One great issue of reforestation is the infestation of pest insects. The pine weevil (Hylobius abietis L.) is one of the economically most important pest insects in Europe and Asia. When insects feed on the trees the plants emit compounds that repel the insects. Methyl salicylate (MeSA) is one substance known to be emitted by conifer seedlings after herbivore feeding. Similar compounds are emitted by fungus isolated from pine weevil frass. Female pine weevils add frass to the egg laying site to protect the eggs from other pine weevils. The goal of the project was to cultivate the fungi Ophiostoma pluriannulatum and analyze the volatile substances. The fungus was cultivated using natural pine weevil frass (NF) as main substrate. Later new batches were prepared using lignin and potato dextrose broth (PDB), as feeding media to compare the results. Two sampling methods were used, SPME and adsorption with Porapak™ polymer column packing material. These samples were analyzed using GC-MS. Methyl salicylate was found in NF fungi cultures, peaking after ten days. Another, unexpected, product of the fungi was a sesquiterpene hydrocarbon. The lignin and the PDB samples did not produce MeSA. However, the sesquiterpene could be found in the lignin samples.

Leptinotarsa decemlineata (Say) is the most important defoliating pest of potato Solanum tuberosum L., in North America and Europe.  Management of this pest relies heavily on chemical control and insecticide resistance is a persistent problem.  This phenomenon has increased the need for developing novel insecticides, resistance evaluation, and the development of alternative control strategies regarding this insect pest.  From 2010 to 2013, field and lab experiments were conducted to evaluate the efficacy of a novel insecticide tolfenpyrad on L. decemlineata.  In leaf-dip assays, tolfenpyrad was highly toxic to L. decemlineata with LC50 values of 0.013 and 0.164 g ai/L for larvae and adults, respectively.  Tolfenpyrad was also toxic to eggs with 0% hatching after being dipped in a field rate concentration.  In field efficacy trials, potato plots treated with tolfenpyrad at rates as low as 153 g ai/ha effectively controlled L. decemlineata. In 2012, populations of L. decemlineata were collected from the Eastern Shore of VA and subjected to toxicity assays to determine current susceptibility to permethrin and oxamyl.  The toxicity assays indicated an increase in toxicity to permethrin in L. decemlineata larvae (LC50 = 3.931 g ai/L) and an increase in toxicity to oxamyl in adult beetles (LC50 = 9.695 g ai/L) compared with LC50 values previously reported in 1990.  In 2012, populations of L. decemlineata from Cheriton, VA, New Church, VA, Painter, VA, and Plymouth, NC were also evaluated for enzyme activity after exposure to sub-lethal concentrations of permethrin, oxamyl, and tolfenpyrad.  Adult beetles were subjected to enzyme assays to measure the activity of cytochrome P450 mono-oxygenase (P450), glutathione-S-transferase (GST), general esterases, and protein content.  Results from the enzyme assays indicated significantly greater esterase activity in beetles from Painter, VA exposed to permethrin [±-naphthol (F= 11.66, df= 4, 20, P<0.0001) and "-naphthol (F= 11.86, df= 4, 20, P<0.0001)], oxamyl [±- naphthol (F= 10.64, df= 4, 20, P<0.0001) and "-naphthol (F= 6.94, df= 4, 20, P=0.0011)], tolfenpyrad [±- naphthol (F= 407.62, df= 1, 8, P<0.0001) and "- naphthol (F= 28.15, df= 1, 8, P= 0.0007)], and the untreated control [±- naphthol (F= 28.14, df= 3, 16, P<0.0001) and "- naphthol (F= 28.86; df= 3, 16, P<0.0001)] compared to most of the other populations tested.  GST activity was significantly greater in tolfenpyrad exposed beetles compared to the non-treated beetles from Painter VA (F= 17.66, df= 5, 24, P< 0.0001). Through laboratory assays and field experiments in potato, the efficacy of a new bio-pesticide derived from the bacterium Chromobacterium subtsugae was evaluated for the control of L. decemlineata.  Results from the laboratory assays showed L. decemlineata feeding was inhibited by the bio-pesticide derived from C. subtsugae.  However, field efficacy trials in 2010, 2011, and 2012, indicated no control of L. decemlineata. Methyl salicylate is an organic compound produced by potato and other plants in response to insect herbivory.  Abundance of predatory arthropods and L. decemlineata life stages were measured in plots treated with and without 5 g slow-release packets of methyl salicylate (95% methyl salicylate (Predalure")).  Methyl salicylate treatment had no impact on predator recruitment or cumulative mortality of L. decemlineata in potatoes. This research has provided us with a new tool for L. decemlineata management, as well as more information about resistance trends and alternative control strategies from which we can build on to reduce resistance development in L. decemlineata and ultimately formulate a stronger integrated pest management strategy for this insect pest. / Ph. D.

Cytosolic protein degradation is crucial for cellular homeostasis as it orchestrates protein turnover by destruction of misfolded, unstable and abnormal proteins. This process has two main stages: (i) an ATP-dependent stage mediated by unfoldases and proteases, and (ii) an ATP-independent stage mediated by various peptidases. The ATP dependent proteases recognise target proteins and cleave them into smaller peptides. These enzymes comprise the ATPase-family-associated-with-various-cellular-activities domain that is important for unfolding target proteins. Subsequently, unfolded proteins enter a barrel-shaped proteolytic chamber, an architecture conserved throughout prokaryotes, archea and eukaryotes, where the peptide bond is hydrolysed in an ATP-independent manner. The smaller peptides released are broken down by ATP-independent peptidases into free amino acids recycled into the cellular pool. In prokaryotes, major cellular protein degradation functions are performed by Clp and Lon proteases. Earlier studies in our laboratory have shown the role of an ATP-independent peptidase, AminopeptidaseN, in sodium salicylate (NaSal) induced growth inhibition. NaSal belongs to the family of Non-Steroidal Anti-Inflammatory Drugs and its acetyl ester, Aspirin, is a very widely used analgesic. It is produced by plants as a defence response and is known to cause different effects, including xenobiotic stress i. e. stress mediated by compounds which are not naturally produced or expected to be present in the organism in bacteria. In bacteria, salicylate modulates outer membrane proteins, virulence factors, and reduces motility. In addition, NaSal is able induce “phenotypic antibiotic resistance” by binding to MarR and de-repressing the mar operon. NaSal promotes the dissociation of MarR from the promoter site leading to transcription of MarA, a transcription factor that induces several genes that encode the AcrAB-TolC pump which effluxes multiple antibiotics from the cell. The present study investigates the possible roles of ATP-dependent proteases, Lon and Clp, during growth reduction of E. coli induced with high (2-3 mM) amounts of NaSal. The growth of the Lon mutant (lon), but not clpP, was found to be greatly reduced with high doses of NaSal in the media. Our hypothesis was that the lack of Lon led to the accumulation of high amounts of substrate proteins, which led to its greater sensitivity with high doses of NaSal. To identify the substrate protein/s and to better understand the mechanism of action, single and double mutants (with lon) of E. coli lacking several prominent Lon substrates, i.e. MarA, RcsA, StpA, SulA and UmuD, were generated and screened for rescue of growth with 2-3 mM NaSal. MarA, a transcription factor, was identified to be important during NaSal-induced growth reduction. It modulates outer membrane proteins and induces the AcrAB-TolC pump that increases the efflux of antibiotics from the cell. Also, RT-PCR analysis revealed that the levels of marA and its targets, acrA and acrB, were higher in the lon strain suggesting that the MarA protein levels were stabilised the cell in the absence of Lon. Further studies using approriate strains demonstrated that one of the effectors of MarA, i.e. the AcrAB-TolC efflux pump, was not involved in the NaSal-induced growth inhibition of lon. Therefore, in presence of higher doses of NaSal, MarA is upregulated due to de-repression of the operon. The levels of MarA are regulated by Lon via degradation but in the absence of Lon, MarA levels are stabilised and lead to upregulation of MarA and its target genes like AcrAB-TolC. This study identifies higher amounts of MarA to be responsible for NaSal-induced growth inhibition of lon. Subsequently, experiments were conducted to demonstrate the role of MarA and its targets in antibiotic resistance with low dose (0.5 mM) NaSal that does not affect growth. This low dose of NaSal was able to upregulate marA and its target genes, acrA, acrB and tolC. Quantification of antibiotic resistance further revealed an induction in resistance by 0.5 mM NaSal in a MarA- and AcrB-dependent fashion. Studies using atomic force microscopy demonstrated that ciprofloxacin-induced cell elongation was lower in lon due to higher levels of MarA. Therefore, low dose of NaSal is capable of upregulating MarA and inducing antibiotic resistance but does not affect cell growth. This part of the study addresses the roles of Lon protease, its substrate MarA and MarA-induced targets, e.g. AcrB, during NaSal-mediated growth reduction and antibiotic resistance. The factors contributing to antibiotic resistance in bacteria are an important area of study for the global public health care system. Antibiotic resistance can be acquired by transmittance of genetic material, accumulation of antibiotic resistant mutations in the target molecule or can be induced by certain compounds present in the environment like NaSal. For rapid identification of compounds that may behave in a similar fashion as NaSal, a 96-well plate based screen was developed utilising the growth inhibition feature of the lon strain. The compounds were selected on the basis of their structural (phenolic compounds) and functional (Non-Steroidal Anti Inflammatory Drugs or NSAIDs) similarity to NaSal. Through this screen, four compounds were identified that lowered the growth of lon more than that of wild type strain and may be important in inducing phenotypic antibiotic resistance: Acetaminophen (anti-pyretic), Ibuprofen (NSAID), and two phenolic uncouplers, Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-dinitrophenol (2,4-DNP). Notably, another compound Phenylbutazone (NSAID), which is used to lower inflammation in animals, did not reduce the growth of E. coli. RNA expression analysis revealed that these four compounds, but not phenylbutazone, induced the expression of marA and its target gene involved in antibiotic efflux, acrB. Furthermore, dose dependent and comparative studies with Nasal demonstrated differential effects of these four compounds in inducing antibiotic resistance with respect to ciprofloxacin, tetracycline and nalidixic acid. The two uncouplers were much more effective in inducing antibiotic resistance at lower doses than the NSAIDs. As NSAIDs are clinically important compounds, the study suggests that it would be desirable to screen them for induction of antibiotic resistance. The approach elucidated in this study has the potential to identify additional compounds present in the environment that may contribute to antibiotic resistance in bacteria. Overall, this study delineates the roles of Lon protease and its substrate, MarA, during NaSal-mediated responses, involving antibiotic resistance and/or growth reduction in E. coli. In addition, four other compounds were identified that could induce phenotypic antibiotic resistance in E. coli in a MarA-dependent manner. These observations may have implications in the adaptation of bacteria under different environmental conditions.

L’hyperacousie est définie comme une hypersensibilité aux sons d’intensité modérée. Il s’agit d’un trouble de la perception auditive qui a des répercussions néfastes sur la qualité de vie des patients. Un mécanisme impliquerait la modification du gain central des neurones auditifs centraux sous l'influence des régions cérébrales non auditives. Toutefois, la question de l'endroit où ces anomalies surviennent dans le système auditif reste ouverte, plus particulièrement lorsque l'hyperacousie survient sans perte auditive neurosensorielle. Nous avons utilisé un modèle animal d’hyperacousie réversible et sans perte audiométrique, induit par injection aigue de salicylate. Nous avons observé un raccourcissement significatif de la latence des ondes III et IV des PEA aux fréquences inférieures à 10 kHz corrélé à une diminution de 15 dB du seuil de réflexe de sursaut acoustique à basses fréquences. La diminution de l’efficacité du réflexe musculaire de l’oreille moyenne suite à l’injection de salicylate semble trop modérée pour expliquer l'hyperacousie, qui semble plutôt être causée par une altération du codage temporo-spatial de la sonie dans le tronc cérébral. L’hyperacousie fait partie des troubles de perception retrouvés dans la migraine, nous avons étudié les changements physiologiques auditifs dans un modèle animal de migraine induit par administration extradurale de soupe inflammatoire. Dans un contexte migraineux, aucun des paramètres modifiés par le salicylate n'est affecté de sorte que l'hypersensibilité au son est plutôt une phonophobie, réponse émotionnelle au son résultant d’une activation anormale du système limbique et du système nerveux autonome. / Hyperacusis is a disorder of loudness perception defined as decreased tolerance to ordinary environmental sounds that are felt as disagreeable, or even, unbearably painful. The idea that hyperacusis is underpinned by an increase in central auditory gain is now widely invoked. Besides, the neural bases of this gain abnormality remain unclear, as well as more generally the mechanisms of loudness perception. We used an animal model of reversible and hearing-loss-free hyperacusis induced by acute salicylate injection. We observed a significant shortening of the latency of ABR waves III and IV at frequencies below 10 kHz correlated to a 15 dB decrease of the threshold of acoustic startle reflex at low frequencies and without sensorineural hearing loss. The decreased effectiveness of middle ear muscle reflex following salicylate injection seems too moderate for explaining hyperacusis, which is more likely produced by impaired temporo-spatial loudness coding with a brainstem origin. Hyperacusis is one of the perceptual disorders found in migraine. We studied auditory physiological changes in an animal model of migraine induced by extradural administration of inflammatory soup. In a migraine context, none of the salicylate-induced changes was observed so that the reaction to sound is likely closer to phonophobia, an emotional response to sound resulting from abnormal activation of the limbic system and the autonomic nervous system.

Inhibitors used as dopant anions in polymer films are responsible for the anticorrosion behaviour of polypyrrole (Ppy). Ppy film doped with MoO42- and TiF62- showed the role of anions in corrosion protection of iron. Ppy doped with 3-nitro salicylate was used for zinc protection. The results of XPS revealed that molybdate exist in two types: [MoO4]2- (62%) and [Mo7O24]6- (28%). Electrochemical Impedance Spectroscopy (EIS) has indicated the change of the resistance RPM and the capacitance CPM of the Ppy film during reduction. Electrochemical Quartz Crystal Microbalance (EQCM) has shown: the mass of the Ppy film decreased in the cathodic region and increased in the anodic region. The anion flux was also observed in Scanning Kelvinprobe (SKP) and X-ray Photoelectron Spectroscopy (XPS) experiments. The release behaviour of molybdate anions depends much on the size of cations in the electrolyte. TiF62- anions has shown good inhibitor properties. However, the mechanism of this action is not yet known. 3-nitro salicylate (3-nisa) was used as inhibitor dopant for zinc in this work. Ppy film could be formed on Zn without any special pretreatment. The dissolution of Zn decreased dramatically in the presence of 3-nisa. The application of Ppy/metal inorganic composites in corrosion protection for mild steel and galvanised steel may be a better selection. Mixture of core-shell particles with matrix polymer was used as primer for protective coatings.

La famille aromatique du végétal dans les vins rouges recouvre une large diversité de nuances. Nos travaux révèlent que celles-ci n’ont pas toujours de lien direct avec les composés connus (2-méthoxy-3-alkylpyrazines, alcools en C6). Aussi, après avoir été définis sensoriellement, les déterminants moléculaires de cette famille d’odeurs ont été recherchés en s’appuyant, à partir de vins représentatifs, sur différentes techniques de fractionnement (CLHP, distillation sous vide) puis MDCPG-O-SM. Une première origine de ces nuances a été développée, celle associée à la maturité des raisins. La présence du 1,8-cinéole (eucalyptol), avec des notes « vertes », selon une origine variétale en lien avec la maturité des raisins a pu être attestée dans les vins de Cabernet Sauvignon. La capacité d’un cépage à en produire des quantités significatives (Fer Servadou) ainsi qu’une origine exogène associée à la présence d’une espèce d’armoise au vignoble (Artemisia verlotiorum) sont aussi établis. Une autre origine d’apparition des nuances végétales a été considérée : le pressurage des marcs. Cette étude montre le lien étroit entre la catégorie aromatique des vins de presse et la famille du végétal. En outre, elle révèle la présence de divers marqueurs odoriférants, en particulier le (Z)-4-heptenol dont l’évolution est corrélée au niveau de pression et la contribution sensorielle établie, à l’inverse de la plupart des alcools en C6 ; de même qu’elle suggère l’implication de plusieurs composés carbonylés à l’odeur caractéristique des vins de presse. L’influence des paramètres technologiques (inertage, pression, SO2, FML) est aussi précisée. La présence du salicylate de méthyle comme marqueur de défense de la plante contre plusieurs maladies cryptogamiques (mildiou, black rot, esca) et comme contributeur à l’odeur végétale de certains vins rouges a enfin pu être démontrée. / The green aromas family includes in red wines a large diversity of nuances. First, this study revealed that these nuances are not directly related to known compounds (e.g. 2-méthoxy-3-alkylpyrazines, C6 alcohols). After a sensory definition, the molecular determinants of these aroma nuances were investigated based on fractionation strategies including HPLC and vacuum distillation then MDGC-O-MS. A first source of nuances associated with grapes ripeness was developed. The presence of 1,8-cineole (eucalyptol) with a varietal origin was evidenced in Cabernet Sauvignon wines. The ability of a grape variety to produce significant amounts (Fer Servadou) and an exogenous origin associated with the presence of Artemisia species in vineyards (Artemisia verlotiorum) were also highlighted. Another origin, related to the pressing of grape marc was then considered. This study shown the connection between the aromatic category of press wines and the green aromas. Furthermore, it revealed the presence of odoriferous molecules including (Z)-4-heptenol, with a sensory contribution, associated to the increase of pressure in the press tank, but not for the majority of C6 alcohols. It also suggested the involvement of several carbonyl compounds in the characteristic odor of press wines. The influence of technological parameters (without oxygen, pressure, SO2, malolactic fermentation) was also specified. Finally, this study revealed the presence of methyl salicylate as a plant defense marker against several fungal diseases (downy mildew, black rot, esca) and as a contributor to fresh green aromas of certain red wines.

Inhibitors used as dopant anions in polymer films are responsible for the anticorrosion behaviour of polypyrrole (Ppy). Ppy film doped with MoO42- and TiF62- showed the role of anions in corrosion protection of iron. Ppy doped with 3-nitro salicylate was used for zinc protection. The results of XPS revealed that molybdate exist in two types: [MoO4]2- (62%) and [Mo7O24]6- (28%). Electrochemical Impedance Spectroscopy (EIS) has indicated the change of the resistance RPM and the capacitance CPM of the Ppy film during reduction. Electrochemical Quartz Crystal Microbalance (EQCM) has shown: the mass of the Ppy film decreased in the cathodic region and increased in the anodic region. The anion flux was also observed in Scanning Kelvinprobe (SKP) and X-ray Photoelectron Spectroscopy (XPS) experiments. The release behaviour of molybdate anions depends much on the size of cations in the electrolyte. TiF62- anions has shown good inhibitor properties. However, the mechanism of this action is not yet known. 3-nitro salicylate (3-nisa) was used as inhibitor dopant for zinc in this work. Ppy film could be formed on Zn without any special pretreatment. The dissolution of Zn decreased dramatically in the presence of 3-nisa. The application of Ppy/metal inorganic composites in corrosion protection for mild steel and galvanised steel may be a better selection. Mixture of core-shell particles with matrix polymer was used as primer for protective coatings.

This study investigated whether a salicylate-restricted diet (eliminating foods containing artificial additives and natural salicylates) could effectively reduce hyperactivity in children more so than a diet not restricting salicylates (ostensibly restricting foods containing refined sugar). Ten hyperactive children, nine boys and one girl, were matched on their pre-treatment activity rates and assigned to either a salicylate-restricted diet (Group I) or a diet not restricting salicylates (Group II). After approximately nine weeks, post-treatment activity rates were obtained, and a significant difference in favor of the salicylate-restricted diet group was found with this diet group exhibiting a significantly lower mean post-treatment activity rate in comparison to the group placed on a diet not restricting salicylates (p<.05). Implications for diagnosis and treatment of hyperactivity in children were discussed.

<p>QCR 20161026</p>

This thesis is based on three inter-related studies: the first part deals with the microbial consortium, the identification of microbes and their volatiles, the second part deals with the study of bio-chemical control methods of two conifer pests; the pine weevil Hylobius abietis (L.) and the root rot fungi Heterobasidion spp., and the third part describes the production of styrene by a fungus using forest waste.The large pine weevil (Hylobius abietis L.) is an economically important pest insect of conifers in reforestation areas of Europe and Asia. The female weevils protect their eggs from feeding conspecifics by adding frass (mixture of weevil feces and chewed bark) along with the eggs. In order to understand the mechanism behind frass deposition at the egg laying site and to find repellents/antifeedants for pine weevils, microbes were isolated from the aseptically collected pine weevil frass. Microbial produced volatile organic compounds (VOCs) were collected by solid phase micro extraction and analyzed by GC-MS after cultivating them on weevil frass broth. The major VOCs were tested against pine weevils using a multi-choice olfactometer. Ewingella sp., Mucor racemosus, Penicillium solitum, P. expansum, Ophiostoma piceae, O. pluriannulatum, Debaryomyces hansenii and Candida sequanensis were identified as abundant microbes. Styrene, 6-protoilludene, 1-octene-3-ol, 3-methylanisole, methyl salicylate, 2-methoxyphenol and 2-methoxy-4-vinylphenol were the VOCs of persistently isolated microbes. In behavioral bioassay, methyl salicylate, 3-methylanisole and styrene significantly reduced the attraction of pine weevils to their host plant volatiles. Heterobasidion spp. are severe pathogenic fungi of conifers that cause root and butt rot in plants. Bacterial isolates were tested for the antagonistic activity against fungi on potato dextrose agar. Bacillus subtilis strains significantly inhibited the growth of H. annosum and H. parviporum. Styrene is an industrial chemical used for making polymeric products, currently produced from fossil fuel. A strain of Penicillium expansum isolated from pine weevil frass was investigated for the production of styrene using forest waste. Grated pine stem bark and mature oak bark supplemented with yeast extract produced greater amounts of styrene compared to potato dextrose broth. / <p>QC 20130507</p>