Date of Award

Spring 2014

Degree Name

Bachelor of Science

Major

Biochemistry

First Advisor

Michelle L. Kovarik

Second Advisor

Janet Morrison

Third Advisor

William Church

Abstract

The phospholipid bilayer of the cell is fluid and allows transport of small, hydrophobic compounds across the membrane. However, larger molecules such as peptides cannot cross the bilayer as easily. Therefore, my goal is to identify and optimize a method for loading exogenous peptides, such as reporters, hormones, or drugs, through the cell membrane. For these studies, we are using Dictyostelium discoideum as a model organism to test four peptide loading methods: pinocytosis, electroporation, cell-penetrating peptides and myristoylation. Pinocytosis uses changes in osmotic pressure to load the peptides through vesicles. The cells were exposed to a hypertonic solution for 10 min, 30 min, 1 hour, 4 hours, 17 hours, or 21 hours to load the peptide. Electroporation uses an electric field to create pores in the cell membrane that allow the diffusion of the peptide into the cell. The three different buffers used were H-50, HEPES, and a sucrose phosphate buffer, which were tested at electric field strengths of 8.50, 1.25, or 3.13 kV/cm, respectively. To follow up on promising initial results, the HEPES and sucrose phosphate buffers were additionally tested at 0.50, 0.63, 0.88, or 1.0 kV/cm. Myristoylation takes advantage of the cell membrane fluidity; the myristoylated peptide inserts itself into the membrane to allow the peptide to flip into the inside of the cell. Similarly, the cell penetrating peptide helps transport cargo into the cell through endocytosis or direct penetration. The incubation times in the loading solutions ranged from 1 minute to 1 hour. Each of these methods was tested for cell viability and highest percent of fluorescent peptide loading, which was measured under fluorescence microscopy. Pinocytosis stresses the cell through the varying osmotic pressures, and loading was very slow. Although electroporation showed efficient loading, this method disturbs the cell membrane and stresses the cells. Incubation of cells in myristoylated peptide and cell penetrating peptides load the peptides more gently. In fact, a 20 minute incubation of cells with the myristoylated peptide showed up to 99% of fluorescent cells with no effect on cell viability. Therefore, future work will use the myristoylated peptides as reporters to learn more about enzymatic activity and better understand signaling pathways implicated in disease.

Comments

Senior thesis completed at Trinity College for the degree of Bachelor of Science in Biochemistry.

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