O uso de microrganismos como promotores de crescimento vegetal tem se apresentado como uma alternativa promissora, sobretudo para a ciclagem de nutrientes, especialmente o fósforo. Dessa forma, objetivou-se avaliar o papel da diversidade microbiana na disponibilização do fósforo, oriundo de fontes de diferente solubilidade, para as plantas de milho no ambiente rizosférico. A hipótese é que quanto mais diverso for o microbioma da rizosfera e do solo, maior será a disponibilidade de fósforo para as plantas e por conseguinte melhor o seu desenvolvimento. O experimento foi instalado em casa de vegetação utilizando a metodologia de diluição para extinção. Foram estabelecidos três fontes de fósforo (superfosfato triplo, fitato de cálcio e fosfato de Araxá) em um gradiente de diversidade microbiana, correspondentes às diluições 10-1, 10-3, 10-6 e 10-9. Adicionalmente foi mantido um tratamento controle sem adição de fósforo (P) e todas as análises compreendiam o ambiente solo e rizosfera. O papel da diversidade microbiana frente a disponibilidade de P foi acessado por meio de metodologias dependentes de cultivo (BIOLOG) e metodologias independente de cultivo (qPCR e PCR-DGGE). As plantas adubadas com fosfato de Araxá apresentaram menor teor de P foliar e os sintomas mais severos de deficiencia de P (raquitismo e arroxeamento), sobretudo nas maiores diluições (menor diversidade). O percentual de P-lábil em relação ao P-total foi bastante variável entre as diluições dentro de cada fonte e ambiente avaliado. Entretanto, o tratamento adubado com fosfato de Araxá apresentou menor proporção de P-lábil (18-55%). Em relação ao gene phoD, no ambiente rizosfera as comunidades menos diversas apresentaram maiores números de cópia nos tratamentos, indicando que a abundância deste gene pode ter sido enriquecida nos tratamentos menos diversos. A atividade enzimática (principalmente para a fosfatase em pH 6,5) diminuiu frente a redução da diversidade da comunidade microbiana. A abundância do gene phoD e a atividade da fosfatase em pH 6,5 se relacionaram apenas no ambiente rizosférico. A estrutura e organização da comunidade bacteriana (gene rrs) e solubilizadora de fósforo (phoD) diferiram entre as diluições (R>0,75; p<0,05). Em relação ao perfil de consumo de fontes de carbono, as fontes avaliadas apresentaram perfis distintos entre si. Foi possivel observar que as fontes de fósforo foram mais atuantes do que o gradiente de diversidade no processo de disponibilidade de P para as plantas. Isso pode estar relacionado à redundância funcional da microbiota. / The use of microorganisms as promoters of plant growth has been presented as a promising alternative, especially for the cycling of nutrients, especially phosphorus. The objective of this study was to evaluate the role of microbial diversity in the availability of phosphorus from sources of different solubility for maize plants in the rhizospheric environment. The hypothesis is that as more diverse the rhizosphere and soil microbiome, the greater will be the availability of phosphorus to the plants and therefore better their development. The experiment was installed in a greenhouse using the dilution methodology for extinction. Three sources of phosphorus (triple superphosphate, calcium phytate and Araxá phosphate) were established in a microbial diversity gradient, corresponding to the dilutions 10-1, 10-3, 10-6 and 10-9. In addition, a control treatment was maintained without addition of P and all analyzes comprised the soil and rhizosphere environment. The role of microbial diversity against P availability was accessed through culture - dependent methodologies (BIOLOG) and independent culture methodologies (qPCR and DGGE). Araxá phosphate fertilized plants had lower leaf P content and P deficiency symptoms (rickets and purplishness), especially in the higher dilutions (lower diversity). The percentage of P-labile in relation to the P-total was quite variable between the dilutions within each source and evaluated environment. However, the Araxá phosphate fertilizer treatment had a lower proportion of P-labile (18-55%). In relation to the phoD gene, in the environment rhizospherein the less diverse communities presented higher copy numbers in the treatments, indicating that the abundance of the gene may have been enriched in the less diverse treatments. Enzymatic activity (mainly for phosphatase at pH 6.5) decreased as the diversity of microbial community decreased. The abundance of the phoD gene and phosphatase activity at pH 6.5 were related only in the rhizosphere environment. The structure and organization of the bacterial community (16S rRNA) and phosphorus solubilizer (phoD) differed at the dilutions (R> 0.75; p <0.05). The consumption of carbon source was variable in relation to evaluated treatments. It was possible to observe that the phosphorus sources were more active than the diversity gradient in the P availability process for the plants. This may be related to the functional redundancy of the microbiota.

A solubilização de compostos não iônicos em micelas de detergentes é uma propriedade intensamente estudada e de grande importância prática. Algumas das aplicações onde a compreensão da solubilização é relevante são detergência, recuperação de solos contaminados e catálise. Em trabalho anterior, o grupo do prof. Quina demostrou que o modelo de relações lineares de energia livre (LSER) é uma poderosa ferramenta para correlação da eficiência de solubilização com parâmetros relacionados com a estrutura do soluto. No presente trabalho, constantes de incorporação micelar, Ks, foram determinadas experimentalmente para uma série de solutos de diferentes estruturas para os detergentes comerciais decil óxido de difenila dissulfonado (DOWFAX C10) e hexadecil óxido de difenila dissulfonado (DOWFAX C16). Verificou-se, para esses detergentes, que a variação do log Ks com o volume molar, Vx, do soluto é distinta para classes homólogas de solutos polares (álcoois e cetonas) e apolares (alcanos, aromáticos e alcinos), indicando a existência de microambientes de solubilização distintos A demonstração, neste trabalho, de que o modelo de LSER não é apropriado para correlacionar dados de Ks, na presença de mais de um microambiente de solubilização, aumenta a validade de estudos prévios, com detergentes de estrutura mais simples, onde a suposição de um único microambiente de solubilização mostrou-se adequada. / The solubilization of non ionic molecules in detergent micelles is a property that has been extensively investigated and that is of great practical importance. Several applications in which solubilization is of relevance are detergency, remediation of contaminated soils and catalysis. In earlier work, the group of Prof. Quina has shown that linear free energy relationships (LSER) are a powerful tool for correlating the efficiency of solubilization with the structure of the solute. In the present work, micellar incorporation coefficients, Ks, were determined for solubilization of a series of solute of diverse structures in two commercial detergents: decyl diphenyl oxide disulfonate (DOWFAX C10) and hexadecyl diphenyl oxide disulfonate (DOWFAX C16). For these detergents it is shown that the dependence of log Ks on solute molar volume is distinct for homologues series of polar (alcohols and ketones) and non-polar (alkanes, alkynes and aromatics) solutes, implying the existence of distinct solubilization microenvironments for these two classes of solutes. The demonstration in the present work that the LSER approach fails to correlate Ks data when more than one solubilization microenvironment is present reinforces the validity of previous studies with detergents of simpler structure where the assumption of a single solubilization microenvironment was found to be adequate.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A solubilidade do fÃrmaco em Ãgua à um fator importante para sua eficÃcia. Entretanto, a maioria dos fÃrmacos apresenta baixa solubilidade em Ãgua. Para resolver este problema, copolÃmeros em bloco com propriedades surfactantes tÃm sido bastante investigados. O objetivo deste trabalho à investigar os copolÃmeros F87 (E62P39E62), E43B14E43 e suas misturas 50/50 a 90/10, com proporÃÃo crescente do copolÃmero mais hidrofÃbico (E43B14E43), a sÃrie de diblocos EmBn para carrear o fÃrmaco modelo griseofulvina atravÃs de diferentes mÃtodos de solubilizaÃÃo e estudar a sua liberaÃÃo, bem como o estudo da solubilizaÃÃo dos fÃrmacos quercetina e mangiferina nos copolÃmeros E65G5 e E65G7E65 visando suas aplicaÃÃes no carreamento e liberaÃÃo controlada de fÃrmacos. Utilizou-se a tÃcnica de inversÃo de tubo para o estudo das propriedades geleificantes dos copolÃmeros F87, E43B14E43 e suas misturas 50/50 a 90/10. Para a determinaÃÃo da concentraÃÃo micelar crÃtica (CMC) utilizou-se o mÃtodo de solubilizaÃÃo de corante medida por fluorescÃncia. A solubilidade dos fÃrmacos griseofulvina, quercetina e mangiferina nos sistemas micelares foi medida por espectrofotometria UV-Vis e para os sistemas dos copolÃmeros E65G5 e E65G7E65 utilizou-se a cromatografia lÃquida de alta eficiÃncia (HPLC). Os copolÃmeros puros e os sistemas contendo os fÃrmacos foram caracterizados por tÃcnicas de espectroscopia de absorÃÃo na regiÃo do infravermelho (FT-IR), espectroscopia RAMAN (FT-RAMAN), raios-X, tamanho de partÃcula por espalhamento de luz dinÃmico (Zetasizer), e experimentos de liberaÃÃo in vitro foram realizados para alguns dos sistemas. Os copolÃmeros F87, E43B14E43, suas misturas, os diblocos da sÃrie EmBn e os copolÃmeros E65G5 e E65G7E65 sÃo promissores para administraÃÃo de fÃrmacos hidrofÃbicos, pois apresentaram bons valores de capacidade de solubilizaÃÃo (Scp). Os copolÃmeros E65G5 e E65G7E65 foram os que mais se destacaram na solubilizaÃÃo da quercetina (HPLC): seu sistema micelar aumentou a solubilidade aquosa da quercetina em 264 vezes a 25 ÂC em ambos os copolÃmeros e 118 e 135 vezes a 37ÂC, no dibloco e tribloco, respectivamente, e suas partÃculas apresentaram total eficiÃncia de encapsulaÃÃo da quercetina. Portanto, o dibloco de glicidil (E65G5) à o melhor copolÃmero para encapsulamento de fÃrmaco. / Most drug candidates fail to progress in their formulations due to its low solubility in water. To resolve this issue, block copolymers with surfactant properties have been well investigated. The objective of this study was to investigate the copolymers F87 (E62P39E62), and mixtures thereof E43B14E43 50/50 to 90/10, with increasing proportion of the more hydrophobic copolymer (E43B14E43), the series of diblock EmBn to carry the model drug through different griseofulvin solubilization methods and study their release, and the study of drug solubilization of quercetin and mangiferin E65G5 the copolymers and their applications in order E65G7E65 Entrainment and controlled release of drugs. We used the technique of inversion of the tube for the study of gelling properties of copolymers F87, and mixtures thereof E43B14E43 50/50 to 90/10. To determine the critical micelle concentration (CMC) used the dye solubilization method measured by fluorescence. The solubility of drugs griseofulvin, quercetin and mangiferin in micellar systems was measured by UV-Vis spectrophotometry and systems of copolymers E65G5 E65G7E65 and used the high performance liquid chromatography (HPLC). The pure copolymers and the systems containing the drugs were characterized by infrared spectroscopy (FT-IR), Raman spectroscopy (FT-Raman), X-rays, particle size by dynamic light scattering (Zetasizer), and experiments in vitro release were carried out for some systems. The copolymers F87, E43B14E43, their mixtures, the number of diblock copolymers and EmBn E65G5 E65G7E65 and are promising for administration of hydrophobic drugs, because they showed good solubilising capacity values &#8203;&#8203;(Scp). The copolymers were E65G7E65 E65G5 and those who stood out in solubilization of quercetin (HPLC): its micellar system increased the aqueous solubility of quercetin in 253 times at 25  C for both copolymers and 124 and 142 times at 37  C for the diblock and triblock, respectively, and its particles showed complete encapsulation efficiency of quercetin

O fósforo é um elemento-chave para a manutenção da vida, desde os processos de transferência energética (ATP) até a estruturação das cadeias de ácidos nucléicos. Frequentemente é o que mais limita o desenvolvimento vegetativo em função da alta reatividade do PO43- na solução do solo. Mais de 80% do P adicionado ao solo pode ser imobilizado na forma de compostos de baixa labilidade, adsorvendo-se as superfícies das argilas e dos oxi-hidróxidos de Fe3+ e Al3+, processo irreversível em condições naturais. A solubilização do P não lábil às plantas é possível mediante a liberação de ácidos orgânicos sintetizados por micro-organismos nativos do solo, os quais também contribuem efetivamente com a mineralização do P orgânico através da produção de enzimas (fosfatases), entretanto, esses mecanismos ainda são pouco conhecidos. O principal objetivo desse estudo foi correlacionar as possíveis mudanças nas frações orgânicas (Po) e inorgânicas (Pi) de fósforo no solo rizosférico de plantas de cana-de-açúcar com a estrutura e a diversidade do seu microbioma. Para tanto, as plantas foram cultivadas durante 180 dias em diferentes microcosmos com fosfato monoamônico (MAP), superfosfato triplo (SFT) e fosfato natural reativo (NAT), tanto na ausência quanto na presença da torta de filtro. As diferentes frações de fósforo no solo rizosférico foram correlacionadas com os dados moleculares baseados no sequenciamento parcial dos amplicons do gene 16S rRNA obtidos do DNA total extraído desses solos. Aos 60 dias, houve uma maior diversidade geral de bactérias, sendo um período com altos teores de P-lábil na rizosfera, beneficiando principalmente as bactérias de hábitos copiotróficas (r-estrategistas): divisão Halophaga/Acidobacterium, Chloracidobacteria e Bacilli. A diminuição da diversidade aos 120 dias ocorreu como resposta da redução dos teores de P-lábil na rizosfera, beneficiando mais as bactérias oligotróficos (k-estrategistas): Actinobacteria e Proteobacterias. A associação da torta de filtro com o MAP revelou fortes indícios do aumento do processo de mineralização na superfície do solo, uma vez que houve rápida liberação de P lábil para a rizosfera. Isso teria aumentado a atividade de micro-organismos envolvidos na mineralização da torta de filtro, havendo forte correlação com o aumento da população de &beta;-Proteobacteria aos 120 dias. Neste período, as Proteobacterias foram mais abundantes nos solos com NAT+torta. Isso explicaria o incremento de P moderadamente lábil desse tratamento aos 180 dias, pois esse filo possui várias espécies capazes de solubilizar o P ligado ao Ca, presente nos fosfatos de rocha. No geral, a torta de filtro alterou a solubilização e disponibilidade do P nas fontes minerais de modo diferente, contribuindo com os incrementos tanto das frações de Po como as de Pi. Esses resultados demonstraram que as bactérias participam ativamente dos processos de disponibilização do P de fontes minerais no solo. Nessas condições, a torta de filtro pode modular essas transferências, em benefício às plantas, por estimular a atividade de micro-organismos solubilizadores de P e mineralizadores da matéria orgânica do solo. / Phosphorus is a key element for the maintenance of life from the energy transfer processes (ATP) to the structuring of chains of nucleic acids. Often, it is more limited vegetative growth due to the high reactivity of PO43- in the soil solution. Up to 80% of P added to the soil can be immobilized as low-labile compounds adsorbing to the clay surfaces and oxy-hydroxides of Fe3+ and Al3+, irreversible process under natural conditions. Solubilization of non-labile P by plants may be carried out for organic acids synthesized by native soil microorganisms, which also effectively contribute for mineralization of organic P through the production of enzymes (phosphatases) although these mechanisms are still poorly understood. The main objective of this study was to correlate the changes in the organic (Po) and inorganic (Pi) fractions of P in the rhizosphere of sugarcane plants with the structure and diversity in their microbiome. Therefore, the plants were grown for 180 days in different microcosms with monoammonium phosphate (MAP), triple superphosphate (TSP) and rock phosphate (NAT), both in absence and in presence of the filter cake. The different P fractions in the rhizosphere soil were correlated with the molecular data based on the partial sequencing of 16S rRNA gene amplicons obtained from total DNA extracted from soils. At 60 days, there was a greater overall diversity of bacteria, it is a period with high labile-P in the rhizosphere, especially benefiting the copiotrophic bacteria (r-strategists): Halophaga/Acidobacterium division, Chloracidobacteria and Bacilli. The decrease in diversity at 120 days occurred in response to the reduction of labile-P content in the rhizosphere, more benefiting oligotrophic bacteria (k-strategists): Actinobacteria and Proteobacteria. The association filter cake with MAP showed strong evidence of increased mineralization process on the soil surface, since there was rapid release of labile P to the rhizosphere. It would have increased the activity of microorganisms involved in the mineralization of the filter cake with a strong correlation with increase in &beta;-Proteobacteria population at 120 days. Then, Proteobacteria were more abundant in soils with NAT+filtercake at 120 days. That would explain the increases in moderately labile P content this treatment at 180 days, because this phylum has several species capable of solubilizing the P linked to Ca present in rock phosphates. In general, filter cake changed solubilization and availability of P in mineral sources of different ways, contributing to the increments of Po and Pi fractions. These results showed that bacteria participate in the processes that afford phosphorus in the soil. Under these conditions, the filter cake can modulate these transfers in beneficial to plants by stimulating the activity of P solubilizing microorganisms and mineralizing the soil organic matter.

Made available in DSpace on 2017-07-10T17:37:13Z (GMT). No. of bitstreams: 1 Katia Kabroski Andrioli.pdf: 1857849 bytes, checksum: 6d9dcc608c1be92e28d7a708d3503ce6 (MD5) Previous issue date: 2016-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Quinoa is a food crop that has received great importance for the high nutritional value in proteins and amino acids. Endophytic bacteria inhabit the interior of plant tissues and promote direct and indirect benefits to plants. The association between quinoa and endophytic bacteria has not been studied. The aim of this study was to evaluate the occurrence of endophytic bacteria with potential for plant growth promotion associated with quinoa plants. The quinoa seeds were seeding in arable and forest soil in Entre Rios do Oeste and Toledo. The isolation was done 40 days after the seed germination, using quinoa roots as baits. The 10-2 to 10-7 dilutions were plated on solid media and inoculated into semi-solid media, both semi-specific and N-free. From the semi-solid media, the count was performed employing the method of the Most Probable Number (MPN). The phosphate solubilization test and the indol-acetic acid (IAA) phytohormone production in the isolates were performed to characterize the plant growth promoting. We obtained 130 colonies of endophytic bacteria, these, seven from the semi-solid media. The MPN ranged from not detected to 1,5x105cells per g-1 root. We observed a large morphological diversity from the phenotypic characterization and there was the formation of 14 groups to 70% of dissimilarity. All bacteria produced IAA in greater or lesser amount with the range of 0.90 to 63.42 μg mg-1 of protein. However, only 40% of the isolates showed phosphate solubilization halo. The morphophysiological characterization enabled to group the isolates with similar traits and the growth promotion tests have identified promising isolates for field tests / A quinoa é um alimento que tem recebido grande importância devido a seu alto valor nutricional em proteínas e aminoácidos. Bactérias endofíticas vivem no interior dos tecidos de plantas e promovem benefícios diretos e indiretos para as plantas. Em quinoa, a associação das bactérias endofíticas ainda não tinha sido estudada. O objetivo deste trabalho foi avaliar a ocorrência de bactérias endofíticas com potencial de promoção de crescimento vegetal associadas a plantas de quinoa. As sementes de quinoa foram semeadas em solo agricultável e de floresta nos municípios de Entre Rios do Oeste e Toledo. O isolamento foi realizado aos 40 dias após a germinação das sementes, utilizando-se raízes de quinoa como iscas. Diluições de 10-2 a 10-7 foram plaqueadas em meios sólidos e inoculadas em meios semissólidos, ambos semi-seletivos e isentos de nitrogênio. A partir dos meios de cultura semissólidos ainda, foi realizada a contagem empregando-se o método do Número Mais Provável (NMP). O teste de solubilização de fosfato e produção do fitohormônio ácido indolacético (AIA) dos isolados foi realizado para caracterização da promoção de crescimento vegetal. Foram obtidas 130 colônias de bactérias endofíticas, destas, sete a partir dos meios semissólidos. O NMP variou de não detectado a 1,5x105 células g-1 de raiz. Da caracterização fenotípica observou-se grande diversidade morfológica com a formação de 14 grupos a 70% de dissimilaridade. Todas as bactérias produziram AIA em maior ou menor quantidade, com variação de 0,90 a 63,42 μg mg-1 de proteína. No entanto, apenas 40% dos isolados apresentou halo de solubilização de fosfato. A caracterização morfofisiológica possibilitou agrupar os isolados de traços semelhantes e os testes de promoção de crescimento identificaram os isolados promissores para testes a campo

A baixa disponibilidade de fósforo (P) na maioria dos solos brasileiros faz com que a produção agrícola seja altamente dependente do uso de fertilizantes fosfatados, os quais geralmente apresentam baixa eficiência agronômica em solos tropicais. Esses fertilizantes são produzidos a partir das rochas apatíticas, um recurso natural não renovável. Em função disso, cientistas alertam para a possibilidade das reservas chegarem ao fim em breve. Diante dessa preocupação, tem-se aumentado a procura por fertilizantes de maior eficiência. Assim, o objetivo deste trabalho foi produzir fertilizantes fosfatados mais eficientes que os atuais solúveis presentes no mercado. Para tanto, foram conduzidos dois estudos; o primeiro avaliou a eficiência agronômica de 12 novos fosfatos obtidos a partir da acidulação parcial (25 e 50% da acidulação total) de fosfatos naturais (FNs) de Araxá (FNA), de Bayóvar (FNB) e de Marrocos (FNM) contendo argilas pilarizadas ou zeólitas na sua formulação, por meio de avaliação de difusão, de solubilização e de cultivo de milho. A incubação em placas de petri por cinco semanas avaliou a difusão e disponibilidade do P proveninte dos novos produtos em solo arenoso e com baixo teor inicial de P. O solo foi amostrado com anéis concêntricos a partir do grânulo aplicado no centro da placa. Independentemente do fertilizante avaliado, a maior parte do P permaneceu no próprio grânulo ou na região próxima a ele, sendo que nenhum dos produtos resultou em maior difusão e disponibilidade de P quando comparados ao superfosfato triplo (SPT). A solubilidade dos fertilizantes foi avaliada em colunas de lixiviação em solo de textura média durante 60 dias, sendo periodicamente coletada solução percolada e amostrado o solo em camadas no final. Foi detectado a presença de P na solução percolada em todos os tratamentos, a exceção dos FNs puros e do controle. Os maiores teores de P percolado no perfil foram com SPT, seguido dos fosfatos reativos (FNB e FNM) com 50% de acidulação contendo zeólitas na formulação. Todos os fertilizantes aumentaram os teores de P lábil e moderadamente lábil no perfil do solo. No experimento de casa de vegetação avaliou-se a eficiência agronômica relativa (EAR) dos fertilizantes, durante 45 dias de cultivo de milho. As fontes produzidas a partir dos fosfatos reativos (FNB e FNM) com 50% de acidulação foram as que se mostraram mais promissoras, com EAR maior que 74% quando comparadas com o SPT. No segundo estudo avaliou-se a difusão e solubilidade de nove produtos, sendo três FNs (FNA, FNB e FNM) misturados a três proporções de substâncias húmicas - SHs (2,5; 5 e 10% (m/m)). Devido a pouca variação observada entre os FNs em ensaios laboratoriais (similares ao estudo um), no cultivo do milho foi avaliado apenas o efeito da associação do FN de Marrocos (FNM) com as SHs. Os resultados comprovaram que a associação dos FNs com as SHs não é uma alternativa viável, devido aos baixos índices de difusão e solubilização e a baixa EAR dessas fontes na produção do milho. / The low availability of phosphorus (P) in most Brazilian soils makes agricultural production highly dependent on the use of phosphate fertilizers, which generally presents low agronomic efficiency in tropical soils. These fertilizers are produced from apatite rocks, a natural non-renewable resource. As a result, scientists warn of the possibility that the reserves will come to an end soon. Concerning about that, there is increased demand for more efficiently fertilizers. Thus, this work aimed to produce phosphate fertilizers more efficient than the solubles ones currently on the market. Therefore, two studies were conducted; the first evaluated the agronomic efficiency of 12 new phosphates obtained from partial acidulation (25 and 50% of total acidulation) of rocks phosphates (RPs) from Araxá (RPA), Bayóvar (RPB) and Morocco (RPM) containing pillarized clays or zeolites in their formulation, by means of evaluating the diffusion, solubilization and corn cultivation. The incubation in petri dishes for five weeks evaluated the diffusion and availability of the P coming from the new products in sandy soil with low initial P content. The soil was sampled with concentric rings from the granule which was applied in the center of the petri dishes. Regardless of the fertilizer, most of the P remained in the granule itself or in the region close to it, and none of the products resulted in greater diffusion and availability of P when compared to triple superphosphate (TSP). The solubility of the fertilizers was evaluated in leaching columns in medium texture soil for 60 days, being periodically collected the solution percolated in the column and it was sampled the soil by layers in the end. The presence of P in percolated solution was detected in all treatments, except for the pure RPs and control. The highest levels of P percolated in the profile were with TSP, followed by the reactive phosphates (RPB and RPM) with 50% of acidification containing zeolites in the formulation. All fertilizers increased the levels of labile and moderately labile P in the soil profile. In the greenhouse experiment the relative agronomic efficiency (RAS) of the fertilizers was evaluated during 45 days of corn cultivation. The sources produced from the reactive phosphates (RPB and RPM) with 50% of acidulation were the most promising, with a RAS greater than 74% when compared to TSP. In the second study the diffusion and solubility of nine products were evaluated, being three RPs (RPA, RPB and RPM) mixed in three proportions of humic substances (HSs) (2,5; 5 and 10% (w/w)). Due to the little variation observed among RPs in laboratory tests (similar to the study one), in maize cultivation it was only evaluated the effect of Morocco source (RPM) with the HSs. The results proved that the association of RPs with HSs is not a viable alternative, due to the low diffusion and solubilization indexes and the low RAS of these sources in corn production.

A baixa disponibilidade de fósforo (P) na maioria dos solos brasileiros faz com que a produção agrícola seja altamente dependente do uso de fertilizantes fosfatados, os quais geralmente apresentam baixa eficiência agronômica em solos tropicais. Esses fertilizantes são produzidos a partir das rochas apatíticas, um recurso natural não renovável. Em função disso, cientistas alertam para a possibilidade das reservas chegarem ao fim em breve. Diante dessa preocupação, tem-se aumentado a procura por fertilizantes de maior eficiência. Assim, o objetivo deste trabalho foi produzir fertilizantes fosfatados mais eficientes que os atuais solúveis presentes no mercado. Para tanto, foram conduzidos dois estudos; o primeiro avaliou a eficiência agronômica de 12 novos fosfatos obtidos a partir da acidulação parcial (25 e 50% da acidulação total) de fosfatos naturais (FNs) de Araxá (FNA), de Bayóvar (FNB) e de Marrocos (FNM) contendo argilas pilarizadas ou zeólitas na sua formulação, por meio de avaliação de difusão, de solubilização e de cultivo de milho. A incubação em placas de petri por cinco semanas avaliou a difusão e disponibilidade do P proveninte dos novos produtos em solo arenoso e com baixo teor inicial de P. O solo foi amostrado com anéis concêntricos a partir do grânulo aplicado no centro da placa. Independentemente do fertilizante avaliado, a maior parte do P permaneceu no próprio grânulo ou na região próxima a ele, sendo que nenhum dos produtos resultou em maior difusão e disponibilidade de P quando comparados ao superfosfato triplo (SPT). A solubilidade dos fertilizantes foi avaliada em colunas de lixiviação em solo de textura média durante 60 dias, sendo periodicamente coletada solução percolada e amostrado o solo em camadas no final. Foi detectado a presença de P na solução percolada em todos os tratamentos, a exceção dos FNs puros e do controle. Os maiores teores de P percolado no perfil foram com SPT, seguido dos fosfatos reativos (FNB e FNM) com 50% de acidulação contendo zeólitas na formulação. Todos os fertilizantes aumentaram os teores de P lábil e moderadamente lábil no perfil do solo. No experimento de casa de vegetação avaliou-se a eficiência agronômica relativa (EAR) dos fertilizantes, durante 45 dias de cultivo de milho. As fontes produzidas a partir dos fosfatos reativos (FNB e FNM) com 50% de acidulação foram as que se mostraram mais promissoras, com EAR maior que 74% quando comparadas com o SPT. No segundo estudo avaliou-se a difusão e solubilidade de nove produtos, sendo três FNs (FNA, FNB e FNM) misturados a três proporções de substâncias húmicas - SHs (2,5; 5 e 10% (m/m)). Devido a pouca variação observada entre os FNs em ensaios laboratoriais (similares ao estudo um), no cultivo do milho foi avaliado apenas o efeito da associação do FN de Marrocos (FNM) com as SHs. Os resultados comprovaram que a associação dos FNs com as SHs não é uma alternativa viável, devido aos baixos índices de difusão e solubilização e a baixa EAR dessas fontes na produção do milho. / The low availability of phosphorus (P) in most Brazilian soils makes agricultural production highly dependent on the use of phosphate fertilizers, which generally presents low agronomic efficiency in tropical soils. These fertilizers are produced from apatite rocks, a natural non-renewable resource. As a result, scientists warn of the possibility that the reserves will come to an end soon. Concerning about that, there is increased demand for more efficiently fertilizers. Thus, this work aimed to produce phosphate fertilizers more efficient than the solubles ones currently on the market. Therefore, two studies were conducted; the first evaluated the agronomic efficiency of 12 new phosphates obtained from partial acidulation (25 and 50% of total acidulation) of rocks phosphates (RPs) from Araxá (RPA), Bayóvar (RPB) and Morocco (RPM) containing pillarized clays or zeolites in their formulation, by means of evaluating the diffusion, solubilization and corn cultivation. The incubation in petri dishes for five weeks evaluated the diffusion and availability of the P coming from the new products in sandy soil with low initial P content. The soil was sampled with concentric rings from the granule which was applied in the center of the petri dishes. Regardless of the fertilizer, most of the P remained in the granule itself or in the region close to it, and none of the products resulted in greater diffusion and availability of P when compared to triple superphosphate (TSP). The solubility of the fertilizers was evaluated in leaching columns in medium texture soil for 60 days, being periodically collected the solution percolated in the column and it was sampled the soil by layers in the end. The presence of P in percolated solution was detected in all treatments, except for the pure RPs and control. The highest levels of P percolated in the profile were with TSP, followed by the reactive phosphates (RPB and RPM) with 50% of acidification containing zeolites in the formulation. All fertilizers increased the levels of labile and moderately labile P in the soil profile. In the greenhouse experiment the relative agronomic efficiency (RAS) of the fertilizers was evaluated during 45 days of corn cultivation. The sources produced from the reactive phosphates (RPB and RPM) with 50% of acidulation were the most promising, with a RAS greater than 74% when compared to TSP. In the second study the diffusion and solubility of nine products were evaluated, being three RPs (RPA, RPB and RPM) mixed in three proportions of humic substances (HSs) (2,5; 5 and 10% (w/w)). Due to the little variation observed among RPs in laboratory tests (similar to the study one), in maize cultivation it was only evaluated the effect of Morocco source (RPM) with the HSs. The results proved that the association of RPs with HSs is not a viable alternative, due to the low diffusion and solubilization indexes and the low RAS of these sources in corn production.

Rinderpest virus is an enveloped Nonsegmented Negative Stranded RNA Virus (NNSV) belonging to the genus Morbillivirus in the Family Paramyxoviridae and the causative organism for “cattle plague”. The virion has a transport component and a replication component. The transport component consists of a lipid membrane with two external membrane-anchored glycoproteins, namely Hemagglutinin (H) and Fusion (F) proteins that are necessary for cell entry and release of newly formed virus particles. The replication component consists of viral genomic RNA encapsidated by the nucleoprotein (N) and a RNA polymerase complex (Large subunit L and phosphoprotein P). These two components are linked together by the matrix protein (M) that is believed to play a crucial role in the assembly and maturation of the virion particle by bringing the two major viral components together at the budding site in the host cell. To perform this function, M protein should be able to interact with the host cellular membrane, especially the plasma membrane in the case of Rinderpest virus, should be able to interact with itself to form multimers as well as with the nucleocapsid core. The function might include the interaction of M protein with the cytoplasmic tail of the other two envelope proteins namely F and H. To understand the role of matrix protein in Rinderpest virus life cycle, the following functions were characterized – 1) Matrix protein association with the host cell membrane. 2) Matrix protein association with nucleocapsid protein. Matrix protein association cellular membranes in rinderpest virus infected cells could be a result of its interaction with the cytoplasmic tails of the viral glycoproteins. Hence, this association was characterized in the absence of other viral proteins. In transiently transfected cells, M protein existed in two isoforms namely the soluble cytosolic form and membrane-bound form. The membrane-bound M protein associated stably with the membranes, most likely by a combination of electrostatic and hydrophobic interactions, which is inhibited at high salt or high pH, but not completely. Confocal microscopy analysis showed the presence of M protein in plasma membrane protrusions. When GFP was tagged with this protein, GFP was absent from nucleus and was present predominantly in the cytosol and the plasma membrane protrusions. However, M protein expression did not result in the release of membrane vesicles (Virus-like particles) into the culture supernatant implicating the requirement of other viral proteins in envelope acquisition. Matrix protein of RPV has been shown to co-sediment with nucleocapsid during mild preparation of RNP from virus-infected cells. This association was further investigated by virus solubilization. The matrix protein could be solubilised completely from virion only in the presence of detergent and high salt. This is in agreement with the previous observation from the laboratory that the purified matrix protein remained soluble in the presence of detergent and 1M NaCl. This suggested that M protein could oligomerise or associate with nucleocapsid. The purified M protein when visualized by Electron microscopy showed the presence of globular structures, which may be due to self association of M protein, which may be due to self-aggregation of M protein. The presence of GFPM in filamentous structures in transfected cells, as visualized by confocal microscopy could also be due to self-assembly of M protein. Interaction of matrix protein RPV nucleocapsid was confirmed using co- sedimentation and floatation gradient analysis. Results obtained from M-N binding assay using C-terminal deletions of nucleocapsid protein suggested that the matrix protein interacted with the conserved N-terminal core of nucleocapsid and non- conserved C-terminus 20% is dispensable. This is in agreement with the report that RPV M protein could be replaced with that of Peste-des-petits-ruminants virus(a closely related morbillivirus). The observation that the nucleocapsid protein interacts with both soluble and membrane-bound form suggests that the matrix protein can possibly interact itself to facilitate the assembly of replication component at the site of budding where the transport component is already assembled. Viral proteins of many RNA viruses interact with detergent-resistant host components that facilitate their transport inside the cell to the sits of assembly or replication. Rinderpest viral proteins acquire detergent resistance in infected cells. This acquisition is mediated by viral N protein. The relevance of this interaction in virus life cycle was studied using small molecule drugs that disrupt host cytoskeleton and lipid raft. The results obtained suggested that the host cytoskeleton, especially actin-filaments facilitate virus release from the plasma membrane. RPV matrix protein acquired detergent resistance in infected cells as well as in transfected cells. The pattern of detergent resistance suggested an association with the cytoskeleton or cytoskeleton associated proteins. However, results obtained from co-localisation studies in the presence of actin inhibitor and cold-ionic detergents are not consistent with the above observation. This property could be due to self-association of matrix protein.

The goal of this project was to develop a non-toxic amphiphilic diblock copolymer nanoparticulate drug delivery system that will solubilize paclitaxel (PTX) and retain the drug in plasma. Methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (MePEG-b-PCL) diblock copolymers loaded with PTX were characterized and their physicochemical properties were correlated with their performance as nanoparticulate drug delivery systems. A series of MePEG-b-PCL was synthesized with PCL blocks ranging from 2-104 repeat units and MePEG blocks of 17, 44 or 114 repeat units. All copolymers were water soluble and formed micelles except MePEG₁₁₄-b-PCL₁₀₄, which was water insoluble and formed nanospheres. Investigation of the effects of block length on the physicochemical properties of the nanoparticles was used to select appropriate copolymers for development as PTX nanoparticles. The critical micelle concentration, pyrene partition coefficient and diameter of nanoparticles were found to be dependent on the PCL block length. Copolymers based on a MePEG molecular weight of 750 g/mol were found to have temperature dependent phase behavior. Relationships between the concentration of micellized drug and the compatibility between the drug and core-forming block, as determined by the Flory-Huggins interaction parameter, and PCL block length were developed. Increases in the compatibility between PCL and the drug, as well as longer PCL block lengths resulted in increased drug solubilization. The physicochemical properties and drug delivery performance characteristics of MePEG₁₁₄-b-PCL₁₉ micelles and MePEG₁₁₄-b-PCL₁₀₄ nanospheres were compared. Nanospheres were larger, had a more viscous core, solubilized more PTX and released it slower, compared to micelles. No difference was seen in the hemocompatibility of the nanoparticles as assessed by plasma coagulation time and erythrocyte hemolysis. Micellar PTX had an in vitro plasma distribution similar to free drug. The majority of micellar PTX associated with the lipoprotein deficient plasma fraction (LPDP). In contrast, nanospheres were capable of retaining more of the encapsulated drug with significantly less PTX partitioning into the LPDP fraction. In conclusion, although both micelles and nanospheres were capable of solubilizing PTX and were hemocompatible, PTX nanospheres may offer the advantage of prolonged blood circulation, based on the in vitro plasma distribution data, which showed that nanospheres retained PTX more effectively.

The goal of this project was to develop a non-toxic amphiphilic diblock copolymer nanoparticulate drug delivery system that will solubilize paclitaxel (PTX) and retain the drug in plasma. Methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (MePEG-b-PCL) diblock copolymers loaded with PTX were characterized and their physicochemical properties were correlated with their performance as nanoparticulate drug delivery systems. A series of MePEG-b-PCL was synthesized with PCL blocks ranging from 2-104 repeat units and MePEG blocks of 17, 44 or 114 repeat units. All copolymers were water soluble and formed micelles except MePEG₁₁₄-b-PCL₁₀₄, which was water insoluble and formed nanospheres. Investigation of the effects of block length on the physicochemical properties of the nanoparticles was used to select appropriate copolymers for development as PTX nanoparticles. The critical micelle concentration, pyrene partition coefficient and diameter of nanoparticles were found to be dependent on the PCL block length. Copolymers based on a MePEG molecular weight of 750 g/mol were found to have temperature dependent phase behavior. Relationships between the concentration of micellized drug and the compatibility between the drug and core-forming block, as determined by the Flory-Huggins interaction parameter, and PCL block length were developed. Increases in the compatibility between PCL and the drug, as well as longer PCL block lengths resulted in increased drug solubilization. The physicochemical properties and drug delivery performance characteristics of MePEG₁₁₄-b-PCL₁₉ micelles and MePEG₁₁₄-b-PCL₁₀₄ nanospheres were compared. Nanospheres were larger, had a more viscous core, solubilized more PTX and released it slower, compared to micelles. No difference was seen in the hemocompatibility of the nanoparticles as assessed by plasma coagulation time and erythrocyte hemolysis. Micellar PTX had an in vitro plasma distribution similar to free drug. The majority of micellar PTX associated with the lipoprotein deficient plasma fraction (LPDP). In contrast, nanospheres were capable of retaining more of the encapsulated drug with significantly less PTX partitioning into the LPDP fraction. In conclusion, although both micelles and nanospheres were capable of solubilizing PTX and were hemocompatible, PTX nanospheres may offer the advantage of prolonged blood circulation, based on the in vitro plasma distribution data, which showed that nanospheres retained PTX more effectively.