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Functional Analysis of Transcriptional Promoters of Macaque Lymph Node Chemokines

Sciullo, Lauren A. (2012) Functional Analysis of Transcriptional Promoters of Macaque Lymph Node Chemokines. Master's Thesis, University of Pittsburgh. (Unpublished)

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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:
CreatorsEmailPitt UsernameORCID
Sciullo, Lauren A.las211@pitt.eduLAS211
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorReinhart, Todd A.reinhar@pitt.eduREINHAR
Committee MemberMartinson, Jeremyjmartins@pitt.eduJMARTINS
Committee MemberWang, Tianyitywang@pitt.eduTYWANG
Committee MemberMurphey-Corb, Michaelmcorb@pitt.eduMCORB
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: Graduate 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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