Graduate Theses & Dissertations

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Expression of Giardia intestinalis flavoenzyme GiOR-1 and characterization of its electron transfer properties
Giardia intestinalis possesses four isotypes of cytochrome b5 (gCYTB-I-IV) that differ from their mammalian counterparts, suggesting different functions in this protozoan parasite. Although the recently discovered Giardia flavoenzyme, GiOR-1, reduces these cytochromes, its properties have not been thoroughly studied, owing to the difficulty in its expression. Here I describe successful conditions for expression of GiOR-1 using autoinduction. GiOR-1 is obtained with flavins bound as indicated by its UV-visible spectrum. Its ability to catalyze electron transfer from donors (NADH, NADPH) to acceptors (oxygen, ferricyanide, cytochrome c, gCYTB5-III) were studied in spectrophotometric rate assays. NADPH is the preferred electron donor, while cytochromes are the preferred electron acceptors. Interestingly, the His-tag used to purify gCYTB5-III decreases its reaction rate with GiOR-1, as an untagged version has slightly faster rates. These findings establish the appropriate conditions for further studies on GiOR-1, including the identification of endogenous electron acceptors. Author Keywords: Autoinduction, Cytochrome b5, Cytochrome P450 oxidoreductase, Giardia intestinalis, GiOR-1, Polyhistidine tag
Immunotherapies Targeting the Amyotrophic Lateral Sclerosis-Associated Protein TDP-43
Transactive response (TAR) DNA-binding protein of 43 kDa (TDP-43) pathology, including fibrillar aggregates and mutations, develops in amyotrophic lateral sclerosis (ALS) and is characterized by hyperphosphorylation and aggregation patterns, a mechanism largely understudied. In addition, ALS remains without a cure. Herein, in vitro aggregation of phosphorylated TDP-43 was explored, and the anti-TDP-43 antibodies tested for their inhibitor efficacies. Additionally, in vitro phosphorylation of TDP-43 by protein kinases was conducted to identify which protein kinases catalyze phosphorylation. The aggregation of phosphorylated and unphosphorylated full-length TDP-43 protein (pS410) was monitored by transmission electron microscopy (TEM), turbidity absorbance, and thioflavin (ThT) fluorescence spectroscopy. The protein aggregates were largely insoluble, ThT-positive and characterized with heterogeneous morphologies. Antibodies specific to epitopes within the RNA-recognition motifs and the C-terminal domains reduced the formation of β-sheets and insoluble aggregates, with outcomes highly dependent on the type of antibodies, indicating dual functionality. The only protein kinase able to phosphorylate TDP-43 at S410 was MARK4, indicating its role in the onset of PTMs in the protein. Thus, targeting TDP-43 epitopes for inhibition of aggregation and in vitro phosphorylation represent viable biochemical assays for screening protein kinase inhibitors as potential drugs against ALS. Author Keywords: aggregation, ALS, antibody-based inhibition, phosphorylation, protein kinase, TDP-43
Electrochemical Biosensors for Neurodegenerative Disease Biomarkers
The onset of neurodegenerative diseases such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) are typically characterised by the aggregation of protein biomarkers into cytotoxic fibrils. Novel means of analysing these biomarkers are needed to expand the literature toward earlier diagnosis of these conditions. Electrochemical sensors could offer the sensitivity and selectivity needed for specialised analysis, including potential point-of-care applications. The AD biomarker Tau, and ALS biomarker TDP-43 proteins are explored here by using a label-free electrochemical sensors. Tau protein was covalently bound to gold electrode surface to study the in vitro mechanisms of aggregation for this protein. An immunosensor to TDP-43 was developed by covalently binding primary TDP-43 antibodies (Abs) on gold electrode surface. A novel direct ELISA sensor for TDP-43 with visual detection and electrochemical quantification was also developed. The results validated the experimental designs toward specialised and selective analysis of these biomarkers and their aggregation mechanisms. Author Keywords: ALS, Alzheimer's, Biosensors, Electrochemistry, Tau, TDP-43
Postclassic Maya Diet
Postclassic (AD 900-1500) Maya diet at Ka’kabish, Belize was examined using stable carbon and nitrogen isotope analysis of human bone collagen, and stable carbon isotope analysis of bone structural carbonate. Isotope data were compared to skeletal and dental indicators of diet and disease, and dietary differences among burials excavated from chultuns (B-2, C-1, C-2, and C-3) at Ka’kabish. Varying in dimensions, chultuns are characterized as multiple subterranean chambers carved into limestone bedrock, where re-entry was permitted through the removal of a capstone placed over a circular entrance. Due to poor preservation and commingling of human remains, diet and its relation to age, sex, and social status could not be explored. The general diet at Ka’kabish is consistent with the consumption of a diverse range of terrestrial plants and animals, in addition to marine resources. Relative to the other chultun burials, Chultun C-2 is an outlier, with a noticeably different diet, evidence for skeletal pathology, and absence of dental modifications. This study demonstrates a lack of significant dietary differences among Postclassic Maya sites in northern Belize, along with an apparent reliance on marine resources, further supporting the notion of a close association, and equal participation in a regional trading system with coastal sites that allowed for populations in this region to thrive during the Postclassic period. Author Keywords: Ancient Maya, Bioarchaeology, Ka'kabish, Osteology, Postclassic, Stable Isotopes
Chew the Fat
Fatty acid analysis was performed on archaeological bone from various fauna from sites in the Canadian arctic to better understand the preservation of fatty acids and their potential applications to palaeoecological and palaeodietary studies. These data were complemented by analyses of modern bone and soft tissue samples from livestock and harp seals (Pagophilus groenlandicus). Results of modern analyses revealed that in terrestrial species, bone has inherently lower concentrations of most fatty acids relative to other soft tissues (adipose, marrow, and muscle). These analyses suggest that the distribution of fatty acids in bone is unique compared to other tissues, and the types and abundances of fatty acids in bone may be linked to dietary sources of lipids. Of the archaeological samples analyzed, terrestrial species (caribou [Rangifer tarandus]) generally exhibited higher concentrations of saturated fatty acids compared to marine species (ringed seals [Pusa hispida] and polar bears [Ursus maritimus]), whereas marine species had higher concentrations of monounsaturated fatty acids compared to terrestrial species. Results of analyses on both modern and archaeological samples provided insight into the degradation of fatty acids in bone, and the rapid loss of polyunsaturated fatty acids in particular. Because the abundances of fatty acids are likely altered in the burial environment, it is recommended that future analyses incorporate compound specific isotope analysis to focus on applications of fatty acids that are typically in the highest abundance and arguably have undergone the least amount of change, including palmitic (C16:0) and stearic acid (C18:0) Author Keywords: Archaeological Science, Bone Lipids, Fatty Acid Analysis, GC-MS, Lipid Preservation, Palaeoecology
Differential expression of cytochrome b5s in Giardia intestinalis during nitrosative stress and encystation
The waterborne protozoan Giardia intestinalis cycles between the environmentally-resistant and infectious cyst and the metabolically-active trophozoite that adheres to the epithelial lining of the small intestine. Adhesion can trigger the innate immune response in epithelial cells, including the synthesis of the free radical nitric oxide (NO) that inhibits cell proliferation and encystation of trophozoites. In this work changes in protein expression of three Giardia isotypes of the redox heme protein cytochrome b5 (gCYTb5 I, II and III) were studied in response to either nitrosative stress or induction of encystation. Two nitrosative stressors, sodium nitrite and the NO donor DETA-NONOate, were used at sub-lethal concentrations (0.5 mM and 0.05 mM, respectively) that do not affect cell proliferation until later time points so that subtle changes in protein expression could be observed in the absence of other confounding factors. Nucleolar gCYTb5-I and nucleoplasmic gCYTb5-III expression patterns were similar in trophozoites exposed to either stressor, showing gradual increases in expression with peaks between 4 and 12 hours, which indicates these cytochromes respond to nitrosative stress and possibly to potential DNA damage in Giardia. In contrast, gCYTb5-II of the peripheral vacuoles, which are part of the endocytic pathway of Giardia, showed little change in expression in response to either stressor. However, changes in gCYTb5-II expression were observed in encysting trophozoites, with a 1.4-fold increase in protein levels at seven hours after induction of encystation, followed by a gradual decrease in expression. These changes are consistent with previous mRNA analysis done in our laboratory and suggest a role for gCYTb5-II in the increase in nutrient uptake during early encystation. Author Keywords: cytochrome, encystation, Giardia, heme, nitrosative, parasite
Biosynthesis and impact of cytokinins on growth of the oyster mushroom, Pleurotus ostreatus
The oyster mushroom, Pleurotus ostreatus, is one of the most widely cultivated edible basidiomycetes. It has gained increased attention for its economic, environmental, and medicinal properties. While a lot is known about cytokinins (CKs) and their actions at the molecular and cellular levels in plants, much less is known about the function of CKs in other kingdoms. Cytokinins, which have been detected in several fungal species, play a role in pathogenic attack against plants or during plant growth promotion by plant beneficial microbes; however, the role of CKs in fungal physiology, separate from plant associations remains largely unknown. This thesis focuses on the occurrence of fungal-derived CKs in P. ostreatus when grown in vitro as submerged or aerial mycelium. Cytokinin profiling by UHPLC-HRMS/MS revealed that P. ostreatus produces CKs and that the tRNA degradation pathway is the main source of these molecules. CK dynamics within fungal growth supported previous evidence, which suggested that tRNA degradation products have a role in the physiological development of fungi for which CKs act as fungal growth regulators. A second component of the thesis demonstrated that P. ostreatus responds to exogenous applications of aromatic and isoprenoid CKs and their effects were dependent on the dose and CK type. N6-Benzyladenine (BAP), Kinetin (KIN), N6-isopentenyladenine (iP), and trans-zeatin (tZ) bioassays revealed hormone-type responses (hormesis: biphasic response). At low doses, mycelium growth could be stimulated, whereas, at high doses only inhibitory effects were observed. This stimulation/inhibition was observed whether the measured response was an increase/decrease of aerial mycelium colony diameter, biomass accumulation or a change in mycelium morphology as compared to the controls. Results indicated there is potential to alter mycelium growth and development of P. ostreatus; thus, CKs may play the role of a “mycohormone” and may be specifically helpful for medicinal fungi by increasing growth and efficiency to produce many biologically active substances with valuable medical and environmental applications. Author Keywords: cytokinins, fungal-derived CKs, hormesis, mycelium, mycohormone, Pleurotus ostreatus
Ligand Binding Properties of Giardia Flavohemoglobin
The parasitic protist Giardia intestinalis possesses flavohemoglobin (gFlHb), an enzyme that detoxifies nitric oxide to the less harmful nitrate, and is a potential target for antigiardial drugs that act as ligands to the iron of its heme cofactor. In this work, the binding constants KD of gFlHb, three active-site variants (Q54L, L58A, Y30F) and the E. coli flavohemoglobin (Hmp) towards cyanide, azide and several substituted imidazoles were measured by optical titration. Certain cases such as gFlHb and Hmp were studied further by isothermal titration calorimetry. Binding constants for cyanide and the imidazoles ranged from 2 to 100 M, with the highest affinities observed with for miconazole, a bulky substituted imidazole. Azide was a poor ligand, with binding constants between 0.48 and 26 mM. Among gFlHb and its mutants, L58A tended to have the highest ligand affinities, as mutation of the distal leucine to a less bulky distal alanine residue facilitates the access of the exogenous ligand to the heme iron. In contrast, the Q54L and Y30F variants had binding affinities that in most cases were similar to wild type, which suggests that the inability of their side chains to form hydrogen bonds to these ligands is not a significant factor in binding of imidazole ligands to the enzyme. Comparative results for Hmp and gFlHb ligand binding affinities revealed slight differences which might be explained by the presence of different residues in their active sites apart from their conserved residues. Author Keywords: Flavohemoglobin, Giardia intestinalis, Imidazole binding, Ligand binding, Nitrosative stress
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
Hydrological and Flooding Effects on Stream Nutrient Levels
Stream solutes are strongly linked to hydrology, and as such, we sought to better understand how hydrology, particularly flooding, influences nitrogen (N) and phosphorus (P) levels. We used a long-term dataset of monthly water quality samples for many Ontario, Canada, catchments to assess the effects of landscape variables, such as land use and physiography, on the export of nutrients during floods, and to characterize overall concentration-discharge patterns. In general, we found that landscape variables could partially explain the export variation in flood waters, but that the importance of specific variables depended on flood characteristics. We also found that overall concentration-discharge relationships for N and P C were positive, but non-linear, with greater concentrations on the rising limb of the hydrograph depending on the nutrient. With these results, we have identified general patterns between nutrients and hydrology, which will be helpful for managing the ecological effects of flooding. Author Keywords: C-Q relationships, Discharge, Export, Flooding, Nutrients, Thresholds
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
NMR and EPR Studies on Cytochrome b5 Isotypes of Giardia intestinalis
The amitochondrial protozoan, Giardia intestinalis, encodes four members of the cytochrome b5 (CYTB5) family of heme proteins of unknown function. While homology models can predict the likely fold of these proteins, supporting experimental evidence is lacking. The small size of the cytochromes (~16 kDa) makes them attractive targets for structural analysis by Electron Paramagnetic Resonance spectroscopy (EPR) and Nuclear Magnetic Resonance spectroscopy (NMR). EPR measurements are particularly useful in defining the geometry of the coordination environment of the heme iron; such measurements indicated that the planar imidazole rings of the invariant histidine axial ligands are nearly perpendicular to each other, rather than in the coplanar orientation observed within mammalian CYTB5s. This may be due to geometrical constraints imposed by a one-residue shorter spacing between the ligand pair in the Giardia cytochromes b5 (gCYTB5s). Following optimization of sample and instrument conditions for NMR experiments, a comparison of the 1D 1H-NMR spectra of gCYTB5 isotype I to those of three of its heme-pocket mutants (Tyr51→Phe, Tyr61→F, and Cys84→Ala) were used to tentatively assign the heme methyl and vinyl protons. Mutant Tyr61→F had the greatest effect on the wild-type spectrum due to maximum through-space contacts with the heme macrocycle and its proximity to the His63 axial ligand. These experiments are a prelude to further NMR experiments that can lead to solving the complete structures of these proteins. Author Keywords: cytochrome b5, heme b, mutant protein, paramagnetic iron, resonant spectroscopy, sequence homology

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