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Quality control systems are critical to improving laboratory practices and contribute significantly to success in human in vitro fertilisation (IVF). The mouse embryo assay (MEA) is commonly used in production quality control, as well as in product research and development. Despite its popularity there are some well-known issues associated with it; these include, but are not limited to, insensitivity to sub-optimal conditions, ethical issues due to animal use and variation in end-point interpretation between operators. The aim of this thesis was to therefore develop a cell-based assay that could facilitate research and development whilst addressing the issues presented above. Firstly, using the pluripotent P19 embryonal carcinoma cell line, 2D and 3D cell-based assays were developed and optimised, and their response to culture perturbations such as varying osmolality and energy source levels were tested. Secondly, given that mammalian cells are generally resistant to changes in culture condition, characteristic features of the mammalian cold stress response, mTOR inhibitor rapamycin and AMPK inhibitor dorsomorphin were incorporated as potential tools for decreasing cellular resistance to altering culture conditions. The P19 assay demonstrated sensitivity to changes in energy substrate availability when treated with rapamycin and dorsomorphin, and was subsequently used to systematically screen different media formulations in order to identify the ranges of pyruvate, glutamine and calcium lactate concentrations required for optimum cell growth. Lastly, the developed 2D assay and the MEA were treated with the same products and their responses were directly compared to highlight similarities, and to identify differences, between the two assays. These assays/comparisons showed that the P19 assay identified differences in human IVF culture media that were not identified by the MEA. The P19 assay may therefore represent a sensitive alternative to the MEA that can detect discrete changes in culture conditions and indicate the extent to which different energy substrates affect cell growth and proliferation. It is important to note that the two assays differ in their robustness and the P19 assay responded differently to repeat experiments with the same treatment. Overall, this novel P19 assay may provide an effective means of validating and testing products used in IVF whilst reducing animal use in research and development.

MicroRNA (miRNA), a class of small, non-coding RNA, are the product of a series of precise processing steps and responsible for regulating the translation of >60% of human protein-coding transcripts. Consequently, the dysregulation of miRNA levels has been linked to many human diseases, including cancers and neurodegenerative and cardiovascular diseases. In turn, RNA-binding proteins (RBPs) have been studied as regulators of miRNA biogenesis. Advancements in large-scale technologies have enabled the identification of proteins that bind specific sequences of precursor (pre-) miRNA and the discovery of disease-relevant miRNAs has inspired efforts to identify small molecule inhibitors of such interactions. To aid the study and identification of inhibitors of RPIs, in vitro and in cellular RPI detection systems have been developed. However, requirements of biochemical and cellular methods limit their utility, particularly for use with small, highly processed RNAs. RiPCA, or RNA interaction with Protein-mediated Complementation Assay, an assay for the direct detection of RPIs in live cells, was developed to enable the validation and manipulation of pre-miRNA-protein interactions. In RiPCA, cells stably expressing the small subunit (SmBiT) of a split nanoluciferase (NanoLuc) fused to HaloTag are transiently co-transfected with a functionalized pre-miRNA probe and a plasmid encoding the RBP-of-interest fused to the large subunit (LgBiT) of NanoLuc. The pre-miRNA probe becomes covalently conjugated to SmBiT via HaloTag and subsequent interaction between the pre-miRNA and RBP drives the reconstitution of functional NanoLuc. Initially optimized using the RPI between the let-7 family of miRNA and the Lin28 RBPs, RiPCA was shown to detect the let-7/Lin28 interaction in both the cytoplasm and the nucleus. Furthermore, RiPCA was capable of discerning sequence-specific binding preference of Lin28 as well as indicating the relative binding affinity of Lin28 and its individual RNA-binding domains. These results encouraged the expansion of RiPCA for the detection of other functional pre-miRNA-protein interactions involving the RBPs hnRNP A1, Msi1, and Msi2. RiPCA was similarly capable of detecting the relative binding affinities of these RBPs for several pre-miRNA sequences, including let-7 family members and pre-miR-18a. In addition, the ability of RiPCA to detect site-specific binding was probed using a small library of pre-miRNA probes. While data reflected site-specific binding for Lin28, it was not shown for hnRNP A1, Msi1, and Msi2. Nevertheless, RiPCA demonstrated broad applicability of detecting pre-miRNA-RBP interactions. Finally, to enable high throughput screening (HTS) of inhibitors of pre-let-7d/Lin28, RiPCA was miniaturized, and a partially automated workflow was optimized. A screen of ~18,000 small molecules derived from a curated library resulted in the identification of seven potential let-7/Lin28 inhibitors. Further characterization of the top hits is required to fully elucidate their mechanism of action and activity against let-7/Lin28 in cells. Future efforts should focus on further engineering RiPCA to enable more precise detection as well as detection of RPIs involving other classes of RNAs, including mRNAs, lncRNAs, and expanded repeats. Overall, the technology reported herein promises to advance the characterization of RPIs and provide a platform for the discovery of RPI inhibitors.

Infection with human papillomavirus (HPV) represents a complex presentation of symptoms, from benign warts to cancer. Prevention options for sexually active individuals outside the nonavalent HPV vaccine range are limited. Thus, the overarching aim of this dissertation work was to prevent HPV at the cellular level by investigating FDA- and internationally-approved drugs with the potential to prevent HPV infection. To this end, I conducted three research studies: a) a review of the literature focusing on human papillomavirus (HPV) biology, pathogenesis, and potential for drug discovery; b) developed a HPV-16 cell based assay for high-throughput screening (HTS) to conduct a pilot screen of 1906 compounds the Small Molecule Discovery Center at UCSF, and c) performed dose-response activity confirmation and investigated mechanisms of action for top candidate drugs. The results of these three studies demonstrate that the identification of novel anti-HPV drugs to be used for HPV prevention in the sexually active population is linked to understanding HPV biology; and using a HTS HPV-16 cell based assay is a valid and reliable tool for identifying potential anti-HPV to prevent HPV infection. Overall, my goal was to identify previously approved FDA- and internationally-approved drugs that have the potential to be repurposed for HPV prevention in sexually active individuals outside the of nonavalent vaccination range.