Heavy metals in the environment are a source of some concern because of their potential reactivity, toxicity, and mobility in the soil. Soil contamination by metals is placing human and environmental health at risk through possible contamination of food chain. / Soil washing can be used to remove metals from the soil. Chemical treatment involves the addition of extraction agents that react with the contaminant and leach it from the soil. The liquid, containing the contaminants, is separated from the soil resulting in a clean solid phase. Six chelating reagents, EDTA, Citric acid, ADA, DTPA, SCMC, and DPTA, were employed to determine the relative extraction efficiencies of the six chelating reagents for the target metals. Recycling of chelating reagent was the main interest of this study. The experiments were divided into four parts: (1) preliminary studies on the preparation and characterization of soil that included grinding, sieving, soil texture measurements, total metals content post digestion and the distribution of metals in different soil fractions as well as (2) a comparison of the extraction efficiencies of six chelating reagents toward Cu, Pb, Zn, Fe, and Mn. Additionally, the chelating reagent was liberated and recycled by treatment of the metal-complexes with disodium diethyl dithiocarbomate (DEDTC). Additionally, supercritical CO2 was used to extract metal-DEDTC complexes using various surfactants to maintain the metal-DEDTC complexes in suspension. Finally, (4) magnesium metal was evaluated as an alternative method for liberating the water-soluble chelating reagent from the complex so as to be able to recycle this reagent as well. / The different approaches were promising in terms of recycling the chelating reagents that suggests a means of optimizing the experimental conditions in future applications.

Thesis (Ph.D.)--Murdoch University, 2005. / CD-ROM contains appendix G. Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 205-218.

Thesis (Ph. D.)--Lehigh University, 1996. / Includes vita. Includes bibliographical references.

Orientador: Sônia Maria Alves Bueno / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-26T10:22:21Z (GMT). No. of bitstreams: 1 Silva_LuanaCristinaAndradeda_D.pdf: 3401459 bytes, checksum: 94b29258ef88b106fed9841a38d11161 (MD5) Previous issue date: 2014 / Resumo: As imunoglobulinas do tipo G humana (IgG) são amplamente empregadas em tratamentos terapêuticos contra câncer, doenças auto-imunes e imunodeficiência adquirida e na área analítica, em testes diagnósticos. Os fragmentos Fab podem ser obtidos por clivagem enzimática da molécula de IgG nativa ou por tecnologia de DNA recombinante. O fragmento Fab é composto por regiões constantes e variáveis, sendo que as regiões variáveis de cadeias leve e pesada compõem o fragmento Fv. Tanto os fragmentos Fab quanto os fragmentos Fv foram citados como segunda e terceira geração de anticorpos, respectivamente, e têm sido crescente o interesse em empregá-los em tratamentos terapêuticos, visto que ambos possuem menor massa molar em relação a IgG intacta e preservam a capacidade de se ligar ao antígeno. Como a utilização destas proteínas nas áreas terapêutica e analítica requer um alto grau de pureza, estratégias de purificação vêm sendo desenvolvidas. No intuito de contribuir para o desenvolvimento de processos de purificação de fragmento Fab, estudou-se o emprego de ligantes alternativos aos bioespecíficos proteínas A, G e L, comumente empregados para este fim. Neste trabalho, a purificação dos fragmentos Fab foi realizada por cromatografia de afinidade com íons metálicos imobilizados (IMAC) e fenil boronato (FB). Quanto a IMAC, avaliou-se o efeito dos agentes quelantes IDA (ácido iminodiacético) e TREN (Tris 2(aminoetil) amina), os íons metálicos Cu2+, Ni2+ e Co2+ e os tampões Tris-HCl, fosfato de sódio e HEPES na ausência e na presença de NaCl para a purificação de Fab a partir de solução de clivagem enzimática de IgG humana policlonal. Os resultados foram analisados conjuntamente por Western blot e imunodifusão radial. No que se refere à pureza dos fragmentos Fab obtidos na etapa de flowthrough, a ordem crescente obtida foi TREN-Ni2+ (100%) > IDA-Ni2 (88,6%) > IDA-Co2+ (71,1%) > IDA-Cu2+ (63,9%). Em relação ao rendimento, obteve-se a seguinte ordem: TREN-Ni2+ (86,2%) = IDA-Co2+ (86,2%) > IDA-Ni2+ (74,3%) = IDA-Cu2+ (73,7%). No tocante à capacidade de adsorção de proteína total por mL de gel, obteve-se a ordem 14,3 mg mL-1 ( TREN-Ni2+) > 11,2 mg mL-1 (IDA-Ni2+) > 5,65 mg mL-1 (IDA-Cu2+) > 4,3 mg mL-1 (IDA-Co2+). O ligante FB adsorveu os fragmentos Fab e Fc e não promoveu a separação destes. Este trabalho possibilitou concluir que os agentes quelantes IDA e TREN com os íons metálicos empregados, possibilitam separar os fragmentos Fab de fragmentos Fc e obtê-los com grau de pureza satisfatório / Abstract: Polyclonal human G immunoglobulins are widely applied in therapeutic treatments for cancer, autoimmune diseases and immunodeficiency and in the analytical area of diagnostic testing. The human IgG Fab fragments can be obtained via enzymatic cleavage of the native molecule or recombinant DNA technology. The Fab is composed of constant regions (CL and CH) and variable (VL and VH). The variable regions of the light and heavy chains comprise the Fv fragment. Fab fragments and Fv fragments were cited as second and third generation of antibodies, respectively, and has been an increasing interest in employing them in therapeutic treatments, as both have molecular weight less than the intact IgG and preserve the affinity for the antigen. The applied these proteins in therapy and analytical investigation require a high degree of purity, so, different purification strategies have been developed. In order to contribute to the development of Fab fragment purification processes, we studied the use of alternative biospecific binding to proteins A, G and L, commonly applied for this purpose. In this work, the purification of the Fab fragments was performed by affinity chromatography with chelated metals (IMAC) and phenyl and boronate (PB) ligand immobilized on agarose gel. In this investigation, the metal chelates Cu 2+, Ni 2+ and Co 2+ were studied when tested in IDA (iminodiacetic acid) and TREN (Tris 2 (aminoethyl) amine) and buffer system Tris-HCl, sodium phosphate and HEPES with or without NaCl for purification of Fab fragments from enzymatic cleavage solution of polyclonal human IgG. The results of Western blot and radial immunodiffusion were analyzed together. For the purity of the Fab fragments obtained in flowthrough, the increasing order was obtained TREN-Ni2+ (100%) > IDA-Ni2+ (88.6%) > IDA-Co2+ (71.1%) > IDA-Cu2+ (63.9%). In relation to yield, we obtained the following order: TREN-Ni2+ (86.2%) = IDA-Co2+ (86.2%) > IDA-Ni2+ (74.3%) = IDA-Cu2+ (73.7% ). With regard to the total capacity protein adsorption per ml of gel, was obtained the order 14.3 mg mL-1 (TREN-Ni2+) > 11.2 mg mL-1 (IDA-Ni2+) > 5.65 mg ml -1 (IDA-Cu2+) > 4.3 mg ml-1 (IDA-Co2+). The binder FB adsorbed Fab and Fc did not cause separation thereof. This work led us to conclude that the chelating agents IDA and TREN with the metal ions used, enable separate the Fab fragments of Fc fragments and get them with satisfactory degree of purity / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutora em Engenharia Quimica

Man has had an intense interest in the role of enzymes in biological reactions for many years. The basic question--how enzymes are able to function so efficiently--remains as yet largely unanswered. Only in recent years has it been discovered that molybdenum is essential for certain biological processes. For example, traces of molybdenum added to certain soils have resulted in spectacular plant growth (1). Four enzymes are known to contain molybdenum: nitrate reductase (2), hydrogenase (3), xanthine oxidase (4), and aldehyde oxidase (5). In each of these enzymes riboflavin is also present as the coenzyme FAD (flavin adeninedinucleotide). Tocatlian, in 1960, showed that a strong complex containing two molybdenum atoms per riboflavin molecule is formed in solution. However, he was unable to determine the formation constants (6).

The problems of phosphate fertilization have attracted the attention of agronomists, nutritionists, and other biological scientists due to the importance of phosphorus in animal and plant nutrition. However, the fundamental behavior of phosphorus in soil systems is not well understood. Because of its far-reaching effects, a solution of a portion of this problem is of importance.

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