To test the hypothesis that red-osier dogwood cuttings from a more stressful edaphic environment (dry site) would have a greater salinity tolerance compared with cuttings from a less stressful environment (moist site) a greenhouse experiment was designed. Cuttings collected from three sites in southeastern Manitoba varying in edaphic conditions (moisture) were exposed to 0, 25, 50, or 100 mM NaCl in hydroponics. After four weeks of treatment, physiological parameters were measured. No differences in salinity tolerance were observed between the sites; nevertheless, this study supported previous results suggesting that red-osier dogwood can limit the transport of Na+ from the root to the shoot. To determine the presence of barriers to ion movement, the roots of red-osier dogwood exposed to NaCl were sectioned and observed using brightfield and fluorescence techniques. A modified outer cortical layer was observed suggesting the presence of an exodermis, which would be an additional barrier to Na+ and Cl- movement. Furthermore, the maturation of the endodermis and exodermis occurring closer to the root tip could also contribute to limiting the transport of Na+ to the shoot.

To test the hypothesis that red-osier dogwood cuttings from a more stressful edaphic environment (dry site) would have a greater salinity tolerance compared with cuttings from a less stressful environment (moist site) a greenhouse experiment was designed. Cuttings collected from three sites in southeastern Manitoba varying in edaphic conditions (moisture) were exposed to 0, 25, 50, or 100 mM NaCl in hydroponics. After four weeks of treatment, physiological parameters were measured. No differences in salinity tolerance were observed between the sites; nevertheless, this study supported previous results suggesting that red-osier dogwood can limit the transport of Na+ from the root to the shoot. To determine the presence of barriers to ion movement, the roots of red-osier dogwood exposed to NaCl were sectioned and observed using brightfield and fluorescence techniques. A modified outer cortical layer was observed suggesting the presence of an exodermis, which would be an additional barrier to Na+ and Cl- movement. Furthermore, the maturation of the endodermis and exodermis occurring closer to the root tip could also contribute to limiting the transport of Na+ to the shoot.

Chilling-related postharvest rind breakdown of navel oranges is a significant economic problem worldwide. Chilling injury (CI) symptoms on navel orange fruit vary, and descriptive classification is generally ad hoc, making inter-study comparisons difficult. In this study, external symptoms of CI were related to patterns of cellular collapse in affected flavedo tissue, and a classification system developed to aid consistent symptom identification and improve communication within the supply chain. Potential markers of senescence were evaluated because older fruit were found to be more susceptible to CI. Electrolyte leakage, moisture content and protein content of flavedo tissue were ineffective indicators of both senescence and chilling stress. Rind colour and internal maturity were generally good indicators of fruit age, but lacked sensitivity over short time periods to be of use. Although there was a strong seasonal effect on CI incidence, pre-storage treatments, including hot water and methyl jasmonate, generally reduced the incidence of CI. Because these treatments elicit defence responses that protect tissue from chilling stress, the response and efficiency of plant defence systems is probably an important factor in chilling tolerance. The concentration of lipid hydroperoxides (LOOH) in flavedo tissue was lower in fruit that were stored at a chilling temperature (1�C) compared to fruit that were stored at a non-chilling temperature (12�C) and lipid peroxidation did not increase during storage at 1�C. There was also a lower concentration of LOOH in the chilling sensitive variety than in the chilling tolerant variety. Therefore, increased lipid peroxidation is not related to chilling stress and subsequent injury but the results do suggest a role for LOOH in stress signalling. Antioxidant activity in the lipophilic fraction of flavedo tissue extracts increased as fruit senesced and was strongly correlated with carotenoid content. LOOH concentrations in flavedo tissue also increased as fruit senesced. The antioxidant activity of both the lipophilic and hydrophilic fractions of flavedo tissue extracts was higher in fruit stored at 12�C than in fruit stored at 1�C.

Microbes have a remarkable ability to adapt to a host of environmental stressors, including low temperature, high pressure and osmotic stresses. The adaptations of resistant microbes to low temperatures are varied, and may include the accumulation of solutes to maintain osmotic balance, the production of antifreeze proteins (AFPs) or ice nucleation proteins (INPs) to manipulate ice growth or formation. AFPs depress the freezing point, inhibit ice recrystallization, and have been reported to inhibit or delay the growth of gas hydrates. Conversely, INPs precipitate ice formation at relatively high subzero temperatures. Collectively, these activities can be described as ‘ice-association’ activities. Here, ice-affinity and/or freeze-thaw cycling were used to either select for isolates with ice association properties or to assess the low temperature resistance of microbial consortia derived from various environments. Ice-affinity successfully selected psychrotolerant microbes from cultured temperate and boreal soils, some of which had been previously reported in glaciers and Arctic/Antarctic sites. Many of the recovered microbes demonstrated ice-association activities. Freeze-thaw selection also greatly decreased the abundance and diversity of consortia from distinct sites, and allowed the recovery of individual isolates, many of which demonstrated ice-association. Freeze-thaw selection was also used to assess the role of cross-tolerance between osmotic and freeze-thaw stresses, based on the common challenge of desiccation. Microbial consortia from lakes with varying degrees of salinity were subjected to freeze-thaw stress, and the consortia from more saline lakes tended to show greater low temperature resistance. While few of the recovered microbes demonstrated ice-association activities, those from the more saline lakes tended to contain a higher intracellular solute concentration and were more likely to form biofilms. This underscores the diversity of resistance strategies and supports the notion of cross-tolerance. To determine if these selective regimes would have applications for hydrate growth inhibition, microbes derived from an oil well sample were subjected to freeze-thaw stress. Selection reduced microbial abundance, shifted the diversity, and resulted in the recovery of microbes with some ice-association activity. Taken together, this thesis demonstrates that the application of low temperature stress can be used to successfully investigate stress resistance mechanisms within microbial communities from distinct environments. / Thesis (Ph.D, Biology) -- Queen's University, 2010-09-21 15:58:14.932

Chilling-related postharvest rind breakdown of navel oranges is a significant economic problem worldwide. Chilling injury (CI) symptoms on navel orange fruit vary, and descriptive classification is generally ad hoc, making inter-study comparisons difficult. In this study, external symptoms of CI were related to patterns of cellular collapse in affected flavedo tissue, and a classification system developed to aid consistent symptom identification and improve communication within the supply chain. Potential markers of senescence were evaluated because older fruit were found to be more susceptible to CI. Electrolyte leakage, moisture content and protein content of flavedo tissue were ineffective indicators of both senescence and chilling stress. Rind colour and internal maturity were generally good indicators of fruit age, but lacked sensitivity over short time periods to be of use. Although there was a strong seasonal effect on CI incidence, pre-storage treatments, including hot water and methyl jasmonate, generally reduced the incidence of CI. Because these treatments elicit defence responses that protect tissue from chilling stress, the response and efficiency of plant defence systems is probably an important factor in chilling tolerance. The concentration of lipid hydroperoxides (LOOH) in flavedo tissue was lower in fruit that were stored at a chilling temperature (1�C) compared to fruit that were stored at a non-chilling temperature (12�C) and lipid peroxidation did not increase during storage at 1�C. There was also a lower concentration of LOOH in the chilling sensitive variety than in the chilling tolerant variety. Therefore, increased lipid peroxidation is not related to chilling stress and subsequent injury but the results do suggest a role for LOOH in stress signalling. Antioxidant activity in the lipophilic fraction of flavedo tissue extracts increased as fruit senesced and was strongly correlated with carotenoid content. LOOH concentrations in flavedo tissue also increased as fruit senesced. The antioxidant activity of both the lipophilic and hydrophilic fractions of flavedo tissue extracts was higher in fruit stored at 12�C than in fruit stored at 1�C.

Due to its unique receptor binding profile and its relationship to clozapine, N-desmethylclozapine (NDMC) has been examined as a possible antipsychotic drug (APD). Clozapine has been trained as discriminative stimulus in our lab, but NDMC has not yet been established as a discriminative stimulus. In experiment 1, 12 C57BL/6 mice were trained to discriminate 10.0 mg/kg NDMC from VEH using a standard-two lever operant procedure to assess antipsychotic substitution. The typical APD clozapine fully substituted for NDMC at 2 doses tested (2.5 and 5.0 mg/kg), while typical APD haloperidol failed to substitute for NDMC. In Experiment 2, 11 mice were given repeated administration of NDMC to assess cross-tolerance development to the discriminative stimulus of clozapine. NDMC was successfully trained as a discriminative stimulus and was also shown to induce cross-tolerance to clozapine’s discriminative stimulus, indicating similar underlying pharmacological mechanisms of action between NDMC and clozapine.

La pollution environnementale constitue un problème majeur pour les écosystèmes naturels et la santé publique. Récemment, la phytoremédiation a émergé comme une stratégie innovante, écologique et à faible coût. Elle consiste à utiliser les capacités des plantes à stocker ou/et à dégrader les polluants. Dans cette étude, des analyses physiologiques, biochimiques et métabolomiques, ont permis de montrer que les halophytes, une catégorie des plantes extrêmophiles, présentent une grande capacité à remédier la pollution engendrée par les Hydrocarbures Aromatiques Polycycliques (HAPs). D'autre part, la salinité modérée améliore leur capacité phytoremédiatrice. Ces résultats apportent de nouvelles données pouvant contribuer à l'amélioration de cette stratégie. / Halopytes, plants naturally adapted to high salt concentrations, have no clear definition, yet. Their cross-tolerance to abiotic stresses was reviewed in this work at the physiological, biochemical, and molecular levels, with a special emphasis on the mechanisms involved in their cross-tolerance to salinity and organic pollutants that could allow them a higher potential of xenobiotic phytoremediation as compared to glycophytes. In our experimental part, we compared in a first step some physiological and antioxidant responses to phenanthrene as well as its accumulation in the two related model plants Arabidopsis thaliana (glycophyte) and Thellungiella salsuginea (halophyte).In a second step, we investigated the effects of moderate salinity on the responses of the two species to phenanthrene considered as model Polycyclic Aromatic Hydrocarbon (PAH) molecule. Obtained results showed an improvement of phenanthrene-induced responses in the two plants, the effect being more marked in the halophyte. This observation was particularly related to higher antioxidant activities and the induction of more adapted metabolism as several accumulated metabolites are known to be involved in signaling and osmotic adjustment processes. In a final step, we studied the potential of the halophyte Cakile maritima to remediate an inert sand (to avoid the degradation of the pollutant by microorganisms or their interaction with the plant) highly contaminated with phenanthrene.

A administração de Ags solúveis via mucosas, antes da sensibilização com o mesmo Ag, leva a tolerância. Na presente tese estudou-se o efeito do LPS i.n. durante a indução de tolerância nasal a ovalbumina (OVA). A maioria dos modelos murinos de asma utilizam a OVA como alérgeno, desse modo também utilizamos o extrato de Blomia tropicalis (Bt), um ácaro prevalente em pacientes asmáticos. Características da Bt impedem o estabelecimento de tolerância e evidências experimentais indicam que a tolerância a um Ag pode induzir tolerância a um Ag não relacionado (tolerância cruzada). Assim, avaliamos se a tolerância a OVA ou lizozima de ovo (HEL) poderia induzir tolerância cruzada a Bt. Observou-se que o LPS i.n. durante a indução de tolerância a OVA previne o estabelecimento da tolerância, resultando em neutrofilia pumonar, IgG2a, diminuição de IgE com aumento de IgG1 anafilática. Observou-se também que a tolerância nasal, a HEL ou OVA, não é eficaz em induzir tolerância cruzada para as respostas contra Bt. Já, a tolerância oral com OVA induziu tolerância cruzada para Bt. / Mucosal administration of soluble Ags, before the sensitization with the same Ag, leads to mucosal tolerance. We studied the effect of i.n. LPS during the induction of ovalbumin (OVA) nasal tolerance. The majority of murine models of asthma used OVA as allergen,thus, we also used Blomia tropicalis (Bt) extract, a mite more common in asthmatic patients. Features of Bt block the nasal tolerance establishment and experimental data indicates that tolerance towards an Ag can promote tolerance to another not related Ag (cross-tolerance). Therefore, we evaluated if OVA or hen-egg white lisozyme (HEL) tolerance could result in cross-tolerance to the Bt. Our data showed that i.n. LPS during induction of tolerance to OVA prevents the establishment of this tolerance, resulting in neutrophil migration, IgG2a production, decreased levels of IgE and increased anaphylactic IgG1. We verify that nasal tolerance to HEL or OVA is not capable in induce cross-tolerance against the Bt. We also observed that OVA oral tolerance is able to induce cross-tolerance against Bt.

O Acidente Vascular Encefálico (AVE) Hemorrágico representa mais de 10% de todos os casos de AVE e possui altas taxas de morbidade e de mortalidade. Os pacientes que sobrevivem a este evento permanecem com alguma disfunção motora, que algumas vezes é incapacitante. O extravasamento de sangue em um AVE hemorrágico ocorre, geralmente, em regiões onde há bifurcação de pequenas arteríolas penetrantes, como na região dos núcleos da base. O estriado, importante componente dessa região, está relacionado a funções motoras superiores, como o planejamento e a execução do movimento. Alguns estudos demonstram que o pré-condicionamento (PC) isquêmico pode gerar a tolerância a outros eventos que acometem o sistema nervoso. O PC é definido como fenômeno decorrente da exposição de um tecido ou órgão a um insulto sub-letal capaz de resultar em adaptações determinantes para a tolerância tecidual. Isso ocorre mesmo quando esses dois estímulos são de origens diferentes; neste caso diz-se que o PC desenvolveu tolerância cruzada. Desta forma, o presente estudo dedicou-se ao estudo de efeitos celulares e funcionais do pré-condicionamento isquêmico, por oclusão bilateral das artérias carótidas durante 10 minutos, sobre o modelo de hemorragia intracerebral (HIC), por administração intraestriatal de colagenase do tipo IV-S em ratos. A hipótese de trabalho era de que o PC causaria tolerância cruzada para a HIC, e consequente neuroproteção avaliada por testes motores, volume de lesão, com envolvimento de astrocitose e de micróglia reativa Foram usados 67 ratos machos Wistar adultos, divididos em 4 grupos: Sham (controle cirúrgico), PC, HIC, PC+HIC. Assim, os animais dos grupos PC e PC+HIC foram submetidos ao pré-condicionamento e 24 horas depois os animais HIC e PC+HIC receberam a injeção de colagenase, enquanto os animais Sham e PC receberam uma injeção de salina. A avaliação motora dos animais foi realizada a partir dos testes do cilindro e do Staircase. Trinta e quatro dias após a HIC os animais foram perfundidos e o estriado ipsilateral à injeção foi dissecada para obtenção de amostras teciduais necessárias à avaliação da perda tecidual e quantificação de intensidade de fluorescência de GFAP (proteína glial fibrilar ácida) e OX-42, importantes marcadores de astrócitos e microglia, respectivamente. Os resultados demonstram que: a) a HIC causa deficits motores em ambos os testes realizados, e que o PC reverte este efeito; b) a HIC causa lesão estriatal que não é revertido pelo pré-condicionamento; c) a HIC causa aumento da intensidade de fluorescência para GFAP e para OX-42, e o PC reverte apenas a reatividade da micróglia. Em conjunto, sugere-se que o pré-condicionamento isquêmico causa tolerância cruzada com a hemorragia intracerebral experimental, resultando em proteção funcional, mas não morfológica, possivelmente associada a uma diminuição da reatividade da microglia após o evento hemorrágico. / Hemorrhagic Vascular Stroke (EVA) represents more than 10% of all stroke cases with high rates of morbidity and mortality. Patients who survive this event, remain with some motor dysfunction, which is sometimes disabling. The extravasation of blood in a hemorrhagic stroke occurs, generally, in regions where there is bifurcation of small vessels, as in the region of striatum. The striatum is related to the higher motor functions, such as the planning and execution of the movement. Some studies have shown that preconditioning (PC) can generate a tolerance to other events that accompany the nervous system. The PC is presented as the source of the exposure of a sub-lethal, resulting in an adaptation of determinants to a tissue tolerance. Thus, the present study aimed shows the ischemic preconditioning effects, by bilateral occlusion of the carotid arteries for 10 minutes, on the intracerebral hemorrhage (ICH) model, by intra- striatum administration of type IV S collagenase in rats. The working hypothesis was tolerance to HIC, and consequent neuroprotection by motor function, lesion volume, astrocytosis and reactive microglia. A total of 84 male Wistar adult rats were divided into 4 groups: Sham (surgical control), PC, HIC, PC + HIC Thus, the animals of the PC and PC + HIC groups were introduced to the preconditioning and 24 hours later, the HIC and PC + HIC animals received a collagenase injection, while the Sham and PC animals received a saline injection. The evaluation of the animal function was performed from cylinder and Staircase tests. Thirty-four days after the surgery, the striatum was dissected and prepared to lesion volume analysis and fluorescence intensity of GFAP quantification (acid glial fibrillary protein) and OX-42, important astrocyte and microglia markers respectively. The results demonstrate that: a) an HIC causes motor deficits in both tests performed, and that the PC reverses this effect; b) an ICH causes a striatal lesion that is not reversed by preconditioning; c) an HIC promoted high fluorescence intensity for GFAP and OX-42, and PC reverses the microglia reactivity. Taken together, we suggest that ischemic preconditioning combined with experimental intracerebral hemorrhage, promotes functional but not morphological protection, being associated with the microglial reactivity decrease after the hemorrhagic event.

O Acidente Vascular Encefálico (AVE) Hemorrágico representa mais de 10% de todos os casos de AVE e possui altas taxas de morbidade e de mortalidade. Os pacientes que sobrevivem a este evento permanecem com alguma disfunção motora, que algumas vezes é incapacitante. O extravasamento de sangue em um AVE hemorrágico ocorre, geralmente, em regiões onde há bifurcação de pequenas arteríolas penetrantes, como na região dos núcleos da base. O estriado, importante componente dessa região, está relacionado a funções motoras superiores, como o planejamento e a execução do movimento. Alguns estudos demonstram que o pré-condicionamento (PC) isquêmico pode gerar a tolerância a outros eventos que acometem o sistema nervoso. O PC é definido como fenômeno decorrente da exposição de um tecido ou órgão a um insulto sub-letal capaz de resultar em adaptações determinantes para a tolerância tecidual. Isso ocorre mesmo quando esses dois estímulos são de origens diferentes; neste caso diz-se que o PC desenvolveu tolerância cruzada. Desta forma, o presente estudo dedicou-se ao estudo de efeitos celulares e funcionais do pré-condicionamento isquêmico, por oclusão bilateral das artérias carótidas durante 10 minutos, sobre o modelo de hemorragia intracerebral (HIC), por administração intraestriatal de colagenase do tipo IV-S em ratos. A hipótese de trabalho era de que o PC causaria tolerância cruzada para a HIC, e consequente neuroproteção avaliada por testes motores, volume de lesão, com envolvimento de astrocitose e de micróglia reativa Foram usados 67 ratos machos Wistar adultos, divididos em 4 grupos: Sham (controle cirúrgico), PC, HIC, PC+HIC. Assim, os animais dos grupos PC e PC+HIC foram submetidos ao pré-condicionamento e 24 horas depois os animais HIC e PC+HIC receberam a injeção de colagenase, enquanto os animais Sham e PC receberam uma injeção de salina. A avaliação motora dos animais foi realizada a partir dos testes do cilindro e do Staircase. Trinta e quatro dias após a HIC os animais foram perfundidos e o estriado ipsilateral à injeção foi dissecada para obtenção de amostras teciduais necessárias à avaliação da perda tecidual e quantificação de intensidade de fluorescência de GFAP (proteína glial fibrilar ácida) e OX-42, importantes marcadores de astrócitos e microglia, respectivamente. Os resultados demonstram que: a) a HIC causa deficits motores em ambos os testes realizados, e que o PC reverte este efeito; b) a HIC causa lesão estriatal que não é revertido pelo pré-condicionamento; c) a HIC causa aumento da intensidade de fluorescência para GFAP e para OX-42, e o PC reverte apenas a reatividade da micróglia. Em conjunto, sugere-se que o pré-condicionamento isquêmico causa tolerância cruzada com a hemorragia intracerebral experimental, resultando em proteção funcional, mas não morfológica, possivelmente associada a uma diminuição da reatividade da microglia após o evento hemorrágico. / Hemorrhagic Vascular Stroke (EVA) represents more than 10% of all stroke cases with high rates of morbidity and mortality. Patients who survive this event, remain with some motor dysfunction, which is sometimes disabling. The extravasation of blood in a hemorrhagic stroke occurs, generally, in regions where there is bifurcation of small vessels, as in the region of striatum. The striatum is related to the higher motor functions, such as the planning and execution of the movement. Some studies have shown that preconditioning (PC) can generate a tolerance to other events that accompany the nervous system. The PC is presented as the source of the exposure of a sub-lethal, resulting in an adaptation of determinants to a tissue tolerance. Thus, the present study aimed shows the ischemic preconditioning effects, by bilateral occlusion of the carotid arteries for 10 minutes, on the intracerebral hemorrhage (ICH) model, by intra- striatum administration of type IV S collagenase in rats. The working hypothesis was tolerance to HIC, and consequent neuroprotection by motor function, lesion volume, astrocytosis and reactive microglia. A total of 84 male Wistar adult rats were divided into 4 groups: Sham (surgical control), PC, HIC, PC + HIC Thus, the animals of the PC and PC + HIC groups were introduced to the preconditioning and 24 hours later, the HIC and PC + HIC animals received a collagenase injection, while the Sham and PC animals received a saline injection. The evaluation of the animal function was performed from cylinder and Staircase tests. Thirty-four days after the surgery, the striatum was dissected and prepared to lesion volume analysis and fluorescence intensity of GFAP quantification (acid glial fibrillary protein) and OX-42, important astrocyte and microglia markers respectively. The results demonstrate that: a) an HIC causes motor deficits in both tests performed, and that the PC reverses this effect; b) an ICH causes a striatal lesion that is not reversed by preconditioning; c) an HIC promoted high fluorescence intensity for GFAP and OX-42, and PC reverses the microglia reactivity. Taken together, we suggest that ischemic preconditioning combined with experimental intracerebral hemorrhage, promotes functional but not morphological protection, being associated with the microglial reactivity decrease after the hemorrhagic event.