Doherty Institute - Theses
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ItemPermanent silencing of HIV transcription using triplex forming oligonucleotides: a novel strategy for an HIV cure( 2018)Combination antiretroviral therapy (cART) for HIV infection has significantly reduced morbidity and mortality, however, treatment is lifelong. The main barrier to a cure for HIV is the persistence of long lived latently infected T-cells. Virus can integrate in the host genome and be transcriptionally silenced however, upon reactivation of transcription virus can re-emerge from these latently infected cells. In individuals on ART, reactivation of virus goes undetected but once ART is stopped, reactivation of virus leads to virus replication and rebound. One strategy to eliminate virus rebound after cessation of ART is to permanently silence HIV transcription. Here we explore an alternative approach to silence HIV transcription in CD4+ T cells using triplex formation oligonucleotides (TFO). We hypothesize that TFOs can bind irreversibly to the integrated provirus in a sequence specific manner with limited off-target effects. We assessed TFO activity against the green fluorescent protein (GFP) and HIV in vitro by using uninfected and latently infected cell lines and determined the effects of gold nanoparticles to enhance nuclear localization.
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ItemThe role of inflammatory stimuli in dendritic cell biology( 2017)Dendritic cells (DC) are professional antigen-presenting cells (APC) that capture and present processed protein antigens to T cells when they are immature and mature, respectively. Infection-induced inflammation drives DC activation by two pathways, termed direct and indirect activation. Direct DC activation occurs when DCs “directly” encounter pathogen- or danger-associated molecular patterns (PAMPs or DAMPs), while “indirect” DC activation occurs when DCs encounter secondary inflammatory signals. Direct activation of DC leads to increased expression of major histocompatibility complex class II (MHCII) and co-stimulator molecules such as CD86, as well as the secretion of cytokines. In contrast, while indirect activation of DC elicits a similar expression level of MHCII and co-stimulator molecules, these cells have impaired cytokine production. However, due to the lack of effective markers to discriminate these two DC populations, studies on the characteristics and immunological roles of the two DC populations are limited. In this thesis, we aim to: i) identify markers that discriminate directly and indirectly activated DCs via next-generation RNA sequencing; and ii) assess the role of directly and indirectly activated DCs in vivo under inflammatory conditions. The major findings are as follows: i) CD38 and CD103 are highly expressed in indirectly activated DCs, while CD205 is elevated in directly activated DCs, and these CD molecules could be used as markers to partially distinguish directly activated DCs from indirectly activated DCs; ii) directly and indirectly activated DCs prime CD4 T cell proliferation at a comparable quantity, but with different effector T cell polarisations; and iii) directly activated DCs induce effector memory CD4 T cells upon a secondary challenge, while indirectly activated DCs preferentially induce central memory CD4 T cells. Based on this study, we can describe the specific phenotypes of directly versus indirectly activated DCs, we were able to assess the immune-functional properties of indirectly activated DCs, and assess the formation of directly and indirectly activated DCs in vivo under both steady-state and inflammatory conditions. The discovery of such murine markers to discriminate these DCs may have direct homologs within humans, which would allow for investigation into these differently activated DC in clinical samples. The results will assist rational designing of vaccines to maximise the immune response and strategies to minimise autoimmunity.