Cheng Tian

Date of Award

12-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Pharmaceutics

Research Advisor

Weikuan Gu, Ph.D.

Committee

David Douglass Brand, Ph.D. Hongsik Jake Cho, Ph.D. MBA Wei Li, Ph.D. Tao L. Lowe, Ph.D.

Keywords

arthritis, gene mutation, genetic, tight skin

Abstract

An autoimmune disease is a condition arising from an abnormal immune response to a normal body part. There are at least 80 types of autoimmune diseases, rheumatoid arthritis and systemic sclerosis are two of them. My study focuses on these two diseases. Our hypothesis is that mutated genes lead to autoimmune diseases that cause the immune system to attack the human body. The mutated genes cause the gene expression levels to change which fail to properly regulate the body's functions, resulting in immune system diseases. I used existing mouse models to improve the understanding of these two diseases.

The first study concerns Arthritis (Chapter 2). This study has been designed to evaluate the molecular mechanism that regulates spontaneous arthritis disease (SAD) in mice that are deficient in production of interleukin 1 receptor antagonist protein (IL-1ra). Mice with a BALB/c genomic background and IL-1Ra deficiency (BALB/c-/-) have shown susceptibility to SAD while those with a DBA/1 background are resistant to SAD. This study was based on our data accumulated over a decade of working with this mouse model of SAD. Our hypothesis is that there are nucleotide mutations in the gene or genes that cause the differences in expression level between BALB/c-/- and DBA/1-/- to regulate resistance or susceptibility to SAD. We obtained the IL-1Ra BALB/c-/- mouse model from the laboratory of Dr. John Stuart in order to test the susceptibility of IL-1Ra deficient mice on different genetic backgrounds. By transferring the IL-1Ra mutation onto the DBA/1 background, it was found that although BALB/c-/- mice spontaneously developed chronic inflammatory arthritis, DBA/1 IL-1Ra–deficient (DBA/1-/-) mice did not. We then determined the location of a genomic component that regulates susceptibility to SAD by identifying a QTL for spontaneous arthritis on mouse chromosome 1 from a F2 population derived from DBA/1-/- and BALB/c-/- mice. We created a congenic strain carrying the DBA/1-/- region of chromosome 1 that contains the QTL on the BALB/c-/- background. We also created a congenic strain carrying the BALB/c-/- region of chromosome 1 that contains the QTL on the DBA/1-/- background. We then tested the susceptibility of these strains to SAD. When the DBA/1-/- fragment was placed on a BALB/c-/- background, arthritis was delayed and became less severe. When the BALB/c-/- fragment was placed on a DBA/1-/ background, arthritis occurred in varying degrees. A study of the whole genome expression profiles of these congenic strains and their parental strains was performed in order to identify a candidate gene. We linked the Ifi204 gene in Ifi200 cluster to the potential causal pathway of spontaneous arthritis in a mouse model. We identified the function of Ifi204 gene in congenic strains and their parental strains to confirm our theory. We used state of the art technology to investigate the molecular mechanism that regulates the susceptibility to SAD. Through this analysis, a strategy to improve the therapeutic outcome of drugs designed based on alternative targets of the interleukin-1 (IL-1) receptor antagonist may be identified.

The second study concerns systemic sclerosis (SSc) (Chapter 3). SSc is a polygenic autoimmune disorder. It is of unknown etiology and is characterized by the excessive accumulation of extracellular matrix (ECM) proteins, vascular alterations, and v

autoantibodies. The tight skin 2 (Tsk2/+) mouse model of SSc demonstrates signs similar to SSc including tight skin and excessive deposition of dermal ECM proteins. Our hypothesis is that ENU caused single nucleotide mutations in one or more genes in the Tsk2/+ locus interval that determines the disease phenotype. From the literature, Tsk2/+ mice have a mutation on chromosome 1 between 42.5 and 52.5 megabases. We used microarray data to analyze the genes’ expression level to narrow down the number of candidate genes. Based on studies of the genes’ functions, we determined that Fhl2 is our primary candidate gene. We performed both RNA sequencing of skin transcripts and DNA sequencing of the region spanning this gene in Tsk2/+ and wild-type littermates. As of now, we have not found any nucleotide polymorphisms between Tsk2/+ and normal mice. We found that there are two transcripts of this gene and they are expressed differently in the various tissues. This can help us explain why there is only fibrosis in skin. We compared the expression levels of the two transcripts in each organ. The high expression level of one of the transcripts in the skin can help us understand the mechanism of skin fibrosis manifested by Tsk2/+ mice. Understanding the mechanism of skin fibrosis in the mouse model will help us and other researchers understand the mechanism in SSc.

ORCID

http://orcid.org/https://orcid.org/0000-0002-0770-4508

DOI

10.21007/etd.cghs.2018.0463

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