Bioanalysis of Pteroyl Derivatives in Various Aspects of Human Health

This dissertation describes the development of bioanalytical strategies for a group of experimental and known therapeutic agents and chemically related essential substances. The array of analytes include the antifolate methotrexate, its various (in)active metabolites and related bio-conjugates, plus several forms of folic acid family, a group of essential substances not synthesized by mammalian species, but endogenously required in numerous biochemical processes. These analytes all share a common structural feature, the heterocyclic pteridine ring system. Analyte identity results from variations of the ptereridine-ring system redox state, a result of minor structural variations of the heterocyclic ring and changes in conjugation status (i.e. polyglutamated or nanoparticle conjugated). Specific and sensitive detection of the individual species was of high interest, as the various chemical forms of these substances may constitute biomarkers for individualizing low dose MTX therapy in autoimmune diseases such as JIA, but is also required for the (pre)clinical studies of novel nano-device MTX drug delivery systems. The dissertation presents state-of-the-art bioanalytical methodology suitable for the specific determination of any specific analyte in the presence of all other analytes. The strategy for each particular analyte possesses its own unique strength and weakness, with the final strategy collectively being based on the specific structure and associated physical-chemical property of a given analyte, the biological environment, and the clinical question to be answered

Nitrification and denitrification kinetics in the activated sludge process

Includes bibliography.The bi-substrate active-site death regeneration model, developed by Dold, Ekama and Marais (1980), to describe the aerobic activated sludge process, was extended to provide a reliable description of the behaviour of the nitrification-denitrification single sludge activated sludge process under constant, cyclic or unsteady conditions of flow and load. The extended model applies to series reactor systems containing aerated and non-aerated reactors and to single reactor systems having alternating aerated and non-aerated periods. The extension describing the behaviour in the anoxic state required no change in the basic equations describing the aerobic state. The numerical values of the kinetic constants in the anoxic state equations were found to be the same as those in the aerobic state equations, except for one: The value of the utilization rate constant for slowly biodegradable material in an anoxic environment is a fraction of approximately 0,38 of the value of this constant in an aerobic environment. Nitrification is affected in so far that growth of nitrifiers takes place in an aerobic environment, whereas death of nitrifiers takes place in both aerobic and anoxic environments. The existing empirical denitrification models (valid for constant flow and load conditions) could be deduced from the solutions of the basic equations. The empirical denitrification rate constants could be interpreted in terms of the expressions for utilization of easily and slowly biodegradable organic substrates in an anoxic environment. These constants were shown to have no fundamental kinetic significance; they are only apparent constants, the result of kinetic reactions which, fortuitously, show little variation under normal operational conditions

The Effects of Aluminium- and Ferric-Based Chemical Phosphorus Removal on Activated Sludge Digestibility and Dewaterability

The uses of Al3+ and Fe3+ salts in chemical phosphorus removal (CPR) in activated sludge plants have increased considerably in recent years and their full impacts on downstream processes such as dewaterability and digestibility are not fully understood. In this research, the effects of CPR on sludge digestibility and dewaterability were investigated in laboratory-scale experiments using sludge samples from a full-scale wastewater treatment plant. The results of the digestibility tests showed a 21% and 36% reduction in the biogas volume generated during anaerobic digestion of surplus activated sludge at 0.1 g/L doses of Al3+ and Fe3+ salts, respectively. This demonstrates that Al3+ dosing for CPR has less of a reduction effect compared with Fe3+ salts on biogas generation during anaerobic digestion of sludge. The dewaterability tests showed that primary sludge dewaterability was improved by up to 25% by Fe3+ and 16% by Al3+, while that of surplus activated sludge was reduced by 64% and 73%, respectively, at a metal salt dose of 50 mg/L. Consequently, a pre-precipitation process during CPR where phosphorus is removed in the primary tank would, therefore, enhance sludge dewaterability

Structure, function and operational stability of peroxidases

This PhD project was started in 1995 and was supported by the dutch Ministry of Economic Affairs through the programme "IOP Catalysis". The main goal of "IOP catalysis" is to obtain clean and more efficient technologies, to improve the quality of the Dutch fine chemistry. Biocatalysis provides a way to minimize non desirable side products, which can have a negative impact on the environment. The objective of this thesis was to investigate the potential of heme-containing peroxidases as natural biocatalysts for industrially relevant conversions. Peroxidases were chosen since they are able to operate under mild conditions using cheap and clean oxidants (hydrogen peroxide).In this work especially the natural production of food flavours was of interest. After initial screening of reactions of potential interest the N-dealkylation of methyl-N-methylanthranilate) (ex citrus leaves) to methylanthranilate (MA), a concord grape flavour was chosen as the mode) reaction in this thesis.In order to obtain better insight in the industrial applications of peroxidases, it is important to understand structure, function and operational stability of these catalysts. Therefore, this project was started by investigating the reaction mechanism of peroxidases in more detail ( chapter 3 ). Horseradish peroxidase (HRP) was used as the model peroxidase, as this enzyme is the best studied enzyme of all peroxidases. Many heme-containing biocatalysts, exert their catalytic action through the initial formation of so-called high-valent- iron-oxo porphyrin intermediates. For HRP the initial intermediate formed has been reported as a high-valent-iron-oxo porphyrinπ-radical cation, called compound I. A strongly hold concept in the field of peroxidase-type of catalysis is the irreversible character of the reaction leading to formation of this compound I. The results of chapter 3, however, indicate that formation of the high-valent-iron-oxo porphyrin intermediate for various heme-containing catalysts, including HRP, might be reversible. This reversible compound I formation results in heme-catalysed exchange of the oxygens of H 2 O 2 with those of H 2 O. The existence of this heme-catalysed oxygen exchange followed from the observation that upon incubation of 18 O labeled H 218 O 2 with heme-containing biocatalysts, significant loss of the 18 O label from the H 218 O 2 was observed, accompanied by the formation of unlabeled H 2 O 2 . Thus, for the heme biocatalysts studied, exchange of the oxygen of their high-valent-iron-oxo intermediate with that of water occurs rapidly. This observation implied the need for an update of the kinetic model for peroxidases. Revaluation and extension of the previous kinetic model for HRP showed the necessity to include several additional reaction steps, taking both reversible compound I formation and the formation of enzyme-substrate complexes into account As a consequence reactions with HRP are saturable, implying that V max and k cat can be measured. This provides possibilities for investigation of structure-activity relationships.Investigation of quantitative structure-activity relationships (QSARs) is a way to obtain more insight in the influence of the structure of a substrate on its conversion by an enzyme of interest. Moreover, QSARs could be of interest for industrial applications. With QSARs the outcomes of conversions may be predicted by simply calculating chemical parameters of structurally related substrates. In this way QSARs could be helpful in facilitating screening procedures for biocatalytic productions, saving time and money.In chapter 4 predictive computer calculation-based QSARs were defined by comparing second order rate constants for the oxidation of a series of model compounds by HRP compound II to computer calculated chemical parameters characteristic for this reaction step. The model compounds studied were a series of structurally related phenols. For the calculation of the chemical parameters characteristic for the reaction step two approaches were used. In the first approach a frontier orbital parameter of the substrate was calculated being the ionisation potential (i.e. minus the energy of their highest occupied molecular orbital (E(HOMO)). In the second approach the relative heat of formation (ΔΔHF) was calculated for the process of one electron abstraction as well as for H·-abstraction from the phenol derivatives. Assuming a reaction of the phenotic substrates in their non- dissociated, uncharged forms, clear correlations were obtained between the natural logarithm of the second order rate constants (ln k app and ln k 2 respectively ) for their oxidation by compound II and their calculated parameters.The computer calculation-based QSARs thus obtained for the oxidation of the various phenol derivatives by compound II from HRP indicate the validity of the approaches investigated, i.e. the frontier orbital approach but also the approach io which the process is described by calculated relative heats of formation. The results also indicated that outcomes from computer calculations on related phenol derivatives can be reliably compared to one another. Since both mechanisms considered, i.e. initial electron abstraction versus initial H·-abstraction provided clear molecular orbital QSARs the results could not be used to discriminate between these two possible mechanisms for phenol oxidation by HRP compound II. Furthermore, as the actual oxidation of peroxidase substrates by compound II is known to be the rate-limiting step in the overall catalysis by HRP, the QSARs described in chapter 4 may have implications for the differences in the overall rate of oxidation of the phenol derivatives by HRP. As a matter of fact similar QSARs should be obtained when the overall rate of oxidation of the respective compounds by HRP is determined.As mentioned above, reactions with HRP are saturable, implying that V max and k cat can be measured. Thus, in chapter 5 the overall conversion of phenols by HRP was investigated, resulting in overall k cat values. These saturating overall k cat values indeed correlated quantitatively with calculated ionisation potentials of the substrates. The observation that the rates and QSARs obtained for the overall rate of conversion in chapter 5 are similar to those described for the rate limiting reaction step in chapter 4, corroborates that phenol oxidation by compound II is the rate limiting step in the reaction cycle of HRP, but even more important, it also illustrates that QSARs for oxidation of substrates by compound II can be used for prediction of the overall rate of oxidation of phenol derivatives by HRP and vice versa. Moreover, QSARs for overall k cat , instead of for individual rate constants, eliminate the need for extensive rapid kinetic analysis to make predictions for HRP-based substrate conversions.Saturating overall k cat values for HRP catalysed conversion of a second series of substrates, i.e. a series of substituted anilines, described in chapter 5, also correlated quantitatively with calculated ionisation potentials of the substrates. However, in the QSAR plots, the correlations for the anilines were shifted to lower k cat values at similar ionisation potentials as compared to those for the phenols. To investigate whether differences in orientation of phenols and anilines within the active site of HRP may be a factor underlying the higher reactivity of the phenols than expected on the basis of their ionisation potential, 1 H NMR T 1 relaxation studies were performed, using 3-methylphenol aod 3-methylaniline as the model substrates. The 1 H NMR T 1 relaxation studies revealed consistently smaller average distances of the phenol than of the aniline protons to the paramagnetic Fe 3+ centre in HRP, may be resulting in differences io the electron transfer process from the aromatic donor substrate to the Compound II of HRP. A shorter distance between the phenol and the heme than between the aniline and the heme may be a factor contributing to the faster rate of electron transfer with phenol as compared to aniline substrates. However, the actual differences in orientation seem small when the difference in oxidation rate at similar calculated ionisation potential phenols or anilines is considered. Since (partial) deprotonation will largely influence, i.e. decrease the ionisation potential of the aromatic substrate, the relatively higher oxidation rates of phenols may be related to their larger extent of deprotonation upon binding to the substrate pocket of HRP, resulting in lower ionisation potentials than actually expected on the basis of calculations on their non-ionised form. Based on the 1 H NMR T 1 relaxation data of chapter 4 and literature data of Hendriksen et al. [1], we put forward the hypothesis that the differential substrate behaviour of phenols and anilines may be due to subtle differences in their binding to the active site substrate pocket of HRP, resulting in i) closer proximity to the heme and ii) larger extent of deprotonation for the phenols than for the aniline substrates. An important conclusion following from the results of chapter 5 is that for each type of reaction and substrate, different QSARs have to be obtained. It is also clear that further investigations are nessecary for succesful application of QSARs to industrial prediction of biocatalysis.Heme-based peroxidases are enzymes with a broad substrate specificity and are capable of catalysing a variety of reactions. However, operational (in)stability limits the use of peroxidases in industrial processes. Chapter 6 describes the possibillities and limitations for using commercially available SP preparations (delivered by QUEST) and other peroxidases (like HRP and microperoxidase-8 (MPS)) for the production of the food flavour methylanthranilate (MA) from methyl-N-methylanthranilate (MNMA). Because W from citrus leaves is a relatively cheap source and MA is more expensive than MNMA, the investigated reaction provides an industrially relevant route for the natural production of an important topnote flavour in concord grape.The tested soybean preparations varied widely with respect to their heme-content and, as a result, their activity for this reaction. Furthermore, the operational stability of purified soybean peroxidase (SP) was at least 25-fold lower than that of HRP and only 5-fold higher than that of MP8, a small peroxidase with a polypeptide chain of only eight amino acids covalently linked to the protoporphyrin IX cofactor. Therefore, the results with SP indicate that the presence of a large protein chain around a porphyrin cofactor in a peroxidase is, by itself, insufficient to explain the observed differences in operational stabifity, and that the inactivation mechanism could be intramolecular. In order to find an explanation for the low operational stability of SP and MP8, it would therefore be interesting to investigate, for example, the shielding of the meso-positions of the heme cofactors of the different peroxidases.All tested peroxidase preparations were able to catalyse the requested N-dealkylation. However, SP proved to be a very efficient biocatalyst for the production of MA with high yield and purity, in spite of its relatively low operational stability. This potential of SP to catalyse the N-demethylation of MNMA to MA more efficiently than HRP and MP8, was especially observed at high temperature and low pH values at which SP appeared to be optimally active. Unfortunately, the prices for MA had dropped by the time this efficient SP catalysed production of MA was developed. As a result the now defined way to produce MA using a peroxidase biocatalyst was no longer of commercial interest for the industrial IOP partner. Nevertheless, the results in chapter 6 clearly define the answer to the initial industrial aim of the preseot IOP project.Biotechnological applicationsPeroxidases, in principle, have remarkable synthetic possibilities, but commerciai processes based on these enzymes have not yet been developed. The inactivation of heme- enzymes by peroxides through oxidation of the porphyrin ring is one of the prime reasoos. Attempts to improve the stability have not been very successful yet. The enzyme stability can be improved by stepwise or continuous addition of the oxidaot, maintaining a low peroxide concentration [2,3]. Hiner et al. [4] tried to increase the resistance of HRP to H 2 O 2 through genetic engineering without satisfying results. Other ways to deal with the problem of poor stability were chemical modification, screening and immobilisation [5].A second reason why commercial processes based on peroxidases have not yet been developed is that the oxidation of some substrates by peroxidases is in competition with their spontaneous chemical oxidation by peroxides. As a consequence, reduction of the purity of products occurs in case enantioselectivity is the target [6]. Maintaming a low peroxide concentration throughout the reaction period, reducing background oxidation, has also been tried here as a solution.Furthermore, in the process of one-electron oxidation by peroxidases free radicals are produced which are difficult to control, thereby reducing the purity of products as well.A fourth reason for the limited scaiing up of peroxidase-catalysed reactions is the low water solubility of most of 8e substrates of synthetic interest. Designing strategies that enhance enzymatic activity in organic solvents [7,8], or the use of hydrophobic matrices that act as a reservoir for both substrates and products are solutions to this general problem.From the above it can be concluded that the problem of (operational) stability of peroxidases is a severe problem for industrial processes and that this problem is difficult to so)ve. On the other hand this thesis shows that a peroxidase exists that, in spite of its low operational stability, can be an efficient biocatalyst for the production of a industrially relevant compound with high yield and purity. Altogether, it can be concluded that peroxidases could represent an interesting tool for industrially relevant reactions, making future research on possibilities and limitations worthwhile.ReferencesHendriksen, A., Schuller, D.J., Meno, K., Welinder, K.G., Smith, A.T., Gaijhede, M. (1998) Biochem.37 : 8054-8060.Colonna, S., Gaggero, N., Casella, L., Carrea, G., Pasta, P., (1992) Tetrahedron asymmetry3 : 95-106.Van Deurzen, M.P.J., Van Rantwijk, F., Sheldon, R.A. (1996) J. Mol. Catal. B. Enzym.2 : 33-42Hiner, A.N.P., Hernandez-Ruiz, J., Arnao, M.B., Garcia-Canovas, F., Acosta, M. (1996) Biotechnol. Bioeng.50 : 655-662Bakker, M., Van de Velde, F., Van Rantwijk, F., Sheldon, R.A. (in preperation) Biotechnol. Bioeng.Colonna, S., Gaggero, N., Richelmi, C., Pasta, P. (1999) Tibtech.17 : 163-168Klibanov, A.M., (1997) Trends Biotechnol.15 : 97-101Osman, A.M., Boeren, S., Boersma, M.G., Veeger, C., Rietjens, I.M.C.M. (1997) Proc. Natl. Acad. Sci. USA94</ol

Influence of phase separator design on the performance of UASB reactors treating municipal wastewater

The objective of anaerobic sewage treatment is to maximize the fraction of influent organic material that is transformed into methane, thus minimizing the COD fractions that are discharged with the effluent or in the  excess sludge production. Experimental data in this paper show that in the case of application of a  UASB reactor for sewage treatment, the phase separator design has an important influence on digestion efficiency. An efficient phase separator leads to retention of a larger sludge mass, which means that the mean solids retention time is increased. The data show that the mean solids retention time or sludge age is the fundamental operational parameter that determines the efficiency of the anaerobic treatment. A simple way to improve the phase separator performance is to apply parallel plates in the settling section of the UASB reactor, above the conventional phase separator design of triangular prisms with an open base.Keywords: anaerobic sewage treatment, UASB reactor, phase separator design, performance optimization, sludge ag

A influência da participação em linha do utilizador na produção das webrádios: um estudo multicaso da Campina FM e da Rádio Comercial

Doutoramento em Informação e Comunicação em Plataformas DigitaisA presente investigação aborda a relação entre a participação em linha do utilizador e a produção de conteúdo na webrádio, tendo como objetivo geral investigar se e como a produção das webrádios é influenciada pela participação em linha dos utilizadores no contexto transmedia. A pesquisa é dividida em duas partes. A primeira é teórica, na qual aborda-se os conceitos fundamentais da investigação e a metodologia adotada para a recolha e análise dos dados. A segunda é prática e se refere às recolhas de dados feitas em duas emissoras (uma brasileira, a Campina FM, e outra portuguesa, a Rádio Comercial) e à apresentação e interpretação desses dados. Em relação ao conteúdo teórico são adotados como conceitos fundamentais: webrádio, radiomorfose, narrativa transmedia, participação em linha do utilizador e produção em webrádio. A metodologia empregue se apresenta no método etnográfico e baseia-se em quatro técnicas de recolhas distintas: observação não-participante, observação participante, inquérito por questionário e inquérito por entrevistas. Tendo por base a análise dos dados recolhidos, as principais conclusões desta tese são: há a potencialização do utilizador nas narrativas transmedia apresentadas, com a rede Facebook sendo o recurso interativo mais importante, mas que, por limites da emissora (sejam conceituais ou técnicos), a participação do utilizador não tem influência direta na produção da webrádio.This research addresses the relationship between user participation and online content production in webradio with the overall objective to investigate whether and how the production of webradios is influenced by online user participation in the transmedia context. The study is divided into two parts. The first is theoretical, in which it approaches the fundamental concepts of research and the methodology used to collect and analyze the data. The second is practical and refers to the data collections made in two stations (one brazilian, Campina FM, and other portuguese, the Rádio Comercial) and the presentation and interpretation of such data. Regarding the theoretical content are adopted as fundamental concepts: webradio, radiomorphosis, transmedia storytelling, online user participation and production in webradio. The methodology used is presented in the ethnographic method and is based on four techniques of distinct collections: non-participant observation, participant observation, questionnaire survey and survey interviews. Based on the analysis of the data collected, the main conclusions of this thesis are: there is the enhancement of the user in transmedia storytelling presented, with the Facebook network being the most important interactive feature, but on the limits of the issuer (whether conceptual or technical), the user participation has no direct influence on the production webradi

AS AÇÕES E IMPLICAÇÕES DA EMERGÊNCIA DOS BAILES SOUL NA PERIFERIA DO RIO DE JANEIRO E NO MERCADO DO DISCO DURANTE A DÉCADA DE 1970

Durante a década de 1970 emergiu o baile soul, que era itinerante em clubes da periferia do Rio de Janeiro, direcionado aos jovens e reunia milhares de pessoas em cada evento, o qual além de veicular músicas para dançar celebrava a cultura negra. A popularização deste tipo de baile coincidiu com a popularização da música pop internacional no Brasil e repercutiu também no rádio e na televisão, a qual incluiu as canções em telenovelas. Tornou-se um sucesso do mercado do disco, o que inclui as coletâneas lançadas pelas próprias equipes de baile por diferentes gravadoras. O objetivo deste presente trabalhar é observar as ações e implicações da emergência dos bailes soul na periferia do Rio de Janeiro e no mercado do disco no período citado