advisor)

Subjects:

Microbiology

Keywords:

Listeria monocytogenes; Listeriolysin O; Pore-forming toxin; Intracellular pathogen

Schlais, Michael J.Detection and Characterization of a Unique Ammonia Oxidizing Archaea; Cultured from Lake Superior
Doctor of Philosophy (Ph.D.), Bowling Green State University, 2014, Biological Sciences
In the past century Lake Superior has seen a 5-fold increase in nitrate levels. Previous research has shown this increase to be due to as of yet undescribed in-lake oxidation processes. It has recently been shown that ammonia oxidizing archaea (AOA) in marine and freshwater environments belonging to the phylum Thaumarchaeota have the ability to oxidize ammonium, and are specifically suited to the low NH4¿ /oligotrophic environment of Lake Superior. In this study isolation and enrichment of these unique freshwater ammonia oxidizing archaea from Lake Superior has enabled the direct measurement of NO2¯ and N2O production and NH4¿ metabolism. In the search for in-lake nitrifying organisms we have identified and described a novel group of freshwater ammonia oxidizing archaea using the ammonia monooxygenase alpha (amoA) subunit gene as a diagnostic tool for microbes capable of ammonia oxidation. Flow cytometry was used to determine cell abundances and ideal incubation times and temperatures for these AOA taken from these Lake Superior. These data along with nitrification measurements enabled us to determine per cell nitrification rates for our AOA enrichment cultures, which have shown that they do indeed account for a major component of in-lake nitrification processes. With the exception of the marine archaeon Nitrosopumilus maritimus, most research has been focused on culture-independent methods for the characterization of nitrifying microbes. This study is among the first of these culture dependent studies to describe methods for culturing a freshwater ammonia oxidizing archaea. iv

Committee:

George Bullerjahn, PhD (Advisor); Michael McKay, PhD (Committee Member); Scott Rogers, PhD (Committee Member); Paul Morris, PhD (Committee Member); John Snyder, PhD (Other)

Subjects:

Biology; Microbiology

Keywords:

ammonia oxidizing archaea; AOA; amoA; Nitrification

Pina-Mimbela, Ruby MelisaAssociation of Polyphosphate (poly P) Kinases with Campylobacter jejuni Invasion and Survival in Human Epithelial Cells
Master of Science, The Ohio State University, 2013, Veterinary Preventive Medicine
The foodborne and zoonotic bacterium Campylobacter jejuni, is one of the major causes of gastrointestinal disorders in humans worldwide. This human pathogen expresses a variety of carbohydrate, protein and lipid structures decorating its outer material (OM) which have been associated with virulence. On the other hand, inorganic polyphosphate (poly P), in many bacteria, is essential for stringent survival response and adaptation, and is considered as a virulence factor. Two kinases in C. jejuni have been associated with virulence: poly phosphate kinase 1 (PPK1) and poly phosphate kinase 2 (PPK2). PPK1 is responsible for poly P generation and PPK2 for GTP biosynthesis from poly P. GTP is a molecule involved in signaling processes as well as protein and capsule synthesis. In previous studies, we demonstrated that C. jejuni delta ppk1 shows reduced stringent survival response, adaptation, and in vivo colonization in chickens, whereas, delta ppk2, in addition to its stress tolerance defect, also displays reduced virulence properties. In the present study, the contribution of component(s) present in the outer material (OM) from the wild-type, delta ppk1 and delta ppk2 to C. jejuni invasion and intracellular survival in human epithelial cells in vitro was investigated. OM from C. jejuni wild type 81-176, delta ppk1 and delta ppk2 was extracted and fractionated into carbohydrates (lipoglycans, poly-oligosaccharides), lipids, and proteins. These fractions were tested for their contribution to C. jejuni invasion, intracellular survival and IL-8 production in INT-407 human embryonic intestinal cells. Different fractions from C. jejuni 81-176 wild type, delta ppk1 and delta ppk2 were pre-incubated with INT-407 human epithelial cells prior to infection with C. jejuni wild type. Results obtained imply that components present in the C. jejuni OM are associated to invasion and intracellular survival in INT-407 cells. Additionally, poly P kinases play a role in intracellular survival in epithelial cells. Alterations in OM from delta ppk1 and delta ppk2 are suggested to be important in C. jejuni intracellular survival in INT-407 cells. Our results suggested that OM proteins mediate C. jejuni invasion and intracellular survival to human epithelial cells whereas lipoglycans are associated with intracellular survival. In addition, alterations present in delta ppk2 lipids are associated with C. jejuni intracellular survival in INT-407 cells. On the other hand, poly- and oligo- saccharides fraction from delta ppk1 and delta ppk2 are likely to play a role in C. jejuni survival within epithelial cells. Further, we evaluated production of IL-8 in INT-407 cells by OM and its fractions from wild type, delta ppk1 and delta ppk2 strains. C. jejuni OM from wild type was not able to induce IL-8 in INT-407 cells, whereas IL-8 secretion was observed when these epithelial cells were exposed to OM from delta ppk1 and delta ppk2. Our results demonstrate that proteins, lipids, and lipoglycans present in C. jejuni OM are associated with its invasion and intracellular survival in human epithelial cells. Additionally, this study provides insights about the role of poly P in modulation of C. jejuni OM composition, thereby contributing to invasion and intracellular survival.

Committee:

Gireesh Rajashekara, Dr. (Advisor); Jordi B. Torrelles, Dr. (Committee Member); Chang-Won Lee, Dr. (Committee Member)

Subjects:

Biochemistry; Cellular Biology; Microbiology; Veterinary Services

Keywords:

Campylobacter jejuni; Poly P kinases; invasion; survival; epithelial cells; INT-407 cells

Othman, Abdulrazzag AbdulazizThe Use of Antibody-Coated Latex Beads To Determine Single Positive and Double Positive Mouse Spleen Cells Expressing CD5 and/or CD19 Glycoproteins.
Master of Science (MS), Wright State University, 2015, Microbiology and Immunology
Flow cytometry is the standard method used to diagnose, stage, and monitor patients’ response to the treatment given by counting the numbers of CD5, CD19 and CD5+ CD19+ B lymphocytes. In this study, a comparison was done between numbers of single CD5+, single CD19+ and dual CD5+ CD19+ mouse spleen B lymphocytes using flow cytometry and antibody-latex beads. The bead method involved antibody-coated latex bead and yielded results similar to those of flow cytometry. For cells exhibiting both markers (CD5+ CD19+), the bead method used antibody-coated beads of two different colors yielded similar results to those of flow cytometry results. These findings show that the antibody-coated beads are adequate to determine the numbers of single CD5+, CD19+positive cells and CD5+ CD 19+double positive B lymphocytes under circumstance where flow cytometry is not available.

Committee:

Nancy Bigley, Ph.D. (Advisor); Barbara Hull, Ph.D. (Committee Member); Cheryl Conley, Ph.D. (Committee Member)

Subjects:

Immunology; Microbiology

Keywords:

microbiology;immunology

Ticak, TomislavAnoxic quaternary amine utilization by archaea and bacteria through a non-L-pyrrolysine methyltransferase; insights into global ecology, human health, and evolution of anaerobic systems
Doctor of Philosophy, Miami University, 2015, Microbiology
Quaternary amines are compounds which are important for every domain of life and play roles as carbon and nitrogen sources but also are known to act as osmoregulants. One quaternary amine, glycine betaine, is considered a key osmoregulatory compound due to its chemical nature and is often the main intersection of choline and carnitine metabolism, both aerobically and anaerobically. Many organisms have the capability of degrading glycine betaine through oxygenases or dehydrogenases aerobically, but there is little literature related to the fate of glycine betaine in anaerobic systems. Many of the reported anaerobic systems for glycine betaine involve a reductase pathway that leads to the formation of trimethylamine and acetate, which are well established methanogenic precursor compounds in anaerobic environments. However, there exist a few reports of acetogens and methanogens with the capability of converting glycine betaine to dimethylglycine, which is a strict deviation from the aformentioned reductase pathway. This suggests a pathway exists for anaerobic glycine betaine metabolism that has largely gone uncharacterized. We used a series of bioinformatic, biochemical, and physiological experiments to examine carbon metabolism in Desulfitobacterium hafniense strain Y51 and demonstrated its ability to perform this novel mechanism of glycine betaine metabolism. We proposed that non-L-pyrrolysine trimethylamine methyltransferases may act as quaternary amine methyltransferases. As a result of this study; we discovered a theoretical key in explaining the evolution of the glycine betaine and trimethylamine methyltransferases regarding incorporation of L-pyrrolysine. The fact that a quaternary amine (e.g., glycine betaine) may bind into the near identical location of the proposed trimethylamine-pyrrolysine adducts may help us to better understand this widespread superfamily of methyltransferases. By using our knowledge of the glycine betaine methyltransferase, we began to investigate anaerobic communities for the presence of these methyltransferase genes by enrichments with quaternary amines resulting in the discovery of methanogens capable of glycine betaine, choline, and tetramethylammonium metabolism. Genomic analysis of these organisms revealed the presence of glycine betaine and trimethylamine methyltransferase-like genes supporting the hypothesis of quaternary amine demethylation by non-L-pyrrolysine methyltransferases. Our future work now points toward the examination of microbial distribution and physiology for anaerobic quaternary amine utilization in human systems, marine and freshwater environments to determine the evolutionary pressure(s) that may have selected for the advent of L-pyrrolysine.

Committee:

Donald Ferguson, Ph.D. (Advisor)

Subjects:

Biochemistry; Ecology; Microbiology; Molecular Biology

Keywords:

quaternary amines; glycine betaine; pyrrolysine; methyltransferase; one-carbon metabolism

Marotta, NicoleMycoplasma pneumoniae protein P30 proline residues: Cytadherence, gliding motility, and P30 stability
Master of Science, Miami University, 2014, Microbiology
Mycoplasma pneumoniae, a pathogen found in the respiratory tract of humans, requires both cytadherence and gliding motility to cause disease. The transmembrane adhesin P30, located at the polar attachment organelle, is essential for these processes. Within the extracellular region of P30 is a stretch of 17 imperfect proline-rich repeats (PRRs) at the C-terminus. Mutants lacking PRRs are unstable, suggesting the stability of P30 depends upon interactions of this region with other proteins. We hypothesize that individual prolines within the PRRs of P30 confer stabilization, allowing P30 to function in cytadherence and gliding motility. We tested this hypothesis by altering selected proline residues and measuring P30 stability levels, cytadherence through a hemadsorption assay, and gliding motility using time-lapse microcinematography. This work demonstrated that alteration of prolines destabilizes P30, with a reduction in gliding speed.

Committee:

Mitchell Balish, PhD (Advisor); Joseph Carlin, PhD (Committee Member); Natosha Finley, PhD (Committee Member)

Subjects:

Microbiology

Keywords:

Mycoplasma; protein; prolines; gliding motility; stability; cytadherence

Balanis, Nikolas GDIVERSE ROLES FOR EGF RECEPTOR SIGNALING IN THE BREAST CANCER TUMOR MICROENVIRONMENT
Doctor of Philosophy, Case Western Reserve University, 2013, Physiology and Biophysics
The ligand/ cell surface receptor interaction is a paradigm for how cells utilize extracellular cues to `signal’ to their intracellular environment. Ligand/receptor interactions are important in almost all biological processes. However, focusing solely on the ligand/receptor interaction excludes the many possible `ligand-independent’ modes of surface receptor action, vis a vis those that occur in `trans’. Our studies sought to uncover the role of extracellular matrix, important molecules in the tumor microenvironment, as they function through integrin receptors to regulate the receptor tyrosine kinase, EGF Receptor (EGFR). This thesis links EGFR/integrin crosstalk to the control of cellular protrusions such as lamellipodia and filopodia, as well as the control of stress fiber formation necessary for cell contractility. We have also linked EGFR/integrin crosstalk to activation of the Signal transducer and activator of transcription 3 (Stat3). We have shown that activation of Stat3 is necessary for EGFR induced transformation of normal mammary epithelial cells. We provide mechanistic insights into how certain breast cancers “switch” from EGFR/Stat3 signaling to Fibronectin/Stat3 signaling following epithelial-to-mesenchymal transition (EMT). We have shown that following EMT, breast cancers are desensitized to EGFR inhibition and become sensitized to Janus kinase 2 (Jak2) inhibitors. This finding may describe why certain cancers are resistant to EGFR directed therapies. Finally, we have identified the EGFR inhibitor protein Mitogen inducible gene 6 (Mig6) as essential to cell survival in Triple-negative breast cancer (TNBC). The observations provided in this thesis uncover the role of EGFR in the tumor microenvironment and provide insight into novel therapies for TNBC.

Committee:

Witold Surewicz (Committee Chair); Cathleen Carlin (Advisor); Kalnay Brady (Committee Member); Tom Egelhoff (Committee Member); Stephen Jones (Committee Member)

Subjects:

Biochemistry; Bioinformatics; Microbiology; Molecular Biology

Keywords:

breast cancer; EGFR; EGF Receptor; Triple Negative Breast Cancer; Epithelial-to-Mesenchymal Transition; EMT; focal adhesion; fibronectin; IL-6; JAK2; MIG6; STAT3; TNBC; FN; PYK2; FAK; NMUMG; Basal-like

Dolhi, Jenna MENVIRONMENTAL IMPACTS ON RUBISCO: FROM GREEN ALGAL LABORATORY ISOLATES TO ANTARCTIC LAKE COMMUNITIES
Doctor of Philosophy, Miami University, 2014, Microbiology
Ribulose-1,5-bisphosphate carboxylase oxygenase (RubisCO) is found in a variety of autotrophic microorganisms ranging from green algae, cyanobacteria, and chemoautotrophic bacteria. As this enzyme has the potential to catalyze carboxylation (carbon fixation) or oxygenation (photorespiration) reactions, it is regulated in response to environmental variables at the levels of transcription, translation, and post-translation by the enzyme, RubisCO activase. A combination of laboratory experiments on green algal isolates and field experiments were utilized to gain insight on carbon fixation in permanently ice-covered Antarctic lakes. RubisCO was investigated as a potential target for cold adaptation of carbon fixation in the psychrophilic green alga, Chlamydomonas raudensis UWO241 (UWO241), isolated from Lake Bonney, Antarctica. RubisCO activity, stability, and whole cell carbon fixation were measured for the psychrophile and compared to a closely related mesophilic alga, C. raudensis SAG49.72 (SAG49.72). The effect of environmental factors including light and temperature on UWO241 and SAG49.72 RubisCO activation state, an indirect measurement of RubisCO activase activity, and abundance was investigated using a modified RubisCO carboxylase assay and immunoblotting, respectively. Lastly, maximum potential RubisCO carboxylase activity was determined using a modified activity assay in multiple ice covered Antarctic lakes including Lake Bonney. This data was complemented with lake depth profiles of enzyme abundance determined by quantitative real-time PCR and RubisCO-harboring organism diversity. While purified RubisCO of the psychrophilic green alga did not function optimally at low temperature, whole cell carbon fixation was greater under such conditions, suggesting that the overall process of carbon fixation is modified to function in UWO241. Increased RubisCO abundance at low temperature may contribute to this phenomenon. Low light levels may be important in regulation of RubisCO via RubisCO activase and should be further investigated. Based on community level RubsiCO activity and enzyme abundance, light and RubisCO harboring organisms including eukaryotic algae and cyanobacteria were positively correlated, but this was variable between lakes. Dark carbon fixation was potentially important in lakes west lobe Bonney and Fryxell and this community was negatively correlated with light. Results of targeted physiology and community level experiments led to development of a carbon fixation model for Lake Bonney.

Committee:

Rachael Morgan-Kiss, PhD (Advisor); Annette Bollmann, PhD (Committee Member); Gary Janssen, PhD (Committee Member); D.J. Ferguson, PhD (Committee Member); Melany Fisk, PhD (Committee Member)

Subjects:

Microbiology

Keywords:

green algae; carbon fixation; RubisCO; ice-covered lakes

Liu, HongyanROLES OF TYPE IV SECRETION EFFECTOR ECH0825 IN EHRLICHIA CHAFFEENSIS INFECTION
Doctor of Philosophy, The Ohio State University, 2013, Comparative and Veterinary Medicine
Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, is an obligatory intracellular bacterium that replicates in the membrane-bound compartment (inclusion) in monocytes/macrophages. In this study, functions of ECH0825, the first identified Type IV secretion effector of Ehrlichia species were studied. The expression of ECH0825 in E. chaffeensis was up-regulated at early exponential growth stage in a human monocytic cell line, THP-1. When the host cytoplasmic ECH0825 was neutralized by anti-ECH0825 delivered by Chariot system, infection of E. chaffeensis was significantly reduced. Secreted or ectopically expressed ECH0825 targeted to the host cell mitochondria. The ectopically expressed ECH0825 inhibited etoposide-induced apoptosis in RF/6A cells, and human Bax–induced apoptosis in yeast. Mitochondrial manganese superoxide dismutase (MnSOD) was stabilized and reactive oxygen species (ROS) levels were reduced in both E. chaffeensis-infected cells and ECH0825-transfected cells than control cells, suggesting that ROS-induced apoptosis is inhibited by ECH0825 translocated to the mitochondria. Thus ECH0825 helps E. chaffeensis to overcome one of important innate immune responses against intracellular infection, cellular apoptosis. Our further study showed that ECH0825 also targeted to the cytosolic side of inclusions in THP-1 cells. Ectopically expressed ECH0825-GFP and GFP-Rab5 surrounded the inclusions. The interaction between ECH0825 and Rab5 was detected by affinity pull-down assay and co-immunoprecipitation. The Rab5-ECH0825 complex contained components of the PI3KC3 complex, Vps34 and Beclin 1. The autophagy inhibitor 3-methyadenine (3-MA) and Spautin-1 inhibited the E. chaffeensis growth and the autophagy inducer rapamycin enhanced the E. chaffeensis infection in infected THP-1 cells. Addition of essential amino acids partially abrogated the growth inhibition caused by 3-MA treatment. The protein GFP-Atg5, but not HA-Atg12 or GFP-LC3 (yeast Atg8 homolog) surrounded the E. chaffeensis inclusions, suggesting that E. chaffeensis inclusions are the precursor of preautophagosomes. The present study reveals a novel mechanism of bacterial protein ECH0825 linking Rab5 to the early stage of autophagy pathway to facilitate E. chaffeensis intracellular growth. Since ECH0825 targets two host cellular sites, the structure of the protein was analyzed. Various truncated and mutated ECH0825 were tagged with GFP and the distribution of the proteins in the transfected cells was examined. The N-terminal amino acids, but not C terminus or coiled-coil domain, was found critical for ECH0825 to target mitochondria. Whether the mutant ECH0825 inhibits host cellular apoptosis and which structure is responsible for Rab5 interaction remain to be studied. In addition to the host cell proteins, ECH0825 may also interact with bacterial proteins other than VirD4 to facilitate the localization on the inclusions. Among several membrane-spanning type IV secretion apparatus proteins, the VirB6-3 was chosen to investigate the interaction with ECH0825, as it has a repeat domain potentially relevant for protein-protein interaction. But the analysis using the bacteria two-hybrid system could not detect the interaction between ECH0825 and the repeat region of VirB6-3. Taken together, this study revealed a novel molecule evolved in Ehrlichia species, and how the obligatory intracellular pathogen uses this molecule to subvert two fundamental innate immune mechanisms of host cells, apoptosis and autophagy, to promote its intracellular growth.

Committee:

Yasuko Rikihisa (Advisor); Xue-feng Bai (Committee Member); Xin Li (Committee Member); Mingqun Lin (Committee Member)

Subjects:

Cellular Biology; Microbiology; Molecular Biology

Keywords:

EHRLICHIA CHAFFEENSIS; TYPE IV SECRETION EFFECTOR; ECH0825

Coffman, Anthony MProduction of Carbohydrases by Fungus Trichoderma Reesei Grown on Soy-based Media
Master of Science in Engineering, University of Akron, 2013, Chemical Engineering
Trichoderma reesei RUT-C30 was cultivated in shaker flasks and pH-controlled, agitated batch fermentations to study the effects of soy-based media on the production of cellulase, xylanase, and pectinase (polygalacturonase) for the purposes of soybean polysaccharide hydrolysis. Growth on defatted soybean flour as sole nitrogen source was compared to the standard combination of ammonium sulfate, proteose peptone, and urea. Carbon source effect was also examined for a variety of substrates, including lactose, microcrystalline cellulose (Avicel), citrus pectin, soy molasses, soy flour hydrolysate, and soybean hulls (both pretreated and natural). Flask study results indicated exceptional enzyme induction by Avicel and soybean hulls, while citrus pectin, soy molasses, and soy flour hydrolysate did not promote enzyme production. Batch fermentation experiments reflected the flask system results, showing the highest cellulase and xylanase activities for systems grown with Avicel and soybean hulls at near-neutral pH levels, and the highest polygalacturonase activity resulting from growth on lactose and soybean hulls at lower pH levels, 4.0 to 4.5. Soy polysaccharides were hydrolyzed with fermentation enzyme broths of varying enzyme composition and concentration. Enzyme mixtures with larger proportions of cellulase and polygalacturonase showed slightly higher sugar release. Hydrolysis was incomplete in all experiments, solubilizing roughly one-third of the polysaccharides to oligomeric products. Additionally, methods for estimation of cell concentration via cell DNA extraction and cell wall chitin hydrolysis and a modified intracellular protein analysis procedure were investigated, showing high levels of error and interference by soy protein from the growth media. Finally, a consistent and reliable assay for polygalacturonase activity was developed and verified.

Committee:

Lu-Kwang Ju, Dr. (Advisor); Gang Cheng, Dr. (Committee Member); Chelsey Monty, Dr. (Committee Member)

Subjects:

Chemical Engineering; Microbiology

Keywords:

enzymes; cellulase; xylanase; fermentation; soybean; Trichoderma reesei; enzymatic hydrolysis

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