Document Type

Dissertation

Date of Degree

Fall 2011

Degree Name

PhD (Doctor of Philosophy)

Degree In

Immunology

First Advisor

Brian K. Martin

Second Advisor

Thomas Waldschmidt

Abstract

The brain and spinal cord make up the central nervous system (CNS), and as an immune-privileged site, it requires special immune surveillance and regulation. The complement system is a component of innate immunity produced locally in the CNS, since size restrictions from the blood brain barrier prevent complement proteins from easily passing through from the rest of the body. The complement pathway contributes to inflammatory cell recruitment, cell lysis, and opsonization, and thus requires regulation to avoid inappropriate activation. Despite its important role in innate immunity, very little is known about complement production, regulation, and function in the CNS of healthy or diseased individuals.

For this dissertation, the central goal was to investigate and characterize the regulation of complement factor H (CFH), a regulator of the alternative pathway of complement activation. CFH polymorphisms have been associated with a number of diseases including atypical hemolytic syndrome, age-related macular degeneration, and Alzheimer's disease, but the regulation of CFH is not well understood, especially in the CNS. To investigate the role of CFH in the CNS, mRNA and protein production in glial cells was first established. The murine CFH (mCFH) promoter was cloned and the transcription start site was identified in astrocytes, microglia, and liver tissue. The mCFH promoter was truncated and different regions were investigated for enhancer or silencer activity. Database mining identified potential transcription factor binding sites, and mutagenesis studies and binding assays identified transcription factor binding candidates. Specifically, the activating protein-1 (AP-1) transcription factors c-Jun and c-Fos bound to a region of the mCFH promoter between – 416 base pairs (bp) and – 175 bp in an electrophoretic mobility supershift assay. Cytokine stimulation increased mCFH mRNA and protein production, as well as the mRNA production of c-Jun and c-Fos and the protein production of c-Jun. These results suggest a relationship between cell cycle and complement regulation, and the investigation of how these transcription factors and CFH affect disease will be a valuable area of research for CNS immune regulation.

Keywords

astrocyte, c-Fos, c-Jun, Complement, Complement Factor H, transcription

Pages

xi, 149 pages

Bibliography

Includes bibliographical references (pages 125-149).

Copyright

Copyright 2011 Laura Anne Fraczek

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