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Abstract

The gastrointestinal tract (GI) represents the largest surface in the human body, the main entry point for nutrients and microbes in food and water. As such, the intestinal epithelial cells (IECs) lining the surface of the GI tract play a critical role in both nutrient absorption and immune surveillance. The intestinal epithelium has undergone morphological adaptations with the development of villi protruding into the lumen and the invagination of the epithelial monolayer at the base of the villi, known as crypt, forming a three-dimensional feature. This adaptation not only maximizes the surface area for nutrient absorption but also restricts the localization of specialized cell types to specific areas in the epithelium. The crypts experience the highest oxygen concentration from the blood flow in the subepithelial mucosa, while the villi protrude into the oxygen-deficit gut lumen. This leads to the formation of a unique oxygen gradient along the crypt-villus axis with the highest oxygen level in the crypt (pO2 of 60 to 80 mmHg) towards the tip of the villus (pO2 of 10 mmHg). During the development in the small intestine, the proliferative stem cells in the crypt differentiate into specialized cell types while they migrate along the crypt-villus axis towards the tips of villi and are eventually shed into the gut lumen. As such, these developing cells experience a steep oxygen gradient while migrating towards the villi. The impact of such oxygen gradient on the development and differentiation process of the IECs still needs to be addressed. In this thesis, I sought to elucidate the effect of varying oxygen levels on developing IECs using a primary, non-transformed human ileum- derived organoid culture. I demonstrated that hypoxia or low oxygen tension negatively impacts stem cell activity while promoting cell differentiation in IECs. Additionally, employing both bulk RNA sequencing and single-cell RNA sequencing, a complex signaling network involving Wnt, BMP, and Notch pathways was shown to be affected under hypoxia through the modulation of their respective regulators and transcription factors. This hypoxia-induced phenotype on cell differentiation could also be a result of metabolic regulation in addition to the modification of cell differentiation pathways. In this work, I sought to demonstrate further how the oxygen gradient along the crypt-villus axis can affect other functional aspects of the IECs such as shaping the early innate immune signaling pathways as the IECs play a pivotal role as the first line of defense in the gut. They must maintain a mutualism relationship with the commensal microbiota present in the lumen while being immune reactive towards possible pathogenic invasions. Here I presented a novel microfluidic device recapitulating the 3D oxygen gradient in the gut in a 2D radial manner. Using a lentivirally transduced T84 cell line expressing a hypoxia sensor, dUnaG, the fluorescence intensity of the reporter can be used as a functional readout of the oxygen concentration. This novel device was proposed as an in vivo model for studying the differential immune response and other aspects of IECs in response to varying oxygen concentrations. Given the life-threatening global pandemic, COVID-19 caused by SARS-CoV-2 causes gastrointestinal distress, especially in patients with severe COVID-19. It is crucial to characterize in details SARS-CoV- 2 infection and the mounted innate immune responses in the human intestinal epithelium. Here, I demonstrated the capability of SARS-CoV-2 infecting the IECs and the mounted interferons (IFNs) antiviral response, emphasizing the efficiency of type-III IFNs in fighting against the infection. Additionally, SPINT2, an endogenous cellular protease inhibitor, was identified and characterized in its ability to control SARS-CoV-2 infection.