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Dopamine exerts an important role in the regulation of motor activity in humans. During the progression of Parkinson’s disease, patients are faced with the progressive neurodegeneration of nigro-striatal dopamine neurons resulting in an array of pathological symptoms characteristic of the disease. Current treatment relies on targeting symptomatic aspects of the disease but currently Parkinson’s disease is incurable. Targeting the regeneration of DA neurons in PD patients could offer an alternative therapeutic approach that could stall and perhaps even revert the progression of the disease and improve the quality of life for patients. Here, I describe the generation of a transgenic zebrafish line for the non-invasive, conditional and specific ablation of dopaminergic neurons in both larval and adult zebrafish. Understanding the endogenous regenerative ability of the zebrafish may in the future contribute to the development of novel therapeutic approaches targeting DA neuron regeneration in humans. The Tg(dat:CFP-NTR) line efficiently labels and ablates most clusters of DA neurons in both the larval and the adult zebrafish brain. Neuronal ablation is followed by a locomotor and tail bend phenotype as well as by an increase in exploratory behavior. Using double transgenic larvae, we showed through live imaging that loss of DA neurons induces an increase in nestin expression; in addition we show an increase in the number of proliferating cells and an up regulation of genes involved in neurogenesis and tissue repair. Adult zebrafish were able to fully recover their DA neuronal population in the olfactory bulb within 45 days post ablation. Overall the Tg(dat:CFP-NTR) zebrafish offers a novel tool for the study of the molecular and cellular mechanisms driving the regeneration of DA neurons in the zebrafish brain and will be a useful tool for the field of regenerative medicine.

The enzyme nitroreductase (NR) catalyzes two-electron reduction of high explosives such as trinitrotoluene (TNT), a wide variety of other toxic nitroaromatic compounds, as well as riboflavin derivatives, using a tightly-bound flavin mononucleotide (FMN) cofactor. It has important environmental and clinical implications. Previous studies have focused on elucidating NRs catalytic mechanism and solving its crystal structure. In this dissertation work, we first develop and implement new strategies for labeling NR with stable isotopes, and report a completely re-designed protocol for NRs purification. Then we report the successful assignment of over half of NRs backbone resonances using 3d-NMR methods. The most significant observation is that we find a well-resolved 2d 1H-15N HSQC NMR spectrum is obtained at 37°C for NR, while the HSQC at 4°C is poorly-dispersed and comprised of sharp overlapped peaks. Thus, it would appear that NR denatures at 4°C. However, as we demonstrate, the non-covalently-bound FMN cofactor is not released at the lower temperature, based on retention of the native flavin visible-CD spectrum. Similarly, far-UV CD spectroscopy shows that the protein retains full secondary structural content at 4C. In addition, near-UV CD and Fluorine-19 NMR experiments demonstrate that under completely native conditions (neutral pH, no additives) NR maintains a high degree of tertiary structure and well-defined hydrophobic side-chain packing, ruling out the possibility of a molten-globule state. Thus, our studies report strong evidence that the dramatic low-temperature (low-T) transition near 20°C observed by NMR is not the result of protein structural changes, but rather, we propose that NR exists as an ensemble of rapidly inter-converting structures, at lower temperature, only adopting a well-defined unique structure above 20°C. Both saturation-transfer from water and solvent proton-exchange measurements support our proposed model in which the unique high-T structure is favored entropically, by release of water molecules; on the other hand, the fluxional low-T state incorporates greater water solvation at 4°C. In the latter part of this study, we present preliminary data suggesting that the flexibility implied by easy water-access to the loosely-structured state plays a role in accommodating binding of diverse substrates. Therefore, the fluxional low-T state may be functionally important. A possible direct link between the enzyme dynamics and its catalytic activity will be an area of future investigation.

A growing number of flavoprotein systems have been observed to bifurcate pairs of electrons. Flavin-based electron bifurcation (FBEB) results in products with greater reducing power than that of the reactants with less reducing power. Highly reducing electrons at low reduction midpoint potential are required for life processes of both aerobic and anaerobic metabolic processes. For electron bifurcation to function, the semiquinone (SQ) redox intermediate needs to be destabilized in the protein to suppress its ability to trap electrons. This dissertation examines SQ suppression across a number of flavin systems for the purpose of better understanding the nature of SQ suppression within FBEB and elucidates potential mechanistic roles of SQ. The major achievement of this work is advancing the understanding of SQ suppression and its utility in flavoproteins with the capacity to bifurcate pairs of electrons. Much of these achievements are highlighted in Chapter 6. To contextualize these mechanistic studies, we examined the kinetic and thermodynamic properties of non-bifurcating flavoproteins (Chapters 2 and 3) as well as bifurcating flavoproteins (Chapters 4 and 5). Proteins were selected as models for SQ suppression with the aim of elucidating the role of an intermediate SQ in bifurcation. The chemical reactions of flavins and those mediated by flavoproteins play critical roles in the bioenergetics of all lifeforms, both aerobic and anaerobic. We highlight our findings in the context of electron bifurcation, the recently discovered third form of biological energy conservation. Bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase I (Nfn) and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin were studied by transient absorption spectroscopy to compare electron transfer rates and mechanisms in the picosecond range. Different mechanisms were found to dominate SQ decay in the different proteins, producing lifetimes ranging over 3 orders of magnitude. The presence of a short-lived SQ alone was found to be insufficient to infer bifurcating activity. We established a model wherein the short SQ lifetime in Nfn results from efficient electron propagation. Such mechanisms of SQ decay may be a general feature of redox active site ensembles able to carry out bifurcation. We also investigated the proposed bifurcating electron transfer flavoprotein (Etf) from Pyrobaculum aerophilum (Pae), a hyperthermophilic archaeon. Unlike other Etfs, we observed a stable and strong charge transfer band (λmax= 724 nm) for Pae’s Etf upon reduction by NADH. Using a series of reductive titrations to probe bounds for the reduction midpoint potential of the two flavins, we argue that the heterodimer alone could participate in a bifurcation mechanism.

NRs are enzymes that catalyze the reduction of nitroaromatics to their corresponding nitroso, hydroxylamine, and, in limited cases, amine They have gathered interest in many scientific communities, and are currently actively researched bioremediation and prodrug activation. Here we attempt to utilize them for the purpose of synthesizing substituted aromatic amines that are found in a number of active pharmaceutical ingredients (APIs). As NRs described in the literature have varying product distribution ranges (from those that produce hydroxylamine to others that yield amine) several similar and different NRs were studied for their selectivity. Additionally, a quantitative structure-activity relationship (QSAR) was determined to characterize the substrate specificity of NRs. To employ the use of flavoenzymes in synthesis, multiple reaction- and protein-engineering approaches were devised. One scheme was to establish an enzymo-chemical synthesis where NRs were paired with reducing agents for a chemical reduction. Another method was to create a monomeric NR through directed evolution from ER scaffolds for future immobilization applications. Protein engineering techniques were also utilized on NADH oxidases which we characterized and developed for nicotinamide cofactor regeneration. As a whole, this dissertation expands our current understanding on NRs and demonstrates the possibility of using several flavoenzymes in the synthesis of organic molecules.

Nitroreductase from Enterobacter cloacae (NR) is a member of a large family of homologues represented in all branches of the tree of life. However the physiological roles of many of these enzymes remain unknown. NR has distinguished itself on the basis the diverse sizes and chemical types of substrates it is able to reduce (Koder et al 1998). This might be an evolved characteristic suiting NR for a role in metabolism of diverse occasional toxins. While there are numerous studies of determinants of substrate specificity, we know less about mechanisms by which enzymes can be inclusive. Therefore, we present a synthesis of NR's dynamics, stability, ligand binding repertoire and kinetic mechanism. We find that NR reduces para-nitrobenzoic acid (p-NBA) via a simple mechanism limited by the chemical step in which the nitro group is reduced (Pitsawong et al 2014). Thus, for this substrate, NR's mechanism dispenses with gating steps that in other enzymes can enforce substrate specificity. Our data demonstrate that substrate reduction is accomplished by rate-contributing hydride transfer from the flavin cofactor coupled to proton transfer from solvent, but do not identify specific amino acids with a role. This is consistent with our crystal structures, which reveal a spacious solvent-exposed active site bounded by a helix that moves to accommodate binding of substrate analogs (Haynes et al 2002). Because it is able to reduce TNT (trinitrotoluene), herbicides and pesticides, NR has important potential utility in bioremediation.

La réduction des liaisons azo (N=N) et des fonctions nitro (NO2) chez les bactéries est liée aux azoréductases et nitroréductases, enzymes catalysant ces réactions. Leur répartition homogène permet de les utiliser pour détecter les bactéries. Cette étude vise à confirmer leur intérêt comme marqueurs métaboliques, à optimiser les réactions enzymatiques pour la détection bactérienne et à approfondir la compréhension du lien entre l’activité nitroréductase et la résistance aux nitrofuranes.L’activité enzymatique est dépendante de la quantité d’enzyme, l’hypothèse d’une augmentation de la quantité de protéines pour accroitre l’activité et optimiser la détection des microorganismes a été testée. Le suivi de la transcription de gènes d’intérêt a permis d’observer une induction de leur transcription par des composés activateurs des mécanismes de régulation et certains substrats. L’ajout d’un inducteur transcriptionnel permet donc d’optimiser l’activité envers certains substrats et par conséquent la détection des bactéries. Ces études ont aussi contribué à identifier des liens entre structure moléculaire des substrats et capacité d’induction.La création et la caractérisation de différents mutants ayant des activités nitro- ou azoréductases altérées a permis de cribler de nouveaux substrats synthétiques en vue de leur utilisation dans des applications de diagnostic. Enfin, l’obtention et l’analyse génomique de souches obtenues par mutations aléatoire et ayant une résistance accrue à la nitrofurantoïne a mis en évidence un nouveau mécanisme de résistance, mécanisme qui apparaît également dans des isolats cliniques / Azo bond (N=N) and nitro reduction in bacteria is linked to the catalytic activities of azoreductase and nitoreductase enzymes, respectively. Their ubiquitous distribution enables their use for bacterial detection. This study aims to confirm their potential as metabolic markers, to optimize the enzymatic activities for improved detection and to generally enhance our understanding of azoreductase and nitroreductase activity . Enzymatic activity relies in part on protein quantity, increasing protein amount was tested as a solution for increased activity and improved bacterial detection. For that, the transcription of selected genes was followed in the presence of regulation system activators and substrates. Some of these compounds promoted an overexpression of selected enzymes and led to a better activity toward some substrates. Consequently, increasing the enzyme amount, through over transcription, can enable a better detection of bacteria. This study also contributed to the determination of structural elements required for induction by the substrate.Mutants with lowered azo or nitro reductase activity were constructed and used to screen new synthetic substrates which could be of use for in vitro diagnostic tests. Finally, strains with a high resistance to nitrofurantoin were obtained by random mutagenesis. Their genome analysis provided evidence for a new resistance mechanism, which can also be detected in clinical isolates

Sleeping sickness, caused by Trypanosoma brucei, is a deadly disease that affects some of the poorest countries in sub-Saharan Africa. Although the disease prevalence is declining, strengthening of the current control efforts, including introduction of more adequate chemotherapeutic options, is needed to prevent the re-emergence of yet another epidemic. Nitroaromatic compounds, such as nifurtimox (in combination with eflornithine) and fexinidazole (in clinical trials), have been recently introduced for the treatment of the second stage of sleeping sickness. These compounds are believed to act as pro-drugs that require intracellular enzymatic activation for antimicrobial activity. Here, the role of the bacterial-like nitroreductase TbNTR as a nitrodrug activating enzyme is examined through overexpression and knock-out studies in T. brucei. Multiple attempts to purify soluble recombinant TbNTR from E. coli were unsuccessful, because the recombinant protein was found to be membrane associated. In keeping with the role of TbNTR in nitrodrug activation, loss of an NTR gene copy in T. brucei was found to be one, but not the only, mechanism that may lead to nitrodrug resistance. Furthermore, in the bloodstream form of T. brucei, resistance was relatively easy to select for nifurtimox, with no concurrent loss of virulence and at clinically relevant levels. More worryingly, nifurtimox resistance led to a decreased sensitivity of these parasites to other nitroaromatic compounds, including a high level of cross-resistance to fexinidazole. Conversely, generation of fexinidazole resistance resulted in cross-resistance to nifurtimox. Should these findings translate to the field, emerging nitrodrug resistance could reverse all recent advances in the treatment of sleeping sickness, made since the introduction of eflornithine 20 years ago. Therefore, all efforts should be made to ensure nitroaromatic drugs are used only in drug combination therapies against sleeping sickness, in order to protect them from emerging resistance.

Submitted by JEFFERSON HONORIO FRANCO null (jeffersonhfranco@gmail.com) on 2016-12-01T15:16:17Z No. of bitstreams: 1 TESE FINAL-IMPRESSÃO CD.pdf: 4568825 bytes, checksum: 666b30b163102c69eb93110502678419 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-12-05T12:50:59Z (GMT) No. of bitstreams: 1 franco_jh_dr_araiq_par.pdf: 1149871 bytes, checksum: 4c942fc016c94499d690f6474dda7078 (MD5) / Made available in DSpace on 2016-12-05T12:50:59Z (GMT). No. of bitstreams: 1 franco_jh_dr_araiq_par.pdf: 1149871 bytes, checksum: 4c942fc016c94499d690f6474dda7078 (MD5) Previous issue date: 2016-11-21 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Corantes sintéticos do tipo azo têm sido um assunto de grande preocupação ambiental devido ao potencial genotóxico e mutagênico dos produtos de biotransformação. Deste modo, nos últimos anos a consequência da ingestão destes corantes presentes na agua potável servida à população é discutida por diversos autores. Este estudo avalia a ação de microssomas de fígado de rato, enzimas redutoras produzidas pela bactéria Escherichia coli (E. coli) e nitroredutase imobilizada na biotransformação de três corantes dispersos que possuem grupos azo, Disperse Red 73 (DR 73), Disperse Red 78 (DR 78) e Disperse Red 167 (DR 167). A técnica de Espectrofotometria de absorção molecular na região do Uv- visível, Cromatografia Líquida de Alta Eficiência com detector de arranjo de diodos (CLAE-DAD) e Cromatografia líquida acoplada à espectrometria de massas (LC-MS/MS) foram técnicas usadas para identificar os principais produtos gerados após os processos de degradação dos corantes. Polímeros de impressão molecular magnéticos (MMIPs) foram investigados usando reações de polimerização por precipitação para pré-concentração do corante DR 73, juntamente com a degradação por fotoeletrocatálise e subsequente análise dos produtos por LC-MS/MS. Os estudos in vitro do metabolismo de biotransformação dos corantes têxteis com microssoma de fígado de rato mostraram que as reações ocorreram preferencialmente no grupo azo e nitro dos corantes, indicando a redução destes grupos pelas enzimas do citocromo P-450. Foram obtidos dois produtos de degradação para cada corante após reação com a bactéria E. coli; o corante DR 73 originou os produtos 3-((4-aminofenil)(etil)amino)propanitrila e 4-nitroanilina, os produtos 3-((4-aminofenil)(etil)amino)propanitrila e 2-cloro-4-nitroanilina foram obtidos após reação com o corante DR78 e o DR 167 originou dimetil 3,3`-((3-acetamido-4aminofenil)azanediyl)dipropanoato e 2-cloro-4-nitroanilina, indicando a clivagem do grupo azo, possivelmente, pela enzima azoredutase, produzida pela bacteria. A enzima nitroredutase, imobilizada em partículas magnéticas modificadas com tosil, mostrou que a redução dos corantes ocorreu preferencialmente no grupo nitro, enquanto que a enzima livre no meio reacional resultou em mais de um produto de biotransformação para cada corante, atuando em mais de um sítio da molécula, comprovando a eficácia da imobilização enzimática para estudos de biotransformação e formação de produtos majoritários. A mutagenicidade dos corantes foi avaliado pelo ensaio de Salmonella/microssoma realizado nas estirpes TA 98 e TA 100, com e sem S9. De acordo com este ensaio, DR 73 foi o mais mutagênico. O MMIP para o corante DR 73 apresentou excelentes valores de religação (16 mg g−1 e 6 mg g−1, para MMIP e MNIP, respectivamente) indicando que o polímero molecularmente impresso formou cavidades específicas para retenção do corante. Através dos resultados obtidos por LCMS/MS, observou-se 100% de degradação do corante em apenas 60 min de tratamento via fotoeletrocatálise para soluções mais diluidas do mesmo, comprovando a eficiência da técnica na degradação de poluentes. Sendo assim, estes resultados sugerem que o MMIP mostrou uma excelente especificidade e seletividade para o corante DR 73 e uma técnica promissora na captação de corantes mutagênicos de águas superficiais, com grande potencial de aplicação e exploração na pré-concentração antes do tratamento. Além disso, a redução destes corantes por sistemas biológicos representa uma grande preocupação ambiental devido ao aumento da genotoxicidade para os seres vivos, em especial a seres humanos, produzindo compostos nocivos, tais como aminas condenadas pela Agência Internacional de Pesquisa sobre o Câncer. / Synthetic azo dyes have been a matter of great concern due to the genotoxic and mutagenic potential of the products originating from azo dye biotransformation. Thus, in recent years the result of the intake of these dyes present in drinking water supplied to a population is discussed by several authors. This work evaluates the action of rat liver microsomes, reducing enzymes produced by the Escherichia coli (E. coli) and nitroreductase immobilized on biotransformation of three disperse dyes bearing azo groups, namely Disperse Red 73 (DR 73), Disperse Red 78 (DR 78), and Disperse Red 167 (DR 167). UV-Vis spectrophotometry, high-performance liquid chromatography with diode array detector (HPLC-DAD), and liquid chromatography coupled to mass spectrometry (LC-MS/MS) were techniques used to identify the main products generated after the process degradation of dyes. Magnetic molecularly imprinted polymers (MMIPs) were investigated using precipitation polymerization reactions for preconcentration of the dye DR 73, together with the photoelectrocatalysis degradation and subsequent analysis of the products by LC-MS/MS. In vitro studies of biotransformation metabolism of textile dyes with rat liver microsome showed that the reactions occur preferentially in the group of azo and nitro dyes, indicating the reduction of these groups by enzymes of the cytochrome P-450. There were obtained two degradation products for each dye after reaction with E. coli; the dye DR 73 gave the product 3 - ((4-aminophenyl) (ethyl) amino) propanitrila and 4-nitroaniline, the product 3 - ((4-aminophenyl) (ethyl) amino) propanitrila and 2-chloro-4-nitroaniline were obtained after reaction with the dye DR78 and DR 167 gave 3,3`-dimethyl-((3-acetamido-4-aminophenyl) azanediyl) dipropanoato and 2chloro-4-nitroaniline; indicating cleavage of the azo group, possibly by azoredutase enzyme produced by bacteria. The nitroreductase enzyme immobilized on modified magnetic particles Tosyl showed that the reduction of dyes occurred preferentially in the nitro group, while the free enzyme in the reaction medium resulted in more than a product of biotransformation for each dye, acting in more than one site of the molecule, proving the efficacy of enzyme immobilization for biotransformation studies and formation of major products. The mutagenicity of the dyes was evaluated by the Salmonella/microsome assay performed on strains TA 98 and TA 100, with and without S9. According to this assay, DR 73 was the most mutagenic. The MMIP to the dye DR 73 showed excellent rebinding values (16 mg g−1 and 6 mg g−1, for MMIP and MNIP, respectively) indicating that the molecularly imprinted polymer formed cavities for specific dye retention. Through the results obtained by LC-MS/MS, it was observed 100% dye degradation in 60 min treatment for more dilute solutions thereof, proving the efficiency of technique in pollutant degradation. Thus, these results suggest that MMIP showed excellent specificity and selectivity for the dye DR 73 and a promising technique in capturing mutagenic dyes of surface water, with great potential for application and operation in the pre-concentration before treatment. Moreover, the reduction of these dyes by biological systems is a major environmental concern due to increased of genotoxicity for living beings, especially humans, producing harmful compounds, such as condemned amines by the International Agency for Research on Cancer.