Graduate Theses & Dissertations

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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
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
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
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
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
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
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:

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Graduate student theses produced at Trent University. You may also browse theses and dissertations by year, program, or author.

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