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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 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 Demonstration of the leukocyte-endothelial cell adhesion cascade involved in lymphocyte recirculation and neutrophil and lymphocyte localization to sites of inflammation(Montana State University - Bozeman, College of Agriculture, 1994) Bargatze, Robert Frost