<b>HOST-PATHOGEN INTERACTIONS IN </b><b><i>CLOSTRIDIOIDES DIFFICILE </i></b><b> INFECTION: SPATIAL TRANSCRIPTOMICS AND NEUROTOXICITY IN BRAIN-ON-A-CHIP MODELS</b>

<p dir="ltr"><i>Clostridioides difficile</i> infection (CDI) ranks as a major cause of antibiotic-associated colitis, with a severe clinical impact from its inconstant presentation as well as systemic complications. This project addresses host-pathogen interactions in CDI through two distinct but complementary studies, with one spatial transcriptomic analysis of colonic tissues and the other exploring <i>C. difficile</i> toxin-induced neurotoxicity in brain cells utilizing a microphysiological system. The initial section of the thesis describes a literature review focusing on using spatial transcriptomics in infectious diseases, highlighting its ability to identify tissue-level dynamics of hosts and pathogens. This review is then supplemented by an empirical research project employing spatial transcriptomics to investigate gene expression in tissues from mice infected with <i>C. difficile. </i>The research describes separate transcription patterns in symptomatic versus asymptomatic mice, illuminating localized epithelial changes and immune reactions potentially responsible for the differential outcome. The second investigation examines <i>C. difficile</i> toxins A and B neurotoxicity in a brain-on-a-chip system that contains primary mouse neurons, astrocytes, and microglia. Electrophysiological recordings with microelectrode arrays show that exposure to toxins induces progressive neuronal dysfunction with different time profiles for each toxin. It substantiates a mechanism for neurocognitive symptoms in some CDI patients, even without overt systemic illness. This appendix presents a step-by-step protocol for isolating and culturing colonic organoids from mouse tissue, providing a valuable tool for future in vitro gut-specific modeling of host-pathogen interactions. These studies offer information on intestinal and neurological aspects of CDI, emphasizing the value of spatial and functional models in infectious disease research.</p>