Many gram-negative bacteria produce and secrete siderophores under iron-deficient conditions. Siderophores are low molecular weight compounds (600-1500 Daltons), which chelate ferric iron with an extremely high affinity, and the complex is actively transported across the outer and inner membranes of gram-negative bacteria. There are two main classes of siderophores: catechol and hydroxamate. Catechol-type siderophores chelate ferric iron via hydroxyl groups, and hydroxamate-type siderophores chelate ferric iron via a carbonyl group with an adjacent nitrogen. Rhizobia fix atmospheric nitrogen symbiotically in leguminous plants using the iron-containing enzyme nitrogenase. To satisfy their iron requirements, many rhizobia are known to produce siderophores. Rhizobium leguminosarum Strain IARI 312 is known to infect pigeon pea plants. R. leguminosarum Strain IARI 312 produces both a catechol-type and a hydroxamate-type siderophore when grown under iron deficient conditions. The catechol-type siderophore has been purified and chemically characterized, and is consistent with that of enterobactin.

Submitted by orjuela@ibilce.unesp.br (orjuela@ibilce.unesp.br) on 2016-02-17T12:16:38Z No. of bitstreams: 1 Isolamento de bactérias degradadoras de hidrocarbonetos aromáticos 1-107.pdf: 3940758 bytes, checksum: c39dfe1dada0d918848470d3f0de5b83 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-02-17T13:16:08Z (GMT) No. of bitstreams: 1 orjuela_gl_dr_rcla.pdf: 3940758 bytes, checksum: c39dfe1dada0d918848470d3f0de5b83 (MD5) / Made available in DSpace on 2016-02-17T13:16:08Z (GMT). No. of bitstreams: 1 orjuela_gl_dr_rcla.pdf: 3940758 bytes, checksum: c39dfe1dada0d918848470d3f0de5b83 (MD5) Previous issue date: 2016-02-11 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Este documento foi organizado em dois capítulos. O Capítulo I é um artigo de revisão intitulado “Metabolic Pathways for Aromatic Compounds Degradation by Bacteria”, no qual são descritas as fontes naturais e antrópicas dos hidrocarbonetos aromáticos e suas características químicas. Relacionaram-se os fatores ambientais que afetam a degradação aeróbia e anaeróbia desses compostos por bactérias e os principais compostos intermediários produzidos. É descrita passo a passo a sequência de preparação e desaromatização do anel benzênico e os compostos finais dessa degradação. O artigo foi publicado no volume 237 da série Reviews of Environmental Contamination and Toxicology em janeiro de 2016 (Springer http://dx.doi.org/10.1007/978-3-319-23573-8_5). O Capítulo II contém os resultados da pesquisa desenvolvida. O objetivo geral foi isolar cepas bacterianas de amostras de solo e avaliar o potencial degradador de fenol e outros hidrocarbonetos aromáticos. Foram realizadas coletas de amostras de solo de cinco postos de combustíveis, para a seleção das cepas bacterianas e para análises químicas e granulométricas. Alíquotas das amostras de solo foram transferidas para meio de cultura seletivo contendo querosene como única fonte de carbono. Testes morfológicos e bioquímicos indicaram que as cepas isoladas são Gram negativas, móveis, catalase positivas, produtoras de cápsula e de biossurfactantes. O sequenciamento do gene 16S rRNA mostrou 99 a 100% de similaridade com o gênero Pseudomonas sp. Todas as cepas degradaram o fenol em concentração de 120 mg L-1 em menos de 24 horas. Testes da atividade enzimática mostraram que algumas das cepas expressaram a catecol 1,2-dioxigenase que catalisa a orto-clivagem do anel benzênico e outras expressaram a catecol 2,3 dioxigenase da meta-clivagem do anel aromático. Uma cepa não apresentou atividade para nenhuma dessas duas enzimas e uma apresentou atividade de ambas. Todas as cepas foram capazes de crescer em presença de fenantreno, fluoranteno e pireno. / This document is organized in two chapters. The Chapter I is a review paper entitled “Metabolic Pathways for Aromatic Compounds Degradation by Bacteria” in which are described natural and anthropogenic sources of aromatic hydrocarbons and their chemical characteristics. There are listed environmental factors that affect the aerobic and anaerobic degradation by bacteria and the central intermediates yielded. It is described step to step of sequence of preparation and dearomatization of benzene ring and the final metabolites of breakdown. The manuscript was publicated in the volume 237 of Reviews of Environmental Contamination and Toxicology in January of 2016 (Springer http://dx.doi.org/10.1007/978-3- 319-23573-8_5). Chapter II has the results of developed research. The general objective was to isolate bacterial strains from samples of soil and to evaluate the potential of them for breakdown hydrocarbons. Soil samples from five gas stations were collected to isolate the bacterial strains and chemical and granulometric analysis was made. Aliquots of the soil samples were cultured with selective media with kerosene as only carbon and energy sources. Morphological and biochemical tests showed that bacterial strains were Gram negatives, motile, positive catalase, capsule-producers and biosurfactant producers. The sequencing of 16S rRNA gene showed 99 to 100% similarity with Pseudomonas sp genera. All bacterial strains were able to degrade phenol 120 mg L-1 in less than 24 hours. Tests of enzymatic activity showed that some bacteria expressed the catecol 1,2-dioxygenase that catalyze ortoclivage of benzene ring, others showed activity for the catecol 2,3-dioxygenase that catalyze the meta-cleavage of the ring. One strain did not show activity for any of these enzymes and one strain had activity for both. All strains were able to growth with fenantrene, fluoranthene and pyrene. / CNPq: 140704/2012-4

We have investigated electropolymerisation for fabrication of a chemically modified working electrode for the determination of dopamine and catechol neurotransmitters in the presence of ascorbic acid. A variety of film compositions were investigated that would allow discrimination of the neurotransmitters through a combination of electrostatic barrier and the film porosity. The films investigated were based on different compositions of () poly-o-toluidine-co-aniline (POT-co-PA), () poly-o-toluidine-co-o-anisidine (POT-co-POA) and () polyacriflavine (PAF). The POT-co-PA and POT-co-POA gave the most promising result although the POT-co-PA was preferred because of higher current enhancement and better separation of dopamine and catechol neurotransmitters in the presence of ascorbic acid. The uses of electropolymerisation make the investigated films attractive candidates for the fabrication of a chemically modified microelectrode with application in capillary electrophoresis separation with electrochemical detection. The active area of nano particle (Au, Pt and Ag) screen printed electrodes was determined using cyclic voltammogram with ferro/ferricyanide couple. The active surface of the nano particle coated electrode was found surprisingly to be 5% - 65% lower than that geometrically calculated surface area for the electrode. This is ascribed to the limitation of the screen printing approach that was used. A low cost high replication approach that would allow development of a capillary electrophoresis microfluidic chip with electrochemical detection (CE-ECD) on a polymer substrate was investigated. A fluidic top layer was fabricated using hot embossing and an electrode bottom layer by metal patterning on a polymer substrate using metallisation and photolithography.

In den vergangenen Jahren wurde vermehrt das Gen, welches für Catechol-O-Methyltransferase codiert, als starker Kandidat für ein erhöhtes Schizophrenierisiko diskutiert. Grund dafür ist die zentrale Rolle der Catechol-O-Methyltransferase beim Katecholaminabbau im menschlichen präfrontalen Cortex. Aufgrund der zunehmend akzeptierten Tatsache, daß die singuläre Betrachtung einzelner Marker bei der komplexen genetischen Textur von Kandidatengenen nur wenig zur Erhellung komplexer Erkrankungen beizutragen vermag (Licinio, 2003), untersuchten wir neben dem Val108/158Met-Polymorphismus (rs4680) vier weitere, die COMT-Gen-Region umspannende SNPs (rs2097603, rs740603, rs4818, rs165599) an einer Stichprobe von 459 Schizophrenen und 150 Kontrollpersonen. Zwar ergab sich für den Marker rs740603 auf Intron 1 eine signifikante Allel- (p = 0.0060) und Genotypassoziation (p = 0.019), der funktionelle Val108/158Met-Polymorphismus (rs4680) zeigte aber keinen signifikanten Zusammenhang mit der Erkrankung. Zudem fand sich in unserer Haplotypanalyse keine Markerkombination, die in überdurchschnittlichem Zusammenhang mit schizophrenen Psychosen stand. Für die Untergruppe der zykloiden Psychosen ließ sich bei einem p-Wert von 0.031 eine 4-Marker-Kombination ermitteln, die die SNPs rs740603, rs4818, rs4680 und rs165599 einschliesst und die Region von Intron 1 bis 3´-UTR umspannt. Zusätzlich ergab sich in der Subgruppe der zykloiden Psychosen ein geschlechtsspezifischer Effekt im Sinne eines signifikanten 3-Marker-Haplotypen (rs4818-rs4680-rs165599) (p = .0044) in der Gruppe der Frauen (n = 27) mit rs165599 als stärkstem Einzelmarker. Aufgrund des komplexen genetischen Zusammenhangs zwischen den untersuchten Markern und der Erkrankung sollte auch in der zukünftigen Forschung eine differenzierte Betrachtung der verschiedenen schizophrenen Zustandsbilder angestrebt werden, wie dies die Klassifikation nach Leonhard ermöglicht. Neben gewebsspezifischen Transkriptionsfaktoren könnten auch epigenetische Faktoren, wie die Cytosinmethylierung von CpG-Stellen in promotorregulierenden Regionen, einen Erklärungsansatz für die Entstehung schizophrener Störungsbilder darstellen. / Since several years, the gene encoding catechol-O-methyltransferase (COMT) at chromosome 22q11 is discussed as a strong candidate for schizophrenia susceptibility due to its key function in degredation of catecholamines in the prefrontal cortex, a critical region of the human brain, involved in cognitive control processes, monitoring of information in working memory and in active judgments on information (Petrides, 2005). To test the association of the COMT gene locus with schizophrenia, we analysed five SNPs (rs2097603, rs740603, rs4818, rs4680, rs165599) spanning from the P2 promotor region (MB-COMT) to the 3´-UTR in 459 index cases, which fulfilled diagnistic criteria of schizophrenia according to DSM IV as well as 150 blood donors as population controls. According to differentiated psychopathology (Leonhard, 1999) probands were categorized into cycloid psychosis, unsystematic schizophrenia and systematic schizophrenia prior to genotyping. In intron 1 the marker rs740603 showed significant allele (p = 0.0060) and genotype (p = 0.019) association, but the functional Val105/158Met variant (rs4680) failed significant association with disease. Considering COMT haplotypes none of the marker combinations showed evidence for an association with schizophrenia. In the subgroup of cycloid psychosis we found 4-locus marker combinations rs740603-rs4818-rs4680-rs165599 associated with disease at p-level 0.031, spanning a region from intron 1 to the 3´-UTR. In conclusion, the genetic interaction of COMT SNPs and haplotypes and schizophrenia susceptibility appears complex across different populations and psychopathological phenotypes. Particularly structures potentially involved in mRNA expression levels need further scrutiny.

Hintergrund: Das visuelle Arbeitsgedächtnis ist eine Leistung des präfrontalen Kortex (PFC) und dient der kurzfristigen Speicherung und Manipulation visueller Information. Ein aufgabenspezifisches Modell des visuellen Arbeitsgedächtnisses unterteilt zwei inhaltlich und örtlich getrennte Subsysteme: Das visuell-räumliche Arbeitsgedächtnis (SWM) scheint vor allem im dorsolateralen PFC (DLPFC) und das visuell-bildliche (OWM) im ventrolateralen PFC (VLPFC) lokalisiert zu sein. Der Dopaminabbau im PFC wird entscheidend von dem Enzym Catechol-O-Methyltransferase (COMT) bestimmt. Es existiert ein funktioneller COMT-Val158Met-Polymorphismus, der aufgrund veränderter Enzymaktivität mit unterschiedlicher dopaminerger Aktivität im PFC verbunden ist. Ziele: Das Aufgabendesign zielte darauf ab, die funktionelle Trennung von SWM und OWM im PFC nachzuweisen und die aufgabenspezifische Hypothese zu testen. Als zweites Ziel der Studie sollte der mögliche Einfluss des COMT-Val158Met-Polymorphismus auf die Arbeitsgedächtnisleistung getestet werden. Methoden: Es wurden 100 körperlich und psychisch gesunde Probanden in einer funktionellen Nah-Infrarot Spektroskopie-Studie (fNIRS-Studie) mit einem visuellen Arbeitsgedächtnisparadigma untersucht. Die NIRS-Messung beruht auf dem Prinzip der neurovaskulären Kopplung. Es wurde eine rechteckige 30*6 cm große Messhaube über dem frontalen Kortex platziert und die Veränderungen von O2Hb und HHb während des Paradigmas bestimmt. Anschließend wurde eine Genotypisierung durchgeführt. Ergebnisse und Diskussion: Zwischen den Arbeitsgedächtnisaufgaben zeigte sich ein deutlicher linearer Zusammenhang für die Verhaltensdaten. Das SWM-Paradigma führte zu einem O2Hb-Anstieg und HHb-Abfall im DLPFC, womit sich eine selektive Aktivierung dieses Areals bestätigte. OWM-Aufgaben hingegen führten zu einem parallelen Anstieg von O2Hb und HHb im VLPFC. Mit der fNIRS-Untersuchung konnten Vorbefunde der funktionellen Bildgebung bestätigt werden, die eine aufgabenspezifische Gliederung des visuellen Arbeitsgedächtnisses postulieren. Ein funktioneller Zusammenhang zwischen dem COMT-Val158Met-Polymorphismus und dem Arbeitsgedächtnis konnte weder in den Verhaltensdaten noch in den Bildgebungsdaten nachgewiesen werden. Falls ein solcher Zusammenhang besteht, lag der fehlende Nachweis am ehesten in Task-Switching-Effekten des Studiendesigns begründet. / Objective: Visual working memory is an accomplishment of the prefrontal cortex (PFC), which serves the maintenance and manipulation of visual information over a short period of time. A ‘domain-specific’ model of visual working memory divides it into two functionally and spatially separated subsystems: Visual location-based memory (SWM) seems to be located in the dorsolateral PFC (DLPFC), visual objective-based memory (OWM) to be located in the ventrolateral PFC (VLPFC). The turnover of dopamine is critically tuned by the enzyme catechol-o-methyl-transferasis (COMT). There is a functional COMT-Val158Met-polymorphism which is associated with different dopaminergic levels in PFC due to a change in enzyme-activity. Aim: The task design was conceived to evaluate the functional dissociation of SWM and OWM in the PFC and to test the ‘dopamin-specific’ hypothesis. Secondly the study aims at testing the possible contribution of COMT-Val158Met-polymorphism to working memory. Methods: Therefore 100 somatically and mentally healthy subjects were assessed by an event-related functional near-infrared-study (fNIRS) with a visual working memory paradigm. The NIRS relies on the principle of neurovascular coupling. A 30*6 cm rectangle probeset was applied on the frontal cortex and changes in O2Hb and HHb were measured during the paradigms. After that, genotyping of the COMT-polymorphism was conformed. Results and Discussion: There was a significant linear dependence of working memory task in regard of behavioral data. SWM paradigm was accompanied by some increase in O2Hb and parallel decrease of HHb in the DLPFC which is in line with a selective activity of this area in SWM. OWM paradigm led to a parallel increase of O2Hb and HHb in the VLPFC. With the NIRS technique it was possible to replicate prior findings of functional imaging studies which postulated a ‘domain specific’ organization of the visual working memory. It was not possible however, to determine any connection between the COMT-Val158Met-polymorphism and the working memory in behavioral or functional imaging data. If there is such an interaction, the missing proof might be due to task-switching-effects in the design of the study.

Der präfrontale Kortex wird im Allgemeinen mit exekutiven Funktionen in Verbindung gebracht, die unter anderem Aufmerksamkeitskontrolle, Inhibition, Arbeitsgedächtnis oder die Erkennung und Bewertung neuartiger Reize beinhalten. Für die situationsgerechte Anpassung der verschiedenen Kontrollmechanismen ist in dieser Hirnregion, wie sich in zahlreichen Studien gezeigt hat, der Neurotransmitter Dopamin entscheidend. Dieser liegt, u.a. bedingt durch den Val108/158Met-Polymorphismus der Catechol-O-Methyl-Transferase, interindividuell in verschiedenem Ausmaß vor und sollte dadurch Unterschiede in präfrontalen Funktionen zwischen einzelnen Individuen maßgeblich beeinflussen. Ziel dieser Arbeit war es, diesen Einfluss mittels EEG und ereigniskorrelierten Potenzialen genauer zu bestimmen und zusätzlich herauszuarbeiten, welche kognitiven Prozesse genau durch den COMT-Genotyp beeinflusst werden. 60 gesunde Probanden absolvierten hierzu unter EEG-Ableitung eine Aufgabe mit variablem Aufmerksamkeitsbedarf. Es wurde zunächst der Zusammenhang zwischen den ereigniskorrelierten Potenzialen N200 und P300 mit präfrontalen Funktionen wie Aufmerksamkeitskontrolle, Inhibition und Konfliktdetektion bzw. –verarbeitung untersucht. Zusätzlich wurden sowohl das Verhalten als auch die elektrophysiologische Aktivität zwischen den sorgfältig gematchten Genotyp-Gruppen Val/Val, Val/Met und Met/Met (je 11 Probanden) verglichen. Es zeigte sich, dass die N200 v.a. durch eine Veränderung der Aufmerksamkeitsrichtung zwischen globalen und lokalen Stimuluseigenschaften beeinflusst wurde. Die P300 wurde dagegen sowohl von der Aufmerksamkeitsrichtung als auch der Konfliktstärke (lokale im Vergleich zu globalen Stimuluseigenschaften sowie erhöhter Konflikt mit vermehrter Beanspruchung von Aufmerksamkeitsressourcen) beeinflusst. Wir konnten keine Auswirkungen der COMT-Ausprägung auf die Performanz (Korrektheit und Reaktionsgeschwindigkeit) im VAC-Test feststellen. Die hirnelektrische Aktivität (N200 und P300) unterlag dagegen einem deutlichen Einfluss des COMT-Genotyps: Met-Allel-Homozygote zeigten ein effizienteres Arbeiten - es lag eine bessere Aktivierung des Kortex (höhere P300) sowie ein geringerer Bedarf an Inhibition bei gleicher Leistung wie bei Val-Allel-Trägern vor (niedrigere N200- Amplitude). Es zeigte sich zudem bei den Met-Allel-Homozygoten kaum Variabilität der Hirnaktivierung über alle Schwierigkeitsstufen hinweg, was möglicherweise Ausdruck einer geringeren kognitiven Flexibilität im Vergleich zu Val-Allel-Trägern ist. / The prefrontal cortex is reported to be associated with executive functions such as attentional control, inhibition, working memory or recognition and evaluation of new stimuli. To the aim of appropriate adaptation of the variable control mechanisms in this brain region, the neurotransmitter dopamine is crucial, as reported in numerous studies. Due to the Val108/158Met polymorphism (amongst others) the neurotransmitter is available in a varying amount and should therefore effect prefrontal functions amongst individuals importantly. The aim of this study was to define this influence using EEG and event related potentials and to identify in addition the cognitive processes influenced by the COMT genotype. 60 healthy individuals therefore passed a variable attentional control task during the recording of an EEG. The association of the event-related potentials N200 and P300 with prefrontal functions (attentional control, inhibition and conflict detection) was analyzed. Additionally performance and electrophysiological activity of the carefully matched genotype groups Val/Val, Val/Met and Met/Met were compared. We found N200 mainly being influenced by an attentional shift between global and local stimulus characteristics - in contrast to P300, which was affected both by attentional focus and amount of conflict (local in comparison to global stimulus characteristics as well as an increased amount of conflict with a higher demand for attentional resources). We found no influence of COMT genotype on performance (reaction time and correctness) in our task whereas electric brain activity is subject to a considerable impact of the COMT genotype: Met allele homozygotes showed more efficient working expressed by a better cortical activation (higher P300) and less inhibitory demand (lower N200) for equal performance compared to Val allele carriers. Furthermore Met allele homozygotes showed only very little variability in cortical activation throughout all conditions – maybe a sign of less cognitive flexibility compared to Val allele carriers.

Nesta dissertação, foram estudados os métodos de síntese de filmes poliméricos como matrizes hospedeiras da enzima tirosinase vislumbrando o desenvolvimento de biossensores mais seletivos e duradouros na detecção de compostos fenólicos. Os polímeros utilizados e preparados por cronoamperometria em acetonitrila foram: polipirrol (PPI), polimetiltiofeno (PMET) e copolímero PPI-PMET. Os filmes PPI-PMET apresentaram características intermediárias às dos filmes PPI e PMET, conforme se verificaram em espectros na região do infravermelho (FTIR), voltametria cíclica e microscopia de força atômica (AFM), evidenciando, assim, a formação de uma nova matriz polimérica. O processo de superoxidação do filme PPI-PMET foi estudado visando a uma imobilização mais eficiente da enzima, quando se obteve uma condição na qual a superoxidação ocorre somente nas cadeias de PPI. Biossensores amperométricos foram preparados a partir do uso da tirosinase extraída do abacate como fonte enzimática por imobilização física e realizada de maneiras diferentes em filmes não superoxidados e superoxidados. Foi feita a otimização das respostas dos filmes variando-se o pH, potencial de trabalho, concentração da enzima e tempo de imobilização da enzima para a detecção de catecol. Os resultados indicaram que a superoxidação do PPI nos filmes PPI-PMET favoreceu a imobilização da enzima, embora não fosse tão eficiente quando se comparou aos filmes com o PPI não superoxidado. Contudo, os biossensores, em geral, apresentaram grande sensibilidade ao catecol, com um limite de detecção baixo, da ordem de 0,12 µmol/L. / The synthesis methods of polymer films as host matrices of the enzyme tyrosinase were studied in this dissertation. It was expected the development of selective and everlasting biosensors for the detection of phenolic compounds. The used polymers were prepared by chronoamperometry in acetonitrile as followed: polypyrrole (PPI), polymethylthiophene (PMET) and copolymer PPI-PMET. The PPI-PMET films showed intermediate characteristics as the PPI and PMET films, as observed in the infrared spectra (FTIR), cyclic voltammetry and atomic force microscopy (AFM), thus indicating the formation of a new polymeric matrix . The process of overoxidized of PPI-PMET film was studied in order to achieve a more efficient immobilization of the enzyme, whilst it was achieved a condition in which overoxidized occurs only in the PPI chain. Amperometric biosensors were prepared from the use of tyrosinase extracted from avocado as an enzyme source by physical immobilization and performed in different ways in both overoxidized and non-overoxidized films. It was performed the optimization of the responses of the films by varying pH, operacuonial potential, enzyme concentration and time in the enzyme immobilization for the detection of catechol. The results showed that the overoxidized PPI in the films PPI-PMET favored the immobilization of the enzyme, although not as efficient when compared to the films with nom overoxidized PPI. Neverthless, the catechol biosensors showed a high sensitivity with a low detection limit in the order of 0.12 µmol/L.

Lam Chun Pong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references. / Abstracts in English and Chinese. / Table of Contents --- p.i / Acknowledgements --- p.v / Abstracts --- p.vi / Abbreviations --- p.viii / Chapter CHAPTER 1. --- SYNTHESIS AND REACTIVITY STUDIES OF MODEL COMPLEXES FOR INTRADIOL DIOXYGENASES WITH BENZIMIDAZOLE- CONTAINING LIGAND / Chapter I.1 --- Introduction / Chapter I.1.1 --- General Background --- p.1 / Chapter I.1.2 --- A General Review on the Modeling Chemistry for Catechol Dioxygenases --- p.3 / Chapter I.1.3 --- Intradiol Dioxygenases --- p.3 / Chapter I.1.3.1 --- Early model studies for intradiol dioxygenases --- p.5 / Chapter I.1.3.2 --- Factors affecting enzymatic reactivity for intradiol dioxygenases --- p.6 / Chapter I.1.3.3 --- Other functional models for intradiol dioxygenases --- p.7 / Chapter I.1.3.4 --- Reactivity studies of model complexes --- p.8 / Chapter I.1.4 --- Extradiol Dioxygenases --- p.8 / Chapter I.1.4.1 --- Early model studies for extradiol dioxygenases --- p.11 / Chapter I.1.4.2 --- Iron(III) complexes with extradiol properties --- p.12 / Chapter I.1.5 --- Objective of This Work --- p.14 / Chapter I.2 --- Results and Discussion / Chapter I.2.1 --- Synthesis of the Ligand Ntb --- p.15 / Chapter I.2.2 --- Synthesis of the Model Complex [Fe(ntb)Cl2]Cl --- p.16 / Chapter I.2.3 --- Synthesis of Enzyme-Substrate Model Complexes --- p.16 / Chapter I.2.4 --- Oxygenation Reactivities of Enzyme-Substrate Model Complexes 2-4 --- p.18 / Chapter I.2.4.1 --- Oxygenation of [Fe(ntb)(dbc)](C104) (2) in DMF --- p.18 / Chapter I.2.4.2 --- Oxygenation of [Fe(ntb)(cat)](Cl04) (3) in DMF --- p.21 / Chapter I.2.4.3 --- Oxygenation of [Fe(ntb)(tcc)](ClO4) (4) in DMF --- p.23 / Chapter I.2.4.4 --- Comparison of the oxygenation reactivities of complexes2-4 --- p.25 / Chapter I.2.5 --- Identification of Oxidative Cleavage Products --- p.27 / Chapter I.2.5.1 --- Isolation of oxidative cleavage products of complex 2 --- p.27 / Chapter I.2.5.2 --- Identification of cleavage products --- p.27 / Chapter I.2.6 --- Physical Characterization of Complexes 1-4 --- p.29 / Chapter I.2.6.1 --- Melting-points --- p.29 / Chapter I.2.6.2 --- Cyclic Voltammograms --- p.30 / Chapter I.2.6.3 --- EPR spectra --- p.31 / Chapter I.2.7 --- Molecular Structures of Complexes 1-4 --- p.34 / Chapter I.2.7.1 --- Molecular structure of [Fe(ntb)Cl2]Cl-4H20 (1) --- p.34 / Chapter I.2.7.2 --- Molecular structure of [Fe(ntb)(dbc)](Cl04)-2Me0H-H20 (2) --- p.36 / Chapter I.2.7.3 --- Molecular structure of [Fe(ntb)(cat)](ClO4) H20 (3) --- p.38 / Chapter I.2.7.4 --- Molecular structure of [Fe(ntb)(tcc)](Cl04).Me2C(0).H20 (4) --- p.41 / Chapter I.2.7.5 --- Comparison of the molecular structures of complexes 1-4 --- p.43 / Chapter I.3 --- Experimentals for Chapter 1 --- p.45 / Chapter I.4 --- References for Chapter 1 --- p.49 / Chapter CHAPTER II --- iron(iii) complexes containing N202 and N3O type ligands as models for INTRADIOL DIOXYGENASES / Chapter II.1 --- Introduction / Chapter II.1.1 --- Brief Remarks on Model Studies of Intradiol Dioxygenases. --- p.53 / Chapter II.1.2 --- Objective of This Work --- p.53 / Chapter II.2 --- Results and Discussion / Chapter II.2.1 --- Synthesis of N202 and N30 Type Ligands --- p.55 / Chapter II.2.2 --- Synthesis of Model Complexes --- p.57 / Chapter II.2.2.1 --- Model complex with ligand L1H --- p.57 / Chapter II.2.2.2 --- Model complex with ligand L2H2 --- p.58 / Chapter II.2.3 --- Synthesis of Enzyme-Substrate Model Complexes --- p.59 / Chapter II.2.3.1 --- Synthesis of enzyme-substrate model complexes from 14.… --- p.59 / Chapter II.2.3.2 --- Attempted synthesis of enzyme-substrate model complexes starting from 15 --- p.61 / Chapter II.2.4 --- Reaction of Complex 16 with Dioxygen --- p.61 / Chapter II.2.4.1 --- Oxygenation of [Fe(L1)(dbc)] (16) in DMF --- p.65 / Chapter II.2.5 --- Identification of Oxidative Cleavage Products --- p.64 / Chapter II.2.5.1 --- Isolation of oxidative cleavage products of complex 16 --- p.64 / Chapter II.2.5.2 --- Identification of cleavage products --- p.65 / Chapter II.2.6 --- "Physical Characterization of L1H, L2H2, Complexes 14-18" --- p.66 / Chapter II.2.6.1 --- NMR spectra --- p.67 / Chapter II.2.6.2 --- Melting-points --- p.69 / Chapter II.2.6.3 --- Mass spectra --- p.69 / Chapter II.2.6.4 --- Cyclic voltammogram --- p.69 / Chapter II.2.6.4 --- EPR spectra --- p.70 / Chapter II.2.7 --- "Molecular Structures of Complexes 14,15 and 18" --- p.71 / Chapter II.2.7.1 --- Molecular structure of [Fe(L1)(MeOH)Cl][BPh4].MeOH (14) --- p.72 / Chapter II.2.7.2 --- Molecular structure of [Fe(L2)Cl].MeOH (15) --- p.75 / Chapter II.2.7.3 --- Molecular structure of [Et3 Nh]3［Fe(tcc)3］.H2O(18) --- p.78 / Chapter II.3 --- Experimentals for Chapter 2 --- p.80 / Chapter II.4 --- References for Chapter 2 --- p.87 / APPENDIX 1 General Procedures and Physical Measurements --- p.89 / "APPENDIX 2 Selected Crystallographic Data for Complexes 1-4, 15,16 and 18.…" --- p.90 / Table A-l.Selected crystallographic data for complexes 1-4 --- p.91 / "Table A-2.Selected crystallographic data for complexes 15, 16 and 18" --- p.92 / "APPENDIX 3 Other Physical Data for Ligand L1H L2H2, Complexes 2 and 16" --- p.93 / Figure A-l.1H NMR spectrum of ligand L1H --- p.94 / Figure A-2.13C NMR spectrum of ligand L1H --- p.94 / Figure A-3.1H NMR spectrum of ligand L2H2 --- p.95 / Figure A-4.13C NMR spectrum of ligand L2H2 --- p.95 / Figure A-5.GC spectrum of the oxidative cleavage products of complex 2 --- p.96 / Figure A-6.- A-l 1.Mass spectra of the oxidative cleavage products of Complex 2 --- p.96 / Figure A-12.GC spectrum of the oxidative cleavage products of complex 16 --- p.99 / Figure A-13.- A-23.Mass spectra of the oxidative cleavage products of Complex 16 --- p.99 / Figure A-24.GC spectrum of dbcH2 standard --- p.105 / Figure A-25.Mass spectrum of dbcH2 standard --- p.106 / Figure A-26.GC spectrum of dbcq standard --- p.106 / Figure A-27.Mass spectrum of dbcq standard --- p.107

Made available in DSpace on 2015-04-22T22:02:08Z (GMT). No. of bitstreams: 1 PRISCILLA OLIVEIRA.pdf: 2800114 bytes, checksum: 0b6a9ea35227863ff51f73dff0623486 (MD5) Previous issue date: 2010-08-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The species Goupia glabra Aublet (Goupiaceae), popularly known as cupiúba is used in popular medicine against several diseases. Their barks are used as dental analgesic, as a vermifuge, to treat malaria, chickenpox and eczema. The leaves are used against syphilis, ocular disorders, migraine, smallpox and as healing. This study aimed to quantify the phenolic compounds and evaluate the antioxidant, inhibiting acetylcholinesterase, brine shrimp toxicity, antitumor and antimicrobial activities of extracts of leaves, thin and thick branches of cupiúba. The dry material was crushed and subjected to Soxhlet extraction using solvents in increasing order of polarity (hexane, ethyl acetate and methanol). The phytochemical screening was performed for different classes of metabolites, observed positive results for sterols in all extracts and terpenoids and some classes of flavonoids in the extracts obtained in ethyl acetate and methanol. The fractionation of the hexane extract of leaves (EHF) resulted in the isolation of a mixture composed by β-sitosterol and stigmasterol. From ethyl acetate extract of leaves (EAF) was isolated catechol, a simple phenol with restricted distribution in nature. The total phenolic content of extracts was determined using the Folin-Ciocalteau and the result was expressed as gallic acid equivalents (GAE). The antioxidant activity was assessed by the ability of capturing the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH ), using quercetin as external standard, expressed as value of the effective concentration (EC50). We observed a positive correlation between total phenolics and EC50, ie as greater the amount of phenolics smaller is the EC50. The ethyl acetate extract of the leaves (EAF) presented the highest antioxidant potential [EC50 = 9.76 ± 0.42 μg/mL (quartz cuvette) and EC50 = 19.87 ± 0.31 μg/mL (microplate)]. The toxicity of the extracts was verified by bioassays with microcrustacea A. salina and expressed in percentual mortality of nauplius. Qualitative analysis of anticholinesterase activity was performed according to the method of Ellman (modified), presenting negative results. The antitumor activity was evaluated against four cell lines (leukemia, breast, colon and glioblastoma) using colorimetric analysis method based on conversion of salt 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2-H-tetrazonium bromide (MTT) into blue formazam by the method of Mossman. The best results were observed for the ethyl acetate extracts, especially for cells HCT-8 (colon) that ranged from 58.35 to 77.12%. The ethyl acetate extract obtained from thick branches (EAGg) showed values of IC50 = 13.73 μg/mL for strain HL-60 (leukemia). This extract was fractionated and the fraction EAGg5 showed the percentage of cell growth inhibition of 85.37 and 101.37% for the lines SF-295 (glioblastoma) and MDAMB-435 (breast), respectively. The antimicrobial activity was conducted using the minimum inhibitory concentration (MIC) that was determined by microdilution in Mueller-Hinton broth for bacteria (Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) and in liquid Sabouraud for fungi (Candida albicans, C. tropicalis and C. parapsilosis). All extracts showed some degree of bacterial and fungicidal activity, especially the ethyl acetate extracts which showed moderate inhibition of microbial growth. The effect of extracts on growth of protozoa in vitro study was evaluated with promastigotes of Leishmania amazonensis and epimastigotes of Trypanosoma cruzi. EAGg extract showed antiproliferative activity against both L. amazonensis and T. cruzi, with IC50 values of 86 and 40 μg/mL, respectively. The significant antioxidant, antitumor and antimicrobial activities observed in the extracts of cupiuba detaches its high potential for obtaining of biotechnological products. / A espécie Goupia glabra Aublet (Goupiaceae), denominada popularmente como cupiúba, é utilizada na medicina popular contra diversas doenças. Suas cascas são utilizadas como analgésico dentário, como vermífugo, no tratamento de malária, catapora e eczema. As folhas são utilizadas contra sífilis, doenças oculares, enxaqueca, varíola e como cicatrizante. O presente trabalho teve como objetivo quantificar compostos fenólicos e avaliar as atividades antioxidante, inibidora de acetilcolinesterase, toxicidade em Artemia salina, antitumoral e antimicrobiana de extratos de folhas, galhos finos e galhos grossos de cupiúba. O material seco foi triturado e submetido à extração em aparelho de Soxhlet com solventes de polaridade crescente (Hexano, AcOEt e MeOH). A prospecção fitoquímica foi realizada para diferentes classes de metabólitos, sendo observados resultados positivos para esteróis em todos os extratos, triterpenóides e algumas classes de flavonóides nos extratos obtidos em acetato de etila e metanol. O fracionamento do extrato em hexano de folhas (EHF) resultou no isolamento de uma mistura de β-sitosterol e estigmasterol. Do extrato em AcOEt de folhas (EAF) foi isolado o catecol, um fenol simples de distribuição restrita na natureza. O conteúdo de fenólicos totais dos extratos foi determinado utilizando o reagente de Folin-Ciocalteau e o resultado foi expresso em equivalentes de ácido gálico (EAG). A atividade antioxidante foi avaliada através da capacidade seqüestrante do radical estável 2,2-difenil-1-picrilhidrazil (DPPH ), utilizando quercetina como padrão externo, expressos como valores de Concentração eficiente (CE50). Foi observada uma correlação positiva entre os fenólicos totais e a CE50, ou seja, quanto maior a quantidade de fenólicos, menor a CE50. O extrato em AcOEt das folhas (EAF) foi o que apresentou maior potencial antioxidante [CE50=9,76 ± 0,42 μg/mL (cubeta) e CE50= 19,87 ± 0,31 μg/mL (microplaca)]. A toxicidade dos extratos foi verificada através de bioensaios com microcrustáceo A. salina e expressa em % de mortalidade das larvas. A análise qualitativa da atividade anticolinesterase foi realizada de acordo com o método de Ellman (modificado), com resultados negativos para inibição da enzima acetilcolinesterase. A atividade antitumoral foi avaliada contra quatro linhagens de células (leucemia, mama, cólon e glioblastoma) utilizando método de análise colorimétrica baseada na conversão do sal 3-(4,5-dimetil-2-tiazol)-2,5-difenil-2-H-brometo de tetrazonium (MTT) em azul de formazam segundo o método de Mossman. Os melhores resultados foram observados para os extratos obtidos em AcOEt, principalmente para células HCT-8 (cólon) que variaram de 58,35 a 77,12%. O extrato obtido em AcOEt dos galhos grossos (EAGg) apresentou valor de CI50 = 13,73 μg/mL para a linhagem HL-60 (leucemia). Esse extrato foi fracionado e a fração EAGg5 apresentou percentual de inibição de crescimento celular de 85,37 e 101,37% para as linhagens SF-295 (glioblastoma) e MDAMB-435 (mama), respectivamente. A avaliação da atividade antimicrobiana foi realizada pelo método da concentração inibitória mínima (MIC) que foi determinada pela técnica da microdiluição em caldo Müeller-Hinton para bactérias (Bacillus subtilis, Staphylococcus aureus, Escherichia coli e Pseudomonas aeruginosa) e em meio líquido Sabouraud para fungos (Candida albicans, C. tropicalis e C. parapsilosis). Todos os extratos demonstraram algum grau de atividade bacteriana e fungicida, com destaque para os extratos obtidos em AcOEt que apresentaram inibição moderada de crescimento microbiano. Para avaliar o efeito dos extratos no crescimento dos protozoários foi realizado estudo in vitro com formas promastigotas de Leishmania amazonensis e epimastigotas de Trypanosoma cruzi. O extrato EAGg mostrou atividade antiproliferativa, tanto contra L. amazonensis quanto para T. cruzi, com valores de IC50 de 86 e 40 μg/mL, respectivamente. A significativa atividade antioxidante, antitumoral e antimicrobiana observada nos extratos de cupiúba destaca seu elevado potencial para a obtenção de produtos biotecnológicos.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Neste trabalho descreve-se a sÃntese, caracterizaÃÃo, reatividade e propriedades de novos complexos de rutÃnio com os ligantes o-fenilenodiamina, o-aminofenol, catecol, naftalenodiol, dopamina e adrenalina a partir do complexo precursor mer-[RuIIICl3(dppb)(H2O)]. Descreve-se tambÃm uma nova rota de desidrogenaÃÃo oxidativa do ligante o-fenilenodiamina a partir da interaÃÃo deste com o complexo mer-[RuIIICl3(dppb)(H2O)]. O complexo mer-[RuIIICl3(dppb)(H2O)] à extremamente versÃtil como precursor. Ao reagir com o ligante o-fenilenodiamina diretamente, forma uma mistura constituÃda por complexos que contÃm as formas oxidada (bqdi) e reduzida (opda) do ligante o-fenilÃnico, os complexos trans-[RuIICl2(dppb)(bqdi)] e trans-[RuIICl2(dppb)(opda)], que foi confirmada pela observaÃÃo de dois singletos em d 47 e d 26 no espectro de RMN 31P{1H}. Uma tentativa de atribuiÃÃo preliminar sugere que o ligante opda à oxidado a bqdi durante a reaÃÃo, conforme mecanismo proposto no presente trabalho. Entretanto, o produto obtido da reaÃÃo entre o ligante o-fenilenodiamina e o complexo mer-[RuIIICl3(dppb)H2O] por meio da lenta adiÃÃo do ligante apresentou apenas um sinal em d 26 no espectro de RMN 31P{1H}, indicando que o complexo trans- [RuIICl2(dppb)(bqdi)] à preferencialmente formado. Tal complexo foi caracterizado por anÃlise elementar, tÃcnicas espectroscÃpicas e eletroquÃmicas e sua estrutura determinada por difraÃÃo de raios X. Com o objetivo de reforÃar o mecanismo proposto, realizou-se a sÃntese entre o o ligante o-fenilenodiamina e o complexo [RuIICl2(dppb)(PPh3)]. Neste caso, o centro metÃlico de rutÃnio jà se encontra no estado reduzido e o metal nÃo promoverà nenhum tipo de mudanÃa no estado de oxidaÃÃo do ligante. Portanto, o produto obtido apresentou apenas um sinal em d 47 no espectro de RMN 31P{1H}, indicando a formaÃÃo do complexo trans-[RuIICl2(dppb)(opda)], caracterizado por microanÃlise, tÃcnicas espectroscÃpicas e eletroquÃmicas e cuja estrutura foi determinada por difraÃÃo de raios X. Os complexos isolados do tipo trans-[RuIICl2(dppb)(X)], onde X = quinona, dopamina e adrenalina e cis-[RuIICl2(dppb)(L)], onde L = o-aminofenol na forma quinonÃide e bqdi, foram caracterizados por espectroscopia eletrÃnica, vibracional e de ressonÃncia magnÃtica nuclear de fÃsforo, alÃm de tÃcnicas eletroquÃmicas, como voltametria cÃclica e de pulso diferencial. / This research work describes the synthesis, characterization, reactivity and properties of new complexes of ruthenium with the ligands o-phenylenediamine, oaminophenol, catechol, naphtalenediol, dopamine and adrenaline and the mer-[RuIIICl3(dppb)(H2O)] complex. Also, it describes a new metal-assisted oxidative dehydrogenation of the interaction o-phenylenediamine ligand and the mer- [RuIIICl3(dppb)(H2O)] complex. The mer-[RuIIICl3(dppb)(H2O)] complex has shown to be a versatile compound as starting material. The reaction of this compound with the o-phenylenediamine ligand produced a mixture of compounds with the bqdi and opda forms of the o-phenylene ligand, the trans-[RuIICl2(dppb)(bqdi)] and trans-[RuIICl2(dppb)(opda)] complexes, that it was confirmed for the observation of two singlet signals at d 47 and d 26 in the 31P{1H} NMR spectrum. One very first assignment suggests that the opda ligand is oxidized to bqdi form during the reaction, according to mechanism proposed in this work. However, the product of the reaction between the o-phenylenediamine ligand and the mer-[RuIIICl3(dppb)H2O] complex by the slow addition of the ligand showed only one signal at d 26 in the 31P{1H} NMR spectrum, indicating that the trans-[RuIICl2(dppb)(bqdi)] complex is preferentially produced. This complex was characterized by the elemental analysis, spectroscopic and electrochemical techniques and its structure has been determined by X-ray crystallography. Aiming to reinforce the proposed mechanism, we conducted the reaction of the o-phenylenediamine ligand with the [RuIICl2(dppb)(PPh3)] complex. Since the ruthenium metal center is already in the reduced state, it will not promote any redox change in the ligand. Thus, the complex produced showed only one signal at d 26 in the 31P{1H} NMR spectrum, indicating the formation of the trans-[RuIICl2(dppb)(opda)] complex that it was characterized by the elemental analysis, spectroscopic and electrochemical techniques and its structure has been determined by X-ray crystallography. The compounds isolated of type trans-[RuIICl2(dppb)(X)], X = quinone, dopamine and adrenaline and cis-[RuIICl2(dppb)(L)], L = o-aminophenol in the quinonoide form and bqdi, were characterized by the spectroscopic and electrochemical techniques.