Gemcitabine is a highly potent chemotherapeutic nucleoside agent used in the treatment of several cancers and solid tumors. However, it is therapeutically limitated because of toxicity to normal cells and its rapid intracellular deamination by cytidine deaminase into the inactive uracil derivative. Modification at the 4-(N) position of gemcitabine's exocyclic amine to an -amide functionality is a well reported prodrug strategy which has been that confers a resistance to intracellular deamination while also altering pharmacokinetics of the parent drug. Coupling of gemcitabine to carboxylic acids with varying terminal moieties afforded the 4-N-alkanoylgemcitabines whereas reaction of 4-N-tosylgemcitabine with the corresponding alkyl amines gave the 4-N-alkylgemcitabines. The 4-N-alkanoyl and 4-N-alkyl gemcitabine analogues with a terminal hydroxyl group on the 4-N-alkanoyl or 4-N-alkyl chain were efficiently fluorinated either with diethylaminosulfur trifluoride or under conditions that are compatible with the synthetic protocols for 18F labeling, such as displacement of the corresponding mesylate with KF/Kryptofix 2.2.2. The 4-N-alkanoylgemcitabine analogues displayed potent cytostatic activities against murine and human tumor cell lines with 50% inhibitory concentration (IC50) values in the range of low nM, whereas cytotoxicity of the 4-N-alkylgemcitabine derivatives were in the low to modest µM range. The cytostatic activity of the 4-N-alkanoylgemcitabines was reduced by several orders of magnitude in the 2'-deoxycytidine kinase (dCK)-deficient CEM/dCK- cell line while the 4-N-alkylgemcitabines were only lowered by 2-5 times. None of the 4-N-modified gemcitabines were found to be substrates for cytosolic dCK, however all were found to inhibit DNA synthesis. As such, the 4-N-alkanoyl gemcitabine derivatives likely need to be converted to gemcitabine prior to achieving their significant cytostatic potential, whereas the 4-N-alkylgemcitabines reach their modest activity without "measurable" conversion to gemcitabine. Thus, the 4-N-alkylgemcitabines provide valuable insight on the metabolism of 4-N-modified gemcitabine prodrugs.

Lectins are multivalent carbohydrate binding proteins that specifically recognise diverse sugar structures and mediate a variety of biological processes, such as cell-cell and host-pathogen interactions, serum glycoprotein turnover and innate immune responses. Lectins have received considerable attention in recent years on account of their properties leading to wide use in research and biomedical applications. Seeds of leguminous plants are mainly rich sources of lectins, but lectins are also found in all classes and families of organisms. Legume lectins have similar tertiary structures, but exhibit a large variety of quaternary structures. The carbohydrate binding site in them is made up of four loops, the first three of which are highly conserved in all legume lectins. The fourth loop, which is variable, is implicated in conferring specificity. Legume lectins which share the same monosaccharide specificity often exhibit markedly different oligosaccharide specificities. This thesis primarily concerns with structure solution and analysis of lectins from the legume and β-prism II fold families using X-ray crystallography. Apart from having the property of specifically and reversibly binding to carbohydrates, lectins are also interesting models to study sequence-structure relationships, especially of how minor change in the sequence may bring about major changes in oligomerization and binding. Chapter 1 gives an overview of different structural types of plant lectins and describes in detail, their carbohydrate binding features. The details of the various experimental procedures employed during the course of this research, are explained in Chapter 2. Chapter 3 describes the crystal structure of a β-prism II fold lectin (RVL), from Remusatia vivipara, an epiphytic plant of traditional medicinal value, and analysis of its binding properties. This lectin was established to have distinct binding properties and has nematicidal activity against a root-knot nematode with the localization site identified as the high-mannose displaying gut-lining in the nematode. The crystal structure of RVL revealed a new quaternary association of this homodimeric lectin, different from those of reported β-prism II lectins. Functional studies on RVL showed that it fails to bind to simple mannose moieties yet showed agglutination with rabbit blood cells (which have mannose moieties on the surface) and some high mannose containing glycoproteins like mucin and asialofetuin. Further, ELISA and glycan array experiments indicated that RVL has high affinity to N-glycans like trimannose pentasaccharide such as in gp120, a capsid glycoprotein of HIV virus, necessary in virus-association with the host cell. The structural basis for this N-glycan binding was revealed through structure analysis and molecular modelling, and it was demonstrated that there are two distinct binding sites per monomer, making RVL a truly multivalent lectin. Evolutionary phylogeny revealed the divergence in the β-prism II fold proteins with regards to the number of sugar-binding regions per domain, oligomerization and specificity. Chapter 4 deals with the structural studies on a galactose-specific legume lectin (DLL-II) from Dolichos lablab, a leguminous plant. The lectin was found to be a planar tetramer in the crystal structures of the native and ligand bound forms, as expected from our solution studies and phylogenetic analysis. The protein is a heterotetramer with subunits differing only in the presence or absence of a C-terminal helical region at the core of the tetramer. Due to the static disorder in all the crystals, the central helix could be oriented in either direction. Structure analysis of DLL-II proved to be an interesting endeavour as static disorder compounded with twinning in the crystal made the data processing and structure solution a challenging process. Subsequent structure and sequence alignments led to the identification of an adenine-binding pocket in the hydrophobic core of the tetramer. Based on this, DLL-II lectin was co-crystallized with adenine and the structure revealed the presence of adenine at the predicted binding site. Chapter 5 describes the identification and analysis of potential plant lectins/lectin-like domains in the genome of Oryza sativa, using bioinformatics approaches. This project was initiated to study the occurrence of legume-lectin like domains (a predominant dicot feature) in O. sativa, which is a monocot. Later, a large scale genome analysis for all types of lectin domains was carried out through exhaustive PSI-BLAST, profile matching by HMMer, CDD and MulPSSM. The final validation was carried out by assessing the carbohydrate binding potential of the domain by examining the sugar binding sites. The primary interest in undertaking this work was to find the occurrence of association of these domains with other domains as in protein receptor kinases, where lectin is the receptor domain. Though primarily initiated as a bioinformatics project, further structural characterization was attempted by cloning, expression and purification of some of the annotated lectin proteins using prokaryotic expression systems. The protein expression was attained in reasonable amounts for a few of the annotated legume lectin homologs, however purification is yet to be achieved as the expressed proteins are insoluble. A part of the results described in this thesis and the other related projects that the author was involved are reported in the following publications. 1) Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity Bhat GG, Shetty KN, Nagre NN, Neekhra VV, Lingaraju S, Bhat RS, Inamdar SR, Suguna K, Swamy BM. 2010. Glycoconjugate J. 27(3):309-320 2) Modification of the sugar specificity of a plant lectin: structural studies on a point mutant of Erythrina corallodendron lectin Thamotharan S, Karthikeyan T, Kulkarni KA, Shetty KN, Surolia A, Vijayan M & Suguna K. 2011. Acta Crystallographica D 67(3):218-227 3) Crystal structure of a β-prism II lectin from Remusatia vivipara Shetty KN, Bhat GG, Inamdar SR, Swamy BM, Suguna K. 2012. Glycobiology 22(1): 56-69. 4) Structure of a galactose binding lectin from Dolichos lablab Shetty KN, Lavanyalatha V, Rao RN, SivaKumar N & Suguna K (Under review) 5) Occurrence of lectin-like domains: Oryza sativa genome analysis. Shetty KN & Suguna K. (Manuscript in preparation)

No description available.

Rift Valley fever virus (RVFV) is a mosquito-borne virus which has the ability to infect a large variety of animals including humans in Africa and Arabian Peninsula. The abortion rate among these animals are close to 100%, and young animals develop severe disease which often are lethal. In humans, Rift Valley fever (RVF) presents in most cases as a mild illness with influenza-like symptoms. However, in about 8% of the cases it progresses into a more severe disease with a high case fatality rate. Since there is such a high abortion rate among infected animals, a link between human miscarriage and RVFV has been suggested, but never proven. We could in paper I for the first time show an association between acute RVFV infection and miscarriage in humans. We observed an increase in pregnant women arriving at the Port Sudan Hospital with fever of unknown origin, and several of the patients experienced miscarriage. When we analysed their blood samples for several viral diseases we found that many had an acute RVFV infection and of these, 54% experienced a miscarriage. The odds of having a miscarriage was 7 times higher for RVFV patients compared to the RVFV negative women of which only 12% miscarried. These results indicated that RVFV infection could be a contributing factor to miscarriage. RVFV is an enveloped virus containing the viral glycoproteins n and c (Gn and Gc respectively), where Gn most likely is responsible for the initial cellular contact. The protein DC-SIGN on dendritic cells and the glycosaminoglycan heparan sulfate has been suggested as cellular receptors for RVFV, however other mechanisms are probably also involved in binding and entry. Charge is a driving force for molecular interaction and has been shown to be important for cellular attachment of several viruses, and in paper II we could show that when the charge around the cells was altered, the infection was affected. We also showed that Gn most likely has a positive charge at a physiological pH. When we added negatively charged molecules to the viral particles before infection, we observed a decreased infection efficiency, which we also observed after removal of carbohydrate structures from the cell surface. Our results suggested that the cellular interaction partner for initial attachment is a negatively charged carbohydrate. Further investigations into the mechanisms of RVFV cellular interactions has to be undertaken in order to understand, and ultimately prevent, infection and disease. There is currently no vaccine approved for human use and no specific treatments for RVF, so there is a great need for developing safe effective drugs targeting this virus. We designed a whole-cell based high-throughput screen (HTS) assay which we used to screen libraries of small molecular compounds for anti-RVFV properties. After dose-response and toxicity analysis of the initial hits, we identified six safe and effective inhibitors of RVFV infection that with further testing could become drug candidates for treatment of RVF. This study demonstrated the application of HTS using a whole-cell virus replication reporter gene assay as an effective method to identify novel compounds with potential antiviral activity against RVFV.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The sustainable development of northern agroecosystems requires, among other factors, the use of species adapted to region the edafoclimtic conditions. Plants able to support water deficits are more indicated, considering to no regular distribution of pluviometric precipitation the high evapotranspiration are characteristic northest region of Brazil. The present work was carried out to determine the ecofisiologic and biochemistry behavoir of three cultivars of basil, Ocimum basilicum L. (Maria Bonita, Mara and Genovese) cultivated under water stress. The ecofisiologics variables were: hidric potential of leaves, photossintesis, transpiration, stomatal conductance; the biochemitry variables were: soluble carbohydrates and proteins, totals cloroplasts, tenor of essential oil and percentage of linalool. The varibles were analyzed in plants with four months of aged, cultivated in 24 vases of 17L, in greenhouse, having two treatments: T0 (control) and T1 (experimental group), with four repetitions. The variables were evaluated at 08:30 to 10:30 hours every two days, during 15 days in October 2005. In base of results it was identifyed that basil presents mechanisms of adaptation under water stress minimizing the water lose. Mara’and Maria Bonita’cultivars presented more adaptaded under water stress in comparison with ‘Genovese’cultivar. In relation to the essential linalool content the three cultivars kept their yield even under water stress. It was observed the geraniol presence in Maria Bonita cultivar. / O desenvolvimento sustentável dos agroecossistemas nordestinos requer, dentre outros fatores, o uso de espécies adaptadas às condições edafomorfoclimáticas da região. Plantas capazes de suportar déficits hídricos são mais indicadas, uma vez que a distribuição irregular da precipitação pluviométrica, aliada a alta evapotranspiração são características do Nordeste brasileiro. O presente trabalho objetiva determinar o comportamento ecofisiológico e análise bioquímica de três cultivares de manjericão, Ocimum basilicum L.(‘Maria Bonita’, ‘Mara’ e ‘Genovese’) quando cultivadas sob estresse hídrico. As variáveis ecofisiológicas foram: potencial hídrico foliar, fotossíntese, transpiração, condutância estomática; e as bioquímicas foram: carboidratos solúveis, proteínas solúveis, cloroplastos totais e teor de óleo essencial e de linalol no óleo essencial. As variáveis ecofisiológicas e bioquímicas foram analisadas quando as plantas apresentavam 4 meses de idade. Cultivadas em 24 vasos de 17 L, em estufa agrícola, sendo 2 tratamentos: T0 (testemunha) e T1 (grupo experimental), com 4 repetições. As variáveis ecofisiológicas foram avaliadas entre 08:30 a 10:30 horas, em dias alternados, durante 15 do mês de outubro de 2005. Com base nos resultados obtidos, constatou-se que o manjericão apresenta mecanismos de adaptação, que sob condição de estresse hídrico, diminuem as perdas de água. As cultivares ‘Mara’ e ‘Maria Bonita’ apresentaram adaptadas às condições de estresse hídrico quando comparadas com a cultivar ‘Genovese’. Em relação ao teor linalol as três cultivares mantiveram as suas produtividades mesmo sob condição de estresse. Observou-se também, a presença de geraniol no óleo essencial da cultivar Maria Bonita.

Fundação de Apoio a Pesquisa e à Inovação Tecnológica do Estado de Sergipe - FAPITEC/SE / The author of the present work aimed at evaluating the influence of the inclusion of soybean hull (SH) upon the apparent digestibility, the physical-chemical characteristics of feces and the postprandial glycemic responses of horses submitted to moderate-intensity exercise. Five horses were used, aging 11 +2 years old and weighing 483 ± 18 kg. The diets consisted of 60% of Tifton 85 hay (Cynodon spp) and 40% of a pelletized commercial concentrate. The treatments were: substitution of the concentrate for 0%, 10%, 20%, 30% and 40% of SH (Kg/Kg). The experimental period lasted 50 days, being 5 ten-day periods: 7 days for the adaptation to the diet and 3 days of total feces collection. Bromatological analyses of both the foods and of the feces samples were performed for the determination of the apparent digestibility coefficients of the nutrients. The evaluation of the characteristics of the feces comprehended consistency, coloration and pH. For the glycemic tests, 5 mL of blood was collected around 30 minutes before, and 30, 60, 90, 120, 180, 240 and 300 minutes after the administration of the soybean hull homogenized to the concentrate. The experimental delineation used was entirely casualized in Latin square (5x5). For the statistical analyses, the data were submitted to variance analysis. The means were compared with Tukey´s test at 5% of significance. The result demonstrated that there were no effects (P>0.05) of the substitution levels of the concentrate for the soybean hull over the apparent digestibility coefficient of dry matter, organic matter, crude protein, ether extract, nonstructural carbohydrates, hemicellulose, rapidly fermented carbohydrate and energy. However, significant statistical differences were observed (P<0.05) for the apparent digestibility coefficient acid detergent fiber and slowly fermented carbohydrate, with better results for the 30% and 40% substitution levels and a drop in hydrolysable carbohydrate at the 40% level of substitution. Regarding the consistency and coloration, 100% of the horses produced normal (score 3) green-colored feces. There were no statistical differences for the pH value, with means of 6.34. The glucose peak and the area below the glycemic curve were not influenced (P<0.05) by the increasing levels of soybean hull in the diet. It is concluded that the soybean hull can be replaced in 30% and 40%, thus allowing the increase in the apparent digestibility of the fibrous fraction, without causing alterations to the physical-chemical characteristics of both the feces and the glycemic responses of horses undergoing moderate-intensity exercises. / O objetivo da autora deste trabalho foi avaliar a influência da inclusão de casca de soja (CS) na digestibilidade aparente, características físico-químicas das fezes e resposta glicêmicas pós-prandial de equinos submetidos a exercício de intensidade moderada. Foram utilizados cinco equinos com idade de 11 +2 anos e peso médio de 455 ± 23 kg. Os tratamentos foram: substituição do concentrado por 0%, 10%, 20%, 30% e 40% de CS (Kg/Kg). O período experimental teve duração de 50 dias, divididos em cinco períodos de 10 dias cada, sendo 7 dias de adaptação à dieta e 3 dias de coleta total de fezes. Realizou-se análises bromatológicas dos alimentos e das amostras compostas de fezes para estimar os coeficientes de digestibilidade aparente dos nutrientes. As avaliações das características das fezes compreenderam consistência, coloração e pH. Para o teste glicêmico foram coletados 5 mL de sangue cerca de 30 minutos antes, 30, 60, 90, 120, 180, 240 e 300 minutos após administração da casca de soja homogeneizada ao concentrado. O delineamento experimental foi inteiramente casualizado em quadrado latino (5x5). Os dados foram submetidos à analise de variância e as médias comparadas pelo teste Tukey a 5% de significância. Os resultados demostraram que não houve efeitos (P>0,05) dos níveis de substituições do concentrado pela casca de soja sobre os coeficientes de digestibilidade aparente da matéria seca, matéria orgânica, proteína bruta, extrato etéreo, carboidratos não estruturais, hemicelulose, energia bruta e carboidratos rapidamente fermentáveis; porém, observaram-se diferenças estatísticas significativas (P<0,05) para os coeficientes de digestibilidade aparente da fibra em detergente ácido e carboidratos lentamente fermentáveis, com aumento para os níveis de 30% e 40% de substituição e queda no coeficiente de digestibilidade aparente dos carboidratos hidrolisáveis ao nível de 40% de substituição Quanto à consistência e coloração, 100% dos equinos apresentaram fezes normal (escore 3) e esverdeada. Não houve diferenças estatísticas para o valor de pH com média de 6,34. O pico de glicose, tempo de pico e área abaixo da curva glicêmica não foram influenciados (P<0,05) pelos níveis crescentes de casca de soja na dieta. Conclui-se que a casca de soja pode ser substituída em 30% e 40% proporcionando aumento na digestibilidade aparente da fração fibrosa, sem causar alterações nas características físico-químicas das fezes e nas respostas glicêmicas de equinos submetidos a exercício moderado.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-01-04T13:24:36Z No. of bitstreams: 1 daniellelobojustopinheiro.pdf: 4869021 bytes, checksum: b4acfc12a41a6f83bf195e5947ae47cc (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-01-25T15:55:49Z (GMT) No. of bitstreams: 1 daniellelobojustopinheiro.pdf: 4869021 bytes, checksum: b4acfc12a41a6f83bf195e5947ae47cc (MD5) / Made available in DSpace on 2016-01-25T15:55:49Z (GMT). No. of bitstreams: 1 daniellelobojustopinheiro.pdf: 4869021 bytes, checksum: b4acfc12a41a6f83bf195e5947ae47cc (MD5) Previous issue date: 2015-03-31 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Carboidratos têm sido utilizados como organocatalisadores em síntese orgânica devido a sua quiralidade intrínseca. Neste trabalho foi sintetizado um novo organocatalisador aproveitando a estrutura da D-galactose como indutor de quiralidade. A síntese ocorreu em cinco etapas, a saber: proteção seletiva das hidroxilas das posições 1, 2, 3 e 4, seguida pela iodação da posição 6, substituição nucleofílica pelo grupo azido, redução à amina e por fim uma reação com anidrido ftálico. O rendimento global foi de 60 %. O organocatalisador foi testado na reação de adição de Michael entre o dibenzilideno acetona e a azalactona derivada da alanina. 20 mol% do catalisador conduziu ao produto com 57 % de rendimento e com total controle da régio- e diasteroseletividade. No escopo, vários produtos com funcionalização no esqueleto de dbas foram preparados e devidamente caracterizados pelas técnicas convencionais de análise. A determinação da estereoquímica relativa foi realizada através do uso de HPLC com fase estacionária quiral e foi atribuída como 1,2-anti após a comparação do tempo de retenção com um padrão já descrito na literatura. De maneira geral, é reportada, pela primeira vez, uma metodologia mais geral para a dessimetrização diasterosseletiva entre dbas e azalactonas catalisadas por ácido de Brønsted. / Carbohydrates have been used as organocatalysts in organic synthesis due to its inherent chirality. In this work, D-galactose was choose as a chiral pool in the catalyst design and it was prepared in five steps: selective ketalyzation of hydroxyl groups, following by an iodination and nucleophilic substitution in the presence of azide. To complete, reduction of the azide to amine and a coupling reaction with phthalic anhydride leading to the catalyst. Overall yield was 60 % for five steps. Then, the catalyst was adopted in the Michael addition reaction between dibenzylidene acetone and azalactone derivative of alanine. The product was obtained in 57% yield and with fully control of both regio- and diastereoselectivity. Next, various funcionalizated dbas were evaluated under the optimized reaction condition and the corresponding final products were fully characterized through conventional elemental analysis. The relative stereochemistry was assigned as being 1,2-anti by using chiral HPLC method. To this end, an authentic sample already described in the literature was prepared in order the retention time. In general, for the first time, a method more general to perform a diastereoselective dessymetrization of dbas in presence of azlactones by using a Brønsted acid as catalyst was described.

O gene TrSNF1, homólogo aos membros da subfamília das proteínas serina-treonina quinases ativadas por AMP (AMPK) e relacionadas a SNF1, foi isolado do fungo filamentoso trichoderma reesei. A seqüência de aminoácidos putativa possui um domínio de quinase com 42% de identidade e 59% de similaridade com outras proteínas quinases da mesma subfamília. Em S. cerevisiae a SNFl é essencial para a expressão de genes reprimidos por glicose, em resposta a privação de glicose do meio de cultura. A expressão de TrSNFl em levedura mutante para SNF1, restaura a função de SNF1. A expressão de um antisense de TrSNFl em T. reesei causa um atraso na expressão do gene regulado por glicose, CBHI. Além disso, em experimento utilizando matrizes de DNA foi possível observar uma alteração da tendência global da expressão gênica entre a cepa selvagem e a cepa antisense. A observação da homologia estrutural com proteínas quinase da mesma subfamília, a similaridade funcional com SNFl de S. cerevisiae, e a alteração no padrão da expressão gênica in vivo, sugerem que TrSNFl pode estar envolvido na regulação do metabolismo de carboidratos em T. reesei. / A gene homologue to the members of the AMP-activated/SNFl protein kinase subfamily, TrSNF1, was isolated from the filamentous fungus Trichoderma reesei. The predicted protein of 692 amino acids has a kinase domain, that share 42 % identity and 59 % similarity to that of serine/threonine protein kinase of this family. In Saccharomyces cerevisiae, the SNFl protein kinase is required for expression of glucose repressed genes in response to withdrawal of glucose from the medium. Expression of the Trichoderma reesei SNF1-related sequence in yeast SNFl mutant restores SNFl function. The TrSNFl antisense expression in T. reesei causes a control alteration in the glucose-regulated gene CBHI. The observed structural identity with the AMP-activated/SNFl protein kinase subfamily, and the functional similarity to the yeast SNFl suggest that the TrSNFl may be involved in the regulation of sugar metabolism in Trichoderma reesei.

A banana é um fruto climatérico que apresenta alta taxa respiratória e alta produção de etileno após a colheita, o que a torna altamente perecível. Acredita-se que o 1-MCP é capaz de ligar-se ao receptor do hormônio etileno, bloqueando sua ação e, consequentemente, retardando o amadurecimento do fruto. Bananas (Musa acuminata AAA cv. Nanicão) com aproximadamente 110 dias pós-antese foram armazenadas em condições controladas de umidade e temperatura. Parte da amostra foi tratada com 1-MCP (100 nl/L), outra parte foi tratada com etileno (100 ppm - 7L/min), e, uma terceira parte, foi mantida como controle. Os frutos foram caracterizados, durante o período de amadurecimento, em relação à produção de etileno e CO2 (por cromatografia à gás), à concentração de amido (pelo método enzimático descrito por Cordenunsi e Lajolo 1995) e açúcares (glicose, frutose, sacarose e maltose - por HPLC-PAD). Também foram analisados os comportamentos das enzimas &#945; e &#946;- amilases, fosforilase, DPE 1 e DPE 2 por atividade enzimática in vitro ou por P.A.G.E. nativo e, quando possível, foram avaliados os comportamentos destas enzimas frente a tradução (Western blotting) e transcrição proteica (Northern blotting). A degradação de amido, assim como a respiração, a produção de etileno e síntese de açúcares foram retardadas nos frutos tratados com o 1-MCP. Como consequência destas mudanças, também houve uma alteração nos perfis das atividades enzimáticas. Os resultados indicaram que o 1-MCP, além de atrasar o climatério respiratório, foi capaz de provocar descompasso no padrão de degradação de amido e síntese de açúcares sugerindo também que outros fatores temporais, necessários ao processo, foram prejudicados. Dentre as enzimas envolvidas no metabolismo de carboidratos de bananas, as &#945;-amilases não demonstraram ser etileno-dependentes (?) as &#946;-amilases mostraram ser enzimas bastante dependentes do etileno e parecem ter importância especial quando se trata do metabolismo da maltose, em conjunto com a DPE 2; atividade fosforolítica foi induzida ao longo do amadurecimento, mas parece ter resposta mais significativa a alterações basais do hormônio etileno e, portanto, sugere-se que esta enzima esteja envolvida com mais de uma forma de regulação; a DPE 1 apresentou maior atividade ao início da degradação do amido, indicando sua atuação sobre glicanos liberados pelas &#945;-amilases. A presença de DPEs na banana torna o seu metabolismo de carboidratos mais próximo daquele presente em folhas. / Banana is a climateric fruit that has a high respiration rate and a high ethylene production after harvest, what makes this fruit very perishable. It is believe that 1- MCP is capable to interact with the hormone ethylene receptors, blocking its action and, as result, delaying the fruit ripening. Bananas (Musa acuminata AAA cv. Nanicão) with 110 days after anthesis were stored in controlled conditions of umidity and temperature. Part of the sample was treated with 1-MCP (100 nl/L), another part was treated with ethylene (100 ppm - 7L1min), and, a third part, was kept as control.The fruits were characterized, along the ripening process, based on the ethylene and CO2 production (by gas chromatography), the starch amount (enzymatic method described by Cordenunsi and Lajolo, 1995) and sugars content (glucose, fructose, sucrose and maltose - by HPLC-PAD). Enzymes behaviors were also followed &#945; and &#946;-amylases, phosphorylase, DPE 1 and DPE 2 by enzymatic activity (in vitro ar native P.A.G .E.) and, when it was possible, the translation (Western blotting) and protein transcription (Northern blotting) were also analyzed. The starch degradation, as well as the CO2 and ethylene production and sugars synthesis were delayed in the fruits treated with the 1-MCP. As consequence of these changes, we also had an alteration in the enzymatic activity profiles. The results indicated that the 1-MCP, besides delaying the respiratory climateric, was capable to change the standard profile of starch degradation and synthesis of sugars, suggesting that other factors, necessary to the process, were damaged. Considering the involved enzymes in the carbohydrates metabolism of bananas, the &#945;-amylases did not demonstrated to be ethylene-dependents (?) &#946;-amylases seem to be ethylene-dependent and seem to have special importance in the maltose metabolism, working on it with the DPE 2; phosphorolytic activity was induced along the ripening, but it seems to have more significant relation to the basal alterations of the hormone ethylene, therefore, it suggests that this enzyme is involved with more than one type of regulation; DPE 1 presented greater activity in the beginning of the starch degradation, indicating its performance on glucans released by the &#945;-amylases activity. The presence of DPEs in bananas approximates its metabolism of carbohydrates to the leaves metabolism.

Objetivou-se com este estudo comparar diferentes métodos de determinação de carboidratos solúveis (CS) em cana-de-açúcar com o intuito de predizer valor nutritivo da forragem: conteúdo de sólidos solúveis (BRIX), conteúdo aparente de sacarose (POL), carboidratos não fibrosos (CNF), carboidratos solúveis em extrato aquoso (CSA) e carboidratos solúveis em extrato alcoólico (CSOH). Foram conduzidos dois experimentos envolvendo amostras da fração colmo e planta inteira. O primeiro avaliou o valor nutritivo da variedade IAC 86-2480 (terceiro corte) em resposta a dois métodos de colheita: colheita manual (MAN) e colheita mecânica (MEC) durante as fases de cultivo: 150, 180, 210, 240, 270, 300, 330 e 360 dias após colheita (DAC). As estimativas do CS, composição bromatológica (MS, CP, FDN, FDA, DIVMS e DIVMO), assim como da relação FDN/CS foram similares (P>0,05) entre os tratamentos, exceto para CSOH e FDN/CSOH para a fração colmo, e FDN/POL (P<0,05) para a planta inteira. O conteúdo de CS da planta inteira representou aproximadamente 80% do teor observado na fração colmo. Foram identificados três padrões de acúmulo de CS: a) POL e CSOH, b) BRIX, c) CNF. O modelo logístico não ajustou na predição de CSA. Até os 240 e 270 DAC, as relações entre FDN/BRIX e FDN/POL seguiram a mesma tendência para os métodos MAN e MEC respectivamente. Relações entre FDN/CS foram altamente correlacionadas com a digestibilidade da forragem, especialmente a relação FDN/CNF (r>0,8). A análise de componentes principais provou ser ferramenta útil para selecionar variáveis com o intuito de caracterizar valor alimentar em cana-de-açúcar. De acordo com o primeiro componente principal, o valor nutritivo é uma resposta de variáveis bromatológicas da fração fibrosa (ex. FDN, FDA), correspondendo a 54,4 e 64% da variância total da fração colmo e planta inteira respectivamente. No segundo experimento, foram avaliadas possíveis relações entre medidas morfológicas e químicas da planta visando explicar o acúmulo de CS e o valor nutricional da cana-de-açúcar (variedade IAC 93-3046), estabelecida sob dois níveis de adubação no plantio (NPK e NPK associada com esterco 120 kg N ha-1) ao longo da fase de maturação da planta em cana de primeiro corte. As amostras foram colhidas manualmente durante os períodos de 300, 360, 420 e 540 dias após plantio (DAP). Não houve efeito (P>0,05) da adubação na concentração de CS, a exceção das folhas secas (FS), CSA e minerais, na fração colmo. Três padrões de acúmulo de CS foram identificados nas duas frações morfológicas da planta: a) BRIX e CSOH; b) CNF E c) CSA. Embora POL não fosse considerada nesse experimento, as análises das curvas de CS (BRIX e CSOH) confirmaram que a cana atingiu a maturidade no mês de setembro como reportado na literatura. Regressões simples e múltiplas foram consideradas para predizer o acúmulo de CS, MS e o valor nutricional (digestibilidade). O modelo univariado apenas considerou variáveis químicas na predição, exceto para o teor de BRIX. Equações do tipo stepwise melhoraram o ajuste dos modelos (R2 e RQMR) ao considerar variáveis morfológicas, sendo a mais importante nas predições, o número de nós da fração colmo (NN). As equações para MS e CSA tiveram o menor poder de predição tanto nas regressões simples quanto nas múltiplas. A fração FDA foi a principal variável que explicou o valor nutritivo em amostras de cana-deaçúcar. / The objective of this research was to compare some potentialities of soluble carbohydrate (SC) methods currently used in sugarcane for animal feeding purposes: soluble solid content (BRIX), apparent sucrose content (POL), non fibrous carbohydrates (NFC), water soluble carbohydrates (WSC) and ethanol soluble carbohydrates (EtOH). Two trials involving stalk and whole plant sugarcane samples were conducted. The first trial evaluated the nutritive value of IAC 86-2480 variety (second growth) in response to two harvesting methods: manual (MAN) and mechanical (MEC) along of crop growing phase: 150, 180, 210, 240, 270, 300, 330 and 360 days after harvesting (DAH). The SC estimative, chemical composition (DM, CP, NDF, ADF, IVDDM and IVOMD) and NDF/SC relationships were similar (P>0.05) across treatments, except for EtOH and NDF/EtOH relationship at stalk and NDF/POL (P<0.05) at whole plant being MAN and MEC of those cases, respectively more advantageous for each. The whole plant SC content reached represents closer to 80% of total content observed in the stalk fraction. Were identified three typical SC accumulation patterns: a) POL and EtOH, b) BRIX, c) NFC. Until 240 (MAN) and 270 (MEC) DAH, the standardized NDF/BRIX and NDF/POL stalk relationships followed the same trend. NDF/SC relationships were highly correlated with forage digestibility, especially NDF/NFC (r > 0.8). Principal Component Analysis proved to be an useful tool to select variables in order to explain sugarcane feeding value. Therefore as described at the first principal component, the sugarcane feed value is in function of forage fibrous fraction (ex. NDF, ADF), corresponding to 54.4% (stalk) and 64.0% (whole plant) of total variance respectively. The second trial studied possible relationships established between both morphological as chemical plant measurements that could explain SC accumulation and feeding value of IAC 93- 3046 variety, under two levels of fertilisation at planting (NPK and NPK associated with cattle manure 120 kg N ha-1) along of ripening phase under plant crop conditions. Forage samples were collected manually at 300, 360, 420 and 540 days after planting (DAP). There was no effect (P>0.05) of fertilisation on SC dynamics, chemical composition and morphological crop variables, with the exception to dry leaves (DLEAF), water soluble carbohydrates (WSC) and minerals (ASH), in stalk fraction (P<0.05). The age of plant was the main factor responsible of the observed variability. Three SC patterns in sugarcane during this phase were identified: a) BRIX and EtOH, b) NFC e c) WSC. Although POL was not considered in this trial, the analysis of SC curves showed that BRIX and EtOH also reached to maturity in September conforming literature. Simple and multiple (stepwise) regressions were considered to predict SC, DM accumulation and feeding value (digestibility). The univariate models only considered chemical variables to prediction, except to BRIX. Stepwise equations have account also morphological crop variables to improve statistical prediction (R2, RMSE), being the most important: number of nodes (NN). Predictions of DM and WSC had the lowest power performed through simple and multiple equations. ADF fraction best fitted to feeding value prediction (IVDMD, IVOMD) in sugarcane samples.