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Title: The impact of inter-strain variation on sputum related phenotypes of Mycobacterium tuberculosis (Review of normalisation procedures)
Authors: Wegrzyn, Malgorzata Grazyna
Supervisors: Barer, Michael
Award date: 1-Mar-2017
Presented at: University of Leicester
Abstract: The properties expressed by expectorated Mycobacterium tuberculosis (Mtb) cells remain poorly defined. Transcriptome and cytological analysis of bacilli in sputum identified signals indicating a slow or non-replicating growth pattern together with lack of aerobic respiration and the accumulation of intracellular triglycerides. Similarly high triglyceride accumulation by non-replicating persistent (NRP) Mtb may serve as a storage form of energy in dormancy. These observations potentially have important implications for our understanding of the transmission and chemotherapy of tuberculosis Two experimental systems were applied in this study with the aim of reproducing the sputum phenotypes; a) exposure of log phase cultures to a host-like tissue fluid and stimuli and b) growth of Mtb in biofilms. These attempts to replicate the sputum transcriptome with laboratory Mtb H37Rv, incompletely replicated the features noted above. We hypothesised that some of the shortcomings of our results might reflect strain specific features thus more recent clinical isolates such as Beijing14 and CH were studied here. Initial work focussed on validation of a suitable normalisation procedure for quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) transcriptional profiles. After investigating a multi-gene procedure, data normalised to sigA were used and compared with the sputum transcriptome. The results confirmed distinct gene expression patterns between strains and also suggested that none of the strains studied fully replicate the previously observed sputum transcriptome. A significant correlation was found with the sputum transcriptome in all strains once normalised to mid-exponential phase of H37Rv, supporting the view that H37Rv may be considered representative in further investigations. However, the normalisation of the individual strain specific transcripts with their own expression profiles in mid-exponential phase altered the correlation, suggesting that the normalisation of sputum transcriptome to mid-exponential of the isolate originated from sputum could affect the obtained pattern and alter conclusions.
Type: Thesis
Level: Doctoral
Qualification: MPhil
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Leicester Theses
Theses, Dept. of Infection, Immunity and Inflammation

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