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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Inhibiting HIV-1 using RNA interference (RNAi) to target novel HIV dependency factors (HDFs)

Blondeel, Mishka Dominique 22 October 2010 (has links)
MSc (Med), Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand / Three separate recent publications used genome-wide RNA interference (RNAi) to screen for novel host factors that are required for HIV-1 infection and replication. This was achieved using small interfering RNAs (siRNAs) to silence the expression of ~21 000 human genes and determining the effect of each gene’s loss of function on HIV-1 replication. Collectively, several hundred genes have now been implicated as novel HIV-1 host factors (termed HIV-1 Dependency Factors, HDFs). However, differences in study design resulted in little overlap and limited interpretive value from the three published datasets. To identify novel HDFs that are potential targets for anti-HIV therapy, five putative HDFs (SPTBN1, TMED2, KIAA1012, PRDM14 and SP110) were chosen for validation. RNAi effecters (both siRNAs and expressed short hairpin RNAs) were used to silence the selected genes. Gene suppression was measured by quantitative RT-PCR assay and two candidate genes were studied further (SPTBN1 and SP110) based on efficient mRNA inhibition (over 90%). As efforts to deliver the RNAi effecters to a T-cell line were unsuccessful, the effect of this knockdown on HIV-1 replication (both early- and late-stage) was assessed in cultured TZM-bl cells, a HeLa-derived cell line that expresses HIV-1 entry receptors and an integrated luciferase reporter for HIV-1 transcriptional activity (also used in the first genome-wide RNAi screen). An initial viral challenge assay with Subtype C-enveloped pseudovirus showed a 60% decrease in TZM-bl luciferase reporter activity in cells with suppressed SPTBN1 function, while knockdown of SP110 showed no effect on reporter activity. The final experiment, using fully-replicating Subtype B virus, showed a 75% decrease in late-stage viral replication when SPTBN1 expression was suppressed. In addition, SP110 suppression was confirmed to have no effect on TZM-bl reporter activity during any stage of HIV-1 replication. In conclusion, SPTBN1, but not SP110, is required for late-stage HIV-1 replication, though these results need to be confirmed in CD4+ T-cells. The absence of several important viral accessory factors from vi the virus used in the genome-wide screen may explain these findings and emphasises the need for using physiologically representative viral and cellular models to study the viral/cellular interactome.
2

The Mechanisms and Consequences of Gene Suppression During the Unfolded Protein Response

Arensdorf, Angela Marie 01 July 2013 (has links)
The endoplasmic reticulum (ER) facilitates the synthesis, assembly and quality control of all secretory, transmembrane, and resident proteins of the endomembrane system. An accumulation of unfolded proteins or a disruption in the specialized folding environment within the organelle causes ER stress, thus impairing the folding capacity of the ER. In response to this stress, the ER initiates a signaling cascade called the unfolded protein response (UPR) in an attempt to restore ER homeostasis. The vertebrate UPR is propagated by three ER-resident transmembrane proteins (i.e., PERK, IRE1α, and ATF6α), each initiating a signaling cascade that ultimately culminates in production of a transcriptional activator. The UPR was originally characterized as a pathway for the upregulation of ER chaperones, and a comprehensive body of subsequent work has shown that protein synthesis, folding, oxidation, trafficking, and degradation are all transcriptionally enhanced by the UPR. However, UPR activation is also accompanied by extensive mRNA suppression. The mechanisms responsible for this suppression and its consequences for physiological processes beyond the realm of ER protein folding and processing are only now beginning to be described. The overall goal of my thesis work was to explore this process of UPR-mediated gene suppression by identifying the mechanisms involved and the cellular processes affected. As a result, I characterized a novel mechanism of UPR-mediated transcriptional repression involving the translational regulation of the transcription factor C/EBPβ resulting in the suppression of the gene Il4ra, encoding an essential subunit of the IL-4/IL-13 receptor. As a consequence of this suppression, a novel effect of ER stress was identified in the impairment of IL-4/IL-13 signaling, a finding of potential significance in the study of inflammatory disease. In addition to this mechanism, I validated a novel approach to the identification of UPR-regulated transcription factors using publically available bioinformatic software. Through this analysis, I identified the transcription factor HNF4α as a novel post-translational UPR-regulated transcription factor, the regulation of which, resulted in the suppression of a number of lipid metabolic genes. This analysis not only identified a novel UPR-regulated transcription factor, but also presented a new tool for the characterization of UPR-mediated gene suppression. My work represents an independent and original investigation into the process of UPR-mediated gene suppression; and reveals that the UPR facilitates transcriptional suppression through the transcriptional, translational, and post-translational regulation of multiple transcription factors, resulting in the coordinated attenuation of physiological pathways. This function of the UPR is likely to contribute to metabolic, inflammatory, and other chronic disease states.

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