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Title: Identification and characterisation of differentially expressed vascular smooth muscle cell genes : the effect of transforming growth factor beta
Author: Adam, P. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1997
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The main aim of this research was to identify TGFβ1-regulated VSMC genes by differential display reverse transcription polymerase chain reaction (DDRTPCR). This required establishing the appropriate techniques for displaying and confirming differentially expressed cDNAs, as well as cloning and sequencing. In the first part of my thesis I established the conditions required for DDRTPCR by comparing mRNAs from high and low passage cultured rat VSMCs, and identified a band unique to high passage cells. I then established the necessary cloning and sequencing techniques to identify this band and also to identify a cDNA found by Dr. C.M. Shanahan to be differentially expressed in contractile rat VSMCs versus proliferating rat VSMCs. The former proved to represent a novel VSMC gene, whilst the latter was identified as polyubiquitin C (UbC); indeed Northern blot analysis showed that UbC was also more highly expressed in freshly dispersed human VSMCs compared with their proliferating cultured counterparts. Having established the differential screening and molecular cloning techniques required, the effect of TGFβ1 on VSMC gene expression was studied. In complementary experiments I studied the regulation of p15INK4B and endoglin in VSMCs. Northern blot analysis and immunohistochemistry was used to show that both endoglin mRNA and protein are present in human VSMCs. Northern blot analysis also demonstrated that endoglin expression was maintained in culture by exogenous TGFβ1 treatment of VSMCs which had been serum stimulated for 24 hours. These studies have identified three novel TGFβ1-regulated VSMC genes whose function in VSMC biology requires further study to determine a better understanding of the role of TGFβ1 in VSMCs in vascular disease.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available