The malaria-causing parasite Plasmodium falciparum 1s dependent on tightly
regulated gene expression for its progression through the intra-erythrocytic life cycle,
pathogenesis and establishment of persistent infection by evasion of the human host's
immune system. Evidence points towards P. falciparum being unusually dependent
on nuclear architecture and genomic organisation for the control of gene expression.
Spatially defined nuclear regions of transcriptional activity have been detected and the
spatial positioning of loci may determine their transcriptional potential. Additionally,
a number of epigenetic markers have been shown to occupy spatially distinct subcompartments
of the nuclear volume. Limitations of microscopic assays used until
now have left us with a stereotyped and incomplete image of the organisation of the
parasite nucleus and the transcriptional and epigenetic factors involved in the
regulation of parasite gene expression, and the possible dynamics thereof.
This work focused on the use of high-content and super-resolution fluorescent
microscopy for the study and graphical representation of the spatial organisation of
various nuclear factors involved in transcriptional regulation in P. falciparum
parasites. The first objective (chapter 2) establishes P. falciparum parasite sample
preparation and fluorescent labeling techniques for microscopy. Immunofluorescent
labeling of var gene associated transcription repressive and permissive histone
modifications, H3K9me3 and H3K9ac, respectively, as well as serine 2-
phosphorylated RNA polymerase II and the putative transcription and splicing factor
PfMyb2, was optimised. DNA fluorescent in situ hybridisation was also optimised for
labeling of var gene exons. In the second objective (chapter 3), the assays established
in the previous chapter are used for high-content combinatorial labeling in thousands
of nuclei, followed by analysis using a bespoke computational algorithm for the
detection and classification of different labeling patterns. This approach revealed a
high level of diversity in the nuclear distributions of each assayed target. Superresolution
stochastic optical reconstruction microscopy was used to further study the
sub-diffraction organisation of selected labeling patterns.
The data presented in this dissertation reveal that the complex spatial organisation of
certain nuclear factors is subject to greater diversity within the nucleus of P.
falciparum parasites than previously thought. / Dissertation (MSc)--University of Pretoria, 2012. / gm2013 / Biochemistry / unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/32818 |
| Date | January 2012 |
| Creators | Griffiths, Caron, A. |
| Contributors | Birkholtz, Lyn-Marie, Mhlanga, Musa M. |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2012 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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