Andre Obua

Date Approved

2017

Degree Type

Open Access Senior Honors Thesis

Department or School

Biology

First Advisor

Michael Angell

Second Advisor

Aaron Liepman

Abstract

The murine alveolar macrophage cell line, MH-S, constitutively expresses the immunoregulatory antigen CD40. Previous experiments have demonstrated that CD40 expression is upregulated following infection with the DNA virus, mouse adenovirus 1 1 Obua (MA V-1 ), suggesting a role for CD40 in MA V-1 infection. The purpose of this experiment is to investigate the effect ofCD40 silencing on MA V-1 replication in MH-S cells. To elucidate the importance ofCD40 signaling in MA V-1 replication, CD40 expression was reduced using a short hairpin RNA (shRNA) RNAi system. Five CD40-silenced lines were generated using shRNA and showed between 26-79 percent reduction in cell surface CD40 expression from wild type. To assess the effect of MA V-1 infection on costimulatory markers, CD40 expression was compared between the silenced cell line that showed the greatest level of CD40 suppression (designated NPBS) and wild type cells at I-day post infection. Flow cytometry revealed CD40 expression was increased in both cell lines following infection with MA V-1. Infected CD40-silenced cultures expressed approximately 50% the level of CD40 as infected wild type cultures. Further characterization of CD40-deficient MH-S cells may reveal novel mechanisms through which MA V-1 infection alters host cell metabolism.

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