Graduate Theses & Dissertations

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SARS-CoV-2 Protein-based Detection Using Localized Surface Plasmon Resonance
During the COVID-19 pandemic, nucleic acid and antibody-based testing methods were heavily relied upon, but can be costly, time-consuming and exhibit high false -negative and -positive rates. Thus, alternative strategies are needed. Viral antigens such as the SARS-CoV-2 spike (S) glycoprotein are critical in the function of the virus and useful as diagnostic biomarkers for viral infections. For biosensing applications, aptamers are suitable high-affinity and cost-effective binding partners for their specific targets. Using localized surface plasmon resonance (LSPR), real-time, rapid acquisition of results can be achieved, essential for improving the efficacy of a sensor. Herein, LSPR aptamer sensors were fabricated for the detection of the SARS-CoV-2 protein. Data indicate that the best performing aptasensor was the streptavidin-biotin sensor, while the current gold aptasensor exhibited lower sensitivity and the fabrication of the carboxyl aptasensor was unsuccessful. The S1 aptamer selectively bound the S1 protein with high binding affinity. Excellent shelf-life stability, reusability, and high recovery in complex matrices was also maintained. Additionally, a receptor binding domain (RBD) functionalized sensor was fabricated to examine the interactions with angiotensin converting enzyme 2 (ACE2), for future assessment of inhibitors used in drug therapies. Overall, LSPR has been demonstrated as a viable tool for measuring SARS-CoV-2 related aptamer-protein and protein-protein interactions, and this strategy may be applied to other viral or non-viral antigen targets. Author Keywords: Antigen-based Detection, Coronavirus, COVID-19, Inhibition, Localized Surface Plasmon Resonance, SARS-CoV-2
Using Fluorescent Carbon Dots for Biosensing Applications of Amino Acids
Amino acids make up proteins, which are the building blocks of life. A balance of amino acids is needed to maintain a healthy state. Tyrosine (Tyr) is synthesized from the metabolism of phenylalanine, which is an essential amino acid, meaning it can only be obtained from the diet. It is related to many metabolic and neurodegenerative diseases. Tyr can undergo post-translational modifications such as phosphorylation and nitration, which are implicated in cancer and nitrative stress, respectively. Although there are many methods to detect Tyr and its analogues, phosphotyrosine (pTyr) and nitrotyrosine (nTyr), these methods are time-consuming, involve expensive instruments and involve tedious process. This research proposes a new type of nanomaterials, carbon dots (CDs), to detect these amnio acids. Data indicate that CDs can be used to detect nTyr with a limit of detection of 34 μM in the linear range of 20 - 105 μM. The amenability of CD-nTyr assay was also tested in various biological matrices and biological molecules and was shown to be sensitive to nTyr. Nitration of Tyr was carried out in the presence of sodium nitrite and hydrogen peroxide catalyzed by either Cu(II) or Fe(III) to mimic biological reactions and CDs were tested as both inhibitors and indicators of Tyr nitration. Although CDs did not inhibit the nitration reaction of Tyr, they did not serve as indicators of Tyr nitration due to the quenching of CDs by the nitrating agents. This shows the importance of using CDs to detect nTyr and further use it for biological applications to detect diseased states. Author Keywords: amino acids, carbon dots, nanomaterials, sensor, spectroscopy, tyrosine
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
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
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
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

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