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Title: Gene expression profiling of quiescent peripheral T lymphocytes
Author: Hughes, B.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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The lymphopenia mutation, a key factor in the development of autoimmune diabetes mellitus in the BB rat, reduces the in vitro life-span of TCRhi single-positive thymocytes and peripheral T cells due to an increased rate of apoptosis. It was proposed that the lymphopenia mutation prevents the accumulation of a normal T cell pool, including regulatory subsets, without preventing the activation and proliferation of reactive T cells, thereby creating conditions appropriate for the development of uncontrolled autoimmune disease. To understand the processes affecting the loss of quiescent peripheral T cells in the lymphopenic rat we must first understand the maintenance and survival of peripheral T cells isolated from ‘normal’ rats. This has broader implications for understanding the genetic maintenance of a healthy peripheral T cell population and avoidance of disease phenotypes (autoimmune and infectious) throughout life. Resting and activated peripheral T cells, isolated from the lymph nodes of the PVG-RT1u, RT7b rat, were separated by FACSDiVa cell sorting. Total RNA isolated from these cell populations was used in subsequent gene expression profiling experiments with the aim to identify genes involved in the maintenance of T cell quiescence. This thesis describes the process of obtaining highly pure populations of resting and activated T cells for gene expression profiling, using high-density macroarrays. The processes of experimental design, sample preparation, hybridisation, image acquisition, normalisation and data analysis are explained. Profiling results were verified using SYBR Green Real Time PCR.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available