Browsing by Author "Muench, Douglas G."

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    Arabidopsis At5g39790 encodes a chloroplast-localized, carbohydrate-binding, coiled-coil domain-containing putative scaffold protein
    (BioMed Central, 2008) Lohmeier-Vogel, Elke M.; Kerk, David; Nimick, Mhairi; Wrobel, Susan; Vickerman, Lori; Muench, Douglas G.; Moorhead, Greg B. G.
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    Biodegradation of Naphthenic Acids by Microorganisms Originating from Alberta's Oil Sands Surface Mining Operations
    (2021-01) Paulssen, Julie Maria; Gieg, Lisa Marie; Muench, Douglas G.; Chua, Gordon
    Billions of liters of tailings water are produced from oil sands surface mining operations each year. Reclamation of tailings ponds is now a legislated process that must occur within 10 years after the end of an oil sands mining operation. To reclaim these waste ponds back to a natural landscape, toxic compounds must first be removed, including naphthenic acids (NAs), which are the primary toxic components. I investigated the biodegradation ability of three different microbial culture sets - photosynthetic algal-bacterial communities, aerobic bacterial communities, and a nitrate-reducing bacterial community - for their ability to metabolize the two model NAs cyclohexanecarboxylic acid (CHCA) and 1-adamantanecarboxylic acid (1-ADCA). Using gas chromatography-mass spectrophotometry, DNA stable isotope probing, and 16S/18S rRNA gene sequencing techniques, the work in this thesis illustrates the metabolism and organisms responsible for the biodegradation of the model NAs studied. All three microbial culture sets successfully biodegraded CHCA, while the structurally more complex 1-ADCA could only be biodegraded by the algal-bacterial communities, suggesting the potential value of such consortia for treating NA in oil sands tailings ponds. The DNA stable isotope probing experiments revealed that of the algae, members of the family Scenedesmaceae and the class Trebouxiophyceae both contribute to the biodegradation of CHCA. Brevundimonas and Rhodococcus were the two primary genera found associated with the biodegradation of CHCA in the aerobic bacterial communities. Sequencing results revealed that the nitrate-reducing bacterial community was primarily composed of known sulfate-reducing microorganisms, an intriguing result that warrants additional study.
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    Characterization of peroxisomal malate dehydrogenase: a microtubule- and RNA-binding protein
    (2005) Freeman, Michelle Catherine; Muench, Douglas G.
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    Characterization of the Derlin protein, CUP-2, in promoting stem cell proliferation in the C. elegans germ line
    (2018-03-27) Singh, Ramya; Hansen, David; Cobb, John A.; Muench, Douglas G.; Childs, Sarah J.; Labbé, Jean Claude
    Understanding of how tissue homeostasis is maintained is not only essential for furthering our understanding of a fundamental area in Biology, but also has important implications for regenerative medicine and the design of therapeutics for human health. The germline of the C. elegans nematode serves as an excellent model to study adult stem cells and how the balance between proliferation and differentiation is maintained in this vital tissue. GLP-1/Notch signalling is the principal pathway that promotes proliferation of Germline Stem Cells (GSCs) in this system. In this thesis, the role of a Derlin (Degradation in the ER) protein, CUP-2, in promoting GLP-1/Notch signalling-mediated proliferation of GSCs is presented. Reducing cup-2 activity strongly suppresses germline tumours caused by increased Notch signalling. However, cup-2(0) is unable to suppress Notch-independent germline tumours, which suggests that cup-2 promotes Notch signalling specifically and not cell proliferation in general. This is further supported by cup-2’s ability to influence the expression of a read-out for Notch signalling, SYGL-1. The cup-2 gene was tagged using CRISPR/Cas9 editing with an epitope tag and CUP-2 was found to be expressed in the germ line, which is consistent with its role in promoting Notch signalling. The ability of cup-2 to promote GLP-1/Notch signalling likely represents a conserved function, since der-2, a Derlin homolog, was also found to contribute to the development of Notch-dependent germ line tumours. In fact, cup-2 and der-2 appear to additively promote GLP-1/Notch signalling. Derlin proteins are known to function in Endoplasmic Reticulum-Associated Degradation (ERAD) of proteins. Therefore, this work sheds light on the importance of ERAD in maintaining the balance between proliferation and differentiation in stem cells. Investigation into the mechanism by which cup-2 promotes GLP-1/Notch signalling led to the discovery that cup-2 is involved in anchoring the niche cell, the Distal Tip Cell, in place in the aging gonad. This is a novel phenotype not reported before in the literature. The CRISPR/Cas9 tagged cup-2 alleles developed as part of this thesis could serve as a useful reagent for further studies in determining the mechanism by which cup-2 promotes GLP-1/Notch signalling.
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    Characterization of the exon-junction complex in plants
    (2007) Park, Nam-il; Muench, Douglas G.
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    Deducing the post-transcriptional role of the Pumilio protein Puf3 in fission yeast.
    (2019-05-29) George, Iain Fraser Scotney; Chua, Gordon; Muench, Douglas G.; Turner, Raymond Joseph; Grewal, Savraj S.; Kothe, Ute
    Pumilio family proteins (Pufs) are a highly-conserved group of post-transcriptional regulators in eukaryotes. Puf proteins contain an arc-shaped Pumilio homology domain with several coiled repeats that bind to potentially predictable sequences in the 3’-UTR of their mRNA targets. The binding of Puf proteins to mRNA targets can influence their stability, translation and cellular localization. In S. pombe, there are seven non-essential genes that encode classical Puf proteins. The biological function of these Puf proteins and their mRNA targets remain unknown. In this study, we investigated the biological role and mRNA targets of Puf3 in S. pombe. Over-expression of puf3+ with the nmt41 promoter results in reduced fitness and an enrichment of elongated cells arrested in mitosis. Phenotypic characterization of puf3 overexpression strains and genetic interaction screens identified a cell cycle regulatory role for Puf3, principally at the G2/M transition and possibly at the G1/S control point. Genetic interaction screening, fluorescence microscopy and tandem mass spectrometry analysis of immunoprecipitated Puf3-HA identified Puf3-interacting proteins in the post-transcriptional machinery of 3’-deadenylation of mRNA and localized to stress granules. Bioinformatic analysis of the Puf proteins in S. pombe identified the Puf repeat domains and these were used to enumerate the predicted Puf binding element in the transcriptome. The enumeration data and annotation data were used to identify putative mRNA targets from microarray, RNA-seq and RNA-immunoprecipitation sequencing experiments. These identified several components of the kinetochore and mitotic checkpoint as putative mRNA targets of Puf3.
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    Diverse Structures and Strategies of 5′ Exon Recognition in Group II Introns
    (2018-06-05) Jarding, Ashley Marie; Zimmerly, Steven; Muench, Douglas G.; Noskov, Sergei Yu; Van Marle, Guido; Cousineau, Benoit
    Group II introns are a family of mobile elements found in bacteria and bacteria-derived organelles, which are thought to have a direct evolutionary link to spliceosomal introns. These introns can self-splice in vitro thanks to their highly organized ribozyme structure. Studying the diverse structural features of group II introns illuminates the breadth of their structural diversity and resultant unique splicing and mobility capabilities. In the first of three projects presented in this dissertation, bioinformatic approaches were used to identify over 800 new introns in a large scale, systematic collection and analysis of group II introns. This allowed inference of class-specific mobility properties and the description of novel intron arrangements in genomes. Additionally, one new class and 15 unique, previously unclassified introns with novel structural features have been identified. The newly collected introns revealed a conserved structural feature for Class A introns: they appear to have two 5′ exon recognition motifs, whereas all other group II introns are reported to have one. The second project investigates the binary splicing hypothesis, which states that either of these two motifs can be used to recognize the exon. This was investigated and confirmed through both experimental and bioinformatic approaches. This strategy appears to expand the set of homing sites available to an intron by permitting splicing from one sequence and reverse-splicing into another. In the third project, the 5′ exon recognition mechanism for a IIC intron was investigated. IIC introns lack the canonical IBS2-EBS2 interactions found for other group II introns, but have a conserved transcriptional terminator in place of EBS2. In B.h.I1 a 5S rRNA upstream of this motif was shown to have an unexpected effect on the splicing reaction in vitro. Mutagenesis of 5S rRNA showed that the A-loop is critical for splicing. RNA protection experiments to characterize the interactions with the 5′ exon support the terminator stem interacting with Domain I while 5S has an auxiliary effect on exon ligation. In summary, this work has increased our understanding of the structural diversity of group II introns and corresponding specialized strategies used to recognize the 5′ exon during splicing.
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    Identification and charaterization of plant RNA- and microtubule-binding proteins
    (2003) Chuong, Simon Dich Xung; Muench, Douglas G.
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    Increasing Reproductive Output of Brassica napus (canola) Through Manipulation of Shoot Architecture
    (2019-11) Stanic, Matija; Samuel, Marcus A.; Ro, Dae-Kyun; Muench, Douglas G.
    Food security and yield increases of important crops is of paramount concern to an ever-increasing human population. A key determinant of yield in plants is shoot architecture which is controlled through a complex interplay between endogenous and exogenous signals and exhibits high plasticity throughout a plant’s lifetime. Strigolactones (SL) are a class of terpenoid lactone plant hormones which have recently been characterized as key regulators of shoot development. Mutants in this pathway exhibit various developmental deviations, most notably a dwarfed and highly branched shoot architecture. Canola transgenic lines suppressed in key SL biosynthetic and signalling components were generated to assess effects on architecture. Lines suppressed in the SL receptor, BnD14, displayed reductions in overall shoot height as well as an increase in lateral branching. This was accompanied by an increase in total flowers per plant. Consistent with this, BnD14 knockout lines generated through a CRISPR/Cas9-mediated system also exhibited a dwarf, highly branched phenotype with significant increases in total flowers per plant. This study provides evidence of the potential use of the SL pathway in future crop modifications to promote increased crop yield.
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    Investigating the Genetics and Physiology of Naphthenic Acid Remediation in Plants
    (2020-05-12) Wong, Jeremy Jordon; Muench, Douglas G.; Samuel, Marcus A.; Turner, Raymond Joseph
    Surface mining of bitumen in the Northern Alberta oil sands produces large volumes of oil sands process-affected water (OSPW). OSPW is toxic to many living organisms, and this toxicity is primarily attributed to a class of organic compounds collectively known as naphthenic acids (NAs). Remediation of NAs is required to meet future water release criteria. This thesis research was aimed at identifying plant genes that are involved in NA tolerance using a novel genetic screen, as well as providing insights into the physiological capacity of plants to take up and possibly biotransform NAs. Six NA tolerant mutants were identified, and the altered region of the genome was identified for four of the mutants. Experiments using labeled NAs were carried out on a native grass species to determine NA uptake efficiency and distribution in roots and shoots. This research provides a framework for identifying NA tolerance genes and quantifying the uptake and biotransformation of NAs in plants.
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    Mass Spectrometry-based Integrative Structural Modeling of the Doublecortin-Microtubule Interaction
    (2021-01-21) Rafiei, Atefeh; Schriemer, David C.; Fraser, Marie Elizabeth; Thurbide, Kevin B.; Politis, Argyris; Muench, Douglas G.
    A comprehensive understanding of protein function requires the structural elucidation of physical contacts with other proteins, known as protein-protein interactions (PPIs). Many PPIs evade classical structural determination techniques for many reasons. One class of PPIs that is challenging to canonical techniques involves large multi-subunit protein complexes comprised of proteins with intrinsically disordered regions. The interaction between doublecortin (DCX) and microtubules (MTs) is a particularly intriguing example of such PPIs. Doublecortin is a critically important protein involved in neuronal development, which appears to function by engaging MTs in a complex interaction pattern involving both ordered and disordered domains. In this work, I developed and used a mass spectrometry-based integrative structural modeling (ISM) approach to generate a unifying structural model of DCX-MT interaction. A crosslinking-mass spectrometry (XL-MS) workflow was developed for the analysis of interactions involving MTs in general. Different crosslinker reagents targeting different amino acid classes were tested and the integrity of the MTs in response to the crosslinking reactions were monitored. Then, a DCX-MT construct was reconstituted in-vitro and XL-MS performed using different reagents. Doublecortin self-association was investigated using isotopically-labeled DCX, to differentiate between inter- and intra- DCX crosslink peptides. It was found that DCX self-associates only in the presence of MTs. The majority of the inter-DCX crosslinks were observed in the C-terminus regions of the DCX sequence. Finally, the structural elucidation of DCX-MT interaction was carried out in a step-wise approach. The residue-based distance restraints from XL-MS along with the cryo-EM map of DCX-MT and the X-ray structures of protein subunits were combined in the Integrative Modeling Platform to identify the MT-binding domain. It was found that the N-terminus doublecortin-like domain is the primary MT binding domain, while the C-terminus doublecortin-like domain and C-tail are MT-dependent oligomerization domains. My modeling results support a DCX-MT interaction model in which DCX can self-associate between all immediate neighbors. Finally, it is shown that fast crosslinking chemistries (i.e. diazirine based photo-crosslinkers) were required to generate a converging model, as the more conventional long-lived crosslinkers are prone to conformational “kinetic trapping”. This has implications for ISM of similar systems, and modeling in general.
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    Oxidative Decorations of the Salvinorin A Backbone: Characterization of Salvia divinorum Cytochrome P450s
    (2019-08-23) Ngo, Iris; Ro, Dae Kyun; Muench, Douglas G.; Ng, Kenneth Kai Sing
    Salvia divinorum is an ethnomedicinal plant belonging to the Lamiaceae (mint) family. The main bioactive compound in S. divinorum is the psychotropic diterpenoid salvinorin A. Salvinorin A was the first naturally occurring, non-nitrogenous, human kappa opioid receptor agonist identified, it thus holds potential for the treatment of addiction and mental disorders. Heterologous production of salvinorin A is desirable as purification from S. divinorum or total synthesis remains arduous. However, only the first step in the biosynthesis of salvinorin A has been elucidated, the formation of (-)-kolavenyl diphosphate (KPP) from the universal diterpenoid precursor, geranylgeranylpyrophosphate by the class II diterpene synthase (diTPS) enzyme, SdCPS2. To date, no class I diTPS which acts upon KPP has been identified, but in Saccharomyces cerevisiae, KPP can be dephosphorylated by endogenous phosphatases to yield kolavenol (KOH), the predicted class I diTPS product. From KOH, a cytochrome P450 monooxygenase (P450) known as crotonolide G synthase (SdCS) can form crotonolide G. Additional oxidative decorations to the salvinorin A backbone are also likely catalyzed by P450s. We thus sought to identify P450s which can catalyze the decorative steps succeeding KPP/KOH and crotonolide G. Since salvinorin A accumulates in the peltate glandular trichomes of S. divinorum, we mined a trichome-specific transcriptome, identifying eight candidate P450s. Of the eight P450s tested, using S. cerevisiae as a platform for heterologous expression and compound production, two P450s, CYP728D25 and CYP728D26, utilized crotonolide G to generate distinctly oxidized compounds. The highest titre of the compounds identified was obtained by having the six pathway genes necessary to synthesize SA intermediates both integrated into the yeast genome using CRISPR-Cas9 and expressed in plasmids. These results advance diterpene biosynthesis knowledge and bring us closer to obtaining therapeutic SA through heterologous means.
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    The Puf family of RNA-binding proteins in plants: phylogeny, structural modeling, activity and subcellular localization
    (BioMed Central, 2010-04-09) Tam, Patrick P. C.; Barrette-Ng, Isabelle; Simon, Dawn M.; Tam, Michael W. C.; Ang, Amanda L.; Muench, Douglas G.
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    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 Dawn
    Floods 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.
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    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.
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    Understanding the processes of oilsands process-affected water phytoremediation using native plant species
    (2022-11-28) Alberts, Mitchell E.; Muench, Douglas G.; Samuel, Marcus A.; Turner, Raymond J.; Germida, James J.; Schriemer, David C.
    Bitumen mining in the oil sands region in Alberta, Canada, generates large volumes of oil sands process-affected water (OSPW). Naphthenic acid fraction compounds (NAFCs) are a major contributor to OSPW toxicity and are a target for removal using remediation approaches. Phytoremediation, the use of plants to remediate the environment, is a biotechnology that has commercial potential as a large-scale treatment strategy for OSPW. Although studies performed thus far demonstrate that phytoremediation is a feasible approach, the mechanisms involved in this process are not well understood. This thesis aims to understand how upland plants can attenuate or modify NAFCs in OSPW and gain insight into the physiological and molecular events that occur when NAFCs are taken up into plant tissue. Lab-scaled hydroponic and soil experiments, greenhouse sub-surface flow mesocosm experiments, and plant NA-tolerance analysis were performed using isotopic NA and NAFC compounds. Plants were shown to take up isotopically-labelled NAs into the root and shoot tissue and localize NA-derived carbon in newly growing tissues. NAFC uptake was observed and was enhanced when plants were grown in media adjusted to lower pH levels naturally found in the rhizosphere. The presence of plants in mesocosm systems resulted in greater decreases in classical O2 NAFC concentrations than in no-plant mesocosms and overall decreases in total NAFC concentrations were observed within 7 days. Additionally, toxicity was reduced after OSPW treatment in plant and no-plant mesocosms. Mass balance analysis revealed that most of the NA-derived carbon taken up by plants remains within the root and shoot tissue, but a small amount was captured in headspace traps, suggesting that limited phytovolatilization processes are involved in plant NA metabolism. Finally, selected NA-tolerant plants displayed NA-derived carbon localization in the vasculature of leaves but no overall difference in the uptake of NAs compared to wildtype plants. The results of this research support the use of phytoremediation using upland plants as a viable approach to OSPW treatment and are foundational for future studies on NAFC metabolism in plants.
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    Utilization of plant Puf RNA-binding proteins to manipulate endogenous mRNA physiology
    (2019-02-01) Wen, Xin; Muench, Douglas G.; Hansen, David V.; Barrette-Ng, Isabelle H.
    RNA-binding proteins have an important role in the regulation of post-transcriptional gene expression. The Puf (Pumilio) family of RNA-binding proteins bind to RNAs in a sequence specific manner. The RNA-binding domain of canonical Puf proteins (the Pumilio homology domain, PUM-HD) consists of eight Puf repeats. Each Puf repeat binds to RNA in a modular fashion, so that one Puf repeat binds to a single nucleotide. The nucleotide binding recognition code of Puf repeats has been well characterized. This one repeat:one nucleotide recognition code has allowed for the alteration of Puf repeats so that the PUM-HD can bind specific RNA targets. These altered Puf repeats have been fused to effector domains so that fusion proteins can affect RNA physiology in the cell. The aim of this thesis was to fuse an Arabidopsis Puf RNA-binding protein with unique binding characteristics to two types of effector domains. These fusion proteins were expressed in onion epidermal cells to determine if they could alter the stability or translation of a reporter mRNA. This research provides foundational evidence that highlights the potential for the utility of plant Puf proteins to alter gene expression for applied purposes