Orientador: Ricardo Antunes de Azevedo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T07:27:10Z (GMT). No. of bitstreams: 1 Cardoso_PatriciaFelippe_D.pdf: 8529922 bytes, checksum: 7d11b42d059e4ce26267792f15c476f6 (MD5) Previous issue date: 2005 / Resumo: A presença de metais pesados no ambiente é atualmente, um dos principais problemas de contaminação ambiental, uma vez que, os metais liberados no ambiente contaminam o solo e entram na cadeia alimentar através das plantas, causando efeitos tóxicos a curto e a longo prazo aos animais e seres humanos. No caso do metal pesado Níquel (Ni), foi constatado que a sua presença nas plantas pode diminuir o crescimento, reduzir a taxa de fotossíntese e provocar alterações, tanto nas atividades enzimáticas quanto metabólicas.Pouca informação está disponível na literatura, com referência a resposta antioxidante das plantas à expressão a esse metal. Neste sentido, os objetivos deste trabalho foram realizar diferentes ensaios para avaliar o efeito fitotóxico do Ni em plântulas de C. juncea. Neste estudo foram analisados parâmetros bioquímicos relativos à atividade das enzimas antioxidantes, Catalase (CAT), Superóxido Dismutase (SOD) e Glutationa Redutase (GR). Constatou-se que, não houve alteração da atividade GR e CAT nas raízes. Entretanto, atividades destas enzimas apresentaram aumento significativo na parte aérea. O aumento na atividade da GR, na parte aérea, pode ser explicado pelo fato de ser o ciclo Halliwell-Asada o principal mecanismo que age na desintoxicação de Espécies Ativas de Oxigênio. Quanto à atividade da SOD, quando comparados ambos tecidos, foi constatado que na parte aérea foi pouco alterada, entretanto, a atividade da SOD foi estimulada nas raízes na presença do Ni. Para aumentar a precisão dos resultados, quantificou-se pela técnica de Fluorescência de Raios X, a concentração de NiCl2 e o seu efeito na absorção de nutrientes nas plântulas de crotalaria. Nesta análise, observou-se o acúmulo do metal nas raízes e baixa translocação para a parte aérea. Também foi analisada a concentração de Malonaldeído (MDA) na parte aérea de plântulas de C. juncea, sendo constatada, a ocorrência de peroxidação lipídica na presença do Ni. Finalmente, determinou-se o efeito do Ni sobre as concentrações de aminoácidos solúveis em raízes e na parte aérea, como estratégia desta planta para superar o efeito do Ni , não tendo sido observadas variações significativas nas concentrações dos mesmos com os diferentes tratamentos com Ni. Palavra¿Chave: Aminoácidos, Atividade Enzimática, Crotalária, Fitotoxicidade, Níquel, Poluição Ambiental / Abstract: The contamination of the environment with heavy metals has become a world-wide problem. Heavy metals can have direct effects on human health and decreases agricultural yields. In the presence of Nickel (Ni), plant growth and the net photosynthetic rate have been shown to be significantly reduced, even at low concentrations. Moreover, Ni can also inhibit the activity of several groups of enzymes and metabolism. The effects of the Ni on the activies of the antioxidant enzymes and metabolism were investigated in C.juncea seedlings. Enzymatic assays for Catalase (CAT) Glutathione Reductase (GR) and Superoxide Dismutase (SOD) were carried out. Response patterns for CAT and GR were similar with low values in the roots and increased activity in the shoots. A significant increase in CAT and GR activity in the shoots suggests that these enzymes play a role in the detoxification process, of reactive oxygen species (ROS) produced by the induced Ni stress, possibly by subquesent dismutase. On the other hand, the activity of SOD was stimulated in the roots, and decreased in the shoots. To gain insight into the response of C. juncea seedlings to the oxidative damage induced by excess Ni, were conducted studies of Malondialdehyde (MDA). It was evident that Ni induced an increase an lipid peroxidation in the shoots during the studied period. The Ni concentration in shoots and roots was determined for C. juncea and demonstrated an increase in the concentration of Ni in the roots, however the shoots did not present an accumulation. Further experiments were carried out on total free amino acids. It was observed that Ni treatment did not cause any change in the accumulation of total free amino acids in the roots. The results suggest that in C. juncea seedlings did not exhibit any major variation following Ni treatment. Key words: Antioxidant enzymes, Amino acids, C. juncea, Nickel, Environmental Pollution / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

The potential of countercurrent chromatography (CCC) as a small footprint bioreactor/separator for manufacture of enantiopure chiral molecules was explored, using as a model reaction the isolation of L-amino butyric acid (L-ABA) from a DL-ABA racemate and the enantioselectivity of D-amino acid oxidase (DAAO). Bioconversion of D-ABA to ketobutyric acid (KBA) by DAAO, immobilised by selective partitioning in the stationary phase of the CCC centrifuge, was accompanied by separation of unreacted L-ABA from KBA by the countercurrent action of the centrifuge. For effective bioreactor/separator action, a high partition of the biocatalyst to the stationary phase was required in order to retain the biocatalyst in the coil, with differing partitions of substrates and products between the stationary phase (SP) and mobile phase (MP) so that these could be separated. Aqueous two-phase systems (ATPS) were the major two-phase systems used to provide SP and MP, as these are well reported to be effective in preserving enzyme activity. The distribution ratios of DL-ABA, KBA and DAAO were measured in a range of phases with polyethylene glycols (PEGs) of different molecular weights, different salts, and different compositions of PEG and salt, using an automated robotic method, developed for the purpose. A system of 14% w/w PEG 1000/ 14% w/w potassium phosphate, pH 7.6, gave the best combination of distributions ratios (CPEG phase/Csalt phase = CSP/CMP) for ABA, KBA and biocatalyst (DAAO) of 0.6, 2.4 and 19.6 respectively. A limited number of aqueous-organic and ionic liquid two-phase systems were also reviewed, but found unsatisfactory. CCC operating conditions such as substrate concentration, biocatalyst concentration, the mobile phase flow rate (residence time in the CCC coil), temperature, rotational speed and operational modes (single flow and multiple-dual flow) and types of mixing (cascade and wave-like) were optimised to produce total conversion of D-ABA to KBA, which was then completely separated from unreacted, enantiomerically pure (>99% ee), LABA. Advantages of the CCC bioreactor over conventional technology include reduced equipment footprint, cheaper running costs, and faster purifications. However, in its current format the drawbacks, such as enzyme instability and excessive optimisation time, reduce its commercial appeal. Additional investigations into the use of whole cell preparations of biocatalyst in the CCC bioreactor showed potential to overcome the problem of enzyme instability and this may in the future give the CCC bioreactor a place in the enantioseparation field.

Several Agrobacterium tumefaciens strains have been isolated for their ability to produce D-amino acids from D, L-substituted hydantoins. The optically pure D-amino acids are used in the synthesis of pharmaceuticals, as food additives and as insecticides. This hydrolysis of D, L-substituted hydantoins is catalysed by two hydantoin-hydrolyzing enzymes, an hydantoinase and an N-carbamyl amino acid amidohydrolase. While the hydantoin-hydrolyzing enzymes have been studied in detail, the mechanisms that control expression of the hyu genes have not. The research reported in this work elucidates some of the mechanisms involved in the regulation of the hyu genes in A. tumefaciens strains. The hydantoin-hydrolyzing enzyme activity from the environmental isolate A. tumefaciens RU-AE01 was characterized. A broad host range vector for the simultaneous analysis of divergent promoters was constructed. The promoter regions responsible for the activation of transcription of hyuH and hyuC were identified by deletion analysis. It was proposed that transcription of hyuH was activated by a putative σ[superscript 54]-dependent promoter or a putative σ[superscript 70]-dependent promoter identified upstream of the hyuH gene. The hyuC gene was activated by a putative σ[superscript 70]-dependent promoter identified upstream of the hyuC gene. The regulation of hydantoinase and N-carbamyl amino acid amidohydrolase enzyme activity was compared to the regulation of transcription from the RU-AE01 hyuH-hyuC region. Expression of the hydantoin-hydrolyzing enzymes was regulated by induction which correlated with reporter enzyme expression from the hyuH and hyuC promoter regions. However, the expression of the hydantoin-hydrolyzing enzymes was also regulated by nitrogen catabolite repression (NCR). This did not correlate to the reporter gene expression of the hyuH promoter region but did compare to the reporter gene expression of the hyuC promoter region. This suggested that NCR of hyuH was at the post-translational level whereas NCR of the hyuC promoter was at the transcriptional level. Pathways involved in the regulation of the hyu genes were characterized. The production of the hydantoin-hydrolyzing enzymes in both A. tumefaciens strains RU-AE01 and RU-OR were regulated by proteins involved in the global ntr pathway. The levels of the hydantoin-hydrolyzing enzymes in strain RU-AE01 were elevated in the presence of increased levels of NtrB and NtrC illustrating the importance of the ntr pathway in the regulation of the levels of the hydantoin-hydrolyzing enzymes. Similarly, in RU-OR the presence of exogenous NtrB and NtrC elevated levels of N-carbamyl amino acid amidohydrolase activity. However, the levels of hydantoinase enzyme activity in strain RU-OR were elevated in the presence of NtrC alone. In addition, the presence of a His6-tagged NtrC molecule abolished the elevation in the levels of the hydantoinase but not the N-carbamyl amino acid amidohydrolase enzyme activity in strain RU-OR. This suggests that NtrC has a direct role in the regulation of the expression of hyuH in RU-OR. In addition, it indicates that the hyu genes in the two A. tumefaciens strains RU-AE01 and RU-OR are different. The presence of the RU-AE01 hyuH-hyuC fragment caused a dramatic increase in the hydantoin-hydrolyzing enzyme activity in strain RU-OR but not strain RU-AE01. This implied the incidence of a possible repressor protein in RU-OR, which is titrated out by the presence of the RU-AE01 hyuH-hyuC fragment. Protein-DNA binding assays suggest that this putative repressor may be 38 kDa in RU-OR cells.

The physico-chemical properties of lecithin:cholesterol acyltransferase were investigated. The amino acid composition analysis showed a relatively high content of glutamic acid, aspartic acid, glycine and leucine. The spectrophotometric titration of phenolic groups in the enzyme showed a large increase in absorbance at 295 nm with an apparent pK of about 12.0. The largest change in molar ellipticity at 222 nm was also observed above pH 11. Circular dichroism studies revealed that human lecithin:cholesterol acyltransferase has a relatively high content of β-pleated sheet structure (48%) with 20% α-helix, and 32% remaining structure. Human lecithin:cholesterol acyltransferase has a high extinction coefficient at neutral pH. Microsequencing of the amino terminal residues of the enzyme revealed a hydrophobic character. Inactivation of lecithin:cholesterol acyltransferase activity was observed using diisopropylfluorophosphate with a stoichiometry of 1 mole of diisopropylphosphate incorporated per mole of enzyme. This suggests the involvement of a serine residue in the active site of the enzyme, possibly for the formation of an acyl-intermediate. A new quicker assay method for lecithin:cholesterol acyltransferase was developed. This assay involved coupling reaction with acyl CoA synthetase, ΡΡᵢ-dependent phosphofructokinase, aldolase, triosephosphate isomerase and α-glycerol-3-phosphate dehydrogenase monitoring a change in the absorbance or fluorescence intensity due to the oxidation of NADH. The activity of each coupling enzyme was accurately determined to establish the optimum assay condition for lecithin:cholesterol acyltransferase. The coupled enzyme assay for lecithin:cholesterol acyltransferase by spectrofluorometry showed a significant change in relative fluorescence intensity whereas a UV absorption spectroscopy method showed no significant absorbance change for the initial rate of lecithin:cholesterol acyltransferase reaction.

My thesis is divided into 4 major sections. The first section is devoted in part to a description of the biochemical abnormalities in the metabolism of homocarnosine (y-aminobutyryl-L-histidine, HCarn) occurring in a patient with homocarnosinosis. The patient studied and two of her siblings have a progressive neurological disorder with grossly elevated concentrations of HCarn in their CSF. HCarn content was four times higher in a biopsy from the patient's frontal cortex than in biopsied cortex from a large group of control subjects. Using new techniques for the measurement of the HCarn synthesizing and catabolizing enzymes, it was found that the activity of HCarn-Carn synthetase was not increased in the patient's biopsy whereas homocarnosinase activity was undetectable. It is concluded that the elevated HCarn in brain and CSF in the homocarnosinosis patient is due to a deficiency of brain homocarnosinase activity. The first description of the regional distribution of the two HCarn metabolizing enzymes in human brain was also obtained. The remainder of the first section deals with a description of the neuropharmacological properties of HCarn. Intraventricular injection of HCarn in the rat produced hyperexcitability and in high doses, convulsions, whereas unilateral intra-striatal injection of HCarn resulted in contralateral myoclonus. The results of these experiments are consistent with the possibility that HCarn may be involved in the neuronal excitability of brain. The second section describes experiments which test the hypothesis that the content of the inhibitory neurotransmitter GABA is altered in the autopsied brains of some patients dying with schizophrenia. The mean content of GABA was reduced by 20-25% in nucleus accumbens, caudate nucleus, frontal cortex and thalamus of the schizophrenic patients as compared to a control group. However, the differences were found to be statistically significant for only the caudate and thalamus. Extraneous factors such as age of patient at death and prolonged drug treatment did not readily explain the observed reduction in GABA content. The results of the investigation suggest an association between a deficiency of GABAergic function in certain brain areas with some forms of schizophrenia. The third section describes experiments which test the hypothesis that a deficiency of aspartate found in the cerebellar cortex of some patients with dominantly inherited cerebellar disorders might be due to reduced activity of two enzymes involved in the synthesis of aspartate, namely, aspartate aminotransferase and pyruvate carboxylase. No deficiency of either enzyme was observed in the cerebellar specimens studied. The results of this investigation suggest that the aspartate deficiency in cerebellar cortex found in some dominantly inherited cerebellar disorders does not result from a deficiency of either of these two brain enzymes. In the final section, experiments are described which study the effects of chronic administration of Y-vinyl GABA and of hydrazine on the contents of GABA and other amino compounds in rat brain. Both of these compounds are presently under consideration for use in clinical trials on patients with disorders involving a brain GABA deficiency. Chronic administration of either y-vinyl GABA or of hydrazine markedly increased brain GABA content in the rat. Prolonged treatment with y-vinyl GABA, but not hydrazine, produced a decrease in the activity of glutamic acid decarboxylase (GAD) in rat brain. Since GAD is localized to a large extent in nerve endings, the possibility exists that y-vinyl GABA might reduce the amount of GABA available for release at synapses, a potentially undesirable effect. The contents of many brain amino compounds other than GABA were markedly altered by both drugs. Since the potential harmful effects of these unexpected biochemical alterations in brain are unknown, the nonspecific effects of Y-vinyl GABA and hydrazine are disturbing. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Ph.D. (Chemistry) / Please refer to full text to view abstract

Les composés fluorés ont montré une importance croissante dans le développement d'agents pharmaceutiques en raison des propriétés exceptionnelles de l'atome de fluor. Dans cette thèse, nous nous sommes concentrés sur la synthèse des composés énantiopurs contenant de l'azote.A partir de l'aldimine trifluorométhylée, une réaction d’aza-Baylis-Hillman asymétrique a été réalisée et a permis d’obtenir un nouvel acide (R)-α-méthylène-β-CF3-β-aminé avec un bon rendement et une excellente diastéréosélectivité. Puis, en utilisant une réaction d’allylation dans les conditions de Barbier, de nouveaux acides aminés trifluorométhylés cycliques à cinq ou six chaînons ont été obtenus avec de bons rendements et d’excellents diastéréosélectivités. En particulier des acides γ et β-aminés, comme la CF3-β-proline, qui est très intéressante dans la synthèse peptidique.Enfin, grâce à l'addition d'éther de vinyle sur l’aldimine trifluorométhylée, différentes amines trifluorométhylées ont été obtenues. Parmi ces composés, les amino-alcools CF3 qui sont très utiles pour la conception de médicaments et dans la synthèse asymétrique, et les amines cycliques trifluorométhylées qui peuvent servir de bon substrat pour synthétiser des composés CF3-cycliques plus fonctionnalisés. / Fluorinated compounds have shown a growing importance in the development of pharmaceutical agents owing to the outstanding properties of the fluorine atom. In this thesis, we focused on the synthesis of enantiopure trifluoromethyl nitrogen-containing compounds.Starting from the trifluoromethyl aldimine, firstly, an asymmetric aza-Baylis–Hillman reaction was realized with high reaction rates, and diastereoselectivities, and the previously unknown enantiomerically pure (R)-α-methylene β-CF3 β-amino acid was obtained in good yield. Then, through aza-Barbier reaction, the five-membered and six-membered trifluoromethyl cyclic amino acids were obtained, including β-amino acid and γ-amino acid, especially CF3-β-proline, which is very interesting in the further peptide synthesis.Finally, through the addition of vinyl ether to imine, different novel trifluoromethylated amines were afforded. Among these compounds, CF3-amino alcohols are very useful in drug design and asymmetric synthesis, and enantiopure CF3-cyclic amine is a very good substrate to synthesize more functionalized CF3-cyclic compounds.

Orientador: Marcos Franke Pinto Pinto / Resumo: O objetivo do presente trabalho foi desenvolver uma barra alimentícia (BA) a base de clara de ovo enriquecida com farinha de casca de uva, otimizando suas características nutricionais, sensoriais e funcionais e avaliar sua viabilidade comercial. Para tal uma formulação inicial foi desenvolvida e submetida à avaliação sensorial por 100 consumidores utilizando a escala do ideal. Com base nos resultados, foram elaboradas BA com diferentes concentrações de clara de clara de ovo: F1-17,93%, F2-20,16% e F3-22,76%, que foram comparadas entre si e com uma barra proteica disponível no mercado denominada F4. As quatro formulações foram avaliadas quanto ao conteúdo total de polifenóis e capacidade antioxidante in vitro, proteína bruta (PB), carboidratos totais (CT), gorduras totais (GT), perfil de aminoácidos (PAT) e perfil de aminoácidos essenciais (PAE). A análise sensorial realizada por 100 praticantes de exercícios físicos, com idade entre 18 e 40 anos, utilizando escala hedônica de cinco pontos, em uma única seção, de forma monádica. A análise estatística foi realizada utilizando o programa GraphPad Prism (versão 5.0). Os dados do conteúdo de polifenóis, atividade antioxidante e análise sensorial foram expressos como média ± padrão, analisados quanto à distribuição e submetidos à análise de variância (ANOVA), seguida do teste de Tukey com nível de significância foi estabelecido em 5%. Os resultados obtidos para PB, CT, GT, PAT e PAE foram expressos em gramas por 100 de BA. Posterio... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of the present work was to develop a food bar (BA) based on egg white enriched with grape husk flour, optimizing its nutritional, sensory and functional characteristics and to evaluate its commercial viability. For such an initial formulation was developed and submitted to sensory evaluation by 100 consumers using the ideal scale. Based on the results, BA were prepared with different egg white concentrations: F1-17.93%, F2-20.16% and F3-22.76%, which were compared with each other and with an available protein bar. in the F4 market. The four formulations were evaluated for total polyphenol content and in vitro antioxidant capacity, crude protein (CP), total carbohydrate (TC), total fat (GT), amino acid profile (PAT) and essential amino acid profile (PAE). Sensory analysis performed by 100 physical exercise practitioners, aged 18 to 40 years, using a five-point hedonic scale, in a single section, in a monadic way. Statistical analysis was performed using GraphPad Prism software (version 5.0). Data on polyphenol content, antioxidant activity and sensory analysis were expressed as mean ± standard, analyzed for distribution and subjected to analysis of variance (ANOVA), followed by Tukey test with significance level set at 5%. Results obtained for PB, CT, GT, PAT and PAE were expressed in grams per 100 BA. Subsequently, the sensory analysis data were evaluated through the Internal Preference Map (MDPREF). The total amount of polyphenols was 97.50 ± 1.55 in formulatio... (Complete abstract click electronic access below) / Mestre

Much of Nature is composed of highly modular and composable nested multicomponent living systems. Synthetic biology and bioengineering exploit this modularity to understand and engineer living things. This thesis explores two projects coupled by these principles, the first utilizing a synthetic genomics approach to probe the evolutionary history, flexibility, and modularity of core metabolism, and the second adapting and engineering components of a living material to generate living architecture and embed add program new behaviors into the living biocomposite. Chapter 1 details the synthetic resurrection of a core metabolic pathway lost from the metazoan lineage millions of years ago. All metazoans are auxotrophic for 9 of the 20 amino acids, the so-called “essential” amino acids. The pressures behind the loss of the 9 are a deep evolutionary puzzle. To investigate this event and probe the limits of core metabolic flexibility, we generated a synthetic valine prototrophic mammalian cell line, restoring valine self-sufficiency to the metazoan lineage. The restoration of this pathway implies the modern mammalian metabolism is still compatible with autogenous valine production, suggests profound modularity in core metabolism, and underscores the potential usefulness of large-scale synthetic genomics approaches in a answering deep evolutionary questions. Chapter 2 describes the engineering of a hybrid fungal-bacterial biocomposite by adapting and leveraging existing behaviors and microbial constituents of a living material. Fungal biocomposites are composed of a particulate lignocellulosic feedstock bound together into a bulk biocomposite by a network of dense fungal mycelium. Using a bioprospecting approach, we designed architectural and design strategies that relied on the natural substrate flexibility and growth patterns of the fungal component of the biocomposite to form origami-inspired human scale folding structures. Similarly, we isolated, characterized, and engineered a natural microbial component of the biocomposite’s own microbiome and used its pre-adapted ability to engraft in the growing biomaterial to embed new genetic functionalities in biocomposite objects. We believe that the strategy of bioprospecting useful components and behaviors holds promise for the development of future biomaterials adapted from living systems.

Herbivorous coral reef fishes play an important role in helping to structure their environment directly by consuming algae and indirectly by promoting coral health and growth. These fishes are generally separated into three broad groups: browsers, grazers, and excavators/scrapers, with these groupings often thought to have a fixed general function and all fishes within a group thought to have similar ecological roles. This categorization assumes a high level of functional redundancy within herbivorous fishes. However, recent evidence questions the use of this broad classification scheme, and posits that there may actually be more resource partitioning within these functional groupings. Here, I use a compound-specific stable isotope approach (CSIA) to show there appears to be a greater diversity of functional roles than previously assumed within broad functional groups. The δ13C signatures from essential amino acids of reef end-members (coral, macroalgae, detritus, and phytoplankton) and fish muscle were analyzed to investigate differences in resource use between fishes. Most end-members displayed clear isotopic differences, and most fishes within functional groups were dissimilar in their isotopic signature, implying differences in the resources they target. No grazers closely resembled each other isotopically, implying a much lower level of functional redundancy within this group; scraping parrotfish were also distinct from excavating parrotfish and to a lesser degree distinct between scrapers. This study highlights the potential of CSIA to help distinguish fine-scale ecological differences within other groups of reef organisms as well. These results question the utility of lumping nominally herbivorous fishes into broad groups with assumed similar roles. Given the apparent functional differences between nominally herbivorous reef fishes, it is important for managers to incorporate the diversity of functional roles these fish play.