Biology ETDs

Publication Date

12-1-2010

Abstract

Nuclear Factor κB (NF-κB) proteins make up a large family of eukaryotic transcription factors that regulate many important cellular functions, including cell signaling, cell growth, development, cell death by apoptosis, and immune and inflammatory responses, and has been shown to be involved in multiple human diseases. NF-κB proteins are subject to careful regulation in the cell, and are regulated at multiple levels. A family of inhibitory proteins exist (called IκB proteins) which bind to NF-κB and cause it to be localized in the cytoplasm. There are multiple signal transduction pathways that can lead to the activation of NF-κB. These signal transduction pathways are induced by contact with a wide variety of molecules, and therefore a huge number of molecules can act as NF-κB activators. One such molecule is lipopolysaccharide (LPS), a bacterial cell wall component, which activates NF-κB via Toll-Like Receptor 4 (TLR4). Following TLR4 activation, IkB is degraded, releasing NF-κB. NF-κB then triggers the transcription of IkB, thus down-regulating its own activity. These complex feedbacks create oscillations that have previously been observed in electrophoretic mobility shift assays (EMSA) and live-cell imaging studies. In order to better understand and characterize NF-κB oscillations, we created macrophage cell lines stably expressing a fluorescently labeled monomer of the NF-κB dimer, RelA-GFP, and conducted time-lapse fluorescence imaging studies of these cells. We examined nuclear NF-κB oscillations in both wild-type and transfected cell lines upon activation by bacterial LPS. LPS chemotypes from various organisms were tested for their effects on oscillatory patterns. Our data show that all LPS were able to elucidate oscillations, a novel finding for RAW 264.7 macrophage cells. The data also show that while the responses to the different LPS chemotypes do lead to different oscillatory dynamics, they do not show any easily discernable patterns. This suggests that the temporal profile of NF-κB may not be stimulus specific (at the chemotype level). Our data leads to the conclusion that the oscillatory dynamics of NF-κB may not play as large of a role in immune response as initially thought.

Project Sponsors

Sandia National Laboratories

Language

English

Keywords

NF-kappaB, lipopolysaccharide, signal transduction, innate immunity, RelA, Yersinia pestis

Document Type

Thesis

Degree Name

Biology

Level of Degree

Masters

Department Name

UNM Biology Department

First Advisor

Nelson, Mary Anne

First Committee Member (Chair)

Natvig, Donald

Second Committee Member

Poschet, Jens

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