Open Theses and Dissertations
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Browsing Open Theses and Dissertations by Department "Biological Sciences"
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- ItemOpen AccessA Biophysical Characterization of Histone detived Antimicrobial Peptides(2013-09-23) Berezowski, Neil Steven; Prenner, ElmarHistones have been recently shown to be integral to the response of the body to invading pathogens. As such, prior work on this project identified segments of several histones or fragments thereof as potential AMPs. This project comprises a continued biophysical characterization of these selected segments of histone. The project investigates the first generation of AMPs with monolayer models and Brewster angle microscopy to screen for the lipids which are most important in AMP-lipid interactions. Subsequently new variant peptides were designed from one parent peptide (H3) and studied through the use of several thermodynamic and spectroscopic techniques. The variant peptides were shown to be more potent than the parent peptide in terms of microbicidal activity, but were not as selective for bacterial membranes. Therefore, further studies are needed to determine the therapeutic value of the variants.
- ItemOpen AccessA Genomic and Proteomic Survey of Traits that Modulate Antimicrobial Resistance in Staphylococcus aureus(2024-04-18) MacKenzie, Colin Campbell; Lewis, Ian; Gregson, Daniel; Turner, RaymondAntibiotic resistance is a growing global public health crisis which threatens to remove our primary treatment against bacterial infections. The mechanisms of antibiotic resistance in bacterial pathogens have been extensively studied, however questions surrounding the regulatory mechanisms of these resistance factors in clinical isolates are yet to be answered. In collaboration with the Broad Institute of MIT and Harvard, The Harvard T.H. Chan School of Public Health, and Alberta Precision Laboratories we completed whole-genome sequencing on 7,997 Staphylococcus aureus genomes from a larger study cohort of over 38,000 blood stream infections over a 16-year period. In addition to whole-genome sequencing, the proteomes of the bacterial isolates were quantitatively assessed using Tandem Mass Tag (TMT) ultra-high-performance liquid chromatography mass-spectrometry (UHPLC-MS) methods. Changes in protein levels and growth in the bacterial isolates are related to the variability in the genetic composition of the resistance operons of the specific clinical strains. This study has resulted in the understanding of a complex coregulatory interaction between two resistance operons of Methicillin Resistant S. aureus related to the mecA and blaZ resistance factors. Further, to better understand the metabolic adaptations of pathogens under antibiotic exposure, kinetic flux profiling of Escherichia coli metabolism under various antibiotic stressors was completed through the addition of fully labelled 13C-glucose. This intracellular flux monitoring via UHPLC-MS analysis, at a scale of seconds, has been used to gain insight into the metabolic alterations within E. coli metabolism under the exposure to twelve antibiotics spanning three common classes of antibiotics: DNA synthesis inhibitors, protein synthesis inhibitors, and cell wall synthesis inhibitors. This study has resulted in the classification of important metabolic adaptations occurring because of specific antibiotic compounds. Further, this intracellular metabolic study has shown evidence of a previously unexpected mevalonate pathway in E. coli. These studies have provided insight into the dynamics of pathogen interactions with antibiotics, and a deeper understanding of the antibiotic resistance mechanisms existing in pathogenic strains.
- ItemOpen AccessAbiK: A Novel Polymerase That Confers Resistance to Phage Infection(2019-05-15) Soufi, Bahar; Zimmerly, Steven; Hynes, Michael F.; Hansen, David V.The abortive infection K (AbiK) system of Lactococcus lactis is a powerful antiviral defense mechanism that acts in the period after phage DNA enters the cell but before new progeny are released. The N-terminal domain of AbiK encodes a novel DNA polymerase that in vitro uses an internal amino acid to synthesize an un-templated DNA. The C-terminal domain of AbiK, on the other hand, has not been biochemically characterized. In the first of two projects presented in this thesis, strategies were devised to identify the amino acid priming site in AbiK. A close examination of data has narrowed down the priming site to two highly-conserved tyrosines in the N-terminal sequence of AbiK. In the second project, the characterization of the AbiK’s C terminal domain was undertaken to identify its function. This study uncovers a novel RNA modification activity, and provides evidence that AbiK’s C-terminal domain has ribonuclease activity.
- ItemOpen AccessAcetic acid formation by acetogens from hydrogen and CO2:dissolution of carbonates and competition by methanogens(2014-07-11) Rollick, Lindsay; Voordouw, GerritMethane-producing archea (methanogens) and bacteria that produce acetic acid from H2 and CO2 (acetogens) exist in many low nutrient environments where other electron acceptors are absent. Methanogens usually outcompete acetogens because of a more powerful energy production but recent evidence suggests that acetogens may be able to compete through greater substrate diversity and energy efficiency (Lever 2012). Acetogenesis could be adapted as a biotechnology to induce carbonate dissolution in carbonate oil reservoirs. Enrichments were conducted on the subsurface production waters from a conventional oil field. Variations in medium composition were tested to promote acetogenesis over methanogenesis. High levels of acetic acid of up to 2400 μmol or 28 mM were produced and the consumed along with various levels of methane. Analysis of 16S rRNA gene sequences show a progression from dominance of acetogenesis to methanogenesis and then a shift to a group of microbes that consume acetate to create biopolymers like polyhydroxybutyrate for storage of carbon in nutrient-limited environments. Factors that increase acetic acid production include ultra-low nutrient environments, sufficient pH buffering, not adding bicarbonate, and possibly increased surface area. Dissolution via microbial acetic acid was tested for powdered CaCO3, crushed and solid carbon rock but results are inconclusive. Acetogens can be competitive with methanogens under the lowest nutrient conditions. The excess of the H2/CO2 energy and carbon substrates used in these experiments along with differences seen in nutrient variation suggest that conditions other than substrate availability can influence this competition. Sampling loss, re-precipitation and other experimental factors make carbonate dissolution mediated by microbial acetic acid difficult to track.
- ItemOpen AccessAcoustics of Mountain Pine Beetle and Lodgepole Pine(2014-07-21) Kaiser, Mathias; Reid, Mary; Yack, JayneMountain pine beetles (Dendroctonus ponderosae) produce acoustic signals in a variety of contexts but more study is needed on the function(s) of their signals and on the acoustic cues beetles use. I studied the context-dependent acoustic signalling of male beetles to elucidate the function(s). When stressed, individuals produced short signals, typical of insect anti-predator signals. Prior to mating, individuals produced longer signals that were related to body size with potential implications for mate selection. I further examined the long-proposed idea that beetles could use acoustic emissions from their host, lodgepole pine (Pinus contorta), for host selection. Emission rates recorded in the field increased with presumed water stress and appeared to be related to lower quality trees. I then tested the effects of acoustic emissions on female host choice in a playback experiment and found that isolated females tended to prefer hosts with high acoustic emission rates.
- ItemRestrictedActivation of latent Escherichia coli virulence factors by Campylobacter jejuni(2014-07-11) Reti, Kristen; Buret, AndreCampylobacter jejuni is the most common cause of bacterial gastroenteritis, and while typically self-limiting, C. jejuni infections are associated with post-infectious intestinal disorders, including inflammatory bowel disease and irritable bowel syndrome. This study sought to determine if C. jejuni induces virulence in commensal, non-invasive E. coli. Expression of adhesin, flagella, hemolysin, biofilm formation, and antibiotic resistance genes were increased in E. coli upon exposure to C. jejuni-conditioned media. Atomic force microscopy demonstrated E. coli was more adherent to human colonic epithelial cells when exposed to C. jejuni-conditioned media. C. jejuni and C. jejuni-conditioned media also induced E. coli flagella expression. In vitro, this altered E. coli phenotype disrupted TLR4 expression and induced IL-8 secretion. These data suggest C. jejuni and C. jejuni-conditioned media can induce virulence in non-invasive, commensal E. coli, and this contributes to host inflammation. These results provide a novel mechanism for post-infectious complications.
- ItemOpen AccessAdverse Effects of Remediated and Unremediated Oil Sands Process-Affected Water on Zebrafish Embryo Morphological Development(2016) Toth, Cameron Graham Andrew; Habibi, Hamid; Chua, Gordon; Gieg, Lisa; Smits, JuditOil sands process-affected water (OSPW) generated from bitumen extraction in the Alberta oil sands is accumulating in on-site tailings ponds. The organic fraction of OSPW is a primary target for remediation as it is toxic to aquatic organisms. Here, robust morphometric biomarkers were identified in developing zebrafish (Danio rerio) embryos for determining the acute toxicities of the acid extracted organics (AEOs) and diluted OSPW. AEOs/OSPW exposures reduced survival and increased cardiovascular, growth, swim bladder and behavioural abnormalities in zebrafish embryos. Phosphate biostimulation of indigenous algal species in OSPW reduced the C5 – C12 cluster of naphthenic acids (NAs) abundance in the AEO fraction by ≥ 50%. Following phosphate biostimulation, embryonic zebrafish endpoints previously sensitive to AEO/OSPW constituents were ameliorated. These findings implicate the acute toxicity of C5 – C12 NAs on zebrafish development and highlight the potential of algal-based NA biodegradation for reducing the impact of OSPW to aquatic organisms.
- ItemOpen AccessAerobic and Anaerobic Naphthenic Acid Biodegradation by Indigenous Tailings Pond Microorganisms(2015-01-07) Clothier, Lindsay Nicole; Gieg, LisaSurface mining of bitumen extraction from Alberta’s oil sands generates large volumes of oil sands process-affected water (OSPW) that is stored in tailings ponds. Naphthenic acids (NAs) are toxic, corrosive, and complex, cyclic carboxylic acids that accumulate in tailings ponds over time and must be removed for effective reclamation. This work has examined microbial NA biodegradation under aerobic and anaerobic conditions as a possible approach for reclamation. Phosphate stimulation of oxic OSPW resulted in growth of algae (Scenedesmus, Chlorella) and bacteria (Porphyrobacter, Planctomyces) capable of biodegrading low molecular weight NAs resulting in small decreases in acute toxicity. Several new NA-biodegrading bacteria were isolated from OSPW and identified. Additionally, this work has now shown that NA can be biodegraded under nitrate-, iron-, sulfate-reducing and methanogenic conditions and has identified microbial community members likely contributing to NA biodegradation. These findings show that the use of microorganisms for NA remediation may be possible.
- ItemOpen AccessAerobic Hydrocarbon-degrading Microbial Communities in Oilsands Tailings Ponds(2016) Rochman, Fauziah; Dunfield, Peter F.; Voordouw, Gerrit; Gieg, Lisa; Hettiaratchi, PatrickOilsands process-affected water (OSPW), produced by the surface-mining oilsands industry in Alberta, Canada, is alkaline and contains salts, various metals, and hydrocarbon compounds. In this thesis, aerobic communities involved in several key biogeochemical processes in OSPW were studied. Degradation of several key hydrocarbons was analyzed in depth. Benzene and naphthalene were used as models for aromatic hydrocarbons, in which their oxidation rates, degrading communities, and degradation pathways in OSPW were researched. The potential oxidation rates were 36.7 μmol L-1 day-1 for benzene and 85.4 μmol L-1 day-1 for naphthalene. Via stable isotope probing (SIP), and high-throughput sequencing of 16S rRNA gene amplicons, it was discovered that strains of the genera Methyloversatilis and Zavarzinia were the main benzene degraders, while Thiococcus and Pseudomonas were the main naphthalene degraders. Cultivated strains of Zavarzinia and Pseudomonas were shown to be growing on benzene and naphthalene. Metagenomics analysis revealed genes encoding oxygenases active against aromatic compounds, as well as catechol oxidases. Although these belonged to many phylogenetically diverse bacteria, only few bacteria were predominant in the SIP experiments. A highly divergent pmoA-like gene was also detected in the metagenome data. Here, the possibility of this gene allowing growth on short alkanes (C1 to C3) was examined. This gene was investigated via SIP and quantitative PCR. Results showed that the monooxygenase encoded by the gene has high affinity toward ethane and mostly propane. For the study of lighter hydrocarbons, methane, ethane, and propane were chosen as model compounds. OSPW was capable of supporting methane oxidation with a rate of 108.2 μmol of CH4 L−1 OSPW d−1, ethane oxidation with a rate of 83.2 μmol of C2H6 L−1 OSPW d−1, and propane oxidation with a rate of 58.6 μmol of C3H8 L−1 OSPW d−1. SIP analysis uncovered Methyloparacoccus to be predominant in methane-incubated samples, whereas Methyloversatilis was predominant in ethane and propane-incubated samples. SIP technique was also employed to study photosynthetic bacterial communities and indigenous aerobic bacterial communities that assimilate methanol, acetate, and protein extracts. All OSPW photosynthetic ‘heavy-DNA’ samples were dominated by unidentified Planctomycetes. Predominant groups in methanol, acetate, and protein extract-SIPs were Betaproteobacteria, Alphaproteobacteria, and Bacteroidetes. Finally, via a modified cultivation technique, a novel Verrucomicrobia was isolated from OSPW. The aerobic bacterium was named Oleiharenicola alkalitolerans gen. nov., sp. nov., and it was studied in depth via phylogenetic, chemotaxonomic and whole-genome sequencing techniques.
- ItemOpen AccessAlternative splicing of an ORF-less group II intron in Clostridium tetani(2013-12-04) McNeil, Bonnie; Zimmerly, StevenGroup II introns are ribozymes that are encoded within all domains of life. They are also capable of mobility through an RNA intermediate. Due to similarities in RNA structure and splicing mechanisms, group II introns are thought to have been the ancestors of nuclear pre-mRNA introns and snRNAs. In this dissertation I report the discovery of a unique ORF-less group II intron, C.te.I1, in the human pathogen Clostridium tetani. The intron is encoded within a surface layer protein region of the C. tetani chromosome and possesses an unusual genomic organization such that a full-length copy of the intron is followed downstream by three copies of the RNA structural domains 5 and 6 (D5/6). This arrangement led to the hypothesis that C.te.I1 is capable of alternative splicing utilizing the downstream copies of D5/6 as alternate 3ʹ splice sites. RNA extractions and RT-PCR support the hypothesis and revealed that the splicing reaction of C.te.I1 links a surface layer protein ORF (CTC00465) in the upstream exon to one of four downstream ORFs that encode transglutaminase-related or protease-related reading frames (CTC00467-CTC00470). Including unspliced transcript, five mRNAs are produced. Sequencing of the exon junctions showed that the 5ʹ splice site utilized by C.te.I1 is shifted 8 nt upstream both in vivo and in vitro. Use of this splice site is critical to alternative splicing as it results in the elimination of the stop codon at the end of CTC00465 and results in the correct ligation of 5ʹ and 3ʹ exon sequences. Site-directed mutagenesis and self-splicing assays for C.te.I1 revealed that the shifted splice site is due to a novel EBS1-IBS1 pairing. Although C.te.I1 is thought to be derived from a mobile Class B (IIB) intron that lost its ORF, the intron was found to have evolved to utilize a IIA-like mechanism of 3ʹ splice site recognition. These changes represent structural adaptations of the intron to its role in alternative splicing. The structural adaptations and splicing of C.te.I1 illustrate the plasticity of group II introns in that they can adapt new RNA structural and catalytic properties which can be utilized to affect gene expression.
- ItemOpen AccessAn Analysis of Enthesis-Bone Microstructure: Implications for Paleontological Soft Tissue Reconstructions.(2021-08) Whitebone, Stephanie Amber; Anderson, Jason; Theodor, Jessica; Matyas, JohnIn the absence of direct preservation, evaluating a fossil organism’s soft tissue anatomy can be a daunting task. Studies that aim to evaluate fossil soft tissues must often rely on clear soft tissue bony correlates, such as bony eminences or concavities in the bone surface. These eminences and concavities are formed at the site of soft tissue attachment to bone, called entheses. However, even in large-bodied, adult organisms, a considerable proportion of soft tissues do not leave these clear indicators. Therefore, the evaluation of bony eminences/concavities is coupled with the use of an extant phylogenetic bracket. However, some fossil taxa are so phylogenetically removed from their extant bracketing taxa that anatomical comparisons are dubious at best. I have shown that scanning electron microscopy and histological staining make it possible to identify areas of soft tissue attachment on the bone surface in the absence of macroscopically visible eminences or concavities. There is also a differentiation between tissue types (muscle, tendon, articular cartilage, and aponeurosis) with collagen fibres incorporated into the bone tissue. At areas of articular cartilages, the surface is relatively smooth but with small, organized hummocky structures. Areas of fleshy muscle attachment are generally planar but with occasional round projections where the collagen fibres have been incorporated into bone tissue. Tendon entheses are areas where long string-like collagen fibres have been incorporated into bone tissue, usually within concave impressions on the bone surface. These areas are generally more organized than other entheses. Lastly, aponeurotic entheses are large areas of thick, high-density, disorganized collagen fibres. All four categories of bone surface microstructure have been observed through broad taxonomic sampling using extant organisms, and the same morphologies are also seen in three-dimensional preserved fossil specimens. These four bone surface microstructures are successfully categorized using image classification programming with novel convolutional neural network architecture. Using polarimetry, I quantify the differences between bone surface microstructures by measuring the orientation of collagen fibres and the homogeneity of collagen fibre density throughout the enthesis bone’s thickness.
- ItemOpen AccessAn Analysis of North American Taeniolabidoid Multituberculate (Mammalia, Allotheria) Dentitions Using Mammalian Dietary Proxies(2018-12-20) Robson, Selina Viktoria; Theodor, Jessica M.; Scott, Craig S.; Cote, Susanne; Syme, Douglas A.; Zelenitsky, Darla K.In this thesis, a set of dietary proxies – dental microwear analysis, cusp row ratios (CRR) (similar to shearing ratios), relief index (RFI), orientation patch count rotated (OPCR), and Dirichlet normal energy (DNE) – was used to infer diets of North American taeniolabidoid multituberculates. Based on the signals recovered by these proxies, taeniolabidoid diets did not vary consistently with body size: small-bodied and large-bodied taeniolabidoids had similar dietary signals for almost all proxies, the only difference being in microwear feature dimensions. Dental microwear signals suggest that taeniolabidoids and non-taeniolabidoid cimolodontans may have had different diets, but all other proxies have recovered equivalent signals between the two groups. Dietary classifications are inconsistent among CRR, RFI, OPCR, and DNE. This suggests that these proxies are not equally good predictors and that their generalizability to non-therian mammals may need to be re-evaluated.
- ItemOpen AccessAnalytical, Dynamic Variable Game Theory Modelling, and Practical Investigations of the Sequential Tactics of Coursing and Tending in Bighorn sheep (Ovis canadensis)(2023-12-11) Tate, Ryan Geoffrey; Ruckstuhl, Kathreen; Neuhaus, Peter; Reid, Mary; Oxoby, RobertAnimals might use different mating tactics to increase their fitness. While some are genetically determined, some are plastic, depending on intrinsic and extrinsic factors. Within the plastic tactics, there can be further temporal structure, in which animals switch tactics once they reach a certain size or competitive ability. This thesis reviews the literature on the terminologies, strategies versus tactics, used in evolutionary game theory, while also making suggestions on improving, standardizing, and clarifying the use of terminology. It then looks at fixed-sequence mating tactics, using an analytical model, to investigate the conditions under which a fixed-sequence tactic can evolve. Then, using a dynamic variable game theory model, the thesis examines various parameters affecting the likelihood of a mating tactic called coursing to involve challenging the dominant male, using another tactic, called tending. Finally, this thesis analyses a long-term data set on bighorn sheep (Ovis canadensis) to see how coursing effort affects coursing and later-in-life tending outcomes.
- ItemOpen AccessAnimal Movement and Distribution at Habitat Edges(2015-02-02) Young, Hilary Catherine; Reid, MaryThe response of organisms to habitat edges is a key factor affecting their distribution across the landscape. Resources are commonly expected to be the primary driver of edge responses, but additional processes may be important. Here I tested resource-based hypotheses and their alternatives by measuring the distribution of organisms and their resources at edges in laboratory and field settings. One non-resource-based process for animal accumulation at edges is a slowing of movement. I recorded the movement paths and distribution of Tribolium confusum beetles in an artificial arena devoid of resources. Beetles took smaller steps with larger turn angles near edges than in the arena’s centre, resulting in a distribution biased towards the edge. The proportion of time spent at edges also depended on the beetles’ propensity to cross the edge. These results suggest that proximate mechanisms such as movement behaviour should be explicitly considered when interpreting animal distributions. To investigate distribution at edges in nature, I examined the response of moose (Alces alces) and deer (Odocoileus hemionus and O. virginianus) to forest-clearcut edges of different ages. Moose responded to differences among habitat types studied but not to distance from edge within habitat types. Clearcuts > 15 years in age contained the most food and cover and were preferred to the adjacent forest and to clearcuts < four years in age. Conversely, differences in deer preferences were detected both among and within habitats. Deer were found more frequently in forest habitats than clearcuts of any age. They were also generally found closer to edges than far from them, although in old clearcuts and the adjacent forests, deer abundance dropped within 50 m of edges. I found evidence for the influence of food and cover availability on deer distribution among habitats, but it was weaker within habitats. These studies suggest that factors other than resources may affect the response of animals to habitat edges, although the field studies were largely consistent with resource tracking. To develop effective models predicting the impact of landscape heterogeneity, researchers should determine both the distribution patterns at habitat edges and the main mechanistic drivers behind these patterns.
- ItemOpen AccessAnion Transport by Proteins: Protein Motifs Involved in Anion Binding(2022-05-07) Damergi, Mirna; Salahub, Dennis R.; Zhekova, Hristina R.; MacCallum, Justin; Fraser, Marie E.Various cellular processes require the participation of specific anions, including Cl-, F-, I-, HCO3- and CO32-. However, their ionic charges impede their permeation across the hydrophobic lipid bilayers of the cells. Cell membranes thus contain specific macromolecular transport proteins, ion channels and transporters, to facilitate and control the passage of anions across the lipid bilayer. Anion-transport proteins need to be able to discriminate between different anions to carry out their function. What drives the protein selectivity for different substrates is a fundamental question that has long fascinated scientists. However, anion recognition in proteins is still poorly understood due to the lack of crystal structures of anion-transporting proteins and the underdevelopment of computational tools for studies of anions. To fill some of these gaps, this thesis elucidates important determinants of ion selectivity in proteins, involved in the transport of Cl-, I-, F-, HCO3-, and CO32- from several different protein families. In chapter 2, we studied carbonate and bicarbonate binding in proteins, through a survey of the preferred coordination numbers, amino acid and atom type composition of the coordination spheres of HCO3- and CO32- in bicarbonate- and carbonate-bound proteins deposited in the Protein DataBank. Some interesting differences in the coordination patterns for HCO3- and CO32- were highlighted. In chapter 3, we evaluated the thermodynamics of anion binding in a series of binding sites constructed from real anion-binding proteins using Quantum Mechanics calculations. The importance of the thermodynamics of binding for the anion selectivity was discussed. In chapters 4 and 5, we assessed the anion dynamics in proteins from the SLC4 and SLC5 families, respectively, using various computational methods, which led to the identification of putative binding sites in the protein matrix for sodium and the transported anions. Our work contributes to the increasing body of knowledge on anion-protein interactions and provides important mechanistic insights into the anion transport and selectivity of several proteins of interest for human physiology.
- ItemOpen AccessAnti-inflammatory Properties of Retinoids and Retinoid Derivatives in Innate Immune Cells(2013-09-06) Duquette, Stephanie; Buret, AndreClearance of apoptotic neutrophils following infection is critical for the resolution of inflammation. Despite demonstrating immunomodulatory properties, the effects of retinoids in neutrophils in the context of an inflammatory response remain unknown. The objective of this project was to evaluate the immunomodulatory properties of retinoic acid (RA) and oxidatively transformed carotene-β (OxC-β) in a model of Mannheimia haemolytica-induced bovine respiratory disease, which is characterized by severe inflammation. Results in vitro and in vivo demonstrate that both RA and OxC-β induce apoptosis in bovine neutrophils and that this phenomenon promotes efferocytosis in bovine macrophages. These effects occur in the absence of antimicrobial properties and any alteration in neutrophil function. The induction of neutrophil apoptosis and their subsequent phagocytic clearance by macrophages are key mechanisms that promote the resolution of inflammation following infection. As such, RA and OxC-β may represent novel nutraceuticals that confer anti-inflammatory benefits following infection.
- ItemOpen AccessAntimicrobial Resistance in Foot rot Bacteria(2014-05-23) Kim, Eunjee; Morck, DouglasThis study set out to determine tetracycline and erythromycin resistance profiles and detect various tetracycline (tetB, tetC, tetL, tetM, tetQ and tetW) or erythromycin (ermA, ermB and ermF) resistance determinants from clinical isolates of bovine foot rot bacteria isolated from affected animals in Alberta. Using agar dilution assay, we determined that 82% (32/39) of Fusobacterium necrophorum were tetracycline resistant and 62% (24/39) were classified intermediate to erythromycin. Approximately 82% (18/22) and 91% (20/22) of Prevotella melaninogenica was sensitive to tetracycline and erythromycin respectively, For Porphyromonas levii, 88% (21/24) were sensitive to tetracycline and 100% (24/24) were erythromycin sensitive. Polymerase chain reaction amplification revealed that tetC, tetL, tetM and ermF were commonly detected in F.necrophorum, and tetQ in Pr.melaninogenica and P.levii. Special resistance determinant patterns were not observed in antibiotic resistant strains compared to sensitive isolates in all three pathogens because as high as 90% of resistance determinants were detected in both resistant and sensitive strains of bacteria.
- ItemOpen AccessArabidopsis thaliana D group MPKs interactions with RLPH2 and PP1(2021-01-08) Toth, Ryan B.; Moorhead, Greg; Samuel, Marcus; Ng, Kenneth; MacDonald, Justin; Moorhead, GregMitogen activated protein kinases (MPK) are key members of the MPK cascade, a pathway that results in an immediate response from (external) stimuli such as infection, cold, or drought. The activation of an MPK relies its activation loop being phosphorylated on a threonine and tyrosine residue. A unique bacteria-like phosphatase Rhizobiales-like phosphatase 2 (RLPH2), which we bioinformatically characterized as a serine/threonine phosphatase, has been discovered to act as a tyrosine phosphatase. In this thesis, data monitoring RLPH2s dephosphorylation of different peptides derived from MPK activation loops would reveal a preference for the dephosphorylation of D group MPKs over non-D group MPKs, and indicate that the central residue in the TXY motif plays a key role in RLPH2 substrate determination. Additionally, RLPH2 would show a preference for the in vitro dephosphorylation of MPK9 (a D group MPK) over MPK3 (a non-D group MPK). An engaging observation is that right next to the TxY motif in D group MPKs is a putative Protein Phosphatase 1 (PP1) regulatory protein binding RVxF motif. This motif is not present in any other characterized MPK, suggesting an interaction between PP1 and D group MPKs. In this thesis, PP1 protein binding assays with MPK9 WT and an MPK9 RASA mutant would show binding occurs between MPK9 and PP1 through the RVXF motif in vitro. Peptide and protein substrate dephosphorylation assays with PP1 would show threonine dephosphorylation on MPK9 and the effect the RVXF motif plays on this interaction. In summary, the specificity of RLPH2 as a D group MPK regulator was examined along with the interaction between PP1 and the D group MPKs via the RVXF motif, with mention of a potential relationship between these three interactions.
- ItemOpen AccessArabidopsis thaliana Protein Phosphatase 1: A Cell Signalling Engine with Many Keys(2016) Sieben, Nicolas Ambrose; Moorhead, Gregory; Ling, Chang-Chun; Muench, Douglas; Prenner, ElmarThroughout all domains of life the strict regulation of signalling pathways allows for the proper control of cellular processes through the action of protein kinases and protein phosphatases. In the model plant Arabidopsis thaliana, one of the major protein phosphatases, PP1, represents a critical regulator of many signalling pathways. Type one phosphatases (PP1) use regulatory subunits to target dephosphorylation and associate with these regulatory proteins via a highly conserved ‘RVxF’ motif. The regulators of PP1 in Arabidopsis are largely unidentified. Using Arabidopsis stable transformant plant extracts, the regulators of PP1 have been identified. Of the previously known PP1 regulators, GEM is a known root hair differentiation checkpoint protein and contains the PP1 docking motif ‘RVxF’. Using a biophysical approach, I have validated the PP1:GEM interaction through the ‘RVxF’ motif. Finally GEM contains a GRAM domain and the analysis of phosphotidyl-inositol lipid binding to this domain was examined.
- ItemOpen AccessAssessing Temperature Tolerance in Ninespine Stickleback (Pungitius pungitius) in Response to Climate Change(2018-07-13) Tufts, Teresa; Rogers, Sean M.; Yeaman, Sam; Vamosi, Jana C.; Murray, Maribeth S.Temperature is the most important environmental factor influencing fish abundance and distribution but, as temperatures rise globally, the consequences for fishes remains largely unknown. I used populations of ninespine stickleback (Pungitius pungitius) from the northern and southern parts of their range in a common environment to characterize thermal traits in association with varying temperatures. Cold tolerance differed between populations but heat tolerance did not. Furthermore, juveniles demonstrated less tolerance for temperature extremes. When incubated at elevated temperatures, populations exhibited higher plasticity in cold tolerance compared to heat tolerance. Plasticity in heat tolerance was associated with a number of trade-offs. The data suggest that cold tolerance is locally adapted and phenotypically plastic while heat tolerance is conserved and phenotypically restricted in this species. My thesis highlights the need for the inclusion of various life stages, time scales, and biotic interactions in climate change research to better predict its impacts on fishes.