ATP Synthase is the fundamental means of cellular energy production in animals, plants, and almost all microorganisms. In order to understand the mechanism of ATP catalysis, critical amino acid residues involved in Pi binding have to be identified. The αVISIT-DG sequence at the interface of α/β subunits that contains residues from 345-351 is highly conserved and αAsp-350 has been chosen because of its negative charge side chain and its close proximity (~2.8 Å) to the known phosphate binding residue αArg-376. The mutant's αD350R, αD350Q, αD350A, αR376A/D, and αG351R/A/D were generated by site directed mutagenesis and several biochemical assays were performed on them to understand the role played by the amino acid residues in Pi binding. Biochemical results suggest that αD350 may be involved in catalysis of ATP synthase and play an important role in Pi binding, whereas αG351 may be involved only in the structural integrity.

Mycobacterium tuberculosis, the principal causative agent of tuberculosis (TB) in humans, is considered to be a successful pathogen owing to the elicitation of multidrug resistance, ability to survive inside macrophage phagosomes by taking nutrients from host cell cytoplasm, and the capacity to alternate between proliferating and dormant (nonproliferating) conditions of growth. Thus, whether one looks at tubercle bacillus from the standpoint of regulation of cell division in the host system, or uptake of nutrients from the host cell cytoplasm or elicitation of drug resistance, the requirement for ATP Binding Cassette (ABC) transporter type protein complexes, which might be involved in the transport of drugs, nutrients or proteins, could be of critical importance to the pathogen. Therefore the present study was initiated to characterize ABC transporter type proteins, FtsE and FtsX of M. tuberculosis (MtFtsE and MtFtsX), and their interaction with FtsZ and FtsQ, which are the septation proteins that are recruited respectively before and after the localization of FtsE and FtsX proteins. The study was carried out in 3 parts. 1. Cloning, overexpression and purification of MtFtsE and MtFtsX proteins and elucidation of ATP binding activity of MtFtsE There exists considerable extent of homology between the FtsE and FtsX proteins of M. tuberculosis and E. coli. Therefore, in order to verify whether the structural homology is reflected in functional homology, complementation of growth defect of E. coli ftsE (Ts) by MtFtsE and MtFtsX was carried out. The MtFtsE protein could partially complement growth defect of E. coli ftsE temperature sensitive strain MFT1181, whereas co-expression of MtFtsE and MtFtsX efficiently complemented growth defect, indicating that the MtFtsE and MtFtsX proteins functionally complement E. coli FtsE and FtsX and that the two proteins together might be performing an associated function. Subsequently, in order to biochemically characterize MtFtsE and MtFtsX proteins of M. tuberculosis, MtftsE gene was cloned in pQE30, overexpressed, purified by Ni2+-NTA agarose affinity chromatography under denaturing conditions and refolded. MtFtsX protein, being toxic to E. coli cells, could not be expressed to sufficient amounts. Western blotting with anti-MtFtsE antibody showed that the recombinant 6xHis-MtFtsE protein and the native MtFtsE protein were localized to the membrane of E. coli and M. tuberculosis cells respectively. 6xHis-MtFtsE protein showed ATP binding in vitro, whereas K42R mutation abolished ATP binding. Thus, like in the case of E. coli FtsE, the K42 residue, which is positionally equivalent to K41 in EcFtsE in Walker A motif, was found to be essential for ATP binding. At 1.3 nM concentration of [α32P] ATP,70 molar excess of ATP, ADP, AMP, and GTP competed out respectively 97%, 87%, 73% and 57% of the [α32P] ATP bound to 6xHis-MtFtsE. 2. Biochemical characterization of MtFtsE protein The functional architecture of an ABC transporter consists of two each of nucleotide binding domain (NBD) and transmembrane domain (TMD), which are either part of a single polypeptide chain or individual subunits. The functional NBD is a ‘nucleotide-sandwich dimer’ with ATP flanked by the Walker A and B motifs of one NBD and the signature motif and D-loop of the other. NBD, through ATPase activity, is involved in energizing the transport of substrates namely drugs, proteins, ions, and solutes across the membrane. Since MtFtsE possesses Walker A and Walker B motifs that constitute NBD, and MtFtsX possesses TMD (four transmembrane segments), the two proteins together might constitute an ABC transporter type complex. Therefore, we wanted to know whether MtFtsE could hydrolyze ATP. MtFtsE not only could bind ATP with high affinity but could hydrolyse it also (Km, 1.5 µM; Vmax, 0.87 nmole/mg/min). It could bind and hydrolyse GTP as well, but not CTP, albeit with lower affinity and rate (Km, 25 µM; Vmax, 0.54 nmole/mg/min). The ATPase activity is strongly dependent on Mg2+ or Mn2+, with a pH optimum of 6.5 – 8.0 and temperature range of 27oC - 40oC. Kinetic analysis of ATPase and GTPase activities indicated nucleotide- dependent cooperativity (Hill coefficient for ATP is 1.7 and for GTP, 2.1). Inhibition of ATPase activity, to almost similar extent, in the presence of 10-fold excess of ATPγS, ADP, AMP, GTP, and CTP, but not TTP, indicated that nucleotide binding is through nitrogenous base of the nucleotide. Inhibition of MtFtsE by orthovanadate classified the enzyme as a P-type ATPase. Partially purified MtFtsE in soluble fraction also showed ATPase activity. The ATPase-active form of MtFtsE is a dimer with the sole cysteine (C84) at the dimer interface. Homology modeling of MtFtsE, using MalK (the NBD component of an ABC transporter for maltose) as the template, supported this observation. Stabilization of the dimer through cys-cys disulphide bond increased ATPase activity by 3.7-fold, although C84 does not have any role in ATPase activity. 3. Identification and elucidation of interaction among cell division proteins FtsE, FtsX, FtsQ and FtsZ of Mycobacterium tuberculosis Septum synthesis in E. coli is mediated by a dozen of proteins, among which the bacterial cytoskeletal protein FtsZ is the first molecule to localise to the mid-cell site, where it forms a scaffold for the localization of downstream cell division proteins namely, FtsA /ZipA < FtsE / FtsX < FtsK < FtsQ < FtsL < FtsB < FtsW < FtsI < FtsN and AmiC. If the above order of recruitment of proteins holds true for M. tuberculosis as well, the immediate proteins recruited to the mid-cell site after MtFtsZ in M. tuberculosis would be MtFtsE and MtFtsX, followed with MtFtsK and MtFtsQ. Thus it is possible that MtFtsE and MtFtsX could be interacting with MtFtsZ and MtFtsQ. Therefore attempts were made to delineate the interaction network among MtFtsE, MtFtsX, MtFtsQ and MtFtsZ of M. tuberculosis. Ni2+-NTA agarose pulldown, co-immunoprecipitation and bacterial two-hybrid assays using wild type and deletion mutants of the proteins showed that MtFtsE interacts with MtFtsQ and MtFtsX through its C-terminus. In addition, MtFtsX could interact with MtFtsZ and MtFtsQ. MtFtsX was found to homodimerise and interact with MtFtsQ in vivo. The ATPase-active of MtFtsE in vivo being a dimer, a hypothetical model for the translocation of MtFtsQ into the membrane at mid-cell site was proposed. According to this model, MtFtsQ might be inserted into the membrane at the mid-cell site by (MtFtsX)2 functioning as the membrane channel for the transport, which could be energized by the ATPase subunit (MtFtsE)2 of the (MtFtsE)2(MtFtsX)2 complex. MtFtsX might have a role in tethering the FtsZ-ring with the membrane at the mid-cell site. An altogether different possibility could be that the (FtsE)2(FtsX)2 complex might have a role in the stabilization or constriction of FtsZ-ring during the inward growth of septum.

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Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The search for solutions to the various problems of power quality, especially to voltage deviation from the standard values, account today, with an extensive range of products aiming above all the dynamic regulation of the supply voltage. Despite this recognition, the challenges to find alternatives technologies that take into account the use of compensators with more attractive operational and economic properties, are still motivating research worldwide. Among the devices based on the principle of consumption or supply of reactive power, in the 60 s came a product design nominated by Reactor Core Saturated Compensator- CERNS. This device is constituted by an inductive unit, comprising a magnetic core and windings with special features, connected in parallel with a capacitor unit. Although this equipment has been widely used in the past, the lack of publications on the subject resulted in a large gap in knowledge domain and dissemination of this technology. This fact contributed to a demand for dedicated efforts in recent years by national and international researchers. In the light of the attractions offered by this philosophy for voltage regulators, the subject has reawakened the interest for research and developments about the project, construction and operation of these compensators, thus aiming the technology domain. On the above, added to efforts previously made by national institutions, arises the present thesis aimed at providing a computational program to assist in the specification and basic design of saturated core reactors and means for determination of the equivalent electrical parameters needed to the model of the equipment proposed and implemented in the ATP simulator. Finally, performance studies are carried out to highlight and to validate the operational efficiency and computational model developed. / A busca por soluções para os distintos problemas da qualidade da energia elétrica, com destaque às variações das tensões de suprimento, conta, na atualidade, com uma extensa gama de produtos visando, sobretudo, a regulação dinâmica da tensão de suprimento. Não obstante tal reconhecimento, os desafios por estratégias alternativas em que pese o emprego de compensadores com propriedades operacionais e econômicas mais atrativas continuam motivando pesquisas em todo o mundo. Dentre os dispositivos fundamentados no princípio do consumo ou fornecimento de potências reativas, nos anos 60 surgiu uma concepção de produto, denominado Compensador a Reator a Núcleo Saturado CERNS. Este é composto, fundamentalmente, por um núcleo magnético com características e enrolamentos especiais e capacitores conectados em paralelo. Embora este equipamento tenha sido amplamente utilizado no passado, a ausência de publicações sobre o tema resultou numa grande lacuna para o conhecimento, domínio e difusão desta tecnologia. Tal fato corroborou para uma demanda de esforços dedicados nos últimos anos no âmbito nacional e internacional e, à luz dos atrativos oferecidos pela presente filosofia para os reguladores, o assunto voltou a despertar interesses para as pesquisas e desenvolvimentos visando o domínio da tecnologia para o projeto, construção e operação destes compensadores. Diante do exposto, somado a esforços já alcançados através de desenvolvimentos anteriores realizados por instituições nacionais, surge esta tese, a qual encontra-se fundamentada no desenvolvimento de uma estratégia computacional para o dimensionamento do compensador, projeto básico do reator saturado e análise de desempenho no domínio do tempo. Para tanto é utilizada a plataforma ATP, de domínio público e aceitação comprovada no cenário nacional. Por fim, estudos diversos de desempenho são conduzidos para a validação da eficácia operacional e do modelo computacional desenvolvido. / Doutor em Ciências

Rare diseases are a heterogeneous group of disorders. Knowledge of their molecular basis is poor and till recently there were no appropriate methodical approaches due to a limited number of patients. Novel genomic techniques, especially the DNA array technology and the next generation sequencing emerging in last few years, enabled studies of these diseases even in small families and sporadic cases. This PhD thesis focuses on application of novel genomic techniques in studies of rare inherited diseases. It describes a use of DNA array technology in linkage analysis, analysis of differential gene expression, analysis of copy number variations and homozygous mapping, and a use of next generation sequencing technology. Combination of these methods was used for identification of molecular basis of adult neuronal ceroid lipofuscinosis, Rotor syndrome, isolated defect of ATP synthase and mucopolysaccharidosis type IIIC.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-31T15:07:36Z No. of bitstreams: 1 lucasvitorfonsecadeoliveira.pdf: 1420167 bytes, checksum: b5075b7ccc33c587012a46901a9b21af (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-02T12:44:37Z (GMT) No. of bitstreams: 1 lucasvitorfonsecadeoliveira.pdf: 1420167 bytes, checksum: b5075b7ccc33c587012a46901a9b21af (MD5) / Made available in DSpace on 2016-07-02T12:44:37Z (GMT). No. of bitstreams: 1 lucasvitorfonsecadeoliveira.pdf: 1420167 bytes, checksum: b5075b7ccc33c587012a46901a9b21af (MD5) Previous issue date: 2012-03-30 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Com a abertura legal do setor elétrico brasileiro na década de 90 para investimentos privados, em especial com a implantação sistemática de leilões para definição dos concessionários de transmissão, diversos estudos foram efetuados com o objetivo de viabilizar projetos de linha de transmissão de modo a concorrem nesses leilões. Grandes transmissoras, como Furnas, Chesf e Eletronorte promoveram pesquisas em consórcio com empresas construtoras de linhas e fabricante de ferragens e isoladores, com o objetivo de desenvolverem projetos com baixa relação Reais/MW transmitido. Neste cenário, muitos projetos de linhas de potência natural elevada foram idealizados e implantados, e como fruto desses projetos, publicações foram realizadas descrevendo os resultados obtidos. Todavia, apesar da quantidade, qualidade e riqueza das informações publicadas, a descrição do processo de otimização dos parâmetros elétricos da linha, em especial da impedância característica, não receberam destaque, sendo muita das vezes suprimida nos artigos. Neste sentido, este trabalho propõe a investigação de um método inovador de otimização da capacidade de transmissão de linhas de potência natural elevada, através da análise indireta dos efeitos da variação do posicionamento dos cabos condutores no feixe sobre a impedância característica da linha, por meio de uma abordagem original que utiliza basicamente o vetor de Poynting como função objetivo do Método de Otimização por Enxame de Partículas, sendo os campos elétricos e magnéticos necessários para definição do vetor de Poynting calculados por meio do Método dos Elementos de Contorno. O método foi validado utilizando como exemplos a otimização das configurações de feixes da linha de 500 kV Interligação Norte/SUL III – Trecho 2 e Linha de 500 kV Presidente Dutra / Teresina / Sobral / Fortaleza. Foram encontrados ganhos na capacidade de transmissão de 7% e 22% respectivamente devido à redução da impedância característica calculada após a otimização através da rotina Line Constants do programa ATP/EMTP, e comparando-a com os valores originais. / With the legal opening for private investments in the Brazilian energy sector in the 90's, in particular the systematic implementation of auctions for defining power transmission concessions, several studies were made with the aim of developing transmission line projects in order to compete in these auctions. Major transmission companies such as Furnas, Eletronorte and CHESF promoted consortium research with line builders and hardware and insulators manufacturers, in order to develop projects with low cost/MW transmitted. In this scenario, many projects of high surge-impedance loading lines were developed and implemented, and as a result of these projects, publications were made describing the results. However, despite the quantity, quality and resourcefulness of published information, the descriptions of the optimization process of electrical line parameters, especially regarding characteristic impedance, were not given prominence, being often suppressed from the articles. Thus, this study proposes the investigation of a method for optimizing the transmission capacity of high surge-impedance loading lines, varying the power cables in the bundle, indirectly reducing its characteristic impedance through an original approach that uses basically the Poynting's vector as objective function of the Particle Swarm Optimization method.The electric and magnetic fields needed for defining the Poynting vector were calculated using the Boundary Element Method. The method was validated through the optimization of bundle configuration, using as a model the characteristics of the 500 kV line North / South Interconnection III - Segment 2 and the 500 kV line Presidente Dutra / Teresina / Sobral / Fortaleza. It was found transmission capacity gains of 7% and 22% respectively, by reducing the characteristic impedance, which was calculated after the optimization using the EMTP/ATP Line Constants Program, comparing it with the original values.

Gout is one form of inflammatory arthritis, which is caused by the precipitation of crystals of monosodium urate (MSU) in the joints. Acute gout is associated with sudden and painful inflammatory episodes characterized by high neutrophil infiltration. In spite of years of study gout treatment remains a challenge due to its relative ineficcacy. Thus, search for new and efficient therapies is necessary. The objective of this study was to investigate the involvement of glibenclamide in a model of acute gout in rats induced by MSU. MSU crystals produced nociception and edema when injected into the ankle joint of rats. Treatment with glibenclamide (3 mg/kg, s.c.) or dexamethasone (8 mg/kg, s.c., used as a positive control) decreased spontaneous nociception (67% ± 11 and 70 ± 7% inhibition, respectively) and edema (28 ± 7% and 77 ± 7% inhibition, respectively) induced 6 hours after MSU injection. The number of leukocyte infiltrates in the synovial fluid as well as the release of interleukin 1β (IL-1β) and prostaglandin E2 (PGE2) significantly increased at 6 hours after injection of MSU joint, but these effects were not reversed by treatment with glibenclamide (3 mg/kg, s.c.). In contrast, dexamethasone reduced the leukocyte infiltration and release of IL-1β and PGE2. To confirm if the dose of glibenclamide was able to block the KATP channels, we determined the levels of glucose in the blood of animals. Glibenclamide decreased (23 ± 2%) and dexamethasone increased the blood glucose of the rats compared to vehicle-treated animals / MSU. Therefoe, the effects of glibenclamide on nociception and edema induced MSU, suggests that this sulfonylurea may be an interesting option as an adjunct therapy in pain observed in acute attacks of gout. / A gota é uma forma de artrite inflamatória, causada pela precipitação de cristais de urato monossódico (MSU) nas articulações. A forma aguda de gota está associada a episódios inflamatórios súbitos e dolorosos caracterizados por uma grande infiltração de neutrófilos. Apesar dos anos de estudo sobre a gota, o seu tratamento ainda é um desafio pela relativa ineficácia dos fármacos disponíveis no mercado. Assim, a busca por novos agentes terapêuticos mais efetivos e seguros se faz necessário. Desta forma, o objetivo deste estudo foi investigar o possível potencial farmacológico da glibenclamida em um modelo de gota aguda induzida por MSU em ratos. Os cristais de MSU produziram nocicepção e edema quando injetados na articulação do tornozelo de ratos. O tratamento com glibenclamida (3 mg/kg, s.c.) ou dexametasona (8 mg/kg, s.c., usada como controle positivo) reduziu a nocicepção espontânea (67 ± 11% e 70 ± 7% de inibição, respectivamente) e o edema (28 ± 7% e 77 ± 7% de inibição, respectivamente) induzidos pelo MSU, 6 horas após a injeção do cristal. O número de leucócitos infiltrados no líquido sinovial, assim como a liberação de interleucina 1β (IL-1β) e de prostaglandina E2 (PGE2) foram consideravelmente aumentados, 6 horas após a injeção de MSU na articulação, porém esses efeitos não foram revertidos pelo tratamento com glibenclamida (3 mg/kg, s.c.). Em contrapartida, dexametasona reduziu a infiltração de leucócitos e a liberação de IL-1β e de PGE2. Para confirmar se a dose utilizada de glibenclamida foi capaz de bloquear os canais de KATP, foi avaliado os níveis de glicose no sangue dos animais. A glibenclamida reduziu (23 ± 2%) e a dexametasona aumentou a glicemia dos ratos quando comparado aos animais tratados com veículo /MSU. Assim, frente aos efeitos desempenhados pela glibenclamida sobre a nocicepção e edema induzidos pelo MSU, sugere-se que esta sulfonilureia possa ser uma opção interessante como um tratamento adjuvante na dor observada em ataques agudos de gota.