A aplicação de ureia em manejos conservacionistas favorece a ocorrência de elevadas perdas por volatilização de NH3. Novas fontes nitrogenadas ou processos que possam ser aplicados à ureia têm sido pesquisados a fim de reduzir esse tipo de perda. Considerando as questões: O uso móvel do coletor semi-aberto exige a alteração das equações de ajuste, que foram desenvolvidas a partir de seu uso na forma estática? Há menor perda de N por volatilização, e influência o estado nutricional, qualidade e a produtividade da cultura com a agregação de tecnologias à ureia, como o revestimento com zeolita ou Cu e B, ou sua mistura com sulfato de amônio, aplicadas em superfície para a cultura de milho em plantio direto? A agregação de tecnologias à ureia pode alterar as transformações do N do fertilizante no solo? A ureia agregada a tecnologias de revestimento apresenta perdas de NH3 por volatilização semelhante ou inferior as obtidas com aplicação de sulfato de amônio? Foram realizados três experimentos com objetivo de elucidá-las: (i) a quantificação de NH3 volatilizada pelo método do coletor semi-aberto usado com base móvel comparado ao estático; (ii) a avaliação do uso de ureia revestida com ácido bórico e sulfato de cobre (UR) ou revestida com zeolita (UZ), além das misturas de sulfato de amônio + ureia (SAU) e sulfato de amônio + ureia revestida com ácido bórico e sulfato de cobre (SAUR) comparadas ao uso da ureia e do nitrato de amônio (NA) na cultura de milho em plantio direto, e (iii) a influência da UR e UZ nas transformações do N proveniente do fertilizante no solo por meio de incubação com 15N. A partir do primeiro experimento concluiu-se que o uso do coletor semi-aberto móvel pode ser utilizado na avaliação de perdas por volatilização de NH3 juntamente com o uso de equações de ajustes desenvolvidos para o modelo estático. No segundo experimento, a UR demonstrou menor pico e perda total de NH3 volatilizado quando as condições climáticas foram favoráveis à volatilização, enquanto em condições de elevada precipitação, as fontes a base de ureia se apresentaram similares. Os tratamentos avaliados influenciaram a produtividade e a quantidade de N exportado nos grãos, porém não diferiram quanto a massa de 1000 grãos e teor de proteína bruta. A fração de N-ISNT não apresentou efeito dos tratamentos, embora tenha apresentado quantidades elevadas de N potencialmente disponíveis em ambas as safras. No terceiro experimento, a perda total de NH3 por volatilização não teve influência dos tratamentos aplicados à ureia. No entanto, com a aplicação de UR foi observado menor quantidade de NH3 perdido nos primeiros dias de incubação, sendo estes resultados consequência do maior tempo de permanência do N do fertilizante na forma de ureia, bem como da alteração no fluxo de N-NH4+, que somados são fatores que permitem maior eficiência de uso do N do fertilizante. / Urea application in no-till is favorable for occurrence of high losses by volatilization of NH3. New nitrogen sources or process that may be applied to urea have been studied to reduce it. Three studies were realized to answer questions as: Is it necessary modifications in adjustment equations with use dynamic of the collector semi-open? Is it possible to get lower losses by volatilization of NH3, nutritional influence, quality and productivity with application of urea coated with zeolite or Cu and B, or urea mix with ammonium sulfate applied in surface for corn in no-tillage? Could these technologies applied in urea modify the dynamic of N from fertilizer in soil? Is it possible the application of coating urea get similar or lower losses of NH3 than ammonium sulfate? The carried out studies were: (i) Evaluation and calibration of the collector semi-open dynamic to quantify losses of NH3 volatilized. (ii) evaluation of urea coated with boric acid and copper sulfate (UC) or with zeolite (UZ), beside the mixes of ammonium sulfate + urea (ASU) and ammonium sulfate + urea coated with boric acid and copper sulfate (ASUC) compared to urea and ammonium nitrate (AN) in corn culture in no-tillage, and (iii) the UC and UZ influence in dynamic of N from fertilizer in soil through 15N incubation. In the first experiment was concluded that dynamic use of collector semi-open can be used in evaluation of losses by volatilization of NH3 with the same adjustment equations developed for static use. In the second experiment, UC showed lower losses of NH3 volatilized when the climatic conditions were favorable to volatilization process occurrence. However, all the treatments with urea were similar when heavy rain occurred. The treatments evaluated influenced the productivity and quantity of N exported by grains; however they had no difference for mass of 1000 grains and crude protein. N-ISNT fraction had no effect of the treatments however they shown high quantities of N potentially available for both harvests. In the third experiment, total losses of volatilization of NH3 had no influence of treatments applied to urea. For other hand, the application of UC showed the lower quantity of losses of NH3 volatilized in the first days of incubation. The results were consequence of the UC effect in the rate of urea hydrolysis, as well as the change on NH4+-N flux for this treatment that could improve N-use efficiency.

Submitted by NATHALIE CARDOSO CABIA null (nccabia@gmail.com) on 2017-01-31T17:19:43Z No. of bitstreams: 1 Nathalie Cardoso Cábia Tese.pdf: 2151127 bytes, checksum: 60d4e1411e5b512e9d366d8cdd972d67 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-02-03T18:41:36Z (GMT) No. of bitstreams: 1 cabia_nc_dr_bot.pdf: 2151127 bytes, checksum: 60d4e1411e5b512e9d366d8cdd972d67 (MD5) / Made available in DSpace on 2017-02-03T18:41:36Z (GMT). No. of bitstreams: 1 cabia_nc_dr_bot.pdf: 2151127 bytes, checksum: 60d4e1411e5b512e9d366d8cdd972d67 (MD5) Previous issue date: 2016-12-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O caqui é um fruto climatérico altamente perecível, e para estender o período de sua comercialização se faz necessário o uso de métodos de conservação pós-colheita. O presente trabalho teve como objetivo avaliar a qualidade físico-química e enzimática de caquis ‘Rama Forte’ e ‘Kyoto’ tratados com 1-metilciclopropeno (1-MCP). Os frutos após a colheita foram selecionados e expostos a diferentes concentrações de 1-MCP (0 ppb, 500 ppb, 1000 ppb e 1500 ppb) e após o procedimento foram armazenados sob refrigeração a 0 ± 0,5ºC e 90 ± 5% de UR. Foram realizadas análises de perda de massa fresca, taxa respiratória, acidez titulável, potencial hidrogêniônico (pH), sólidos solúveis, índice de maturação (“Ratio”), firmeza, índice de adstringência, ácido ascórbico, açúcares redutores, coloração, capacidade antioxidante, compostos fenólicos totais, atividade das enzimas polifenoloxidase (PFO), poligalacturonase (PG) e pectinametilesterase (PME). As análises foram realizadas nos frutos a cada 7 dias, ao longo de 35 dias de armazenamento refrigerado. O delineamento utilizado foi inteiramente casualizado, com 3 repetições por tratamento. Nas condições em que o trabalho foi realizado pode-se concluir que a aplicação de 1-MCP associada ao armazenamento refrigerado foi eficiente no controle do amadurecimento dos caquis ‘Rama Forte’ e ‘Kyoto’. Em caquis ‘Rama Forte’ não destanizados a dose que apresentou melhores resultados foi a de 1500 ppb de 1-MCP. Em caquis ‘Rama Forte’ submetidos à destanização o 1-MCP, independente da dose, mostrou-se eficiente. Já em caquis ‘Kyoto’, a dose que apresentou melhores resultados foi a de 1500 ppb de 1-MCP. / Persimmon is a highly perishable climacteric fruit, and to extend the commercialization period, is necessary to apply post-harvest conservation techniques for its marketing. The objective of this work was to evalueate the physical-chemical and enzymatic quality of ‘Rama Forte’ and ‘Kyoto’ persimmons using 1-metylcyclopropene (1-MCP). Harvested fruits were selected and exposed to different concentrations of 1-MCP (0 ppb, 500 ppb, 1000 ppb and 1500 ppb) and then, they were stored under refrigeration at 0 ± 0,5°C and 90 ± 5% RH. Analyses were conducted to determine the weight loss, respiration, titratable acidity, pH, soluble solids, maturation index (“Ratio”), firmness, astringency index, reducing sugar, ascorbic acid, antioxidant activity for DPPH, total phenolic compounds, polyphenol oxidase activity, poligalacturonase and pectin. The analyzes were carried out on the fruits every 7 days, during 35 days of refrigerated storage. The experiment was completely randomized, with three replicates each treatment. Under conditions in wich the work performed can be concluded that the application of 1-MCP associated witch cold storage was effective in ripening controlling of 'Rama Forte' and 'Kyoto' persimmons. In persimmons 'Rama Forte' witch tannin, the dose that presented the best results was 1500 ppb of 1-MCP. In ‘Rama Forte’ persimmons submitted to destanizacion 1-MCP, regardless of the dose, was efficient. In ‘Kyoto’ persimmons, the dose that presented the best results was 1500 ppb os 1-MCP.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Acridinas sÃo molÃculas policÃclicas aromÃticas planares que possuem a capacidade de intercalar no DNA nuclear. Muitos dos seus representantes apresentam propriedades antibacterianas, antiparasitÃrias e antitumorais. O presente estudo avaliou o potencial citotÃxico de 22 novos compostos acridÃnicos em linhagens de cÃlulas tumorais humanas. Dentre esses, quatro compostos [5-(acridin-9-il-metileno)-3-(4-metil-benzil)-tiazolidina-2,4-diona, AC4; 5-(acridin-9-il-metileno)-3-(4-bromo-benzil)-tiazolidina-2,4-diona, AC7; 5-(acridin-9-il-metileno)-3-(4-cloro-benzil)-tiazolidina-2,4-diona, AC10; e 5-(acridin-9-il-metileno)-3-(4-flÃor-benzil)-tiazolidina-2,4-diona, AC23] foram ativos, especialmente em HCT-8 (cÃlon) e SF-296 (glioblastoma), com valores de CI50 variando de 2,3 a 5,3 Âg/mL. Os compostos apresentaram seletividade para cÃlulas tumorais, desde que nÃo inibiram (IC50 > 25 Âg/mL) a proliferaÃÃo de cÃlulas monocucleares de sangue perifÃrico humano (CMSPH), bem como, nÃo foram capazes de induzir dano ao DNA dessas cÃlulas. Nenhum dos compostos mostrou atividade hemolÃtica contra eritrÃcitos de camundongos (EC50 > 200 Âg/mL), o que sugere uma citotoxicidade por mecanismos mais especÃficos. A fim de determinar o mecanismo envolvido na citotoxicidade seletiva dos compostos, foi realizada uma seqÃÃncia de experimentos in vitro, usando a linhagem HCT-8 como modelo. As cÃlulas foram tratadas em diferentes concentraÃÃes dos compostos (2,5; 5 e 10 Âg/mL) por 12 e 24 horas. Todos os compostos foram capazes de reduzir a viabilidade (teste do azul de tripan) e a proliferaÃÃo (ensaio do BrdU) de cÃlulas HCT-8 apÃs o tratamento. A induÃÃo de apoptose pelos derivados acridÃnicos foi determinada por citometria de fluxo (integridade da membrana, fragmentaÃÃo do DNA internucleosomal e potencial transmembrÃnico) e por anÃlise morfolÃgica das alteraÃÃes celulares (brometo de etÃdeo/laranja de acridina e hematoxilina/eosina). A anÃlise por citometria de fluxo revelou que os compostos avaliados promoveram despolarizaÃÃo mitocondrial, o qual evidencia a ativaÃÃo da apoptose pela via intrÃnseca nas cÃlulas HCT-8. Na anÃlise do screening em 3 diferentes linhagens mutantes de Saccharomyces cerevisiae, foi observado que a linhagem Top1Δ (sem topoisomerase I) mostrou moderada resistÃncia aos compostos acridÃnicos testados na concentraÃÃo de 50 Âg/mL. AlÃm disso, as acridinas inibiram parcialmente o relaxamento do DNA por topoisomerase I, sugerindo que os compostos AC4, AC7, AC10, AC23 tem o potencial antiproliferativo, em parte, relacionado a inibiÃÃo da atividade catalÃtica desta enzima. Esses dados apontam o potencial anticÃncer dos compostos testados. / Acridines are planar aromatic polycyclic molecules that have the ability to progress in nuclear DNA. Many of its representatives have antibacterial, antiparasitic and antitumor. This study evaluated the cytotoxic potential of 22 new acridine compounds in strains of human tumor cells. Among these, four compounds [5-(acridin-9-yl-methilene)-3-(4-methyl-benzyl)-thiazolidine-2,4-dione, AC4; 5-(acridin-9-yl-methilene)-3-(4-bromine-benzyl)-thiazolidine-2,4-dione, AC7; 5-(acridin-9-yl-methilene)-3-(4-chloro-benzyl)-thiazolidine-2,4-dione, AC10; and 5-(acridin-9-yl-methilene)-3-(4-fluor-benzyl)-thiazolidine-2,4-dione, AC23] were active, especially in HCT-8 (colon) and SF-296 (glioblastoma), with IC50 values ranging from 2.3 to 5.3 mg / mL. The compounds were selective for tumor cells, provided they do not inhibit (IC50 > 25 Âg/mL) cell proliferation monocucleares human peripheral blood (CMSPH) and were not able to induce DNA damage to these cells. None of the compounds showed hemolytic activity against erythrocytes of mice (EC50 > 200 Âg/mL), suggesting a cytotoxicity by more specific mechanisms. In order to determine the mechanism involved in the selective cytotoxicity of compounds was carried out a sequence of in vitro experiments, using the line HCT-8 as a model. The cells were treated in different concentrations of compounds (2.5, 5 and 10 Âg/mL) for 12 and 24 hours. All compounds were able to reduce the viability test (trypan blue) and proliferation (BrdU assay) of HCT-8 cells after treatment. Induction of apoptosis by acridine derivatives was determined by flow cytometry (membrane integrity, DNA fragmentation and internucleosomal transmembrane potential) and morphological analysis of cellular changes (ethidium bromide/acridine orange, and hematoxylin-eosin). The analysis by flow cytometry showed that the compounds evaluated promoted mitochondrial depolarization, which shows the activation of apoptosis by intrinsic pathway in HCT-8 cells. In the analysis of screening in 3 different mutant strains of Saccharomyces cerevisiae, it was observed that the line Top1Δ (without topoisomerase I) showed resistance to acridine compounds tested at a concentration of 50 Âg/mL. In addition, the acridines partially inhibited the relaxation of DNA by topoisomerase I, suggesting that the compounds AC4, AC7, AC10, AC23 has the potential antiproliferative partly related to inhibition of catalytic activity of this enzyme. These data indicate the potential anticancer compounds tested.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Cinco inibidores de tripsina da famÃlia Kunitz (JB1, JB2, JB3-1, JB3-2 e JB4) foram purificados de sementes de Pithecellobium dumosum, uma Ãrvore da subfamÃlia Mimosoideae, por precipitaÃÃo com Ãcido tricloroÃcetico (TCA), cromatografia de afinidade sobre tripsina imobilizada em Sepharose e coluna de fase reversa em sistema de CLAE. Os cinco inibidores possuem massa molecular entre 18 e 20 kDa formados por uma cadeia polipeptÃdica como determinado por SDS-PAGE na presenÃa ou ausÃncia de -mercaptoetanol. JB1, JB3-1 e JB3-2 tÃm massas moleculares de 19,70, 19,69 e 19,69 kDa, respectivamente, por MALDI-TOF. JB2 e JB4 tÃm massa molecular de 18,08 e 20,85 kDa, respectivamente. A seqÃÃncia N-terminal de JB1, JB3-1 e JB3-2 mostrou identidade com outros inibidores da famÃlia Kunitz. Os cinco inibidores foram estÃveis Ãs variaÃÃes de temperatura e pH. A inibiÃÃo da tripsina por JB1, JB2 e JB4 foi do tipo competitivo. JB1, JB2, JB3-1 e JB3-2 tiveram Kis de 3,56 x 10-8 M, 1,65 x 10-8 M, 4,20 x10-8 M, 2,88 x 10-8 M, respectivamente para a tripsina bovina. Em comparaÃÃo com os outros inibidores JB4, com Ki de 5,70 x 10-10 M, apresentou maior afinidade para tripsina. Entre os inibidores purificados apenas JB4 inibiu moderadamente a atividade da quimotripsina. A atividade da elastase e bromelaÃna nÃo foi inibida efetivamente por esses inibidores. A inibiÃÃo de JB1, JB2, JB3-1 e JB3-2 sobre a papaÃna variaram entre 32,93 a 48,82% e foi indicativo de sua bifuncionalidade, com exceÃÃo de JB4 que inibiu fracamente essa atividade (9,93% de inibiÃÃo). A inibiÃÃo da papaÃna por JB1 e JB2 foi do tipo nÃo competitiva e os valores de Ki foram de 7,6 x 10-7 e 5,1 x 10-7 M, respectivamente. Ensaios in vitro sobre as proteinases digestÃrias de Lepidoptera, Diptera e Coleoptera foram feitos. Esses inibidores foram efetivos para enzimas digestÃrias semelhantes à tripsina desses insetos em diferentes graus. As enzimas digestivas de Zabrotes subfasciatus e Ceratitis capitata foram inibidas por JB1 em 68,87 e 65,53%, respectivamente, e as enzimas de Callosobruchus maculatus, Alabama argillaceae e Plodia interpunctella foram inibidas entre 29,18 e 44,35%. Enzimas digestÃrias de Z. subfasciatus, C. maculatus e C. capitata foram inibidas por JB2 entre 70,04 e 74,54% e as enzimas de larvas de A argillaceae e P. interpunctella foram suprimidas em 13,58 e 48,67%, respectivamente. A atividade semelhante à tripsina de larvas de Z. subfasciatus foi suprimida em 67,33 e 56,93% por JB3-1 e JB3-2, respectivamente, e a atividade de C. maculatus, A argillaceae, P. interpunctella e C. capitata foram suprimidas por esses inibidores entre 5,17 e 49,00%. JB4 inibiu entre 54,53 a 66,15 % as enzimas digestivas de C. maculatus, Z. subfasciatus e A argillaceae e inibiu as enzimas digestivas de larvas de P. intepunctella e C. capitata em 8,97 e 37,47%, respectivamente. A inibiÃÃo de proteinases semelhantes à tripsina e à papaÃna presentes no intestino de vÃrios insetos sugere que esses inibidores possam afetar o crescimento e sobrevivÃncia desses insetos pragas quando incorporados em sementes artificiais e esta bifuncionalidade à indicativo de que estes inibidores possam ser fortes candidatos para os programas de melhoramento de plantas via transgenia. / Five Kunitz-type trypsin inhibitors (JB1, JB2, JB3-1, JB3-2 and JB4) were purified from Pithecellobium dumosum seeds, a tree of the sub-family Mimosoideae, by TCA precipitation, affinity chromatography on immobilized trypsin-Sepharose and reverse phase HPLC using Vydac C-18 column. The five inhibitors had Mr between 18 and 20 kDa with a single polypeptide chain as determined by SDS-PAGE with and without reduction. JB1, JB3-1 and JB3-2 had Mr of 19.70, 19.69 and 19.69 kDa, respectively, by MALDI-TOF. JB2 and JB4 had Mr of 18.08 and 20.85, respectively, by SDS-PAGE. The N-terminal sequences of JB1, JB3-1 and JB3-2 showed identity with others Kunitz-type inhibitors. The five inhibitors were stable over a wide range of temperature and pH. The inhibition of trypsin by JB1, JB2 and JB4 was competitive. JB1, JB2, JB3-1 and JB3-2 showed Ki values of 3.56 x 10-8 M, 1.65 x 10-8 M, 4.20 x 10-8 M, 2.88 x 10-8 M, respectively, against bovine trypsin. In comparison with others inhibitors JB4, with Ki of 5.70 x 10-10 M, showed a high affinity toward trypsin. Among the inhibitors purified only JB4 inhibited chymotrypsin activity. The activities of elastase and bromelain were not inhibited for these inhibitors. The inhibition of JB1, JB2, JB3-1 and JB3-2 on papain varied between 32.93 to 48.82% of inhibition and was indicative of its bifunctionality with exception of JB4 that inhibited this activity in 9.9%. The papain inhibition by JB1 and JB2 were noncompetitive type and the Kivalues were 7.6 x 10-7 and 5.1 x 10-7 M, respectively. In vitro assays against digestive proteinases from Lepidoptera, Diptera and Coleoptera pests were carried out. These inhibitors were effective towards trypsin-like digestive enzymes of the insect in different degrees. The digestive enzymes from Zabrotes subfasciatus and Ceratitis capitata were inhibited by JB1 in 68.87 and 65.53% respectively, and Callosobruchus maculatus, Alabama argillaceae and Plodia intepunctella enzymes were inhibited in the range of 29.18 to 44.35%. Digestive enzymes from Z. subfasciatus, C. maculatus and C. capitata were inhibited by JB2 in the range of 70.04 to 74.54%, and the enzymes of A. argillaceae and P. intepunctella were suppressed in 13.58 and 48.67%, respectively. Trypsin-like activities of larval from Z. subfasciatus were suppressed in 67.33 and 56.93% by JB3-1 and JB3-2, respectively, and the activities of C. maculatus, A. argillaceae, P. intepunctella and C. capitata were inhibited by these inhibitors in the range of 5.17-49.00%. JB4 inhibited around 54.53 to 66.15% the digestive enzymes of C. maculatus, Z. subfasciatus and A. argillaceae and the digestive enzymes from P. intepunctella and C. capitata larvae in 8.97% and 37.47%, respectively. The inhibition of trypsin-like and papain-like proteinases of several insects suggested that these inhibitors may affect the growth and survival of these insect pests when incorporated into artificial diet and their bifunctionality are indicative that these inhibitors could be strong candidates to plant management programs cross transgenia.

A matriz extracelular e as células musculares lisas das vias aéreas estão intimamente interrelacionadas. Poucos estudos porém, avaliaram a composição dos diferentes componentes da matriz extracelular e seus reguladores na camada do músculo liso brônquico na asma. Utilizando um programa de análise de imagens, a área fracionada do colágeno total e das fibras elásticas foi quantificada no interior do músculo liso brônquico de 35 indivíduos que faleceram devido a um ataque de asma (Asma Fatal), e comparada com 10 casos de indivíduos com asma e que faleceram de outras causas (Asma Não Fatal), e com 22 indivíduos controles sem patologia pulmonar. Expressão dos colágenos I e III, fibronectina, versicam, metaloprotease (MMP)-1, 2, 9 e 12, e inibidores de metaloprotease 1 e 2 foram quantificados no interior do músculo liso brônquico de 22 casos de asma fatal e 10 controles. Nas grandes vias aéreas dos casos de asma fatal, a área fracionada das fibras elásticas foi significativamente maior na camada do músculo liso brônquico quando comparada com os grupos de Asma Não Fatal e Controle. Semelhantemente, fibronectina, MMP-9 e MMP-12 estavam aumentadas no músculo liso das grandes vias aéreas nos casos de asma fatal quando comparadas aos controles. Apenas aumento das fibras elásticas foi observado nas pequenas vias aéreas na Asma Fatal, e somente quando comparadas aos casos de Asma Não Fatal. O conjunto dos resultados mostra que há uma composição alterada dos elementos da matriz extracelular e um ambiente de degradação protéica no músculo liso brônquico de indivíduos que morreram por asma, o qual pode acarretar importantes conseqüências nas funções sintéticas e mecânicas do músculo liso das vias áreas. / There is an intimate relationship between the extracellular matrix (ECM) and smooth muscle cells within the airways. Few studies have comprehensively assessed the composition of different ECM components and its regulators within the airway smooth muscle (ASM) in asthma. With the aid of image analysis, the fractional area of total collagen and elastic fibres was quantified within the ASM of 35 subjects with Fatal Asthma (FA) and compared with 10 Nonfatal Asthma (NFA) patients and 22 nonasthmatic control cases. Expression of collagen I and III, fibronectin, versican, matrix metalloprotease (MMP)-1, 2, 9 and 12 and tissue inhibitor of metalloprotease-1 and 2 was quantified within the ASM in 22 FA and 10 control cases. In the large airways of FA cases, the fractional area of elastic fibres within the ASM was increased compared with NFA and controls. Similarly, fibronectin, MMP-9 and MMP-12 were increased within the ASM in large airways of FA cases compared with controls. Elastic fibres were increased in small airways in FA only in comparison with NFA cases. The results shower that, there is altered extracellular matrix composition and a degradative environment within the airway smooth muscle in fatal asthma patients, which may have important consequences for the mechanical and synthetic functions of airway smooth muscle.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-08-02T11:48:25Z No. of bitstreams: 1 2011 - Wellington Oliveira da Cruz.pdf: 7968929 bytes, checksum: 9dd68ee567386fa437d5f3438c9db428 (MD5) / Made available in DSpace on 2016-08-02T11:48:25Z (GMT). No. of bitstreams: 1 2011 - Wellington Oliveira da Cruz.pdf: 7968929 bytes, checksum: 9dd68ee567386fa437d5f3438c9db428 (MD5) Previous issue date: 2011-09-16 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior, CAPES. / CRUZ, Wellington Oliveira. Characterization of Alphitobius diaperinus (Coleoptera, Tenebrionidae) ?-amylase: Effect of Different Birds Diet and Inhibitors from Phaseolus vulgaris. 2011. 50 p Disserta??o (Mestrado em Qu?mica, ?rea de Concentra??o Bioqu?mica). Instituto de Ci?ncias Exatas, Departamento de Qu?mica, Universidade Federal Rural do Rio de Janeiro, Serop?dica, RJ, 2011. Alphitobius diaperinus known as mealworm of poultry litter, is one of the main poultry pests. It uses high protein and carbohydrates substrate of birds as a food source, colonizing all the production area finding a favorable environment for its proliferation. This insect causes sanitary and economic problems, affecting not only healthy and growth of the poultry but also acting as a microorganisms transmitter like bacterias, protozoas and virus. In this work it was identified an ?-amylase activity in crude extracts of A. diaperinus larvae and adults. This enzyme is responsible for starch degradation used as a energy source extremely important for survival of many insects. The ?-amylase was partially purified by ammonium sulfate precipitation (25-75%) in a chromatographic Sephadex column G (50-80), polyacrylamide gel revealed only one band with molecular weight of approximately 30 kDa, its purity was confirmed by the method of enzyme activity assay dinitrosalicylic 3.5-alkaline (DNS) was increased 7.2 times when compared with the crude extract. The ?-amylase assays presented a maximum activity at a pH 5.0 and pH 5.6 and an optimal activity temperature of 50 ? C. The maximum activity of ?-amylase A. diaperinus was achieved in the presence of 0.02 mM CaCl2, and inhibited by increasing this reaction concentration. The enzymatic activity of A. diaperinus ?-amylase was significantly inhibited by amylase inhibitor ?-AIs (lectin-like inhibitor) of Phaseolus vulgaris, suggesting that this inhibitors can be used as an important tool in biological control and development of these pests by reducing the digestion reducing amylase activity which plays an important role in the starch digestion / CRUZ, Wellington Oliveira. Caracteriza??o de ?-amilase de Alphitobius diaperinus (Coleoptera, Tenebrionidae): Efeito de Diferentes Ra??es de Aves e dos Inibidores de Phaseolus Vulgaris. 2011. 50 p Disserta??o (Mestrado em Qu?mica, ?rea de Concentra??o Bioqu?mica). Instituto de Ci?ncias Exatas, Departamento de Qu?mica, Universidade Federal Rural do Rio de Janeiro, Serop?dica, RJ, 2011. Alphitobius diaperinus, conhecido como cascudinho de cama avi?ria, ? uma das principais pragas av?colas. Ele utiliza o substrato das aves rico em prote?nas e carboidratos como fonte de alimento, colonizando toda a ?rea de produ??o, encontrando um ambiente favor?vel para sua prolifera??o. Este inseto causa problemas sanit?rios e econ?micos, afetando a sa?de e o crescimento das aves e atuando tamb?m como transmissor de microrganismos tais como baact?rias, protozo?rios e v?rus. Neste trabalho foi identificada a presen?a de uma ?-amilase no extrato bruto de larvas e adultos de Alphitobius diaperinus. Esta enzima digestiva ? respons?vel pela degrada??o de amido utilizado como combust?vel na obten??o de energia, sendo extremamente importante para sobreviv?ncia de muitos insetos. A ?-amilase foi parcialmente purificada por precipita??o em sulfato de am?nio (25-75 %) em coluna cromatogr?fica Sephadex G (50-80), revelando em gel de poliacrilamida uma ?nica banda com peso molecular de aproximadamente 30 KDa. Sua pureza enzim?tica foi confirmada atrav?s de ensaio de atividade pelo m?todo 3,5-dinitrosalic?lico alcalino (DNS), observando-se um aumento de 7.2 vezes em rela??o ao extrato bruto. A ?-amilase ensaiada apresentou uma atividade m?xima em pH 5.0 e pH 5.6 e uma temperatura de atividade ?tima de 50?C. A atividade m?xima da ?-amilase de A. diaperinus foi alcan?ada na presen?a de 0.02 mM de CaCl2, e inibida com o aumento da concentra??o deste reagente. A atividade enzim?tica da ?-amilase de A. diaperinus, foi significativamente inibida pelo inibidor ?-AIs, (inibidores tipo lectina) de Phaseolus vulgaris, sugerindo que estes podem ser utilizados como importantes ferramentas biol?gicas no controle e desenvolvimento de A. diaperinus atrav?s da redu??o da atividade digestiva destas pragas devido a atua??o desses inibidores sobre a a??o catalisadora de amilases, enzimas fundamentais para a digest?o de amido

Made available in DSpace on 2017-07-10T17:36:53Z (GMT). No. of bitstreams: 1 2013_Diss_Luiz_Neri_Berte.pdf: 2155538 bytes, checksum: d732fa38ae96b3be68ba89a5652e4659 (MD5) Previous issue date: 2013-05-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Due to the expansion of the cultivation of Jatropha curcas, in Brazil studies are needed covering agronomic management, especially in weed management, due to the susceptibility of Jatropha curcas the competition and lack of information. Thus, the objective was to quantify the effects of increasing doses of clomazone in the initial development of the Jatropha curcas to identify selective herbicide doses for culture. To this end, two experiments were conducted in the randomized factorial 2 x 5 in randomized blocks with 4 replications. The first factor representing the combination of two formulations of the herbicide clomazone being used CS (suspension of encapsulated) formulations and EC (emulsifiable concentrate) the commercial product GAMIT and 360 CS ® 500 EC ® and five dosages 0; 250; 500; 1000 and 2000 g ha-1. In the first experiment we studied the application of clomazone reemergence and second applying post-emergence. To identify the effects of clomazone on culture biometric, physiological and biochemical parameters were investigated. In trials of pre and post emergence, application of clomazone herbicide symptoms caused the higher dosages used, as evidenced by symptoms of leaf chlorosis. In both trials and for all doses and formulations used, plant height and stem diameter showed a linear increase during the trial period. In pre emergence, application of clomazone hindered the development of biometric parameters, however, the post emergence test the changes were less dramatic. The biochemical and physiological parameters were affected by the application of clomazone in pre and post emergence. The application of clomazone in pre and post-emergence in the culture of Jatropha, the suspension formulations of encapsulated (GAMIT 360 CS ®) and emulsifiable concentrate (EC GAMIT 500 ®) to a dose of 2000 g ha-1 produced an intermediate symptoms phytointoxication and negatively affect early development and dry matter accumulation by plants. The recovery of plants after 51 days when applied before emergence, and 42 days in the post application emergency suggest the existence of clomazone selectivity for this crop in both formulations until the dose of 500 g ha-1. However, further studies needed for longer duration and are contemplating the crop to verify the negative effects of the herbicide in the production of jatropha aspects / Devido à expansão do cultivo de pinhão-manso no Brasil são necessários estudos abrangendo o manejo agronômico, especialmente no manejo de plantas daninhas, devido à susceptibilidade do pinhão-manso a competição e à escassez de informações. Assim, objetivou-se quantificar os efeitos de doses crescentes de clomazone no desenvolvimento inicial do pinhão-manso visando identificar doses seletivas do herbicida para a cultura. Para tal foram conduzidos dois ensaios sob o delineamento em esquema fatorial 2 x 5, em blocos casualizados com 4 repetições. O primeiro fator representou a combinação de duas formulações do herbicida clomazone, sendo utilizadas as formulações CS (suspensão de encapsulado) e CE (concentrado emulsionável) do produto comercial GAMIT 360 CS® e GAMIT 500 CE®, respectivamente, e cinco dosagens: 0; 250; 500; 1000 e 2000 g ha-1. No primeiro ensaio estudou-se a aplicação do clomazone em pré emergência e no segundo a aplicação em pós-emergência. Para a identificação dos efeitos do clomazone sobre a cultura foram investigados parâmetros biométricos, bioquímicos e fisiológicos. Nos ensaios de pré e pós emergência, a aplicação do clomazone ocasionou sintomas de fitointoxicação nas maiores dosagens utilizadas, evidenciados por sintomas de clorose foliar. Em ambos os ensaios e para todas as doses e formulações utilizadas, a altura de plantas e diâmetro do caule apresentaram incremento linear no decorrer do período de avaliações. Em pré emergência, a aplicação do clomazone prejudicou o desenvolvimento dos parâmetros biométricos, porém, no ensaio de pós emergência as alterações foram menos expressivas. Os parâmetros bioquímicos e fisiológicos foram afetados pela aplicação do clomazone em pré e pós emergência. A aplicação de clomazone em pré e pós emergência na cultura do pinhão-manso, nas formulações de suspensão de encapsulado (GAMIT 360 CS®) e concentrado emulsionável (GAMIT 500 CE®) até a dosagem de 2000 g ha-1 promoveu sintomas intermediários de fitointoxicação e interferiu negativamente no desenvolvimento inicial e no acúmulo de massa seca pelas plantas. A recuperação das plantas após 51 dias quando da aplicação em pré emergência, e 42 dias na aplicação em pós emergência sugerem a existência de seletividade do clomazone para essa cultura em ambas formulações até a dose de 500 g ha-1. No entanto, são necessários novos estudos de maior duração e que contemplem a produtividade da cultura para verificar os efeitos negativos do herbicida nos aspectos produtivos do pinhão-manso

Haematopoietic stem cells (HSCs) are capable of self-renewing and multi-lineage reconstitution of the haematopoietic system of irradiated recipient mice. In the mouse embryo, HSCs originate in a step-wise manner from the haematogenic endothelium. The first HSC precursor has been detected at E9.5 in the dorsal aorta, while HSCs emerge in the aorta-gonad-mesonephros (AGM) region around E11. To date, the molecular mechanisms regulating these events are poorly characterized. Through the activating role of Angiopoietin1 (Ang1) on Tie2 receptor, the Ang-Tie signalling pathway plays a critical role in HSC maintenance in the adult bone marrow niche. Tie2 ligand Angiopoietin2 (Ang2) is described as being a Tie2 inhibitor, however its role is unknown. The aim of this thesis was to characterise the role of Ang-Tie signalling pathway in HSC formation in the mouse embryo. First, I used an ex vivo aggregate system to culture with angiopoietins cells derived from the AGM region at stages of development preceding HSC formation (E9.5-E11). Ang2- treated cells were able to reconstitute the peripheral blood of recipient mice to a higher extent compared to control, indicating a role for Ang2 in promoting HSC maturation. Then, I characterized the expression pattern of Ang-Tie molecules in the AGM region. Ang2-expressing cells were identified as perivascular and sub-aortic mesenchymal cells located in the ventral side of the aorta and in proximity of intra-aortic haematopoietic clusters. Finally, I performed an RNA-seq analysis with the aim of unravelling the molecular mechanisms involved in Ang2-mediated HSC maturation. Pre-HSC-I were cultured in presence or absence of Ang2 and their transcriptional profiles were compared, revealing a number of genes and pathways up-regulated or down-regulated in presence of Ang2, which might indicate a role for Ang2 in increasing cell proliferation, favouring cell migration, and regulation of other signalling pathways involved in HSC development. All together, these data support Ang2 as a novel regulator for HSC formation.

Upon exposure to heat or cold, Arabidopsis thaliana seedlings undergo rapid transcriptional reprogramming of several hundreds of genes that promote stress tolerance. Despite extensive characterisation of the transcriptional responses to these stimuli, however, relatively little is known about the mechanisms by which temperature signals are perceived and transduced in plant cells. High or low seasonal temperatures have large impacts on crop productivity and are expected to intensify given current global climatic projections. It is therefore of agricultural importance to better understand temperature signalling pathways in plants in order to find solutions to this problem. In this thesis, a chemical genomics screen for molecules activating or repressing heat-inducible genes in A. thaliana was performed in collaboration with Syngenta and the biological targets of these chemicals were predicted based on structural similarities to compounds with known modes of action. Many molecules that affect the function of chloroplasts or mitochondria either activate or repress heat-responsive genes, thus implicating these organelles in the regulation of plant temperature responses. In addition, the translation inhibitor cycloheximide was identified as a repressor of heat-inducible genes and an activator of early cold-inducible genes. Diverse translation inhibitors trigger a cytosolic influx of calcium ions and several inhibitors of translation elongation were found to strongly activate cold-inducible gene expression in a calcium-dependent manner. Furthermore, it was demonstrated that cold shock causes rapid translation repression in A. thaliana seedlings and that the elongation factor LOS1 is involved in cold- or cycloheximide-induced gene expression, thus implicating translational machinery in the regulation of temperature signalling in plants. Finally, one of the chemicals identified in the screen, S01A463859Y, was found to improve heat resilience in A. thaliana and may therefore be of potential use in enhancing crop productivity during thermal stress.

Terminally differentiated B cells are responsible for antibody generation, a key component of adaptive immunity. IgG antibodies play an important role in defence against infection but can be pathogenic in some autoimmune diseases and in solid organ transplantation. In addition to antibody generation, there is increasing interest in the antibody-independent functions of B cells, including their ability to regulate immune responses via the production of IL10. In this thesis I firstly explored the therapeutic potential of belimumab, an anti-BLyS antibody, in an experimental medicine study in kidney transplant recipients. The rationale for this study was based on published studies showing that B cells activate alloreactive T cells and secrete human leukocyte antigen (HLA) and non-HLA antibodies that negatively affect graft function and survival, but may also play a protective role by regulating alloimmune responses promoting transplant tolerance. B-Lymphocyte Stimulator (BLyS) is a cytokine that promotes B cell activation and survival. We performed the first randomized controlled trial using belimumab as early maintenance immunosuppression in kidney transplantation. In belimumab-treated subjects, we demonstrate a reduction in naïve and activated memory B cells, plasmablasts, IgG transcripts in peripheral blood and new antibody formation as well as evidence of reduced CD4 T cell activation and of a skewing of the residual B cell compartment towards an IL10-producing regulatory phenotype. This experimental medicine study highlights the potential of belimumab as a novel therapeutic agent in transplantation. In the second part of my project I performed a preclinical study investigating the potential efficacy of bromodomain inhibitors in reducing antibody-mediated immune cell activation. Immune complexed antigen can activate mononuclear phagocytes (MNP), comprising macrophages and dendritic cells (DCs), via ligation of Fc gamma receptors (FcγR), that bind the Fc region of IgG. FcγR-dependent MNP activation results in profound changes in gene expression that mediate antibody effector function in these cells. The resulting inflammatory response can be pathological in the setting of autoimmune diseases, such as systemic lupus erythematosus and in antibody-mediated rejection in transplantation. BET proteins are a family of histone modification 'readers' that bind acetylated lysine residues within histones and function as a scaffold for the assembly of complexes that regulate gene transcription. Bromodomain inhibitors (I-BET) selectively inhibit the transcription of a subset of inflammatory genes in macrophages following toll-like receptor stimulation. Since MNPs make a key contribution to antibody-mediated pathology, we sought to determine the extent to which I-BET inhibits macrophage and DC activation by IgG. We show that I-BET delays phagolysosome maturation associated with build-up of immune complex (IC) whilst selectively inhibiting IC induced cytokine production. I-BET changed MNP morphology, resulting in a less adherent phenotype, prompting an assessment of its impact on DC migration. In vitro, in a three-dimensional collagen matrix, IgG-IC induced augmentation of DC chemotaxis to chemokine (C-C motif) ligand 19 (CCL19) was abrogated by the addition of I-BET. In vivo, two photon imaging showed that systemic I-BET treatment reduced IC-induced dermal DC mobilisation. Tissue DCs and transferred DC also had reduced migration to draining lymph nodes following I-BET treatment. These observations provide mechanistic insight into the potential therapeutic benefit of I-BET in the setting of antibody-associated inflammation.