Methods for fast, simple detection of biomarkers to detect and differentiate closely related Bacillus endospores including Bacillus anthracis (BA), Bacillus thuringiensis (BT), Bacillus atrophaeus (BG), and Bacillus cereus (BC) using thermochemolysis and methylation (TCM), coiled wire filament (CWF), solid phase micro extraction (SPME) and gas chromatography-mass spectrometry (GC-MS) were developed. The main biomarkers detected and used for differentiation include dipicolinic acid methyl ester (DPAME), fatty acid methyl esters (FAMEs), 3-methyl-2-butenoic acid methyl ester (3M2BAME), 2-butenoic acid methyl ester (2BAME), and several methylated sugars. TCM of endospores was performed based on hydrolysis and methylation at elevated temperature after the endospores were mixed with sulfuric acid (H2SO4) with or without addition of tetramethylammonium hydroxide (TMAH) in methanol (MeOH). TCM products were then introduced into a heated GC injector port using a coiled wire filament (CWF) or solid phase microextraction (SPME) for detection and differentiation of the endospores by GC-MS.The CWF, which consisted of a tiny platinum helical wire coil attached to a retractable plunger that moved the coil in and out of a syringe needle housing, allowed for sampling to be accomplished by dipping the CWF in an endospore sample suspension, evaporating the suspension liquid, and then introducing the CWF into the injection port to enable on-line TCM. New SPME techniques, including half-half extraction, coated-needle extraction (CNE), and a new home-made polymer coated needle were used to speed up solid phase micro extraction of biomarkers produced from TCM. These simplified the detection of anthrose and other biomarkers. TCM with a CWF and TCM with SPME produced high intensity profiles of DPAME, FAMEs, 2BAME, 3M2BAME and methylated sugars. While the presence of DPAME can be used for the general detection of endospores (Bacillus and Clostridium) and the presence of 3M2BAME for the detection of BA, specific saturated and unsaturated C15, C16, and C17 fatty acid methyl esters and methylated sugars provide additional information for differentiating various Bacillus species grown at different temperatures and in different media. DPAME was detected in samples containing as few as 2,500 and 6,000 endospores using TCM-CWF with and without a concentration step, respectively. GC-MS peak area percent reproducibility for FAMEs using TCM and CWF varied from 3 to 13% (RSD). Better than 97% correct predictability of Bacillus species identity was obtained from a blind experiment consisting of 145 samples using DPAME and specific FAMEs. Conventional SPME and a modified form of "in-needle" extraction allow for detection of the biomarkers in less than 35 min. The detection limits with SPME sample introduction injection were approximately 5 x 103 endospores.Using these approaches, differentiation of Bacillus endospores and other biological agents grown under different conditions were based on the following characteristics: (1) presence of DPAME and specific FAMEs (iso or anteiso C15:0 and iso or anteiso C17:0) in Bacillus endospores, (2) unique presence of 3M2BAME (anthrose by-product) in BA, (3) absence of 2-butenoic acid methyl ester in BG, and (4) presence and absence of specific methylated monosaccharides in various Bacillus species. Clostridium endospores and non-sporulating bacteria, such as Yersinia pestis (YP) and Francisella tularensis (FT) could also be easily distinguished from Bacillus endospores based on the presence and absence of several specific sugar derivatives and fatty acid methyl esters (FAMEs), such as iso or anteiso C15:0 and iso or anteiso C17:0, and > C18 FAMEs which were simultaneously produced during TCM.

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Tesis por compendio / [ES] Actualmente, hay un progresivo interés por una dieta equilibrada y saludable, rica en frutas y verduras con un efecto positivo en la salud y, particularmente, con alto contenido en antioxidantes. Paralelamente existe una demanda, por parte del consumidor, de fruta de alta calidad que tenga un sabor equilibrado y que recuerde a las variedades tradicionales. Todo ello ha propiciado que los programas de mejora genética también tengan como objetivo la mejora de la calidad de la fruta, tanto en propiedades fisicoquímicas del fruto como de su perfil nutricional. Desde este punto de vista, el albaricoque es un fruto muy completo ya que es una fuente extraordinaria de fibra, vitaminas, compuestos fenólicos y ácidos orgánicos. Sus propiedades hacen posible su consumo tanto en fresco o como procesado, lo que es aprovechado por las industrias agroalimentaria, farmacéutica y cosmética para la elaboración de una amplia variedad de productos. En el Instituto Valenciano de Investigaciones Agrarias (IVIA) se está llevando a cabo un programa de mejora para la obtención de nuevas variedades de albaricoquero que, además de solucionar factores que afectan directamente a la producción, como son la autocompatibilidad o resistencia a Sharka, también tiene por objetivo la mejora de la calidad de la fruta. Como primer paso en todo programa de mejora, en este trabajo se han identificado fuentes de variación entre la colección de accesiones del banco de germoplasma del IVIA, quedando detallada la composición del perfil para los principales azúcares, ácidos orgánicos, compuestos fenólicos y de vitamina C. Además, se ha identificado una mayor concentración de compuestos en la piel del fruto que en su pulpa, lo que hace de este tejido una fuente de compuestos de interés para la industria. Por otro lado, para un mejor conocimiento del control genético del metabolismo de azúcares, se estudió la expresión de los genes clave implicados en esta ruta (SUS, SPS y FK), relacionados con QTLs previamente descritos en materia de sólidos solubles. Además, también se reforzó el estudio con el correspondiente análisis filogenético entre diferentes especies, observándose el grado de conservación entre las mismas y confirmándose el alto grado de sintenia en el género Prunus. Con relación a los compuestos fenólicos, se ha analizado la expresión de genes implicados en puntos críticos de la ruta metabólica (DRF, PAL y FLS). Nuestros resultados revelaron que las variedades más rojizas se asocian a una mayor expresión de ParDFR y ParPAL2, así como también que las diferencias de expresión entre parálogos podría deberse a la presencia de un BOXCOREDLPAL, relacionado también con los genes implicados en la síntesis de antocianinas (ParDFR, ParFLS2 y ParPAL2). Paralelamente, también se estudió el efecto genético que ejerce sobre su descendencia la variedad 'Goldrich', uno de los principales genitores empleados en el programa de mejora para la introgresión de la resistencia a Sharka, concluyendo que favorece la concentración de neoclorogénico y clorogénico, así como en la expresión génica de ParPAL1. / [CA] Actualment, hi ha un progressiu interés per una dieta equilibrada i saludable, rica en fruites i verdures amb un efecte positiu en la salut i, particularment, amb alt contingut en antioxidants. Paral·lelament existeix una demanda, per part del consumidor, de fruita d'alta qualitat que tinga un sabor equilibrat i que recorde a les varietats tradicionals. Tot això, ha propiciat que els programes de millora genètica també tinguen com a objectiu la millora de la qualitat de la fruita, tant en propietats fisicoquímiques del fruit com del seu perfil nutricional. Des d'aquest punt de vista, l'albercoc és un fruit molt complet ja que és una font extraordinària de fibra, vitamines, compostos fenòlics i àcids orgànics. Les seues propietats fan possible el seu consum tant en fresc com processat, sent també aprofitat per les indústries agroalimentària, farmacèutica i cosmètica per a l'elaboració d'una àmplia varietat de productes. A l'Institut Valencià d'Investigacions Agràries (IVIA) s'està duent a terme un programa de millora per a l'obtenció de noves varietats d'albercoquer que, a més de solucionar factors que afecten directament la producció, com són l'autocompatibilitat o resistència a Sharka, també té per objectiu la millora de la qualitat de la fruita. Com a primer pas en qualsevol programa de millora, en aquest treball s'han identificat fonts de variació entre la col·lecció d'accessions del banc de germoplasma de l'IVIA, quedant detallada la composició del perfil per als principals sucres, àcids orgànics, compostos fenòlics i de vitamina C. A més, s'ha identificat una major concentració de compostos en la pell del fruit que en la seua polpa, convertint aquest teixit en una font de compostos d'interés per a la indústria. D'altra banda, per a un millor coneixement del control genètic del metabolisme de sucres, es va estudiar l'expressió dels gens clau implicats en aquesta ruta (SUS, SPS i FK), relacionats amb QTLs prèviament descrits en matèria de sòlids solubles. A més, també es va reforçar l'estudi amb la corresponent anàlisi filogenètica entre diferents espècies, observant-se el grau de conservació entre les mateixes i confirmant-se l'alt grau de sintenia al gènere Prunus. En relació amb els compostos fenòlics, s'ha analitzat l'expressió de gens implicats en punts crítics de la ruta metabòlica (DRF, PAL i FLS). Els nostres resultats van revelar que les varietats més vermelles s'associen a una major expressió de ParDFR i ParPAL2, així com també que les diferències d'expressió entre paràlogs podria deure's a la presència d'un BOXCOREDLPAL, relacionat també amb els gens implicats en la síntesi de antocianines (ParDFR, ParFLS2 i ParPAL2). Paral·lelament, també es va estudiar l'efecte genètic que exerceix sobre la seua descendència la varietat 'Goldrich', un dels principals genitors empleats al programa de millora per a la introgressió de la resistència a Sharka, concloent que afavoreix la concentració de neoclorogènic i clorogènic, així com en l'expressió gènica de ParPAL1. / [EN] Nowadays, there is a growing interest in a balanced and healthy diet, rich in fruit and vegetables with a positive effect on health and, particularly, with high antioxidant content. Simultaneously exists a consumer demand for high quality fruit with a balanced flavour and reminiscent of traditional varieties. As a result, breeding programmes are also aimed at improving the quality of the fruit, both in terms of the physicochemical properties of the fruit and its nutritional profile. From this point of view, the apricot is a complete fruit as far it is an extraordinary source of fibre, vitamins, phenolic compounds, and organic acids. Apricot properties make it possible to consume fruits both fresh and processed, which is exploited by the agri-food, pharmaceutical and cosmetic industries for the production of a wide variety of products. At the Instituto Valenciano de Investigaciones Agrarias (IVIA) a breeding program is carrying out to obtain new apricot varieties but also to solve factors that directly affect production, such as self-compatibility or resistance to Sharka, but also is aimed to improve fruit quality. As the first step in any breeding program, this work has identified sources of variation among the collection of accessions in the IVIA's apricot collection, detailing the composition profiles for the main sugars, organic acids, phenolic compounds, and vitamin C. In addition, a higher concentration of compounds has been identified in the peel than in the flesh, which makes this tissue a source of compounds of interest for the industry. On the other hand, for a better knowledge of the genetic control of sugar metabolism, the expression of the key genes involved in this pathway (SUS, SPS and FK), related to QTLs previously described in soluble solids, was studied. In addition, the study was also supported with the corresponding phylogenetic analysis among different species, revealing the level of conservation among them and confirming the high level of synteny in the genus Prunus. Regarding phenolic compounds, the expression of genes involved in critical points of the metabolic pathway (DRF, PAL and FLS) was analysed. Our results revealed that the red-blushed accessions are associated with a higher expression of ParDFR and ParPAL2, and the expression differences among paralogues could be due to the presence of a BOXCOREDLPAL, also related to genes involved in anthocyanin biosynthesis (ParDFR, ParFLS2 and ParPAL2). Simultaneously, the genetic effect in the offspring of 'Goldrich', one of the main genitors used at the breeding program for the introgression of Sharka resistance, was also studied. Results revealed that it improves the concentration of neochlorogenic and chlorogenic acids, as well as the genetic expression of ParPAL1. / Este trabajo ha sido posible gracias a la ayuda predoctoral FPI-INIA-2015, del Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), bajo el proyecto FPI2015-0042 con la denominación ‘CPD2015-0245 Mejora sostenible de albaricoquero y melocotonero’ y desarrollado en el Instituto Valenciano de Investigaciones Agrarias (IVIA), dependiente de la Generalitat Valenciana. / Gómez Martínez, H. (2021). Variation of Fruit Quality Traits in Apricot as Sources for Nutraceutical Breeding [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/178973 / Compendio

Le procédé innovant d’hydrodiffusion par micro-onde a été testé et validé pour la fabrication de jus de fruit sur des raisins, abricots et prunes. Ce procédé présente des avantages au niveau technique : rapidité d’extraction, pas de préparation de l’échantillon, aucun auxiliaire de fabrication. Il permet l’obtention de jus à partir de fruits difficiles à presser, avec une pasteurisation « in line » et des produits se différenciant nettement des jus commerciaux par leur couleur et texture.Le jus est expulsé du fruit sous l’effet de la vapeur produite in situ. A partir du fruit congelé, la déstructuration liée à la croissance des cristaux de glace facilite l’extraction. Les rendements le plus élevés sont obtenus à partir de fruits congelés, et à basse puissance. Une partie de l’eau des fruits est convertie en vapeur, mais peut être récupérée en utilisant un réfrigérant, ce qui permet d’atteindre des rendements proches des rendements de jus obtenus avec un prétraitement enzymatique et pressurage. Globalement, le jus a une composition proche de celle du jus présent dans le fruit ou obtenus par pressurage. La composition du jus expulsé des fruits change au cours de l’extraction. La concentration des composés présents dans la chair et hydrosolubles (sucres, acides, acides phénoliques) baisse nettement en fin d’extraction, tandis que les anthocyanes, présentes dans l’épiderme des raisins ou des prunes, montrent un pic de concentration lors de la phase d’expulsion rapide des jus. La teneur en procyanidines est nettement plus élevée que pour des jus obtenus par pressurage / The hydrodiffusion process was tested and validated for production of juices from grapes, apricots and plums. Its major advantages are speed, no sample preparation, no processing aid. It allows production of juices from hard-to-press fruits with an in line pasteurisation. The juices present original characteristics notable bright colours, texture, and a fresh fruit aroma.The juice is expulsed from the fruit by the pressure due to in situ vapour generation. The destructuration occuring in frozen fruits due to growth of ice crystals facilitate the juice expulsion. The highest yields are obtained from frozen fruits and at low power densities. Part of the water from the fruits is vaporised, and can be collected by use of a cooler, allowing to reach yields comparable to those obtained by pressing after an enzymatic pre-treatment. Juice composition is close to that of juice extracted by pressing, though with slightly lower concentrations of most soluble solids. The composition of juice changes along the extraction. The highly soluble compounds present in the fruit flesh mostly show a clear exhaustion at the end ofextraction. Anthocyans, present exclusively in the peel of the test fruits, present a maximum during the fast juice extraction phase. Procyanidin concentrations is much higher in juices obtained by hydrodiffusion than in conventional juices

UDP-sugar metabolizing pyrophosphorylases provide the primary mechanism for de novo synthesis of UDP-sugars, which can then be used for myriads of glycosyltranferase reactions, producing cell wall carbohydrates, sucrose, glycoproteins and glycolipids, as well as many other glycosylated compounds. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase) and UDP-N-acety lglucosamine pyrophosphorylase (UAGPase), which can be discriminated both by differences in accepted substrate range and amino acid sequences. This thesis focuses both on experimental examination (and re-examination) of some enzymatic/ biochemical properties of selected members of the UGPases and USPases and UAGPase families and on the design and implementation of a strategy to study in vivo roles of these pyrophosphorylases using specific inhibitors. In the first part, substrate specificities of members of the Arabidopsis UGPase, USPase and UAGPase families were comprehensively surveyed and kinetically analyzed, with barley UGPase also further studied with regard to itspH dependency, regulation by oligomerization, etc. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Frc-1-P, whereas USPase reacted with arange of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P, β-L-Ara-1-P and α-D-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P and, to some extent, with D-Glc-1-P. A structure activity relationship was established to connect enzyme activity, the examined sugar-1-phosphates and the three pyrophosphorylases. The UGPase/USPase/UAGPase active sites were subsequently compared in an attempt to identify amino acids which may contribute to the experimentally determined differences in substrate specificities. The second part of the thesis deals with identification and characterization of inhibitors of the pyrophosphorylases and with studies on in vivo effects of those inhibitors in Arabidopsis-based systems. A novel luminescence-based high-throughput assay system was designed, which allowed for quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The assay was then used to screen a chemical library (which contained 17,500 potential inhibitors) to identify several compounds affecting UGPase and USPase. Hit-optimization on one of the compounds revealed even stronger inhibitors of UGPase and USPase which also strongly inhibited Arabidopsis pollen germination, by disturbing UDP-sugar metabolism. The inhibitors may represent useful tools to study in vivo roles of the pyrophosphorylases, as a complement to previous genetics-based studies. The thesis also includes two review papers on mechanisms of synthesis of NDP-sugars. The first review covered the characterization of USPase from both prokaryotic and eukaryotic organisms, whereas the second review was a comprehensive survey of NDP-sugar producing enzymes (not only UDP-sugar producing and not only pyrophosphorylases). All these enzymes were discussed with respect to their substrate specificities and structural features (if known) and their proposed in vivo functions.

Dans la dernière décennie, plusieurs hectares de terre agricole ont été convertis à la culture intensive sur courtes rotations (CICR) de saules dans le sud du Québec (Canada). Peu d’études ont été réalisées afin de déterminer comment se comporte la dynamique du carbone organique (Corg) dans le sol suivant cette conversion. Nous avons donc comparé la quantité du Corg et de deux pools labiles de carbone (carbone extractible à l’eau chaude et les sucres aminés) entre des CICR en phase initiale d’établissement (1-2 ans) et des parcelles appariées représentant le système de culture qui prévalait avant la transformation en culture de saules (culture fourragère) et d’autres cultures d’intérêt. La même chose a été faite pour une CICR en exploitation (depuis 9 ans) à un autre site. La quantité de Corg du sol n’était pas différente entre les CICR et les parcelles sous culture fourragère. Une plus haute concentration de sucres aminés dans le Corg total des CICR en établissement, par rapport aux autres parcelles sur le même site, permet de soupçonner que les perturbations liées à l’établissement ne mènent pas à une minéralisation accrue du Corg à court terme. La proportion de sucres aminés fongiques, qui diminue théoriquement lors de perturbations, était aussi plus élevée sous la plus jeune culture. Sous la CICR de neuf ans, le Corg était redistribué dans le profil vertical et les pools labiles étaient de plus petite taille (à une profondeur de 20-40 cm) comparativement à une parcelle témoin. La conversion d’une culture fourragère en plantation de saules en CICR n’a pas mené à la formation d’un puits de carbone. L’étude laisse entrevoir qu’un tel puits pourrait être créé si la conversion se faisait à partir d’un aménagement impliquant la culture en rotation de plantes annuelles et des labours. / Over the last ten years, many hectares of short-rotation willow coppice (SRWC) have been established on abandoned agricultural lands in southern Quebec (Canada). However, few studies were conducted to determine if these changes from a conventional crop to SRWC of willows would affect the soil carbon dynamic. A preliminary paired-site study was conducted to assess the impact of this land-use conversion on soil carbon stocks and dynamics shortly after they were established (1-year and 2-year) at one site and after multiple rotations (9-year) at another site. Apart from the total soil organic carbon pool, two labile carbon pools were investigated: the hot-water extractable carbon (HWC) pool and the amino sugars (AS) pool. Willow establishment and exploitation for nine years did not bring any changes in total organic carbon compared to an abandoned hay culture. The mature SRWC displayed a redistribution of TSOC in the vertical soil profile. We also measured a higher proportion of AS, compared to other plots on the same site, in total organic carbon under the 1-year-old SRWC; especially the proportion in AS of fungi origin that theorically decrease with perturbation intensity. Proportion of AS was also elevated under 2-year-old SRWC. A possible interpretation is that establishment-linked perturbations did not shift carbon dynamic toward an increased mineralization. The mature willow plantation exhibited depletion of HWC and the more labile amino sugar (muramic acid) in the deeper soil layer (20-40cm). This case study shows that conversion from an abandoned hay culture to a SRWC did not create a carbon sink. It was also found that the 9- year old willow plantation contained higher TSOC and had better soil quality than an adjacent short-term no-till crop rotation culture.

A babosa (Aloe vera (L.) Burm. f.), família Asphodelaceae, reconhecida e explorada mundialmente pela indústria farmacêutica e cosmética devido aos princípios medicinais de seus compostos fenólicos e principalmente ao gel de polissacarídeos específicos, foi submetida a dois experimentos interligados. O primeiro, refere-se a micropropagação no qual foram avaliados tratamentos de desinfestação de ápices caulinares, multiplicação in vitro e condições de aclimatação ex vitro. O segundo experimento refere-se ao cultivo das plantas em areia lavada e irrigada com solução nutritiva, em condições controladas de casa de vegetação, onde foram testados os efeitos de doses (105; 210 e 315 ppm) de nitrogênio avaliados aos 90; 180 e 270 dias de cultivo. Os efeitos foram avaliados em função dos teores foliares dos macronutrientes e dos micronutrientes boro, cobre, ferro manganês e zinco, de proteínas totais solúveis (PTS), de açúcares redutores (AR) e açúcares totais solúveis (ATS) e sobre o crescimento por meio do índice de área foliar (IAF), da taxa de assimilação líquida (TAL), taxa de crescimento relativo (TCR), taxa de crescimento absoluto (TCA) e incrementos de massas de matérias fresca (IMMF) e seca (IMMS). Todos os dados foram analisados estatisticamente. Em relação a micropropagação, a eficiência de desinfestação foi aumentada em torno de 40% na obtenção de ápices caulinares verdes em início de brotação quando as plantas colhidas a campo foram previamente desinfestadas por lavagem com solução de hipoclorito de sódio com 0,5% de cloro ativo ou com solução de dicloroisocianurato de sódio (Sumaveg®) 0,66%. p.v-1 e os ápices caulinares submetidos a imersões alternadas nas soluções dos dois produtos utilizados. A fase multiplicativa da microproagação em meio MS apresentou um rendimento de 1:5,3 a cada intervalo de 30 dias de multiplicação. A partir de 136 ápices caulinares desinfestados, verdes, em início de brotação, foram obtidas 40.495 microplantas. Classificadas em pequenas, médias e grandes, foram submetidas a condições de aclimatação observando-se que bandejas de 64 células com 40 cm3 de substrato apresentaram economia em torno de 50% de substrato e em espaço físico na casa-de-vegetação com micro-aspersão e exaustão de ar em sistema \"pad-house\", e durante o processo de aclimatação e transporte das microplantas aclimatadas. Em relação ao cultivo das plantas em doses de nitrogênio, apesar de algumas variáveis responderem melhor à dose de 105 ppm e outras à dose de 315 ppm, a dose de 210 ppm de nitrogênio favoreceu as melhores respostas para os teores de açúcares totais solúveis (504,21 mgATS.g-1 de MMS), que são diretamente relacionados ao conteúdo de polissacarídeos específicos, de interesse comercial da cultura de Aloe vera. Esses teores, por sua vez, foram propiciados pelos melhores valores de IAF, TCA, IMMF e IMMS, todos observados aos 270 dias de cultivo, ratificando a significância do fator tempo e da dose de 210 ppm de nitrogênio no cultivo dessa espécie vegetal. / Aloe vera (L.) Burm. f., family Asphodelaceae, worldwide renowned and explored by pharmaceutics and cosmetics industries due to its phenolics bearing medicinal principles and mainly to the specific polysaccharides present in the gel, was submitted to two interlinked experiments. The first one refers to apical shoot micropropagation evaluating different disinfection treatments of the explants, the in vitro bud multiplication and ex vitro acclimatization of the microplants. The second one refers to cultivation in greenhouse of the micropropagated plants in washed sand and irrigated with nutritive solution, in the presence of three nitrogen rates (105, 210 and 315 ppm); the plant material was harvested at 90-, 180- and 270-day. All data were statistically analyzed. The effects of nitrogen were evaluated on the content of the macronutrients, the following micronutrients: B, Cu, Fe, Mn and Zn and total soluble proteins, reducing sugars, total soluble sugars; the growth of the Aloe vera plants was measured through the foliar area index, the rate of liquid assimilation, rates of relative and absolute growth and increases in the fresh and dry weights. In regards to micropropagtion, the efficiency of the disinfection process was increased by 40% when the plants harvested in the field were previously washed either with sodium hypochloride (0.5% active chlorine) solution or sodium dichloroisocyanurate (Sumaveg®) 0.66% w.v-1 solution and the apical shoots explants were afterwards alternatively treated with the two disinfectants. The multiplication phase in MS medium showed a rate of 1:5.3 of microplant production at each 30-day interval with a production of 40.495 microplants out of the 136 initial disinfected apical shoots. The microplants were classified as small, medium and large plants and acclimatized in polyethylene trays bearing 64 cells with 40 cm3 of substrate each cell, a 50% saving in terms of substrate amount and free space in the greenhouse equipped with micro-aspersion irrigation, pad-house and air exhaustion systems and also in the transport of the acclimatized microplants. In regards to the effect of nitrogen rates on the development of Aloe vera plants, besides the fact that the best responses were observed either to 105 ppm or 315 ppm nitrogen by some variables, at 210 ppm nitrogen rates the best result was obtained for total soluble sugars (504.21 mg.g-1DW); the sugars are directly related to specific polysaccharides of Aloe vera and are of great importance for the industries. On the other hand, these values were favored by the best values reached by the physiological variables studied in this work at 270-day validate the significance of the time factor and the 210 ppm N rates in the Aloe vera production system.

Made available in DSpace on 2017-07-21T18:53:09Z (GMT). No. of bitstreams: 1 Mary Dias Queji.pdf: 909281 bytes, checksum: 0364cd27c13b09019850a0944168a898 (MD5) Previous issue date: 2008-04-05 / The agro industry of the apple generates, during the processing, the pomace which is considered the main by-product. Studies show that of the total amount of fruit that is processed to obtain the apple juice, 20 to 40 correspond to this by-product, which, is usually destined as a complement in animal feed or delivered onto the soil as organic fertilizer. The chemical composition, in moist base, is constituted by moisture (80), fibers (5) and soluble solids (14) represented, mainly, by fructose, glucose and sucrose, as well as, by organic acids, represented, in the most part by malic acid. The apple pomace also presents phenolic compounds that are target nutrients of great sensory and nutritional importance. Therefore, the quantification of its constituents represents an important source of data in the characterization of apple pomace for biotechnological purposes, seeking to attribute an appropriate use for this by-product. The conventional methodologies used for the quantification of sugars, organic acids and total phenols, although being part of the routine analyses in the quality control laboratories, are onerous, time consuming and generate residues. The aim of this study was to develop a fast, versatile analytical technique, of low cost and no pollutant, using the diffuse reflectance infrared spectroscopy (DRIFTS) allied to methods of multivariate calibration (PLSR). For the construction of the multivariate models, the averages of the concentrations of the simple sugars, malic acid and total phenols were used, obtained by the conventional methodologies, as well as the data in the medium infrared spectroscopy (MID) and near (NIR), obtained in duplicate, and of the 52 spectra obtained for the samples of apple pomace, 47 made part of the set calibration and 5 of the set validation. The regression models for the prediction of the concentration of fructose, sucrose, total phenols and malic acid obtained better results in MID, with averages of relative standard errors of 3.9 (with 5 Latents Variable), 6.6 (with 5 Latents Variable), 6.4 (with 4 Latents Variable) and 5.9 (with 5 Latents Variable), respectively. Already the best capacity prediction for glucose concentration was obtained by NIR, in which the average of relative standard error was 7.4 (with 6 Latents Variable). The obtained results demonstrate the good capacity of prediction of the multivariate models based in infrared spectroscopy and characteristic advantages of the association DRIFTPLSR. / A agroindústria da maçã gera, durante o processamento, o bagaço que é considerado o principal subproduto deste setor. Levantamentos mostram que da quantidade total de fruta que é processada para a obtenção do suco de maçã, 20 a 40 correspondem a este subproduto, que, normalmente, é destinado como complemento na alimentação animal ou dispensado no solo como adubo orgânico. Sua composição química, em base úmida, é constituída por umidade (80), fibras (5) e sólidos solúveis (14), estes últimos representados, principalmente, por frutose, glucose e sacarose, bem como, ácidos orgânicos, representados, majoritariamente pelo ácido málico. O bagaço da maçã também apresenta compostos fenólicos, que são fito-nutrientes de grande importância sensorial e nutricional. Portanto, a quantificação desses constituintes representa uma importante fonte de dados na caracterização do bagaço de maçã para finalidades biotecnológicas, visando atribuir um fim mais nobre a este subproduto. As metodologias convencionais empregadas para a quantificação de açúcares, ácidos orgânicos e fenóis totais, embora façam parte das análises de rotina nos laboratórios de controle de qualidade, são onerosas, demoradas e geram resíduos. O objetivo deste trabalho foi desenvolver uma técnica analítica rápida, versátil, de baixo custo e não poluente. Para tal, utilizou-se a espectroscopia no infravermelho por refletância difusa (DRIFTS) aliada a métodos de calibração multivariada (Regressão de Mínimos Quadrados Parciais - PLSR). Para a construção dos modelos multivariados, foram utilizadas as médias das concentrações dos açúcares simples, ácido málico e fenóis totais, obtidas pelas metodologias convencionais, bem como, os dados de espectroscopia no infravermelho médio (MID) e próximo (NIR). Os espectros foram obtidos em duplicata, sendo que dos 52 espectros das amostras de bagaço de maçã, 47 fizeram parte do conjunto de calibração e 5 do conjunto de validação externa. Os modelos de regressão para a previsão da concentração de frutose, sacarose, fenóis totais e ácido málico obtiveram melhores resultados no MID, com médias de erro padrão relativo de 3.9 (com 5 Variáveis Latentes), 6.6 (com 5 Variáveis Latentes), 6.4 (com 4 Variáveis Latentes) e 5.9 (com 5 Variáveis Latentes), respectivamente. Já a melhor capacidade de previsão para a concentração de glucose foi obtida pelo NIR, na qual a média de erro padrão relativo foi de 7.4 (com 6 Variáveis Latentes). Os resultados obtidos demonstram a boa capacidade de previsão dos modelos multivariados fundamentados em espectroscopia no infravermelho e vantagens características da associação DRIFT-PLSR.

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-04-07T12:30:41Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_felipe_koch.pdf: 799099 bytes, checksum: 5d61cb7ae229b25b533d3f35001c2acc (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-04-12T18:07:39Z (GMT) No. of bitstreams: 2 dissertacao_felipe_koch.pdf: 799099 bytes, checksum: 5d61cb7ae229b25b533d3f35001c2acc (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-04-12T18:09:51Z (GMT) No. of bitstreams: 2 dissertacao_felipe_koch.pdf: 799099 bytes, checksum: 5d61cb7ae229b25b533d3f35001c2acc (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-12T18:10:04Z (GMT). No. of bitstreams: 2 dissertacao_felipe_koch.pdf: 799099 bytes, checksum: 5d61cb7ae229b25b533d3f35001c2acc (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-09-28 / Sem bolsa / Este trabalho teve por objetivo avaliar o crescimento de plantas de trigo submetidas a aplicação de regulador de crescimento vegetal e adubação para altos rendimentos, assim como, a composição química e a qualidade fisiológica de sementes produzidas. A cultivar utilizada foi a OR Topázio e o regulador de crescimento foi o trinexapac-ethyl, aplicado via foliar. No capítulo I, o experimento foi conduzido em casa de vegetação e o delineamento experimental foi o inteiramente casualisado, em esquema fatorial 5x8 (cinco doses e oito épocas de coleta) com quatro repetições. Foram avaliados a matéria seca total, a taxa de produção de matéria seca, as taxas de crescimento relativo e assimilatória liquida, a razão de área e massa foliar, o índice de área foliar. Foi determinada a eficiência de conversão da energia solar, a partição de assimilados, o índice de colheita, a altura das plantas, a emergência e o índice de velocidade de emergência de plântulas. No capítulo II, o experimento foi conduzido em delineamento experimental inteiramente casualisado com cinco tratamentos e quatro repetições, sendo avaliados a germinação e primeira contagem de germinação, índice de velocidade de germinação, matéria seca de plântulas, condutividade elétrica e envelhecimento acelerado. No capítulo I, a alocação de matéria seca total, a taxa de crescimento relativo, a altura de plantas e o índice de colheita foram reduzidos, com intensidade variável, conforme a dose do regulador de crescimento. A taxa assimilatória líquida foi reduzida em plantas submetidas a doses de 400, 600 e 800 mL ha-1, a razão de massa e de área foliar foram superiores quando utilizadas as doses de 400 e 800 mL ha-1. A emergência e índice de velocidade de emergência não foram afetados pela ação do regulador de crescimento. No capítulo II, a primeira contagem de germinação aumentou até a maior dose. A condutividade elétrica após três horas de embebição foi reduzida, a partir da dose de 600 mL ha-1. Os teores de amido aumentaram até a dose de 448 mL ha-1; o de aminoácidos até a dose 243 mL ha-1 e os teores de proteína até a dose de 417 mL ha-1. Houve redução no teor de açúcares solúveis totais ao aumentar a dose do regulador. Portanto, plantas submetidas à aplicação do trinexapac-ethyl apresentam respostas diferenciais no crescimento e partição de assimilados, menor índice de colheita e altura de plantas. Já, as sementes possuem maior vigor e teores de amido, proteína e aminoácidos, contudo, menor teor de açúcares solúveis totais. / This work aimed to evaluate the growth of wheat plants subjected to the application of growth regulator and fertilization for high yields, as well as, chemical composition and physiological quality of seeds produced. The variety used was OR Topázio and the growth regulator was trinexapac-ethyl, via leaf application. On chapter I, the experiment was performed on greenhouse and the experimental design was completely randomized, factorial 5x8 (five doses and eight harvest dates) with four repetitions. Total dry matter, dry matter production rate, relative growth and net assimilation rates, leaf mass ratio, leaf area ratio and leaf area index were evaluated. The conversion efficiency of solar energy, assimilate partitioning, harvest index, plant height, seedling emergence and speed of emergence index were determined. On chapter II, the experiment was performed under completely randomized experimental design using five treatments and four repetitions, being evaluated germination and first count of germination, speed of germination index, seedling dry matter, electrical conductivity and accelerated aging. On chapter I, total dry matter allocation, relative growth rate, plant height and harvest index were reduced, with variable intensity, according to the growth regulator dose. The net assimilation rate was reduced in plants subjected to 400, 600 e 800 mL ha-1; leaf mass ratio and leaf area ratio were superior when doses of 400 and 800 mL ha-1 were used. The seedling emergence and the speed of emergence index were not affected by the action of the growth regulator. On chapter II, germination and first count of germination increased until the higher dose. Electrical conductivity after three hours of imbibition was reduced starting from the dose of 600 mL ha-1. The starch content increased until the dose of 448 mL ha-1; the amino acids up to the dose of 243 mL ha-1 and the protein content to the dose of 417 mL ha-1. There was a decrease on total soluble sugars content with the increase of growth regulator dose. Therefore, plants subject to the application of trinexapac-ethyl feature differential responses on growth and assimilate partitioning, lower harvest index and plant height. Seeds produced by these plants possess higher germination, vigor expression and starch, protein and amino acids contents, however, lower content of total soluble sugars.

Proteins that recognise the sugar surface structures on cells have an enormous potential to be used as tools in the characterisation of these structures. A group of proteins, called lectins, have been identified that can bind to carbohydrate complexes on the receptors of cells. The crude extract from Grevillea robusta seeds was found to contain lectin-like proteins that were different from most other lectins, as they would specifically target the receptors of white blood cells and not those found on red blood cells. Therefore, the lectin isolated from G.robusta could be used as a tool to identify the specific surface structures on white blood cells. The lectin was isolated using affinity chromatography where a complex (oligosaccharide) matrix was used. Agglutination, binding and sugar inhibition assays confirmed the isolated protein was a lectin. The lectin was found in low amounts (up to 5% of the total protein content) within the seeds of G.robusta. As a result of this low yield, the identification of the lectin by PAGE was difficult because the levels of protein were beyond the detection limit of the commercial staining reagents. The lectin was called the GR2 protein and was characterised as a monocot mannose binding lectin based on its sugar specificity for only mannose. A serine protease inhibitor was isolated from the seeds of G.robusta using two different chromatography methods, reverse phase HPLC (GR1.HPLC) and gel filtration chromatography (GR1.GF). Ion exchange chromatography was used to initially separate the proteins in the crude extract and the fraction containing the GR1 protein was further purified using reverse phase HPLC (GR1.HPLC). N-terminal sequencing results of the GR1.HPLC protein, showed evidence of proteolytic cleavage during the extraction process, which lead to the second purification method being established. Protease inhibitors were added to the buffers prior to being purified by gel filtration chromatography, which resulted in the GR1 protein being isolated from the crude extract without the presence of the contaminating protein. Mass spectroscopy identified the molecular weight of the GR1 protein to be 6669Da and the full amino acid sequence was derived by cDNA techniques. Sequence alignment studies of the GR1 protein showed significant similarities with the Bowman-Birk inhibitor. The positioning of the cysteine residues were conserved throughout the Bowman-Birk superfamily, however these residues were not conserved within the GR1 protein. Competitive inhibition assays on the GR1 protein revealed the protein could inhibit both trypsin and chymotrypsin at similar levels to that seen for the Bowman-Birk inhibitor. Therefore, the GR1 protein was characterised as a member of the Bowman-Birk superfamily of serine protease inhibitors. The three-dimensional structure of the GR1 protein was determined using two-dimensional NMR spectroscopy. Computer programs such as XEASY, DYANA and SYBYL® were used to tabulate the information taken from the 2D experiments, generate structures and minimise these structures respectively. The solution structure of the GR1 protein was found to contain a region of antiparallel β-sheet structure that corresponded to the trypsin binding site and the remainder of the protein consisted of loops and turns that were held together by disulfide bridges (the chymotrypsin-binding region). Structural similarities between the GR1 protein and the Bowman-Birk inhibitor existed only in the trypsin-binding site of the Bowman-Birk inhibitor. The GR1 protein is the first member of the Proteaceae family to be characterised as a Bowman-Birk inhibitor. This thesis outlines the isolation and biochemical characterisation of the two proteins found within Grevillea robusta and also describes the steps involved and results obtained in determining the three-dimensional structure of the GR1 protein.