Natural products are biologically active metabolites produced by fungi, bacteria, and plants that have an extended application in pharmaceutical and chemical industries. Because of their chemical versatility, flavoenzymes are commonly involved in natural product biosynthetic pathways. This has given rise to the identification of flavoenzymes that are promising candidates for biomedical and biotechnical applications. This dissertation discusses the characterization of three flavoenzymes involved in natural product biosynthesis. The class B flavin-dependent monooxygenases S-monoooxygenase from Allium sativum (AsFMO) and N-hydroxylating monooxygenase from Streptomyces sp. XY332 (FzmM) were studied. Both enzymes perform heteroatom oxidation as part of allicin or fosfazinomycin biosynthesis respectively. AsFMO was predicted to oxidize S-allyl-L-cysteine (SAC) to alliin in allicin biosynthesis. Surprisingly, AsFMO exhibited negligible activity with SAC, and instead was highly active with allyl mercaptan and NADPH. This contradicted the initial proposal and suggested that AsFMO is involved in an alternative path producing allicin directly from allyl mercaptan. FzmM was identified to perform multiple N-oxidations which lead to the formation of a nitro group. FzmM performed a highly coupled and specific reaction with L-aspartate and NADPH to produce nitrosuccinate. Both AsFMO and FzmM followed a kinetic mechanism representative of class B flavin-dependent monooxygenases with a rapid pro-R stereospecific reduction and the formation of a C(4a)-hydroperoxyflavin intermediate during oxidation. In addition, the AsFMO structure was obtained and consisted of two domains for FAD and NADPH binding signature of class B monooxygenases. The biochemical and structural study of the Acinetobacter baumannii siderophore interacting protein (BauF) was also accomplished. This enzyme is essential in acinetobactin mediated iron assimilation and is important for virulence. The characterization of the binding and reduction of acinetobactin-ferric iron complex revealed that BauF is specific for this substrate and does not utilize NAD(P)H as an electron donor. The unique activity and structure of BauF can aid future drug design. / Doctor of Philosophy / Plants, fungi, and bacteria synthesize and excrete unique chemicals called secondary metabolites or natural products. These compounds are used for many applications including dyes, flavorings, fragrances, and medicine. To make natural products, organisms use enzymes to perform complex reactions. Studying the enzymes that are involved in natural product pathways is important for understanding how secondary metabolites are made. Additionally, these enzymes can be engineered to perform reactions relevant to biotechnical applications. Our lab specializes in the study of flavoenzymes which use flavin chemistry for catalysis. Flavin is a yellow coenzyme that contributes to wide array of reactions by performing 1 or 2 electron transfers. This dissertation discussed the characterization of three flavoenzymes. The first enzyme is a S- monooxygenase from Allium sativum (garlic) called AsFMO. Reported here is the kinetic and structural characterization of AsFMO. We demonstrated that AsFMO was cabable of performing an unexpected reaction with allyl mercaptan likely converting it into allicin, the main flavor ingredient of garlic. Secondly, we reported the kinetic characterization of a nitro- forming enzyme termed FzmM. Nitro- formation is a valuable process as nitro- compounds are used in industrial organic synthesis. It was shown that FzmM performs nitro- formation with high efficiency and is specific for the substrate L-aspartate. Lastly, this work described the characterization of the the siderophore-interacting protein from Acinetobacter baumannii, BauF, which was predicted to be involved in iron acqusition. A. baumannii is a serious human pathogen with multidrug resistance, and inhibiting iron acquisition has been shown to prevent its survival. The characterization of the enzymes involved in this pathway is essential for developing new treatments for A. baumannii infection. We report the structure and function of BauF confirming its role in A. baumannii iron uptake and providing information that will aid in future drug design.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-14T16:00:03Z No. of bitstreams: 1 Kesia Ferreira Alves.pdf: 466143 bytes, checksum: 66630edcc98b43dfb320ee8189581aa0 (MD5) / Made available in DSpace on 2017-03-14T16:00:03Z (GMT). No. of bitstreams: 1 Kesia Ferreira Alves.pdf: 466143 bytes, checksum: 66630edcc98b43dfb320ee8189581aa0 (MD5) Previous issue date: 2008-02-28 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Anthracnose is considered an important disease of bell pepper (Capsicum annuum L.) in the state of Pernambuco and in various parts of the world, causing significant losses for fruit commercialization. Actions directed to preventive measures such as alternative control, are of great importance through the exploration of secondary compounds present in medicinal plants with antimicrobial activity. This study aimed to, assess the efficiency of crude extracts in the control of anthracnose in bell pepper, and analyze the control stability of promising treatments in relation to different extract, inoculum, temperature and pathogen isolates concentrations. In preliminary selection, 16 plant species were used in order to obtain aqueous and hidroalcoólico extracts. The aqueous extracts of garlic (Allium sativum L.), Mallow (Malva sp.), and ginger (Zingiber officinale Roscoe.) showed greater than 97% reductions in disease severity levels. The greatest value of CL75 was found for the garlic extract (3.5%), while the lowest value of CL75 was verified with the application of the ginger extract (2.1%). The lowest rate of efficiency reduction of the anthracnose control (TRE) with the pathogen inoculum increase occurred with garlic extract. With regard to different C. gloeosporioides isolates, significant interaction was found between the different extracts and isolates of the pathogen. The plant extracts were effective in controlling the disease in temperatures of 20, 25 and 30 ° C, while temperatures in the 10, 15and 35 ° C there was no disease. There were no differences in the efficiencies of garlic and ginger extracts, while the extract of hollyhock had the lower efficiency at 20 ° C, differing from the other temperatures, and other extracts at this temperature. The results obtained in this study indicate the great potential for use of the garlic extract (6%) in anthracnose control of bell pepper, considering its stability in different situations. / A antracnose é considerada uma importante doença do pimentão (Capsicum annuum L.) no Estado de Pernambuco e em várias partes do mundo, causando perdas significativas à produção de frutos para comercialização. Ações voltadas para medidas preventivas como o controle alternativo, são de grande relevância por meio da exploração de compostos secundários presentes em plantas medicinais com atividade antimicrobiana. O presente trabalho teve por objetivo, avaliar a eficiência de extratos vegetais brutos no controle da antracnose em pimentão, bem como analisar a estabilidade do controle dos tratamentos promissores em relação a.diferentes concentrações dos extratos, concentrações do inoculo e isolados do patógeno e, temperaturas. Na seleção preliminar foram utilizadas 16 espécies vegetais para obtenção de extratos aquosos e etanólicos. Os extratos aquosos de alho (Allium sativum L.), malva (Malva sp.) e gengibre (Zingiber officinale Roscoe.) propiciaram reduções superiores a 97% nos níveis de severidade. O maior valor de CL75 foi constatado para o extrato de alho (3,5%), enquanto o menor valor de CL75 foi verificado com a aplicação de extrato de gengibre (2,1%). A menor taxa de redução da eficiência do controle (TRE) da antracnose com o incremento do inóculo do patógeno ocorreu no extrato de alho. Com relação a diferentes isolados de C. gloeosporioides, foi constatada interação significativa entre os tipos de extratos e os isolados do patógeno. Os extratos vegetais foram eficientes no controle da doença nas temperaturas de 20, 25 e 30 oC, enquanto nas temperaturas 10, 15 e 35 °C não houve doença. Não houve diferenças nas eficiências dos extratos de alho e gengibre, enquanto o extrato de malva apresentou a menor eficiência a 20 oC, diferindo das outras temperaturas, bem como dos demais extratos nessa temperatura. Os resultados obtidos nesse estudo indicam o grande potencial de utilização do extrato de alho a 6% no controle da antracnose em pimentão, considerando sua estabilidade em diferentes situações.

Made available in DSpace on 2014-06-11T19:26:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-06-19Bitstream added on 2014-06-13T19:13:47Z : No. of bitstreams: 1 fernandes_ljc_me_botfca.pdf: 381482 bytes, checksum: e32cf2712ac5f36fe4ffd164adeb9b78 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Foram desenvolvidos dois experimentos com o objetivo de avaliar o crescimento, estado nutricional e produtividade de plantas de alho livres de vírus, em função de doses crescentes de nitrogênio. Os tratamentos foram 0, 20, 40, 80, 160, e 320 kg N ha-1, utilizando, como fonte, nitrato de amônio. Foi utilizada a cultivar Caçador L. V. (livre de vírus), os bulbilhos foram obtidos mediante cultura de tecido e termoterapia no Departamento de Produção Vegetal – Defesa Fitossanitária FCA / UNESP, multiplicados por quatro gerações em telado, no município de Guarapuava - PR. No experimento I, conduzido no município de Guarapuava – PR, foi utilizado o delineamento experimental de blocos ao acaso, com cinco repetições, com doses aplicadas em cobertura, na proporção de 20% da dose, quando as plantas apresentavam de 5 a 6 folhas; 30% quando apresentavam de 7 a 8 folhas; e 50% após a fase de diferenciação dos bulbos. A colheita ocorreu 149 dias após o plantio. O experimento II foi conduzido no município de Santa Juliana – MG, utilizando o delineamento de blocos ao acaso, com seis repetições, com doses aplicadas em cobertura, na proporção de 25% da dose, quando as plantas apresentavam de 3 a 4 folhas; 25% com 5 a 6 folhas; e 50% após a fase de diferenciação dos bulbos. A colheita ocorreu 112 dias após o plantio. As doses crescentes de N não influenciaram o crescimento das plantas, a média do comprimento na diferenciação foi de 92,8 cm para o experimento I e de 88,9 cm para o experimento II. Os valores do índice relativo de clorofila (IRC) foram aumentados com a elevação das doses de N apenas na fase de diferenciação e 15 dias após, para ambos os experimentos. Em relação aos teores foliares dos macronutrientes, no experimento I as doses de N influenciaram na absorção de N, K e S; já no experimento II, na absorção de N e na diminuição da absorção... / Two experiments were developed with the objective to evaluate the growth, plant nutrition and productivity of garlic without virus, submitted to nitrogen fertilization. The treatments used were 0, 20, 40, 80, 160, and 320 kg N ha-1, using as source ammonium nitrate. The cultivar used was Caçador L.V. (free of virus), the cloves were gotten through tissue culture and thermotherapy in Vegetal Production Department - Plant Protection FCA/UNESP, multiplied for four generations in greenhouse environment in Guarapuava - PR city. The experiment I was developed in Guarapuava - PR city, the experimental delineation used was randomized blocks, with five repetitions, the doses were applied in covering, divided: 20% of the dose when the plants have 5 or 6 leaves, 30% when the plants have 7 or 8 leaves and 50%, after the bulbs differentiation, the harvest occurred 149 days after planting. The experiment II was developed in Saint Juliana - MG city, using randomized blocks delineation with six repetitions, the doses were applied in covering divided: 25% of the dose when the plants have 3 or 4 leaves, 25% when the plants have 5 or 6 leaves and 50%, after the bulbs differentiation, the harvest occurred 112 days after planting. The increasing doses of N had not influenced the growth of the plants, the average length of garlic plants at differentiation was 92,8 cm in the experiment I and 88,9 cm in the experiment II. The values of relative index of chlorophyll (IRC) were increased with the rise of the N doses only at differentiation and 15 days after, for both experiments. About macronutrients concentration in leaves, in experiment I the doses of N had influenced in N, K and S absorption, and in experiment II, in N absorption and in the reduction of the P, K and S absorption. The micronutrients absorption was influenced only in experiment II, for the nutrients B, Mn and Zn...(Complete abstract click electronic access below)

Submitted by (edna.saturno@ufrpe.br) on 2017-04-11T16:02:29Z No. of bitstreams: 1 Elton Lima Santos.pdf: 629199 bytes, checksum: 4a4e2175cbcad51125cd66f7f4ae9359 (MD5) / Made available in DSpace on 2017-04-11T16:02:30Z (GMT). No. of bitstreams: 1 Elton Lima Santos.pdf: 629199 bytes, checksum: 4a4e2175cbcad51125cd66f7f4ae9359 (MD5) Previous issue date: 2010-02-23 / The research was developed in Laboratory of Ponderal Evaluation in aquatic Animals (LaAqua), located in the Department of Fisheries and Aquaculture of the UFRPE, with the aim to study the effect of the natural promoter growth additive in feed for Nile tilapia, through the evaluation of the digestibility of the dry matter, crude protein and crude energy of the rations, the performance and the morfometry of the mucosa gut of Nile tilapia. The used natural additives had been the alcoholic extract of propolis and garlic powder in the levels of 0.0, 0.5, 1.0 and 1.5 % and 0.0, 1.0, 2.0 and 3.0% of inclusion in the ration, respectively, arranged in a design randomized with four treatments and six repetitions. In the performance, that was carried through in aquariums of 70L, in a system of closed water circulation with the use of biofilter, the profit of weight, apparent alimentary conversion, consumption of apparent ration, hepatossomatic index, protein efficiency tax, viscerosomatic fat index, digestives somatic index and the morfometry of the mucosa gut had been evaluated, on the basis of the intestinal villus height. In the digestibility experiment had been used 320 juveniles of Nile tilapia with initial average weight of 20,00 ±5,0g The rations hadbeen supplied “ad libitum” (of 45 in 45 minutes) in small portions, in the period of the 8:00 to 17:00 h. The excrements had been collected in the deep one of the aquariums, daily, to each six hours. In the condition where the experiment was carried through, the use of the alcoholic extract of propolis that as additive promoter of growth for Nile tilapia, did not imply in improvement in the performance, however it provided one better energy exploitation of the ration. The garlic powder as additive in rations for youthful the Nile tilapia, did not provide positive effect as promotional of growth, in the studied levels. / Os experimentos foram conduzidos no Laboratório de Avaliação Ponderal em Animais Aquáticos (LaAqua), localizado no Departamento de Pesca e Aqüicultura da UFRPE, com o objetivo de estudar os efeitos da adição de aditivos naturais promotores de crescimento na alimentação de tilápia do Nilo, através da avaliação da digestibilidade da matéria seca, da proteína e da energia das rações, do desempenho produtivo e da morfometria da mucosa intestinal de tilápia do Nilo. Os aditivos naturais utilizados foram o extrato alcoólico de própolis e alho em pó nos níveis de 0,0; 0,5; 1,0 e 1,5,% e 0,0; 1,0; 2,0 e 3,0 % de inclusão na ração, respectivamente, arranjados num delineamento inteiramente casualizado com quatro tratamentos e seis repetições, tanto para digestibilidade, quanto para o desempenho. No desempenho, que foi realizado em aquários de 70L, em um sistema de circulação fechada de água com a utilização de biofiltro, foram avaliados o ganho de peso, conversão alimentar aparente, consumo de ração aparente, índice hepato-somático, taxa de eficiência protéica, índice de gordura viscero-somática, índice digestivo-somático e amorfometria da mucosa intestinal, com base na altura das vilosidades. No experimento de digestibilidade foram utilizados 320 juvenis de tilápia do Nilo com peso médio inicial de 20,00 ± 5,0g. As rações foram fornecidas “ad libitum” (de 45 em 45 minutos) em pequenas porções, no período das 8h às 17h. As fezes foram coletadas no fundo dos aquários, diariamente, a cada seis horas. Nas condições em que foram realizados os experimentos, pode-se concluir que o uso do extrato alcoólico de própolis, como aditivo promotor de crescimento para tilápia do Nilo, não implicou em melhora no desempenho, porém, proporcionou um melhor aproveitamento energético da ração. O alho em pó, como aditivo em rações para juvenis de tilápia do Nilo, não proporcionou efeito positivo como promotor de crescimento, nos níveis estudados.

Intimal hyperplasia is associated with graft failure and vascular sutures in the first year after surgery and in postangioplasty restenosis. Allium sativum (common garlic) lowers cholesterol and has antioxidant effects; it also has antiplatelet and antitumor properties and, therefore, has great potential to reduce or inhibit intimal hyperplasia of the arteries. Attempts have been made to inhibit intimal hyperplasia of the arteries with cilostazol and other drugs. Therefore, it is important to address the following research question: what is the difference between the mean of post-angioplasty myointimal thickening in the iliac artery of rabbits treated with A. sativum and that treated with cilostazol? Our objective is to determine the mean difference of post-angioplasty myointimal hyperplasia in the external iliac artery of rabbits with induced atherosclerosis, and compare the outcome between treatment with A. sativum and treatment with cilostazol. This is a randomized preclinical trial study conducted in experimental animals. We used female New Zealand rabbits (Oryctolagus cuniculus), submitted to an atherosclerotic diet and angioplasty of the external iliac artery. The animals were divided into the following groups (n = 10 each) according to treatment: group A, A. sativum, 800 μg⋅kg-1⋅day-1, orally; group C, cilostazol, 50 mg/day, orally; group S, 10 mL of 0.9% physiological saline solution, orally. Our primary variable is the difference of the mean myointimal hyperplasia. The secondary variable is the mean plate thickness of the arterial wall. Complementary data include frequency of limb ischemia, limb loss frequency, frequency of hematoma or bruising, frequency of infection, frequency of bleeding, frequency of animal death, and mean of lipid levels. The sample size was arbitrated in 30 rabbits. Statistical analysis was performed by using ANOVA and Tukey tests, as well as the χ2 test. We calculated the 95% confidence interval (CI) for each point estimate, and the P value was set as < 0.05. Group S had a mean hyperplasia rate of 35.74% (95% CI, 31.76–39.71%); group C, 16.21% (95% CI, 13.36–19.05%); and group A, 21.12% (95% CI, 17.26–25.01%); P < 0.0001. In conclution the Allium sativum quite as effective in inhibiting myointimal hyperplasia compared with those treated with cilostazol. / A hiperplasia intimal está relacionada à falha de enxertos e suturas vasculares no primeiro ano pós-cirurgia e nas reestenoses pós-angioplastia. O Allium sativum (alho comum) reduz o colesterol e tem efeito antioxidante, antiplaquetário e antitumoral; logo havia grande possibilidade de reduzir ou inibir a hiperplasia da íntima das artérias, a qual sua inibição vem sendo tentada com o Cilostazol e outros tratamentos. Sendo assim é relevante responder a pergunta de pesquisa: qual a diferença de média de hiperplasia miointimal pós-angioplastia na artéria ilíaca de coelhos tratados com Allium sativum comparada aos tratados com Cilostazol? O objetivo é determinar a diferença de média de hiperplasia miointimal pós-angioplastia na artéria ilíaca externa de coelhos com aterosclerose induzida e tratados com Allium sativum comparada aos tratados com Cilostazol. O estudo foi um ensaio pré-clínico aleatório em animais de experimentação por 35 dias. Os animais foram coelhos fêmeas (Oryctolagus cuniculus), da linhagem Nova Zelandia, submetidos à dieta ateroslcerótica e à angioplastia da artéria ilíaca externa direita. Os animais foram divididos em: Grupo A (n=10) coelhos tratados com a Allium sativum (800 μg/kg/dia), em doses diárias, por via oral. Grupo C (n=10): coelhos tratados com Cilostazol em doses diárias de 50 mg/dia, por via oral. Grupo S (n= 10) coelhos tratados com 10 mL de soro fisiológico 0,9%, por via oral, que foi nosso controle negativo. A variável primária foi a diferença de frequência da média de hiperplasia miointimal. Variáveis secundárias: a média de espessura da placa na parede arterial. Dados complementares: a frequência isquemia do membro, a frequência de perda do membro, a frequência de hematoma ou equimose, a frequência de infecção, a frequência de sangramento, a frequência de morte do animal e a média dos níveis lipídicos. O tamanho da amostra foi arbitrado em 30 coelhos. A análise estatística foi realizada com o teste ANOVA, Qui-quadrado e Tukey. Foi calculado o intervalo de confiança de 95% para cada ponto estimado. Os resultados foram: grupo S teve um índice médio de hiperplasia de 35,74% IC de 95% (31,76% a 39,71%); grupo C teve um índice médio de hiperplasia de 16,21% IC de 95% (13,36% a 19,05%); grupo A teve um índice médio de hiperplasia de 21,12% IC de 95% (17,26% a 25,01%); com P < 0,0001. Sendo assim, o Allium sativum tem a mesma eficácia na inibição da hiperplasia miointimal comparada aos tratados com Cilostazol

Philosophiae Doctor - PhD / Fusarium verticil/ioides (Sacc) Nirenberg a common phytopathogen of maize and maize-based products produces fumonisin B (FB) mycotoxins that have been related to several diseases such as equine leukoencephalomalacia (ELEM), porcine pulmonary edema (PPE), liver toxicity in several animals and esophageal and liver cancer in humans. In one of our studies we hypothesize that aqueous extracts of indigenous South African wild garlic species (Tulbaghia violacea, T. alliacea and T. simmleri) may enhance the efficacy of the fungicides, SporekilPu, Thiram, Itraconazole and Fluconazole against F. verticil/ioides (MRC 826). Data analysis from in vitro results indicates that for the 16 different mixtures of each plant extract and fungicide combination, several significantly (P<O.05) higher growth inhibition responses were produced. More synergistic interactions were observed for the combinations of sporekill with T. violacea (62%) and T. alliacea (75%) than for T. simmleri (25%) .. Mixtures between the azole fungicides and T. simmleri produced 94 % synergistic interactions. Combination of fungicides and plant compounds offers the opportunity to find synergistic mixtures and may validate disease control strategies with increased biological activity and low dose rate application. Modulation studies of hepatic drug metabolizing enzymes and oxidative properties of AI/ium sativum, Tulbaghia violacea and T. alliacea in male Fischer rats were also evaluated. Due to its complex phytochemical composition a battery of assays were used to evaluate antioxidant potential. The extracts exhibited no adverse effects in the liver and kidneys of the rats. Total plasma iron was not affected showing no evidence for iron catalyzed lipid peroxidation. An increase was noted in hepatic ORAC values for rats consuming T. violacea and T. al/iacea. However, no correlation was observed between the phenolic intake by the rats and the increased hepatic ORAC levels. In this study, pre-treatment with aqueous extracts of T. violacea, T. al/iacea and A. sativum resulted in a significant elevation in GSH levels, induction of GST -IJ and UDP-GT and modulation of CAT and SOD. This modulated oxidative status and phase II drug metabolizing enzymes in the liver may protect the liver against the adverse effects related to oxidative damage and mutagenesis. The chemoprotective properties of crude aqueous extracts of A. sativum, T. violacea and T. alliacea were investigated on preneoplastic foci formation promoted by culture material of F. verticil/ioides MRC 826 utilizing diethylnitrosamine (DEN) as cancer initiator. Clinical chemical parameters related to liver and kidney function and decreased body weight gain suggesting that severe, acute liver injury had been induced in the positive control (DEN-eMF) rats, while the levels were mostly reduced by the garlic treatments. This study further indicates that T. alliaceae (2 % w/v) and A. sativum (1% w/v) treatment suppressed GST-P+ foci formation with the modulation of GST-0 phase II detoxification enzymes, as well as the antioxidant enzyme, SOD (T. alliaceae) and decreased GSH levels as being possible mechanisms of protection. These results provide new evidence showing the modulation of phase II drug metabolizing enzymes and the oxidative status in the liver of rats by the wild garlic species as well as A. sativum.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2018-09-14T17:56:45Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_EssentialOilsComponents.pdf: 4263439 bytes, checksum: c1189e55f12d63ca94f4a519200746ce (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2018-09-14T17:57:50Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_EssentialOilsComponents.pdf: 4263439 bytes, checksum: c1189e55f12d63ca94f4a519200746ce (MD5) / Made available in DSpace on 2018-09-14T17:57:50Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_EssentialOilsComponents.pdf: 4263439 bytes, checksum: c1189e55f12d63ca94f4a519200746ce (MD5) Previous issue date: 2017-03-31 / Óleos essenciais derivados do metabolismo secundário de plantas podem ter atividades contra nematóides das galhas, Melodogyne incognita. Devido à necessidade de nematicidas mais eficientes e menos tóxicos para o homem e para o ambiente do que os disponíveis para o controle de Meloidogyne incognita, assim, neste trabalho objetivou-se estudar os óleos essenciais de Cinnamomum cassia e de Allium sativum, que foram descritos como ativos in vitro contra o nematóide Bursaphelenchus xylophilus. O óleo essencial de ambas as espécies foi obtido por hidrodestilação, dissolvidos em solução aquosa de Tween 80 a 0,01 g mL-1 e testado in vitro contra M. incognita. A 62 μg mL-1 as emulsões dos óleos foram mais ativas sobre ovos e juvenis de segundo estagio (J2) do que o Carbofuran a 173 μg mL-1. De acordo com a análise de cromatografia gasosa-espectrometria de massas, o constituinte majoritário de C. cassia é o (E)-cinamaldeído (83,3%), enquanto para o óleo de alho foram trisulfeto de dialila (66.7%) e dissulfeto de dialila (21.3%). Esses constituintes explicaram a atividade nematicida in vitro do óleo essencial de cada espécie de planta. A emulsão do óleo de C. cassia (500 μg mL-1), (E)-cinamaldeído (416 μg mL-1) e alho (250 μg mL-1) reduziram o numero de galhas e de ovos em plantas de soja e tomateiro, respectivamente, a valores estatisticamente iguais àquele obtido pelo Carbofuran (416 μg mL-1). Vapores dos óleos essenciais e do (E)-cinamaldeído foram ativos tanto quanto o nematicida fumigante Basamid usado nos ensaios in vitro contra M. incognita. A infectividade e a reprodução de M. incognita em plantas de soja e de tomate cultivadas em substrato infestado artificialmente com ovos do nematóide e tratado com 0,2 mL (alho), 1,0 mL (E)-cinamaldeído e 0,25 g de Basamid foram estatisiticamente iguais entre si na redução da população do nematoide. Esses resultados inequivocamente provam a atividade nematicida dos óleos essenciais e da substância (E)-cinamaldeído contra M. incognita, portanto, tanto o óleo quanto o (E)-cinamaldeído são muito promissores ao desenvolvimento de novos nematicidas fumigantes para o controle de nematóides em plantas de soja e de tomate. / Essential oils derived from secondary plant metabolism may have activities against root-knot nematodes, Meloidogyne incognita. Because nematicides that are more efficient and less toxic to humans and the environment than those available are desirable to control this pathogen, this work aimed at studying the essential oil of Cinnamomum cassia and Allium sativum, which were described as active in vitro against the nematode Bursaphelenchus xylophilus. The essential oils of both species were obtained by hydrodistillation, initially dissolved in aqueous solution of Tween 80 at 0.01 g mL-1 and tested in vitro against M. incognita. At 62 μg mL-1 the oil emulsions were more active on eggs and second stage juveniles (J2) than Carbofuran at 173 μg mL-1. According to gas chromatography-mass spectrometry analysis, the major constituent of C. cassia is (E) -cinnamaldehyde (83.3%), while for garlic oil were diallyl trisulfide (66.7%) and diallyl disulfide (21.3%). These constituents explained the in vitro nematicidal activity of the essential oil of each plant species. The emulsion of the C. cassia (500 μg mL-1), E)-cinnamaldehyde (416 μg mL-1) and garlic (250 μg mL -1) reduced the number of galls and eggs in roots soybean and tomatoes to values statistically equal to those obtained with Carbofuran at 415 μg mL-1. Vapors from the essential oils and (E)-cinnamaldehyde were active as much as the basamid fumigant nematicide used in the in vitro assays against M. incognita. Infectivity and reproduction of M. incognita in soybean and tomato plants grown on substrate artificially infested with nematode eggs and treated with 0.2 mL (garlic), 1.0 mL (E)-cinnamaldehyde and 0.25 g of Basamid were statistically equal among themselves in reducing the nematode population. These results unequivocally proofs of the nematicidal activity of the essential oils and of the (E)-cinnamaldehyde substance against M. incognita, therefore, both oil (E)-cinnamaldehyde are very promising to the development of new fumigant nematicides for the control of nematodes in soybean and tomato plants. / UFPA/Altamira

Lectins are a class of proteins that bind to carbohydrates with a high degree of specificity. They are involved in various cellular processes such as, host - pathogen interactions, targeting of proteins within cells, cell - cell interaction, cellular segregation and development. They serve as important tools for probing the carbohydrate structures in biological systems such as cell membranes and also as model systems for elucidating protein - carbohydrate interactions. Lectins are distributed ubiquitously in nature ranging from microorganisms to the plants and animals. Plant lectins are a group of proteins that according to a recently updated definition comprise all plant proteins possessing at least one non-catalytic domain that binds reversibly to specific mono- or oligosaccharide. The majority of all currently known plant lectins may be classified into four major groups - (1) Legume lectins, (2) Chitin-binding lectins, (3) Type 2 Ribosome inactivating proteins and the (4) Monocot mannose binding lectins. The monocot mannose binding lectins are an extended superfamily of structurally and evolutionarily related proteins. Till now these proteins have been isolated from the following families, namely, Amaryllidaceae, Affiaceae, Araceae, Orchidaceae, Iridaceae and Li/iaceae. They exhibit marked sequence homology and a unique specificity for mannose. At present there is a wide interest in the monocot mannose-binding lectins because of: (1) their exclusive specificity towards mannose, (2) their anti - retroviral activity and (3) their potent entomotoxic properties. Of particular interest are lectins from the bulbs of garlic (Allium sativum) and ramson (A. ursinum), which contain more than one type of lectin. The first report of the presence of lectins in the bulbs of garlic {Allium sativum agglutinin, ASA) was made by Van Damme et al in 1991. Bulbs of garlic are known to accumulate two types of mannose binding lectins, the heterodimeric, ASAI and the hornodimeric, ASAII. Though these two lectins differ in the lengths of their polypeptide chains, they exhibit marked similarities with respect to their primary sequence, post translational modifications, serological properties, immunochemical attributes as well as carbohydrate binding properties. This thesis describes the successful cloning of the ASAI gene from the garlic genomic DNA and expression of the functional recombinant protein in insect cell lines. ASAI was subsequently characterized for its carbohydrate binding specificity by means of a sensitive enzyme based assay. Finer insights into this sugar binding topology of ASAI for its complementary ligands was obtained from the surface plasmon resonance studies. Lastly, the folding behaviour as well as an estimate of its conformational stability was investigated by differential scanning calorimetric and equilibrium solution denaturation studies. Chapter 1 provides a comprehensive review on lectins pertaining to their definition, historical background, occurrence in nature, three dimensional structure and architecture, modes of bonding, biological functions and implications as well as their applications in biomedical research. Chapter 2 describes the isolation and purification of the heterodimeric lectin, ASAI in two steps using affinity chromatography followed by gel filtration chromatography from the bulbs of garlic. The purified ASAI was then characterized for their serological, physico- and immuno-chemical properties by means of capillary electrophoresis, hemagglutination activity and generation of antisera against ASAI in rabbits. Chapter 3 revolves around the cloning of the gene encoding ASAI by PCR amplification from garlic genomic DNA. The authenticity of the ASA gene was established by means of gene sequencing, which in turn provided us with the primary sequence of this lectin. With the ASAI clone established innumerable attempts, as highlighted in the chapter, were made to express the functional protein in bacteria. All attempts yielded pure recombinant garlic lectin with no detectable activity. This prompted us to shift our efforts into expression of the recombinant protein in the baculovirus expression system using the Sf21 insect cell lines and the Autographa californica nuclear polyhedrosis virus (AcNPV). The choice of this system proved beneficial as we obtained functional recombinant garlic lectin with its hemagglutinating activity comparable to the native protein. Chapter 4 highlights the design of an elegant coupled enzyme-based colorimetric assay (Enzyme Linked Lectin Adsorbent Assay) for elucidation of the carbohydrate binding specificity of ASAI. This expansive and extensive study involved the assay of a wide range of mannooligosaccharides in order to gain an insight into the sugar binding details of ASAI. ASAI recognizes monosaccharides in the mannosyl configuration. The potencies of the ligands for ASAI is shown to increase in the following order: Mannobiose < Mannotriose Mannopentaose Man9 oligosaccharide. Mannononase glycopeptide (Man9GlcNAc2Asn), the highest oligomer studied exhibited the greatest binding affinity suggesting ASAI to possess a preference for cluster of terminal αl-2-linked mannosyl residues at the non-reducing end. This kind of exquisite specificity is unique in the lectins described so far. Among the glycoproteins assayed, invertase, soyabean agglutinin and ovalbumin displayed high binding affinity. Chapter 5 unravels the fine specificity of the mannose containing carbohydrate moieties for binding to ASAI with emphasis on their kinetics of binding. This has been achieved by invoking the principle of surface plasmon resonance allowing measurement of bimolecular interactions in real time. This investigation corroborates our earlier study about the special preference of garlic lectin for terminal a α1-2 linked mannose residues. Increase in binding propensity can be directly correlated to the addition of αl-2 linked mannose to the mannooligosaccharide at its non-reducing end. An analyses of these data reveals that the α1-2 linked terminal mannose on the α1-6 arm to be the critical determinant in the recognition of mannooligosaccharides by the lectin. While kI increases progressively from Man3 to Man7 derivatives, and more dramatically so for Man8 and Man9 derivatives, k-1 decreases relatively much less gradually from Man3 to Man9 structures. An unprecedented increase in the association rate constant for interaction with ASAI with the structure of the oligosaccharide ligand constitutes a significant finding in protein-sugar recognition. Chapter 6 deals with the thermal unfolding of ASAI, characterized by differential scanning calorimetry and circular dichroism which shows it to be highly reversible and can be defined as a two-state process in which the folded dimer is converted directly to the unfolded monomers (A2 2U). Moreover, its conformational stability has been determined as a function of temperature; GdnCl concentration and pH using a combination of thermal and isothermal GdnCl induced unfolding monitored by DSC, far-UV CD and fluorescence, respectively. Analysis of these data yielded the heat capacity change upon unfolding (∆CP) as also the temperature dependence of the thermodynamic parameters, namely, ∆G, ∆H, ∆S. The protein appears to attain a completely unfolded state irrespective of the method of denaturation. The absence of any folding intermediates suggests the quaternary interactions to be the major contributor to the conformational stability of the protein, which correlates very well with its X-ray structure. The final chapter summarizes the findings reported in the thesis.

Submitted by FÁBIO YOMEI TANAMATI null (fabioyomei@hotmail.com) on 2016-12-21T18:02:23Z No. of bitstreams: 1 Tese- Fábio Final.pdf: 1752648 bytes, checksum: f9b77e3ee591fdbadfb4e16551e1bfc3 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-12-22T13:34:23Z (GMT) No. of bitstreams: 1 tanamati_fy_dr_bot.pdf: 1752648 bytes, checksum: f9b77e3ee591fdbadfb4e16551e1bfc3 (MD5) / Made available in DSpace on 2016-12-22T13:34:23Z (GMT). No. of bitstreams: 1 tanamati_fy_dr_bot.pdf: 1752648 bytes, checksum: f9b77e3ee591fdbadfb4e16551e1bfc3 (MD5) Previous issue date: 2016-06-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O alho é uma hortaliça utilizada como condimento e como planta medicinal. O Brasil é um importante produtor/consumidor desta hortaliça, entretanto a produção nacional é insuficiente e portanto importa de quase 60% do alho consumido. Práticas precisas de manejo na produção e pós-colheita precisas podem resultar em economia na produção, aumento da produtividade e qualidade. Entre as práticas de manejo destaca-se a fertilização destaca-se pela influência direta na produção do alho e constituir um dos principais itens do custo de produção. Após a colheita a deterioração do alho minimamente processado (produto com maior valor agregado) pode ser diminuída através do armazenamento em temperatura controlada e atmosfera modificada. Com o objetivo de avaliar e documentar as melhores fontes e doses de nitrogênio e fontes potássio, bem como o melhor método para o armazenamento do alho minimamente processado foram conduzidos três ensaios que serão apresentados em três capítulos, sendo o primeiro intitulado “Fontes de nitrogênio e potássio sob a produtividade e nutrição mineral do alho”, o segundo capítulo intitulado “Produtividade e nutrição mineral do alho sob diferentes doses de nitrogênio e fontes de potássio”, e o terceiro capítulo intitulado “Efeito de diferentes temperaturas e atmosfera modificada sob as características qualitativas do alho minimamente processado”. Concluiu- se a) Diferentes fontes de N e K podem ser combinadas para a obtenção de máxima produção de alho comercial, b) o teor de nutrientes do alho não é influenciado pelas fontes de N e K ao ponto de constituir um fator crítico à produtividade do alho, c) Recomenda-se a dose 80 kg ha -1 de N como forma de obtenção de máxima eficiência do fertilizante e produtiva do alho vernalizado livre de vírus cv. Caçador, d) Não houve diferenças significativas na produção do alho acima da dose 80 kg ha -1 de N, a partir desta dose, o acúmulo de nutrientes no bulbo, de forma geral, foi inferior nos tratamentos K2SO4, e) baixa temperatura e atmosfera modificada contribuem significativamente para a manutenção da qualidade do alho minimamente processado, f) embalagem a vácuo mantém uma atmosfera consistentes em uma maior gama de temperaturas, g) as atmosferas modificadas com alto teor de CO2 podem influenciar a diminuição do valor de L* durante o armazenamento, h) a pungência do alho é mais bem conservada em embalagens a vácuo. / Garlic is used as a spice and as a medicinal plant, Brazil is an important producer/ consumer of this herb, however small domestic production stimulates import of about 60% the garlic consumed in this country. Management practices in the production and accurate post-harvest can result in savings in production and increasing in yield and quality. Among the management practices, fertilization is distinguished by its direct influence on the production of garlic and constitute one of the main cost items of production and postharvest deterioration of minimally processed garlic (product with higher added value) can be reduced by storing in controlled and modified atmosphere temperature. In order to assess and document the best sources and doses of nitrogen and potassium sources, as well as the more suitable storage method for fresh-peeled garlic we carried out three tests that will be presented in three chapters, the first entitled "Yield and mineral nutrition of vernalized virus free garlic in response to nitrogen and potassium sources topdressing" the second chapter entitled "Yield and mineral nutrition of garlic in response to nitrogen doses and potassium sources topdressing”, "Quality changes in fresh-peeled garlic cloves in relation to storage temperatures and modified atmospheres". From these assays we concluded that a) Different sources of N and K may be combined to obtain maximum production of commercial garlic, b) the garlic mineral content is not influenced by the sources of N and K to be considered a critical factor to garlic productivity, c) 80 kg ha -1 of N is the best way to obtain both maximum fertilizer efficiency and productive of virus free vernalizated garlic cultivar Caçador, d) from the dose 80 kg ha -1 , the lowest accumulation of nutrients in the bulb in SKO treatments suggests that potassium sulfate, alleviates possible effects of toxicity caused by excess NH4 + and reduces minerals export for bulb and thereby the ground, e) both low temperature and modified atmosphere significantly contribute to the maintenance of the quality of fresh-peeled garlic, f) vacuum packaging maintains an atmosphere consistent across a wider range of temperatures, g) modified atmospheres with high CO2 content can influence the decrease in L* value during storage, h) garlic pungency is better preserved in vacuum packaging.

Resumo: Foram desenvolvidos dois experimentos com o objetivo de avaliar o crescimento, estado nutricional e produtividade de plantas de alho livres de vírus, em função de doses crescentes de nitrogênio. Os tratamentos foram 0, 20, 40, 80, 160, e 320 kg N ha-1, utilizando, como fonte, nitrato de amônio. Foi utilizada a cultivar Caçador L. V. (livre de vírus), os bulbilhos foram obtidos mediante cultura de tecido e termoterapia no Departamento de Produção Vegetal - Defesa Fitossanitária FCA / UNESP, multiplicados por quatro gerações em telado, no município de Guarapuava - PR. No experimento I, conduzido no município de Guarapuava - PR, foi utilizado o delineamento experimental de blocos ao acaso, com cinco repetições, com doses aplicadas em cobertura, na proporção de 20% da dose, quando as plantas apresentavam de 5 a 6 folhas; 30% quando apresentavam de 7 a 8 folhas; e 50% após a fase de diferenciação dos bulbos. A colheita ocorreu 149 dias após o plantio. O experimento II foi conduzido no município de Santa Juliana - MG, utilizando o delineamento de blocos ao acaso, com seis repetições, com doses aplicadas em cobertura, na proporção de 25% da dose, quando as plantas apresentavam de 3 a 4 folhas; 25% com 5 a 6 folhas; e 50% após a fase de diferenciação dos bulbos. A colheita ocorreu 112 dias após o plantio. As doses crescentes de N não influenciaram o crescimento das plantas, a média do comprimento na diferenciação foi de 92,8 cm para o experimento I e de 88,9 cm para o experimento II. Os valores do índice relativo de clorofila (IRC) foram aumentados com a elevação das doses de N apenas na fase de diferenciação e 15 dias após, para ambos os experimentos. Em relação aos teores foliares dos macronutrientes, no experimento I as doses de N influenciaram na absorção de N, K e S; já no experimento II, na absorção de N e na diminuição da absorção ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Two experiments were developed with the objective to evaluate the growth, plant nutrition and productivity of garlic without virus, submitted to nitrogen fertilization. The treatments used were 0, 20, 40, 80, 160, and 320 kg N ha-1, using as source ammonium nitrate. The cultivar used was Caçador L.V. (free of virus), the cloves were gotten through tissue culture and thermotherapy in Vegetal Production Department - Plant Protection FCA/UNESP, multiplied for four generations in greenhouse environment in Guarapuava - PR city. The experiment I was developed in Guarapuava - PR city, the experimental delineation used was randomized blocks, with five repetitions, the doses were applied in covering, divided: 20% of the dose when the plants have 5 or 6 leaves, 30% when the plants have 7 or 8 leaves and 50%, after the bulbs differentiation, the harvest occurred 149 days after planting. The experiment II was developed in Saint Juliana - MG city, using randomized blocks delineation with six repetitions, the doses were applied in covering divided: 25% of the dose when the plants have 3 or 4 leaves, 25% when the plants have 5 or 6 leaves and 50%, after the bulbs differentiation, the harvest occurred 112 days after planting. The increasing doses of N had not influenced the growth of the plants, the average length of garlic plants at differentiation was 92,8 cm in the experiment I and 88,9 cm in the experiment II. The values of relative index of chlorophyll (IRC) were increased with the rise of the N doses only at differentiation and 15 days after, for both experiments. About macronutrients concentration in leaves, in experiment I the doses of N had influenced in N, K and S absorption, and in experiment II, in N absorption and in the reduction of the P, K and S absorption. The micronutrients absorption was influenced only in experiment II, for the nutrients B, Mn and Zn...(Complete abstract click electronic access below) / Orientador: Leonardo Theodoro Büll / Coorientador: Marcelo Agenor Pavan / Banca: Rosemary Marques de Almeida Bertani / Banca: Francisco Maximino Fernandes / Mestre