Restricted Theses and Dissertations
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Browsing Restricted Theses and Dissertations by Department "Biological Sciences"
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Item Embargo Anaerobic Methanotrophy in an Oil Sands End Pit Lake(2024-08-23) Davidson, Hanna Elise; Dunfield, Peter; Siddique, Tariq; Hubert, Casey; Stein, LisaAlbertan oil sands, and even oil sands reclamation projects, emit large volumes of greenhouse gases. Methane is one of the most potent greenhouse gases, and aerobic methane oxidation has been a subject of interest for methane mitigation. However, anaerobic oxidation of methane (AOM) may be just as influential in reducing methane emissions, as concerns with excess methane emissions and sediment disturbance by ebullition remain. Oil sands end pit lakes, made to reclaim tailings as well as mined-out pits, may be prime environments for AOM. This project determined if AOM was occurring in the first demonstration-scale oil sands end pit lake and attempted to determine the microbial communities facilitating this process. The potential of adding electron acceptors to sediment to increase rates of AOM was also investigated. We determined that AOM was occurring in the oil sands end pit lake’s sediment, using 13CH4-spiked incubations. We estimate that overall, AOM rates fall between 0.07-0.51 nmol mL-1 d-1 in the natural sediment. Incubations showed that AOM was most active within the first 100 days of methane-rich incubation. Electron acceptor amendments did not affect rates universally, although ferrihydrite amendments did stimulate 13CO2 production rates of up to 0.15 nmol mL-1 d-1 and may extend the longevity of high AOM rates. Effects of electron acceptor addition appear to be very site- and depth- specific and this may indicate that there are microhabitats ideal for AOM. The microbial communities responsible for AOM remain elusive. Known anaerobic methane-oxidizing taxa were identified only in minute abundances and did not detectably increase in abundance, even after 150 days of incubation. Methane oxidation rates recorded in incubations were comparable to the methane oxidation rates known for both methanogenic trace methane oxidation and anaerobic methane oxidation. This work is an initial assessment of AOM in oil sands end pit lakes, providing a base for the next steps for understanding AOM. Studying AOM rates to better illustrate methane emissions will be instrumental in managing tailings ponds and end pit lakes. This work could also have a substantial impact on microbial biodiversity knowledge, particularly in the understanding of the communities impacting freshwater AOM.Item Open Access Benzylisoquinoline Alkaloid Biosynthesis in Sacred Lotus (Nelumbo nucifera)(2022-08-29) Menendez Perdomo, Ivette M.; Facchini, Peter J.; Ng, Kenneth K.S.; Yeaman, Samuel J.; Back, Thomas G.; Ober, Dietrich, O.Benzylisoquinoline alkaloids (BIA) constitute a family of plant specialized metabolites comprising numerous bioactive compounds. BIA metabolism has been extensively studied in the opium poppy and related species in the Ranunculales order, but limited research has been conducted in non-model plants. Sacred lotus (Nelumbo nucifera), in the order Proteales, is an ancient aquatic plant rich in proaporphine, aporphine, and bisbenzylisoquinoline alkaloids, with a prevalence of unusual R-enantiomers. However, BIA biosynthetic genes and cognate enzymes in this plant remain unknown.Considering the proposed monophyletic origin of BIA biosynthesis in Angiosperms and sacred lotus’ reported alkaloid profile, opium poppy’s well-established pathways could be envisaged as a blueprint to predict the corresponding biosynthetic genes in N. nucifera. This thesis describes the isolation and characterization of several genes involved in Nelumbo’s BIA metabolism, including those purportedly encoding for norcoclaurine synthase, O- and N-methyltransferases, proaporphine synthase, bisbenzylisoquinoline synthase, and aporphine methylenedioxy bridge synthase. Through a combination of in vitro assays with recombinant enzymes and plant total protein extracts and organ-specific correlational analysis between transcript levels and alkaloid content, we demonstrate that NnOMT1 acts as the main 6-O-methyltransferase (favoring the conversion of (R)-norcoclaurine), whereas NnOMT5 and NnOMT7 are non-stereospecific 7-O-methyltransferases acting on several 1-benzylisoquinolines. Additionally, in vivo assays performed in engineered yeast strains allowed the preliminary characterization of three novel P450 catalysts, NnCYP80Q1 acting as a proaporphine synthase (R-stereospecific intramolecular C-C phenol coupling), NnCYP80Q2 as a bisbenzylisoquinoline synthase (R-stereospecific intermolecular C-O phenol coupling), and NnCYP719A22, the aporphine methylenedioxy bridge synthase. Furthermore, we describe how the absence of 3'-hydroxylase activity radically changes the alkaloid profile in Proteales versus Ranunculales.In addition, via in vivo deuterium labeling experiments, this work provides the first empirical evidence to support that L-tyrosine is the primary precursor for lotus BIA. Based on a consistent absence of norcoclaurine synthase activity for the recombinant enzyme candidates and plant total protein extracts, we suggest that a non-enzymatic spontaneous Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde produces racemic norcoclaurine in lotus, in opposition to the enzyme-catalyzed (S)-norcoclaurine formation in the Ranunculales. These results support a possible convergent evolution of BIA biosynthesis in sacred lotus.Item Embargo Biochemical characterization of human dihydroxyacetone phosphate acyltransferase, the first step in ether lipid biosynthesis(2024-09-11) Li, Victoria; Zaremberg, Vanina; Edwards, Robert A.; Shutt, Timothy; Moorhead, Gregory B. G.Ether lipids are a subclass of glycerophospholipids where an alkyl tail is attached to the sn-1 position of the glycerol backbone. They are found enriched in the central nervous system. Defects in ether lipid synthesis lead to severe human conditions including Zellweger syndrome and rhizomelic chrondrodysplasia punctata. The latter manifests with congenital cataracts, skeletal abnormalities, facial deformities, and high childhood mortality, underlining the critical role of the ether lipid biosynthesis. The first acylation step in the synthesis of ether lipids occurs in peroxisomes and is catalyzed by the enzyme dihydroxyacetonephosphate acyltransferase (DHAPAT EC2.3.1.42). DHAPAT has a peroxisomal targeting signal type 1 (PTS1) at the C-terminus, lacks predicted membrane domains or lipid binding motifs. A functional assay in yeast was devised where full length DHAPAT was able to support life of a double knockout yeast devoid of endogenous acyltransferases. This was dependent on DHAPAT activity and the presence of the residues encoded by the first three exons. Despite comparable expression levels to the wild type, a catalytically dead mutant failed to support life of the knockout yeast. Furthermore, an alternatively spliced variant lacking exon 2 (V2) also resulted in the inability to support life of double knockout yeast. Unexpectedly, full length DHAPAT localizes to both peroxisomes and lipid droplets (LDs) in yeast, while V2 localizes exclusively to peroxisomes. Interestingly, putative amphipathic helices (AHs) were identified within the region encoded by exons 2 and 3 which may aid in association with LDs. A systematic series of truncations in the first 146 amino acids revealed that the minimum sequence required for LD association relies in the region encoded by exon 3. A colorimetric DHAPAT activity assay was developed and validated by detecting activity of the full-length enzyme but not of the catalytically dead H162A mutant. Consistent with its failure to support the life of the double knockout yeast, no activity was detected for V2, at least in its soluble form. Insights from in silico modelling and non-reducing SDS-PAGE analysis support a role of the region encoded by exons 1-3 in dimerization of the enzyme. Together, these results indicate that the amino end of DHAPAT modulates its targeting priority to peroxisomes when the PTS1 signal is present and regulates its activity.Item Embargo Characterization of genes for outer core lipopolysaccharide synthesis in salmonella typhimurium LT(1990) MacLachlan, Philip Ronald; Sanderson, Kenneth E.Item Embargo Effect of density, microhabitat and food on growth and survival of rainbow trout fry, oncorhynchus(1990) Beers, Chris E.; Culp, Joseph M.Item Embargo Farnesylation-mediated control of skotomorphogenesis and seedling establishment in Arabidopsis thaliana(2024-05-21) Hickerson, Neil Murray; Samuel, Marcus; Muench, Douglas; Chua, Gordon; Arcellana-Panlilio, Mayi; Belmonte, MarkSkotomorphogenesis is growth in the dark, a critical process in the establishment of seedlings for all crop systems in which the seed is sown below the soil surface. Regulation of skotomorphogenesis, seedling growth, plant development, and stress tolerance has been shown to involve the interplay and negative interactions between the signalling and synthesis of the stress hormone abscisic acid (ABA) and the growth hormone brassinosteroid (BR). Mutations or modifications resulting in reduced BR synthesis have been shown to enhance perception of ABA for improved stress responses and overall drought tolerance. A previous study demonstrated that the farnesyltransferase, Enhanced Response to ABA 1 (ERA1), is required for BR synthesis through farnesylation of the terminal enzyme of the BR biosynthesis pathway. However, new evidence suggests that the absence of ERA1 can directly impact the BR-dependent skotomorphogenic response as farnesylation may be important in the activity of downstream genes associated with BR-induced signalling. Screening BR-regulated gene expression for potential substrates for farnesylation has yielded three likely candidate proteins: a bHLH-type transcription factor of the Phytochrome-Interacting Factor (PIF) group, PIF3; and a pair of cell-wall modification enzymes of the Xyloglucan endotransglycosylase/hydrolase (XTH) family, XTH22 and XTH23. Complementation experiments revealed that PIF3 lacking farnesylation was unable to rescue the skotomorphogenic defects observed in the multigenic pifq mutant background. PIF3 farnesylation was further shown to be required for transcriptional activity of PIF3 in the transgenic seedling. This study has directly linked PIF3 farnesylation with transcriptional control during skotomorphogenesis. Additional experiments highlighted the complexity of farnesylation-mediated regulation among downstream effectors. Methods and tools developed in this study can be used to verify additional targets responsible for the impact of farnesylation on seedling growth.Item Open Access Identification and characterization of a J-domain containing protein as a novel co-modifier of Self-incompatibility response in Brassica napus(2021-08-20) Kumar, Abhinandan; Samuel, Marcus A; Moorhead, Gregory BG; Muench, Douglas GSelf-incompatibility (SI) is a mechanism that plants utilize to prevent inbreeding and promote outcrossing. In Brassicaceae, this is a genetic mechanism that is controlled by S-locus in which haplotype-specific rejection of self-pollen is achieved through recognition of the pollen ligand SCR/SP11 by the S-receptor kinase of the papillary cells. The receptor-ligand activation converges on an E3 ligase, ARC1, which targets degradation of compatibility factors that result in blocking of delivery of resources required for pollen germination, leading to SI response. In this thesis, I have used the CRISPR-Cas9 gene editing platform to confirm the role of ARC1 during SI signaling and have also identified JDP1 as an interactor and modifier of ARC1, required for full manifestation of SI response.The role of ARC1 in the self-incompatibility pathway in Brassicaceae has remained a point of contention within the scientific community for over 20 years. Through gene-editing to create loss-of-function of alleles of ARC1 in Brassica napus, I was able to show that loss of ARC1 resulted in complete breakdown of SI in two different haplotype combinations proving the necessity of ARC1 for successful manifestation of SI.I next explored whether there are other positive regulators of SI at or downstream of ARC1 through a cytosolic interaction assay that identified J-domain containing protein (JDP1) as a potential cytosolic interactor of ARC1. This HSP-40 family protein interacted with ARC1 only when Tyr8 was modified or absent suggesting likely post-translational in-vivo regulation of this Tyr8 of JDP1. Through transgenic approaches to suppress JDP1, I was able to demonstrate that JDP1 is a positive regulator of SI and overexpression of the JDPY8F form led to constitutive rejection response irrespective of upstream signals demonstrating that the ARC1 interacting JDPY8F was sufficient to confer SI response.Further biochemical characterization of JDP1 revealed that the X-domain of JDP1 which is necessary for binding ARC1, harbored E3 ligase activity. Both JDPY8F and JDP?J (X-domain) were able to enhance ARC1-dependent ubiquitination of previously identified ARC1 substrates GLO1 and EXO70A1 suggesting that interaction of ARC1 with Tyr8 modified JDP1 may be essential for full functionality of ARC1.Item Embargo Improving the therapeutic potential of staphylokinase, a potent thrombolytic agent(2023-05-26) Baharian, Azin; Vogel, Hans J.; MacDonald, Justin A.; MacCallum, JustinStaphylokinase (Sak) is a small bacterial-derived protein (15.5 kDa) that can hydrolyze fibrin-rich blood clots by activating the conversion of plasminogen into plasmin. Several studies have shown the effectiveness of Sak as a thrombolytic agent that could be used to treat stroke or cardiac arrest. However, its short half-life in blood circulation and immunogenicity are the main issues that prevent Sak from clinical applications. Herein, we explore two modifications of Sak to improve its therapeutic potential. Covalently attaching PEG (polyethylene glycol) polymers to therapeutic proteins has been widely applied to improve their pharmacokinetic properties, resulting in several FDA-approved protein-based drugs. Although PEGylation of Sak has previously been investigated, the effects of PEGylation on the 3D structure of Sak have not been studied in detail. In the first part of this research project, site-specific PEGylation of the truncated version of Sak was performed in the immunogenic region of the protein, and three covalent PEGylated derivatives of the protein were prepared. NMR studies showed a slight structural perturbation upon PEGylation, mostly close to the PEGylation site, and a direct relationship between the hydrodynamic radius of the PEGylated protein and the PEG size. Together, the results suggest that PEG and Sak∆10 move relatively independently from each other. As a second approach, we introduce cyclic Sak (cyc-Sak), a novel form of staphylokinase with higher stability and improved plasminogen activation activity. Using an Ssp GyrB split intein, the N- and C-terminal ends of the linear Sak were connected by a peptide bond, rendering the protein into a cyclic form (cyc-Sak). This structural modification was generated at the protein expression level in Escherichia coli, and the cyclic protein could be purified by common chromatography techniques. Successful backbone cyclization was confirmed by NMR spectroscopy of the 13C,15N-labeled cyc-Sak and by chemical cleavage assays. Our studies show that mono-PEGylation and intein-mediated backbone cyclization of Sak are powerful strategies to improve its therapeutic potential. These approaches could be combined to facilitate the design of protein-based medications when the stability of therapeutic proteins is an obstacle to their clinical application.Item Embargo Lend Me Your Ear: Testing Hypotheses of Tylopod (Artiodactyla) Relationships Using Otic Region Morphology(2024-09-03) Robson, Selina Viktor; Theodor, Jessica Madeleine; Scott, Craig S.; Cote, Susanne Meredith; Summers, Mindi; Bertram, John Edward Arthur; Uhen, Mark D.Tylopoda are an artiodactyl suborder that includes camelids and their putative extinct relatives. A vast number of extinct species have been referred to Tylopoda, but the composition of the suborder remains unclear—few characters unite all purported tylopods, and those that do are ancestral features. Furthermore, the position of the suborder within Artiodactyla has recently come into question because of conflicting results from morphological and molecular phylogenetic analyses. Given that morphological and molecular data are unable to resolve these taxonomic problems, new lines of evidence are needed. The otic region, which includes the petrosal and bony labyrinth, is thought to be relatively slow-evolving, likely to retain phylogenetic characters important for resolving deep evolutionary relationships; these data have historically been inaccessible without destructive sampling, but with the proliferation of μCT scanning, the otic region is becoming increasingly easy to study. In this thesis, I describe the otic region of several early camelids and purported tylopods, including the dichobunoid Bunomeryx, two protoceratids, and two oromerycids. I used these data in conjunction with a larger unpublished dataset to code a taxon-character matrix primarily composed of otic region characters, which then analyzed with both parsimony and Bayesian analysis. All my analyses recovered a monophyletic Camelidae with Eotylopus, an oromerycid, the sister to Camelidae; I consider Tylopoda to include Camelidae and Oromerycidae. No other clade was consistently recovered with camelids, although oreodonts, anthracotheriids, and protoceratids all held that position in the results of some analyses. The placement of anthracotheriids was unexpected, but this appears to be the result of homoplasy; with additional dental, cranial, and postcranial characters, anthracotheriids are recovered sister to suoids. Based on my morphological descriptions and phylogenetic analyses, the otic region—particularly the petrosal—is not evolutionarily static. While the relative amount of homoplasy in the otic region compared to other skeletal regions was not tested, a large amount of intraspecific variation was observed in camelids, and all recovered (parsimony) topologies had a homoplasy index of over 0.70. This suggests that the petrosal is not as “slow evolving” as previously thought, an avenue of research that bears further investigation.Item Embargo Let There be Morphine: Structural Insights into Functional and Evolutionary Relationships of Morphine Biosynthesis in Opium Poppy(2023-11-30) Carr, Samuel Clyde; Facchini, Peter; Ng, Kenneth; Ro, Dae-Kyun; Moorhead, Gregory; Noskov, SergeiBenzylisoquinoline alkaloids are a large and diverse class of plant specialized metabolites known for their pharmaceutical properties, including the tumor suppressant noscapine, vasodilator papaverine, antimicrobial sanguinarine, and the important analgesics codeine, morphine, and their semisynthetic derivatives. The latter group of analgesics are known as morphinan alkaloids or opiates and are produced solely in select members of the genus Papaver, most importantly Papaver somniferum commonly known as opium poppy. This thesis highlights the use of structural biology in multidisciplinary approaches to understand the biosynthesis of morphinan alkaloids in opium poppy and related species. Dehydroreticuline reductase and codeinone reductase are closely related enzymes from the aldo-keto reductase superfamily catalyzing the second and second-last steps, respectively, in morphine biosynthesis in opium poppy. The elucidation of the crystal structure of codeinone reductase reveals novel structural features allowing for the in-depth analysis of substrate binding and catalysis leading to the engineering of substrate specificity. This structure also provides the means for the homology modeling of dehydroreticuline reductase giving insight into its novel catalytic mechanism. Transcriptomic analysis of representative Papaver species reveals putative morphinan biosynthetic enzyme orthologues and the ubiquitous distribution of dehydroreticuline reductase and codeinone reductase, among other morphinan biosynthetic enzymes. Structural analysis and functional/kinetic characterization of aldo-keto reductases demonstrates the ubiquitous conservation of dehydroreticuline reductase and codeinone reductase activity in the genus Papaver. These results challenge the current evolutionary narrative of morphinan biosynthesis suggesting: [1] a more ancient neofunctionalization of early enzymatic steps of morphinan biosynthesis than previously believed and [2] that morphinan biosynthesis evolved via the patchwork evolutionary model. Enzymatic latency and ligand binding affinity both provide a platform for the neofunctionalization of novel enzymatic activities. The alkaloid binding capabilities of pathogenesis related 10 proteins provides a means to understand the neofunctionalization of the morphine biosynthetic enzymes thebaine synthase and neopinone isomerase, alongside a biological role in alkaloid storage for abundant non-catalytic pathogenesis related 10 proteins. Their binding to alkaloids is predicted to promote the formation of protein-alkaloid aggregates based on binding-induced conformational changes observed through X-ray crystallography and dramatic changes in sucrose gradient fractionation.Item Embargo Methanotrophs in an Oil Sands End-Pit Lake and their Potential Co-oxidation of Naphthenic Acids(2023-06) Nwosu, Felix Chinweije; Dunfield, Peter; Gieg, Lisa; Chua, Gordon; Turner, RayMethanotrophs possess the highly promiscuous enzyme, methane monooxygenase (MMO) which has been characterized to co-oxidize many substrates in conjunction with methane. As such, methanotrophs are hereby explored in the remediation of environmental pollutants such as naphthenic acids (NAs) in Base Mine Lake (BML), the first oil sands end-pit lake. BML is known to harbor an active methanotrophic population and can be explored for the co-oxidation or co-metabolism of compounds of concern in BML like NAs. We report on the dynamics of the methanotrophic population in the BML water column and sediment interface over time was explored using both 16S rRNA gene amplicon sequencing and pmoA gene (encoding particulate MMO) abundance by qPCR quantification. The results showed that methanotrophs can be up to 2.7 × 104 cells mL-1 of water by qPCR and are delineated by season, with Methylobacter dominating the water columns in winter and Methylococcus being the most abundant in the summer seasons based on 16S rRNA gene amplicon sequencing. Conversely, Methylococcus was most abundant in winter BML ice cover compared to Methylobacter spp. in this preliminary analysis of the methanotroph community associated with the ice cover in winter. In a mixed culture of methanotrophs enriched from BML water, we demonstrated that a surrogate NA, cyclohexane carboxylic acid (CHCA), could be biodegraded in a methane-dependent manner to non-detectable levels. Similarly, a tailings ponds methanotroph isolate, Methylicorpusculum oleharenae, was also able to degrade CHCA, strongly suggesting that MMO is implicated in the hydroxylation of NA. Overall, these findings may suggest that methanotrophs can play a role in the bioremediation and reclamation of organic compounds found in BML.Item Embargo Molecular genetic studies of the inner core region of the lipopolysaccharide of salmonella typhimu(1990) Sirisena, Dassanayake M.; Sanderson, Kenneth E.Item Embargo Multi-faceted Approach for Engineering Novel Herbicide Resistance in Canola(2023-07-11) Scott, Robert Joseph; Samuel, Marcus; Yeung, Edward Chee-Tak; Muench, DouglasCanola, Brassica napus, is a plant species of vital importance to both the Canadian economy and cultural history. For canola to remain the powerhouse commodity crop that it is, effective herbicide resistance technologies will need to be advanced in order to combat the growing threat of herbicide resistant weeds. In order to control weed populations with effective herbicides without causing harm to the planted crop, growers often utilize herbicide resistant cultivars. While resistant cultivars of canola already exist that are tolerant to highly effective herbicides such as Roundup and Liberty, overuse of few herbicides will only lead to greater likelihood of resistant weed populations developing. Cellulose Biosynthesis Inhibitors (CBIs) are known pre-emergent herbicides with no known field resistance but have currently had very limited application in commodity farming. These herbicides directly interfere with a plant’s ability to produce cellulose, necessary for their cell walls as a vital structural component. Current literature agrees that CBIs likely interfere directly with CELLULOSE SYNTHASE (CESA), an enzyme known to play a vital role in cellulose biosynthesis. If canola cultivars were to be developed with resistance, either GMO or non-GMO, to CBI herbicides such as isoxaben or flupoxam, it could represent a major economic boon for canola growers. In the present work, I utilize Ethyl methanesulfonate (EMS) mutagenized B. napus seed to screen for novel alleles conferring isoxaben resistance. By Whole Genome Sequencing (WGS) candidate resistant seedlings, I was able to find three potentially causal mutations in BnCESA3 that may independently confer isoxaben resistance. Transgenic lines of B. napus were also developed containing a synthetic version of BnCESA1 containing three previously characterized flupoxam-resistant alleles on a single transgene. Pot screening of these transgenic lines revealed moderate level of tolerance to flupoxam. Lastly, CRISPR/Cas9-deaminase lines of B. napus were also developed to base edit BnCESA1 and BnCESA3 to generate novel alleles that could confer tolerance to CBI herbicides.Item Embargo Nitrogen metabolism during somatic embryogenesis in Picea glauca and Daucus carota : a NMR study(1991) Joy, Richard W.; Thorpe, Trevor A.Item Metadata only Plant preference of the alfalfa leafcutter bee, megachile rotundata (fabricius) (hymenoptera : meg(1990) Horne, Marjorie L.; Owen, Robin E.Item Embargo Regulation of Fatty Acid Biosynthesis by Protein Phosphorylation of the α-CT Subunit of ACCase in Arabidopsis thaliana(2024-04-30) Wong, Lana; Moorhead, Gregory; Zaremberg, Vanina; Burkinshaw, BrianneReversible protein phosphorylation, the most common post-translational modification, is essential in mediating most cellular functions in living organisms. With recent advances in omics-based technologies and its applications to studying the phosphoproteome of particular organisms, many previously uncharacterized phosphoproteins, protein kinases and protein phosphatases have been identified. Shewanella-like protein phophatase 1 (SLP1), a novel Arabidopsis thaliana protein phosphatase localized to the chloroplast, is a protein of interest as it is predicted to play an antagonistic role to the constitutively active chloroplast localized kinase, casein kinase 2α4 (CK2α4). Through a quantitative mass spectrometry based phosphoproteomics study carried out by previous members of the Moorhead Lab, many putative substrates of AtSLP1 were identified, one of them being the alpha-carboxyltransferase (α-CT) subunit of heteromeric acetyl-CoA carboxylase (htACCase). HtACCase catalyzes the first committed step of de novo fatty acid biosynthesis, and like AtSLP1, is chloroplast localized. HtACCase has been known to interact with the chloroplast envelope membrane through association with an integral membrane protein, however, the identity of this protein has been a mystery until recently. Carboxyltransferase interactors (CTIs), a group of small plastidal proteins of the inner chloroplast envelope have been identified to interact with the α-CT subunit of htACCase in a light-dependent manner. The focus of the research presented here was to characterize α-CT as a substrate of both CK2α4 and SLP1 and to determine the role of protein phosphorylation in lipid metabolism. Here, initial steps were taken in employing phosphospecific antibodies against the S741 site on α-CT, with immunoblot analysis confirming previous findings from a phosphoproteomics study indicating hyperphosphorylation of α-CT in the absence of SLP1. To explore the relationship between protein phosphorylation and the membrane partitioning of ACCase with CTI, recombinant proteins were cloned for subsequent protein-protein interaction studies. By studying the effects of this post-translational modification, we will gain a better understanding of the role of protein phosphorylation in regulating fatty acid biosynthesis, which will ultimately uncover new ways to increase bio-oil production in crops.Item Embargo RNA-binding proteins implicated in the anaerobic response of Arabidopsis thaliana and rice(2024-08-06) De la Torre Espinoza, Daniela Naomi; Muench, Douglas G.; Chua, Gordon; Samuel, Marcus; Singer, Stacy DawnFloods and waterlogging significantly impact economically important crops by inducing anaerobic stress, leading to severe agricultural losses. When soils become waterlogged, oxygen levels drop, creating anaerobic conditions that impair root respiration and metabolic functions. Crops can suffer from reduced growth, nutrient uptake inefficiencies, and increased susceptibility to diseases when exposed to anaerobic stress conditions, resulting in decreased yields, and, in severe cases, complete crop failure. The economic ramifications that result from flooding include reduced farm income, higher food prices, and increased costs for recovery and mitigation efforts, thus posing a substantial threat to food security and agricultural sustainability. To further analyze post-transcriptional gene expression under anaerobic stress, the RNA interactome capture (RIC) technique was applied to Arabidopsis roots and rice coleoptiles exposed to anaerobic stress. This advanced method involves UV-crosslinking to stabilize RNA-protein interactions, followed by oligo(dT) capture under denaturing conditions and subsequent protein identification via mass spectrometry. Optimizing the UV crosslinking dosage ensures effective capture of RNA-protein complexes without damaging the tissues. The application of RIC revealed distinct RNA-binding protein (RBP) compositions in both Arabidopsis roots and rice coleoptiles under normal and anaerobic conditions. These findings highlighted significant changes in RNA-protein interactions due to oxygen availability, offering insights into the molecular mechanisms that plants use to adapt to anaerobic stress. This research enhances our understanding of plant resilience and provides potential targets for improving crop tolerance to flooding and waterlogging.Item Embargo Sex ratios, sexual dimorphism and population dynamics in the dioecious shrub shepherdia canadensis(1990) Lewis, Glennis M.; Chinnappa, C. C.Item Embargo Spectrometric analysis of the metabolism and adsorption of naphthenic acid fraction compounds in a phytoremediation treatment system(2024-08-14) Charriere, Camryn; Muench, Douglas G.; O'Sullivan, Gwen; Gieg, Lisa M.Bitumen mining in northern Alberta produces large volumes of oil sands process-affected water (OSPW) that require treatment before being released into the environment. Naphthenic acid fraction compounds (NAFCs) are considered primary contributors to OSPW toxicity; therefore, remediation efforts often target these compounds. Phytoremediation is proposed as a feasible treatment for OSPW using a constructed wetland treatment system (CWTS) strategy due to the cost-effective and low-maintenance nature of this technology. A CWTS uses plants and their associated microorganisms to take advantage of natural metabolic processes for the uptake and biotransformation of environmental contaminants. While CWTSs for OSPW treatment have demonstrated success in attenuating NAFCs and reducing toxicity in mesocosm and pilot-scale studies, the fate of NAFCs in a CWTS is not well understood. This thesis research aimed to gain insight into NAFC fate in CWTSs by exploring NAFC biotransformation processes in plant tissues and the adsorption characteristics of NAFCs onto sediment substrates. A method for extracting NAFCs and their metabolites from plant tissues was developed and used to extract the isotopically labelled model compound 1-adamantanecarboxylic acid (¹³C-AdCA) and NAFCs from OSPW. This method, in combination with high-resolution Orbitrap mass spectrometry, was used to track the uptake, translocation, and biotransformation within plant roots and shoots. These experiments showed a general decrease in compound abundance over time, indicative of transformation events. The OSPW treatments further demonstrated that the majority of NAFCs were completely transformed in root and shoot tissues. Additionally, incubation studies were conducted to test parameters associated with NAFC adsorption onto various sediments. The adsorption of NAFCs from OSPW onto a sediment substrate from the oil sands region appeared to be impacted by the properties of the OSPW itself, including NAFC concentration, class differences, or shifts in carbon number and double bond equivalents. Other factors, such as water quality characteristics, may also impact adsorption. Overall, the results of this research provide insight into the fate of NAFCs in phytoremediation systems, guiding future metabolomics studies and considerations for the large-scale implementation of plant-based technologies in OSPW treatment strategies.Item Embargo The Role of Corticotropin-Releasing Hormone Receptor 1 in the Heat Shock-Mediated Behavioural and Metabolic Response in Zebrafish(2024-08-16) Shvartsburd, Zachary Skyler; Vijayan, Mathilakath; Lucas, Kelsey; Kurrasch, Deborah; Lukowiak, KennethActivation of the primary stress response, the hypothalamic-pituitary-adrenal (HPA)-axis in mammals or the hypothalamic-pituitary-interrenal (HPI)-axis in fish, requires the binding of corticotropin-releasing hormone (CRH) to CRH receptor 1 (CRHR1), though CRHR1 activity is also implicated in behavioural adjustment and peripheral signalling not associated with the HPI-axis. With global water temperatures rising and daily temperature fluctuations becoming more extreme, the stress response may be a critical mechanism of aquatic species to withstand shifts in temperature. By using a ubiquitous CRHR1 knockout (CRHR1KO) model, I investigated the hypothesis that CRHR1 is involved in the behavioural and metabolic modulation of the developmental and adult life stage stress response during acute heat shock in zebrafish. Following a 1-hour +5°C heat shock, wildtype (WT) fish larvae showed an increase in swimming activity but CRHR1KO larvae failed to respond to the heat shock. Basal oxygen consumption between WT and CRHR1KO larvae were similar, but CRHR1KO had elevated oxygen consumption in non-mitochondrial and ATP-linked processes. Cardiac performance was unchanged between WT and CRHR1KO larvae, indicating differences in behaviour were not due to modulation of heart function. Adult WT zebrafish showed the same increase in swimming activity when exposed to acute heat shock, while CRHR1KO fish had no change in swimming behaviour. The metabolic rate (MO2) of WT adult fish also increased with heat shock, but this was not associated with increased circulating cortisol, as exposure to metyrapone, an inhibitor of cortisol synthesis, failed to attenuate the heat shock-induced rise in MO2. The MO2 of CRHR1KO fish did not increase following heat shock, suggesting CRHR1 action independent of the HPI-axis. Swimming performance was impaired in the CRHR1KO, with a lower aerobic scope and Ucrit compared to WT. Transcript and metabolite analysis in the muscle revealed potential futile cycling of glycogen in CRHR1KO fish. In summary, my study shows CRHR1 as a key integrator of the behavioural and metabolic rate response which improves performance following an acute heat shock, implicating CRHR1 as a metabolic thermal sensor for ectotherms.