Flavonoids are the C-15 phenolic compounds containing two phenyl rings and a heterocyclic ring. The majority of the flavonoids accumulated in grapefruit are flavonol, flavanone, flavone, dihydroflavonol, and chalcone glycosides. Most flavonoids are present in glucosylated form and the glucosylation is mediated by a class of enzymes called glucosyltransferases that transfer glucose from a high energy sugar donor to the acceptor aglycone at a particular position. A clone encoding a flavonol-specific 3-O-glucosyltransferase (Cp-3-O-GT) from Citrus paradisi has been previously characterized in our lab. The study of structure and function of flavonoid GTs is an important aspect of our research that contributes to the synthesis of novel glucosides by changing the glucosylation patterns of GTs. Our study focuses on the structural and functional analysis of Cp-3-O-GT through site directed mutation and analysis of mutated enzyme in terms of substrate specificity and regiospecificity. Multiple sequence alignment and homology modeling was used to identify candidate areas for mutation. For this study, Cp-3-O-GT was modeled with a flavonoid 3- O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GT – P145T mutant to test the hypothesis that that mutation of key amino acid residues (proline) in Cp-3-O-GT by position specific amino acids of VvGT (threonine) could alter substrate specificity or regiospecificity of Cp-3-O-GT. Initial screening results suggested that the mutant P145T glucosylates flavanones and flavones in addition to flavonols. This is significant because flavanones and flavonols do not contain a 3-OH group for glucosylation. HPLC was performed to identify the reaction products. Early results indicate that the P145T mutant glucosylates naringenin at 7-OH position forming naringenin-7-O-glucoside and this is being confirmed. Product identification with other substrates is also being conducted. Results are being used to revisit and refine the structure model.

The study aimed to investigate the following: 1. Investigate a putative glutathione peroxidase gene (Glyma17g34110) within Glycine max by an in silico analysis and spatial expression. 2. Determine the effects of exogenously applied nitric oxide on the expression of Glyma17g34110. 3. Investigate the antioxidant mechanism with attention to Glyma17g34110,reactive oxygen species and cell death in the response to salt stress. 4. Establish whether Glyma17g34110 is a glutathione peroxidase or thioredoxindependent peroxidase gene. / Magister Scientiae - MSc

The production of reactive oxygen species (ROS) is prominent in all aerobic metabolisms including plants. For this reason, the redox homeostasis of the production and scavenging of these intermediates is imperative for growth, development and survival during unfavourable conditions. In this study, a putative glutathione peroxidase gene (Glyma17g34110) from Glycine max (soybean) was identified and analyzed. The successful characterisation of Glyma17g34110 provided evidence of it being a glutathione peroxidase using glutathione as its preferred electron donor and substrate. Furthermore, it is known that antioxidant enzymes such as GPX exist in various tissues, performing a diverse set of functions. By a bioinformatic analysis of Glyma17g34110 and its promoter region, it was indicated that Glyma17g34110 could be a putative chloroplast protein that could play an important role in photosynthesis.One of the major factors affecting plant growth and development worldwide is abiotic stresses such as salinity. In the presence of salinity the production of harmful ROS is increased, resulting in detrimental reactions with important biological features (DNA, protein and lipid membranes), leading to cell death. The analysis of Glyma17g34110 under salt stress revealed that it is a salt sensitive gene and thus, the down-regulation of Glyma17g34110 could be due to the lack of known defence and response cis-acting elements present in the promoter region. Furthermore, it was proven in previous studies that the application of exogenous nitric oxide (NO) increases the activity of antioxidant enzymes. In this thesis it was observed that the presence of exogenously applied NO increased the expression of Glyma17g34110 tremendously in all soybean tissues (leaves, roots and nodules) investigated.Studies have found numerous cis-acting elements to be NO responsive, however, none of these elements were found in the promoter region upstream of glyma17g34110. This suggests that novel cis-acting elements could be present in the promoter region of Glyma17g34110.Thus, increasing the expression of Glyma17g34110 during salinity in the presence of NO, as well as the identification of these novel cis-acting elements, could lead to the enhancement of the defence mechanisms against ROS, which could lead to increasing plant tolerance to stress. / >Magister Scientiae - MSc

Die humanen Noroviren (Familie Caliciviridae, Genus Norovirus) bilden den Haupterreger der nichtbakteriellen Gastroenterotiden. Seit Beginn der 1990er Jahre – besonders seit dem Herbst 2002 – kann weltweit eine sehr starke Zunahme der Norovirus-Infektionen beobachtet werden. Die Mehrheit der Erkrankungen (ca. 80%) ist dabei durch den Genotyp II.4 verursacht, wobei innerhalb dieses Genotyps kontinuierlich „neue“ genetisch veränderte Norovirus-Stämme auftreten, die kontinuierlich in „neue“ Subgenotypen eingeordnet werden. Bis dato wird vermutet, dass die hohe Zunahme der Infektionen durch die subgenotypspezifische Evolution des strukturellen Proteins VP1 bewirkt wurde, da dadurch die Bindung der Viruspartikel an die Rezeptoren der Wirtszelle – die HBGAs – verbessert sein könnte bzw. ein breiteres Spektrum an Rezeptoren erkannt werden könnte. Bisher ist aber weitgehend unbekannt, ob im Bereich der nichtstrukturellen Proteine auch eine subgenotypspezifische Evolution stattgefunden hat und ob diese durch eine verbesserte Enzymaktivität zu der Zunahme der Infektionen beigetragen haben könnte. Im Rahmen dieser Arbeit wurden demnach aus unterschiedlichen Stuhlproben die beiden Enzyme NS6pro und NS7pol von jeweils einem Stamm der epidemiologisch relevanten Subgenotypen II.4-1995, II.4-2002, II.4-2004, II.4-2006a und II.4-2006b rekombinant hergestellt und betreffend ihrer Evolution und Enzymaktivität untersucht. Anhand der durchgeführten Untersuchungen konnte festgestellt werden, dass bei den epidemiologisch relevanten Subgenotypen des Genotyps II.4 im Bereich der NS6pro und der NS7pol keine subgenotypspezifische Evolution stattgefunden hat und daher keine subgenotypspezifische Enzymaktivität vorliegt. Somit hatte die Enzymaktivität der NS6pro und der NS7pol keinen Einfluss auf die Zunahme der Infektionen. Dennoch konnte aber beobachtet werden, dass durch die Mutation einer einzelnen Aminosäure – entsprechend der Position in der räumlichen Struktur der NS6pro bzw. der NS7pol – die Enzymaktivität um bis zu 100 Prozent erhöht bzw. reduziert werden kann. Bei diesen bedeutenden Mutationen ist entweder die Substratbindung oder die Koordination bzw. die Zufuhr der Reaktionskomponenten betroffen. Somit konnte bestätigt werden, dass die Zunahme der Infektionen vermutlich doch durch die Evolution des VP1 bedingt ist. Dies ist auch nachvollziehbar, da das VP1 den Eintritt der Viruspartikel in das Zellplasma erlaubt, in dem danach erst die Virusvermehrung durch die Enzyme stattfindet. Hierbei muss aber erwähnt werden, dass die Evolution des VP1 durch die Mutationsrate der NS7pol bestimmt ist, sodass die NS7pol letztendlich doch für die Zunahme der Infektionen verantwortlich ist. Um aber definitiv abzuklären, ob durch die Evolution des VP1 tatsächlich die Bindung der Viruspartikel an die HBGAs verbessert bzw. ein breiteres Spektrum an Rezeptoren erkannt wird, müssten bspw. Bindungs-Assays zwischen den entsprechenden VLPs (virus-like particles) und bereits typisierten Serum- oder Speichelproben – ähnlich den Untersuchungen von Lindesmith et al. (2008) – durchgeführt werden. Darüber hinaus müsste die Evolution und die Funktionalität der restlichen nichtstrukturellen Proteine untersucht bzw. deren Einfluss auf die Zunahme der Infektionen überprüft werden.

ABSTRACT Covalent Immobilization of Glucose Isomerase on Poly (2-hydroxyethyl methacrylate) Particles Yildiz, Hakan &Uuml / mit M.S., Department of Chemistry Supervisor: Prof. Dr. Nesrin Hasirci July 2004, 54 pages In this study, poly (2-hydroxyethyl methacrylate), P(HEMA), particles were prepared by suspension polymerization of the monomer 2-hydroxyethyl methacrylate with addition of ethylene glycol dimethyacrylate, EGDMA, as cross linker. Glucose isomerase, GI, enzyme was covalently immobilized on the prepared P(HEMA) particles after activation of the particles with cyanuric chloride. The activities of the free and immobilized enzymes were measured with Ethanol-Carbazole method. The immobilization of GI on P(HEMA) particles promoted enzyme stability and as a result, the enzyme became more stable to temperature, storage, and reuse. For maximum substrate conversion, optimum temperature was determined as 70 oC for free GI and this value shifted to 60 oC for immobilized enzyme. Optimum pH for maximum substrate conversion was found to be 7.0 for free GI and 8.0 for immobilized GI. The change of enzyme activity with substrate concentration were determined to calculate Km and Vmax values of the free and immobilized enzymes. Km values were found to be 1.7x10-2 mol/L and 3.1x10-1 mol/L while Vmax values were 1.01x10-4 mol/L.min, 1.65x10-3 mol/L.min for free and immobilized GI, respectively. Reuse capability of immobilized GI on P(HEMA) particles was measured and compared with commercial GI. Both systems retained 80 % of their original activities after 40th use, within 6 days. The change of enzyme activities upon storage were detected at certain time intervals for the samples stored in buffer solution at 4 oC. Immobilized enzyme was retained 60% of its original activitiy in 60 days of storage at 4 oC. Immobilized GI and commercial GI both retained 90% of their activities under continuous flow after 180 mL of substrate solution passed through the column.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Submitted by ALINE MENDES DE SOUSA GOUVEIA null (alinemendgouv@hotmail.com) on 2016-03-11T18:04:01Z No. of bitstreams: 1 Gouveia_Aline M Sousa_ dissertação final.pdf: 1929135 bytes, checksum: afef42c043c33f7c7a9cdcb09f7f4188 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-03-14T17:13:34Z (GMT) No. of bitstreams: 1 gouveia_ams_me_bot.pdf: 1929135 bytes, checksum: afef42c043c33f7c7a9cdcb09f7f4188 (MD5) / Made available in DSpace on 2016-03-14T17:13:34Z (GMT). No. of bitstreams: 1 gouveia_ams_me_bot.pdf: 1929135 bytes, checksum: afef42c043c33f7c7a9cdcb09f7f4188 (MD5) Previous issue date: 2016-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O rabanete pode ser cultivado entre o período de safra de outras culturas de ciclo mais longo, sendo uma opção interessante aos pequenos produtores rurais que enxergaram nesta hortaliça uma possibilidade de incremento na rentabilidade das suas áreas. Devido a poucas informações na literatura que mostrem os efeitos do potássio na produção, qualidade e nas características bioquímicas do rabanete, objetivou-se com este trabalho avaliar os efeitos da adubação potássica aplicado em cobertura na produção e qualidade pós-colheita da cultura do rabanete armazenado sob refrigeração. O experimento foi conduzido na Fazenda Experimental da FCA/UNESP, em São Manuel/SP e as avaliações foram realizadas no Departamento de Horticultura da Faculdade de Ciências Agronômicas (FCA/UNESP). O delineamento experimental foi em blocos ao acaso, composto por cinco tratamentos de doses de potássio (K2O) (0; 22,5; 45; 67,5 e 90 kg ha-1) na forma de cloreto de potássio em cobertura, com cinco repetições. Foram avaliadas as características vegetativas da planta, acúmulo de nutrientes na raiz e produtividade. Para as características de qualidade (físico-química e bioquímica) as raízes colhidas foram armazenadas a temperatura de 5 ± 1 °C e UR de 85 ± 5 % durante 28 dias, sendo avaliadas a cada 7 dias, quanto a perda de massa, pH, acidez titulável, sólidos solúveis, açúcares redutores, ácido ascórbico (vitamina C), compostos fenólicos, pigmentos (carotenoides e antocianinas totais) e atividade enzimática (peroxidase). Para as características de produção do rabanete, obtiveram-se respostas lineares significativas em função das doses de potássio aplicadas em cobertura para: altura de plantas, massas da matéria fresca da parte aérea, raiz e total, com incremento de 0,46 cm, 0,61 g, 1,05 g, e 2,02 g por planta para cada 10 kg ha-1 de K2O aplicados, respectivamente. Para produtividade, houve u¬¬¬¬¬¬m incremento em 1,05 t ha-1 a cada 10 kg ha-1 de K2O aplicados. Para a porcentagem de raízes comerciais, houve ajuste da equação quadrática, sendo a dose 52,4 kg ha-1 K2O apresentou maior percentual (97,7 %) de raízes comercializáveis. A ordem decrescente dos macronutrintes acumulados pela raiz em média foi de: K > N > Ca > P > S > Mg. Para as caraterísticas ¬¬físico-químicas e bioquímicas, observou-se aumento em função do aumento das doses de potássio para acidez titulável e diminuição para perda de massa, pH, sólidos solúveis, açúcares redutores, pigmentos (carotenoides e antocianinas totais), compostos fenólicos e na atividade da peroxidase, mantendo-se estável os teores de ácido ascórbico. Já em função do período de armazenamento, observou-se aumento para perda de massa, acidez titulável e pigmentos (carotenoides e antocianinas totais), diminuição para sólidos solúveis, açúcares redutores e manteve-se estável os teores de ácido ascórbico, compostos fenólicos e atividade da peroxidase. / The radish is grown in consortium with other longer-cycle crops, but now small farmers have seen in the vegetable, a chance to increase the profitability of their areas. The literature has little information as to potassium’s effects on the production, quality and the biochemical Radish characteristics. This study is aimed to evaluate the effects of potassium fertilizer applied in coverage in the production and culture of post-harvest quality radish under refrigeration. The experiment was conducted at the Experimental Farm of FCA/UNESP in San Manuel / SP and the evaluations were carried out in the Department of Horticulture, Faculty of Agricultural Sciences (FCA / UNESP). The experimental design was in randomized blocks, comprising of five treatments of potassium (K2O) (0; 22,5; 45; 67,5 and 90 kg ha-1) in coverage, with five repetitions. We evaluated the vegetative characteristics of the plant, nutrient accumulation in the shoots, roots and productivity. For the quality features, physical chemistry and biochemistry harvested roots were stored at 5 ± 1 °C and RH 85 – 90 % for 28 days were evaluated every 7 days, the mass loss, pH, titratable acidity, soluble solids, reducing sugars, ascorbic acid (vitamin C), phenolic compounds, pigments (carotenoids and anthocyanins) and enzyme activity (peroxidase). For radish production characteristics, we obtained significant responses as a function of potassium doses on coverage for plant height, mass of fresh matter of the aerial part, root and total, an increase of 0,46 cm, 0,61 g, 1,05 g and 2,02 g per plant per 10 kg ha-1 K2O applied, respectively. The decreasing order of macronutrintes accumulated by the root is: K> N> Ca> P> S> Mg. For productivity, there was an increase by 1,05 t ha-1 every 10 kg ha-1 K2O applied and the dose 52,4 kg ha-1 K2O with the highest percentage (97,7 %) in marketable yield. For the physicochemical and biochemical characteristics as a function of potassium doses, were obtained rise to acidity, decreased to mass loss, pH, soluble solids, reducing sugars, pigments (carotenoids and anthocyanins), phenolic compounds and activity peroxidase and remained stable ascorbic acid content. Already a function of storage time, we obtained increased to weight loss, titratable acidity and pigments (carotenoids and anthocyanins), decreased to soluble solids, reducing sugars and remained stable ascorbic acid content, phenolic compounds and activity peroxidase.

Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2015-12-17T09:13:12Z No. of bitstreams: 0 / Made available in DSpace on 2015-12-17T09:13:12Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2015-03-26T13:53:16Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2894312 bytes, checksum: 8e4e6d33bfbb1f07f987dfb380479055 (MD5) Previous issue date: 2010-07-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The coffee tree is a demanding plant in micronutrients, especially Zn, and the P x Zn interaction is an important issue for the management of tropical soils, due to the use of high doses of P in soils with low availability of this nutrient. In this work was studied the seedlings response of Coffea arabica L. cv. Catuaí to doses of Zn and P, including the supply of P corresponding to a high availability of P, therefore, a high antagonism potential to the availability of Zn. The experiment was carried out in a greenhouse in the period between the October/2009 and January/2010 (122 days of conducting the experiment), using 4 × 4 factorial design, distributed in randomized blocks, with three replications, being four doses of Zn (0, 5, 15, and 45 mg / dm 3 of soil) and P (0, 200, 600 and 1,800 mg / dm 3 of soil). On the soil, having as a source ZnSO 4 .7 H 2O, the doses of Zn were applied trough a soil solution and P levels were incorporated, being the source the triple superphosphate. It was used samples of a Red Yellow sandy-loam originating from Três Marias - MG and coffee seedlings with four pairs of leaves, which were transplanted to pots (2 dm 3 of soil), maintaining soil moisture near to field capacity. It was evaluated the total content of Zn in plant parts (leaf, stem and root), the soluble fraction of Zn (leaf), the levels of total P (leaf, stem and root), the enzyme superoxide dismutase (SOD) , plant height, number of nodes per plant, average length of internodes and dry matter production (leaf, stem and root). There were no significant changes in levels of total Zn in leaves with increasing doses of P added to soil, although the levels of soluble Zn (active) have fallen, indicating, in this case, antagonistic interaction between the variables. The SOD activity was reduced with increasing doses of Zn added to soil. The growth of coffee seedlings was influenced by the interaction between P and Zn. / O cafeeiro é uma planta exigente em micronutrientes, especialmente, em Zn, e a interação P x Zn é uma questão importante para o manejo de solos tropicais, haja vista a utilização de altas doses de P em solos com baixa disponibilidade deste nutriente. Neste trabalho foi estudada a resposta de mudas de Coffea arabica L. cv. Catuaí às doses de Zn e P, incluindo-se o suprimento de P que correspondeu a uma alta disponibilidade de P, portanto, a um alto potencial de antagonismo em relação à disponibilidade de Zn. O experimento foi conduzido em casa-de-vegetação entre o período de outubro/2009 a janeiro/2010 (122 dias de condução do experimento), utilizando esquema fatorial 4 × 4, distribuído em blocos casualizados, com três repetições, sendo quatro doses de Zn (0, 5, 15, e 45 mg/dm3 de solo) e quatro doses de P (0, 200, 600 e 1.800 mg/dm3 de solo). No solo, tendo como fonte ZnSO4.7H2O, as doses de Zn foram aplicadas em solução via solo, e as doses de P foram incorporadas, sendo a fonte o superfosfato triplo. Utilizaram-se amostras de um Latossolo Vermelho Amarelo textura franco-arenosa originário de Três Marias – MG e mudas de café com quatro pares de folhas, que foram transplantadas para vasos (2 dm3 de solo), mantendo-se a umidade do solo próxima à capacidade de campo. Avaliaram-se o teor total de Zn nos órgãos das plantas (folha, caule e raiz) e a fração solúvel de Zn (folha), os teores totais de P (folha, caule e raiz), atividade da enzima superóxido dismutase (SOD), altura de plantas, número de nós por planta, comprimento médio dos entrenós e produção de matéria seca (folha, caule e raiz). Não houve alterações significativas nos teores de Zn total nas folhas com o aumento das doses de P adicionadas ao solo, embora os teores de Zn solúvel (ativo) tenham diminuído, indicando, neste caso, interação antagônica entre as variáveis. A atividade da SOD foi reduzida com o aumento nas doses de Zn adicionadas ao solo. O crescimento das mudas de café foi influenciado pela interação P e Zn.

Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2015-12-17T09:13:12Z No. of bitstreams: 0 / Made available in DSpace on 2015-12-17T09:13:12Z (GMT). No. of bitstreams: 0 / A enzima conversora de angiotensina (ECA) catalisa a conversão de angiotensina I (Ang I) no vasoconstritor angiotensina II (Ang II) e hidrolisa a bradicinina (BK). ECA somática (sECA) possui dois domínios homólogos (N e C) que têm 60% de identidade. Embora estas duas regiões tenham homologia grande, o sítio catalítico C-domínio exibe uma atividade três vezes maior do que o N-domínio na hidrolise de Ang I in vivo. Este fato torna interessante o desenvolvimento de novos estudos de inibidores ou a melhoria dos já existentes. O objetivo deste estudo foi obter a região Ala959 até Ser1066 do Cdomínio da sECA (c-sECA), em uma estrutura conformacional semelhante à estrutura nativa. Nós amplificamos a sequência correspondente ao sítio catalítico da c-sECA com 324pb e clonamos esta sequência no vetor pET 28a(+). O segmento (nomeado de pET28_c-sECA) foi expresso em sistema bacteriano. A proteína foi expressa na forma solúvel e a purificação foi feita em uma única etapa utilizando a coluna de afinidade His-tag, a qual produziu a proteína pura. Análises estruturais por dicroísmo circular e fluorescência confirmaram que a proteína recombinante estava na conformação correta, e os ensaios de atividade mostraram que a c-sECA possui atividade enzimática e é inibida por lisinopril. / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP