3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Reticuloendothelial ferritin messenger RNA in inflammatory states(1989) Lapinsky, Stephen. E.Ferritin is an iron storage protein, made up of heavy (H) and light (L) subunits. Ferritin synthesis is regulated at a post transcriptional level by iron, which induces a redistribution of ferritin mRNA from a free cytoplasmic pool to polyribosomes. Inflammatory states influence iron metabolism, causing a decrease in serum iron levels associated with an increase in reticuloendothelial ferritin synthesis and iron storage.Item The role of small RNAs in susceptibility and tolerance to cassava mosaic disease(2016) Rogans, Sarah JaneCassava (Manihot esculenta, Crantz) is considered to be an important food security crop consumed by over a billion peoples globally, many who subsist on it. Cassava mosaic disease (CMD) is one of the main biotic and economically important constraints to cassava cultivation in sub-Saharan Africa. Geminiviruses are the casual agents of CMD and cause disease to many staple food and cash crops of great economic importance worldwide. There are currently 11 species of Begomoviruses that belong to the Geminiviridae family. South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite (DNA A and DNA B components) begomovirus belonging to the family Geminiviridae, and is one of the causal agents of cassava mosaic disease (CMD) endemic to southern Africa. Various strategies to control CMD are currently being investigated, one of which is cis-genics, which involves manipulation of endogenous host genes to combat viral pathogens. In order to achieve this, it is imperative to elucidate molecular mechanisms involved in host-virus interactions. Endogenous small RNAs (sRNAs), including microRNAs (miRNAs), have been found associated with gene regulatory mechanisms in response to virus infection. Amongst the non-coding host sRNAs targeting viruses are small interfering RNAs (siRNAs) associated with posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS), which are involved in the host RNA silencing pathway. The RNA silencing pathway is a highly conserved basal immunity pathway involved in host defence against plant viruses. The aim of this study was to identify siRNAs and miRNAs associated with gene regulatory mechanism in response to SACMV infection and to determine if they a play a role in the susceptible or recovery phenotype observed in SACMV tolerant cassava landrace TME3 or T200, respectively. Furthermore, virus-derived siRNA (vsRNA) populations targeting the DNA A and B components of SACMV were also investigated. MicroRNAs (miRNAs) are an important class of endogenous non-coding single-stranded small RNAs (21-24 nt in length), which serve as post-transcriptional negative regulators of gene expression in plants. Despite the economic importance of Manihot esculenta Crantz (cassava) only 153 putative cassava miRNAs (from multiple germplasm) are available to date in miRBase (V.21). Therefore, both conserved and novel miRNAs needed to be identified in cassava before we could determine what association they had with SACMV infection. In this part of the study, mature sequences of all known plant miRNAs were used as a query for homologous searches against cassava EST and GSS databases, and additional identification of novel and conserved miRNAs were gleaned from next generation sequencing (NGS) of two cassava landraces (T200 from southern Africa and TME3 from West Africa) at three different growth stages post explant transplantation and acclimatization. EST and GSS derived data revealed 259 and 32 conserved miRNAs in cassava, and one of the miRNA families (miR2118) from previous studies has not been reported in cassava. NGS data collectively displayed expression of 289 conserved miRNAs in leaf tissue, of which 230 had not been reported previously. Of the 289 conserved miRNAs identified in T200 and TME3, 208 were isomiRs. Thirty-nine novel cassava-specific miRNAs of low abundance, belonging to 29 families, were identified. Thirty-eight (98.6%) of the putative new miRNAs identified by NGS have not been previously reported in cassava. Several miRNA targets were identified in T200 and TME3, highlighting differential temporal miRNA expression between the two cassava landraces. This study contributes to the expanding knowledge base of the micronome of this important crop. MicroRNAs play a crucial role in stress response in plants, including biotic stress caused by viral infection. Viruses however can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of miRNAs. This study aimed to understand the regulation of miRNAs in tolerant (TME3) and susceptible (T200) cassava landraces infected with SACMV. Next-generation sequencing was used for analysing small RNA libraries from infected and mock-inoculated cassava leaf tissue collected at 12, 32 and 67 dpi (days post-inoculation). The total number of differentially expressed miRNAs (normalized against mock-inoculated samples) across all three time points was 204 and 209 miRNAs, in TME3 and T200 infected plants, respectively, but the patterns of log2fold changes in miRNA families over the course of infection differed between the two landraces. A high number were significantly altered at 32 dpi when T200 and TME3 plants showed severe symptoms. Notably, in T200 69% and 28 (100%) of miRNA families were upregulated at 12 and 32 dpi, respectively. In contrast, TME3 showed an early pre-symptomatic response at 12 dpi where a high number (87%) of miRNAs showed a significant log2fold downregulation. Endogenous targets were predicted in the cassava genome for many of the identified miRNA families including transcription factors, disease resistance (R)-genes and transposable elements. Interestingly, some of the miRNA families (miR162, miR168 and miR403) that were significantly affected in both T200 and TME3 upon SACMV infection were shown to target proteins (DCL1, AGO1 and AGO2) that play important roles in the RNA silencing pathway. From results, we suggest that the early (12 dpi) miRNA response to SACMV in TME3 appears to involve PTGS-associated AGO1, DCL2 and a cohort of R genes belonging to the miR395 family which may prime the plant for tolerance and recovery downstream, while in T200, SACMV suppresses AGO1, AGO2 (at 32 and 67 dpi), and DCL2 (32 dpi) mediated RNA silencing, leading to severe persistent disease symptoms. This study provides insights into miRNA-mediated SACMV cassava interactions and may provide novel targets for control strategies aimed at developing CMD-resistance cassava varieties Endogenous small RNAs (sRNAs) associated with gene regulatory mechanisms respond to virus infection, and virus-derived small interfering RNAs (vsRNAs) have been implicated in recovery or symptom remission in some geminivirus-host interactions. Transcriptional gene silencing (TGS) (24 nt vsRNAs) and post transcriptional gene silencing (PTGS) (21-23 nt vsRNAs) have been associated with geminivirus intergenic (IR) and coding regions, respectively. In this Illumina deep sequencing study, we compared for the first time, the small RNA response to South African cassava mosaic virus (SACMV) of cassava landrace TME3 which shows a recovery and tolerant phenotype, and T200, a highly susceptible landrace. Interestingly, different patterns in the percentage of SACMV-induced normalized total endogenous sRNA reads were observed between T200 and TME3. Notably, in T200 there was a significant increase in 21 nt sRNAs during the early pre-symptomatic response (12 dpi) to SACMV compared to mock, while in TME3, the 22 nt size class increased significantly at 32 dpi. While vsRNAs of 21 to 24 nt size classes covered the entire SACMV DNA- A and DNA-B genome components in T200 and TME3, vsRNA population counts were significantly lower at 32 (symptomatic stage) and 67 dpi in tolerant TME3 compared with T200 (non-recovery). It is suggested that the high accumulation of primary vsRNAs, which correlated with high virus titres and severe symptoms in susceptible T200, may be due to failure to target SACMV-derived mRNA. In contrast, in TME3 low vsRNA counts may represent efficient PTGS of viral mRNA, leading to a depletion/sequestration of vsRNA populations, supporting a role for PTGS in tolerance/recovery in TME3. Notably, in TME3 at recovery (67 dpi) the percentage (expressed as a percentage of total vsRNA counts) of redundant and non-redundant (unique) 24 nt vsRNAs increased significantly. Since methylation of the SACMV genome was not detected by bisulfite sequencing, and vsRNA counts targeting the IR (where the promoters reside) were very low in both the tolerant or susceptible landraces, we conclude that 24 nt vsRNA-mediated RNA directed genome methylation does not play a central role in disease phenotype in these landraces, notwithstanding recognition for a possible role in histone modification in TME3. This work represents an important step toward understanding variable roles of sRNAs in different cassava genotype-geminivirus interactions. Also, by comparing the differences between a tolerant and susceptible host the aim is to achieve better understanding of the effect of pathogens on host sRNAome, an area that is deserving of me attention in plant systems. The expectation is that these findings presented in the PhD will contribute to the long-term goals of devising new methods of disease control against SACMV and understanding the complex interconnected mechanisms involved in virus-host interactome.Item Optimisation of expressed RNA interference mimics using predicted stem length(2016) Van den Berg, Fiona TaylorPrimary microRNA (pri-miRNA) mimics have been shown to mediate effective gene silencing and are well-suited for therapeutic applications. Pri-miRNA mimics, like natural pri-miRNA, are processed in the endogenous microRNA (miRNA) biogenesis pathway. Elements of the secondary RNA structure are crucial for processing by the Drosha-DGCR8 microprocessor, including a basal stem of - 11 bp. However, structural variation is common and the exact determinants of pri-miRNA processing have been elusive. The aim of this project were to explore the use of natural pri-miRNAs with exceptional basal stem in the design of correspondingmimics and to identify optimal stem features.[Abbreviated Abstract. Open document to view full version]Item Ribosomal RNA mutations to rifampicin resistance(2012-01-17) Macheke, Rulane GlendaIn prokaryotes, transcription and translation are coupled and as a result, the beginning of the messenger RNA is translated by the ribosome while the 3' end is still synthesized. How exactly this occurs is still not clear. One possibility is that RNA polymerase and the ribosomes may be in physical contact with each other at some stage during gene expression or RNA polymerase has a binding site in the ribosomes. Mutational analysis is one method to explore how coordination between these two moieties occurs in bacteria. An Escherichia coli strain with all seven chromosomal ribosomal RNA operons deleted, replaced by a single rrnB plasmid-borne operon, was used to isolate ribosomal RNA mutants with increased rifampicin resistance, two of which were studied further. The altered rrnB operon in pGM1 was obtained by spontaneous whilst in pGM2 by EMS mutagenesis. The mutated rrnB operon in pGM1 conferred resistance to 25μg/ml of rifampicin while in pGM2 resistance of 30μg/ml was observed. A base substitution of T to A at position 355 of the 23S rRNA was detected in pGM1and no nucleotide change was detected in pGM2. The successful isolation of ribosomal RNA mutants with rifampicin resistance is consistent with the hypothesis of interaction between the RNA polymerase and the ribosomes and suggests the part of this interaction is with the large ribosomal subunit.Item Inhibiting HIV-1 using RNA interference (RNAi) to target novel HIV dependency factors (HDFs)(2010-10-22) Blondeel, Mishka DominiqueThree 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.Item Inhibiting HIV-1 gene expression and replication with expressed long hairpin RNAs(2010-09-22) Saayman, Sheena MegThe vast potential of the RNA interference (RNAi) pathway as a new tool for the development of therapeutic modalities has been quickly realised since its discovery in 1998. RNAi effector mimics have been developed to successfully silence an array of disease-causing genetic elements. However, because of the rapidly mutating genome of viruses such as the human immunodeficiency virus (HIV), inhibition of replication cannot be sustained with single RNAi effector mimics. Instead, a combinatorial approach is required, analogous to the cocktail of drugs necessary for successful highly active antiretroviral therapy (HAART). Pioneering studies utilizing long hairpin RNAs (lhRNAs) showed that the long double-stranded RNA stem region acts as a Dicer substrate and is processed into multiple siRNA species. This intrinsic combinatorial property of lhRNAs was exploited in this thesis by attempting to incorporate three non-contiguous potent siRNA sequences within a single lhRNA stem expressed from an RNA Pol III promoter. Although significant knockdown of three independent HIV target sequences was possible, the limitations of this approach became apparent when it was observed that human Dicer does not function efficiently as a multiple turnover enzyme. The generation of siRNA products therefore occurred in a gradient, with higher levels of siRNA produced from the base of the hairpin stem and decreasing quantities generated towards the loop. Modifications to the configuration of integrated siRNA sequences within the stem region enabled augmented RNAi activity of siRNAs in the second position of the hairpin stem. This led to the notion that further manipulation of the structural design of the stem duplex may improve efficacy of up to two siRNAs. Dual-targeting anti-HIV lhRNAs encoding only two highly effective siRNAs targeted against non-contiguous sites within the tat, nef, LTR and int viral genes were therefore propagated. The spatial arrangement of two siRNA sequences was extensively characterised within dual-targeting lhRNAs by inserting up to three random base pairs at the junctions of siRNA encoding sequences and 5 bp preceding the terminal loop sequence. A universally optimal hairpin design was identified which contained a single mismatched base pair between two 19 bp + 2 nt siRNA sequences, as well as a terminal extension. Two powerful dual-targeting lhRNA species, lhRNA-tat-nef +1 and lhRNA-LTR-int +1, each capable of producing two potent anti-HIV siRNA products in equal quantities were selected for incorporation into a combinatorial RNAi system. These two effective dual-targeting lhRNAs were combined, adjacent to one another within a single RNA Pol III-expressed transcript to create a novel lhRNA-based combinatorial RNAi structure. This double lhRNA (dlhRNA) construct served as a precursor for four discrete highly functional RNAi effector sequences which were capable of simultaneously silencing four unique HIV target sites within the tat, nef, LTR and int genes. Furthermore, the ectopic expression of dlhRNAs did not elicit activation of the interferon response, nor did it cause saturation of the endogenous miRNA biogenesis pathway in vitro. In conclusion, the inherent combinatorial RNAi properties of long hairpin RNAs were evaluated and the detailed analysis is presented in this thesis. Structurally optimised dualtargeting lhRNAs subsequently formed the core components of a novel dlhRNA precursor which meets all the requirements for an effective combinatorial RNAi strategy and therefore holds great promise for mediating an effective and sustained gene therapy against HIV.