Avaliação da ação bioinseticida de SBTI e vicilina de Erythrina velutina em enzimas digestivas e membrana peritrófica de larvas de Plodia interpunctella (Lepidoptera: Pyralidae)

Plodia interpunctella (Indian meal moth) is a cosmopolitan pest that attacks not only a wide range of stored grain as well other food products. Due to its economic importance several researches have focused in a method with ability to control this pest with few or no damage to the environment. The study of digestive enzymes inhibitors, lectins and chitin-binding proteins, has often been proposed as an alternative to reduce insect damage. In this study we report the major classes of digestive enzymes during larval growth in P. Interpunctella, being those proteinases actives at pH 9.5 and optimum temperature of 50 oC to both larvae of the 3rd instar and pre-pupal stage of development. In vitro and zymogram assays presented the effects of several inhibitors, such as SBTI, TLCK and PMSF to intestinal homogenate of 3rd instar larvae of 62%, 92% and 87% of inhibition and In pre-pupal stage of 87%, 62 % and 55% of inhibition, respectively. Zymograms showed inhibition of two low molecular masses protein bands by TLCK and that in presence of SBTI were retarded. These results are indicative of predominance of digestive serine proteinases in gut homogenate from Plodia interpunctella larvae. This serine proteinase was then used as a target to evaluate the effect of SBTI on larvae in in vivo assay. Effect of SBTI on mortality and larval mass was not observed at until 4% of concentration (w/w) in diets. Chitin, another target to insecticidal proteins, was observed by chemical method. Moreover, optic microscopy confirmed the presence of a peritrophic membrane. Established this target, in vivo effect of EvV, a chitin binding vicilin, evaluated during the larval development of P. interpunctella and was obtained a LD50 of 0,23% and WD50 of 0,27% to this protein. Mechanism of action was proposed through of the in vivo digestibility of EvV methodology. During the passage through the larval digestive tract was observed that EvV was susceptible to digestive enzymes and a reactive fragment, visualized by Western blotting, produced by digestion was recovered after dissociation of the peritrophic membrane. The bound of EvV to peritrophic membrane was confirmed by immunohystochemical assays that showed strong immunofluorescent signal of EvV-FITC binding and peritrophic membrane. These results are a indicative that vicilins could be utilized as potential insecticide to Plodia interpunctella and a control methods using EvV as bioinsecticide should be studied to reduce lost caused by storage insect pests

Análise de falhas em martelos oscilantes dos desfibradores da indústria sucroalcooleira da Região Norte-Nordeste

The failure of materials is always an unwelcome event for several reasons: human lives are put in danger, economic losses, and interference in the availability of products and services. Although the causes of failures and behaviour of materials can be known, the prevention of such a condition is difficult to be guaranteed. Among the failures, wear abrasion by the low voltage is the kind of failure that occurs in more equipment and parts industry. The Plants Sucroalcooleiras suffer significant losses because of such attrition, this fact that motivated their choice for the development of this work. For both, were considered failures in the swing hammers desfibradores stopped soon after the exchange provided in accordance with tonnage of cane processed, then were analyzed by the level of wear testing of rubber wheel defined by the standard ASTM G65-91.The failures were classified as to the origin of the cause and mechanism, moreover, were prepared with samples of welding procedures according to ASME code, sec. IX as well, using the technique of thermal spraying to analyze the performance of these materials produced in laboratories, and compares them with the solder used in the plant. It was observed that the bodies-of-proof prepared by the procedure described as welding, and the thermal spraying the results of losing weight have been minimized significantly compared to the preparations in the plant. This is because the use of techniques more appropriate and more controlled conditions of the parameters of welding. As for the thermal spraying, this technique has presented a satisfactory result, but requires the use of these coatings in the best condition for real affirmation of the results

Levantamento e avaliação de critérios para a ampliação de escala da produção de biossurfactantes utilizando melaço como substrato

Biosurfactants are amphiphilic molecules synthesized by microorganisms such as bacteria, yeast or filamented fungi cultivated in various carbon sources among sucrose and hydrocarbons. These molecules are composed by a hydrophilic and hydrophobic part. They operate mostly at interfaces of fluids of different polarities. Because of this characteristic, they are potentially employed in numerous industries, such as the textile, medical, cosmetics, food and mainly in the petrochemical ones. Therefore industry has interest in developing new biosurfactant production processes in high scale, in order to become them economically competitive when compared to synthetic biosurfactants. This work aims to evaluate the biosurfactant production applying a non-conventional substrate sugar cane molasses proceeding from the sugar industry thus reducing the production costs. The strain identified as AP029/GLIIA, isolated from oil wells in Rio Grande do Norte state and used in these experiments belongs to the culture collection of Antibiotics Department of UFPE. The fermentation were carried out using different conditions according to a factorial planning 24 with duplicate at center point, in which the studied factors were molasse concentration, nitrate concentration, agitation and aeration ratio. The experiments were performed in a shaker at 38ºC of temperature. Samples were withdrawn in regular periods of time of up to 72 hours of fermentation in order to analyze substrate consumption, cellular concentration, superficial tension, critical micelle dilution (CMD-1 e CMD-2) as well as extracelullar protein production. The results showed a production of 3,480 g/L of biomass, a reduction of 41% on superficial tension, 67% of substrate consumption and 0,2805 g/L of extracellular protein

Análise de falhas em martelos oscilantes dos desfibradores da indústria sucroalcooleira da Região Norte-Nordeste

The failure of materials is always an unwelcome event for several reasons: human lives are put in danger, economic losses, and interference in the availability of products and services. Although the causes of failures and behaviour of materials can be known, the prevention of such a condition is difficult to be guaranteed. Among the failures, wear abrasion by the low voltage is the kind of failure that occurs in more equipment and parts industry. The Plants Sucroalcooleiras suffer significant losses because of such attrition, this fact that motivated their choice for the development of this work. For both, were considered failures in the swing hammers desfibradores stopped soon after the exchange provided in accordance with tonnage of cane processed, then were analyzed by the level of wear testing of rubber wheel defined by the standard ASTM G65-91.The failures were classified as to the origin of the cause and mechanism, moreover, were prepared with samples of welding procedures according to ASME code, sec. IX as well, using the technique of thermal spraying to analyze the performance of these materials produced in laboratories, and compares them with the solder used in the plant. It was observed that the bodies-of-proof prepared by the procedure described as welding, and the thermal spraying the results of losing weight have been minimized significantly compared to the preparations in the plant. This is because the use of techniques more appropriate and more controlled conditions of the parameters of welding. As for the thermal spraying, this technique has presented a satisfactory result, but requires the use of these coatings in the best condition for real affirmation of the results

Estratégias para a organização da pesquisa em cana-de-açúcar: uma análise de governança em sistemas de inovação

PEDRO, Edilson da Silva. Estratégias para a organização da pesquisa em cana-de-açúcar: uma análise de governança em sistemas de inovação. 2008. 226f. Tese (Doutorado em Política Científica e Tecnológica) - Universidade Estadual de Campinas, Campinas, 2008.

Caracterização e comparação de genes expressos em ápices meristemáticos de cana-de-açúcar cultivados em SP e RN

In this work, we used sugarcane as a model due to its importance for sugar and ethanol production. Unlike the current plant models, sugarcane presents a complex genetics and an enormous allelic variation. Here, we report the analysis of SAGE libraries produced using the shoot apical meristem from contrasted genotypes by flowering induction (non-flowering vs. early-flowering varieties) grown under São Paulo state conditions. The expression pattern was analyzed using samples from São Paulo (SP) and Rio Grande do Norte (RN) states. These results showed that cDNAs identified by SAGE libraries had differential expression only in São Paulo state samples. Furthermore, the cDNA identified CYP (Citocrome P450) was chosen for in silico and genome characterization because it was found in SAGE libraries and subtractive libraries from samples from RN. Phylogenetic trees showed the relationship for these sequences. Furthermore, the qRT-PCR for CYP showed a potential role as flowering indutor for RN samples considering different isophorms. Considering the results present here, it can be consider that CYP gene may be used as molecular marker

Efeitos da microgravidade em plantas de cana-de-açúcar

Sugarcane (Saccharum spp.) is a plant from Poaceae family that has an impressive ability to accumulate sucrose in the stalk, making it a significant component of the economy of many countries. About 100 countries produce sugarcane in an area of 22 million hectares worldwide. For this reason, many studies have been done using sugarcane as a plant model in order to improve production. A change in gravity may be one kind of abiotic stress, since it generates rapid responses after stimulation. In this work we decided to investigate the possible morphophysiological, biochemical and molecular changes resulting from microgravity. Here, we present the contributions of an experiment where sugarcane plants were submitted to microgravity flight using a vehicle VSB-30, a sounding rocket developed by Aeronautics and Space Institute teams, in cooperation with the German Space Agency. Sugarcane plants with 10 days older were submitted to a period of six minutes of microgravity using the VSB-30 rocket. The morphophysiological analyses of roots and leaves showed that plants submitted to the flight showed changes in the conduction tissues, irregular pattern of arrangement of vascular bundles and thickening of the cell walls, among other anatomical changes that indicate that the morphology of the plants was substantially influenced by gravitational stimulation, besides the accumulation of hydrogen peroxide, an important signaling molecule in stress conditions. We carried out RNA extraction and sequencing using Illumina platform. Plants subjected to microgravity also showed changes in enzyme activity. It was observed an increased in superoxide dismutase activity in leaves and a decreased in its activity in roots as well as for ascorbate peroxidase activity. Thus, it was concluded that the changes in gravity were perceived by plants, and that microgravity environment triggered changes associated with a reactive oxygen specie signaling process. This work has helped the understanding of how the gravity affects the structural organization of the plants, by comparing the anatomy of plants subjected to microgravity and plants grown in 1g gravity

Análise filogenética e funcional de dois genes de reparo homólogos a AP endonuclease em cana-de-açúcar: ScARP1 e ScARP3

The genome of all organisms constantly suffers the influence of mutagenic factors from endogenous and/or exogenous origin, which may result in damage for the genome. In order to keep the genome integrity there are different DNA repair pathway to detect and correct these lesions. In relation to the plants as being sessile organisms, they are exposed to this damage frequently. The Base Excision DNA Repair (BER) is responsible to detect and repair oxidative lesions. Previous work in sugarcane identified two sequences that were homologous to Arabidopsis thaliana: ScARP1 ScARP3. These two sequences were homologous to AP endonuclease from BER pathway. Then, the aim of this work was to characterize these two sequence using different approaches: phylogenetic analysis, in silico protein organelle localization and by Nicotiana tabacum transgenic plants with overexpression cassette. The in silico data obtained showed a duplication of this sequence in sugarcane and Poaceae probably by a WGD event. Furthermore, in silico analysis showed a new localization in nuclei for ScARP1 protein. The data obtained with transgenic plants showed a change in development and morphology. Transgenic plants had slow development when compared to plants not transformed. Then, these results allowed us to understand better the potential role of this sequence in sugarcane and in plants in general. More work is important to be done in order to confirm the protein localization and protein characterization for ScARP1 and ScARP3

Caracterização funcional de dois cDNAs de cana-de- açúcar : PKCI e SHAGGY

Flowering is controlled by several environmental and endogenous factors, usually associated with a complex network of metabolic mechanisms. The gene characterization in Arabidopsis model has provided much information about the genetic and molecular mechanisms that control flowering process. Some of these genes had been found in rice and maize. However, in sugarcane this processe is not well known. It is known that early flowering may reduce its production up to 60% at northeast conditions. Considering the impact of early flowering in sugarcane production, the aim of this work was to make the gene characterization of two cDNAs previously identified in subtractive cDNA libraries: scPKCI and scSHAGGY. The in silico analysis showed that these two cDNAs presented both their sequence and functional catalytic domains conserved. The results of transgenic plants containing the overexpression of the gene cassette scPKCI in sense orientation showed that this construction had a negative influence on the plant development as it was observed a decrease in plant height and leaf size. For the scPKCI overexpression in antisense orientation it was observed change in the number of branches from T1 transgenic plants, whereas transgenic T2 plants showed slow development during germination and initial stages of development. The other cDNA analyzed had homology to SHAGGY protein. The overexpression construct in sense orientation did not shown any effect on development. The only difference observed it was an increase in stigma structure. These results allowed us to propose a model how these two genes may be interact and affect floweringdevelopment.

Análise de cDNAs identificados em bibliotecas substrativas de cDNA para floração de variedades de cana-de-açúcar cultivadas no Rio Grande do Norte(RN): fator de transição NAC, Calmodulina e Fosfatidiltransferase

The flowering is a physiological process that it is vital for plants. This physiological process has been well studied in the plant model Arabidopsis, but in sugarcane this process is not well known. The transition of the shoot apical meristem from vegetative to flowering is a critical factor for plant development. At Brazil northeastern region, the transition to flowering in sugarcane has an important effect as it may reduce up to 60% its production. This is a consequence of the sugar translocation from stalks to the shoot apical meristem which is necessary during the flowering process. Therefore, the aim of this work was to explore and analyze cDNAs previously identified using subtractive cDNA libraries. The results showed that these cDNAs showed differential expression profile in varieties of sugarcane (early x late flowering). The in silico analysis suggested that these cDNAs had homology to calmodulin, NAC transcription factor and phosphatidylinositol, a SEC14, which were described in the literature as having a role in the process of floral development. To better understand the role of the cDNA homologous to calmodulin, tobacco plants were transformed with overexpression cassettes in sense and antissense orientation. Plants overexpressing the cassette in sense orientation did not flowered, while plants overexpressing the cassette in the antissense orientation produced flowers. The data obtained in this study suggested the possible role from CAM sequence, SEC14 and NAC in the induction/floral development pathway in sugarcane, this is the first study in order to analyze these genes in the sugarcane flowering process.

Avaliação de parâmetros de estresse oxidativo em plantas de cana-de-açúcar tratadas com peróxico de hidrogênio

The genus Saccharum belongs to Poaceae family. Sugarcane has become important monocultures in Brazil due to their products: ethanol and sugar. The production may change between different regions from Brazil. This difference is related to soil, climatic conditions and temperature that promotes oxidative stress that may induce an early flowering. The aim of this work was to identify the effects of oxidative stress. In order to analyse this, sugarcane plants were submitted to oxidative stress using hydrogen peroxide. After this treatment, the oxidative stress were analyzed Then, the plant responses were analyzed under different approaches, using morphophysiological, biochemical and molecular tools. Thus, sugarcane plants were grown under controlled conditions and until two months they were subjected first to a hydroponics condition for 24 hours in order to acclimation. After this period, these plants were submitted to oxidative stresse using 0 mM, 10 mM, 20 mM and 30 mM hydrogen peroxide during 8 hours. The histomorphometric analysis allowed us to verify that both root and leaf tissues had a structural changes as it was observed by the increased in cell volume, lignin accumulation in cell walls. Besides, this observation suggested that there was a change in redox balance. Also, it was analyzed the activity of the SOD, CAT and APX enzymes. It was observed an increase in the SOD activity in roots and it was also observed a lipid peroxidation in leaves and roots. Then, in order to identify proteins that were differently expressed in this conditions it was used the proteomic tool either by bidimensional gel or by direct sequencing using the Q-TOF EZI. The results obtained with this approach identified more than 3.000 proteins with the score ranging from 100-5000 ions. Some of the proteins identified were: light Harvesting; oxygenevolving; Thioredoxin; Ftsh-like protein Pftf precusor; Luminal-binding protein; 2 cys peroxiredoxin e Lipoxygenase. All these proteins are involved in oxidative stress response, photsynthetic pathways, and some were classified hypothetical proteins and/or unknown (30% of total). Thus, our data allows us to propose that this treatment induced an oxidative stress and the plant in response changed its physiological process, it made changes in tissue, changed the redox response in order to survival to this new condition

Competitividade dos produtores rurais de cana-de-açúcar da região agreste do Rio Grande do Norte

Deregulation of the sugar-alcohol sector in the 90s meant that agribusiness firms begin a process of reorganization of their production structures to enhance their abilities to become competitive in the new scenario. This study aims to examine the Production Chain of Sugar cane in Agreste region of Rio Grande do Norte, with studies to contribute to the competitiveness of farmers giving grants for planning and implementing policies, programs and projects, public and / or private. The method used in the study was researching the literature on the history of sugar cane in Brazil, agribusiness management, production chains and competitiveness, in field research used a questionnaire to collect information from farmers. It is concluded that the rural producers of cane sugar in newborns are not competitive and therefore it is necessary policies and / or for private gain in competitiveness of producers and consequently the production chain. It is hoped that the study serves as a subsidy for those policies proposição

Estudo da influência de pré-tratamentos de dois resíduos lignocelulósicos (bagaço do pedúnculo de caju e casca de coco) utilizados como substrato na indução à síntese de enzimas celulolíticas

Nowadays generation ethanol second, that t is obtained from fermentation of sugars of hydrolyses of cellulose, is gaining attention worldwide as a viable alternative to petroleum mainly for being a renewable resource. The increase of first generation ethanol production i.e. that obtained from sugar-cane molasses could lead to a reduction of lands sustainable for crops and food production. However, second generation ethanol needs technologic pathway for reduce the bottlenecks as production of enzymes to hydrolysis the cellulose to glucose i.e. the cellulases as well as the development of efficient biomass pretreatment and of low-cost. In this work Trichoderma reesei ATCC 2768 was cultivated under submerged fermentation to produce cellulases using as substrates waste of lignocellulosic material such as cashew apple bagasse as well as coconut bagasse with and without pretreatment. For pretreatment the bagasses were treated with 1 M NaOH and by explosion at high pressure. Enzyme production was carried out in shaker (temperature of 27ºC, 150 rpm and initial medium pH of 4.8). Results showed that T.reesei ATCC 2768 showed the higher cellulase production when the cashew apple bagasse was treated with 1M NaOH (2.160 UI/mL of CMCase and 0.215 UI/mL of FPase), in which the conversion of cellulose, in terms of total reducing sugars, was of 98.38%, when compared to pretreatment by explosion at high pressure (0.853 UI/mL of CMCase and 0.172 UI/mL of Fpase) showing a conversion of 47.39% of total reducing sugars. Cellulase production is lower for the medium containing coconut bagasse treated with 1M NaOH (0.480 UI/mL of CMcase and 0.073 UI/mL of FPase), giving a conversion of 49.5% in terms of total reducing sugars. Cashew apple bagasse without pretreatment showed cellulase activities lower (0.535 UI/mL of CMCase and 0,152 UI/mL of FPase) then pretreated bagasse while the coconut bagasse without pretreatment did not show any enzymatic activity. Maximum cell concentration was obtained using cashew nut bagasse as well as coconut shell bagasse treated with 1M NaOH, with 2.92 g/L and 1.97 g/L, respectively. These were higher than for the experiments in which the substrates were treated by explosion at high pressure, 1.93 g/L and 1.17 g/L. Cashew apple is a potential inducer for cellulolytic enzymes synthysis showing better results than coconut bagasse. Pretreatment improves the process for the cellulolytic enzyme production

Floração precoce em cana-de-açúcar - um estudo utilizando ferramentas de análise in silico e proteômica

Sugarcane is one of the most important products of the world and Brazil is responsible for 25 % of the world production. One problem of this culture at northeast of Brazil is the early flowering. In our laboratory, it has been made before four subtractive libraries using early and late flowering genotypes in order to identify messages related to the flowering process. In this work, two cDNAs were chosen to make in silico analysis and overexpression constructs. Another approach to understand the flowering process in sugarcane was to use proteomic tools. First, the protocol for protein extraction using apical meristem was set up. After that, these proteins were separated on two bidimensional gels. It was possible to observe some difference for some regions of these gels as well as some proteins that can be found in all conditions. The next step, spots will be isolated and sequence on MS spectrometry in order to understand this physiological process in sugarcane

Prospecção e caracterização de genes associados ao processo de indução floral em cana-de-açúcar

The northeastern region is responsible to 14.32% of sugarcane national production. This lowered contribution is due to edaphoclimatic condition. Flowering is a vital process to plant which consumes lots of energy and it culminates in a process called isoporization. This one can give in a decreasing of 60% on alcohol and water production. It may consider that cropped sugarcane has a hibrid with octaploid genome, there are varieties with a flowering standard until of non flowering. Using this natural genetic potential on different croppings of sugarcane, the aim of this work was to understand as this process occurs by the usage of subtractive approaches. The total RNA was extracted using Trizol of peaks of merisematics of croppings with induced flowering and other with late flowering. From this total RNA were built four subtractives libraries (B1- induced early flowering subtracted on late flowering not induced; B2- late flowering not induced subtracted induced early flowering; B3- induced early flowering subtracted of not induced early flowering; B02- not induced early flowering subtracted from induced early flowering) using kits Super Smart cDNA synthesis and BD Clontech kit select cDNA subtraction (Clontech). This material was clone don vector pGEM T-easy(Promega) and changed in competent cells of E.coli DH10B. Given analysis sequence was carried out a program BLASTn against database of NCBI and genome of Arabidopsis thaliana, rice and maize. Clones were grouped in 9 different classes according to function. Some factors already related as couples of flower induction were identified at different libraries. And grouped proteins with cell cycle and it controls were presents, mainly kinases proteins. Related factors to proteic sinthesis, metabolism, defence, cell communication were also given in both libraries .Some identified genes did not show similarity on database or homology with hypothesis function, and it can represents new genes to be deposited in international database. These results offers that some identified on sugarcane, classified as on factors classes, cell cycle and cell communication, trough unknown genes, can be linked with genetic changing to the flowering process found in the northeastern region