Date of Award

5-2008

Level of Access

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemical Engineering

Advisor

David J. Neivandt

Second Committee Member

Igor Prudovsky

Third Committee Member

Michael Mason

Abstract

The secretion of a variety of growth factors occurs early in the initiation of angiogenesis, restenosis, and certain cancers. Once secreted in response to cellular stress these growth factors lead to the initiation of inflammation and the production of blood vessels to provide oxygen and nutrients to growing tumors. Since the secretion of these growth factors is an early event in the disease progression it would appear an attractive point to design novel therapeutics against these disorders. However, the mechanism by which many of these growth factors are transported across the cell membrane during secretion is poorly understood. Proteins destined to be secreted typically contain a signal peptide which allows them to utilize the endoplasmic reticulum/Golgi apparatus classical release pathway. Several signal peptide-less (SPL) proteins have been discovered and shown to be secreted during cellular stress without the direction of a signal peptide. Fibroblast growth factor 1 (FGF1) is one of these SPL proteins known to be transported through a non-classical mechanism. A better understanding of the non-classical transport process would provide crucial information to the scientific community towards the elucidation of therapeutics targeted against heart disease, Alzheimer's disease, and cancer. In this work a variety of techniques were employed to study the behavior of the FGFl protein and its release complex with regard to their interaction with the plasma membrane. Experiments were performed to determine how FGFl is able to bind to the plasma membrane and in what form it is able to permeate it. These experiments along with a mutation analysis identified a critical phospholipid binding domain within FGFl. Tertiary structure analysis discovered that, unlike classically transported proteins, FGFl is able to pass through the cell membrane in a locked tertiary state. To further understand the biophysics underlying non-classical transport events sum frequency generation vibrational spectroscopy (SFS) was employed. Utilizing this technique FGFl induced deformation of a model membrane was observed providing the first evidence recorded of such interactions via SFS. Final work involves the design and characterization of hydrogel supported lipid bilayer substrates which provide a better physiological model for study of the transport process.

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