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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:
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:
Mutation of the B10 Tyrosine and E11 Leucine in Giardia intestinalis Flavohemoglobin
The flavohemoglobin in Giardia intestinalis (gFlHb) is the only known protozoan member of a protein class typically associated with detoxifying nitric oxide (by oxidation to nitrate) in bacteria and yeast. Mutants of the B10 tyrosine (Y30F) and E11 leucine (L58A), conserved residues thought to influence ligand binding, were expressed and studied using Resonance Raman (RR) spectroscopy. In the wild type protein, RR conducted using a carbon monoxide probe detects two distinct Fe-CO stretches associated with two different active site configurations. In the open configuration, CO does not interact with any polar side chains, while in the closed configuration, CO strongly interacts with one or more distal residues. Analysis of the Y30F mutant provided direct evidence of this tyrosine’s role in ligand stabilization, as it had only a single Fe-CO stretching mode. This stretching mode was higher in energy than the open conformer of the wild type, indicating a residual hydrogen bonding interaction, likely provided by the E7 glutamine (Q54). In contrast the L58A mutant had no effect on the configurational nature of the enzyme. This was unexpected, as the side chain of L58 sits atop the heme and is thought to regulate the access of distal residues to the heme-bound ligand. The similar spectroscopic properties of wild type and L58A suggest that any such regulation would involve rapid conformational dynamics within the heme pocket. Author Keywords: B10 Tyrosine, Catalytic Globin, E11 Leucine, Flavohemoglobin, gFlHb, Giardia intestinalis
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
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
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
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
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
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
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
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
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

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