Restricted Theses and Dissertations
Permanent URI for this collection
This collection is the result of a joint project between the Faculty of Graduate Studies and Libraries and Cultural Resources which provides Graduate students with the opportunity to archive their thesis with University Archives in our digital repository.
If you are a Graduate student submitting your final thesis to PRISM, please ensure you have read and submitted all required documents: http://grad.ucalgary.ca/current/thesis
If you require technical assistance please contact thesis@ucalgary.ca
The electronic theses and dissertations on this site are for the personal use of students, scholars and the public. Any commercial use, publication or lending of them in libraries is strictly prohibited.
Browse
Browsing Restricted Theses and Dissertations by Department "Biological Sciences"
Now showing 1 - 20 of 20
Results Per Page
Sort Options
- ItemOpen AccessBenzylisoquinoline 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.
- ItemEmbargoCharacterization of genes for outer core lipopolysaccharide synthesis in salmonella typhimurium LT(1990) MacLachlan, Philip Ronald; Sanderson, Kenneth E.
- ItemOpen AccessCharacterizing Hydrological Balance and Hydrochemistry of a Chronosequence of Newly Formed Wetlands within the Athabasca Oil Sands Region using Isotopic and Geochemical Tracers(2023-09-06) Wendlandt, Michael Edwin; Dr. Ciborowski, Jan; Dr. Birks, Jean; Dr. Wrona, Fred; Piercey, JessicaWetlands account for approximately 65% of the land area of the oil sands surface mining region, many of which have been lost due to resource extraction. Mined areas are reclaimed using saline-sodic materials present in both surficial geology and residual tailings, affecting wetlands constructed or naturally (“opportunistically”) forming within reclaimed landscapes. Newly-forming wetlands’ hydrological properties and ecological trajectories are poorly known. As part of the Boreal Wetland Reclamation Assessment Program, a chronosequence study design was used to infer the trends in water balance and geochemistry of 80 wetlands aged 2 to 40 years and varying in hydroperiod, salinity, and degree of riparian disturbance. Half of the wetlands were in reclamation landscapes of oil sands lease areas. The other half, located outside of the mining lease, which were considered ‘reference’ comparators. The region’s low-relief and wetland-dominated landscapes rendered traditional hydrological methods difficult to implement, so stable isotopes and geochemical tracers were used to infer the water balances. Isotope hydrological balances measured as Evaporation: Inputs ratio (E/I) varied significantly among wetlands on the two types of landscapes. Ratios for wetlands found on-lease were significantly higher (0.08-1.23) than those found off-lease (0.01- 0.46). Radon concentration (a proxy for groundwater inputs) in surface water of wetlands showed no significant difference between disturbance type and age. Analysis of hydrochemical facies revealed that NaCl and Mixed-water types dominated on-lease wetland waters, while in off-lease wetlands, Ca/MgHCO3 water type was dominant. Total dissolved solids concentrations were significantly higher in on-lease wetlands (2239 ± 1437 mg/L) than off-lease wetlands (766 ± 484 mg/L). ANCOVA analysis indicated that the largest effects on both TDS and Na:Ca ratios were landscape type and E/I(18O), while age played a less significant role. In contrast, Cl:HCO3 ratios were only influenced significantly by wetland age. Thus, surficial geology and evaporation effects seem to drive the large differences between wetlands found on and off lease; wetland age may play a role in decreased salinity found on and off the mining landscape. These findings may provide a useful comparative metric for reclamation success in landscapes possessing similar hydrological and hydrochemical facies.
- ItemOpen AccessComputer-aided drug design of activators of the cardiac Kv7.1 potassium channel(2023-08) Castro Gonzalez, Laura Maria; Tieleman, Peter; Zaremberg, Vanina; Derksen, Darren Jason; Fraser, Marie ElizabethLong QT syndrome is a heart disease that ends the lives of numerous children and young adults every year. This condition is triggered by the malfunction of some ion channels, including the voltage-gated potassium Kv7.1. In this thesis, multiple computational tools were employed to identify compounds that potentiate the activity of this ion channel. First, a Kv7.1 computational model was created from the Xenopus laevis structure. Then, suitable binding sites in the channel were identified using libraries of known activators of the channel: polyunsaturated fatty acids and endocannabinoids. Selected sites were in silico screened against a large library of small molecules. Multiple criteria such as binding energies, bioavailability and selectivity were employed to finally select compounds for experimental evaluation. This work identified two compounds with high potency and specificity for each binding site studied in the Kv7.1 channel.
- ItemEmbargoEffect of density, microhabitat and food on growth and survival of rainbow trout fry, oncorhynchus(1990) Beers, Chris E.; Culp, Joseph M.
- ItemOpen AccessEffects of Landscape Age and Salinity on Plant Community Composition and Productivity in Opportunistic and Constructed Wetlands in the Athabasca Oil Sands Region, Alberta(2023-08) Mombourquette, Ashlee Dawn; Ciborowski, Jan; Chasmer, Laura; Hornung, Jon; Vamosi, Jana; Galpern, PaulWetlands comprise 65% of the Athabasca Oil Sands Region (AOSR) surface mineable area and thus support diverse flora (approximately 400 species in Alberta). Due to increased anthropogenic land disturbance activities such as bitumen extraction, reclamation of surface mineable areas will also increase. The resulting reclaimed areas will tend to be sodium-enriched compared to pre-disturbance landscapes. In this thesis, forty young (<40 years old) stratified-randomly selected wetlands were sampled on reclaimed landscapes at Syncrude’s Mildred Lake lease and from reference wetlands in adjacent areas in the AOSR to determine how salinity and age influence the vegetation community composition, and the biomass of six dominant wetland plant species (Carex aquatilis, Calamagrostis canadensis, Carex atherodes, Carex utriculata, Schoenoplectus tabernaemontani, and Typha latifolia) found on reclaimed and reference landscapes. Wetland vegetation communities on reclaimed landscapes differed from those on reference landscapes; however, landscape type had no impact on the biomass of the dominant plant species. Vegetation communities varied along a salinity gradient; species richness was negatively associated with salinity, and vegetative species abundance (percent cover) was lowest in freshwater wetlands and highest in moderately brackish wetlands. Species richness did not differ among wetland classes. However, vegetative species abundance was significantly different among age classes. The biomass produced by each dominant species did not vary with respect to a gradient of salinity or among wetlands of different age classes. These findings may provide a useful frame of reference against which to compare vegetation communities that may be observed in wetlands forming on reclaimed landscapes.
- ItemEmbargoFarnesylation-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.
- ItemOpen AccessIdentification 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.
- ItemEmbargoImproving 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.
- ItemEmbargoLet 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.
- ItemEmbargoMethanotrophs 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.
- ItemEmbargoMolecular genetic studies of the inner core region of the lipopolysaccharide of salmonella typhimu(1990) Sirisena, Dassanayake M.; Sanderson, Kenneth E.
- ItemEmbargoMulti-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.
- ItemOpen AccessMunicipal Wastewater Effluent and Stormwater impacts the acute stress-induced Metabolic Rate and Plasma Metabolome in Rainbow Trout(2023-09-22) Young, Graham Patrick Murray; Vijayan, Matt (Mathilakath); Munkittrick, Kelly Roland; Syme, Douglas A.Municipal wastewater effluent (MWWE) and urban runoff (stormwater) are complex contaminant mixtures that have the potential to impact aquatic biota including fish. Stormwater consists of compounds such as herbicides, pesticides, nutrients, metals, hydrocarbons, and emerging contaminants that wash off surfaces within urban catchments and into aquatic environments, while MWWE often includes nutrients and pharmaceuticals that can be resistant to current treatment methods. Previous studies have identified obesogenic and reproductive effects on wild fish populations within the Bow River, however the influence of these effects on stress performance, swimming ability, and energy metabolism have received less attention. The objective of this thesis was to use a metabolomic approach to investigate whether metabolic costs associated with MWWE and stormwater exposure altered stress response, swimming ability, and metabolic rate of an economically important sportfish, rainbow trout (Oncorhynchus mykiss), in the Bow River as it flows through Calgary, Alberta. We hypothesized that metabolic costs for detoxification would reduce the ability of rainbow trout to cope with an acute secondary stressor (Chapter 2), and that these costs would also reduce swimming ability and energy availability during a strenuous swim performance test (Chapter 3). Our findings indicate that cortisol release following activation of the hypothalamus-pituitary-interrenal (HPI) axis by an acute 1-min air exposure stressor was maintained following in vivo MWWE and stormwater exposure. Plasma metabolomics analysis identified metabolic disruptions that were amplified during stress response resulting in a distinct plasma metabolome response after exposure to MWWE. Metabolite concentrations and activities of pyruvate kinase and lactate dehydrogenase suggest an increased glycolytic capacity and more reliance on anaerobic metabolism following MWWE and stormwater exposure. The maximum metabolic rate (MMR) measured during a ramp swim velocity test was reduced in fish exposed to MWWE and stormwater, and this contributed to a reduced metabolic scope in fish exposed to MWWE. Our results suggest that metabolic disruption caused by MWWE and stormwater reduces energy availability for energy demanding activities such as predator avoidance and migration.
- ItemEmbargoNitrogen metabolism during somatic embryogenesis in Picea glauca and Daucus carota : a NMR study(1991) Joy, Richard W.; Thorpe, Trevor A.
- ItemMetadata onlyPlant preference of the alfalfa leafcutter bee, megachile rotundata (fabricius) (hymenoptera : meg(1990) Horne, Marjorie L.; Owen, Robin E.
- ItemEmbargoRegulation 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.
- ItemEmbargoSex ratios, sexual dimorphism and population dynamics in the dioecious shrub shepherdia canadensis(1990) Lewis, Glennis M.; Chinnappa, C. C.
- ItemOpen AccessThe effect of maternal stress on early life history traits and performance in salmonids(2023-11-23) Lazaro-Côté, Analisa; Vijayan, Mathilakath M.; Harder, Lawrence; Post, John RobertDuring the upstream spawning migration of Pacific salmon, circulating cortisol concentration increases to mobilize energy stores. Pacific salmon contend with a gauntlet of stressors that can further elevate cortisol concentrations in response to stressful stimuli. Consequently, plasma cortisol concentrations measured in maternal fish that have reached their spawning grounds may reflect their recent stress history. Despite studies mimicking the maternal transfer of cortisol to the eggs, how natural variation in maternal cortisol concentrations shapes offspring phenotypes is unclear. Therefore, I assessed whether stress in maternal salmonids alters the growth and stress performance of their offspring via increased cortisol deposition in the eggs. Specifically, I tested whether high concentrations of circulating cortisol in maternal fish reduced embryo survival, and fry growth and stress performance. Eggs from 101 wild coho salmon (Oncorhynchus kisutch) females were collected from three rivers in the interior Fraser River watershed, British Columbia, Canada. The eggs were fertilized and reared until the fry stage at a hatchery. High egg cortisol concentration increased offspring mortality at the eyed stage. Interestingly, the surviving free-swimming fry had enhanced growth, resulting in larger size, and heightened cortisol stress response. The corresponding increase in the transcript abundance of genes related to growth and stress axis functioning in the fry suggest that high maternal cortisol concentrations programmed offspring for faster growth and heightened stress reactivity during early development. To test whether high egg cortisol concentrations specifically causes the phenotypic variation such as that observed in coho salmon, I manipulated steroid concentrations in the eggs of 10 maternal rainbow trout (Oncorhynchus mykiss) by immersion in water-borne cortisol during fertilization. Cortisol treatments increased egg steroid concentrations; however, neither the developmental trajectory nor the stress performance of fry differed statistically among treatments. Therefore, the utility of egg cortisol concentration as a biomarker of offspring developmental performance may be dependent on the life history of the animal. Overall, maternal cortisol concentration seems to act as a cue that prepares the offspring of wild coho salmon for stressful environments.
- ItemOpen AccessThe Role of Nuclear Envelope Lipids in Nuclear Shape and Transcription(2023-04-23) Sosa Ponce, Maria Laura; Zaremberg, Vanina; Cobb, Jennifer A.; Chua, Gordon; Moorhead, Greg; Grewal, Savraj; Siniossoglou, Symeon; Jackson, LelandThe nuclear envelope (NE) is a double membrane structure contiguous with the endoplasmic reticulum (ER) encompassing the nucleus. The NE is important in maintaining genome organization, as silenced domains are sequestered at the nuclear periphery, while active domains associate with the nuclear pore complex for rapid export of mRNA. Budding yeast in particular tether silenced telomeres at the NE to prevent aberrant recombination of the chromosome ends. In this thesis, the role of lipids in communication between the NE and telomere regulation was investigated, including how changes in lipid composition impact gene expression and overall nuclear architecture. Yeast cells were treated with the non-metabolizable lysophosphatidylcholine analog edelfosine, a lipid drug known to accumulate at the perinuclear ER. Edelfosine induced NE deformation and disrupted telomere clustering but not anchoring. In addition, the association of Sir4 of the Silent Information Regulator complex with telomeres decreased. RNA-seq analysis showed upregulation of Sir-dependent genes located at sub-telomeric regions, and downregulation of ribosomal protein genes. Transcriptomic analysis also revealed that two lipid metabolic circuits are activated in response to edelfosine, one mediated by the membrane sensing transcription factors, Spt23/Mga2, and the other by a transcriptional repressor, Opi1. Activation of these transcriptional programs resulted in higher levels of unsaturated fatty acids and the formation of nuclear lipid droplets. Interestingly, cells lacking Sir proteins displayed resistance to edelfosine and unsaturated fatty acids. Characterization of the edelfosine mechanism of toxicity in cells lacking SIR4 found that while these cells display similar NE deformations and formation of nuclear lipid droplets as seen in wild type cells, they have less internalization of sterols from the plasma membrane, with a concomitant maintenance of the proton pump Pma1 at the plasma membrane and a reduced acidification of the cytosol. Altogether this body of work presents evidence supporting a cross-talk between NE lipids and nuclear architecture and function.