Undergraduate Summer Research Internship 2010
Project Proposal
Title: Inflammation in Islet -Cells
By: Renee C Taylor-Sapp
Faculty Mentor: Dr. Jason Collier
Proposed Dates of Work: Monday May 24, 2010 – Friday July 16, 2010
Background
Diabetes mellitus is a disease affecting the body’s ability to self-regulate blood
glucose levels. Due to insufficient insulin or ineffective insulin receptors cells within the
body, particularly adipose and muscle cells, are unable to take up glucose from the blood
stream. There are two forms of diabetes mellitus both with different etiologies, both
producing inflammation and eventually -cell death. The proposed project will examine
in detail the two main mechanisms of death proposed for beta-cells in type one diabetes
Type one diabetes (T1D) is an autoimmune disease with onset usually in
childhood or early adolescence. T1D is characterized by the selective destruction of
pancreatic beta cells located within the islet of Langerhans. The process of beta-cell
damage and eventually death is facilitated by the attack of white blood immune cells (t-
Cells and macrophages) in to the pancreas. Once these cells infiltrate the pancreas they
release proinflammatory cytokines, such as interlukin (IL)-1 and -interferon (-INF),
that have cytotoxic effects. It is known that these cytokines activate inducible nitric oxide
synthase (iNOS) and NO production but the exact mechanism of death is still debated.
The two main mechanisms being pursued are apoptosis and necrosis.
Apoptosis requires the activation of a series of capsases. Once activated capsases
cleave intracellular substrates causing cell death. Apoptosis as a mode of regulated cell
death has been extensively studied and is therefore clearly defined. In Dr. Jason Colliers
lab studies have already been conducted comparing true apoptosis with cytokine
mediated -cell death. Key discoveries have already been made to negate the theory of
apoptosis and promote a theory of non-apoptotic -cell death. A few key points include:
1) cell lines selected for resistance to cytokine-induced toxicity are equally susceptible to
known apoptotic inducer camptothecin 2) over expression of known anti-apoptotic
kinases provide protection against camptothecin yet not cytokine-mediated death 3)
suppression of proapoptotic proteins increase viability in apoptotic conditions but not
cytokine conditions 4) Capsase activity is clear with camptothecin but not in cytokine
induced death 5) in apoptotic conditions ATP levels are know to rise however in cytokine
mediated cell death ATP levels fall. All of these conclusions point to a different
mechanism of -cell death. The current goal is to define the exact mechanism of -cell
death and to understand methods that could possibly stop the chain of events that leads to
T1D.
Proposed Project
My role within the lab this summer would be to help contribute to a better
understanding of the mechanism of death and measures that could prevent it by applying
multidisciplinary approaches.
The main focus of the project is to work in conjunction with the chemistry
department to profile the different metabolites present within the different mechanisms of
cell death (apoptosis and cytokine mediated) in various cell lines. Once the metabolite
profiles are understood the similarities and differences can be explored and further
experiments designed. The goal is to discover key components within cytokine mediated
-cell death that will help lead to a clearer understanding of the steps that lead to T1D
and possible methods of prevention.
The project will include working with several cell lines, including the original
cytokine sensitive cell line (832/13) and cytokine resistant cell line (832/15) as well as
two new lines from European study groups each selected for either cytokine sensitivity or
resistance. I will be working to pass the cells, grow them, treat them with the various
conditions, harvest them and centrifuge them down in to small pellets that will be treated
and profiled in a mass spectrometer. I will also be working to assist in performing nitrite
and viability assays.
This project will enhance my educational program by allowing me to learn
various lab techniques and protocols. It will also allow me to experience the dynamics of
working in a cutting edge research facility, explore the dimensions of analyzing and
interpreting data in a research study, and help design new experiments based of found
data. This is a completely different experience than in undergraduate course labs where
all experiments are predesigened to give an expected outcome. My goal is to make a
meaningful contribution to the overall project allowing me to be a coauthor on an article
to be submitted to a peer-reviewed scientific journal.