AN INTEGRATED MULTI-LAYERED APPROACH USING HUMANIZED SINGLE-CELL TRANSCRIPTOMICS AND IN VIVO MODELS TO DEFINE THE ROLE OF CHOLINERGIC RECEPTOR NICOTINIC ALPHA 7 SUBUNIT (CHRNA7) IN VASCULAR AND NEUROINFLAMMATORY DISEASE

English Abstract Background: Atherosclerosis is a chronic inflammatory disease driven by the activation of innate and adaptive immune cells. The Chrna7 gene, encoding the α7 nicotinic acetylcholine receptor (α7‑nAChR), has emerged as a potential regulator of myeloid and microglial functions. Growing evidence points to its involvement in neuroinflammatory processes, particularly in subarachnoid hemorrhage (SAH), yet its contribution to atherosclerosis remains poorly understood. Aim: This thesis develops a computational pipeline for species‑resolved single‑cell analysis in humanized mouse models and uses its conceptual framework to investigate the immunological and inflammatory consequences of Chrna7 deletion, integrating transcriptomic, metabolic, and proteomic approaches to advance understanding of immune regulation. Materials and Methods: A single‑cell RNA‑seq analysis pipeline was implemented on AWS and optimized for species‑specific cell identification in mixed human–mouse samples. Proteomic analyses were then employed to characterize Chrna7‑associated pathways in neuroinflammatory responses following SAH. Finally, a Chrna7‑/‑/Ldlr‑/‑ mouse model was used to assess the gene’s impact on peripheral immune regulation and atherosclerotic plaque development. Results: The cloud‑based pipeline enabled accurate and reproducible discrimination of human and murine cell populations, demonstrating high scalability. Proteomic profiling revealed a potential involvement of Chrna7 in modulating microglial activation after SAH. However, the Chrna7‑/‑/Ldlr‑/‑ model showed no significant alterations in peripheral immune composition or in the extent of atherosclerotic lesions. Conclusions: These findings indicate that Chrna7 may play a context‑dependent role in microglia‑mediated neuroinflammation during SAH, while exerting minimal influence on systemic immune responses and atherogenesis. Moreover, the single‑cell pipeline developed in this work provides a robust framework for future investigations in humanized models and complex inflammatory conditions.