Dissertation - Open Access
Doctor of Philosophy (PhD)
Chemistry and Biochemistry
Douglas E. Raynie
Proteomics is a high-throughput approach to study protein expression, structure, function, interaction, post-translational modifications, and localization in a cell or tissue. Proteomic shotgun approach was used to analyze changes in proteins in bovine follicular fluid (FF) and plasma (PL) from cows with high estradiol (HE2) or low E2 (LE2) during the preovulatory period. FF creates a unique microenvironment in follicles necessary for follicle growth and oocyte maturation, and pre-ovulatory concentrations of E2 have been reported to impact several processes involved with fertility. Initial steps in the analysis involved development of an immunodepletion protocol for high abundant proteins (HAPs) in bovine PL, FF, epididymis sperm and ejaculated sperm. The determined depletion rates for the HAPs albumin, IgG, and IgA ranged from 98.7 to 99.9%. Similar depletion rate was observed for alpha-1-antitrypsin based on its gel band. Peptides were labeled with iTRAQ reagents and quantified using 2-dimentional liquid chromatography (LC) electrospray (ES)-based mass spectrometry. E2 was associated with protein changes in PL and FF. Protein expression changes between FF HE2 and FF LE2 were higher than PL HE2 and PL LE2. There were 15 up-regulated proteins and ten down-regulated proteins in FF HE2 compared to FF LE2. Seven proteins were up-regulated and nine proteins down-regulated in PL HE2 compared to PL LE2. Proteins were more predominant in PL than in FF but the extent of protein changes with HE2 was greater in FF than in PL. Several of the differentially expressed proteins function in follicle development and were mainly categorized under cellular process and metabolic process. Pathway analysis identified the up and down-regulated proteins were predominantly associated with the complement and coagulation cascades which support the view that folliculogenesis and ovulation are hemorrhagic events. The data demonstrates E2 regulates a wide range of reproductive associated proteins in bovine PL and FF and forms the basis for further investigation of specific processes involved in such regulation. This work was also interested in characterization and quantification of atherosclerotic lesions in three regions of aortic root of ApoE-/- mice. Atherosclerosis is the underlining cause of heart attack and stroke, the two leading cause of cardiovascular death worldwide. The three regions of the aortic root examined were the ascending aorta region (AAR), region showing the orifices of the coronary arteries marking the start of the ascending arch (OCAR) and aortic sinus region (ASR) in ApoE-/- male and female mice at different ages. 67 ApoE-/- and 27 wild type C57BL/6J mice (controls) were fed with a high fat diet (HFD) until age 8, 12, 18, or 24 weeks. Through systematic classification and quantification of lesions in each region and statistically data analysis, we found that the complexity and total atherosclerotic lesion areas in ApoE-/- mice was location and age dependent. It was slowest in the AAR with lesions progressing from dominant type I at 8 weeks, type II at 12 weeks, types III at 18 and types III and IV at 24 weeks of age. Lesion development was comparable in the OCAR and ASR regions; types II and III lesions dominated in mice at 8 and 12 weeks of age respectively while types IV and types IV and V dominated at 18 and 24 weeks of age, respectively. Average percentage of atherosclerotic lesions typically increased from the AAR to the OCAR to the ASR at a specific age, and from 8 to 24 weeks of ApoE-/- mice at each region, correlating with the histological data. These findings would be beneficial in experimental design and targeting of lesion types in aortic roots of the popular ApoE-/- murine atherosclerosis model.
Library of Congress Subject Headings
Cattle -- Fertility
South Dakota State University
In Copyright - Non-Commercial Use Permitted
Afedi, Patience Akosua, "Mass Spectrometry-Based Quantitative Proteomic Analysis of Biological Fluids" (2019). Electronic Theses and Dissertations. 3156.