Graduate Theses & Dissertations

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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
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
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
Electrochemical Characterization of Giardia Intestinalis Cytochromes b5
Giardia intestinalis is a protozoan parasite that causes waterborne diarrheal disease in animals and humans. It is an unusual eukaryote as it lacks the capacity for heme biosynthesis; nonetheless it encodes heme proteins, including three cytochrome b5 isotypes (gCYTB5s) of similar size. Homology modelling of their structures predicts increased heme pocket polarity compared to mammalian isotypes, which would favour the oxidized state and lower their reduction potentials (E°’). This was confirmed by spectroelectrochemical experiments, which measured E°’ of -171 mV, -140 mV and -157 mV for gCYTB5-I, II, III respectively, compared to +7 mV for bovine microsomal cytochrome b5. To explore the influence of heme pocket polarity in more detail, five gCYTB5-I mutants in which polar residues were replaced by nonpolar residues at one of three positions were investigated. While these substitutions all increased the reduction potential, replacement of a conserved tyrosine residue at position-61 with phenylalanine had the most significant effect, raising E°’ by 106 mV. This tyrosine residue occurs in all gCYTB5s and is likely the greatest contributor to their low reduction potentials. Finally, complementary substitutions were made into a bovine microsomal cytochrome b5 triple mutant to lower its reduction potential. These not only lowered the E°’ by more than 140 mV but also weakened the interaction of heme with the protein. The lower reduction potentials of the gCYTB5s may indicate that these proteins have different roles from their more well-known mammalian counterparts. Author Keywords:
Expression and characterization of cytochrome b5 from Giardia lamblia
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
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
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
Hormonal Algae
Based on an endogenous hormone study, three cytokinin type phytohormones; benzyladenine (BA), trans-zeatin (tZ) and methylthiol trans-zeatin (MeSZ), as well as abscisic acid (ABA) were exogenously added at three concentrations (10-7, 10-6 and 10-5 M) to cultures of Chlorella vulgaris in an attempt to alter growth rate, total lipid and fatty acid yields and fatty acid profile. Growth stimulation was highest at 10-6 M for BA, MeSZ and ABA and 10-5 M for tZ. All treatments caused changes in total lipid and fatty acid content, with BA causing an increase to lipid content. The most significant change in the fatty acid profile was observed with the addition of MeSZ at 10-7 and 10-6 M causing increases of 204% and 457% in linolenic acid respectively above the control. These results are novel and potentially highly impactful, as MeSZ has never been added exogenously to algae and may be used to stimulate overproduction of linolenic acid for pharmaceutical or industrial purposes. Author Keywords: Abscisic Acid, Chlorella vulgaris, Cytokinin, Fatty acid, Linolenic Acid, Methylthiol trans-Zeatin
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
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
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
Mercury and Persistent Organic Pollutants in Remote Acid Sensitive Irish Lake Catchments
A catchment-based study was carried out at three remote acid sensitive Irish lakes to determine concentrations of Hg and POPs and to investigate the factors governing the partitioning of these pollutants in various environmental matrices. Both Hg and POPs are an environmental concern due to their ability to travel long distances via atmospheric transport and their tendency to accumulate in biota and in various environmental compartments. Concentrations of POPs and Hg measured in this study were relatively low and consistent with concentrations measured at background levels around the world. Mercury concentrations appeared to be influenced by various site characteristics, specifically organic matter. Many of the POPs examined in this study appeared to be present as a result of long-range transport and more specifically; the physico-chemical properties of POPs appeared to dictate their distribution within soils, moss and sediment at each of the study catchments. Author Keywords:

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