PhD, Faculty of Health Sciences, University of the Witwatersrand / The 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.

Defects in BRCA1 and BRCA2 tumor suppressors predispose one to breast and ovarian cancer. The current treatment for BRCA-deficient cancers is mastectomy. Because both copies of the tumor suppressor need to be defective for cancer to occur, identifying cellular mechanisms that specifically target BRCA-deficient cells is of paramount importance. Luckily, recent experiments have shown that depletion of a protein named RAD52 in BRCA1 or BRCA2 cancer cells causes them to die. Therefore, we can use small molecules to stop the RAD52 protein from functioning. We need, however, to know which of the RAD52 activities to inhibit and how. One function of RAD52 that likely underlies all cellular activities is its ability to bind single-stranded DNA (ssDNA). To identify if small molecules could inhibit the RAD52-ssDNA complex, I screened a small library of compounds and found 13 potential inhibitors. We validated that these small molecules bind to RAD52 and inhibit RAD52 DNA binding and annealing activities. The identification of these small molecules is important because we can use them to dissect the function of RAD52 in normal and malignant cells, which to date remains elusive. In an attempt to further advance our understanding of RAD52 function and regulation we are also investigating how a novel binding partner, DSS1, interacts with RAD52 and modulates its activities. My data show that this protein enhances the way RAD52 finds separate complementary DNA templates and anneals them to make a double-stranded product. At least in part, these studies have identified some residues likely involved in the binding site of DSS1 on RAD52. In aggregate, the outcome of the two projects deepens our understanding of the complex and interconnected cellular pathways that support the integrity of genomes.

The classical Nuclear Factor kappa B (NF-B) signaling pathway is an important regulator of inflammation and innate immune responses. Poxviruses, including vaccinia virus, encode multiple immune evasion proteins, including a growing number of NF-B inhibitors. To determine if additional vaccinia virus gene products disrupted NF-B signaling, we utilized VV811, a mutant virus missing 55 open reading frames and devoid of the known inhibitors of TNF-induced NF-B activation. NF-B nuclear translocation was inhibited in VV811 infected cells stimulated with TNF. Furthermore, VV811 infection suppressed IB degradation and resulted in accumulation of phosphorylated IB in cells stimulated with TNF. Coimmunoprecipitation assays demonstrated that the inhibitory IB-p65-p50 complex was intact in VV811 infected cells, and, significantly, treatment with AraC revealed the involvement of late protein synthesis in stabilization of IB. This work indicates that unidentified inhibitors of NF-B exist in vaccinia virus and illustrates the importance of NF-B activation in the antiviral response. / Virology

Histone Deacetylase Inhibitors (HDACi) are a new class of chemotherapeutics which have shown promise in pre-clinical and clinical settings. HDACi have been shown to act by re-programming gene expression, with the transcription of some genes such as p21WAF1 being activated, while others like SRC and BCL2L1 are repressed. The mechanism behind HDACi gene expression changes remains unknown; although it has been shown to involve a direct interaction with gene promoters. Using a quantitative qRT-PCR approach, the effect of various HDACi on the transcription of p21WAF1, SRC and BCL2L1 was examined. TSA and apicidin led to an up regulation of p21WAF1 mRNA levels while c-Src and Bcl-xL mRNA levels were downregulated. Short c-Src mRNA transcripts were unaffected following TSA and apicidin treatments, despite the full length transcripts being repressed. Repression of full length c-Src and Bcl-xL mRNA transcripts was not seen following treatment with MS-275 and MGCD0103, although p21WAF1 mRNA expression was induced. ChIP experiments revealed that following HDACi treatment, histone acetylation levels and RNA Polymerase II occupancy increased in the promoter regions of both the SRC and BCL2L1 genes. RNA Polymerase II occupancy lasted less than 15 minutes in the 3’ regions of the gene following treatment with apicidin and TSA, but was more long-term following MS-275 and MGCD0103 treatment. The protein phosphatase inhibitor Calyculin A completely blocked HDACi mediated repression of c-Src and Bcl-xL mRNA, suggesting a role for protein phosphatases in the mechanism behind HDACi. It is therefore hypothesized that HDACi work through at least two different mechanisms. Whether or not an HDACi leads to gene repression depends on its ability to disrupt an HDAC/protein phosphatase complex and not on their HDAC specificities. The disruption of the complex leads to the release of an active protein phosphatase. The released phosphatase can then presumably act on various factors changing a gene from an active to paused state, possibly through promoter proximal pausing. HDACi unable to disrupt this complex are unable to induce gene repression. Collectively, these studies highlight not only the complexity of HDACi mediated effects within the cell, but also present a new explanation behind HDACi mediated gene repression.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 183-192). Also available on the Internet.

Thesis--Lund. / Extra t.p. with thesis statement inserted.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 96-102). Available also in a digital version from Dissertation Abstracts.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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