Nrf2 (NF-E2-related factor 2) is a transcription factor that regulates a battery of downstream genes that contain an antioxidant response element (ARE) in their promoters, including intracellular redox-balancing proteins, phase II detoxifying enzymes, and transporters. These Nrf2-dependent proteins work in collaboration to protect against many diseases where oxidative stress plays an essential role in disease onset and progression. Consequently, it is imperative to understand the basic molecular mechanisms of how Nrf2 is regulated so we can target this pathway for disease prevention and treatment.Nrf2 is mainly regulated at the protein level by the ubiquitin proteasome system. Under basal conditions Nrf2 is constantly ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and subsequently degraded by the 26S proteasome. Currently, regulation of the Nrf2-Keap1 pathway by ubiquitination is largely understood. However, the mechanism responsible for removal of ubiquitin conjugated to Nrf2 or Keap1 remains unknown. In this dissertation, we identified two molecular mechanisms that are important in understanding how the Nrf2-Keap1 pathway is regulated: (i) USP15 negatively regulates the Nrf2-Keap1 pathway by deubiquitinating Keap1 and (ii) deubiquitinated-Keap1 binds in the Cul3-Keap1-E3 ligase complex more tightly than ubiquitinated-Keap1. Additionally, (iii) we demonstrated the importance of the Nrf2-Keap1 pathway in USP15-dependent paclitaxel-chemoresistance.Under oxidative stressed or induced conditions the ability of the E3-ligase to target Nrf2 for degradation becomes impaired. As a result, Nrf2 is stabilized and free Nrf2 translocates to the nucleus and initiates transcription of ARE-bearing genes. Activation of this pathway is advantageous for chemoprevention. In Chapters 4 and 5, we identified and characterized two activators of the Nrf2 cytoprotective pathway, oridonin and cinnamic aldehyde. These compounds inhibit Cul3-Keap1-dependent degradation of Nrf2, stabilize Nrf2 protein levels, and activate the antioxidant response. Furthermore, both compounds are able to protect against cytotoxic and genotoxic stress-induced cell death. Moreover, our study on USP15 has elucidated an additional mechanism that allows small molecules, such as oridonin, to activate Nrf2 by causing a switch in ubiquitination from Nrf2 to Keap1. Taken together, these findings further our understanding of how the Nrf2-Keap1 pathway is regulated, which is imperative in targeting this pathway for chemoprevention or chemotherapy.

Avaliou-se o efeito de tratamentos químicos, revestimentos comestíveis e irradiação na conservação de mamões processados minimamente. Após seleção, lavagem e sanitização, os mamões foram descascados e cortados em meias rodelas, as quais foram submetidas a diferentes tratamentos, embaladas e armazenadas a 5±1°C e 90±2%UR. Os mamões PM foram avaliados após 1, 3, 6, 9, 12 e 15 dias. As análises microbiológicas foram fundamentadas na quantificação de coliformes totais e termotolerantes, bactérias psicrotróficas, bolores e leveduras, assim como na verificação da presença de Salmonella. As avaliações físico-químicas basearam-se na determinação da concentração de CO2 no interior das embalagens, perda de massa, cor, firmeza, sólidos solúv eis, acidez titulável, ratio e pH. As características sensoriais aparência, aroma, sabor e textura foram avaliad as por meio de testes com escala hedônica. No primeiro experimento os tratamentos foram: controle, aldeído cinâmico a 0,1%, cloreto de cálcio a 0,75% e combinação de aldeído cinâmico a 0,1% com cloreto de cálcio a 0,75%. O uso de tratamentos químicos em mamões PM resultou em: maior controle de coliformes totais para os mamões PM tratados com aldeído cinâmico e com a combinação de aldeído cinâmico e cloreto de cálcio; menor concentração de CO2 e maior manutenção da firmeza para mamões PM tratados com a combinação de aldeído cinâmico e cloreto de cálcio; e maior concentração de CO2 para os mamões PM tratados apenas com aldeído cinâmico. A imersão nos tratamentos químicos resultou em maior descoloração da polpa dos mamões PM e redução do teor de sólidos solúveis ao longo do armazenamento. No segundo experimento foram utilizados os tr atamentos: controle, amido de arroz a 3%, alginato de sódio a 0,5% e carboximetilcelulose a 0,25%. O uso desses três tipos de revestimento resultou em maior controle de coliformes totais do que o observado no controle. Mamões PM revestidos com amido de arroz e carboximetilcelulose apresentaram redução e aumento da concentração de CO2, respectivamente. Os mamões PM revestidos apresentaram menores teores de sólidos solúveis e seus valores de pH se tornaram menores após 9 dias de armazenamento refrigerado. O r evestimento com carboximetilcelulose proporcionou maior firmeza da polpa no 15° dia. No terceiro experimento foram utilizados os tratamentos: controle, radiação nas doses de 2kGy e 4kGy. O uso de radiação gama em mamões PM resultou em: maior controle de coliformes totais; menor concentração de CO2 nos mamões PM tratados com 2kGy; maior concentração de CO2 e maior descoloração da polpa nos mamões PM tratados com 4kGy; redução da firmeza nos mamões PM tratados com 2kGy e 4kGy; ligeira redução do teor de sólidos solúveis e pequenas variações da acidez titulável em todos os tratamentos. As características sensoriais dos mamões PM tratados com radiação gama não diferiram significativamente do controle durante os 15 dias de armazenamento / The effect of chemical treatments, edible coatings , and irradiation on fresh-cut papaya conservation was evaluated. After selection, washing, and sanitation the papayas were peeled and cut into half slices, which were submitted to different treatments, packed, and stored at 5±1°C and 90±2%RU. The fresh-cut papayas were evaluated after 1, 3, 6, 9, 12, and 15 days. The microbiological analyses were based on the count of total coliform, thermotolerant and psychrotrophic bacteria, molds and yeasts, as well as on the presence of Salmonella. The physicochemical evaluations were based on the determination of CO2 concentration inside the package, weight loss, color, firmness, soluble solids, titratable acidity, ratio, and pH. The sensory characteristics appearance, aroma, flavor, and texture were evaluated using a hedonic scale. In the first experiment, the treatments tested we re: control, cinnamic aldehyde 0.1%, calcium chloride 0.75%, and the combination of cinnamic aldehyde 0.1% and calcium chloride 0.75%. Using chemical treatments to preserve fresh cut papaya resulted in: higher control of total coliforms in fresh-cut papayas treated with cinnamic aldehyde and with the combination of cinnamic aldehyde and calcium chloride; lower CO2 concentration and increased maintenance of firmness in freshcut papayas treated with the combination of cinnamic aldehyde and calcium chloride; and increased in the CO2 concentration in fresh-cut papayas treated only with cinnamic aldehyde. Immersion in chemical treatments caused higher pulp discoloration and reduction in solu ble solids during storage. In the second experiment, the treatments tested were: control, rice starch 3%, sodium alginate 0.5%, and carboxymethylcellulose 0.25%. The use of these three coatings resulted in higher control of total coliforms compared to the control treatment. The fresh-cut papayas coated with rice starch and carboxymethylcellulose presented reduction and increase in the CO2 concentration, respectively. Coated fresh-cut papayas presented lower soluble solids and pH values were lower after 9 days of cold storage. Carboxymethylcellulose coating increased firmness maintenance at day 15. In the third experiment, the following treatments were used: control, radiation at the doses of 2kGy and 4kGy. The use of Gamma radiation in fresh-cut papaya resulted in: higher control of total coliforms; lower CO2 concentration in fresh-cut treated with 2kGy; increased CO2 concentration and increased pulp discoloration in fresh-cut papayas treated with 4kGy; reduction in firmness in fresh-cut papayas treated with 2kGy and 4kGy; slight reduction in soluble solids and small changes in titratable acidity in all treatments. The sensory characteristics of fresh-cut papayas treated with gamma radiation did not significantly differ from the control during the 15 days of storage

The large pine weevil, Hylobius abietis L., is a major insect pest on conifer seedlings in northern Europe. Due to its feeding newly planted trees get girdled, resulting in high seedling mortality (up to 80%). As a consequence great financial losses to the forest industry occur. Today the seedlings are protected with the pyrethroid insecticide cypermethrin. This insecticide is toxic to aquatic organisms and is, from 2010, prohibited for use in Sweden by the Swedish Chemicals Inspectorate. An alternative to insecticides is to protect the seedlings with antifeedants, compounds that, either through taste or smell or both, deter the weevils from feeding. This thesis describes the search for and the synthesis of such antifeedant compounds. Bark extracts of several woody species, known to be non-palatable to the weevil, were prepared and found to display antifeedant activity against H. abietis. The major chemical constituents of the extracts were tested for antifeedant activity. Antifeedants such as eugenol, 2-phenylethanol and benzylalcohol, but also feeding stimulants such as β-sitosterol and linoleic acid, were identified. An extract of linden bark, Tilia cordata, was shown to contain nonanoic acid, a highly active antifeedant. Other aliphatic carboxylic acids were also found to display high antifeedant activities against the weevil, both in laboratory and in field tests. The enantiomers of dihydropinidine, a piperidine alkaloid present in several conifer species, were prepared by dimethylzinc mediated allylation of 2- methyltetrahydropyridine-N-oxide. When tested in micro feeding assays, no difference in antifeedant activity was found for the enantiomers. In a field test high antifeedant activity, comparable with that of the presently used insecticide cypermethrin, was found for (±)-dihydropindine. Other naturally occurring piperidine alkaloids were synthesised and also found to display high antifeedant activities in laboratory tests. Structure-activity relationships were evaluated for methoxy substituted benzaldehydes, benzoic acids and cinnamic aldehydes, -acids, -esters and -alcohols. While the carboxylic acids were inactive or even feeding stimulants, the aldehydes were the most active antifeedants / QC 20110124

<p>The large pine weevil, Hylobius abietis L., is a major insect pest on conifer seedlings in northern Europe. Due to its feeding newly planted trees get girdled, resulting in high seedling mortality (up to 80%). As a consequence great financial losses to the forest industry occur. Today the seedlings are protected with the pyrethroid insecticide cypermethrin. This insecticide is toxic to aquatic organisms and is, from 2010, prohibited for use in Sweden by the Swedish Chemicals Inspectorate. An alternative to insecticides is to protect the seedlings with antifeedants, compounds that, either through taste or smell or both, deter the weevils from feeding. This thesis describes the search for and the synthesis of such antifeedant compounds.</p><p>Bark extracts of several woody species, known to be non-palatable to the weevil, were prepared and found to display antifeedant activity against H. abietis. The major chemical constituents of the extracts were tested for antifeedant activity. Antifeedants such as eugenol, 2-phenylethanol and benzylalcohol, but also feeding stimulants such as β-sitosterol and linoleic acid, were identified. An extract of linden bark, Tilia cordata, was shown to contain nonanoic acid, a highly active antifeedant. Other aliphatic carboxylic acids were also found to display high antifeedant activities against the weevil, both in laboratory and in field tests.</p><p>The enantiomers of dihydropinidine, a piperidine alkaloid present in several conifer species, were prepared by dimethylzinc mediated allylation of 2- methyltetrahydropyridine-N-oxide. When tested in micro feeding assays, no difference in antifeedant activity was found for the enantiomers. In a field test high antifeedant activity, comparable with that of the presently used insecticide cypermethrin, was found for (±)-dihydropindine. Other naturally occurring piperidine alkaloids were synthesised and also found to display high antifeedant activities in laboratory tests.</p><p>Structure-activity relationships were evaluated for methoxy substituted benzaldehydes, benzoic acids and cinnamic aldehydes, -acids, -esters and -alcohols. While the carboxylic acids were inactive or even feeding stimulants, the aldehydes were the most active antifeedants</p>

Avaliou-se o efeito de tratamentos químicos, revestimentos comestíveis e irradiação na conservação de mamões processados minimamente. Após seleção, lavagem e sanitização, os mamões foram descascados e cortados em meias rodelas, as quais foram submetidas a diferentes tratamentos, embaladas e armazenadas a 5±1°C e 90±2%UR. Os mamões PM foram avaliados após 1, 3, 6, 9, 12 e 15 dias. As análises microbiológicas foram fundamentadas na quantificação de coliformes totais e termotolerantes, bactérias psicrotróficas, bolores e leveduras, assim como na verificação da presença de Salmonella. As avaliações físico-químicas basearam-se na determinação da concentração de CO2 no interior das embalagens, perda de massa, cor, firmeza, sólidos solúv eis, acidez titulável, ratio e pH. As características sensoriais aparência, aroma, sabor e textura foram avaliad as por meio de testes com escala hedônica. No primeiro experimento os tratamentos foram: controle, aldeído cinâmico a 0,1%, cloreto de cálcio a 0,75% e combinação de aldeído cinâmico a 0,1% com cloreto de cálcio a 0,75%. O uso de tratamentos químicos em mamões PM resultou em: maior controle de coliformes totais para os mamões PM tratados com aldeído cinâmico e com a combinação de aldeído cinâmico e cloreto de cálcio; menor concentração de CO2 e maior manutenção da firmeza para mamões PM tratados com a combinação de aldeído cinâmico e cloreto de cálcio; e maior concentração de CO2 para os mamões PM tratados apenas com aldeído cinâmico. A imersão nos tratamentos químicos resultou em maior descoloração da polpa dos mamões PM e redução do teor de sólidos solúveis ao longo do armazenamento. No segundo experimento foram utilizados os tr atamentos: controle, amido de arroz a 3%, alginato de sódio a 0,5% e carboximetilcelulose a 0,25%. O uso desses três tipos de revestimento resultou em maior controle de coliformes totais do que o observado no controle. Mamões PM revestidos com amido de arroz e carboximetilcelulose apresentaram redução e aumento da concentração de CO2, respectivamente. Os mamões PM revestidos apresentaram menores teores de sólidos solúveis e seus valores de pH se tornaram menores após 9 dias de armazenamento refrigerado. O r evestimento com carboximetilcelulose proporcionou maior firmeza da polpa no 15° dia. No terceiro experimento foram utilizados os tratamentos: controle, radiação nas doses de 2kGy e 4kGy. O uso de radiação gama em mamões PM resultou em: maior controle de coliformes totais; menor concentração de CO2 nos mamões PM tratados com 2kGy; maior concentração de CO2 e maior descoloração da polpa nos mamões PM tratados com 4kGy; redução da firmeza nos mamões PM tratados com 2kGy e 4kGy; ligeira redução do teor de sólidos solúveis e pequenas variações da acidez titulável em todos os tratamentos. As características sensoriais dos mamões PM tratados com radiação gama não diferiram significativamente do controle durante os 15 dias de armazenamento / The effect of chemical treatments, edible coatings , and irradiation on fresh-cut papaya conservation was evaluated. After selection, washing, and sanitation the papayas were peeled and cut into half slices, which were submitted to different treatments, packed, and stored at 5±1°C and 90±2%RU. The fresh-cut papayas were evaluated after 1, 3, 6, 9, 12, and 15 days. The microbiological analyses were based on the count of total coliform, thermotolerant and psychrotrophic bacteria, molds and yeasts, as well as on the presence of Salmonella. The physicochemical evaluations were based on the determination of CO2 concentration inside the package, weight loss, color, firmness, soluble solids, titratable acidity, ratio, and pH. The sensory characteristics appearance, aroma, flavor, and texture were evaluated using a hedonic scale. In the first experiment, the treatments tested we re: control, cinnamic aldehyde 0.1%, calcium chloride 0.75%, and the combination of cinnamic aldehyde 0.1% and calcium chloride 0.75%. Using chemical treatments to preserve fresh cut papaya resulted in: higher control of total coliforms in fresh-cut papayas treated with cinnamic aldehyde and with the combination of cinnamic aldehyde and calcium chloride; lower CO2 concentration and increased maintenance of firmness in freshcut papayas treated with the combination of cinnamic aldehyde and calcium chloride; and increased in the CO2 concentration in fresh-cut papayas treated only with cinnamic aldehyde. Immersion in chemical treatments caused higher pulp discoloration and reduction in solu ble solids during storage. In the second experiment, the treatments tested were: control, rice starch 3%, sodium alginate 0.5%, and carboxymethylcellulose 0.25%. The use of these three coatings resulted in higher control of total coliforms compared to the control treatment. The fresh-cut papayas coated with rice starch and carboxymethylcellulose presented reduction and increase in the CO2 concentration, respectively. Coated fresh-cut papayas presented lower soluble solids and pH values were lower after 9 days of cold storage. Carboxymethylcellulose coating increased firmness maintenance at day 15. In the third experiment, the following treatments were used: control, radiation at the doses of 2kGy and 4kGy. The use of Gamma radiation in fresh-cut papaya resulted in: higher control of total coliforms; lower CO2 concentration in fresh-cut treated with 2kGy; increased CO2 concentration and increased pulp discoloration in fresh-cut papayas treated with 4kGy; reduction in firmness in fresh-cut papayas treated with 2kGy and 4kGy; slight reduction in soluble solids and small changes in titratable acidity in all treatments. The sensory characteristics of fresh-cut papayas treated with gamma radiation did not significantly differ from the control during the 15 days of storage