• Identification and characterization of retinoic acid-induced morphological and electrophysiological changes in an invertebrate nervous system

      Vesprini, Nicholas; Department of Biological Sciences (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.
    • Evolutionary Origin and Maintenance of Sociality in the Small Carpenter Bees

      Rehan, Sandra; Department of Biological Sciences (2012-07-31)
      Many arthropods exhibit behaviours precursory to social life, including adult longevity, parental care, nest loyalty and mutual tolerance, yet there are few examples of social behaviour in this phylum. The small carpenter bees, genus Ceratina, provide important insights into the early stages of sociality. I described the biology and social behaviour of five facultatively social species which exhibit all of the preadaptations for successful group living, yet present ecological and behavioural characteristics that seemingly disfavour frequent colony formation. These species are socially polymorphic with both / solitary and social nests collected in sympatry. Social colonies consist of two adult females, one contributing both foraging and reproductive effort and the second which remains at the nest as a passive guard. Cooperative nesting provides no overt reproductive benefits over solitary nesting, although brood survival tends to be greater in social colonies. Three main theories explain cooperation among conspecifics: mutual benefit, kin selection and manipulation. Lifetime reproductive success calculations revealed that mutual benefit does not explain social behaviour in this group as social colonies have lower per capita life time reproductive success than solitary nests. Genetic pedigrees constructed from allozyme data indicate that kin selection might contribute to the maintenance of social nesting -, as social colonies consist of full sisters and thus some indirect fitness benefits are inherently bestowed on subordinate females as a result of remaining to help their dominant sister. These data suggest that the origin of sociality in ceratinines has principal costs and the great ecological success of highly eusociallineages occurred well after social origins. Ecological constraints such as resource limitation, unfavourable weather conditions and parasite pressure have long been considered some of the most important selective pressures for the evolution of sociality. I assessed the fitness consequences of these three ecological factors for reproductive success of solitary and social colonies and found that nest sites were not limiting, and the frequency of social nesting was consistent across brood rearing seasons. Local weather varied between seasons but was not correlated with reproductive success. Severe parasitism resulted in low reproductive success and total nest failure in solitary nests. Social colonies had higher reproductive success and were never extirpated by parasites. I suggest that social nesting represents a form of bet-hedging. The high frequency of solitary nests suggests that this is the optimal strategy when parasite pressure is low. However, social colonies have a selective advantage over solitary nesting females during periods of extreme parasite pressure. Finally, the small carpenter bees are recorded from all continents except Antarctica. I constructed the first molecular phylogeny of ceratinine bees based on four gene regions of selected species covering representatives from all continents and ecological regions. Maximum parsimony and Bayesian Inference tree topology and fossil dating support an African origin followed by an Old World invasion and New World radiation. All known Old World ceratinines form social colonies while New World species are largely solitary; thus geography and phylogenetic inertia are likely predictors of social evolution in this genus. This integrative approach not only describes the behaviour of several previously unknown or little-known Ceratina species, bu~ highlights the fact that this is an important, though previously unrecognized, model for studying evolutionary transitions from solitary to social behaviour.
    • Investigating the role of retinoic acid in the vertebrate and invertebrate nervous system

      Dmetrichuk, Jennifer M.; Department of Biological Sciences (2012-12-13)
      Although abundant in the developing central nervous system (CNS) of vertebrates, the precise roll of all-trans retinoic acid (RA) is neuronal development and regeneration is undetermined. This study suggests that all-trans RA acts via its RAR Beta receptor to stimulate neurite outgrowth from adult newt spinal cord explants, and may represent an important chemotropic molecule for nerve dependent limb regeneration. All-trans RA's effects are not limited to the vertebrate species, as it was found that all-trans RA induces neurite outgrowth and retains electrical excitability in isolated adult molluscan invertebrate neurons. Using analytical chemistry techniques, both all-trans RA and its isoform, 9-cis RA, were identified in the invertebrate CNS. 9-cis RA showed a similar neurotrophic role to that of all-trans RA on cultured molluscan neurons. Further, all-trans and 9-cis RA were capable of inducing growth cone chemoattraction. This occurred in the absence of the neuronal cell body, unlike the growth promoting and increased survival effects of all-trans RA, suggesting a novel signaling mechanism for all-trans RA in the invertebrate and vertebrate species. Investigating the factors involved in promoting neurite outgrowth may ultimately aid in designing strategies to prevent nerve degeneration and developing approaches to support axonal regeneration in humans.
    • Cellular Mechanisms of Resveratrol's Interaction with Mitochondrial Reactive Oxygen Species Metabolism

      Robb, Ellen; Department of Biological Sciences (Brock University, 2013-05-14)
      Resveratrol, a polyphenol found naturally in red wines, has attracted great interest in both the scientific community and the general public for its reported ability to protect against many of the diseases facing Western society today. While the purported health effects of resveratrol are well characterized, details of the cellular mechanisms that give rise to these observations are unclear. Here, the mitochondrial antioxidant enzyme Mn superoxide dismutase (MnSOD) was identified as a proximal target of resveratrol in vitro and in vivo. MnSOD protein and activity levels increase significantly in cultured cells treated with resveratrol, and in the brain tissue of mice given resveratrol in a high fat diet. Preventing the increase in MnSOD levels eliminates two of resveratrol’s more interesting effects in the context of human health: inhibition of proliferative cell growth and cytoprotection. Thus, the induction of MnSOD is a critical step in the molecular mechanism of resveratrol. Mitochondrial morphology is a malleable property that is capable of impeding cell cycle progression and conferring resistance against stress induced cell death. Using confocal microscopy and a novel ‘cell free’ fusion assay it was determined that concurrent with changes in MnSOD protein levels, resveratrol treatment leads to a more fused mitochondrial reticulum. This observation may be important to resveratrol’s ability to slow proliferative cell growth and confer cytoprotection. Resveratrol's biological activities, including the ability to increase MnSOD levels, are strikingly similar to what is observed with estrogen treatment. Resveratrol fails to increase MnSOD levels, slow proliferative cell growth and confer cytoprotection in the presence of an estrogen receptor antagonist. Resveratrol's effects can be replicated with the specific estrogen receptor beta agonist diarylpropionitrile, and are absent in myoblasts lacking estrogen receptor beta. Four compounds that are structurally similar to resveratrol and seven phytoestrogens predicted to bind to estrogen receptor beta were screened for their effects on MnSOD, proliferative growth rates and stress resistance in cultured mammalian cells. Several of these compounds were able to mimic the effects of resveratrol on MnSOD levels, proliferative cell growth and stress resistance in vitro. Thus, I hypothesize that resveratrol interacts with estrogen receptor beta to induce the upregulation of MnSOD, which in turn affects cell cycle progression and stress resistance. These results have important implications for the understanding of RES’s biological activities and potential applications to human health.
    • Defence of Agaricus bisporus against toxic secondary metabolites from Trichoderma aggressivum.

      Sjaarda, Calvin; Department of Biological Sciences (Brock University, 2013-09-12)
      Trichoderma spp are effective competitors against other fungi because they are mycoparasitic and produce hydrolytic enzymes and secondary metabolites that inhibit the growth of their competitors. Inhibitory compounds produced by Trichoderma aggressivum, the causative agent of green mold disease, are more toxic to the hybrid off-white strains of Agaricus bisporus than the commercial brown strains, consistent with the commercial brown strain’s increased resistance to the disease. This project looked at the response of hybrid off-white and commercial brown strains of A. bisporus to the presence of T. aggressivum metabolites with regard to three A. bisporus genes: laccase 1, laccase 2, and manganese peroxidase. The addition of T. aggressivum toxic metabolites had no significant effect on MnP or lcc1 transcript abundance. Alternatively, laccase 2 appears to be involved in resistance to T. aggressivum because the presence of T. aggressivum metabolites results in higher lcc2 transcript abundance and laccase activity, especially in the commercial brown strain. The difference in laccase expression and activity between A. bisporus strains was not a result of regulatory or coding sequence differences. Alteration of laccase transcription by RNAi resulted in transformants with variable levels of laccase transcript abundance. Transformants with a low number of lcc transcripts were very sensitive to T. aggressivum toxins, while those with a high number of lcc transcripts had increased resistance. These results indicated that laccase activity, in particular that encoded by lcc2, serves as a defense response of A. bisporus to T. aggressivum toxins and contributes to green mold disease resistance in commercial brown strains.
    • 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.
    • METHOXYPYRAZINES AND LADYBUG TAINT IN WINES

      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.
    • HUMAN GENOME VARIATIONS AND EVOLUTION WITH A FOCUS ON THE ANALYSIS OF TRANSPOSABLE ELEMENTS

      Ahmed, Musaddeque; Department of Biological Sciences (Brock University, 2014-02-19)
      Genome sequence varies in numerous ways among individuals although the gross architecture is fixed for all humans. Retrotransposons create one of the most abundant structural variants in the human genome and are divided in many families, with certain members in some families, e.g., L1, Alu, SVA, and HERV-K, remaining active for transposition. Along with other types of genomic variants, retrotransponson-derived variants contribute to the whole spectrum of genome variants in humans. With the advancement of sequencing techniques, many human genomes are being sequenced at the individual level, fueling the comparative research on these variants among individuals. In this thesis, the evolution and functional impact of structural variations is examined primarily focusing on retrotransposons in the context of human evolution. The thesis comprises of three different studies on the topics that are presented in three data chapters. First, the recent evolution of all human specific AluYb members, representing the second most active subfamily of Alus, was tracked to identify their source/master copy using a novel approach. All human-specific AluYb elements from the reference genome were extracted, aligned with one another to construct clusters of similar copies and each cluster was analyzed to generate the evolutionary relationship between the members of the cluster. The approach resulted in identification of one major driver copy of all human specific Yb8 and the source copy of the Yb9 lineage. Three new subfamilies within the AluYb family – Yb8a1, Yb10 and Yb11 were also identified, with Yb11 being the youngest and most polymorphic. Second, an attempt to construct a relation between transposable elements (TEs) and tandem repeats (TRs) was made at a genome-wide scale for the first time. Upon sequence comparison, positional cross-checking and other relevant analyses, it was observed that over 20% of all TRs are derived from TEs. This result established the first connection between these two types of repetitive elements, and extends our appreciation for the impact of TEs on genomes. Furthermore, only 6% of these TE-derived TRs follow the already postulated initiation and expansion mechanisms, suggesting that the others are likely to follow a yet-unidentified mechanism. Third, by taking a combination of multiple computational approaches involving all types of genetic variations published so far including transposable elements, the first whole genome sequence of the most recent common ancestor of all modern human populations that diverged into different populations around 125,000-100,000 years ago was constructed. The study shows that the current reference genome sequence is 8.89 million base pairs larger than our common ancestor’s genome, contributed by a whole spectrum of genetic mechanisms. The use of this ancestral reference genome to facilitate the analysis of personal genomes was demonstrated using an example genome and more insightful recent evolutionary analyses involving the Neanderthal genome. The three data chapters presented in this thesis conclude that the tandem repeats and transposable elements are not two entirely distinctly isolated elements as over 20% TRs are actually derived from TEs. Certain subfamilies of TEs themselves are still evolving with the generation of newer subfamilies. The evolutionary analyses of all TEs along with other genomic variants helped to construct the genome sequence of the most recent common ancestor to all modern human populations which provides a better alternative to human reference genome and can be a useful resource for the study of personal genomics, population genetics, human and primate evolution.
    • 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.
    • The Fast Regulation of Photosynthesis in Diatoms: an inquiry into the physiological and physical origins of non-photochemical chlorophyll fluorescence quenching.

      Derks, Allen Kimbell; Department of Biological Sciences (Brock University, 2015-01-06)
      Diatoms are renowned for their robust ability to perform NPQ (Non-Photochemical Quenching of chlorophyll fluorescence) as a dissipative response to heightened light stress on photosystem II, plausibly explaining their dominance over other algal groups in turbulent light environs. Their NPQ mechanism has been principally attributed to a xanthophyll cycle involving the lumenal pH regulated reversible de-epoxidation of diadinoxanthin. The principal goal of this dissertation is to reveal the physiological and physical origins and consequences of the NPQ response in diatoms during short-term transitions to excessive irradiation. The investigation involves diatom species from different originating light environs to highlight the diversity of diatom NPQ and to facilitate the detection of core mechanisms common among the diatoms as a group. A chiefly spectroscopic approach was used to investigate NPQ in diatom cells. Prime methodologies include: the real time monitoring of PSII excitation and de-excitation pathways via PAM fluorometry and pigment interconversion via transient absorbance measurements, the collection of cryogenic absorbance spectra to measure pigment energy levels, and the collection of cryogenic fluorescence spectra and room temperature picosecond time resolved fluorescence decay spectra to study excitation energy transfer and dissipation. Chemical inhibitors that target the trans-thylakoid pH gradient, the enzyme responsible for diadinoxanthin de-epoxidation, and photosynthetic electron flow were additionally used to experimentally manipulate the NPQ response. Multifaceted analyses of the NPQ responses from two previously un-photosynthetically characterised species, Nitzschia curvilineata and Navicula sp., were used to identify an excitation pressure relief ‘strategy’ for each species. Three key areas of NPQ were examined: (i) the NPQ activation/deactivation processes, (ii) how NPQ affects the collection, dissipation, and usage of absorbed light energy, and (iii) the interdependence of NPQ and photosynthetic electron flow. It was found that Nitzschia cells regulate excitation pressure via performing a high amplitude, reversible antenna based quenching which is dependent on the de-epoxidation of diadinoxanthin. In Navicula cells excitation pressure could be effectively regulated solely within the PSII reaction centre, whilst antenna based, diadinoxanthin de-epoxidation dependent quenching was implicated to be used as a supplemental, long-lasting source of excitation energy dissipation. These strategies for excitation balance were discussed in the context of resource partitioning under these species’ originating light climates. A more detailed investigation of the NPQ response in Nitzschia was used to develop a comprehensive model describing the mechanism for antenna centred non-photochemical quenching in this species. The experimental evidence was strongly supportive of a mechanism whereby: an acidic lumen triggers the diadinoxanthin de-epoxidation and protonation mediated aggregation of light harvesting complexes leading to the formation of quencher chlorophyll a-chlorophyll a dimers with short-lived excited states; quenching relaxes when a rise in lumen pH triggers the dispersal of light harvesting complex aggregates via deprotonation events and the input of diadinoxanthin. This model may also be applicable for describing antenna based NPQ in other diatom species.
    • 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.
    • Cell-selective modulation of the neuromuscular system in Drosophila

      Ormerod, Kiel; Department of Biological Sciences
      The capacity for all living cells to sense and interact with their environment is a necessity for life. In highly evolved, eukaryotic species, like humans, signalling mechanisms are necessary to regulate the function and survival of all cells in the organism. Synchronizing systemic signalling systems at the cellular, organ and whole-organism level is a formidable task, and for most species requires a large number of signalling molecules and their receptors. One of the major types of signalling molecules used throughout the animal kingdom are modulatory substances (e.x. hormones and peptides). Modulators can act as chemical transmitters, facilitating communication at chemical synapses. There are hundreds of circulating modulators within the mammalian system, but the reason for so many remains a mystery. Recent work with the fruit fly, Drosophila melanogaster demonstrated the capacity for peptides to modulate synaptic transmission in a neuron-specific manner, suggesting that peptides are not simply redundant, but rather may have highly specific roles. Thus, the diversity of peptides may reflect cell-specific functions. The main objective of my doctoral thesis was to examine the extent to which neuromodulator substances and their receptors modulate synaptic transmission at a cell-specific level using D. melanogaster. Using three different modulatory substances, i) octopamine - a biogenic amine released from motor neuron terminals, ii) DPKQDFMRFa - a neuropeptide secreted into circulation, and iii) Proctolin - a pentapeptide released both from motor neuron terminals and into circulation, I was able to investigate not only the capacity of these various substances to work in a cell-selective manner, but also examine the different mechanisms of action and how modulatory substances work in concert to execute systemic functionality . The results support the idea that modulatory substances act in a circuit-selective manner in the central nervous system and in the periphery in order to coordinate and synchronize physiologically and behaviourally relevant outputs. The findings contribute as to why the nervous system encodes so many modulatory substances.
    • 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.
    • 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.
    • 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.
    • Functional antagonism of the mesolimbic dopaminergic system on mesolimbic cholinergic system in the vocal expression of an emotional state

      Silkstone, Michael; Department of Biological Sciences
      The overarching goal of this thesis was to determine if the initiation of a positive emotional state could antagonize the expression of a negative emotional state in rats. The hypothesis of the thesis argued that the initiation of a positive emotional state would ameliorate the vocal expression of a negative emotional state. The subjective emotional state of the rat was indexed by the quantity and type of pharmacologically induced ultrasonic vocalizations (USVs). Adolescent and adult rats can emit vocalizations above the upper threshold of human hearing (>20 kHz) termed ultrasonic vocalizations (USVs). These USVs are broadly divided into 50-kHz, reflective of a positive emotional state, and 22-kHz USVs, reflective of a negative emotional state. Pharmacologically, injection of dopamine agonists into the nucleus accumbens shell is sufficient for the initiation of 50-kHz USVs, while injection of cholinergic agonists into the anterior hypothalamic-medial preoptic area (AH-MPO) or the lateral septum (LS) can initiate 22-kHz USVs. In chapter two of the thesis, I demonstrated that microinjection of the dopamine agonist, apomorphine, into the medial shell of the nucleus accumbens attenuated the extent of carbachol-induced 22-kHz USVs from the AH-MPO. I also demonstrated that this effect was dependent upon the microinjection of apomorphine into the central region of the nucleus accumbens shell. In chapter three, I demonstrated that apomorphine could also decrease the extent of carbachol-induced 22-kHz USVs from the LS providing evidence that the effect reported in chapter two was not isolated to the AH-MPO, but rather extending along the medial cholinoceptive vocalization strip. In the third chapter. I also demonstrated that the magnitude of the reduction in the number of 22-kHz USVs was correlated to the number of emitted frequency-modulated (FM) 50-kHz USVs induced by apomorphine. In the fourth chapter, I investigated whether blocking dopamine receptors, either systemically using the typical D2-antipsychotic agent, haloperidol, or microinjection of the D2 antagonist, raclopride, into the nucleus accumbens shell could increase the emission of carbachol-induced 22-kHz USVs from the LS. The results showed that antagonism of dopamine receptors, either systemically or intracerebrally, did not increase the number of 22-kHz USVs. Interestingly, it was also observed that after the prolonged recording of carbachol-induced 22-kHz USVs, some 50-kHz USVs spontaneously appeared after roughly 300 s into the recording. I argued that these 50-kHz USVs, which I defined as “rebound 50-kHz USVs” are not initiated by carbachol since they occurred when the carbachol-response weaned. It was also demonstrated these rebound 50-kHz USVs were dependent upon dopamine release within the nucleus accumbens since both systemic, and intracerebral application of dopamine antagonists into the central division of the nucleus accumbens shell blocked the occurrence of rebound 50-kHz USVs. Altogether, the data supports the thesis that activation of a positive emotional state decreases the expression of the negative emotional state in rats when measured using ultrasonic vocalizations.
    • 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.
    • 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.
    • 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.
    • ENDOCANNABINOID REGULATION OF ADOLESCENT DEVELOPMENT IN MALE AND FEMALE RATS

      Simone, Jonathan; Department of Biological Sciences
      The present thesis investigated the contributions of adolescent endocannabinoid signalling to brain and behaviour development in male and female rats. In chapter 2, daily administration of the CB1 antagonist AM251, alone or in tandem with a psychological stressor, increased social interactions, reduced dorsal hippocampal CB1 expression, and increased mPFC GAD67 expression in female rats 24-48 h after treatment, with no effects in males. In chapter 3, adolescent CB1 antagonism reduced anxiety in adult males, with no effects in females. Conversely, adolescent AM251 increased contextual fear in adult females, with no effects in males. In chapter 4, AM251 females spent more time initiating social interactions after a 5-day drug washout period than vehicle females, with no effects in males. To identify brain regions underlying the effects of AM251 on social behaviours, I repeated social interaction testing in vehicle and AM251 females and collected brains for immunohistochemical labelling of EGR-1 as a marker of neural activation in the CA1, CA2, and CA3 subfields of the dorsal hippocampus and the shell and core divisions of the nucleus accumbens (NAc). Consistent with my previous findings, AM251 females spent more time initiating social interactions and had greater EGR-1 cell counts in the NAc shell than vehicle females, with no group differences in the NAc core or in any of the hippocampal subfields investigated. EGR-1 cell counts in the dCA2 were negatively correlated with social interactions in vehicle and AM251 females. A positive correlation between NAc shell EGR-1 cell counts and social interactions was observed only in AM251 females. Regression analysis using drug treatment and EGR-1 cell counts in dCA2 and NAc shell resulted in a model with an adjusted R2 of 0.90. Both drug treatment and EGR-1 cell counts in the dorsal CA2 emerged as unique predictors of individual differences in social interaction, and drug and NAc shell EGR-1 cell counts interacted to significantly predict social interactions in AM251 females only. Together, these studies provide support for sex-specific contributions of endocannabinoid signalling to the development of brain and behaviour in adolescence in male and female rats.