Cellular function is dependent upon the correct translation of genomic information encoded in DNA into functioning products, usually proteins. Prior to protein translation, DNA is transcribed into messenger RNA (mRNA). In eukaryotes such as human, this process must almost always undergo an intermediate step, termed pre-mRNA splicing, in which non-protein-coding regions (introns) are removed from the mRNA, yielding mature message (exons). Defects in pre-mRNA splicing are responsible for various human disorders including retinitis pigmentosa, spinal muscular atrophy, and myotonic dystrophy. In order to work towards a cure for these diseases, it is necessary to understand how pre-mRNA splicing works normally. One potentially useful tool for this is small molecule inhibitors of splicing, which have previously been shown to inhibit catalytic RNAs the additional benefit of being candidates for therapeutics. Only two papers (Hertweck et al., 2003; Kaida et al. 2007) have explored the effects of small molecules on nuclear splicing. Previous work has investigated inhibition of human splicing. In this work, I have examined the effect of small molecules on yeast splicing in order to make use of the powerful genetic and biochemical tools available for yeast. The main goal of this thesis was to identify small molecule inhibitors of yeast pre-mRNA splicing and to characterize the step at which they exert their inhibitory effects. Thirty-two different small molecules were tested. Ten of them were found to completely inhibit pre-mRNA splicing. IC₅₀ values were measured for each of the inhibitory small molecules, and neomycin was found to be the strongest inhibitor with an IC₅₀ of 80~M, while cefoperazone was the weakest inhibitor with an IC50 of 6.1 mM. Native gel analysis was used to establish the step at which splicing was inhibited. Four of the ten inhibitors showed a complete block in spliceosome assembly with accumulation of spliceosomal complex H; one accumulated spliceosomal complex A; two accumulated both spliceosomal complexes A and B; and three accumulated spliceosomal complexes B and C. I anticipate that these inhibitors will be useful tools for the studying the mechanism of pre-mRNA splicing. By characterizing the splicing complexes that accumulate in the presence of these inhibitors it will be possible to map out the path by which splicing complexes assemble. Furthermore, several of the inhibitors are previously uncharacterized, and consequently have potential to be useful in a variety of other context. In the long term, these inhibitors may lead to novel therapies for splicing related diseases.
Biomass ashes are potential soil amendments that reduce soil acidity and provide plant nutrients, but trace elements in ash may be leached from the solid phase, thereby posing environmental concerns. We determined the leachability of major and trace elements as influenced by ash pre-treatments, the presence of soil, and the pH of the receiving environment. Weathering was simulated by serial batch extraction where pH was uncontrolled, and by single extraction under controlled pH conditions. We found that hardening reduced the solubility of ash, and reduced the leachability of Al, Ba, Ca, Cu, Mo, Sr, and V, as determined by ICP-MS. In a separate experiment, extractions of ash samples showed that when pH was lowered the leachability of most elements increased while a few decreased. The results of the weathering experiments support the use of ash as a soil amendment. --Leaf ii.
Worldwide, many fungal species have been used for their medicinal properties by various cultures for centuries. Major challenges with cancer chemotherapy, which includes toxicity, non-specificity and several adverse effects have motivated researchers to search for novel anti-cancer compounds from natural sources. Several studies have identified various anti-cancer compounds from mushrooms in many parts of the world, but mushrooms occurring in British Columbia remain unstudied for their anti-cancer activities. In this thesis, five mushroom species were collected, extracted, and assessed for anti-proliferative and immuno-stimulatory activities. Eleven extracts from these species showed anti-proliferative or immuno-stimulatory activity or both. Sodium hydroxide extract from Paxillus obscurisporus exhibited potent anti-proliferative activity and was further purified using size-exclusion and anion-exchange chromatography. Analysis of the relatively pure anti-proliferative fractions suggests the presence of protein and glycoprotein moieties. Further purification and characterisation is needed before these compounds can be tested for biomedical purposes in the future.
