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Glucose Uptake by the Cellulolytic Rumen Anaerobe Bacteroides Succinogenes
(North Dakota State University, 1986) Franklund, Clifton Victor
Glucose uptake by the cellulclytic rumen anaerobe, Bacteroides succinogenes S85, was measured under conditions that maintained anaerobiosis and osmotic stability. This organism was found to possess a highly specific, active transport mechanism for glucose. Evidence for a phosphoenol-pyruvate:g1ucose phosphotransferase system was not detected. Compounds that inhibit electron transport systems (non-heme iron chelators, and sulfhydryl reagents) were effective inhibitors of glucose uptake. The strongest inhibitors were compounds (proton and metal ionophores) that interfere with maintenance of the proton motive force. Compounds which interfere with ATP synthesis also inhibited glucose uptake, but a role for ATP in energizing uptake could not be inferred from these results. Oxygen prevented glucose uptake (75% inhibition), reflecting possible active sulfhydryl centers (above) or autooxidation of electron transport components. The results suggest the fumarate reductase-coupled electron transport system of B. succinogenes can generate a proton motive force that is used to energize glucose uptake. Na+ and Li+. but not K+, stimulated glucose uptake and may partly account for the growth requirement of B. succinogenes for Na+. However, the data were insufficient to conclude that glucose uptake occurs by a Na+ symport mechanism. Spheroplasts of B. succinogenes transported glucose as well as whole cells, indicating glucose uptake is not dependent on a periplasmic glucose binding protein. A variety of sugars including the nonmetabolizable analog, [inversely proportional symbol]-methylglucoside. did not inhibit glucose uptake. Only cellobiose and 2-deoxyglucose were active and neither behaved as a competitive inhibitor. Metabolism of both sugars was probably responsible for the inhibition. Cellobiose-grcwn B. succinogenes showed a reduced ability to transport glucose compared to glucose-grown cells. This may indicate regulation of synthesis of the glucose carrier protein by cellobiose through a mechanism other than catabolite repression. Differences in the ability to transport glucose were detected between transition cells (transition from lag to log phase of growth) and log-phase cells. However, the differences were not due to different glucose transport mechanisms. Alterations in the structural integrity of the cell envelope, as reflected by osmotic- and cold-sensitivity features of transition and log cells, may have affected the glucose uptake abilities in these cell types.
Characterizing Humidity, Sex, and B-Cell Gene Regulation in Fungal Allergic Asthma
(North Dakota State University, 2020) Kusick, Emma Claire
Asthma is a debilitating lung disease that affects nearly 300 million people worldwide. Environments with high humidity and subsequent mold exposure often trigger allergic asthma. Sex differences have been reported in the incidence, prevalence, and severity of asthma. B-lymphocytes are recruited in high numbers to the allergic lung in response to the inhalation of Aspergillus fumigatus mold spores (conidia). In this work, we used a mouse model of allergic fungal asthma to assess environmental humidity, sex, and B-lymphocytes in an inhalational model of allergic fungal asthma. Our results showed that animals sensitized in low humidity conditions had no airway hyperresponsiveness (AHR), inflammation, but an increase in IgG3 antibody production. Males weighed more than females, female mice had more fibrosis and produced more IgG3 Ab, but sex showed no impact on low humidity. C19+ B-lymphocytes differentially downregulated multiple genes related to allergic asthma returning the body to homeostasis.
Investigations in Asthma Heterogeneity: The Roles of Aspergillus Fumigatus-Derived Eicosanoid Synthases and Occupational Exposures to Grain Dusts on the Development of Fungal Allergic Asthma
(North Dakota State University, 2013) Sharma, Akshat
Allergic asthma is an inflammatory syndrome of the respiratory system which changes the airway wall architecture. Using an aeroallergen, murine model of A. fumigatus-mediated asthma, the two studies herein examine the development of asthma in the contexts of host-allergen interactions via A. fumigatus knock-outs of eicosanoid synthases and occupational exposures to corn and soybean dusts. The lack of difference between control and treatment groups seen in post-methacholine airway responses, goblet cell metaplasia, peribronchial inflammation, and fibrosis in the first study show that fungus-derived eicosanoid synthases are dispensable in the development of fungal allergic asthma. However, the same set of respiratory parameters in the grain dust study reveals an increase in BAL neutrophilia and serum IgE titer. The study also underscores a need for modifications of dust exposure times and of time-points of data analysis. These two studies represent unique perspectives on asthma pathogenesis and emphasize the heterogeneity of the syndrome.
The Link Between Gut Microbiota Metabolism and Host Gluconeogenesis by Vasoactive Intestinal Peptide
(North Dakota State University, 2022) Dawlaty, Razia
The gut microbiota (GM) plays a beneficial role in host metabolism. In mammals, the GM ferments dietary fiber into short chain fatty acids (SCFA), like propionate, that improves glucose metabolism. Rats fed a propionate diet increased intestinal gluconeogenic (IGN) gene expression, which was blocked by treatment with the neurotoxin, capsaicin, suggesting a neuronal-dependent mechanism. We hypothesized that the gut neuropeptide, vasoactive intestinal peptide (VIP), links GM derived propionate to IGN expression. We fed VIP deficient mice a 5% propionate chow diet (n=60) for 2 weeks and measured mRNA levels for GN genes by dPCR. Basel intestinal and liver GN mRNA expression was dysregulated by the loss of VIP. GN mRNA levels in liver were differentially altered in males versus females fed a propionate diet in a VIP-dependent manner. We conclude that VIP regulates basal intestinal and hepatic GN mRNA expression and mediates propionate induced GN mRNA changes in liver.
16S Ribosomal RNA and Phylograms: Characterizing Student Reasoning to Learning Outcomes from the American Society for Microbiology Curriculum
(North Dakota State University, 2017) Grassie, Chelsey Lee
The American Society for Microbiology (ASM) has established a suggested curriculum for introductory microbiology courses that includes a focus on evolution. However, no data is published to describe how proficiently students address the learning outcomes, in part because validated assessments do not exist. Thus, the goal of this project was to develop assessment prompts that capture student understanding about fundamental statement five under the core concept of evolution. In total, 167 written responses were collected from upper-division microbiology courses, with pre-pharmacy and microbiology majors comprising the majority of students (74.6%). Two coders coded all written responses, and five student interviews were conducted. Results indicate that students have not retained instruction on 16S rRNA, or have not been exposed to it in their classes. Additionally, most students have not been exposed to phylograms, and are unfamiliar with genetic distance being represented on a phylogenetic tree. Emergent reasoning techniques are described.
Studies in Pathogenesis of a Novel Isolate of Cronobacter Sakazakii using an In Vitro Blood Brain Barrier Model
(North Dakota State University, 2013) Welker, Elliott Weston
Genus Cronobacter is a member of the family Enterobacteriaceae consisting of several opportunistic species. The primary focus of this study was to utilize an in vitro co-culture model of the blood brain barrier to investigate a bovine fecal strain of C. sakazakii to investigate pathogenicity. The strain was found to have the same effect on the barrier’s integrity as the positive Escherichia coli control. Additionally, C. sakazakii strain BAA-894 was found to have the same effect as the negative E. coli control. This study also focused on the development of a site-specific mutagenesis procedure for C. sakazakii. A procedure using linear transformation was able to replace the putative virulence gene zpx (zinc-containing metalloprotease) in C. sakazakii. A future virulence study would involve using this mutagenesis procedure to induce a mutation in genes of C. sakazakii speculated to play a role in BBB translocation followed by challenge in the BBB model.
The Response of Salmonella enterica and Enterohemorrhagic Escherichia coli in Low-Moisture Environments
(North Dakota State University, 2021) Lauer, Jessica Rae
The recent increase in outbreaks of low-moisture foods due to contamination with foodborne pathogens has led to an increase in interest of the response of these pathogens to low-moisture environments. In addition, knowledge of the response of foodborne pathogens to immediate desiccation stress is sparce. The first objective was to evaluate the long-term survival of enteric pathogens on wheat grain over the course of a year. Hard red spring wheat was inoculated with strains of Salmonella enterica and enterohemorrhagic Escherichia coli (EHEC) and their survival was monitored for a year. Strains of Salmonella enterica were detected over the course of 52 weeks, while all strains of EHEC passed below the limit of detection by 44 weeks. The second objective was to evaluate the tolerance of various enteric pathogens to sudden desiccations stress and wide variation was seen between species and among strains and growth conditions.
Variability in Plant Growth Promoting Properties Among Clinical and Environmental Isolates of Stenotrophomonas Maltophilia
(North Dakota State University, 2015) Domfeh, Yayra Ekui
Stenotrophomonas maltophilia has both negative and positive attributes by being a human pathogen and plant growth promoting rhizobacterium. This study sought to determine if environmental and clinical isolates of S. maltophilia are phenotypically distinct. A total of 18 S. maltophilia isolates from clinical and environmental sources were investigated. Under normal growing conditions, S. maltophila isolates did not enhance growth of canola seedlings. However, under sodium chloride stress (6 decisiemens per meter or 0.33% NaCl), canola seedlings inoculated with S. maltophilia isolates had significantly (P < 0.05) higher number of root branches (isolate D457), root length (D457, CDC 2004-33-01-01 and CDC 2007-23-08-03) and stem length (D457, CDC 2005-37-11-04 and CDC 2011-01-42) than the “no bacteria” control. A number of S. maltophilia isolates protected canola plants from the growth limiting effects of Leptosphaeria maculans and Burkholderia cenocepacia. No clear evidence was found between clinical and environmental isolates based on phenotypic data.
ß-phenylethylamine: A Novel Chemo-Attractant Agent
(North Dakota State University, 2021) Burg, August Henry
Bacterial metabolism and physiology are finely tuned mechanisms that maintain homeostasis for the bacterium and allow for responses to environmental signals. Responses could include anything from regulation of cell division to the expression of virulence factors leading to serious infection. This thesis explores the role of neurotransmitter molecules and ß-phenylethylamine (a structurally similar molecule to catecholamines) on the physiological characteristics of flhD expression, biofilm formation, and chemotactic behaviors in the E. coli organism. We observed changes in physiology leading to chemotactic changes in the presence of ß-phenylethylamine through the Plug Agarose Assay as well as the Microfluidic Assay. In a comparison with serine, the amino acid and documented chemoattractant agent, ß-phenylethylamine was revealed to be a novel chemoattractant agent with comparable influence on the bacterium.
Cronobacter Sakazakii Characterization and Analysis of Cytotoxicity in Microvascular Endothelial Cells
(North Dakota State University, 2014) Hafner, Hilary Jayne
Contamination of powdered infant formulas by the bacteria Cronobacter sakazakii can pose serious risk to infants and neonates who consume the formula and subsequently develop C. sakazakii related illnesses such as sepsis and meningitis (1). The Gibbs’ lab assesses C. sakazakii isolates’ ability to cross the blood brain barrier and cause meningitis. This thesis research investigated C. sakazakkii cytotoxicity towards microvascular endothelial cells which comprise the first cell line encountered in the barrier. Understanding the mechanisms used to affect these cells will contribute to our understanding of early stages of invasion. Cytotoxicity assays performed for this research found that the cell line used could not sustain confluency when co-cultured with C. sakazakii isolates over periods beyond 24 hours of incubation. In addition, cell-free cytotoxicity assays found that live cells are not necessary to cause damage suggesting a toxin mediated effect.

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