• Identification and characterization of a Catharanthus roseus mutant altered in monoterpenoid indole alkaloid biosynthesis

      Thamm, Antje MK; Department of Biological Sciences (Brock University, 2014-09-09)
      The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially important horticultural flower species and is the only source for several pharmaceutically valuable monoterpenoid indole alkaloids (MIAs), including the powerful antihypertensive ajmalicine and the antineoplastic agents vincristine and vinblastine. While biosynthesis of MIA precursors has been elucidated, conversion of the common MIA precursor strictosidine to MIAs of different families, for example ajmalicine, catharanthine or vindoline, remains uncharacterized. Deglycosylation of strictosidine by the key enzyme Strictosidine beta-glucosidase (SGD) leads to a pool of uncharacterized reaction products that are diverted into the different MIA families, but the downstream reactions are uncharacterized. Screening of 3600 EMS (ethyl methane sulfonate) mutagenized C. roseus plants to identify mutants with altered MIA profiles yielded one plant with high ajmalicine, and low catharanthine and vindoline content. RNA sequencing and comparative bioinformatics of mutant and wildtype plants showed up-regulation of SGD and the transcriptional repressor Zinc finger Catharanthus transcription factor (ZCT1) in the mutant line. The increased SGD activity in mutants seems to yield a larger pool of uncharacterized SGD reaction products that are channeled away from catharanthine and vindoline towards biosynthesis of ajmalicine when compared to the wildtype. Further bioinformatic analyses, and crossings between mutant and wildtype suggest a transcription factor upstream of SGD and ZCT1 to be mutated, leading to up-regulation of Sgd and Zct1. The crossing experiments further show that biosynthesis of the different MIA families is differentially regulated and highly complex. Three new transcription factors were identified by bioinformatics that seem to be involved in the regulation of Zct1 and Sgd expression, leading to the high ajmalicine phenotype. Increased cathenamine reductase activity in the mutant converts the pool of SGD reaction products into ajmalicine and its stereoisomer tetrahydroalstonine. The stereochemistry of ajmalicine and tetrahydroalstonine biosynthesis in vivo and in vitro was further characterized. In addition, a new clade of perakine reductase-like enzymes was identified that reduces the SGD reaction product vallesiachotamine in a stereo-specific manner, characterizing one of the many reactions immediately downstream of SGD that determine the different MIA families. This study establishes that RNA sequencing and comparative bioinformatics, in combination with molecular and biochemical characterization, are valuable tools to determine the genetic basis for mutations that trigger phenotypes, and this approach can also be used for identification of new enzymes and transcription factors.
    • Identification and characterization of retinoic acid-induced morphological and electrophysiological changes in an invertebrate nervous system

      Vesprini, Nicholas; Department of Biological Sciences (Brock University, 2012-07-31)
      The vitamin A metabolite, retinoic acid (RA) is known to play an important role in the development, patterning and regeneration of nervous tissue, both in the embryo and in the adult. Classically, RA is known to mediate the transcription of target genes through the binding and activation ofits nuclear receptors: the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Recently, mounting evidence from many animal models has implicated a number of RA-mediated effects operating independently of gene transcription, and thus highlights nove~ nongenornic actions of RA. For example, recent work utilizing cultured neurons from the pond snaa Lymnaea stagnalis, has shown that RA can elicit a regenerative response, growth cone turning, independently of "classical" transcriptional activation While this work illustrates a novel regeneration-inducing effect in culture, it is currently -unknown whether RA also induces regeneration in situ. This study has sought to determine RA's regenerative effucts at the morphological and molecular levels by utilizing an in situ approach focusing on a single identified dopaminergic neuron which possesses a known "mapped" morphology within the CNS. These studies show, for the first time in an invertebrate, that RA can increase neurite outgrowth of dopaminergic cells that have undergone a nerve-crush injury. Utilizing Western blot analysis, it was shown that this effect appears to be independent of any changes in whole CNS expression levels of either the RAR or RXR. Additionally, utilizing immunohistochemistry, to examine protein localization, there does not appear to be any obvious changes in the RXR expression level at the crush site. Changes in cell morphology such as neurity extension are known to be modulated by changes in neuronal firing activity. It has been previously shown that exposure to RA over many days can lead to changes in the electrophysiological properties of cultured Lymnaea neurons; however, no studies have investigated whether short-term exposure to RA can elicit electrophysiological changes and/or changes in firing pattern of neurons in Lymnaea or any other species. The studies performed here show, for the first time in any species, that short-tenn treatment with RA can elicit significant changes in the firing properties of both identified dopaminergic neurons and peptidergic neurons. This effect appears to be independent of protein synthesis, activation of protein kinase A or phospholipase C, and calcium influx but is both dose-dependent and isomer-dependent. These studies provide evidence that the RXR, but not RAR, may be involved, and that intracellular calcium concentrations decrease upon RAexposure with a time course, dose-dependency and isomer-dependency that coincide with the RA-induced electrophysiological changes. Taken together, these studies provide important evidence highlighting RA as a multifunctional molecule, inducing morphological, molecular and electrophysiological changes within the CNS, and highlight the many pathways through which RA may operate to elicit its effects.
    • Identification of novel retinoid receptors and their roles in vertebrate and invertebrate nervous systems

      Charter, Christopher J; Department of Biological Sciences (Brock University, 2012-07-30)
      In vertebrates, signaling by retinoic acid (RA) is known to play an important role in embryonic development, as well as organ homeostasis in the adult. In organisms such as adult axolotls and newts, RA is also important for regeneration of the CNS, limb, tail, and many other organ systems. RA mediates many of its effects in development and regeneration through nuclear receptors, known as retinoic acid receptors (RARs) and retinoid X receptors (RXRs). This study provides evidence for an important role of the RA receptor, RAR~2, in ,( '. regeneration ofthe spinal cord and tail of the adult newt. It has previously been proposed that the ability of the nervous system to regenerate might depend on the presence or absence of this RAR~2 isoform. Here, I show for the very first time, that the regenerating spinal cord of the adult newt expresses this ~2 receptor isoform, and inhibition of retinoid signaling through this specific receptor with a selective antagonist inhibits tail and spinal cord regeneration. This provides the first evidence for a role of this receptor in this process. Another species capable of CNS ~~generation in the adult is the invertebrate, " Lymnaea stagnalis. Although RA has been detected in a small number of invertebrates (including Lymnaea), the existence and functional roles of the retinoid receptors in most invertebrate non-chordates, have not been previously studied. It has been widely believed, however, that invertebrate non-chordates only possess the RXR class of retinoid receptors, but not the RARs. In this study, a full-length RXR cDNA has been cloned, which was the first retinoid receptor to be discovered in Lymnaea. I then went on to clone the very first full-length RAR eDNA from any non-chordate, invertebrate species. The functional role of these receptors was examined, and it was shown that normal molluscan development was altered, to varying degrees, by the presence of various RXR and RAR agonists or antagonists. The resulting disruptions in embryogenesis ranged from eye and shell defects, to complete lysis of the early embryo. These studies strongly suggest an important role for both the RXR and RAR in non-chordate development. The molluscan RXR and RAR were also shown to be expressed in the adult, nonregenerating eNS, as well as in individual motor neurons regenerating in culture. More specifically, their expression displayed a non-nuclear distfibution, suggesting a possible non-genomic role for these 'nuclear' receptors. It was shown that immunoreactivity for the RXR was present in almost all regenerating growth cones, and (together with N. Farrar) it was shown that this RXR played a novel, non-genomic role in mediating growth cone turning toward retinoic acid. Immunoreactivity for the novel invertebrate RAR was also found in the regenerating growth cones, but future work will be required to determine its functional role in nerve cell regeneration. Taken together, these data provide evidence for the importance of these novel '. retinoid receptors in development and regeneration, particularly in the adult nervous system, and the conservation of their effects in mediating RA signaling from invertebrates to vertebrates.
    • Impact of different irrigation strategies on grapes and wine quality of four grapevine cultivars (Vitis sp.) in cool climate conditions. An investigation into the relationships among ABA, water status, grape cultivar and wine quality

      Balint, Gabriel; Department of Biological Sciences (Brock University, 2012-04-04)
      Niagara Peninsula of Ontario is the largest viticultural area in Canada. Although it is considered to be a cool and wet region, in the last decade many water stress events occurred during the growing seasons with negative effects on grape and wine quality. This study was initiated to understand and develop the best strategies for water management in vineyards and those that might contribute to grape maturity advancement. The irrigation trials investigated the impact of time of initiation (fruit set, lag phase and veraison), water replacement level based on theoretical loss through crop evapotranspiration (ETc; 100,50 and 25%) and different irrigation strategies [partial root zone drying (PRD) versus regulated deficit irrigation (RD!)] on grape composition and wine sensory profiles. The irrigation experiments were conducted in a commercial vineyard (Lambert Vineyards Inc.) located in Niagara-on-the-Lake, Ontario, from 2005 through 2009. The two experiments that tested the combination of different water regimes and irrigation time initiation were set up in a randomized block design as follows: Baco noir - three replicates x 10 treatments [(25%, 50% and 100% of ETc) x (initiation at fruit set, lag phase and veraison) + control]; Chardonnay - three replicates x seven treatments [(25%, 50% and 100% of ETc) x (initiation at fruit set and veraison) + control]. The experiments that tested different irrigation strategies were set up on two cultivars as follows: Sauvignon blanc - four replicates x four treatments [control, fully irrigated (100% ETc), PRD (100% ETc) and RDI (25% ETc)]; Cabemet Sauvignon - four replicates x five treatments [control, fully irrigated (100% ETc), PRD (100% ETc), RDI (50% ETc) and RDI (25% ETc)]. The controls in each experiment were nonirrigated. The irrigation treatments were compared for many variables related to soil water status, vine physiology, berry composition, wine sensory profile, and hormone composition [(abscisic acid (ABA) and its catabolites]. Soil moisture profile was mostly affected by irrigation treatments between 20 and 60 em depth depending on the grapevine cultivar and the regime of water applied. Overall soil moisture was consistently higher throughout the season in 100 and 50% ETc compare to the control. Transpiration rates and leaf temperature as well as shoot growth rate were the most sensitive variables to soil water status. Drip irrigation associated with RDI treatments (50% ETc and 25% ETc) had the most beneficial effects on vine physiology, fruit composition and wine varietal typicity, mainly by maintaining a balance between vegetative and reproductive parts of the vine. Neither the control nor the 100 ETc had overall a positive effect on grape composition and wine sensory typicity. The time of irrigation initiation affected the vine physiology and grape quality, the most positive effect was found in treatments initiated at lag phase and veraison. RDI treatments were overall more consistent in their positive effect on grape composition and wine varietal typicity comparing to PRD treatment. The greatest difference between non-irrigated and irrigated vines in most of the variables studied was found in 2007, the driest and hottest season of the experimental period. Soil water status had a greater and more consistent effect on red grapevine cultivars rather than on white winegrape cultivars. To understand the relationships among soil and plant water status, plant physiology and the hormonal profiles associated with it, abscisic acid (ABA) and its catabolites [phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy-ABA (TOH-ABA), 8' -hydroxy-ABA, neophaseic acid and abscisic acid glucose ester (ABA-GE)] were analyzed in leaves and berries from the Baco noir and Chardonnay irrigation trials over two growing seasons. ABA and some of its catabolites accurately described the water status in the vines. Endogenous ABA and some of its catabolites were strongly affected in Baco noir and Chardonnay by both the water regime (i.e. ET level) and timing of irrigation initiation. Chardonnay grapevines produced less ABA in both leaves and berries compared to Baco noir, which indicated that ABA synthesis is also cultivar dependant. ABA-GE was the main catabolite in treatments with high water deficits, while PA and DPA were higher in treatments with high water status, suggesting that the vine produced more ABA-GE under water deficits to maintain rapid control of the stomata. These differences between irrigation treatments with respect to ABA and catabolites were particularly noticeable in the dry 2007 season. Two trials using exogenous ABA investigated the effect of different concentrations of ABA and organs targeted for spraying, on grape maturation and berry composition of Cabemet Sauvignon grapevines, in two cool and wet seasons (2008-2009). The fIrst experiment consisted of three replicates x three treatments [(150 and 300 mg/L, both applications only on clusters) + untreated control] while the second experiment consisted in three replicates x four treatments [(full canopy, only clusters, and only leaves sprayed with 300 ppm ABA) + untreated control]. Exogenous ABA was effective in hastening veraison, and improving the composition of Cabemet Sauvignon. Ability of ABA to control the timing of grape berry maturation was dependant on both solution concentration and the target organ. ABA affected not only fruit composition but also yield components. Berries treated with ABA had lower weight and higher skin dry mass, which constitutes qualitative aspects desired in the wine grapes. Temporal advancement of ripening through hormonal control can lead to earlier fruit maturation, which is a distinct advantage in cooler areas or areas with a high risk of early frost occurrence. Exogenous ABA could provide considerable benefits to wine industry in terms of grape composition, wine style and schedule activities in the winery, particularly in wet and cool years. These trials provide the ftrst comprehensive data in eastern North America on the response of important hybrid and Vitis vinifera winegrape cultivars to irrigation management. Results from this study additionally might be a forward step in understanding the ABA metabolism, and its relationship with water status. Future research should be focused on ftnding the ABA threshold required to trigger the ripening process, and how this process could be controlled in cool climates.
    • Influence of adolescent social instability stress on the intake of ethanol and sucrose in a rodent model

      de Lima Marcolin, Marina; Department of Biological Sciences
      Adolescence is a sensitive period in which the effects of stress and alcohol can have long-lasting impacts. Social instability stress in adolescent rats (SS; postnatal day 30-45, daily 1 hour isolation + new cage partner) alters behavioural responses to psychostimulants and increases anxiety-like behaviour, but differences in voluntary consumption of natural and drug rewards are unknown. The main goal of my thesis was to investigate the effects of adolescent social instability stress (SS) on immediate and long-lasting changes on reward-related behaviours in male rats using voluntary alcohol intake paradigms. Another goal was to investigate the influence of social context on the propensity to drink alcohol, as well as the influence of these factors on sucrose intake. In chapter 2, I found that adolescent SS increased alcohol intake irrespective of social context, and adolescents drank more alcohol than adults. The intake of sucrose was not altered by stress, except during context of competition. In chapter 3, I found that history of alcohol drinking reduced synaptic plasticity markers in the dorsal hippocampus and prefrontal cortex, and this reduction was sometimes further reduced by SS. The propensity to drink alcohol was found not to differ between SS and CTL rats in the first experiment, and reduced among SS rats in the second experiment. After nine days of alcohol absence, the propensity to drink alcohol was not increased by previous alcohol access, and SS increased intake only in alcohol-naïve rats. History of alcohol drinking reduced anxiety-like behaviours and blunted SS-induced reduction in social interactions. Both SS and alcohol decreased corticosterone levels at baseline and after fear recall without changing freezing behaviour. My findings indicate that using a model of mild social stressor can have great impact on adolescent rats, but moderate effects in adult rats. The behavioural changes caused by stress can be enhanced later in life by history of alcohol drinking, but that does not necessarily cause an increase in the propensity to drink during adulthood, as other studies have shown. Adolescent stressed rats drink more alcohol than other groups, but they don’t seem to continue drinking more when they reach adulthood. These results indicate that the effects of social instability stress are transient in regards to propensity to drink, and can be the basis for alterations caused by both alcohol and stress.
    • Intracellular antioxidant and DNA repair enzymes as correlates of stress resistance and longevity in vertebrates

      Page, Melissa Maire; Department of Biological Sciences (Brock University, 2011-10-14)
      In animals, both stress resistance and longevity appear to be influenced by the insulin/insulin-like growth factor-l signaling (lIS) pathway, the basic organization of which is highly conserved from invertebrates to vertebrates. Reduced lIS or genetic disruption of the lIS pathway leads to the activation of forkhead box transcription factors, which is thought to upregulate the expression of genes involved in enhancing stress resistance, including perhaps key antioxidant enzymes as well as DNA repair enzymes. Enhanced antioxidant and DNA repair capacities may underlie the enhanced cellular stress resistance observed in long-lived animals, however little data is available that directly supports this idea. I used three. experimental approaches to test the association of intracellular antioxidant and DNA base excision repair (BER) capacities with stress resistance and longevity: (1) a comparison of multiple vertebrate endotherm species of varying body masses and longevities; (2) a comparison of long-lived Snell dwarf mice and their normallittermates; and (3) a comparison of hypometabolic animals undergoing hibernation or estivation with their active counterparts. The activities of the five major intracellular antioxidant enzymes as well as the two rate-limiting enzymes in the BER pathway, apurininc/apyrimidinic (AP) endonuclease and polymerase ~, were measured. These measurements were performed in one or more of the following: (1) cultured dermal fibroblasts; (2) brain tissue; (3) heart tissue; (4) liver tissue. My results indicate that antioxidant enzymes are not universally upregulated in association with enhanced stress resistance and longevity. I also did not find that BER enzyme activity was positively correlated with longevity, in an inter-species context, though there was evidence for enhanced BER in long-lived Snell dwarf mice. Thus, while there were instances in which enhanced antioxidant and BER enzyme activities were associated with increased stress resistance and/or longevity, this was not universally the case, indicating that other mechanisms must be involved. These results suggest the need to re-examine existing 'oxidative stress' hypotheses of longevity and probe further into the molecular physiology of longevity to discover its mechanistic basis.
    • Investigating the Effect of Cell Culture Compositions on Mitochondrial Metabolism, Dynamics, and Transcriptome and Proteome of cells

      Moradi, Fereshteh; Department of Biological Sciences
      The phytoestrogen Resveratrol (RES) is a natural polyphenol that has been detected in more than 70 plant species. RES has structural similarity to mammalian estrogens and can bind to estrogen receptors, eliciting genomic and non-genomic effects. Both RES and physiological estrogens like 17-β-estradiol (E2) have wide-ranging effects on mitochondria. In this thesis, I began by investigating RES’s effects on mitochondrial network dynamics (Chapter 2) and discovered a pro-fusion activity apparently mediated by the mitofusin enzyme Mfn2. RES stimulated mitochondrial network hyper-fusion morphology in all three cell lines investigated (C2C12 (mouse myoblast), PC3 (prostate cancer), and MEFs (mouse embryonic fibroblast)), but the effect was absent in Mfn2-null MEFs. As this work was being completed; several research groups introduced ‘physiologic cell culture media’ that are modeled on the human plasma metabolome. I co-authored a study (not in this thesis) demonstrating that RES’s effects on mitochondrial dynamics are dependent on cell culture conditions. To follow up on this, I investigated whether E2’s mitochondrial effects might also be dependent on the cell culture environment, and showed conclusively that this is indeed the case, using C2C12 cells as a model system (Chapter 3). These results and those published by others in 2017-2019 suggested that medium composition can profoundly affect cellular functions. In Chapter 4, I followed this up by studying how culture conditions affect mitochondrial bioenergetics and network morphology using four cancer cell lines and showed that this is a significant issue. Finally, to gain a more complete understanding of this phenomenon, I completed a full transcriptomic and proteomic analysis of media effects using MCF7 breast cancer cells as a model (Chapter 5). I showed that hundreds of transcripts and proteins are affected according to culture conditions. Taken together, the results presented in this thesis emphasize the significant extent to which the cell culture environment affects experimental outcomes, particularly with respect to mitochondrial bioenergetics and dynamics. This information contributes to the development of cell culture experiments providing results that can be translated in vivo.
    • Investigating the role of apoptosis regulator EndoG on exogenous DNA uptake, stability, replication and recombination

      Misic, Vanja; Department of Biological Sciences (Brock University, 2013-11-05)
      Endonuclease G (EndoG) is a well conserved mitochondrial nuclease with dual lethal and vital roles in the cell. It non-specifically cleaves endogenous DNA following apoptosis induction, but is also active in non-apoptotic cells for mitochondrial DNA (mtDNA) replication and may also be important for replication, repair and recombination of genomic DNA. The aim of our study was to examine whether EndoG exerts similar activities on exogenous DNA substrates such as plasmid DNA (pDNA) and viral DNA vectors, considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA and nuclease activity in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four day time-course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus (a cationic liposome transfection reagent), targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances it may non-specifically cleave intracellular DNA regardless of its origin. To investigate possible effects of EndoG on viral DNA vectors, we constructed and evaluated AdsiEndoG, a first generation adenovirus (Ad5 ΔE1) vector encoding a shRNA directed against EndoG mRNA, along with appropriate Ad5 ΔE1 controls. Infection of HeLa cells with AdsiEndoG at a multiplicity of infection (MOI) of 10 p.f.u./cell resulted in an early cell proliferation defect, absent from cells infected at equivalent MOI with control Ad5 ΔE1 vectors. Replication of Ad5 ΔE1 DNA was detected for all vectors, but AdsiEndoG DNA accumulated to levels that were 50 fold higher than initially, four days after infection, compared to 14 fold for the next highest control Ad5 ΔE1 vector. Deregulation of the cell cycle by EndoG depletion, which is characterized by an accumulation of cells in the G2/M transition, is the most likely reason for the observed cell proliferation defect. The enhanced replication of AdsiEndoG is consistent with this conclusion, as Ad5 ΔE1 DNA replication is intimately related to cell cycling and prolongation or delay in G2/M greatly enhances this process. Furthermore, infection of HeLa with AdsiEndoG at MOI of 50 p.f.u./cell resulted in an almost complete disappearance of viable, adherent tumour cells from culture, whereas almost a third of the cells were still adherent after infection with control Ad5 ΔE1 vectors, relative to the non-infected control. Therefore, targeting of EndoG by RNAi is a viable strategy for improving the oncolytic properties of first generation adenovirus vectors. In addition, AdsiEndoG-mediated knockdown of EndoG reduced homologous recombination between pDNA substrates in HeLa cells. The effect was modest but, nevertheless demonstrated that the proposed role of EndoG in homologous recombination of cellular DNA also extends to exogenous DNA substrates.
    • Investigating the Role of MicroRNAs in Regeneration and Axonal Pathfinding

      Walker, Sarah; Department of Biological Sciences
      During both development and regeneration, axons must navigate through a complex and changing environment to reach their proper synaptic target. To do so, axons utilize a specialized structure, the growth cone, which senses and interprets guidance cues in its surrounding environment to change the direction of axonal outgrowth. MicroRNAs, which regulate mRNA translation, have recently been shown to regulate both neurite outgrowth and growth cone guidance in response to classical guidance cues during vertebrate development. However, little is known of their regulation of neuronal regeneration in an invertebrate. Thus, the main aim of this thesis was to study the role of microRNAs during CNS regeneration of the pond snail, Lymnaea stagnalis. Specifically, I determined the expression patterns and relative abundance of microRNAs in the regenerating CNS in response to retinoic acid (RA). Using miRNA-Sequencing, I identified one neuronally enriched microRNA, miR-124, that was up-regulated in RA-induced regenerating CNS. Using PCR and in situ hybridization, I characterized its distribution in the snail CNS, and discovered it shared similar expression patterns to that of vertebrates. In cell culture, I found miR-124 was abundant within regenerating motorneurons and was localized to their growth cones. I next determined that miR-124 contributed to RA-induced growth cone turning behaviour. During attractive growth cone turning to RA, the abundance and distribution of miR-124 was altered, in both a cue and context-dependent manner. Finally, I demonstrated that miR-124 targeted the Rho kinase, ROCK, during turning responses to RA, likely to promote the formation of a neurite shaft, or to maintain growth cone polarity. Together, these findings provide the first evidence for a role of microRNAs in mediating growth cone behaviours to RA in regenerating motorneurons.
    • Metarhizium robertsii interactions with Phaseolus vulgaris (Haricot Bean)

      Hu, Shasha; Department of Biological Sciences
      Metarhizium is an insect pathogenic fungus, as well as a plant root symbiont. During symbiotic interactions, it can benefit the plant by improving plant growth, antagonizing plant pathogens and herbivores, and enhancing plant tolerance to abiotic stresses. In this thesis, the interactions between Metarhizium robertsii and Phaseolus vulgaris (haricot bean) were studied from two aspects. First, a phenotypically degenerated (low conidia production) strain of Metarhizium was serially passaged through bean plant. Second, the immune responses of haricot bean during endophytic colonization were assessed. Commercial application of Metarhizium for insect biocontrol requires optimal production of conidia as infective propagules. It was demonstrated that conidial production and virulence of phenotypically degenerated Metarhizium were restored by serial passages through bean roots, as well as switchgrass roots, and wax moth larvae. A decrease in the expression of fungal DNA methyltransferase was observed in the phenotypically degenerated Metarhizium strain through bean passages. Whole genome bisulfite sequencing analysis showed differences in the distribution of differentially methylated regions in the degenerated and subsequently recovered strains. Metarhizium can antagonize the plant pathogen, Fusarium solani f. sp. phaseoli during bean root colonization. Using comprehensive plant hormone analysis, transcriptional expression, and stomatal size analysis, bean immune responses to colonization by Metarhizium and/or Fusarium were assessed. In comparison to un-inoculated bean, root colonization by Metarhizium resulted in reduction of abscisic acid (ABA), increased stomatal size, and decreased expression of plant immunity genes in bean leaves, which is different from those in bean colonized by Fusarium. Furthermore, exogenous application of ABA resulted in reduction of bean root colonization by Metarhizium but increased colonization by Fusarium, compared to corresponding plants without ABA application. Therefore, ABA was implicated in differential responses of bean plants to root colonization by Metarhizium and Fusarium. In conclusion, this thesis provided new insights into the study of the interactions between Metarhizium and haricot bean. Some novel findings were that fungal DNA methyltransferase was implicated in the recovery of phenotypically degenerated Metarhizium and a plant hormone, abscisic acid was implicated in differential interactions of endophytic colonization by Metarhizium when compared to a pathogenic interaction by Fusarium.

      Botezatu, Andreea Ioana; Department of Biological Sciences (Brock University, 2013-11-05)
      Methoxypyrazines are aroma active compounds found in many wine varietals. These compounds can be of either grape-derived nature or can be introduced into wines via Coccinellidae beetles. Regardless of their origin, methoxypyrazines can have either a beneficial role for wine quality, contributing to the specificity of certain wine varietals (Cabernet sauvignon, Cabernet franc, Sauvignon blanc) or a detrimental role, particularly at higher concentrations, resulting in overpowering green, unripe and herbaceous notes. When methoxypyrazines of exogenous nature are responsible for these unpleasant characteristics, wines are considered to be affected by what is generally known as Ladybug taint (LBT). This is work is a collection of studies seeking to create a sensitive analytical method for the detection and quantification of methoxypyrazines in wines; to investigate the role of different Coccinellidae species in the tainting of wines with LBT and identify the main compounds in ladybug tainted wines responsible for the typical green herbaceous characteristics; to determine the human detection threshold of 2,5-dimethyl-3-methoxypyrazine in wines as well as investigate its contribution to the aroma of wines; and finally to survey methoxypyrazine concentrations in a large set of wines from around the world. In the first study, an analytical method for the detection and quantitation of methoxypyrazines in wines was created and validated. The method employs multidimensional Gas Chromatography coupled with Mass Spectrometry to detect four different methoxypyrazines (2,5-dimethyl-3-methoxypyrazine, isobutyl methoxypyrazine, secbutyl methoxypyrazine and isopropyl methoxypyrazines) in wine. The low limits of detection for the compounds of interest, improved separation and isolation capabilities, good validation data, as well as the ease of use recommend this method as a good alternative to the existing analytical methods for methoxypyrazine detection in wine. In the second study the capacity of two Coccinellidae species, found in many wine regions – Harmonia axyridis and Coccinella septempunctata - to taint wines is evaluated. Coccinella septempunctata is shown to be as capable as causing LBT in wines as Harmonia axyridis. Dimethyl methoxypyrazine, previously thought to be of exogenous nature only (from Coccinellidae haemolymph), is also detected in control (untainted) wines. The main odor active compounds in LBT wines are investigated through Aroma Extract Dilution Assay. These compounds are identified as isopropyl methoxypyrazine, sec- and iso- butyl methoxypyrazine. In the third study, the human detection threshold for dimethyl methoxypyrazine in wine is established to be 31 ng/L in the orthonasal modality and 70 ng/L retronasally. After wines spiked with various amounts of dimethyl methoxypyrazine are evaluated sensorally, dimethyl methoxypyrazine causes significant detrimental effects to wine aroma at a concentration of 120 ng/L. The final study examines methoxypyrazine (dimethyl methoxypyrazine, isopropyl methoxypyrazine, secbutyl methoxypyrazine and isobutyl methoxypyrazine) concentrations in 187 wines from around the world. Dimethyl methoxypyrazine is detected in the majority of the red wines tested. Data are interpreted through statistical analyses. A new measure for predicting greenness/herbaceousness in wines - methoxypyrazine “total impact factor” is proposed.
    • Molecular ecology and social evolution of the eastern carpenter bee, Xylocopa virginica

      Vickruck, Jessica L; Department of Biological Sciences
      Bees are extremely valuable models in both ecology and evolutionary biology. Their link to agriculture and sensitivity to climate change make them an excellent group to examine how anthropogenic disturbance can affect how genes flow through populations. In addition, many bees demonstrate behavioural flexibility, making certain species valuable models with which to study the evolution of social groups. This thesis studies the molecular ecology and social evolution of one such bee, the eastern carpenter bee, Xylocopa virginica. As a generalist native pollinator that nests almost exclusively in milled lumber, anthropogenic disturbance and climate change have the power to drastically alter how genes flow through eastern carpenter bee populations. In addition, X. virginica is facultatively social and is an excellent organism to examine how species evolve from solitary to group living. Across their range of eastern North America, X. virginica appears to be structured into three main subpopulations: a northern group, a western group and a core group. Population genetic analyses suggest that the northern and potentially the western group represent recent range expansions. Climate data also suggest that summer and winter temperatures describe a significant amount of the genetic differentiation seen across their range. Taken together, this suggests that climate warming may have allowed eastern carpenter bees to expand their range northward. Despite nesting predominantly in disturbed areas, eastern carpenter bees have adapted to newly available habitat and appear to be thriving. This is in marked contrast to many other bee species, particularly in the genus Bombus, who appear unable to shift their ranges along with climate change. Facultatively social organisms are interesting species to study the evolution of social groups, and the remaining chapters address questions of sociality in X. virginica. I used observation nests and genetic relatedness to examined how females behave towards one another in the spring prior to the establishment of dominance hierarchies in social nests. In spring, females directed fewer aggressive behaviours and more cooperative behaviours towards familiar rather than related individuals, indicating that females use nestmate recognition rather that kin recognition when interacting with conspecifics. Overwintering groups often contain both related and unrelated individuals, indicating that many bees interacting with one another in the fall prior to overwintering may be unrelated, emphasizing the importance of recognizing nestmates. Within social carpenter bee nests three different types of female have been described: primary, secondary and tertiary. Primary females are the dominant foragers and egg layers in the nest while secondary and tertiary females appear to join a reproductive queue behind the primary. To understand the nature and flexibility of this reproductive queue I performed removal experiments across three different years. This study showed that secondary females always assumed the role of replacement primary, while tertiary females rarely opted to forage and reproduce even if they were the only female in the nest. Removal experiments demonstrated that social groups in X. virginica are complex and comprise two different reproductive strategies (breed in the current year or delay reproduction) as well as form dominance hierarchies among primary and secondary females. Several tertiary females were able to become primary or solitary females in their second summer, providing evidence for how each type of female may have evolved in social nests. Finally, I examined how competition influences the evolution and maintenance of social groups in eastern carpenter bees. In conditions of high population density significantly more social nests were present in the population, indicating that competition for limiting nesting resources drives individuals together into social groups. Within social groups relatedness was low, and siblings actually dispersed away from one another to other nests in the population, reducing competition among kin. Eastern carpenter bees appear to demonstrate an interesting evolutionary route to sociality, where very high levels of competition among kin lead to dispersal, while limited nesting substrate forces individuals back into unrelated social groups. While predicted by kin selection, social groups of this nature are previously undescribed in the Hymenoptera, and further study of eastern carpenter bees can provide novel insights into alternate routes to sociality.
    • Phage-mediated biological control of Erwinia amylovora: The role of CRISPRs and exopolysaccharide

      Yagubi, Abdelbaset; Department of Biological Sciences
      Fire blight, caused by bacterium Erwinia amylovora, is a very serious disease affecting apple, pear and other fruit plants. The development of phage-based biopesticides is currently in progress in our lab. Emergence of phage-resistant bacteria is a valid concern. Two attributes of the bacterial host that may contribute to the development of resistance were studied, the Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated (CRISPR/Cas) system and exopolysaccharide (EPS) interaction with phages. The structure of E. amylovora CRISPR/Cas system was determined in 8 E. amylovora isolates from different geographical regions. Three CRISPR-array sets named CR1, CR2 and CR3 were detected in 4 isolates, and only 2 arrays were determined in the rest of the isolates. No significant similarity was found between spacers in any of these systems to phage DNA sequenced in this study or from GenBank. Also the Cas level of expression was not stimulated during phage infection. Introduction of extra copies of Cas genes to enhance expression did not result in phage resistance. Nevertheless, E. amylovora CRISPR/Cas system was found to be efficient in blocking the transformation of plasmids carrying protospacers matched spacers in CRR1 and CRR2. Among phages that have been sequenced in this study are ΦEa9-2 and ΦEa35-70. ΦEa9-2 (Podoviridae) genome is 75,568 bp, and found to be related to coliphage N4. ΦEa35-70 (Myoviridae) genome is 271,084 bp, and found to carry a potential EPS depolymerase gene. Activity of ΦEa35-70 EPS depolymerase was only detected when cloned and expressed in E. coli, but His-tagged purified protein did not exhibit any EPS-depolymerase activities. This study offers critical information for the design of novel and effective phage-based biopesticides for the control of E. amylovora. It provides a new knowledge on the molecular structure and function of CRISPR/Cas system and EPS-phage interaction.
    • Proximate influence on eusocial caste behaviour

      Awde, David N; Department of Biological Sciences
      Queens and workers of eusocial sweat bee species are morphologically and developmentally similar, which means that each female is capable of behaving as a queen or a worker. However, few females lay eggs and behave as queens, while the majority of females provision the queen’s offspring, rarely lay eggs, and behave as workers. This makes eusocial sweat bee species, such as Lasioglossum laevissimum, excellent models to study the underling environmental (social) and genetic factors that contribute to variation in caste behaviours. My research focused on describing some of the proximate mechanisms that influence caste behaviours in L. laevissimum females. The social environment of a sweat bee colony, specifically the behaviour of a queen, can have a dramatic impact on worker behaviour. Queens suppress worker reproduction by physically bullying their workers. In a nesting aggregation at Brock University, almost half of L. laevissimum nests became queenless, which provided me with a natural experiment to assess the direct influence by queens on worker behaviour. Dissection data showed that a small proportion (17%) of workers developed their ovaries in both queenright and queenless nests. This suggests that L. laevissimum queens exert an early, negative, and strong influence on worker egg-laying behaviour, which lasts after she is gone. Next I assessed the relationship between gene expression and L. laeivsismum caste behaviours. I predicited that queens would express a gene associated with egg-laying, vitellogenin, more than workers, and that workers would express genes associated with foraging, the foraging gene, more than queens. Lasioglossum laevissimum queens had higher vitellogenin expression levels than workers, and females with high ovarian development had high vitellogenin expression, regardless of caste. On the other hand, queens and workers had similar foraging expression levels. Gene expression comparisons between queens and workers highlight two important behavioural characteristics of sweat bee castes. First, in eusocial sweat bees, both queens and workers actively provision brood at some point during the breeding season, which is reflected in their similar foraging expression levels. Secondly, queens lay eggs while a small proportion of workers have queen-like ovarian development, reflected in vitellogenin expression differences between castes.
    • Responses to Reflection in Two Invertebrate Species

      May, Holly; Department of Biological Sciences
      The present thesis investigates the responses to reflection in both the crayfish Procambarus clarkii and the fruit fly Drosophila melanogaster. Responses to reflection in crayfish depend on social status and the current work suggests that learning and memory consolidation are required for these responses to be altered. Crayfish were treated to either massed or spaced training fights prior to reflection testing. The results show that subordinate crayfish treated to spaced training display a response typical of subordinate crayfish but subordinate crayfish treated to massed training exhibit a response typical of dominant crayfish. Fruit flies are shown to be attracted to reflection and responses to reflection are described here for the first time. Responses in fruit flies are shown to be dependent on social status. The frequency of behaviours were altered in isolated flies but not socialized flies. The addition of pheromones cVA and 7,11-HD were used to investigate how the addition of chemical cues altered responses to reflection in fruit flies. Socialized fruit flies treated with cVA exhibited an increase in the frequency of behaviours on both mirrored and clear glass walls, while isolated flies exhibited a decrease. Socialized flies treated with 7,11-HD spent more time on mirrored walls compared to glass walls, whereas the frequency of all behaviours were decreased in isolated flies treated with 7,11-HD.
    • The role of microRNAs and retinoid signaling during spinal cord regeneration in the adult newt.

      Lepp, Amanda; Department of Biological Sciences
      The molecular events after spinal cord injury that lead to the establishment of a permissive environment and epimorphic regeneration remain unclear. Two molecular pathway regulators that may converge to create a spinal cord regeneration-permissive environment in the urodele are retinoic acid (RA) and microRNAs (miRNAs). Recent evidence suggests that RARβ-mediated signaling is necessary for tail and caudal spinal cord regeneration in the adult newt. MicroRNAs are attractive candidates as mediators of retinoid signaling during regeneration, as their pleiotropic effects are vital in situations where global changes in gene expression are required. Thus, the overall aim of this thesis was to determine if miRNAs are involved in tail and caudal spinal cord regeneration in the adult newt, and if they act as regulators and/or effectors of retinoid signaling during this process. I have demonstrated here, for the first time, that multiple miRNAs are dysregulated in response to spinal cord injury in the adult newt, as well as in response to inhibition of retinoid signaling. Two of these miRNAs, miR-133a and miR-1, appear to target RARβ2 transcripts both in vivo and in vitro. Inhibition of RA signaling via RARβ with a selective antagonist, LE135, alters the pattern of expression of these miRNAs, which leads to an inhibition of tail regeneration. These data are indicative of a negative feed back loop, albeit potentially an indirect one. I also aimed to examine which miRNAs are affected by inhibiting RA synthesis during regeneration, and provided a long list of miRNAs that are dysregulated. These data provide the foundation for future studies on the putative roles of these miRNAs, as well as their function in retinoid signaling. Overall, these studies provide the first evidence for a role for miRNAs as mediators of retinoid signaling during caudal spinal cord regeneration in any system.
    • The role of mobile elements in recent primate genomes

      Tang, Wanxiangfu; Department of Biological Sciences
      Mobile elements (MEs), which constitute ~50% of the primate genomes, have contributed to both genome evolution and gene function as demonstrated by ample evidence discovered over the last few decades. The three studies in this thesis aims to provide a better understanding of the evolutionary profile and function of MEs in the primate genomes by taking a computational comparative genomics approach. The first study represents a comprehensive analysis of the differential ME transposition among primates via identification of species-specific MEs (SS-MEs) in eight primate genomes from the families of Hominidae and Cercopithecidae using a comparative genomics approach. In total, 230,855 SS-MEs are identified, which reveal striking differences in retrotransposition level in the eight primate genomes. The second study represents a more focused analysis for the identification of a new type of MEs, which we term “retro-DNA” for non-LTR retrotransposons derived from DNA transposons, in the recent primate genomes. By investigating biallelic DNA transposons that have both the insertion and pre-integration alleles in ten primate genomes, a total of 1,750 retro-DNA elements representing 750 unique insertion events are reported for the first time. The third study provides an analysis of the mechanism underlying the differential SINE transposition in the primate genomes. In this study, Alu profiles are compared and the Alu master copies are identified in six primate genomes in the Hominidae and Cercopithecidae groups. The results show that each lineage of the primates and each species owns a unique Alu profile exclusively defined by the AluY transposition activity, which is determined by the number of Alu master copies and their relative activity. Overall, work in this thesis provides new insights about MEs and their impact on the recent primate genomes by revealing differential ME transposition as an important mechanism in generating genome diversity among primate lineages and species through discovering a new type of MEs and preliminary analysis of the mechanism underlying the differential ME transposition among primates. Furthermore, taking advantage of the recently available primate genomes and transcriptomes data, the work in this thesis demonstrates the great potential of the comparative genomic approach in studying MEs in primate genomes.
    • The role of retinoic acid in long-term memory formation and synaptic plasticity in the mollusc Lymnaea stagnalis

      Rothwell, Cailin; Department of Biological Sciences (Brock University, 2015-02-04)
      The active metabolite of vitamin A, retinoic acid (RA), is involved in memory formation and hippocampal plasticity in vertebrates. A similar role for retinoid signaling in learning and memory formation has not previously been examined in an invertebrate species. However, the conservation of retinoid signaling between vertebrates and invertebrates is supported by the presence of retinoid signaling machinery in invertebrates. For example, in the mollusc Lymnaea stagnalis the metabolic enzymes and retinoid receptors have been cloned from the CNS. In this study I demonstrated that impairing retinoid signaling in Lymnaea by either inhibiting RALDH activity or using retinoid receptor antagonists, prevented the formation of long-term memory (LTM). However, learning and intermediate-term memory were not affected. An additional finding was that exposure to constant darkness (due to the light-sensitive nature of RA) itself enhanced memory formation. This memory-promoting effect of darkness was sufficient to overcome the inhibitory effects of RALDH inhibition, but not that of a retinoid receptor antagonist, suggesting that environmental light conditions may influence retinoid signaling. Since RA also influences synaptic plasticity underlying hippocampal-dependent memory formation, I also examined whether RA would act in a trophic manner to influence synapse formation and/or synaptic transmission between invertebrate neurons. However, I found no evidence to support an effect of RA on post-tetanic potentiation of a chemical synapse. Retinoic acid did, however, reduce transmission at electrical synapses in a cell-specific manner. Overall, these studies provide the first evidence for a role of RA in the formation of implicit long-term memories in an invertebrate species and suggest that the role of retinoid signaling in memory formation has an ancient origin.

      Mansour, Hayam; Department of Biological Sciences (Brock University, 2012-06-04)
      Hepatitis C virus (HCV) is the causative agent of Hepatitis C, a serious global health problem which results in liver cirrhosis and hepatocellular carcinoma. Currently there is no effective treatment or vaccine against the virus. Therefore, development of a therapeutic vaccine is of paramount importance. In this project, three alternative approaches were used to control HCV including a DNA vaccine, a recombinant viral vaccine and RNA interference. The first approach was to test the effect of different promoters on the efficacy of a DNA vaccine against HCV. Plasmids encoding HCV-NS3 and E1 antigens were designed under three different promoters, adenoviral E1A, MLP, and CMV ie. The promoter effect on the antigen expression in 293 cells, as well as on the antibody level in immunized BALB/c mice, was evaluated. The results showed that the antigens were successfully expressed from all vectors. The CMV ie promoter induced the highest antigen expression and the highest antibody level. Second, the efficiency of a recombinant adenovirus vaccine encoding HCV-NS3 was compared to that of a HCV-NS3 plasmid vaccine. The results showed that the recombinant adenovirus vaccine induced higher antibody levels as compared to the plasmid vaccine. The relationship between the immune response and miRNA was also evaluated. The levels of mir-181, mir-155, mir-21 and mir-296 were quantified in the sera of immunized animals. mir-181 and mir-21 were found to be upregulated in animals injected with adenoviral vectors. Third, two recombinant adenoviruses encoding siRNAs targeting both the helicase and protease parts of the NS3 region were tested for their ability to inhibit NS3 expression. The results showed that the siRNA against protease was more effective in silencing the HCV-NS3 gene in a HCV replicon cell line. This result confirmed the efficiency of adenovirus for siRNA delivery. These results confirmed that CMV ie is optimum promoter for immune response induction. Adenovirus was shown to be an effective delivery vector for antigens or siRNAs. In addition, miRNAs were proved to be involved in the regulation of immune response.
    • Thermal tasting: methodological considerations and implications for alcohol behaviour.

      Thibodeau, Margaret; Department of Biological Sciences
      Thermal tasting is a phenomenon whereby some individuals perceive thermally-induced taste sensations when their tongue is warmed or cooled. These individuals, known as thermal tasters (TT), report a variety of thermally-induced tastes and the tastes reported can vary with temperature regime used and location on the tongue tested. TT are typically compared to thermal non-tasters (TnT), individuals who do not experience thermally-induced sensations. The literature suggests that TT give higher intensity ratings to orosensory stimuli than TnT; however, small sample sizes and differences in classification schemes between studies confound our understanding of TTS (thermal taste status). It is unknown whether the increased orosensory responsiveness of TT is universal or whether it is driven by a subgroup of TT. Furthermore, up to 50% of individuals are non-classifiable (NC). The largest database of individuals who have undergone TTS screening was compiled to address the literature gaps. Findings indicate that TT are more responsive than TnT to orosensory stimuli, regardless of the classification scheme used. The orosensory responsiveness of NC is not homogeneous, suggesting that NC are not a separate group but rather misclassified TT and TnT. Sweet TT are more likely than non-sweet TT to experience thermally-induced sensations during lingual warming. Similarly, sour TT are more likely than non-sour TT to report thermally-induced tastes during cooling. However, no differences in orosensory responsiveness based on these or other subgroups are identified, suggesting that the heightened orosensory responsiveness of TT is universal across this phenotype. The final study sought to characterize the binary interactions between ethanol and four orosensory stimuli (fructose, quinine, tartaric acid and alum sulphate) both overall and by comparing TT and TnT. In general, TT are more responsive than TnT to all stimuli in the study. Few interactions between TTS and stimulus intensity exist suggesting that TT and TnT perceive the sensations elicited by alcoholic beverages similarly, albeit at different intensities. Together, the thesis helps inform best practices for TTS screening and classification, provides insights into TTS mechanisms and furthers our understanding of alcoholic beverage perception.