Leveraging metabolomics for pathobiology: Advancements and applications of mass spectrometry-based approaches for chemical biology.
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Leveraging metabolomics for pathobiology: Advancements and applications of mass spectrometry-based approaches for chemical biology.


Metabolism occurs in a spatial-temporal fashion and represent the product of cellular functions. Mass spectrometry-based metabolomics enables the analysis of hundreds to thousands of analytes that can be used to study biological processes in the context of disease. Here, we leverage novel mass spectrometry approaches- both high-resolution mass spectrometry coupled to liquid chromatography, as well as matrix assisted laser desorption ionization mass spectrometry imaging- to exam neurometabolic related questions.Chapter one characterizes the biochemical, behavioral, and cytological in the brain in long-evans rats following neonatal, postnatal day six (P6), exposure to DMSO, the widely used vehicle (VEH) solvent. First, neurometabolic changes were profiled 24 hours after exposure in four distinct brain regions- the cortex, hippocampus, basal ganglia, and cerebellum- using hydrophilic interaction liquid chromatography. Second, behavioral tests were performed between P21-40 to investigate the chronic alterations to behavior following short term, early exposure. Lastly, immunofluorescence and immunohistochemistry were used to assess cytological changes in microglia, astrocytes, and neurons at P40. These findings show that short term exposure of DMSO, regardless of dose, at neonatal stages alters key regulatory metabolites and alters neurochemistry, results in chronic hypoactive behavior and decreased social habits, and in dose dependent increase in microglia and astrocytes. Chapter two investigates the role of p73a1, a p73 C-terminal isoform, in regulating lipid metabolism. Characterization of this novel protein isoform was described by Dr. Kyra Laubach, and cell lines were generated using CRIPSR to remove E12 in cancer cell lines. A multi-omic approach was integrated with molecular biology techniques to identify lipid classes and lipid metabolism-associated genes that were altered by the expression of p73a1. Furthermore, the tumor suppressive function of p73a1 was found to be mediated in part through lipid metabolism-associated genes. Through these findings, a previously unidentified p73 target was established, in addition to determining a role for p73a1 in lipid metabolism. Chapter three provides a framework and application for spatial metabolomics using matrix assisted laser desorption ionization- mass spectrometry imaging (MALDI-MSI). I demonstrate that spatial metabolomics enables data driven segmentation that provides unique clusters that resemble and map to anatomical structures. Next, I show that lipids in the brain are highly organized, and these lipids can be identified in tissue through MS/MS experiments. Additionally, I perform manual segmentation to extract ion intensities for regions of interest and show that MSI enables histopathological analysis of small molecules. Finally, I apply this workflow to a case study of litter-matched matched rats, one that expresses transgenes that induce an Alzheimer-like pathology, and its wild-type, healthy control litter-mate. I show that several phosphatidylcholine (PC) lipids are altered in the entire brain and specific regions of interest. Other PC lipids are changed only in clear ROIs when comparing WT and AD brains but are not changed in the whole brain. This workflow is a critical step necessary to enable histopathological analysis using metabolomics.

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