Theses and Dissertations at Montana State University (MSU)
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Item The interaction of heavy metals with the mammalian gut microbiome(Montana State University - Bozeman, College of Agriculture, 2022) Coe, Genevieve Lea; Chairperson, Graduate Committee: Seth Walk; This is a manuscript style paper that includes co-authored chapters.Heavy metals are for the most part, naturally occurring elements found in the environment. Some are essential, meaning they are involved in critical biochemical pathways, in all branches of life. Other heavy metals are non-essential and disrupt metabolic functions in most organisms rendering them toxic. The following research explored the interactions of the mammalian gut microbiome with an essential heavy metal, iron, and a non-essential heavy metal, methylmercury, acquired through the diet. The overarching goal was to define and characterize the influence of gut microbial interactions with heavy metals on host health. Novel experimental designs using murine models were designed to examine 1) the consequences of low iron challenge on the murine gut microbiome and whether host iron availability was affected and 2) the potential influence of the gut microbiome in methylmercury elimination rate and demethylation in conventional, germ-free, gnotobiotic, and humanized mice. Culturing in vitro and toxicity assays, 16S sequencing, deep metagenomic sequencing of human stool, bioinformatic analysis, transcriptional analysis of iron biomarkers, quantification of iron and mercury by ICP-MS and HPLC-ICP-MS methods were performed as well as the use of mouse models to examine iron and methylmercury interactions with the gut microbiome in vivo. Our results from this project indicate that the gut microbiome is significantly affected by loss of iron from the diet, and does not fully recover post-iron repletion, while the host is relatively unaffected by low-iron challenge to the gut microbiome. Methylmercury elimination and demethylation is significantly faster and higher, respectively, in mice with a gut microbiome, providing novel evidence in support of a role for the gut microbiome in methylmercury demethylation and elimination. However, exact mechanisms of microbial interactions with methylmercury in the gut have yet to be elucidated. Our data also suggests the possibility of host-mediated mechanisms of methylmercury demethylation, by yet unknown mechanisms that warrant further exploration.Item Aerobic bacterial methane synthesis in the human gastrointestinal tract(Montana State University - Bozeman, College of Agriculture, 2023) Jackson, Thomas Robert; Chairperson, Graduate Committee: Seth WalkAerobic bacterial methane synthesis constitutes a paradigm-shifting novel metabolism recently described in aquatic environments. It challenges the traditional model of methanogenesis as being a strictly anaerobic process carried out by archaeal methanogens. To date, the presence of aerobic bacterial methane synthesis has not been studied within the context of the human gastrointestinal tract. The goal of this work was to investigate the possibility of the presence of such metabolisms in the human gut microbiome. To investigate this, fecal samples from six individuals were first screened for the ability to produce methane under aerobic conditions. Bacteria from two of those fecal samples were isolated and evaluated for their ability to utilize methylamine, a known substrate involved in aerobic bacterial methane synthesis, as a sole nitrogen source. The ability of those isolates to produce methane under aerobic conditions from methylamine was then evaluated. Additionally, a flask-independent culture-based assay was developed in order to screen larger numbers of future isolates for the ability to utilize methylamine as a sole nitrogen source. This work demonstrates the first evidence of aerobic bacterial methane synthesis from members of the human gastrointestinal tract, finding two isolates capable of producing methane under aerobic conditions. Such findings broaden the understanding of methane-generating pathways that may have implications for the development of dysbiosis and atherosclerosis in human hosts.Item Microbiomes and zoonoses: dynamics of the black flying fox (Pteropus alecto) gastrointestinal microbiome(Montana State University - Bozeman, College of Agriculture, 2022) Jones, Devin Nicole; Chairperson, Graduate Committee: Raina K. Plowright; This is a manuscript style paper that includes co-authored chapters.Land-use change is increasingly recognized as a driver of spillover of zoonotic pathogens. Australian black flying foxes (Pteropus alecto) are experiencing extensive loss of habitat which reduces available food, particularly in winter. Hendra virus (HeV, family: Paramyxoviridae) was isolated from horses and humans in 1994 and P. alecto was later identified as the reservoir host. As habitat loss threatens these bat populations, and Hendra virus continues to spill over to horses annually, it is important to understand factors that influence bat health and viral shedding. Because gastrointestinal tract (GIT) microbiomes are important for host health and are understudied in flying foxes, the goal of this research was to understand the natural dynamics of the P. alecto GIT microbiome and its associations with diet, body composition, markers of inflammation, and viral shedding. We sampled Pteropus alecto near Brisbane from 2018-2020. We captured bats returning from foraging and collected rectal swabs to determine the GIT microbiome using 16S rRNA amplicon sequencing. In addition to feces for dietary analysis, we also collected samples to measure health and infection, including blood to measure neutrophil-to-lymphocyte ratios, urine to detect Hendra virus, and bioelectrical impedance analysis to measure body fat. These data enabled us to determine how the P. alecto GIT microbiome varied within individuals over time and in the context of physiological, ecological, and dietary shifts. Lastly, we asked if we could predict health outcomes using the GIT microbiome. We found that P. alecto GIT microbiomes are highly dynamic over time, through different life stages, between foraging strategies, and that the type of diet is associated with GIT microbiome diversity. Bats consuming native foods had lower GIT microbiome diversity compared to those consuming introduced and cultivated foods. Despite associations between body fat and HeV infection, the GIT microbiome was not able to predict these health outcomes. These results suggest that P. alecto GIT microbiomes are highly dynamic and may not contribute significantly to host health. Future research should incorporate more health metrics or other approaches to microbiome profiling to determine if the GIT microbiome could be used as a biomarker of health.Item Diet-induced alterations to postprandial metabolism and the gut microbiota(Montana State University - Bozeman, The Graduate School, 2022) Wilson, Stephanie Michelle-Gandia; Chairperson, Graduate Committee: Mary P. Miles; This is a manuscript style paper that includes co-authored chapters.Obesity is a key component of a cluster of metabolic risk factors for chronic disease which include dyslipidemia, hyperglycemia, and hypertension. Stark changes in diet and lifestyle contribute to growing metabolic disorder prevalence. Many Americans regularly consume foods low in fiber and rich in fat and sugar, which can negatively influence glucose and lipid metabolism over time. A nutritionally poor diet exerts deleterious effects on the gastrointestinal microbial community which has larger host health implications. As a modifiable risk factor, diet can be part of the solution to counter the rise of chronic disease. However, dietary responses as it pertains to glucose and lipid metabolism display high interindividual variability. This interindividual variability with diet can also be observed at the microbial level in our gastrointestinal system. In metabolically at-risk adults, we examined postprandial responses and the efficacy of a long-term lentil dietary intervention in improving postprandial glycemic responses. We further assessed the effect of an antioxidant-rich juice from the Aronia melanocarpa berry and its anti-inflammatory potential against a high fat diet in a gnotobiotic mouse model to evaluate how specific gut microbial communities transferred from metabolically at-risk adults with different inflammatory profiles may impact dietary responses. Postprandial triglyceride responses in adults with overweight and obesity can be partially explained by central adiposity, insulin resistance, and the ability to switch between glucose and fat metabolism to a metabolic stress. A long-term dietary intervention with lentils, a fiber-rich pulse crop, worked in a dose-dependent manner to reduce insulin resistance in adults with increased central adiposity without an increase in gastrointestinal symptoms, a common deterrent to incorporating fiber-foods in the diet. Our gnotobiotic mouse experiments revealed donor- dependent changes in dietary responses. We observed protective effects of Aronia juice in mice, particularly in mice from the low inflammation stool donor. Metabolomic changes in phospholipids such as phosphatidylcholine and sphingomyelin were further detected, with changes respectively unique to Aronia juice and high-fat diet. Taken together, this dissertation provides an improved understanding of our metabolic responses and microbial alterations to the foods we consume, and how these responses influence the progression of metabolic diseases.Item Using gastrointestestinal organoids to study infectious diseases in humans and bats(Montana State University - Bozeman, College of Agriculture, 2021) Hashimi, Marziah; Chairperson, Graduate Committee: Diane Bimczok; This is a manuscript style paper that includes co-authored chapters.The gastrointestinal epithelium plays a critical role in protecting the gastrointestinal mucosa from invading microorganism such as bacteria or a viruses. Helicobacter pylori (H. pylori) infection of human gastric epithelium causes gastric cancer, which is the third leading cause of cancer-related mortality worldwide. Dendritic cells (DCs)--which are antigen presenting cells--are responsible for the activation of T cells. However, the mechanism by which DCs are recruited to the gastric epithelium is still unknown. We hypothesized that the DCs are recruited to the gastric epithelium in a chemokine- dependent manner. For my thesis work, I utilized human primary gastric epithelial organoids cells to test this hypothesis and evaluate the recruitment of DCs to the epithelium under normal conditions and upon H. pylori infection. Using monocyte-derived DCs in a chemotaxis assay, I showed that these cells are recruited to H. pylori-infected organoid supernatant. I showed that this recruitment is chemokine- dependent, as it was significantly decreased when a chemokine receptor inhibitor was included in the chemotaxis assay. COVID-19 is caused by severe respiratory syndrome coronavirus-2 (SARS-CoV-2). In addition to respiratory symptoms, COVID-19 patients can also have diarrhea and vomiting, indicating that SARS-CoV-2 may infect the gastrointestinal tract. Bats are thought to be the natural reservoirs for SARS-CoV-2, however there is no known bat gastrointestinal model to study SARS-CoV-2 infection. In the second part of my thesis, I developed Jamaican fruit bat (JFB), Artibeus jamaicensis) gastrointestinal organoids (JFB organoids). I successfully developed organoids from JFB stomach, proximal and distal intestine. I showed via histology and gene expression that developed organoids do indeed recapitulate their corresponding tissues from which they were derived. I also tested whether the JFB distal intestinal organoids were susceptible to SARS-CoV-2 infection. While they do not support the active replication of SARS-CoV-2 infection, they did show antiviral and pro-inflammatory responses. My results also showed that SARS-CoV-2 does not induce programmed cell death in the organoids.Item Scoping out intestinal epithelium differentiation, proliferation, and homeostasis through the lenses of interleukin-10 and aryl hydrocarbon receptor signaling pathways(Montana State University - Bozeman, College of Agriculture, 2020) Jenkins, Brittany Rene; Co-Chairs, Graduate Committee: Douglas Kominsky and Seth Walk; Nathan A. Blaseg, Heather M. Grifka-Walk, Benjamin Deuling, Steve D. Swain, Eric L. Campbell, Seth T. Walk and Douglas J. Kominsky were co-authors of the article, 'Loss of interleukin-10 receptor disrupts intestinal epithelial cell proliferation and skews differentiation towards the goblet cell fate' submitted to the journal 'The Federation of American Societies for Experimental Biology (FASEB) journal' which is contained within this dissertation.; Heather M. Grifka-Walk, Steve D. Swain, Trevor R. Zahl, Andrew Gentry, Seth T. Walk and Douglas J. Kominsky were co-authors of the article, 'Aryl hydrocarbon receptor modulation of intestinal epithelial cell fate is sex-dependent and exhibits variability among allelic variants' which is contained within this dissertation.; Dissertation contains two articles of which Brittany Rene Jenkins is not the main author.Intestinal epithelial cells (IEC) are crucial for maintaining proper digestion and overall homeostasis of the gut mucosa. IEC proliferation and differentiation are tightly regulated by well described pathways, however, relatively little is known about the influence of interleukin (IL)-10 and aryl hydrocarbon receptor (AHR) signaling pathways on these processes or whether AHR can regulate IL-10R expression in IECs. IL-10 signaling suppresses inflammation. AHR is a ligand activated transcription factor largely known for downstream activation of xenobiotic-metabolizing enzymes but also exerts a diverse range of responses in the host that can be modulated by gut microbe metabolites. Both IL-10 and AHR signaling are shown to promote IEC barrier function, and thus, they may also regulate other critical homeostatic functions like IEC lineage fate and regenerative capacity. These gaps in knowledge were addressed in Chapters 2 and 3. Techniques such as reverse-transcription quantitative polymerase chain reaction (RT-qPCR), western blotting, and histology staining techniques were used to assess changes in expression of target genes and proteins between control and either IL-10R- or AHR-deficient models. Loss of IL-10R or AHR demonstrated substantial impacts exhibiting nearly opposite patterns on lineage fate outcomes and on the proliferative compartment. In Chapter 4, we showed that activation of AHR by microbe-derived tryptophan metabolites increased IL-10R1 expression in IECs, and these metabolites ameliorated disease in a murine model of colitis. Findings from Chapters 2-4 add to a growing body of evidence for the importance of IL-10R and AHR signaling pathways in inflammatory bowel disease (IBD) and gastrointestinal (GI) cancers. Organoid models provide an additional study system to test these gaps in the field and hold great promise for advancing disease research. However, limitations exist for accessing the luminal surface to recapitulate the GI environment. In Chapter 5, we developed the GOFlowChip to solve this problem. This platform applies long-term, steady-state flow through to the organoid and can be modified to serve different research goals. These studies culminate in a deeper understanding of how IEC homeostasis is maintained and how innovative technologies can be developed for advancing this field of research.Item The influence of an iron deficient diet on the murine gut microbiome(Montana State University - Bozeman, College of Letters & Science, 2019) Coe, Genevieve Lea; Chairperson, Graduate Committee: Jennifer DuBoisIron is an essential nutrient for mammals 1. It is involved in multiple redox reactions that are essential for the survival of most organisms 2. There are two main types of iron that are absorbed from the diet: inorganic iron and heme 3. Dietary iron ingested by mammals is mostly absorbed in the small intestine; however, it is unclear whether the gut microbiome is involved in iron homeostasis or whether iron in the diet influences the microbiome. The goal of this project is to characterize the change in microbial composition in response to iron deficiency and iron repletion in conventional mice and define a baseline model for future studies involving the more complex human gut microbiome.Item Gastrointestinal organoid structure and transport(Montana State University - Bozeman, College of Engineering, 2019) Sidar, Barkan; Chairperson, Graduate Committee: James Wilking; Thomas A. Sebrell was an author and Rachel Bruns, Royce A. Wilkinson, Blake Wiedenheft, Paul J. Taylor, Brian A. Perrino, Linda C. Samuelson, James N. Wilking and Diane Bimczok were co-authors of the article, 'Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation, and fusion events' in the journal 'Cell and tissue research' which is contained within this dissertation.; Thomas A. Sebrell, Bengisu Kilic, David Brown, Mert Aytac, Brian A. Perrino, Linda C. Samuelson, Henry Fu, Diane Bimzcok, James N. Wilking were co-authors of the article, 'Rupturing of human gastric organoids' which is contained within this dissertation.; Brittany R. Jenkins, Sha Huang, Jason R. Spence, Seth T. Walk and James N. Wilking were co-authors of the article, 'Flow through human intestinal organoids with the gut organoid flow chip (GOFlowChip)' submitted to the journal 'Lab on a Chip' which is contained within this dissertation.; Dissertation contains two articles of which Barkan Sidar is not the main author.Organoids are three-dimensional (3D) self-assembled, mammalian tissue cultures derived from stem cells that differentiate to contain multiple cell types. These cells spatially organize within the 3D structure and are capable of recapitulating the structure and function of a particular organ. Organoids offer a variety of existing and potential applications in medicine and biotechnology, including drug formulation testing, regenerative medicine, and microbiome research. Despite their value, knowledge of how organoid structure impacts dynamics, mechanics, and transport is lacking. This is particularly true for gastrointestinal organoids, which are composed of a monolayer-thick epithelial sheet wrapped into a closed sphere. The primary goals of this dissertation are to understand the impact of gastrointestinal organoid structure on organoid function, develop a millifluidic chip platform to improve their viability and reliability as a model system and to explore their uses as model co-culture systems. To achieve this, we use a combination of time-lapse microscopy, image analysis, modeling, and fluidics fabrication techniques to develop an understanding of organoid growth and development in addition to expanding current uses as model systems. Our observations revealed that human gastric organoid growth was associated with cyclic rupture of the epithelial shell, rotational movement around their axes within the Matrigel matrix and luminal fusion by adjacent organoids. Furthermore, the rupture events are an indirect result of osmotic swelling carried out by the diffusion of water due to osmolyte concentration regulation by the epithelial shell. To overcome the advection limitation due to the topologically closed spherical structure of the organoids, we developed a millifluidic device called the Gut Organoid Flow Chip (GOFlowChip). This represents the first demonstration of established liquid flow through the luminal space of a gastrointestinal organoid. Given that organoids show great potential as model systems, established co-culture system consisting of dendritic cells (DC) with infected human gastric organoids shows the gastric epithelium actively recruits DCs for immunosurveillance with increased recruitment upon active Helicobacter pylori infection. Finally, investigation on CD103 attachment protein in gastric DCs revealed that CD103 engages in DC-epithelial cell interactions upon contact with epithelial E-cadherin but is not a significant driver of DC adhesion to gastrointestinal epithelia.Item Analysis of human host defense mechanisms against the opportunistic pathogen, Candida albicans, using in vitro model systems(Montana State University - Bozeman, College of Agriculture, 1999) Edens, Heather Amy