京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18461号 / 医博第3916号 / 新制||医||1005(附属図書館) / 31339 / 京都大学大学院医学研究科医学専攻 / (主査)教授 村井 俊哉, 教授 竹内 理, 教授 長田 重一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Neuroprogenitor cells are an important resource because of their potential to replace damaged cells in the brain caused by trauma and disease. It is of great importance to better understand which factors influence the differentiation and migration of these cells. Previously it has been reported that neuroprogenitor cells undergoing apoptotic stress have increased levels of Amyloid precursor protein (APP) and increased APP expression results in glial differentiation. APP activity was also shown to be required for staurosporine induced glial differentiation of neuroprogenitor cells. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is expressed during inflammatory. The binding of MCP-1 to its chemokine receptor induces expression of novel transcription factor MCP-1 induced protein (MCPIP). MCPIP expression subsequently leads to cell death. Previous studies have shown that pro apoptotic factors have the ability to induce neural differentiation. Therefore, we investigated if MCPIP expression leads to differentiation of NT2 neuroprogenitor cells. Results showed that MCPIP expression increased glial fibrillary acid protein expression and also caused distinct morphological changes, both indicative of glial differentiation. Similar results were observed with MCP-1 treatment. Interestingly, APP expression decreased in response to MCPIP. Instead, we found APP activity regulates expression of both MCP-1 and MCPIP. Furthermore, inhibition of either p38 MAPK or JAK signaling pathways significantly reduced APP's effect on MCP-1 and MCPIP. These data demonstrate the role APP has in glial differentiation of NT2 cells through MCP-1/MCPIP signaling. It is possible that increased APP expression after CNS injury could play a ii role in MCP-1 production, possibly promoting astrocyte activation at injured site. We next investigated the effect that MCP-1 has on NT2 cell migration. Studies have shown that when neuroprogenitor cells are transplanted into the brain they migrate towards damaged areas, suggesting that these areas express factors that recruit migrating cells. Generally, after neuronal injury there is a neuroinflammatory response that results in increased chemokine production. We demonstrate that MCP-1 significantly induces the migration of NT2 neuroprogenitor cells. Activation of intracellular cyclic adenosine monophosphate (cAMP) or protein kinase C with forskolin and phorbol 12-myristate 13-acetate (PMA), respectively, was able to completely abolish the MCP-1 induced migration. Contrarily, neither extracellular signal-regulated kinase or p38 mitogen activated protein kinase was required for NT2 cells to respond to MCP-1. Interestingly, APP's ability to activate MCP-1 expression was shown to play a role in NT2 cell migration. We showed that NT2 cells expressing APP were capable of inducing migration of other neuroprogenitor cells. Utilizing a MCP-1 neutralizing antibody we discovered that APP induced migration was not caused solely by increased MCP-1 production. Interestingly, APP increased expression of several C-C chemokines: MCP-1, Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES), and macrophage inflammatory protein alpha (MIP-1 alpha). This demonstrates the unique role APP has in regulating chemokine production, which directly affects cell migration. Taken together, this study provides us with a greater understanding of the mechanisms involved in both glial differentiation and migration of NT2 neuroprogenitor cells.

Monocyte chemotactic protein-1 (MCP-1) plays a critical role in the development of cardiovascular diseases. How MCP-1 contributes to the development of heart disease is not understood. We present evidence that MCP-1 causes death in cardiac myoblasts, H9c2 by inducing oxidative stress, ER stress and autophagy via a novel Znfinger protein, MCP-1 induced protein (MCPIP). MCPIP expression caused cell death and knockdown of MCPIP, attenuated MCP-1 induced cell death. Expression of MCPIP resulted in induction of iNOS and production of reactive oxygen (ROS). It caused induction of NADPH oxidase subunit phox47 and its translocation to the cytoplasmic membrane. Oxidative stress led to the induction of ER stress markers HSP40, PDI, GRP78 and IRE1α. ER stress lead to autophagy as indicated by beclin-1 induction, cleavage of LC3 to LCII and autophagolysosome formation. Here, MCPIP-induced processes lead to apoptosis as indicated by caspase 3 activation and TUNEL assay. This cell death involved caspase 2 and caspase 12 as specific inhibitors of these caspases prevented MCPIP-induced cell death. Inhibitors of oxidative stress inhibited ER stress, and cell death. Specific inhibitors of ER stress inhibited autophagy and cell death. Inhibition of autophagy inhibited cell death. Microarray analysis showed that MCPIP expression caused induction of a variety of genes known to be involved in cell death. MCPIP caused activation of JNK and p38 and induction of p53 and PUMA. These results collectively suggest that MCPIP induces ROS/RNS production that causes ER stress which leads to autophagy and apoptosis through caspase 2/12 and IRE1α –JNK/p38-p53-PUMA pathway. These results provide the first molecular insights into the mechanism by which elevated MCP-1 levels associated with chronic inflammation may contribute to the development of heart failure. A role for inflammation and MCP-1 in obesity and diabetes has been implicated. Adipogenesis is a key process involved in obesity and associated diseases such as type 2 diabetes. This process involves temporally regulated genes controlled by a set of transcription factors, C/EBPβ, C/EBPδ, C/EBPα, and PPARγ. Currently PPARγ is considered the master regulator of adipogenesis as no known factor can induce adipogenesis without PPARγ. We present evidence that a novel Zn-finger protein, MCPIP, can induce adipogenesis without PPARγ. Classical adipogenesis-inducing medium induces MCP-1 production and MCPIP expression in 3T3-L1 cells before the induction of the C/EBP family of transcription factors and PPARγ. Knockdown of MCPIP prevents their expression and adipogenesis. Treatment of 3T3-L1 cells with MCP-1 or forced expression of MCPIP induces expression of C/EBPβ, C/EBPδ, C/EBPα, PPARγ and adipogenesis without any other inducer. Forced expression of MCPIP induces adipogenesis in PPARγ-/- fibroblasts. Thus, MCPIP is a newly identified master controller that can induce adipogenesis without PPARγ. Heart failure is a major cause of death in diabetic patients. Hyperglycemia is a major factor associated with diabetes that causes cardiomyocyte apoptosis that leads to diabetic cardiomyopathy. Cardiomyoycte apoptosis is a key event involved in the pathophysiological progression of diabetic cardiomyopathy. We have recently found that in ischemic hearts, MCP-1 can induce the zinc-finger protein, MCP-1 induced protein (MCPIP) that causes cardiomyocyte apoptosis. Although there is evidence that inflammation may play a role in diabetic cardiomyopathy, the underlying mechanisms are poorly understood. In this study, we show that treatment of H9c2 cardiomyoblasts and Neonatal Rat Ventricular Myocytes (NRVM) with 28mmol/L glucose concentration results in the induction of both transcript and protein levels of MCP-1 and MCPIP. Inhibition of MCP-1 interaction with CCR2 via specific antibody or with the G-coupled receptor inhibitors propagermanium and pertussis toxin attenuated glucose-induced cell death. Knockdown of MCPIP with specific siRNA yielded similar results. Treatment of cells with 28mmol/L glucose resulted in increased ROS production and phox47 activation. Knockdown of MCPIP attenuated these effects. The increased ROS production observed in H9c2 cardiomyoblasts and NRVM's resulted in increased ER stress proteins GRP78 and PDI. Knockdown of MCPIP attenuated expression of both GRP78 and PDI. Inhibition of ER stress with TUDC and 4'PBA prevented high glucoseinduced cell death death. Treatment of cells with 28mmol/l glucose resulted in autophagy as determined by an increase in expression of beclin-1 and through increased cleavage of LC3I to LC3II. Knockdown of MCPIP attenuated expression of beclin-1 and prevented cleavage of LC3. Addition of the autophagy inhibitors 3'methyladenine and LY294002 attenuated high glucose-induced H9c2 cardiomyoblast death. We conclude that high glucose-induced H9c2 cardiomyoblast death is mediated via MCP-1 induction of MCPIP that results in ROS that leads to ER stress that causes autophagy and eventual apoptosis.

In this dissertation, we propose a computational approach using a resampling based permutation test as an alternative to MCP-Mod (a hybrid framework integrating the multiple comparison procedure and the modeling technique) and gMCP-Mod (generalized MCP-Mod) [11], [29] in the step of identifying significant dose-response signals via model selection. We name our proposed approach RMCP-Mod or gRMCP-Mod correspondingly. The RMCP-Mod/gRMCP-Mod transforms the drug dose comparisons into a dose-response model selection issue via multiple hypotheses testing, an area where not much extended researches have been done, and solve it using resampling based multiple testing procedures [38]. The proposed approach avoids the inclusion of the prior dose-response knowledge known as "guesstimates" used in the model selection step of the MCP-Mod/gMCP-Mod framework, and therefore reduces the uncertainty in the significant model identification. When a new drug is being developed to treat patients with a specified disease, one of the key steps is to discover an optimal drug dose or doses that would produce the desired clinical effect with an acceptable level of toxicity. In order to nd such a dose or doses (different doses may be able to produce the same or better clinical effect with similar acceptable toxicity), the underlying dose-response signals need to be identified and thoroughly examined through statistical analyses. A dose-response signal refers to the fact that a drug has different clinical effects at many quantitative dose levels. Statistically speaking, the dose-response signal is a numeric relationship curve (shape) between drug doses and the clinical effects in quantitative measures. It's often been a challenge to nd correct and accurate efficacy and/or safety dose-response signals that would best describe the dose-effect relationship in the drug development process via conventional statistical methods because the conventional methods tend to either focus on a fixed, small number of quantitative dosages or evaluate multiple pre-denied dose-response models without Type I error control. In searching for more efficient methods, a framework of combining both multiple comparisons procedure (MCP) and model-based (Mod) techniques acronymed MCP-Mod was developed by F. Bretz, J. C. Pinheiro, and M. Branson [11] to handle normally distributed, homoscedastic dose response observations. Subsequently, a generalized version of the MCP- Mod named gMCP-Mod which can additionally deal with binary, counts, or time-to-event dose-response data as well as repeated measurements over time was developed by J. C. Pinheiro, B. Bornkamp, E. Glimm and F. Bretz [29]. The MCP-Mod/gMCP-Mod uses the guesstimates" in the MCP step to pre-specify parameters of the candidate models; however, in situations where the prior knowledge of the dose-response information is difficult to obtain, the uncertainties could be introduced into the model selection process, impacting on the correctness of the model identification. Throughout the evaluation of its application to the hypothetical and real study examples as well as simulation comparisons to the MCP-Mod/gMCP-Mod, our proposed approach, RMCP-Mod/gRMCP-Mod seems a viable method that can be used in the practice with some further improvements and researches that are still needed in applications to broader dose-response data types. / Statistics

The mango (Mangifera indica L.) is an appealing subtropical fresh fruit with a pleasant flavor and taste, high nutritional value, beneficial medicinal properties and various processing options. However, as is the case with most subtropical fruit, it is a sensitive commodity, prone to losses postharvestly. The South African mango industry is highly dependent on a hot water and cold prochloraz dip treatment, to control postharvest anthracnose and soft brown rot on fruit destined for export. However, negatve public perceptions of synthetic fungicides and its use on fresh produce for disease control has been increasing in major export markets such as the European Union. This growing concern from a public point of view is forcing industry to consider more environmentally acceptable methods to maintain quality of mangoes during extended export periods. ‘Tommy Atkins’ and ‘Kent’ mangoes either uninoculated or artificially inoculated with Colletotrichum gloesporioiedes, Botryosphaeria parva or sterile agar, were used to evaluate softer, greener alternatives, in this study. Fruit were subjected to either a hot, Bacillus amyloliquefaciens (PPCB004) containing dip treatment for two minutes or a 24 hour 1-methylcyclopropene (1-MCP) gas treatment at 16 ºC or no treatment. Fruit were then stored at 10 ºC under either 5% O2 and 5% CO2 (CA-1) or 3% O2 and 8% CO2 (CA-2) controlled atmospheres (CA) for 18 days and allowed to ripen for five days at 25 ºC. Similarly, uninoculated or artificially inoculated fruit subjected to B. amyloliquefaciens, 1-MCP or a combination of the two treatments was stored at 10 ºC for 18 days under conventional storage. ‘Tommy Atkins’ fruit were packed into bags made from four different film types, untreated or after being subjected to a cold B. amyloliquefaciens dip treatment and stored for 23 days at 10 ºC. Overall, ‘Kent’ fruit were more susceptible to anthracnose and SBR after artificial inoculation. In vivo inoculated ‘Tommy Atkins’ fruit, storage under CA-1 gave the best control of soft brown rot whilst CA-2 storage gave the best control of anthracnose. For quality retention no definite conclusion could be made for both cultivars after CA storage or the combination of 1-MCP pre-treatment and CA storage. The combination of 1-MCP pre-treatment and B. amyloliquefaciens maintained the quality of ‘Kent’ mangoes under conventional storage the best. Anthracnose severity on both cultivars was reduced with 1-MCP treated fruit combined with the biocontrol pre-treatment. Modified atmosphere packaging in this study was found to be ineffective in maintaining quality of mangoes. Copyright / Dissertation (MSc(Agric))--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

Impulsionado pela mecânica de leilões de energia, o aproveitamento energético de recursos eólicos no Brasil atravessa um momento de expansão em participação na matriz de energia elétrica nacional. Não obstante, o desempenho da geração dos parques eólicos que estão em operação foi monitorado e apresentou, em média, resultados aquém daquilo que fora confiado ao Sistema Interligado Nacional, revelando que as estimativas de geração projetadas e declaradas por alguns dos projetos vencedores dos processos licitatórios podem ter sido supervalorizadas. Tal cenário provocou a exigência de medidas mais conservadoras para participação nos leilões de energia, como a já vigente adoção do P90 no cálculo da Garantia Física e o aumento da duração da campanha de medição anemométrica, a entrar em rigor a partir de 2017. Sendo o vento uma variável estocástica, existem incertezas intrínsecas à Avaliação de Recursos Eólicos que influenciam no processo de estimação da geração por um parque eólico e que devem, desta forma, ser identificadas, quantificadas e reduzidas, na medida do possível. Nesse sentido, este trabalho estuda a influência da duração da campanha de medição anemométrica na Avaliação de Recursos Eólicos com base na aplicação do método MCP ferramenta imprescindível no processo de caracterização do regime eólico no longo prazo com vistas para aprimorar a exatidão das previsões de geração pela fonte eólica. Para tanto, foram utilizadas quatro bases de dados contendo séries temporais de velocidade e direção do vento referentes a uma região de interesse. Inicialmente, nove diferentes métodos MCP foram testados e comparados, sendo que o método Vertical Slice aplicado com auxílio do software Windographer destacou-se dos demais e mostrou-se mais aderente aos dados utilizados conforme as métricas de Erro Absoluto Médio e Raiz Quadrada do Erro Quadrático Médio. Posteriormente, as bases de dados foram configuradas para simular campanhas de medição anemométricas com durações que variavam de 2 a 6 anos, de modo a avaliar o comportamento da incerteza relativa à caracterização histórica de recursos eólicos e analisar em que medida esta incerteza impacta no cálculo da estimativa de geração de eletricidade por um conjunto de aerogeradores hipoteticamente dispostos naquele local de interesse. Foi possível verificar que, para os dados e casos analisados, à medida que se aumentou a duração da campanha de medição anemométrica, a incerteza da caracterização histórica de recursos eólicos sofreu queda significativa; determinando, por conseguinte, redução da incerteza total que permeia a geração eólica. Ademais, a quantidade de energia estimada para o parque eólico hipotético exemplificado também decresceu, permitindo melhora na acurácia da previsão de geração e beneficiando a confiabilidade da fonte eólica no sistema elétrico brasileiro. / Driven by the energy auctions system, the energetic harnessing of wind resource in Brazil is now going through a phase of expansion in participation in the national electric energy mix. Nevertheless, the performance of power generation of in-operation wind farms was monitored and the results proved to be, on average, below what was initially entrusted to the National Grid System, indicating that the energy production estimations projected by some energy auctions winners could have been overestimated. This scenario has caused the requirements for participating in the energy auctions to be more conservative, with measures such as the adoption of the P90 on the calculation of the physical guarantee and the increase of the wind measurement campaigns time span the latter to be enforced as of 2017. The wind is a stochastic resource, hence there are uncertainties intrinsic to the Wind Resource Assessment that influence a wind farms power generation estimation and that need to be properly identified, quantified and reduced, as far as possible. In this respect, the influence of a wind measurement campaigns time span on the Wind Resource Assessment based on MCP methods an important tool in the process of characterizing the long-term wind regime was studied in order to detect the potential of enhancing the accuracy of wind power generation forecasts. For this purpose, four databases containing time series of wind speed and direction belonging to a target site were used. Firstly, nine different MCP methods were tested and compared, of which the Vertical Slice method implemented on the software Windographer outperformed all the others according to the Mean Absolute Error and Root Mean Square Error metrics. Subsequently, the databases were set to simulate campaigns with time spans varying from 2 to 6 years, in such a way to evaluate the behavior of the uncertainty in the long-term wind speed and to analyze how this uncertainty impacts the calculation of the energy production estimation of an array of wind turbines hypothetically placed on that target site. From the analyzed data and cases, it was verified that, as the wind measurement campaigns time span was increased, the uncertainty in the long-term wind speed was significantly diminished, thereby reducing the overall uncertainty that pervades the wind power harnessing. Furthermore, the energy production estimation of the exemplified hypothetical wind farm also decreased, allowing an improvement on the accuracy of the energy generation prediction and benefiting the reliability of wind power in the Brazilian electric system.

Tomatoes are among the most cultivated and used vegetables in the world. They are very succeptible to post harvest losses due to high perishability, therefore the use of postharvest treatments may contribute to conservation of this fruit, however the treatments might affect significantly physico-chemical, sensory and nutritional characteristics of tomatoes. Given the perishability of tomato and the economic importance of small tomato fruits, the purpose of the present study was to determine the effect of gamma radiation, carnauba coating and 1-MCP treatments on tomato fruit quality during storage. The study may be divided into two parts. In the first, mini tomatoes cv. Sweet Grape were harvested at breaker stage, divided into 4 grous and treated with gamma radiation (0.6 kGy), carnauba coating (1 L 1000 kg-1) and 1-MCP (500 nL L-1) and then stored at 25±2°C for 30 days with a control group of tomatoes. In the seconnd part, tomatoes harvested at light-red stage were submitted to the same treatments and storage period. Every 6 days tomatoes were evaluated for color modifications, fruit firmness, souble and total pectin (only for light-red tomatoes), mass loss, titratable acidity (TA), soluble solids (SS), SS/TA ratio, carotenoids profile, formation of lycopene isomers, total phenolic compounds, ascorbic acid and antioxidant capacity. For tomatoes harvested at breaker stage and submitted to the treatments the results showed mass loss was delaying mainly by carnauba wax, and to a lesser extend by 1-MCP. Fruit firmness were better retained for 1-MCP treated fruits and carnauba treatment showed a transient effect in preserving fruit firmness. SS/TA of tomatoes treated with gamma radiation and carnauba presented no differences from control values, and were lower with the application of 1-MCP. Color was negatively affected by 1-MCP and earlier changed (6th day) when gamma radiation was applied. In relation to bioactive compounds of tomatoes harvest at breaker stage, results indicated gamma radiation and 1-MCP decreased the final content of lycopene and produced more (Z)-isomers of lycopene. Gamma radiation also induced a decreased in ?-carotene and an increased in phenolic compounds by the end of storage period. 1-MCP treatment promoted a slow down increase in ascorbic acid content during storage. Antioxidant capacity of the hydrophilic fraction was not dramatically affected by treatments and the lipophilic fraction was lower, especially for 1-MCP fruits. In addition, contents of ?-carotene, lycopene, (Z)-isomers of lycopene, ascorbic acid and antioxidant capacity increased during the period of storage while contents of lutein and phenolic compounds tended to decrease. Regarding tomatoes harvest at light-red stage, the most effective treatments for delaying fruit firmness and mass loss was carnauba and 1-MCP, while gamma radiation was the treatment with higher mass loss and the less fruit firmness, which could be associated with the higher solubilization of pectins promoted by radiation treatment. Color (L* and Hue) was mainly affected by 1-MCP treatment which delayed color development, however, by the end of storage, the values were not different from the other treatments. SS/TA ratio was lower for fruits treated with 1-MCP and TA was not so dramatically affected by treatments. Furthermore, mini tomatoes harvested at light-red stage, demonstrated irradiation induced changes in the final content of lycopene, increasing it, and formed less (13Z)-lycopene, while 1-MCP and carnauba coating slow down the increase in lycopene and slown down the decrease of ascorbic acid and phenolic compounds. Antioxidant capacity of lipophilic fraction was not affected by treatments and the hydrophilic fraction was lower for irradiated fruits only on day 0 as well as phenolic compounds. In the other days, no differences among treatments were observed for hydrophilic antioxidant capacity. Considering the results, the best combination of SS and TA and fruit preservation for mini tomatoes harvest at breaker stage was promoted by carnauba coating, which seems to be the treatment that causes fewer changes in bioactive compounds of breaker tomatoes. However, when mini tomatoes were harvested at light-red stage, SS/TA ratio and color were better and, to preserve the quality of these fruits, besides carnauba coating, 1-MCP also could be indicated / Os tomates estão dentre as hortaliças mais cultivadas e consumidas no mundo, porém os frutos do tomateiro são muito sucetíveis a perdas após a colheita devido a alta perecibilidade, por isso, o uso de tratamentos pós-colheita pode contribuir para a conservação dos frutos. Entretanto, o uso destes tratamentos pode afetar significativamente características físico-químicas, sensorias e nutricionais dos tomates. Dada a perecibilidade e a importância econômica dos mini tomates, o objetivo deste estudo foi determinar o efeito da radiação gama, revestimento à base de cera de carnaúba e 1-metilciclopropeno (1-MCP) na qualidade do tomate durante o armazenamento. O estudo pode ser dividido em duas partes. Na primeira, mini tomates cv. Sweet grape colhidos no estádio de maturação breaker foram divididos em quarto grupos e tratados com radiação gama (0.6 kGy), cera de carnaúba (1 L 1000 kg-1) e 1-MCP (500 nL L-1) e então armazenados a 25±2°C por 30 dias, juntamente com um grupo de frutos controle. Na segunda parte, tomates colhidos no estádio de maturação vermelho-claro foram submetidos aos mesmos tratamentos e período de armazenamento. A cada seis dias os frutos foram avaliados para modificações nas características físico-químicas: coloração, firmeza, pectina solúvel e total (somente para os frutos vermelho-claro), perda de massa, acidez titulável (AT), sólidos solúveis (SS), relação SS/AT; e compostos bioativos: perfil de carotenoides, formação de isômeros de licopeno, compostos fenólicos totais, ácido ascórbico, e capacidade antioxidante. Para os tomates colhidos no estádio breaker, os resultados mostraram que a perda de massa foi retardada, principalmente pelo uso de cera de carnauba e de 1-MCP, este último em menor proporção. A firmeza dos frutos foi melhor retida para os frutos tratados com 1-MCP e, a cobertura com carnauba mostrou um efeito transitório em preservar a firmeza dos frutos. A relação SS/AT dos frutos irradiados e cobertos com carnauba não apresentaram diferenças dos valores do controle, mas foram menores com a aplicação de 1-MCP. A coloração foi negativamente afetada pelo uso do 1-MCP e precocemente modificada (no 6° dia) quando a radiação gama foi aplicada. Em relação aos compostos bioativos destes mesmos frutos, os resultados indicaram que a radiação gama e o 1-MCP diminuiram o conteúdo final de licopeno e produziram mais (Z)-isômeros de licopeno nos frutos. A radiação gama também induziu a diminuição de ?-caroteno e o aumento dos compostos fenólicos no final do período de armazenamento. A aplicação de 1-MCP promoveu desaceleração no aumento do conteúdo de ácido ascórbico durante o armazenamento. A capacidade antioxidante da fração hidrofílica não foi muito afetada pelos tratamentos e, a fração lipofílica foi menor para os frutos tratados com 1-MCP. Além disso, os teores de ?-caroteno, licopeno, (Z)-isômeros de licopeno, ácido ascórbico e capacidade antioxidante aumentaram durante o armazenamento, enquanto que, o teor de luteína e compostos fenólicos tenderam a diminuir. A respeito dos tomatates colhidos no estádio de maturação vermelho claro, os tratamentos mais efetivos para retardas a perda demassa e a firmeza foram a cobertura de carnauba e 1-MCP, enquanto o tratamento com radiação gama apresentou a maior perda de massa e de firmeza de frutos, o que pode ser associado a maior solubilização de pectinas promovida pela radiação gama. A cor (L* e Hue) foi principalmente afetada pelo tratamento com 1-MCP, que retardou o desenvolvimento da mesma, porém no final do período de armazenamento, os valores não diferiram dos outros tratamentos. A relação SS/AT foi menor para os frutos tratados com 1-MCP e a AT não foi muito afetada pelos tratamentos. Além disso, mini tomates colhidos no estádio vermelho-claro demosntraram que a irradiação induziu modificações no teor final de licopeno, aumentando-o e houve menor formação de (13Z)-licopeno, enquanto que 1-MCP e carnauba desaceleraram o aumento no teor de licopeno e desaceleraram a diminuição de ácido ascórbico e compostos fenólicos. A capacidade antioxidante da fração lipofílica não foi afetada pelos tratamentos e a fração hidrofílica foi menor para os tomates irradiados somente no dia 0, assim como para compostos fenólicos. Nos demais dias não houve diferenças entre os tratamentos. Considerando os resultados, a melhor combinação de SS/AT e preservação de mini tomates colhidos no estádio breaker foi promovida pela cobertura de carnaúba, a qual pareceu ser o tratamento que causou menores mudanças nos compostos bioativos dos tomates beaker. Entretanto, quando os mini tomates são colhidos no estádio vermelho-claro, SS/AT e coloração são melhor desenvolvidas e, para preservar a qualidade destes frutos, além da cera de carnaúba, o 1-MCP também pode ser indicado

A lesão tecidual é um dos efeitos locais descritos nos envenenamentos botrópicos. Toxinas isoladas, como: a jararagina (JAR) e bothropstoxina-I (BthTX-I), obtidas dos venenos de B. jararaca e B. jararacussu, respectivamente, induzem intensa resposta inflamatória e lesão tecidual, porém apresentam diferentes mecanismos de ação. Como descrito, a resolução da lesão tecidual envolve a interação entre mecanismos de reparo do tecido e o sistema imune. Neste contexto, a resposta inflamatória é iniciada por meio da detecção de sinais de dano tecidual agudo devido a distúrbios da homeostasia resultantes ou não de agentes microbianos (DAMPs) e/ou por reconhecimento de padrões moleculares associados a patógenos (PAMPs). Uma vez induzida, a resposta inflamatória está envolvida tanto no processo de lesão visando a eliminação do agente indutor, assim como no reparo tecidual. Diversos receptores estão envolvidos no reconhecimento de PAMPs e DAMPs como os transmembrânicos representados pelos do tipo Toll, e os citosólicos que compreendem complexos proteicos que formam os inflamassomas. Estes complexos multiproteicos citosólicos podem participar da indução da resposta imune inata por ativação de caspase-1 com consequente liberação de IL-1&#946, que pode resultar em morte celular. Considerando o exposto, o objetivo deste trabalho foi estudar a participação de inflamassoma na resposta inflamatória no tecido muscular de injeção das toxinas, a capacidade da BthTX-I e JAR de induzir a ativação de inflamassoma em macrófagos e os mecanismos moleculares envolvidos nesse processo. O estudo da migração de neutrófilos e macrófagos para o músculo gastrocnémio de animais C57BL/6 ou deficientes em caspase 1/11 (Caspase 1/11-/-) ou NLRP3 (NLRP3-/-) injetados com JAR e BthTX-I permitiu verificar que o inflamassoma NLRP3 participa da migração destas células inflamatórias para local de injeção das toxinas. A análise da produção de IL-1&#946 nas culturas de macrófagos peritoneais incubados com JAR e BthTX-I (6 e 24h) mostrou que somente a BthTX-I foi capaz de induzir a secreção desta citocina por um mecanismo dependente de caspase 1/11, ASC e NLRP3 e independente de IPAF. A incubação de macrófagos humanos com as toxinas permitiu verificar que ambas as toxinas induziram a secreção de IL-1&#946 dependente de caspase 1, porém esta produção foi significativamente maior em resposta à BthTX-I. Nos macrófagos peritoneais de camundongos observamos a relação entre a secreção de IL-1&#946 e morte celular em ensaio de incorporação do brometo de etídio nas culturas incubadas com BthTX-I por 24h e não com a JAR. Visto que ambas as toxinas injetadas via intramuscular induzem resposta inflamatória intensa, foi analisado o efeito delas sobre a viabilidade e secreção de IL-6 e MCP-1 em miotubos C2C12. Os resultados mostraram que somente a BthTX-I induz efeito miotóxico sobre esta linhagem celular. Além disso, pudemos verificar que BthTX-I induz altos níveis de IL-6 e MCP-1 quando comparados aos obtidos com a JAR. Visto que a BthTX-I induziu alta secreção de MCP-1 pelos miotubos C2C12, em experimento in vivo realizado em camundongos C57BL/6 ou deficientes em CCR2 (CCR2-/-) pode ser observada a dependência da interação entre MCP-1 e o CCR2 para a migração dos macrófagos em resposta a injeção de BthTX-I. Em culturas de miotubos incubados com as toxinas pôde ser observada alta liberação de ATP induzida pela BthTX-I in vitro. Em outros experimentos foi estudada a capacidade do sobrenadante de miotubos incubados com BthTX-I de induzir a secreção de IL-1&#946 pelos macrófagos in vitro. Os resultados mostraram a produção de IL-1&#946 nessas culturas de macrófagos, assim como a produção desta citocina em macrófagos incubados com BthTX-I juntamente com ATP. A hidrólise do ATP no sobrenadante da cultura de C2C12 estimulada com BthTX-I aboliu a secreção de IL-1 pelos macrófagos in vitro. Além disso, foi observada a inibição da produção de IL-1&#946 nas culturas de macrófagos primados com LPS e incubados com a BthTX-I em condições de altas concentrações de KCl sugerindo papel relevante do efluxo de K+ na produção de IL-1&#946 induzida pela BthTX-I. Em conjunto, os resultados acrescentam novas informações sobre o potencial inflamatório da JAR e BthTX-I, quanto à ação destas toxinas em macrófagos, ativação de inflamassoma e células musculares. / Tissue damage is one of the local effects described in bothropic envenomations. Isolated toxins, such as jararhagin (JAR) and bothropstoxin-I (BthTX-I), obtained from B. jararaca and B. jararacussu venoms, respectively, induce intense inflammatory response and tissue injury, however mediated by distinct mechanisms. As described, resolution of tissue injury involves the interaction between mechanisms of tissue repair and the immune system. In this context, the inflammatory response is initiated by detecting of signs of acute tissue damage due to disorders of homeostasis resulting from distinct agents (DAMPs) and / or recognition of pathogens associated molecular patterns (PAMPs). Once induced, the inflammatory response is involved both in the injury process for the elimination of the pathogenic agent, as well as in the tissue repair. Several receptors are involved in the recognition of PAMPs and DAMPs as the transmembrane receptors such as the Toll-like, and the cytosolic ones that comprise protein complexes - inflammasomes. These cytosolic multiprotein complexes may participate in the induction of the innate immune response by activation of caspase-1 with consequent release of IL-1&#946, which may result in cell death. Thus, we aimed to study the role of inflammasome on the inflammatory response in muscular tissue of JAR and BthTX-I injection, the ability of BthTX-I and JAR to induce the activation of inflammasome in macrophages and the molecular mechanisms involved in this process. The analyses of the neutrophils and macrophages migration in gastrocnemius muscle of C57BL/6 or Caspase 1/11 (Caspase 1/11-/-) or NLRP3 (NLRP3-/-) deficient mice injected with JAR e BthTX-I allow us to verify that NLRP3 inflammasome participates in these cells migration for the local of the toxins injection. The detection of IL-1&#946 on supernatants from macrophage cultures incubated with JAR or BthTX-I (6 e 24h) showed that only BthTX-I was able to induce this cytokine secretion by a mechanism dependent of caspase 1/11, ASC and NLRP3 and independent of IPAF. The incubation of human macrophages with the toxins demonstrated that both toxins induced IL-1&#946 secretion, however this production was significantly higher in response to BthTX-I. On murine macrophage cultures it was verified the correlation between the IL-1&#946 secretion and the cell death in the incorporation of ethidium bromide assay of cultures incubated with BthTX-I during 24h and not with JAR. Since that both toxins injected in the muscle induced intense inflammation, it was analyzed the effect of both toxins on the viability and the secretions of IL-6 and MCP-1 by C2C12 myotubes. The results showed that only BthTX-I induces a myotoxic effect on this cell line. Furthermore, it was verified that BthTX-I induces high secretion of IL-6 and MCP-1 when compared with those induced by JAR. Considering that BthTX-I induces high levels of MCP-1, in in vivo experiment using C57BL/6 and CCR2 deficient (CCR2-/-) mice it was observed that the interaction of MCP-1 and CCR2 is essential for the macrophage recruitment for the toxin injection. High release of ATP was detected in C2C12 myotube cultures incubated with BthTX-I but not with JAR. In another experiments it was studied the ability of the supernatants of C2C12 myotubes incubated with BthTX-I to induce the IL-1&#946 secretion by peritoneal macrophages in vitro. The results showed the IL-1&#946 secretion in the macrophage cultures as well as the cytokne secretion in macrophages incubated with BthTX-I and ATP independent of the priming with LPS. The hydrolysis of the ATP on the supernatants of C2C12 incubated with the BthTX-I abolished the IL-1&#946 production by macrophages in vitro. In addition, it was not observed IL-1&#946 production by macrophages primed with LPS and incubated with BthTX-I in the presence of high concentration of KCl suggesting a relevant role of K&#43 efflux for this cytokine secretion in response to BthTX-I. Taken together, the results show new findings about the inflammatory effect of JAR and BthTX-I, concerning about the action of these toxins on macrophages, inflammasome activation and muscle cell.

O sistema glicosinolato-mirosinase faz parte do mecanismo de defesa das plantas, quando o tecido é danificado, os glicosinolatos são degradados pela mirosinase e os compostos tóxicos são liberados. No mamão, o principal composto liberado pela enzima é o benzilisotiocianato (BITC), a partir da degradação de benzilglicosinolato (BG). Altos teores de BG e BITC, presentes no início da formação do fruto, diminuem durante o seu desenvolvimento. A semente, é o tecido que mais acumula estes compostos, seguido da casca e da polpa e estes teores parecem ser afetados pelo 1-MCP, mas não pelo etileno. Além disso, foi observado neste trabalho, que mesmo a mais baixa atividade da mirosinase parece ter sido suficiente para liberar o BITC, que nestas quantidades, poderia exercer ação contra as moscas-das-frutas e outros microorganismos. A seqüência parcial da mirosinase do mamão mostrou alto grau de similaridade com Arabidopisis (67%), mostarda branca (62%) e canola (51 %), plantas modelo no estudo de glicosinolatos, sendo encontradas muitas regiões e resíduos altamente conservados. O perfil de transcritos da mirosinase mostrou que ela está presente somente na semente, e a partir dos 90 dpa, apresentando um significativo aumento no ponto de colheita, concomitante ao aumento de atividade. / The glucosinolate-myrosinase system is part of the defense mechanism in plants, when tissue is damaged, the glucosinolates are degraded by myresinases and toxic compounds are released. In papaya fruit, the major compounds released are the benzylisothiocyanates, frem the degradation of benzylglucosinolate (BG). High levels of BG and BITC are present in the beginning of the fruit formation and they decreased during the development. The seed is the tissue that accumulates higher contents of these compounds, followed by skin and pulp and these levels seems to be disturb by 1-MCP, but not by ethylene. Moreover, it was observed in this work, that exactly the lowest activity of myresinase seems to have been enought to liberate the BITC, that in these amounts, could to act against fruit f1ies and other microorganisms. The partial sequence of myresinase of the papaya fruit showed high degree of similarity with with Arabidopisis (67%), white mustard (62%) and rape (51 %), plants model in the study of glicosinolatos, being found a lot of regions and residues highly conserved. The transcript prefile of myresinase showed that it is present only in the seed, and frem the 90 dpa, having a significant increase in the point of harvest, concomitant to the activity increase.

Tomatoes are among the most cultivated and used vegetables in the world. They are very succeptible to post harvest losses due to high perishability, therefore the use of postharvest treatments may contribute to conservation of this fruit, however the treatments might affect significantly physico-chemical, sensory and nutritional characteristics of tomatoes. Given the perishability of tomato and the economic importance of small tomato fruits, the purpose of the present study was to determine the effect of gamma radiation, carnauba coating and 1-MCP treatments on tomato fruit quality during storage. The study may be divided into two parts. In the first, mini tomatoes cv. Sweet Grape were harvested at breaker stage, divided into 4 grous and treated with gamma radiation (0.6 kGy), carnauba coating (1 L 1000 kg-1) and 1-MCP (500 nL L-1) and then stored at 25±2°C for 30 days with a control group of tomatoes. In the seconnd part, tomatoes harvested at light-red stage were submitted to the same treatments and storage period. Every 6 days tomatoes were evaluated for color modifications, fruit firmness, souble and total pectin (only for light-red tomatoes), mass loss, titratable acidity (TA), soluble solids (SS), SS/TA ratio, carotenoids profile, formation of lycopene isomers, total phenolic compounds, ascorbic acid and antioxidant capacity. For tomatoes harvested at breaker stage and submitted to the treatments the results showed mass loss was delaying mainly by carnauba wax, and to a lesser extend by 1-MCP. Fruit firmness were better retained for 1-MCP treated fruits and carnauba treatment showed a transient effect in preserving fruit firmness. SS/TA of tomatoes treated with gamma radiation and carnauba presented no differences from control values, and were lower with the application of 1-MCP. Color was negatively affected by 1-MCP and earlier changed (6th day) when gamma radiation was applied. In relation to bioactive compounds of tomatoes harvest at breaker stage, results indicated gamma radiation and 1-MCP decreased the final content of lycopene and produced more (Z)-isomers of lycopene. Gamma radiation also induced a decreased in ?-carotene and an increased in phenolic compounds by the end of storage period. 1-MCP treatment promoted a slow down increase in ascorbic acid content during storage. Antioxidant capacity of the hydrophilic fraction was not dramatically affected by treatments and the lipophilic fraction was lower, especially for 1-MCP fruits. In addition, contents of ?-carotene, lycopene, (Z)-isomers of lycopene, ascorbic acid and antioxidant capacity increased during the period of storage while contents of lutein and phenolic compounds tended to decrease. Regarding tomatoes harvest at light-red stage, the most effective treatments for delaying fruit firmness and mass loss was carnauba and 1-MCP, while gamma radiation was the treatment with higher mass loss and the less fruit firmness, which could be associated with the higher solubilization of pectins promoted by radiation treatment. Color (L* and Hue) was mainly affected by 1-MCP treatment which delayed color development, however, by the end of storage, the values were not different from the other treatments. SS/TA ratio was lower for fruits treated with 1-MCP and TA was not so dramatically affected by treatments. Furthermore, mini tomatoes harvested at light-red stage, demonstrated irradiation induced changes in the final content of lycopene, increasing it, and formed less (13Z)-lycopene, while 1-MCP and carnauba coating slow down the increase in lycopene and slown down the decrease of ascorbic acid and phenolic compounds. Antioxidant capacity of lipophilic fraction was not affected by treatments and the hydrophilic fraction was lower for irradiated fruits only on day 0 as well as phenolic compounds. In the other days, no differences among treatments were observed for hydrophilic antioxidant capacity. Considering the results, the best combination of SS and TA and fruit preservation for mini tomatoes harvest at breaker stage was promoted by carnauba coating, which seems to be the treatment that causes fewer changes in bioactive compounds of breaker tomatoes. However, when mini tomatoes were harvested at light-red stage, SS/TA ratio and color were better and, to preserve the quality of these fruits, besides carnauba coating, 1-MCP also could be indicated / Os tomates estão dentre as hortaliças mais cultivadas e consumidas no mundo, porém os frutos do tomateiro são muito sucetíveis a perdas após a colheita devido a alta perecibilidade, por isso, o uso de tratamentos pós-colheita pode contribuir para a conservação dos frutos. Entretanto, o uso destes tratamentos pode afetar significativamente características físico-químicas, sensorias e nutricionais dos tomates. Dada a perecibilidade e a importância econômica dos mini tomates, o objetivo deste estudo foi determinar o efeito da radiação gama, revestimento à base de cera de carnaúba e 1-metilciclopropeno (1-MCP) na qualidade do tomate durante o armazenamento. O estudo pode ser dividido em duas partes. Na primeira, mini tomates cv. Sweet grape colhidos no estádio de maturação breaker foram divididos em quarto grupos e tratados com radiação gama (0.6 kGy), cera de carnaúba (1 L 1000 kg-1) e 1-MCP (500 nL L-1) e então armazenados a 25±2°C por 30 dias, juntamente com um grupo de frutos controle. Na segunda parte, tomates colhidos no estádio de maturação vermelho-claro foram submetidos aos mesmos tratamentos e período de armazenamento. A cada seis dias os frutos foram avaliados para modificações nas características físico-químicas: coloração, firmeza, pectina solúvel e total (somente para os frutos vermelho-claro), perda de massa, acidez titulável (AT), sólidos solúveis (SS), relação SS/AT; e compostos bioativos: perfil de carotenoides, formação de isômeros de licopeno, compostos fenólicos totais, ácido ascórbico, e capacidade antioxidante. Para os tomates colhidos no estádio breaker, os resultados mostraram que a perda de massa foi retardada, principalmente pelo uso de cera de carnauba e de 1-MCP, este último em menor proporção. A firmeza dos frutos foi melhor retida para os frutos tratados com 1-MCP e, a cobertura com carnauba mostrou um efeito transitório em preservar a firmeza dos frutos. A relação SS/AT dos frutos irradiados e cobertos com carnauba não apresentaram diferenças dos valores do controle, mas foram menores com a aplicação de 1-MCP. A coloração foi negativamente afetada pelo uso do 1-MCP e precocemente modificada (no 6° dia) quando a radiação gama foi aplicada. Em relação aos compostos bioativos destes mesmos frutos, os resultados indicaram que a radiação gama e o 1-MCP diminuiram o conteúdo final de licopeno e produziram mais (Z)-isômeros de licopeno nos frutos. A radiação gama também induziu a diminuição de ?-caroteno e o aumento dos compostos fenólicos no final do período de armazenamento. A aplicação de 1-MCP promoveu desaceleração no aumento do conteúdo de ácido ascórbico durante o armazenamento. A capacidade antioxidante da fração hidrofílica não foi muito afetada pelos tratamentos e, a fração lipofílica foi menor para os frutos tratados com 1-MCP. Além disso, os teores de ?-caroteno, licopeno, (Z)-isômeros de licopeno, ácido ascórbico e capacidade antioxidante aumentaram durante o armazenamento, enquanto que, o teor de luteína e compostos fenólicos tenderam a diminuir. A respeito dos tomatates colhidos no estádio de maturação vermelho claro, os tratamentos mais efetivos para retardas a perda demassa e a firmeza foram a cobertura de carnauba e 1-MCP, enquanto o tratamento com radiação gama apresentou a maior perda de massa e de firmeza de frutos, o que pode ser associado a maior solubilização de pectinas promovida pela radiação gama. A cor (L* e Hue) foi principalmente afetada pelo tratamento com 1-MCP, que retardou o desenvolvimento da mesma, porém no final do período de armazenamento, os valores não diferiram dos outros tratamentos. A relação SS/AT foi menor para os frutos tratados com 1-MCP e a AT não foi muito afetada pelos tratamentos. Além disso, mini tomates colhidos no estádio vermelho-claro demosntraram que a irradiação induziu modificações no teor final de licopeno, aumentando-o e houve menor formação de (13Z)-licopeno, enquanto que 1-MCP e carnauba desaceleraram o aumento no teor de licopeno e desaceleraram a diminuição de ácido ascórbico e compostos fenólicos. A capacidade antioxidante da fração lipofílica não foi afetada pelos tratamentos e a fração hidrofílica foi menor para os tomates irradiados somente no dia 0, assim como para compostos fenólicos. Nos demais dias não houve diferenças entre os tratamentos. Considerando os resultados, a melhor combinação de SS/AT e preservação de mini tomates colhidos no estádio breaker foi promovida pela cobertura de carnaúba, a qual pareceu ser o tratamento que causou menores mudanças nos compostos bioativos dos tomates beaker. Entretanto, quando os mini tomates são colhidos no estádio vermelho-claro, SS/AT e coloração são melhor desenvolvidas e, para preservar a qualidade destes frutos, além da cera de carnaúba, o 1-MCP também pode ser indicado