Sciullo, Lauren A.
(2012)
Functional Analysis of Transcriptional Promoters of Macaque Lymph Node Chemokines.
Master's Thesis, University of Pittsburgh.
(Unpublished)
Abstract
Inflammation has been shown to play a part in the disease processes of human immunodeficiency virus type-1 (HIV-1) and simian immunodeficiency virus (SIV) infections. The chemokines CCL20, CCL21, and CXCL13 all play important roles in the immune response and have each been, in some manner, linked to HIV-1 and SIV infection. Expression of these chemokine genes has been shown to affect disease state and it is, therefore, important to study the transcriptional regulation of these genes as a possible mechanism of controlling their expression. Transcriptional regulation occurs primarily via the promoter region of a gene. This promoter is the target for binding by transcription factors that can either activate or repress the expression of that specific gene. In this study, the promoter regions of the macaque chemokines CCL20, CCL21, and CXCL13 were analyzed. These promoter regions were characterized via sequence alignments and analyses, as well as by mapping putative transcription factor binding sites. Not only were differences between multiple clones of each chemokine promoter identified, but the homology between the rhesus macaque, cynomolgus macaque, and human promoter regions were explored. By cloning the three promoters into the pGL2-Basic vector, a promoterless luciferase expression plasmid, transcriptional control was measured from each promoter via levels of luciferase expression. A dual-luciferase reporter assay was designed and optimized as a method of quantitating transcriptional control from macaque chemokine promoters. After stimulation, an increase in CCL20 transcriptional levels, but no change in CCL21 and CXCL13 transcriptional levels, was observed. Possible methods of inhibiting transcription from the CCL20 promoter were explored by testing the effects of glycerol monolaurate (GML), (-)-epigallocatechin gallate (EGCG), and ethyl gallate (EG) on transcriptional levels. In summary, we have amplified three macaque chemokine promoters, characterized their basal and induced transcriptional control of the luciferase gene, and have identified potential inhibitors of their upregulation. The public health relevance of this study is its ability to potentially pave the way for additional approaches to modulating chemokine expression as a method of combating the inflammation associated with HIV-1-driven disease.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
21 September 2012 |
| Date Type: |
Publication |
| Defense Date: |
21 June 2012 |
| Approval Date: |
21 September 2012 |
| Submission Date: |
23 July 2012 |
| Access Restriction: |
5 year -- Restrict access to University of Pittsburgh for a period of 5 years. |
| Number of Pages: |
110 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Public Health > Infectious Diseases and Microbiology |
| Degree: |
MS - Master of Science |
| Thesis Type: |
Master's Thesis |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
CCL20, CCL21, CXCL13, Promoter, Transcription |
| Date Deposited: |
21 Sep 2012 19:54 |
| Last Modified: |
21 Sep 2017 05:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/13079 |
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