Keywords

Wnt7a; WB; western blot; HEK; IF; Wnt

Abstract

Fetal Alcohol Spectrum Disorders (FASDs) refer to a set of development abnormalities affecting a fetus that can result from prenatal alcohol exposure (PAE). Studies performed by the National Institute of Health estimate that the pervasiveness of FASDs may number as high as 1 to 5 per 100 school children. Congenital heart defects (CHDs) are a subset of these abnormalities and have been observed to occur in 38% of children with FASDs. While there is an association between PAE and CHDs, the exact molecular mechanism as to how it occurs remains unclear. A 2022 RNA sequencing study points to the Wnt7a gene as one of several others to have its mRNA expression significantly decrease in the heart in response to PAE at a critical developmental time point. This gene codes for the Wnt7a protein that can play a role in activating the Wnt signaling pathways. This critical pathway plays a role in cell differentiation, cell proliferation, morphogenesis, embryonic development, and adult tissue homeostasis. While mRNA expression in response to alcohol has been studied, the change in protein expression of Wnt7a is yet to be determined. This thesis serves to demonstrate assay techniques and antibodies that can be used to reliably detect Wnt7a protein. We hypothesize that Wnt7a protein expression can be reliably detected in Human Embryonic Kidney 293 (HEK 293) cells through Western blot and immunofluorescence assays when the protein is overexpressed. Fluorescent, and more effectively, chemiluminescent western blot procedures produced a positive signal for detection of Wnt7a protein at the expected 40-42 kDA molecular weight range. Through Immunofluorescence, the ii Wnt7a+ HEK cells were confirmed to express the protein through Alexa Fluor probing of an anti-Wnt7a antibody, and the DYK HEK cells failed to produce a signal for expression of the protein as expected. The methods and techniques used in this study can be used to detect Wnt7a protein in embryonic hearts and determine how much it is affected by alcohol exposure. This serves toward the larger goal of identifying Wnt7a as a potential biomarker for helping to diagnose alcohol-induced CHDs. Additionally, these future directions can help direct attention to this gene as a useful therapeutic target for preventing and treating CHD formation.

Thesis Completion Year

2024

Thesis Completion Semester

Fall

Thesis Chair

Ebert, Steven

College

College of Medicine

Department

Biomedical Sciences

Thesis Discipline

Medicine

Language

English

Access Status

Open Access

Length of Campus Access

None

Campus Location

Orlando (Main) Campus

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Rights Statement

In Copyright