The long held goal of malaria eradication faces increasing adversity due to limited treatment options and growing drug resistance. New drug scaffolds are needed to both improve treatment outcomes, and expand our knowledge of parasite-drug interaction. In the following dissertation, we utilize fungi as a chemical source for antiplasmodial screening. Through these endeavors, over 6,000 fungal extracts have been screened, and more than 100 compounds with antiplasmodial activity have been identified. In the coming pages, we outline the work performed on two prominent compound classes identified: cyclic tetrapeptide HDAC inhibitors and peptaibols. Our investigation of a potent cyclic tetrapeptide scaffold developed the structure activity relationship of 24 inhibitors that show markedly improved selectivity for the malaria parasite over previous analogs. These new inhibitors demonstrated nanomolar potency against the plasmodial class I HDAC, PfHDAC1, and caused hyperacetylation of histone H4 in vitro. Semi-synthetic modifications to the zinc binding group resulted in a dramatic change in killing permeance, potentially linked to early life cycle inhibition. While chemical optimization is needed for in vivo implementation, this analysis generated valuable data regarding the class as a whole, and the factors that govern their killing profile. In our work with peptaibols, we break ground on a completely unexplored class in the malaria field. These inhibitors show incredible antiplasmodial potency, and more than two hundred fold selectivity. Additionally, they demonstrate potential as dual stage inhibitors for both prophylaxis and treatment. Their blood stage activity appears linked to a change in membrane permeability, specifically at the level of the parasites digestive vacuole. This finding is further supported by an in-depth transcriptome analysis and resistance generation. In summary, the original research produced as part of this dissertation expands the current boundaries of knowledge in the field of malaria therapeutics, furthering drug discovery and development efforts.

Economically viable fisheries for the American blue crab, Callinectes sapidus, occur in the USA from Delaware Bay to the Gulf of Mexico. Unfortunately, a number of factors, including overexploitation, degraded habitats, and disease, have contributed to the decline of blue crab populations in the USA. In April 2008, the Secretary of Commerce declared the blue crab fishery in the Chesapeake Bay a "commercial fishery disaster." One factor potentially contributing to the decline of blue crab populations in high salinity waters is infection with Hematodinium sp., a parasitic dinoflagellate, which can cause significant mortalities in blue crab populations. Since the first report of this parasite infecting blue crabs in 1975, few studies have used molecular techniques to examine life history characteristics, population structure, geographic range, and host specificity of the species of Hematodinium that infects this host. to examine host specificity, a variety of crustaceans were collected from the Delmarva Peninsula, a region that has high prevalence of Hematodinium sp. in blue crabs. Sequence data obtained from the first internal transcribed spacer (ITS1) region of the ribosomal RNA (rRNA) gene complex from Hematodinium sp. in blue crabs and potential alternate hosts was used to determine that this species of Hematodinium is a host generalist, capable of infecting many decapods and possibly amphipods along the Delmarva Peninsula. Additional sequence data obtained from Hematodinium sp. from infected blue crabs collected from Georgia, the Gulf Coast of Florida, and Texas showed that one species of Hematodinium, the same species found in Virginia, infected blue crabs in all of these locations. Due to the lack of variation observed in the ITS1 region sequences, an enriched microsatellite library was developed using a non-clonal primary culture of Hematodinium sp. from an infected blue crab. From this library, eleven microsatellite markers were identified and optimized for Hematodinium sp. to assess intra-specific variation. Eight microsatellite markers were polymorphic and these were used to genotype Hematodinium sp. from infected blue crabs from six locations along the Delmarva Peninsula. Allele frequencies from these microsatellite markers, along with analyses of the multi-locus genotypes recovered from single genotype infections, indicated that the Delmarva Peninsula contains a single population of this parasite. Microsatellite data also indicate that this parasite is haploid in the hemolymph of the blue crab and that multiple infections are common. The observed genotypic variation was extremely high, indicating that sexual reproduction occurs at some point in the life cycle of this parasite, though the exact life history stage that undergoes sexual reproduction is unknown. This is the first report for evidence of ploidy, multiple infections, population structure, and sexual reproduction for this species of Hematodinium .

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Elongate hemlock scale (Fiorinia externa) is an invasive species from Japan that was first detected in the United States in 1908 and has established in most states where eastern hemlock (Tsuga canadensis ) occurs. Scale density is much higher in the US (21 to >400 scales per 100 needles) compared to Japan (<1 scale per 100 needles) despite the presence in both regions of the parasitoid Encarsia citrina that has been assumed to be responsible for low densities in Japan. I have found that phenology of the vulnerable scale stage was asynchronous with E. citrina flight throughout the eastern United States. This asynchrony was the result of overlapping life stages and generations of elongate hemlock scale throughout the growing season. This overlap appears to result from delayed senescence of first generation adult females. To investigate the impact of E. citrina on elongate hemlock scale, natural enemy exclusion experiments were conducted. I found that E. citrina exerts some control on elongate hemlock scale, but insufficient to maintain density at levels seen in its native Japan. Existing records of parasitoids of elongate hemlock scale in Japan are limited to the Kyoto area. I sampled the parasitoid community of elongate hemlock scale and other scales on hemlock throughout Japan. Sequence data from two gene loci, 28SD2 and COI, were used to identify the number of unique parasitoid species attacking scales on Japanese hemlocks. This sequence data was also used to identify possible cryptic species within E. citrina in Japan. Twenty-two genetically distinct parasitoids were identified (seven singletons). Eleven (three singletons) of these were from elongate hemlock scale. With the exception of E. citrina, parasitoids tended to be host species specific. No evidence of cryptic species within E. citrina was found. The parasitoids identified dramatically increase the number of elongate hemlock scale parasitoids previously reported and this study may lead to the identification of several potential biological control agents of elongate hemlock scale for introduction to the United States.

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Trypanosomatid parasites are unicellular eukaryotes characterised by the presence of a subpellicular array of microtubules, a single flagellum, and a kinetoplast (containing the condensed mitochondrial DNA). The majority of trypanosomatid species undergo complex life-cycles, alternating between a mammalian host and an insect vector. Progression through this life-cycle requires the differentiation of trypanosomatids into distinct, niche adapted developmental forms. Differentiation into each life-cycle stage involves important biochemical and morphological changes, including the remodelling of the subpellicular cytoskeleton that defines cell shape. In higher eukaryotes, proteins from the calpain superfamily are involved in developmentally- and environmentally-regulated remodelling of the cytoskeleton and the dynamic organisation of signal transduction cascades. Interestingly, trypanosomatids contain unusually large families of calpain-related proteins, but there is little knowledge about the functional roles of these molecules during the life-cycle of trypanosomatid parasites. In this thesis, I present the results of the bioinformatic analysis of calpain-like proteins in three trypanosomatid parasites, Trypanosoma brucei, Leishmania major and Leishmania infantum. From this analysis, I selected several calpain-related proteins tor RNAi functional analysis, on the bases of their domain composition and conservation across different species. The detailed analysis of the resulting RNAi phenotypes revealed the essential function of some calpain-like proteins for the correct morphogenesis of specific developmental forms of T. brucei, shedding some light on the mechanisms that regulate this parasite differentiation and cytoskeletal remodelling, and providing new putative therapeutic targets for African sleeping sickness.

No description available.