CO2 fixation is a thermodynamically and kinetically challenging process, but nature has its own way of transforming CO2 into diverse organic molecules. Of our particular interest is 2-oxoacid:ferredoxin oxidoreductase (OFOR) that catalyzes the anaerobic, reversible inter-conversion of 2-oxoacids and CO2, making use of a small electron-transfer protein, ferredoxin (Fd), as the redox partner. This dissertation characterizes OFORs and Fds from organisms that exhibit different metabolic patterns and investigates how the interplay of OFOR and Fd could impact the fate of CO2 metabolism, asking the question What controls the catalytic bias of OFOR for CO2 evolution versus fixation? The study of OFORs and Fds from Desulfovibrio africanus and Hydrogenobacter thermophilus through an electrocatalytic assay reveals that the reduction potential of Fd is possibly associated with the biological function of OFOR and that CO2 fixation requires a low-potential electron donor. The Fd from H. thermophilus (HtFd1) is used as a model to probe the factors that govern iron-sulfur cluster potential. The dependence of OFOR activity on Fd potential is systematically studied with HtFd1 and its molecular variants through the electrocatalytic assay and a coupled enzyme assay. The results suggest there is a Fd “potential optimum” for OFOR-catalyzed CO2 fixation. The study of a 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) and three Fds from Magnetococcus marinus MC-1 further highlights other factors such as the intramolecular electron-transfer within Fd and the electrostatic and hydrophobic interactions at the protein-protein interface in determining OFOR-Fd interaction. The characterization of an OGOR from M. marinus MC-1 (MmOGOR) also provides kinetic, structural and spectroscopic details for a CO2-fixing OFOR that contains only one iron-sulfur cluster. Overall, this work furthers the scientific understanding of how nature achieves CO2 fixation through supplying reducing equivalents and with enzymes as efficient catalysts, and how intermolecular electron-transfer mediated by protein-protein interaction could regulate enzyme catalysis. / 2019-10-08T00:00:00Z

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-11-07T10:28:17Z No. of bitstreams: 1 gisetyulianasanchezdelgado.pdf: 4087455 bytes, checksum: 74e12bffed9f492497ef465546d6ac98 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-11-09T14:36:17Z (GMT) No. of bitstreams: 1 gisetyulianasanchezdelgado.pdf: 4087455 bytes, checksum: 74e12bffed9f492497ef465546d6ac98 (MD5) / Made available in DSpace on 2017-11-09T14:36:17Z (GMT). No. of bitstreams: 1 gisetyulianasanchezdelgado.pdf: 4087455 bytes, checksum: 74e12bffed9f492497ef465546d6ac98 (MD5) Previous issue date: 2017-08-11 / Na última década, os complexos de Au(III) tornaram-se uma classe importante de compostos com promissores efeitos anticancerígenos. Geralmente, em condições fisiológicas, os complexos de Au(III) são bastante reativos devido à alta afinidade dos íons de ouro em relação aos nucleófilos celulares e devido à instabilidade através das reações redox intracelulares. Métodos da teoria do funcional da densidade (DFT) foram aplicados no presente trabalho para calcular o potencial de redução padrão de nove complexos organometálicos de Au(III) do tipo [Au3+(R-C^N^C)L]n, usando dados experimentais disponíveis como referência. Em geral, os métodos DFT levaram a resultados satisfatórios com um erro médio absoluto inferior a 170 mV. O funcional CAM-B3LYP em combinação com o modelo de solvatação SMD, foi superior aos resultados de wB97xD, M11-L e B3LYP, com um erro médio absoluto de 81 mV em relação ao par redox ferroceno/ferrocênio (Fc+/Fc). No entanto, apesar do maior erro encontrado para os resultados B3LYP, a tendência qualitativa encontrada na série de moléculas estudadas foi a mais próxima daquela observada experimentalmente, o que permitiu propor um modelo de escalonamento usando o potencial de redução experimental como variável dependente e a energia livre de Gibbs calculada como variável independente. A regressão linear foi estatisticamente aceitável (R2=0,8) no nível B3LYP levando a um erro de apenas 35 mV. Além disso, a metodologia “the variable-temperature H-atom addition/abstraction (VT-HAA)” foi aplicada para os pares redox com cargas não equivalentes (complexos contendo cloreto como ligante), levando a uma melhora significativa na previsão do potencial de redução. O erro médio absoluto foi de apenas 87 mV, inferior ao encontrado com o protocolo padrão (144 mV). Na sequencia, a reatividade dos complexos de Au(III) foi estudada abordando dois diferentes caminhos para a redução do complexo [Au3+(C^N^C)Cl], os quais incluem reações de troca de ligantes associadas a processos de redução na presença de bionucleófilos de interesse (H2O, CH3SH CH3SeH e meim-4). Os resultados DFT mostraram que o caminho de reação mais favorável passa em primeiro lugar através da troca do cloreto ( =2,59 M-1 s-1 para CH3SeH) e, em seguida, o metabólito de Au(III) é reduzido ao análogo correspondente de Au(I) implicando na abertura do quelato (ɛ˚=+0,33 V para o derivado de CH3SeH). / In the last decade, Au(III) complexes become an important class of compounds with promising anticancer effects. Generally, under physiological conditions the Au(III) complexes are quite reactive due to the high affinity of gold ions toward cellular nucleophiles, and due to the instability through the intracellular redox reactions. Density functional theory (DFT) methods were herein applied to calculate the standard reduction potential of nine Au(III) organometallic complexes of the type [Au3+(R-C^N^C)L]n, using available experimental data as benchmark. Overall, the DFT methods lead to satisfactory results, with absolute error lower than 170 mV. The CAM-B3LYP functional in combination with the SMD solvation model was superior to wB97xD, M11-L and B3LYP results, with absolute error of 81 mV relative to ferrocene/ferrocenium (Fc+/Fc) redox couple. However, in spite of the larger error found for B3LYP results, the qualitative trend was closer to the observed one, which allowed the proposal of a scaling model using the experimental reduction potential as dependent variable and the calculated reaction Gibbs free energy as independent variable. The linear regression was statistically acceptable (R2=0.8) at B3LYP level and lead to an average error of only 35 mV. Besides, the variable-temperature H-atom addition/abstraction (VT-HAA) approach was applied for the redox couples with non-equivalent charges (complexes containing chloride as ligand), leading to a significant improvement in the reduction potential prediction. The mean absolute error was only 87 mV without any scaling procedure, which is much lower than found for standard approach, 144 mV. Moreover, the reactivity of Au(III) complexes was studied including two different pathways to the reduction of the [Au3+(C^N^C)Cl] complex, which involve ligand exchange reactions associated with reduction processes in the presence of bionucleophiles of interest (H2O, CH3SH CH3SeH e meim-4). The DFT results show that the most favorable reaction path passes firstly through chloride-exchange ( =2.59 M-1 s-1 for CH3SeH) and then the Au(III) metabolite is promptly reduced to the corresponding Au(I) analogue with ring-opening (ɛ˚=+0.33 V for CH3SeH derivative).

Submitted by ANA KARLA PEREIRA RODRIGUES (anakarla_@hotmail.com) on 2017-08-04T17:21:10Z No. of bitstreams: 1 arquivototal.pdf: 15443881 bytes, checksum: a81c2afaef7e1fccdeb2543bc340d5a1 (MD5) / Made available in DSpace on 2017-08-04T17:21:10Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 15443881 bytes, checksum: a81c2afaef7e1fccdeb2543bc340d5a1 (MD5) Previous issue date: 2015-08-10 / Nitroaromatic compounds derived from Morita-Baylis-Hillman reaction (RMBH) have been tested in the treatment of most neglected diseases such as malaria, Chagas disease and leishmaniasis. An important experimental observation is the relation between biological activity (measured by IC50) and reduction potential of these compounds (estimated by the cathodic and anodic peak potentials determined by electroanalytical techniques), the latter directly connected to the reduction of the nitro group (-NO2 ). For this reason, electrochemical methods have been used in order to mimic the enzymatic bioreduction of these compounds, as reported by Vasconcellos et al. (J. Braz. Chem. Soc. 23:894, 2012). The objective of this work was to develop a computational protocol to predict the reduction potential in aprotic media to support the molecular modeling of new compounds with desired pharmacological activity. The developed direct protocol (for aprotic solvents) consists of performing DFT calculations with B98, PBE1PBE or M06-2X functionals with 6-31+G(d,p) basis set and C-PCM solvation method (with standard cavitation method UFF/VdW). The results show that it is possible to predict the experimental variation of the reduction potential of at least 70 % of confidence (in a range of experimental data of only 140 mV) with absolute average errors less than 45 mV (much less than the experimental uncertainty of the absolute reaction potential of hydrogen electrode, approximately 400 mV) and standard deviation of about 35 mV (inferior to 1,0 kcal/mol). The application of direct protocol for a series of 65 uncorrelated molecules, whose reduction potentials vary in a range of more than 6 V, provided a model with more than 99% of predictive power. From the application of the protocol to a series of 40 molecules, for which experimental results are not available, it was possible to predict that some of these structures may have more favorable potentials to bioreduction process than the systems used in the calibration step, which makes them candidates for new drugs. / Compostos nitroaromáticos derivados da reação de Morita-Baylis-Hillman (RMBH) vêm sendo testados no tratamento de doenças extremamente negligenciadas, tais como malária, doença de Chagas e leishmanioses. Uma importante observação experimental consiste na relação entre a atividade biológica (medida pelo IC50) e o potencial de redução (estimado pelos potencias de pico anódico e catódico determinados por técnicas eletroanalíticas) destes compostos, este último diretamente ligado à redução do grupo nitro (-NO2). Por esta razão, métodos eletroquímicos têm sido utilizados com o intuito de simular a biorredução enzimática destes compostos, como reportado por Vasconcellos e colaboradores (J. Braz. Chem. Soc. 23:894, 2012). O objetivo deste trabalho foi o de desenvolver um protocolo computacional para a predição de potenciais de redução em meio aprótico para auxiliar a modelagem molecular de novos compostos com a atividade farmacológica desejada. O protocolo direto desenvolvido (para solventes apróticos) consiste na realização de cálculos DFT com os funcionais B98, PBE1PBE ou M06-2X, com o conjunto de funções de base 6-31+G(d,p) e método de solvatação C-PCM (com o método de cavitação padrão UFF/VdW). Os resultados mostram que é possível prever a variação experimental do potencial de redução com pelo menos 70 % de confiança (em uma faixa de valores experimentais de apenas 140 mV) e erros médios absolutos inferiores a 45 mV (muito inferior à incerteza experimental do potencial de redução absoluto do eletrodo de hidrogênio, que é de cerca de 400 mV) e desvio-padrão de cerca de 35 mV (inferior a 1,0 kcal/mol). A aplicação do protocolo direto a uma série de 65 moléculas não-correlacionadas, cujos potenciais de redução variam em uma faixa de mais de 6 V, forneceu um modelo com mais de 99 % de poder preditivo. A partir da aplicação do protocolo a uma série de 40 moléculas, para as quais ainda não estão disponíveis resultados experimentais, foi possível prever que algumas destas estruturas podem possuir potenciais mais favoráveis ao processo de biorredução que os estudados na etapa de calibração, o que as tornam candidatos à novos fármacos.

No description available.

Constructed wetlands and the soil filters are water treatment devices using the filter material that in most cases consists of natural river gravel, coarse sand or crushed stone. Master´s thesis deals with the research of cleaning effect of unconventional filtration materials, or more precisely with examination to load real waste water in a filtration column, to prove their ability to be used as an alternative medium that can ensure better cleaning effect. For the research work following metallurgical industry materials were used: fine slag and coarse slag 8-16 mm, materials from the natural character: zeolite, keramzit, a plastic material: PUR-foam. Also the traditional materials of fine gravel and coarse gravel were tested to compare the cleaning efficiency. Both the hydraulic and the cleaning efficiency of all above mentioned materials were tested. The examined hydraulic parameters are particularly porosity, hydraulic conductivity and suspended solids, turbidity and oxidation-reduction potential of both the inflow and outflow of the filter columns were determined. Accredited laboratory set the inflow and outflow of the filter columns BOD5, CODCr, total phosphorus, total nitrogen, nitrite, nitrate, ammonia and dissolved oxygen. By evaluating the results of the measurements it is determined which materials are suitable to be applied in the constructed wetlands and the soil filters. This filtration column was modeled by program HYDRUS and the result was compared with real results.

Philosophiae Doctor - PhD / The aim of this thesis is first, to evaluate various culture conditions to improve embryo development, and secondly, to analyze spent culture media for any biomarkers that may be predictive of embryo health. Single-step and sequential culture media were compared in both Planer and EmbryoScope™ incubators. Single-step media resulted in better blastocyst development compared to sequential media and the EmbryoScope™ incubation system showed slight improvements in embryo development than the Planer system. The benefits of supplementing the culture medium with either insulin or insulin-like growth factor 1 (IGF-1) or culturing in a 2% O2 environment, using two different strains of mice (hybrid and C57), as well as the suitability of these strains for quality control were compared. In insulin, hybrid embryos were slower to blastulate and had a lower blastocyst rate, whereas C57 embryos were slower to the morula and faster to blastocyst stages, and lower blastocyst rate than the controls. IGF-1 showed no difference in time-lapse morphokinetics (TLM) or blastocyst rates compared to controls in both hybrid and C57 embryos. Under 2% O2, hybrid embryos showed no significant difference in TLM up to the 8-cell stage, but slowed down afterwards, resulting in blastocysts with significantly lower cell counts than the 6% O2 group. The C57 embryos were slower to reach morula and expanded blastocyst, and had lower blastocyst rates in 2%O2 vs 6%O2. The C57 strain had significant slower overall embryo development for all time points than hybrid embryos in insulin, IGF-1 and ultra-low O2, as well as lower blastocyst rates. Measurement of growth differentiation factor 9 (GDF-9) and oxidation-reduction potential (ORP) in spent media as markers for embryo health were evaluated. Day 5 human blastocysts yielded higher pregnancy rates and GDF-9 levels in spent media compared to Day 6 blastocysts, but TLM parameters showed no impact on pregnancy outcome. In Day 6 blastocysts, the non-pregnant group showed significantly faster embryo development compared to the clinically pregnant group up to the 8-cell stage and start of blastulation. GDF-9 did not show any significant differences between non-pregnant and pregnant groups of Day 5 or Day 6 embryo transfers. ORP in spent media from good quality Day 3 embryos that developed into blastocysts were significantly higher than from those that did not, with no difference in control medium ORP. Spent media from arrested embryos showed lower ORP than their corresponding controls. Arrested embryos had slower development at syngamy, morula, blastulation and blastocyst stages. The single step medium in the EmbryoScope™ is the preferred choice for embryo culture. Insulin or IGF-1 media supplementation or 2% O2 culture did not provide any benefit to embryo development. The C57 mouse strain is more sensitive and may be better to detect changes in culture conditions, and therefore better model for quality control assays. GDF-9 values decrease from Day 5 to Day 6 which gives new insight to understanding the role of GDF-9 during embryogenesis. ORP in spent media indicate that embryos that developed into blastocysts did not contribute to ROS, but maintained ORP balance.