Graduate Theses & Dissertations

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Expression optimization and NMR spectroscopy of Giardia intestinalis cytochrome b5 isotype III
The parasitic protist Giardia intestinalis does not synthesize heme and lacks many common eukaryotic heme proteins, yet it expresses four cytochrome b5 (gCYTB5) isotypes of unknown function. These have low reduction potentials and distinct subcellular locations that are consistent with structural features and biological functions that differ from their mammalian counterparts. Isotype III (gCYTB5-III) is particularly fascinating for its unusual location in the nuclei of Giardia. This thesis reports the optimization of recombinant gCYTB5-III overexpression for structural studies by NMR spectroscopy. Vital optimization factors for isotope labelling were first identified, finding that auto-induction promotes the optimization of many other conditions, such as colony selection, starter cultures, media components, temperature, pH and aeration. Optimized conditions were then applied to the expression and NMR spectroscopy of isotope-labelled gCYTB5-III and bovine cytochrome b5 as a control. These results can be extended to other heme proteins and will expand our biochemical knowledge of Giardia. Author Keywords: Auto-induction, Cytochrome b5, Giardia intestinalis, Isotope Labelling, Nuclear Magnetic Resonance Spectroscopy, Recombinant Protein
Effect of Nitrosative Stress on Heme Protein Expression and Localization in Giardia Intestinalis
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. Author Keywords:
Characterization of a Zn(II)2Cys6 transcription factor in Ustilago maydis and its role in pathogenesis
Ustilago maydis (D.C.) Corda is a biotrophic pathogen that secretes effectors to establish and maintain a relationship with its host, Zea mays. In this pathosystem, the molecular function of effectors is well-studied, but the regulation of effector gene expression remains largely unknown. This study characterized Zfp1, a putative U. maydis Zn(II)2Cys6 transcription factor, as a modulator of effector gene expression. The amino acid sequence of Zfp1 indicated the presence of a GAL4-like zinc binuclear cluster as well as a fungal specific transcription factor domain. Nuclear localization was confirmed by tagging Zfp1 with enhanced green fluorescent protein. Deletion of zfp1 resulted in attenuated hyphal growth, reduced infection frequency, an arrest in pathogenic development, and decreased anthocyanin production. This phenotype can be attributed to the altered transcript levels of genes encoding predicted and confirmed U. maydis effectors in the zfp1 deletion strain during pathogenic growth. Complementation of zfp1 deletion strain with tin2, an effector involved in anthocyanin induction, suggested this effector is downstream of Zfp1 and its expression is influenced by this transcription factor during in planta growth. When wild-type zfp1 was ectopically inserted in the zfp1 deletion strain, pathogenesis and virulence were partially restored. This, coupled with zfp1 over-expression strains having a similar phenotype as the deletion strains, suggested Zfp1 may interact with other proteins for full function. These findings show that Zfp1, in conjunction with one or more binding partners, contributes to U. maydis pathogenesis, virulence, and anthocyanin production through the regulation of effector gene expression. Author Keywords: effector, pathogenesis, transcription factor, Ustilago maydis, Zea mays, zinc finger
Evaluation of silver nanoparticles (AgNPs) and anti-GD2-AgNP antibody-drug conjugates as novel neuroblastoma therapies
Neuroblastoma (NB) has one of the highest mortality rates in pediatric oncology due to relapsed and refractory disease. Current aggressive multi-modal treatments are inhibited by dose-limiting toxicities and are associated with late-effects and secondary malignancies, emphasizing the necessity for novel therapeutics. Uniquely, most NB cells highly express disialoganglioside (GD2) a cell surface glycolipid that can provide a target for tumour-specific delivery. This study demonstrates a comprehensive evaluation of silver nanoparticles (AgNPs) and the first preliminary evaluation of anti-GD2-AgNP antibody-drug conjugates (ADCs) against NB in vitro. This present study validates the potential for AgNPs as an anti-cancer agent against NB as AgNPs demonstrated preferential toxicity towards NB cells through metabolic inhibition and indicative morphological alterations, while a less tumorigenic cell line demonstrated resistance to AgNP treatment. Therefore, this work identified an AgNP cell-type-dependent cytotoxicity effect. Low conjugation efficiency of the anti-GD2 monoclonal antibody, 14.G2a, to NHS-activated AgNPs failed to exert greater toxicity than the AgNPs alone. Collectively, this thesis provides novel information regarding the anti-cancer effects of AgNPs against NB with recommendations for anti-GD2-AgNP ADCs. Author Keywords: ADC, Chemotherapy, GD2, Neuroblastoma, Silver nanoparticles
Functional Investigation of A Ustilago maydis Xylose Metabolism Gene and its Antisense Transcripts
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
Effect of the neonicotinoid imidacloprid on embryogenesis and anuran survivorship in frog virus 3 infected tadpoles
Exposure of pre-metamorphic amphibians to neonicotinoid insecticides may be contributing to the global decline in amphibian populations. In this study, anuran embryos and tadpoles of the African clawed frog (Xenopus laevis) and the North American leopard frog (Lithobates pipiens) were used to determine the effects of embryonic exposure to neonicotinoids. In addition, Xenopus was used to determine if prolonged exposure to neonicotinoids influenced tadpole sensitivity to frog virus 3 (FV3). Exposure of anuran embryos to concentrations of the neonicotinoid insecticide, imidacloprid, ranging from 1 -20 ppm induced a concentration dependent increase in malformations of the retina in Xenopus embryos. However, similar responses were not observed with embryos of leopard frogs. Exposure of Xenopus tadpoles to 500 ppb concentration of imidacloprid followed by challenge with FV3 showed that pesticide exposure unexpectedly decreased the rates of mortality, although total mortalities by the end of the experiment were not significantly different from controls. This unexpected observation may be attributed to a reduced inflammatory response induced by exposure to imidacloprid. Despite the low acute toxicity of neonicotinoid insecticides to vertebrates, these studies indicate that exposure to this class of insecticides causes sublethal effects in anuran species during early life stages. Author Keywords: embryogenesis, Lithobates pipiens, neonicotinoid, ranavirus, tadpole, Xenopus laevis
Interactome Study of Giardia Intestinalis Cytochromes B5
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
Pathogen vs. Predator
Stressors are often an inescapable part of an organism’s life. While the effects of many stressors have been well studied individually, potential interactions between stressors exist that may result in greater than additive negative effects. Stressors may be linked by conflicting demands on energy budgets, interfering with important physiological pathways, or necessitating incompatible adaptive responses. Using Ranavirus (FV3) and larval dragonfly predators (Anax spp.) in a 2x2 factorial experiment on green frog (Lithobates clamitans) tadpoles, I investigate the interactions in behaviour, morphology, and metabolism when both stressors were applied in concert. I demonstrate that activity and feeding are reduced additively by both stressors, and tadpoles increase distance between conspecifics in FV3-exposed tanks, but only in the absence of predators. I also note decreases in mass, and a non-significant marginal increase in metabolic rate of tadpoles exposed to FV3. Interestingly, I provide evidence that FV3 can compromise morphometric responses through antagonistic interactions with perceived predation risk exposure, which may result in significantly elevated mortality even when either stressor is present in sub-lethal quantities. Thus, I conclude that sub-lethal exposure to stressors can nonetheless have substantial impacts on organisms and a more integrative approach to examining the impacts of stressors on individual physiology and fitness is necessary. Author Keywords: Behaviour, Interaction, Morphology, Predation Risk, Ranavirus, Tadpoles
Studies of the Giardia intestinalis trophozoite cell cycle
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
Flavohemoglobin expression in Giardia intestinalis exposed to nitrosative stress
The parasitic protist Giardia intestinalis lacks most heme proteins yet encodes a flavohemoglobin (gFlHb) that converts nitric oxide to nitrate and likely protects the cell from nitrosative stress. In this work an antibody raised against gFlHb was used to examine both changes in gFlHb expression levels and intracellular localization in Giardia in response to nitrosative stress. Giardia trophozoites exposed to stressors which either directly release nitric oxide (diethyltriamine NONOate, 1 mM) or are sources of other reactive nitrogen intermediates (sodium nitrite 20 mM or S-nitrosoglutathione, 1 or 5 mM) exhibited a 2 to 9-fold increase of gFlHb after 24 hours. Increased expression levels of gFlHb were detectable by 8 hours in S-nitrosoglutathione and diethyltriamine-NONOate-treated trophozoites, and by 12 hours after sodium nitrite exposure; these differences were likely due to differences in the rates of release of RNS from these compounds. In addition to a band of the expected size for gFlHb (52 kDa), western blots detected a second, higher molecular weight band (72 kDa) with comparable or higher intensity upon treatment with these RNS donors, which is consistent with sumoylation of gFlHb. Immunofluorescence microscopy of Giardia trophozoites detected gFlHb diffused throughout the cytoplasm and more punctuated staining along the cell membrane and between the nuclei. The punctuated staining may be due to the association of gFlHb with either peripheral vacuoles or basal bodies. Author Keywords: Flavohemoglobin, Giardia intestinalis, Nitrosative stress
Characterization of frog virus 3 and its binding partner LITAF
Iridoviruses are large (120-200nm) double stranded DNA viruses that contain an icosahedral capsid. The iridoviridae family is composed of five genera that infect a wide range of poikilothermic vertebrates (Lymphocystivirus, Ranavirus and Megalocyivirus) and invertebrate hosts (Iridovirus, Chloriridovirus). Frog virus 3 (FV3) is a member of the Ranavirus genus, and is commonly used as a model system to study iridoviruses. I was interested in understanding virus-host interaction in FV3. I studied two viral genes, FV3 97R and FV3 75L. Here I demonstrate that 97R localizes to the endoplasmic reticulum (ER) at 24 hours post-transfection. However, at 35 hours post-transfection 97R localizes to the ER but also begins to form concentrated pockets, continuous with the nuclear membrane This study found that 97R possess a unique phenotype and that its localization to the ER is mediated through its C-terminus transmembrane domain. FV3 75L encodes an 84 amino acids protein. I showed that FV3 75L localizes to the early endosomes, while its cellular binding partner, LITAF, localizes to late endosome/lysosome. Interestingly, when FV3 75L and LITAF are co-transfected into cells, LITAF can alter the subcellular localization of FV3 75L to late endosome/lysosomes. A physical interaction between LITAF and FV3 75L was demonstrated through a pull-down assay and that a highly conserved domain found in both proteins may mediate the interaction. LITAF has been proposed to function in protein degradation, but there is still uncertainty on LITAF's specific role. I was interested in further characterizing LITAF and its implications in protein degradation and a neurodegenerative disorder. At least 9 mutations of LITAF are associated with Charcot-Marie-Tooth disease type 1C (CMT1C), which belongs to the group of most common heritable neuromuscular disorders, affecting approximately one in 2500 people. We show that LITAF mutants G112S and W116G mislocalize from the late endosome/lysosome to the mitochondria while the T49M and P135T mutants show partial mislocalization with a portion of the protein present in the late endosome/lysosome and a portion of the protein localized to the mitochondria. Since LITAF is believed to play a role in protein degradation, it is possible that the specific characteristics of CMT1C may occur though impaired degradation of Schwann cell membrane proteins, such as PMP22. I was able to show that when WT LITAF is present, there is a decrease in the PMP22 intracellular levels, which suggest that LITAF plays an important role in protein degradation, and also in other types of CMT. Insight into how mutations in LITAF cause CMT1C may not only help better understand cellular pathways, but also further elucidate the role LITAF's viral homolog FV3 75L during viral infection. Author Keywords: 75L, Charcot-Marie-Tooth, CMTC1, ER, FV3, LITAF
Nutrient Metabolism of an Aquatic Invertebrate and its Importance to Ecology
Aquatic consumers frequently face nutritional limitation, caused in part, by imbalances between the nutrients supplied by primary producers and the metabolic demands of the consumers. These nutritional imbalances alter many ecological processes including consumer life-history traits, population dynamics, and food web properties. Given the important ecological role of organismal nutrition, there is a need to have precise and specific indicators of nutritional stress in animals. Despite this need, current methods used to study nutrition are unable to distinguish between different types of nutritional limitation. Here I studied nutritional metabolism in the freshwater zooplankter, Daphnia. A greater understanding of nutritional metabolism would allow for the development of dietary bio-indicators that could improve the study of the nutritional ecology of animal consumers. Specifically, I addressed the question: What affects the biochemical composition of a generalist aquatic consumer? My overall hypothesis was that the quantity and quality of the diet affects the biochemical composition in a nutrient specific manner. To test this hypothesis, I examined various response variables involved in nutrient metabolism such as alkaline phosphatase activity, whole metabolome, and free amino acid composition. For each response variable, I grew Daphnia under various nutritional stressors and determined if responses are nutrient specific or are a general stress response. I found the current method of measuring alkaline phosphatase was not a phosphorus specific indicator, as activity increased in all nutrient stressed treatments. Analyzing the whole metabolome resulted in nutritional stressors being separated in multivariate space, with many identified metabolites being significantly different from nutrient rich Daphnia. Upon further examination the daphnids free amino acids profiles are caused by differences between the supply of amino acids from the algae and the demand within the Daphnia. These differences in supply and demand resulted in the ability to classify the nutritional status of Daphnia with the use of discriminant analysis, a classification multivariate model. In addition to a deeper understanding and advanced knowledge of the physiological changes caused by nutrient limitation, this research has provided strong evidence for the application of nutritional biomarkers/profiles to identified the nutritional status of Daphnia. Author Keywords: Bio-indictor, Ecological stoichiometry, Metabolism, Nutritional limitation, Nutritional status

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