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This thesis investigated mitochondrial transfer in Saccharomyces cerevisiae, between respiratory compromised B18p⁰ recipient and respiratory competent donor cells. The respiratory compromised strain had three red fluorescent proteins tagged to the membrane, nucleus and cytoplasm (triple RFP-B18p⁰) and is referred to as the B18p⁰ strain. B18p⁰ cells did not contain mitochondrial DNA, causing it to be respiratory compromised and required a fermentable carbon source, such as glucose/dextrose, for proliferation. The respiratory competent strain used had a green fluorescent protein tagged to the Tom70 mitochondrial protein (Tom70-GFP) and is referred to as the Tom70 strain. The Tom70 cells contained the nuclear encoded URA3 cassette, allowing for negative selectivity of this strain using 5-FOA. S. cerevisiae strains were co-cultured together in media containing only non-fermentable carbon sources (YPGE), plated on YPGE plates containing 5-FOA and colonies grown were distinguished post-co-culture based on their distinct phenotypic and genotypic characteristics. Fluorescent analysis of co-culture colonies revealed the presence of 5-FOA resistant Tom70 cells and some red B18p⁰ cells that had acquired the ability to grow on non-fermentable carbon sources. Genotypic analysis revealed that the majority of these red colonies had acquired mtDNA as well as the nuclear encoded, Tom70 specific URA3 cassette. Several permutations of co-cultures were performed, using different ratios of recipient and donor cells and single-gene deletion donor cells. Purified mitochondria from Tom70 cells were tried to be transferred into B18p⁰ cells using centrifugation forces to induce a higher occurrence frequency of mitochondrial transfer. Metabolic support experiments were conducted to investigate if the Tom70 strain could provide metabolic support to the B18p⁰ strain without mitochondrial transfer. Results indicate that no permutation induced potential mitochondrial transfer at a higher rate tha

Drug discovery is a multi-disciplinary field incorporating both chemistry and biology to create novel pharmaceuticals. Nature synthesizes a diverse range of chemical entities that can demonstrate a wide range of biological interactions, though often produces these compounds in small amounts. Using natures structural diversity as a template, organic synthetic chemistry can tap into the structures of natural products and provide novel structures as well as overcome supply issues through large-scale synthetic chemical processes. A novel benzopyran library was synthesised by Sandile Simelane by reacting 3,4,6,-tri-O-acetyl-D-galactal with various phenols to create a novel focused library of bridged benzopyrans. Each molecule has unique functional groups at defined points in the structure due to varying the functional groups on the phenol, allowing for variation within the library whilst retaining the core scaffold. In this thesis, the bioactivity of this novel benzopyran library was explored using a phenotypic screen measuring growth inhibition. A compound, S13, was determined to be the most potent in the library, therefore genome-wide screening was performed using S13. High-throughput microscopy of 4,100 strains, each with a different GFP-tagged protein, was utilized to determine proteins that increased in abundance or changed localization in response to perturbation with S13. Following treatment with S13, the yeast vacuole increased in size due to an aggregation of proteins in the vacuolar lumen. The increase in vacuole size was coincident with a decrease in vacuolar acidity, potentially disrupted autophagy and the upregulation of several proteins involved in ergosterol biosynthesis. Together, these results reveal a novel bridged benzopyran that increases vacuolar size and pH through an epistatic mechanism involving ergosterol biosynthesis.

Mutations in either the Niemann-Pick type C1 or C2 (NPC1/NPC2) gene result in a fatal lysosomal storage disorder, Niemann-Pick type C (NP-C) disease, for which there is no effective cure. The disease is characterized by systemic and neurodegenerative symptoms arising from toxic accumulation of unesterified cholesterol within the late endosome and lysosome, with a common cause of death for patients being respiratory failure or recurrent infection of pulmonary tissue. Interestingly, the disease symptoms are heterogeneous, with age of onset and severity varied, even among siblings with the same mutations in the NPC1 or NPC2 gene causing this monogenic disease. To date there is no clear explanation for disease severity in siblings with the same mutation. As siblings are raised in the same environment, the major hypothesis of this thesis is that there are genetic modifiers that explain variation in disease severity within siblings. To determine if there are genetic variants associated with disease severity, exomes were sequenced from five sibling pairs exhibiting divergent onset and progression of NPC disease. Out of 23,105 genes, 26 variants were identified that were predicted to have functional consequences in NP-C patients, of which homozygous MUC5B and MARCH8 variants segregated across siblings that exhibited increased and decreased severity of disease, respectively. A cluster of variants was discovered on chromosome 11 belonging to the matrix metalloproteinase (MMP) family. Further investigation of one of these variants, a frameshift insertion in MMP-12, confirmed that this locus regulates the accumulation of unesterified cholesterol in primary neurons derived from a murine model of NPC disease. However, this region on chromosome 11 did not have any statistically significant copy number alteration detectable through a depth of coverage analysis. Overall, these results provide groundwork into the sequence variants mediating disease severity, which with further invest

Nature has been a rich source of pharmaceutical compounds, producing 80% of our currently prescribed drugs. The feijoa plant, Acca sellowiana, is classified in the family Myrtaceae, native to South America, and currently grown worldwide to produce feijoa fruit. Compounds with anticancer, anti-inflammatory, antibacterial and antifungal activities have been isolated from feijoa; however, the diversity of these compounds is not known nor is the mechanism of action of any of these compounds. I hypothesized that identifying compounds in novel feijoa cultivars would improve our understanding of the chemical diversity of antifungal compounds in feijoa and determining the antifungal mechanism of action of feijoa compounds would provide insight into the pharmaceutical potential of these compounds. First, GC-MS analyses were used to obtain an unbiased profile of 151 compounds from 16 cultivars of feijoa, of which six were novel cultivars. Multivariate analysis distinguished 18 compounds that were significantly and positively correlated to antifungal activity based on growth inhibition of Saccharomyces cerevisiae, of which seven had not previously been described from feijoa. Two novel cultivars were identified as the most bioactive cultivars, and the compound 4-cyclopentene-1,3-dione found in a couple of cultivars was potently antifungal against human pathogenic isolates of four Candida species. Second, chemical genetic analyses were used to investigate the mechanism of action of estragole, an antifungal compound previously isolated from feijoa. The chemical genetic profile of estragole was distinct from that of other known antifungal compounds, suggesting the mechanism of action of estragole has a novel antifungal mechanism. Third, chemical genetic analyses were used to investigate the mechanism of action of an ethanol adduct of vescalagin (EtOH-vescalagin) isolated from feijoa. We showed EtOH-vescalagin is antifungal against human pathogenic strains. Genome-wide chemical gen

Transition-state structure analogues are among the most powerful chemical inhibitors discovered to date with picomolar efficacy for enzymes. The nucleoside analogue methylthioadenosine-DADMe-immucillin A (MTDIA) is an inhibitor of the enzyme methylthioadenosine phosphorylase (MTAP) in polyamine biosynthesis. The recently approved forodesine (Mundesine®) is an inhibitor of purine nucleoside phosphorylase (PNP) and purine synthesis. Although the targets of these drugs were known at the time of drug design, it is important to know the compendium of cellular perturbations resulting from use of these inhibitors. Several suspected mechanisms of MTDIA and forodesine in progression of apoptotic cell death have been identified but the underlying mechanisms initiating apoptosis remain elusive. We hypothesize that numerous cellular processes are affected in MTDIA and forodesine treatments given the importance of polyamine and purine synthesis in cancer cells. To elucidate the unsuspected mechanisms mediating anti-cancer activity, unbiased genomic analyses were employed using Saccharomyces cerevisiae. First, gene-gene interactions with MEU1 (the MTAP orthologue in yeast) were determined using Synthetic Genetic Array methodology followed by assessment of drug-gene interactions with MTDIA treatment under a MEU1 essential condition with MTA as the sole source of sulphur. Disruptions to suspected mechanisms of amino acid metabolism, carbohydrate metabolism, response to starvation, vesicle-mediated transport, vacuole fusion, lipid homeostasis, chromatin organisation, transcription, and translation were implicated well as unsuspected mechanisms of NAD+ dependent cellular processes, multi-vesicular body formation, endosomal transport, ion homeostasis, mitochondrion organisation, and cell cycle progression. Induction of autophagy was subsequently confirmed with MTDIA to validate the disruptions to vesicle-mediated transport, response to starvation, multi-vesicular body formation and va

Alzheimer’s disease (AD) is a neurodegenerative disease that is responsible for 50-80% of dementia cases and is characterised by lack of visuospatial perception, impairment of language and memory. One of the main physiological attributions towards this disease is the accumulation of large insoluble deposits of amyloid beta, a toxic peptide, which results in the generation of amyloid plaques found in between neurons in the brain. Currently no therapeutic treatments are available. Clusterin (CLU) is an apolipoprotein that when defective is the second highest genetic risk factor for AD. It has been strongly debated whether CLU counteracts or promotes AD pathology. With the roles of CLU including but not limited to acting as a chaperone for cholesterol transport and aiding autophagy functionality in cancer models, this thesis investigates these two specific functionalities by overexpressing CLU in an in vitro SH-SY5Y and in an in vivo AD model of Drosophila melanogaster (fruit fly). Conclusions from this study reveal that within D. melanogaster, CLU reduced Aβ42 levels and increased cholesterol effect through the blood brain barrier. Additionally, in human cells, CLU ameliorated the defective flux in autophagy. This thesis sheds light into how CLU plays a protective role within an Alzheimer’s disease mammalian system.

Niemann-Pick type C (NPC) disease is a rare neuro-visceral, lysosomal storage disorder for which no effective therapy yet exists. A recessive mutation in the late endosomal/lysosomal cholesterol transport genes NPC1 (95%) or NPC2 (5%) are the causative factors which leads to an accumulation of unesterified cholesterol and sphingolipids in the late endosome/lysosome. It is a build-up of these lipids that, in the majority of cases, ultimately leads to premature death prior to adolescence. In recent years, an imbalance of histone acetylation in a yeast model of NPC disease and subsequently an increased expression of histone deacetylase genes in NPC patient fibroblasts relative to healthy controls was discovered. This led to the finding that Vorinostat (suberoylanilide hydroxamic acid (SAHA); Zolinza®) a histone deacetylase inhibitor (HDACi) drug, rescued unesterified cholesterol accumulation in NPC patient fibroblasts. From these findings in NPC patient fibroblasts, a Phase I clinical trial testing the efficacy of orally-administered Vorinostat in adult NPC disease patients commenced in 2014; however, the therapeutic efficacy of Vorinostat in a whole animal model of NPC disease has not been investigated and is thus unknown. In this thesis, the therapeutic efficacy of intra-peritoneal administered 150 mg/kg Vorinostat in the Npc1nmf164 mouse was explored. This internationally approved HDACi reduced liver disease by decreasing lipid accumulation without increasing expression of NPC1; however, the treatment did not delay weight loss, onset of ataxia and premature death, possibly due to insufficient concentrations penetrating through the blood brain barrier. Transcriptome analysis suggested Vorinostat improved liver disease in a pleiotropic manner, not surprising given the epigenetic nature of HDACi at the gene expression level. Overall, the results herein are of particular importance to the current clinical trial where the therapeutic efficacy is being investigated withou

Niemann-Pick type C (NP-C) disease is a neurodegenerative disease with a typically paediatric onset, for which no satisfactory cure is available. Inheritance of two dysfunctional copies of either cholesterol transport gene NPC1 or NPC2 perpetrates substantial accumulation of sterols and secondary lipids within the lysosome, eventuating in devastating and progressive visceral and neurological symptoms. Although the disease is ostensibly monogenic, there is an impressive diversity among sufferers of the disease in regards to onset of disease, specific symptoms imposed and prognosis. Recently, genetic modifiers of the NP-C disease have been reported in several models, which have improved understanding of the disease and identified new targets for treatment. Accordingly, the relevance of the LPIN1 gene and the phosphatidic acid phosphatase activity of its enzyme, were investigated for their ability to modify the NP-C phenotype in a fibroblast and mouse model. Treatment of NP-C fibroblasts with inhibition of phosphatidic acid-generating enzymes DGK and PLD was ineffective in reducing the lysosomal accumulation of cholesterol. Likewise, Npc1-/-Lpin1-/- mice were largely indistinguishable from Npc1-/-, Lpin1-/- and wild-type mice at P0 for brain and liver lipid profiles, cerebellar histology and liver autophagy.

Natural products are a robust source of drug leads, and medicinal plants have been the source of natural products that are important pharmaceuticals in modern medicine. Samoan medicinal plants have been investigated in the past, but their potential as a source of new drug leads has not been realized. I hypothesized that determining the mechanism of action of Samoan medicinal plant extracts would provide insight into their pharmaceutical potential. The work described herein was carried out on 11 Samoan medicinal plants, from which 22 extracts were prepared. A bioactivity rate of 68% was determined when 15 of the 22 extracts inhibited the growth of yeast (Saccharomyces cerevisiae). The medicinal plant Psychotria insularum was the most potent, thus genome-wide analyses were completed using the haploid deletion mutant library of S. cerevisiae. Yeast strains deficient in iron transport were hypersensitive to the P. insularum aqueous extract. Further investigations showed that exogenous iron supplementation rescued the growth defect induced by P. insularum extracts, suggesting that P. insularum reduced intracellular iron. Fittingly, yeast cells treated with P. insularum extracts contained less intracellular iron than control cells. Paraxodically, the expression levels of iron transporter proteins were upregulated upon extract treatment. When we investigated iron-requiring cellular processes, we found that yeast cells treated with P. insularum extracts exhibited a respiratory deficient phenotype and reduced heme synthesis, indicative of an impaired cellular iron status. These findings suggested that P. insularum reduced bioavailable iron leading to the induction of the low iron response, and indeed the extracts of P. insularum were shown to chelate iron via the iron-chelating CAS assay. To translate results from yeast to mammalian cells, we treated primary murine macrophages with P. insularum extracts and detected an anti-inflammatory response, which we found to correlate

Heparan sulphate proteoglycans (HSPG) are central to numerous processes of the mammalian cell. The highly charged negative side chains of the heparan sulphate (HS) oligosaccharides are essential for the regulatory and structural functions of the proteoglycan. Synthetic HS compounds have potential therapeutic value due to their ability to mimic naturally occurring HS. Niemann-Pick disease type C (NPC) is a fatal childhood neurodegenerative disease with characteristic cholesterol and sphingolipid accumulation in the late endosome or lysosome. Alzheimer’s disease, another neurodegenerative disorder, shares alterations of cholesterol and amyloid β metabolism with NPC. In this study,a set of novel heparan sulphate compounds with a range of structures and oligosaccharide side groups with a variety of degrees of sulphation was investigated with regards to their effects on cholesterol and amyloid β metabolism in cell line models of these two diseases. Fluorescent staining of cholesterol and confocal microscopy showed highly sulphated compounds reduce the accumulation of cholesterol in the perinuclear lysosomal storage organelles in patient fibroblast cell lines. The compounds had no effect on secreted amyloid β levels or amyloid precursor protein levels in a neuronal cell line model of early onset Alzheimer’s disease. The mechanism of cholesterol reduction is unclear but may be related to a reduction in HSPG-associated endocytosis of LDL/cholesterol.