Electrical and Computer Engineering ETDs

Author

Julia Kleven

Publication Date

7-12-2014

Abstract

The innate immune system enables cellular response to infectious agents, and protein interactions are essential for this response. However, the protein interactions involved in cellular response to pathogens are not completely understood. Clarifying the manner in which proteins bind and respond to infectious agents is necessary for development of potential therapeutics or preventative measures. Fluorescent probes and fluorescent microscopy are used to aid in the visualization of these components, but proteins associated with or spanning the cellular membrane are on the nanometer scale, smaller than some microscopes can image, which makes it difficult to accurately localize the proteins of interest. To further the understanding of protein dynamics, a higher resolution form of optical microscopy had to be developed that allowed for multi-component cellular imaging without the need for harsher fixatives like those required for electron microscopy. To this end, optical super resolution techniques rely on the blinking attributes of fluorophores currently utilized in protein labeling in conjunction with specialized post processing to enable sub-diffraction limit v localization. These techniques allow the visualization of protein dynamics on the scale in which they occur. It is through these methods that we clarify the protein interactions involved in response to the extracellular stimuli provided by a variety of bacterial lipopolysaccharides (LPS), known stimulants of the innate immune system. It has been shown that LPS-induced TLR4 dimerization and clustering correlate to an appropriate innate immune response. Imaging the degree of TLR4 clustering after exposure to different gram negative LPS can further the understanding of TLR4 pathway dynamics. By studying the internalization of TLR4, it can be determined whether cells have had adequate time to react and form clusters as a result of being exposed to LPS. These experiments will focus on imaging the LPS from E. coli as well as of Y. pestis 21C on several microscopes.

Keywords

TLR4, E. coli LPS, Y. pestis LPS, fluorescent antibody

Sponsors

Sandia National Laboratories

Document Type

Thesis

Language

English

Degree Name

Electrical Engineering

Level of Degree

Masters

Department Name

Electrical and Computer Engineering

First Advisor

Schamiloglu, Edl

First Committee Member (Chair)

Timlin, Jerilyn

Second Committee Member

Portillo, Salvador

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