All ETDs from UAB

Advisory Committee Chair

Helen Kim

Advisory Committee Members

Lori L McMahon

Stephen Barnes

Rakesh Patel

Giuseppe Squadrito

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Estrogen replacement therapy (ERT) has been shown to improve memory function in menopausal women, but increases hormone sensitive cancers, prompting a more targeted investigation of estrogen's action in learning and memory to develop more targeted therapeutics. Estrogen improves performance on the novel object recognition task by increasing phosphorylation of extracellular signal-regulated kinase (pERK) within minutes in the hippocampus. Dr. Lori McMahon's lab showed that the improvement in novel object recognition can be seen 24 hours after subcutaneous injection of proestrous levels of estrogen (referred to as E24). Whether a corresponding increase in pERK occurs at the E24 time point had not been determined. We hypothesize pERK will be increased at E24. Western blot analysis of specific hippocampal region (CA1) showed that pERK was increased by estrogen. Tamoxifen, which is thought to act as an antagonist to estrogen receptor alpha in the brain, blocked the estrogen-enhancement of pERK, suggesting that the phosphorylation of ERK seen in E24 is downstream of genomic actions of estrogen. Estrogen may also enhance cognitive function by protecting against molecular events that lead to age-related dementias. Estrogen was shown to reduce the ratio of amyloid beta 42/40 peptides, the highest known correlate of Alzheimer's disease progression. The most widely used assay for measuring amyloid beta 42/40 is an ELISA. Unfortunately, ELISAs have been reported to give inaccurate results when measuring amyloid beta due to giving both false positive and false negative signal. An alternative method for measuring amyloid beta is using mass spectrometry. The second half of this dissertation is a systematic optimization of analytical methods for detecting and quantifying amyloid beta 42/40, using mass spectrometry-based approaches from cell culture media at biologically relevant concentrations without the use of antibodies, while minimizing manipulations that could result in loss of amyloid beta peptides, which are notorious for aggregation and nonspecific adsorption to plastics. We found that we could detect amyloid beta 42/40 at concentrations as low as 5 ng/ml, which is the reported concentration for amyloid beta in cell culture media. Our findings should be applicable to cell culture media as well as artificial cerebrospinal fluid and possibly brain tissue samples.

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