Date of Award

2022

Degree Type

Thesis

Department

Pharmaceutical Sciences

First Advisor

Fatemeh Akhlaghi

Abstract

Background and Objectives:Non-alcoholic fatty liver disease (NAFLD) encompasses the progressive hepatic pathologies from steatosis, non-alcoholic steatohepatitis (NASH), fibrosis, and irreversible cirrhosis in populations with little to no alcohol consumption. The disease is prevalent in societies dominated by Western dietary patterns and is associated with obesity, hypertension, dyslipidemia, type-2 diabetes mellitus, and metabolic syndrome. The pathogenesis of NAFLD is multifaceted and not completely understood. A proteomic analysis of a human liver tissue repository (n = 106) was used to gain insight into dysregulated pathways and significantly altered protein expression in NAFLD without or with fibrosis as well as NAFLD and diabetes mellitus.

Methods:A bottoms-up proteomic approach was used for label-free protein quantitation of whole human liver tissue homogenate (n = 106) through sequential window acquisition of all theoretical mass spectra (SWATH-MS) based data-independent acquisition (DIA) method. Human proteome (UniProtKB) and mitochondrial (MitoCarta 3.0) databases were used for protein identification. Differential expression of proteins was conducted using limma (linear models of microarray data), followed by enrichment and pathway analysis using tools such as ShinyGO and MitoCarta 3.0.

Results:A total of 3,334 proteins were identified in liver tissue samples (n = 106) with 338, 615, and 122 differentially expressed proteins in NAFLD, NAFLD and diabetes, and fibrosis disease groups, respectively. Enrichment analysis uncovered affected biological processes related to mRNA translation, movement to the endoplasmic reticulum, and extracellular matrix integrity; cellular components were localized to the mitochondria and extracellular matrix; and molecular function of fatty acid metabolism, collagen binding, and extracellular matrix. Exploratory analysis of mitochondrial proteins (n = 619) resulted in 98, 166, and 12 that were differentially expressed in NAFLD, NAFLD and diabetes, and fibrosis disease groups, respectively. Enrichment analysis pointed to mitochondrial dysfunction related processes, including acyl-CoA metabolism and oxidative phosphorylation. Evaluation of mitochondrial pathways (n = 10) demonstrated up and down regulation of proteins in the associated pathways from significantly altered proteins (-log10 Benjamini-Hochberg adjusted p-values), and that NAFL and diabetes, NASH and diabetes, and NAFL disease states had the most impact in modifying protein expression when compared to their respective normal condition. Analysis in limma uncovered several proteins (AADAT, ACADSB, MRPL17, MRPL38, NDFUFA12, and SLC25A24) that were significantly different (p-value < 0.05 and Benjamini-Hochberg adjusted p-values < 0.1) and identified in several disease groups (i.e., NAFLD, NAFLD and diabetes, and fibrosis).

Conclusions:Proteome analysis of human liver tissue in NAFLD, NAFLD and diabetes, and fibrosis disease groups identified proteins and pathways associated with mitochondrial dysfunction. Further studies of mitochondrial liver fractions will better elucidate the impact of NAFLD disease progression on mitochondrial protein expression and pathway dysregulation that may have been confounded with the analysis of whole liver homogenate samples.

Available for download on Friday, September 06, 2024

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