Graduate Theses & Dissertations

Mfsd8 regulates growth and multicellular development in Dictyostelium discoideum
The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of inherited neurodegenerative lysosomal storage disorders. CLN7 disease is a subtype of NCL that is caused by mutations in the MFSD8 gene. MFSD8 encodes a lysosomal transmembrane protein that is predicted to play a role in transporting small substrates across membranes. However, little is known about its role and substrate specificity. Previous work identified an ortholog of human MFSD8 in the social amoeba Dictyostelium discoideum and reported its localization to endocytic compartments. In this study, the effects of mfsd8 loss during Dictyostelium growth and multicellular development were further characterized. Dictyostelium mfsd8- cells displayed increased rates of proliferation and pinocytosis in liquid media. During growth, loss of mfsd8 altered lysosomal enzymatic activities and reduced the intracellular and extracellular levels of autocrine proliferation repressor A. mfsd8- cells grown on a lawn of bacteria formed plaques in a shorter period of time compared to WT cells, providing additional support for the enhanced growth of mfsd8- cells. Upon starvation, the aggregation of mfsd8- cells was delayed, and mfsd8- cells formed more mounds that were smaller in size, which may be attributed to the reduced cell-substrate adhesion and altered lysosomal enzymatic activities observed for mfsd8- cells. Following aggregation, tipped mound formation was delayed, however, loss of mfsd8 did not affect the timing of slug/finger and fruiting body formation. Additionally, slug migration was reduced in mfsd8- cells. These aberrant phenotypes, excluding fruiting body formation, were effectively or partially rescued when Mfsd8-GFP was introduced into mfsd8- cells. Overall, these results show that Mfsd8 plays a role in regulating growth and developmental processes in Dictyostelium via lysosomal-associated functions. Author Keywords: CLN7, Dictyostelium discoideum, Lysosomes, MFSD8, Neuronal Ceroid Lipofuscinoses
role of Cln5 in autophagy, using a Dictyostelium discoideum model of Batten disease
This thesis investigated the role of the neuronal ceroid lipofuscinosis protein, Cln5, during autophagy. This was accomplished by performing well-established assays in a Dictyostelium cln5 knockout model (cln5-). In this study, cln5- cells displayed a reduced maximum cell density during growth and impaired cell proliferation in autophagy-stimulating media. cln5- cells had an increased number of autophagic puncta (autophagosomes and lysosomes), suggesting that autophagy is induced when cln5 is absent. cln5- cells displayed increased amounts of ubiquitin-positive proteins but had no change in proteasome protein abundance. During the development of cln5- cells, fruiting bodies developed precociously and cln5- slug size was reduced. Lastly, when cln5- cells were developed on water agar containing ammonium chloride (NH4Cl), a lysosomotropic agent, the formation of multicellular structures was impaired, and the small slug phenotype was exaggerated. In summary, these results indicate that Cln5 plays a role in autophagy in Dictyostelium. The cellular processes that regulate autophagy in Dictyostelium are similar to those that regulate the process in mammalian cells. Thus, this research provides insight into the undefined pathological mechanism of CLN5 disease and could identify cellular pathways for targeted therapeutics. Author Keywords: Autophagy, Batten disease, Cln5, Dictyostelium discoideum, NCL
Investigating wheat rust virulence evolution through transcriptome analysis of a recently emerged race of Puccinia triticina
Puccinia triticina, wheat leaf rust (WLR), is the most economically damaging fungal rust of wheat on a global scale. This study identified transcriptome changes in a recently emerged race of WLR in Ontario with a new virulence type relative to a possible ancestor race. Also, this study focused on detecting variation in candidate virulence genes and uncovering novel insight into WLR virulence evolution. Various race-by-variety interactions were evaluated using RNA-seq experiments. A list of genes with statistically significant expression changes in each comparison was prepared and predicted effectors were retained for further analysis. Proteins with nonsynonymous substitutions were run through BLASTx to identify potential orthologs. Over 100 candidate effectors with a 2-fold or higher change in transcript level were identified. Seven of these candidate effector genes were recognized to contain single nucleotide polymorphisms (SNPs) which altered the amino acid sequence of the resulting protein. The information gained may aid in targeted breeding programs to combat new WLR races as well as provide the basis for functional analysis of WLR using potential orthologs in a model basidiomycete. Author Keywords: effectors, RNA-seq, rust fungi, SNPs, transcriptome, wheat leaf rust

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