Yee, Janet

Giardia intestinalis is the causative agent of a diarrheal disease in mammals, but the mechanisms of disease pathogenesis are unclear. While proteins secreted by Giardia affect the host cells, the potential of hormone secretion has not been investigated to date. Cytokinins (CKs) are classified as phytohormones, but little is known about their role beyond plants. Mass spectrometry-based intracellular analysis revealed CKs typical of tRNA degradation, and extracellular analysis showed CK-riboside scavenging by Giardia with concurrent secretion of CK-free bases. Metabolomics profiling of culture supernatants showed similar trends where nucleosides were up taken, and nucleobases were secreted. The dynamics of amino acids, nucleosides and nucleobases were altered by CK-supplementation during encystation, along with inhibition of encystation. In summary, this is the first study to report CK synthesis and metabolism by Giardia along with the effects of CKs on the metabolite secretome of Giardia, while establishing a link between CK and nucleoside metabolism.

Author Keywords: Cytokinins, Giardia, mass spectrometry, metabolomics, parasite, secretome

The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of fatal neurodegenerative disorders that primarily affect children. Several subtypes of NCLs have been reported, each being caused by a mutation in a distinct ceroid lipofuscinosis neuronal (CLN) gene; this results in aberrant lysosome function and the accumulation of lipoprotein aggregates (known as ceroid lipofuscin) within cells. Several innate cellular pathways exist to alleviate the stress caused by the buildup of aggregates. The endoplasmic reticulum (ER) is an essential organelle in this process because it is responsible for maintaining cellular homeostasis through protein production, quality control, and regulating several signalling pathways. The unfolded protein response (UPR) consists of several conserved pathways devoted to attenuating ER stress caused by an accumulation of misfolded proteins or aggregates; at the center of this stress response is GRP78, a molecular chaperone that binds to misfolded proteins to facilitate proper folding. The social amoeba Dictyostelium discoideum is an excellent model system for studying NCLs as it encodes more CLN-like proteins when compared to other classical model organisms (e.g., yeast, worm, fruit fly). In this study, D. discoideum was used to elucidate the effects of ER stress and build an understanding of how cells cope with increased stress. Beyond this, ER stress in D. discoideum models for CLN3 disease and CLN5 disease were evaluated. First and foremost, during the induction of ER stress by tunicamycin, there was an increase in intracellular and extracellular amounts of Grp78 accompanied by an increase in stress-related changes to the ER. Furthermore, models of CLN3 disease and CLN5 disease displayed increased amounts of Grp78 as well as a disrupted ER morphology. Interestingly, wildtype D. discoideum, AX3 cells, treated with tunicamycin displayed a similarly disrupted ER when compared to CLN models. Finally, when subjected to tunicamycin-induced ER stress, these NCL models displayed a trend towards increased Grp78 amounts, however, these cells appear to have a reduced sensitivity to tunicamycin-induced stress compared to wild-type cells. In summary, this study highlights D. discoideum as a model for studying ER stress through the conserved role of Grp78 in the stress response and concludes that an aberrant ER stress underlies the pathology of the NCLs.

Author Keywords: Batten disease, Dictyostelium discoideum, ER stress, GRP78, neuronal ceroid lipofuscinoses (NCLs)

Amino acids make up proteins, which are the building blocks of life. A balance of amino acids is needed to maintain a healthy state. Tyrosine (Tyr) is synthesized from the metabolism of phenylalanine, which is an essential amino acid, meaning it can only be obtained from the diet. It is related to many metabolic and neurodegenerative diseases. Tyr can undergo post-translational modifications such as phosphorylation and nitration, which are implicated in cancer and nitrative stress, respectively. Although there are many methods to detect Tyr and its analogues, phosphotyrosine (pTyr) and nitrotyrosine (nTyr), these methods are time-consuming, involve expensive instruments and involve tedious process. This research proposes a new type of nanomaterials, carbon dots (CDs), to detect these amnio acids. Data indicate that CDs can be used to detect nTyr with a limit of detection of 34 μM in the linear range of 20 - 105 μM. The amenability of CD-nTyr assay was also tested in various biological matrices and biological molecules and was shown to be sensitive to nTyr. Nitration of Tyr was carried out in the presence of sodium nitrite and hydrogen peroxide catalyzed by either Cu(II) or Fe(III) to mimic biological reactions and CDs were tested as both inhibitors and indicators of Tyr nitration. Although CDs did not inhibit the nitration reaction of Tyr, they did not serve as indicators of Tyr nitration due to the quenching of CDs by the nitrating agents. This shows the importance of using CDs to detect nTyr and further use it for biological applications to detect diseased states.

Author Keywords: amino acids, carbon dots, nanomaterials, sensor, spectroscopy, tyrosine

Giardia intestinalis is a waterborne enteric parasite that lacks mitochondria and the capacity for heme biosynthesis. Despite this, Giardia encodes several heme proteins, including four cytochrome b5 isotypes (gCYTB5-I – IV) of unknown function. The aim of this thesis is to gain insight into the function of the Giardia cytochrome b5 isotype III (gCYTB5-III) that is found in the nucleus, as first reported by our laboratory using immunofluorescence microscopy experiments with an isotype-III specific antibody. Nuclear localization of isotype-III is supported by two of my experiments: i) immunoblot analysis of crude cytoplasmic and nuclear enriched fractions of Giardia trophozoites; ii) association of gCYTB5-III with the insoluble fraction of Giardia lysates crosslinked with formaldehyde is reversed by DNase I treatment. To gain an understanding of the possible roles of gCYTB5-III, I performed immunoprecipitation (IP) experiments on lysates from Giardia trophozoites to identify its protein partners. Mass spectroscopy analysis of the immunoprecipitate identified proteins localized to the nucleus (RNA polymerase, DNA topoisomerase, histones, and histone modifying enzymes). Intriguingly, over 40% of the known mitosomal proteome, which functions in iron-sulfur (Fe-S) cluster assembly was also associated with gCYTB5-III. One of these proteins, the flavoenzyme GiOR-1, has been shown to mediate electron transfer from NADPH to recombinant gCYTB5-III. These IP results provide evidence that GiOR-1 and gCYTB5-III interact in vivo, and furthermore, suggest that some proteins in the mitosome could interact with those in the nucleus. I also found that DNA stress, caused by low concentrations of formaldehyde (0.1 – 0.2%) resulted in the increased expression of gCYTB5-III. Collectively these findings suggest a role of gCYTB5-III in Giardia's response to DNA stress and perhaps the formation of Fe/S clusters.

Author Keywords: cluster, cytochrome, heme, iron, mitosome, nuclear

Ustilago maydis (DC) Corda is the causal agent of 'common smut of corn'. Completion of the U. maydis lifecycle is dependent on development inside its host, Zea mays. Symptoms of U. maydis infection include chlorosis and the formation of tumours on all aerial corn tissues. Within the tumours, thick-walled diploid teliospores form; these are the reproductive and dispersal agent for the fungus. U. maydis is the model to study basidiomycete biotrophic plant-pathogen interactions. It holds this status in part because of the completely sequenced 20.5 Mb genome; however, thorough genome annotation is required to fully realize the value of this resource. The research presented here improved U. maydis genome annotation through the analysis of cDNA library sequences and comparative genomics. These analyses identified and characterized pathogenesis-related genes, and identified putative meiosis genes. This enabled the use of U. maydis as a model for investigating 'host-induced' meiosis. Further, the cDNA library analyses identified non-coding RNAs (ncRNAs) and natural antisense transcripts (NATs). NATs are endogenous RNA molecules with regions complementary to a protein-coding transcript. Although NATs have been identified in a wide variety of mammals, plants, and fungi, very few have been functionally characterized. Over 200 U. maydis NATs were annotated by analyzing full-length cDNA sequences. NAT structural features were characterized. Strand-specific RT-PCR was used to detect NATs in U. maydis and in a related smut fungus, U. hordei. The data supported a common role for NATs in smut teliospore development, independent of the RNA interference pathway. Analysis of the expression of one U. maydis NAT, as-um02151, in haploid cells, led to a model for NAT function in U. maydis during teliospore dormancy. This model proposed NATs facilitate the maintenance of stored mRNAs through the formation of double-stranded RNA. In testing this model, it was determined that the deletion of two separate upstream regulatory regions, one of which contained a ncRNA (ncRNA1), altered NAT levels and decreased pathogenesis. These studies strengthened U. maydis as a model organism, and began the functional investigation of NATs in U. maydis, which identified a new class of fungal pathogenesis genes.

Author Keywords: cDNA library analysis, genome annotation, mRNA stability, natural antisense transcripts, pathogenesis, Ustilago maydis

The parasitic protist Giardia intestinalis has five heme proteins: a flavohemoglobin and several isotypes of cytochrome b5. While the flavohemoglobin has a role in counteracting nitric oxide, the functions of the cytochromes (gCYTb5s) are unknown. In this study, the protein level and cellular localization of three gCYTB5 isotypes (gCYTb5-I, II and III) and flavohemoglobin were examined in Giardia trophozoites exposed to three nitrosative stressors at two different concentrations: nitrite (20 mM, 0.5 mM); GSNO (2 mM, 0.25 mM) and DETA-NONOate (2 mM, 0.05 mM). An increase in protein levels was observed for gCYTb5-II with all stressors at both concentrations. However, the effects of these nitrosative stressors on gCYTb5-I and III were inconclusive due to the variation among the replicates and the poor detection of gCYTb5- III on western blots. The protein level of the flavohemoglobin also increased in response to the three stressors at the low concentrations of stressors that were tested. Only the cellular localization of gCYTb5-I changed in response to nitrosative stress, where it moved from the nucleolus to the nucleus and cytoplasm. This response was extremely sensitive and occurred at the lower doses of the three stressors, suggesting that gCYTb5-I may be involved in a nucleolar- based stress response.

Giardia intestinalis is an anaerobic protozoan that lacks common eukaryotic heme-dependent respiratory complexes and does not encode any proteins involved in heme biosynthesis. Nevertheless, the parasite encodes several hemeproteins, including three members of the Type II cytochrome b5 sub-group of electron transport proteins found in anaerobic protist and amitochondriate organisms. Unlike the more well-characterized cytochrome b5s of animals, no function has been ascribed to any of the Type II proteins. To explore the functions of these Giardia cytochromes (gCYTB5s), I used bioinformatics, immunofluorescence microscopy (IFM) and co-immunoprecipitation assays. The protein-protein interaction in silico prediction tool, STRING, failed to identify relevant interacting partners for any of the Type II cytochromes b5 from Giardia or other organisms. Differential cellular localization of the gCYTB5s was detected by IFM: gCYTB5-I in the perinuclear space; gCYTB5-II in the cytoplasm with a staining pattern similar to peripheral vacuole-associated protein; and gCYTB5-III in the nucleus. Co-immunoprecipitation with the gCYTB5s as bait identified potential interacting proteins for each isotype. The most promising candidate is the uncharacterized protein GL50803_9861, which was identified in the immunoprecipitate of both gCYTB5-I and II, and which co-localizes with both. Structural analysis of GL50803_9861 using Swiss Model, Phyre2, I-TASSER and RaptorX predicts the presence of a nucleotide-binding domain, which is consistent with a potential redox role involving nicotinamide or flavin-containing cofactors. Finally, the protein GL50803_7204 which contains a RNA/DNA binding domain was identified a potential partner of gCYTB5-III. These findings represent the first steps in the discovery of the roles played by these proteins in Giardia.

Author Keywords: Cytochrome b5, Giardia intestinalis, Heme, Interactome, Protein structure prediction

To study the Giardia intestinalis cell cycle, counterflow centrifugal elutriation (CCE) was used to separate an asynchronous trophozoite culture into fractions enriched for cells at the different stages of the cell cycle. For my first objective, I characterized the appearance of a third peak (Peak iii) in our flow cytometry analysis of the CCE fractions that initially suggested the presence of 16N cells that are either cysts or the result of endoreplication of Giardia trophozoites. I determined that this third peak consists of doublets of the 8N trophozoites at the G2 stage of the cell cycle that were not removed effectively by gating parameters used in the analysis of the flow cytometry data. In the second objective, I tested the use of a spike with RNA from the GS isolate of Giardia as an external normalizer in RT-qPCR on RNA from CCE fractions and encystation cultures of Giardia from the WB isolate. My results showed that the GS RNA spike is as effective as the use of previously characterized internal normalizer genes for these studies. For the third objective, I prepared two sets of elutriation samples for RNA seq analysis to determine the transcriptome of the Giardia trophozoite cell cycle. I confirmed the results of the cell cycle specific expression of several genes we had previously tested by RT-qPCR. Furthermore, our RNA-seq identified many genes in common with those identified from a microarray analysis of the Giardia cell cycle conducted by a collaborator. Finally, I observed an overall <4 fold change in differentially expressed genes during the G1/S and G2/M phase of the cell cycle. This is a modest change in gene expression compared to 10 - 30 fold changes for orthologous genes in mammalian cell cycles.

Author Keywords: Cell cycle, Counterflow Centrifugal Elutriation, Flow Cytometry, RNA-sequencing, RT-qPCR

Ustilago maydis is a biotrophic fungal plant pathogen that causes 'common smut of corn' disease. During infection, U. maydis develops a metabolic dependency on its host, relying on uptake of the carbon molecules provided within Zea mays tissues. The research presented indicated a requirement for metabolism of the pentose sugar D-xylose through functional investigation of a U. maydis xylitol dehydrogenase (uxm1), an enzyme involved in the bioconversion of D-xylose. This work is the first to outline the importance of pentose metabolism during biotrophic plant pathogenesis, as U. maydis haploid cells lacking this gene were impaired in their ability to cause disease and grow on medium containing only D-xylose. This thesis also explored the possibility that expression of this carbon-related gene is controlled by antisense RNAs (asRNAs), endogenous molecules with complementarity to mRNAs. Previous investigation of U. maydis asRNAs identified some that are exclusively expressed in the dormant teliospore, suggesting they have a functional role within this cell-type. A subset of these asRNAs at the uxm1 locus were investigated, with the purpose of identifying the mechanism(s) by which they influence U. maydis pathogenesis. This investigation involved the creation and functional analysis of a series of U. maydis deletion and expression strains. Together, these findings provided additional knowledge regarding the possible functions of U. maydis asRNAs, and their involvement in controlling important cellular processes, such as carbon metabolism and pathogenesis.

Author Keywords: antisense transcripts, fungal carbon metabolism, non-coding RNAs, pathogenesis, Ustilago maydis, xylitol dehydrogenase

Giardia lamblia is an intestinal parasite found globally in freshwater systems that is responsible for endemic outbreaks of infectious diarrhea. As a unicellular parasite that lacks mitochondria, a respiratory chain and lives in the anaerobic environment of its host's intestine, Giardia was assumed for decades to lack heme proteins. However, its genome encodes several putative heme proteins, including three with sequence similarity to the cytochrome b5 family, referred to as Giardia cytochromes b5 (gCYTb5). Recombinant expression of one of these genes (gCYTb5-I), results in a protein (17-kDa) that is isolated with noncovalently bound heme. Resonance Raman and UV-visible spectra of gCYTb5-I in oxidized and reduced states resemble those of microsomal cytochrome b5, while sequence alignment and homology modelling supports a structure in which a pair of invariant histidine residues act as axial ligands to the heme iron. The reduction potential of gCYTb5-I measured by cyclic voltammetry is -165 mV vs the standard hydrogen electrode and is relatively low compared to those of other family members. The amino- and carboxy-terminal sequences that flank the central heme-binding core of the gCYTb5 are highly charged and do not occur in other family members. An 11-kDa core gCYTb5-I variant lacking these flanking sequences was also able to bind heme; however, we observe very poor expression of this truncated protein as compared to the full-length protein.

Author Keywords: b-type cytochrome, cytochrome b5, electron transfer protein, Giardia intestinalis, heme/heam protein, spectroelectrochemistry