Is GABA involved in regulating plant growth and development? /

Rapid and large accumulation of GABA (y-aminobutyric acid) in response to a number of plant stresses has been well documented. But the role(s) of GABA in plants is not well defined. In recent years, the possibility of GABA involvement in regulating plant growth and development has been raised. In the present study, this possibility was examined. First, to rapidly and accurately determine GABA levels in plant tissues, a spectrometric method for GABA determination was developed based on a commercially available enzyme Gabase. Seventy mM LaCb almost completely removed water-soluble pigments from plant tissues which greatly interfere with the absorbance reading at 340nm. Inactivation of GAD (glutamate decarboxylase) by immediately adding methanol to a frozen plant tissue powder was suggested to prevent GABA production during extraction. The recovery of GABA with this method was approximately 100%. Second, the relationship between GABA levels and hypocotyl elongation in soybean seedlings was analyzed using different approaches to regulate in vivo GABA levels and the elongation of hypocotyls. The following major observations were made. (1) Mechanical stimulation by stroking elevated GABA levels and concurrently induced a rapid and significant reduction in hypocotyl elongation. (2) External GABA was demonstrated to penetrate into the hypocotyls using '*C-GABA. Application of external GABA elevated in vivo GABA levels, but failed to inhibit hypocotyl elongation. (3) LaCla and blue light irradiation caused an inhibition in the elongation of dark-grown hypocotyls, whereas GABA levels were not significantly affected. (4) Ca^was suggested to be involved in the signal transduction pathway leading from mechanical stimulation to GABA production, as indicated by the ability of La'* to inhibit GABA production in stimulated hypocotyls. (5) Bicuculline, saclofen and baclofen (agonists and antagonists of GABA receptors in animals) had no effect on hypocotyl elongation. It might indicate that GABA-binding components which are structurally similar to animal GABA receptors and functionally capable of regulating plant growth may not exist in plants. Therefore, the conclusion was drawn that GABA alone is not sufficient to inhibit hypocotyl elongation. Third, chloride influx in isolated Asparagus cells was enhanced by lOmM GABA during a 3 hour incubation, but the effect was not specific for GABA. Chloride efflux was not influenced by GABA. Both influx and efflux of chloride were significantly inhibited by NPPB, a chloride channel blocker. These results suggest that GABA does not influence the activity of plant chloride channels.

Primary characterization of genes involved in the colony development of the insect pathogenic fungus Metarhizium anisopliae /

Strain improvement of the insect pathogenic fungus Metarhizium anisopUae is necessary to increase its virulence towards agricultural pests and thus improve its commercial efficacy. Nevertheless, the release of genetically modified conidia in crop fields may negatively affect the ecosystem. Controlling conidiation is a potential means of limiting the release of engineered strains since conidia are the infective propagules and the means of dispersal. The purpose of this study was to research the colony development of M. anisopUae to identify potential targets for genetic manipulation to control conidiation. Following Agrobacterium tumefaciem insertional mutagenesis, phenotypic mutants were characterized using Y-shaped adaptor dependent extension PCR. Four of 1 8 colony development recombinants had T-DNA flanking sequences with high homology to genes encoding known signaling pathway proteins that regulate pathogenesis and/or asexual development in filamentous fungi. Conidial density counts and insect bioassays suggested that a Serine/Threonine protein kinase COTl homolog is not essential for conidiation or virulence. Furthermore, a choline kinase homolog is important for conidiation, but not virulence. Finally, the regulator of G protein signaling CAG8 and a NADPH oxidase NoxA homolog are necessary for conidiation and virulence. These genes are candidates for further investigation into the regulatory pathways controlling conidiation to yield insight into promising gene targets for biocontrol strain improvement.

The Catharanthus roseus 16-hydroxytabersonine O-methyltransferase involved in vindoline biosynthesis /

Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex dimeric anticancer alkaloids vinblastine and vincristine that are derived by coupling vindoline and catharanthine monomers. This thesis describes the novel application of carborundum abrasion (CA) technique as a tool for large scale isolation of leaf epidermis enriched proteins. This technique was used to facilitate the purification to apparent homogeneity of 16-hydroxytabersonine-16-0-methyltransferse (l60MT) that catalyses the second step in the 6 step pathway that converts tabersonine into vindoline. This versatile tool was also used to harvest leaf epidermis enriched mRNAs that facilitated the molecular cloning of the 160MT. Functional expression and biochemical characterization of recombinant 160MT enzyme showed that it had a very narrow substrate specificity and high affinity for 16-hydroxytabersonine, since other closely related monoterpene indole alkaloids (MIAs) did not act as substrates. In addition to allowing the cloning of this gene, CA technique clearly showed that 160MT is predominantly expressed in Catharanthus leaf epidermis, in contrast to several other OMTs that appear to be expressed in other Catharanthus tissues. The results provide compelling evidence that most of the pathway for vindoline biosynthesis including the 0- methylation of 16-hydroxytabersonine occurs exclusively in leaf epidermis, with subsequent steps occurring in other leaf cell types. Small molecule O-methyltransferases (OMTs) (E.C. 2.1.1.6.x) catalyze the transfer of the reactive methyl group of S-adenosyl-L-methionine (SAM) to free hydroxyl groups of acceptor molecules. Plant OMTs, unlike their monomeric mammalian homologues, exist as functional homodimers. While the biological advantages for dimer fonnation with plant OMTs remain to be established, studies with OMTs from the benzylisoquinoline producing plant, Thalictrum tuberosum, showed that co-expression of 2 recombinant OMTs produced novel substrate specificities not found when each rOMT was expressed individually (Frick, Kutchan, 1999) . These results suggest that OMTs can fonn heterodimers that confer novel substrate specificities not possible with the homodimer alone. The present study describes a 160MT model based strategy attempting to modify the substrate specificity by site-specific mutagenesis. Our failure to generate altered substrate acceptance profiles in our 160MT mutants has lead us to study the biochemical properties ofhomodimers and heterodimers. Experimental evidence is provided to show that active sites found on OMT dimers function independently and that bifunctional heterodimeric OMTs may be fonned in vivo to produce a broader and more diverse range of natural products in plants.

Immuno-cytochemical localization of glycoproteins involved in recognition and attachment in a mycoparatism

Polyclonal antibodies prepared against the two glycoproteins (Mr 100 and 85 kDa) involved in recognition and attachment of the mycoparasite, Piptocephalis virginiana, to its hosts, Mortierella pusilla and Phascolomyces articulosus, susceptible and resistant, respectively, were employed to localize the antigens at their cell surfaces. Indirect immunocytochemical technique using secondary antibodies labelled with either FITC or gold particles as probes, were used. FITC-Iabelled antibodies revealed a discontinous pattern of fluorescence on the hyphae of MortlerelLa pusilla and no fluorescence on the hyphae of Phascolomyces articulosus. Intensity of fluorescence was high in the germinating spores of both the fungi. Fluoresence could be observed on P. articulosus hyphae pretreated with a commercial proteinase. Fluorescence was not observed on either hyphae or germinating spores of the nonhost M0 r tie re11 a ca ndelabrum and the mycoparasite P. virginiana. Antibodies labelled with gold conjugate showed a different pattern of antigen localization on the hyphal walls of the susceptible and resistant hosts. Patches of gold particles were observed allover the whole cell wall of the susceptible host but only on the inner cell wall layer of the resistant host. Cell wall fragments of the susceptible host but not those of the resistant host, previously incubated with the antibodies inhibited attachment of the mycoparasite. Implications of preferential localization of the antigen in the resistant host and its absence in the nonhost are described.

Niagara alternative food projects : networks, discourses and nature /

This thesis undertakes an exploration of the nature of alternative food projects in Niagara. A review of various theoretical approaches to the study of food and agriculture, suggests that actor-network theory offers the most useful lens through which to understand these projects. In particular, actor-network theory facilitates non-dualistic theorisations of power and scale and a commitment to the inclusion of non-humans in the 'social' sciences. The research is based on 19 in-depth interviews with actors involved in various urban and rural projects including community supported agriculture, community gardens, chefs using local seasonal food, a winery that grows organically, the good food box, a value-added small business, and organic producers. The analysis consists of four themes. The first analytical section pays special attention to the prominence of agri-tourism in Niagara, and examines the ways in which the projects in the sample interact with agri-tourist networks. In the second section the discussion focuses on the discourses and practices of resistance among Niagara alternative food actors. The participants' interviews suggest there are more discourses of resistance toward agri-tourist than toward dominant food networks. The third section questions commodity chain theorisations of alternative food projects. In particular, this section shows how the inclusion of non-human actors in an analysis confounds conceptualisations of 'short' and 'local' chains. The final analytical section assesses relations of power in Niagara alternative food projects. Three important conclusions arise from this research. First, Niagara alternative food projects cannot be conceptualised as operating at the 'local' scale. Broadening the scope of analysis to include non-human actors, it becomes apparent that these projects actually draw on a variety of extra-local actors. They are at once local and global. Second, the projects in this sample are simultaneously part of alternative, dominant and agri-tourist networks. While Niagara alternative food projects do perform many of the roles characteristic of alternative food systems, they are also involved in practices of development, business, and class distinction. Thus, alternative food networks should not be understood as separate from and in direct opposition to dominant food networks. Despite the second conclusion, this research determines that Niagara alternative food projects have made significant strides in the reworking of power. The projects represented in this thesis do engage in resistant practices and are associated with increased levels ofjustice.

Personality and neuropsychological factors involved in females' relational aggression

The personality and neuropsychological factors associated with relational aggression were examined in a group of30 grade 6,7, and 8 girls identified through cluster analysis as being highly, yet almost exclusively, relationally aggressive and a group of 30 nonaggressive matched controls. Parents of the students in both groups completed the Coolidge Personality and Neuropsychological Inventory (1998), a 200- item DSM-IV -TR aligned, parent-as-respondent, standardized measure of c.hildren' s psychological functioning. It was found that high levels of relational aggression, in the absence of physical and verbal aggression, were associated with symptoms of DSM-IV - TR Axis I oppositional defiant disorder and conduct disorder and a wide variety of personality traits associated with DSM-IV -TR Axis II paranoid, borderline, narcissistic, histrionic, schizotypal, and passive aggressive personality disorders. Implications of these findings for theory, practice, and further research are discussed.

Attenuation of skeletal muscle FFA-induced insulin resistance by the polyphenol resveratrol. Elucidation of the mechanisms involved

Excess plasma free fatty acids (FFA) are correlated with insulin resistance and are a risk factor for the development of type 2 diabetes. In this study we examined the effect of the polyphenol resveratrol on FF A-induced insulin resistance in skeletal muscle cells and the mechanisms involved. Incubation of L6 myotubes with the FF A palmitate significantly decreased the insulin-stimulated glucqse uptake. Importantly, the effect of palmitate was ameliorated by resveratrol. Palmitate significantly increased serine phosphorylation of IRS..; 1 and reduced insulin-stimulated Akt phosphorylation, an effect that was abolished by resveratrol. We then investigated the effect of palmitate and resveratrol on the expression and phosphorylation of JNK, mTOR, p70-S6K, and AMPK kinases. The results demonstrated that our treatments had no effect on the expression of these proteins. However, palmitate increased the phosphorylation of mTOR and p70- S6K, whereas resveratrol abolished this effect and increased the phosphorylation of AMPK. Furthermore, all effects of resveratrol were abolished with sirtuin inhibitors, sirtinol and nicotinamide. These results indicate that resveratrol ameliorated FF A-induced insulin resistance by regulating mTOR and p70-S6K phosphorylation in skeletal muscle cells, through a mechanism involving sirtuins.

Benefits, challenges, and limitations of fair trade as a social justice movement

This thesis answers some important questions about how Fair Trade is experienced and perceived by some Northern sellers, consumers, activists, advocates, practitioners, and an importer. As it relates to sellers, I focus only on small scale independent businesses (i.e. I do not include large corporate businesses in my interview sample). Fair Trade works to establish a dignified livelihood for many producers in the South. Some of the most important actors in the Fair Trade movement are the people who buy, sell, and/or advocate for Fair Trade in the North. Fair Trade is largely a consumer movement which relies on the purchase of Fair Trade products. Without consumers purchasing Fair Trade products, retailers providing the products for sale, and activists raising awareness of Fair Trade, the movement, as it is presently constituted, would be non-existent. This qualitative research is based on 19 in-depth i.nterviews with nine interviewees involved with Fair Trade in Canada. I focus on benefits, challenges, and limitations of Fair Trade in the context of their involvement with it. I describe and analyze how people become involved with Fair Trade, what motivates them to do so, what they hope to achieve, and the benefits of being involved. I also describe and analyze how people understand and deal with any challenges and limitations associated with their involvement with Fair Trade. I also explore whether involvement with Fair Trade influences how people think about other products that they purchase and, if so, in what ways. I focus mainly on the commodity of coffee, but my discussion is not limited to this single commodity. Interviewees' experiences with and participation in Fair Trade vary in terms of their level of involvement and interest in the broader Fair Trade movement (as opposed to just participating in the market component). This research reveals that while Fair Trade is a small movement, sellers, consumers, and activists have had much success in the advancement of Fair Trade. While challenges have not deterred interviewees from continuing to participate in Fair Trade, analysis and explanation of such challenges provides the opportunity for Fair Trade practitioners to develop effective solutions in an effort to meet the needs of various Fair Trade actors.

TIME COURSE OF SIGNALING PROCESSES INVOLVED WITH EXTRACELLULAR OSMOTIC - INDUCED GLUCOSE UPTAKE IN MAMMALIAN SKELETAL MUSCLE

Extracellular hyper-osmotic (HYPER) stress increases glucose uptake to defend cell volume, when compared to iso-osmotic (ISO) conditions in skeletal muscle. The purpose of this study was to determine a time course for changes in common signaling proteins involved in glucose uptake during acute hyper-osmotic stress in isolated mammalian skeletal muscle. Rat extensor digitorum longus (EDL) muscles were excised and incubated in a media formulated to mimic ISO (290 ± 10 mmol/kg) or HYPER (400 ± 10 mmol/kg) extracellular condition (Sigma Media-199). Signaling mechanisms were investigated by determining the phosphorylation states of Akt, AMPK, AS160, cPKC and ERK after 30, 45 and 60 minutes of incubation. AS160 was found to be significantly more phosphorylated in HYPER conditions compared to ISO after 30 minutes (p<0.01). It is speculated that AS160 phosphorylation increases glucose transporter 4 (GLUT4) content at the cell surface thereby facilitating an increase in glucose uptake under hyper-osmotic stress.

Elucidation of the components involved in the antioxidant activity of honey

Canadian honeys were analyzed for sugar concentration, honey colour, total phenolic content, the level of brown pigments, and antioxidant activity in order to elucidate the main components involved in the antioxidant activity of honey. By employing size-exclusion chromatography in combination with activity-guided fractionation, it was demonstrated that the antioxidant components are of high molecular weight (HMW), brown in colour and absorb at both 280nm and 450nm. The presence of brown HMW antioxidant components prompted an investigation on the influence of heattreatment on the Maillard reaction and the formation of melanoid ins. Heat-treatment of honey resulted in an increase in the level of phenolics in the melanoidin fractions which correlated with an increase in antioxidant activity. The preliminary results of this study suggest for the first time that honey melanoidins underlie the antioxidant activity of unheated and heat-treated honey, and that phenolic constituents are involved in the melanoidin structure and are likely incorporated by covalent or non-covalent interaction.

Elucidation of the Retinoid Signalling Pathway Involved in Axon Guidance in Lymnaea stagnalis and Xenopus laevis

The vitamin A metabolite, retinoic acid (RA), is known to play a crucial role in several developmental processes including axial patterning and differentiation. More recently, RA has been implicated in the regenerative process acting through its classical signaling pathway, the nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), to mediate gene transcription. Moreover, RA has been shown to act as a guidance molecule for growth cones of regenerating motorneurons of the pond snail, Lymnaea stagnalis. Our lab has recently shown that RA can induce this morphological response independent of nuclear transcription, however, the role of the retinoid receptors in RA-induced chemoattraction is still unknown. Here, I show that the retinoid receptors, RXR and RAR, may mediate the growth cones response to the metabolically active retinoic acid isomers, all-trans and 9-cis RA, in Lymnaea stagnalis. Data presented here show that both an RXR and RAR antagonist can block growth cone turning in response to application of both isomers. Because no prior investigations have shown growth cone turning of individual vertebrate neurons, I aimed to show that both retinoic acid isomers were capable of inducing growth cone turning of embryonic spinal cord neurons in the frog, Xenopus laevis. For the first time in Xenopus, I showed that both all-trans and 9-cis RA were able to induce significantly more neurite outgrowth from cultured embryonic spinal cord neurons and induce positive growth cone turning of individual growth cones. In addition, I showed that the presence of the RXR antagonist, HX531, blocked 9-cis RA-induced growth cone turning and the RARβ antagonist, LE135, blocked all-trans RA-induced growth cone turning in this species. Evidence provided here shows for the first time, conservation of retinoic acid-induced growth cone turning in a vertebrate model system. In addition, these data show that the receptors involved in this morphological response may be the same in vertebrates and invertebrates.

Neuromotor Mechanisms Involved in the Recovery from Local Muscular Fatigue

The phenomenon of over-recovery consists of a participant’s maximal force levels returning to values above initial levels. The present study examined the presence and causes of over-recovery following local muscular fatigue. Fourteen males completed two fatigue protocols consisting of maximal isometric dorsiflexion contractions. Upon completion of the fatigue protocol participants’ force was monitored over a 15 minute recovery period. Dorsiflexion force and surface electromyography (sEMG) from the tibialis anterior and soleus were monitored concurrently. Following the two fatigue conditions (10 and 20% force decrement) force recovered to 100.5 and 99.5% of initial levels for each condition, respectively. Surface EMG root-mean-square amplitude and MPF exhibited changes consistent with a warm-up effect. It was concluded that over-recovery was not present in the tibialis anterior following a local muscular fatigue. However, the return in force to initial values, rather than a persistent decrement as normally observed, was mediated by the warm-up effect.

Priming the Governance System for Climate Change Adaptation: The Application of a Social Ecological Inventory (SEI) to Engage Actors in Niagara, Canada

Adaptive systems of governance are increasingly gaining attention in respect to complex and uncertain social-ecological systems. Adaptive co-management is one strategy to make adaptive governance operational and holds promise with respect to community climate change adaptation as it facilitates participation and learning across scales and fosters adaptive capacity and resilience. Developing tools which hasten the realization of such approaches are growing in importance. This paper describes explores the Social Ecological Inventory (SEI) as a tool to 'prime' a regional climate change adaptation network. The SEI tool draws upon the social-ecological systems approach in which social and ecological systems are considered linked. SEIs bridge the gap between conventional stakeholder analysis and biological inventories and take place through a six phase process. A case study describes the results of applying an SEI to prime an adaptive governance network for climate change adaptation in the Niagara Region of Canada. Lessons learned from the case study are discussed and highlight how the SEI catalyzed the adaptive co-management process in the case. Future avenues for SEIs in relation to climate change adaptation emerge from this exploratory work and offer opportunities to inform research and adaptation planning.

Novel glutathione disulfide transferase function of CC-glutaredoxins involved in disease resistance and flower development

Glutaredoxins are oxidoreductases capable of reducing protein disulfide bridges and glutathione mixed disulfides through the process of deglutathionylation and glutathionylation. Lately, redox-mediated modifications of functional cysteine residues of TGA1 and TGA8 transcription factors have been postulated. Namely, GRX480 and ROXY1 glutaredoxins have been previously shown to interact with TGA proteins and have been suggested to regulate redox state of these proteins. TGA1, together with TGA2, is involved in systemic acquired resistance (SAR) establishment in the plant Arabidopsis thaliana through PR1 (Pathogenesis related 1) gene activation. They both form an enhanceosome complex with the NPR1 protein (non-expressor of pathogenesis related gene 1) which leads to PR1 transcription. Although TGA1 is capable of activating PR1 transcription, the ability of the TGA1 NPR1 enhanceosome complex to assembly is based on the redox status of TGA1. We identified GRX480 as a glutathionylating enzyme that catalyzes the TGA1 glutathione disulfide transferase reaction with a Km of around 20μM GSSG (oxidized glutathione). Out of four cysteine residues found within TGA1, C172 and C266 were found to be glutathionylated by this enzyme. We also confirmed TGA1 glutathionylation in vivo and showed that this modification takes place while TGA1 is associated with the PR1 promoter enzymatically via GRX480. Furthermore, we show that glutathionylation via GRX480 abolishes TGA1's interaction with NPR1 and consequently prevents the TGA1-NPR1 transcription activation of PR1. When glutathionylated, TGA1 is recruited to the PR1 promoter and acts as a repressor. Therefore, glutathionylation is a mechanism that prevents TGA1 NPR1 interaction, allowing TGA1 to function as a repressor of PR1 transcription. Surprisingly, GRX480 was not able to deglutathionylate proteins demonstrating the irreversible nature of the reaction. Moreover, we demonstrate that other members of CC-class glutaredoxins, namely ROXY1 and ROXY2, can also catalyze protein glutathionylation. The TGA8 protein was previously shown to interact with NPR1 analogs, BOP1 and BOP2 proteins. However, unlike the case of TGA1 NPR1 interaction, here we demonstrate that TGA8-BOP1 interaction is not redox regulated and that TGA8 glutathionylation by ROXY1 and ROXY2 enzymes does not abolish this interaction in vitro. However, TGA8 glutathionylation results in TGA8 oligomer disassembly into smaller complexes and monomers. Our results suggest that CC-Grxs are unable to reduce mixed disulfides, instead they efficiently catalyze the opposite reaction which distinguishes them from traditional glutaredoxins. Therefore, they should not be classified as glutaredoxins but as protein glutathione disulfide transferases.

List of Employees to be involved in the extension of the Port Dalhousie and Thorold Railway

List of Employees to be involved in the extension of the Port Dalhousie and Thorold Railway (1 page, handwritten). This is signed by S.D. Woodruff, Nov. 25, 1856.