FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / The aim of this work was to study the electrochemical behavior of antifungal ketoconazole on polished Silver Solid Amalgam Electrode (p-AgSAE).The study began with the evaluation of the best electrolyte for ketoconazole (KC) electrochemical reduction and the BrittonâRobinson buffer solution at pH 12 was selected as the best support electrolyte. In the following, with a constant ketoconazole concentration (1.0x10-5 mol L-1) the square wave voltammetric parameters were optimized for the p-AgSAE. The best experimental responses were achieved with 100 s-1 frequency, 25 mV of amplitude and 2 mV of scan increment. The results showed that ketoconazole is reduced in a quasi-reversible process, with one reduction peak at -1.50 V vs. Ag/AgCl/Cl- reference system. After optimized the SWV parameters, the analytical studies were developed in accord to ANVISA for validation the methodology. Linearity range, detection and quantification limits, precision, robustness and accuracy were evaluated. An analytical curve was constructed, that presented a linear region to 4.97x10-7 a 4.30x10-6 mol L-1 with a correlation coefficient to 0.9989. The detection limits for the concentration range were determined as 63.4 μg L-1 while the quantification limits were to 211.5 μg L-1. Studies of accuracy of the methodology were made in different formulations: tablet, cream and shampoo. The values obtained were to 100.8% for the electrolyte, 91% for the tablet, 104% for the cream and 103% for the shampoo. The Relative Standard Deviation (RSD) for the accuracy was less than 5.0%. Quantum chemical calculations were also made with the aim of studying the electronic characteristics of the molecule, and predicting which region of the molecule occurs a electrochemical reduction. It was investigated the distribution of charge on the molecule and the molecular orbital (HOMO and LUMO), which are possible sites of oxidation and reduction, respectively. The LUMO was located on the imidazole ring. In the sequence of the mechanistic elucidation, it was made an exhaustive electrolysis (E = -1,8V during six hours) with monitoring by UV-Vis, being used in the studies that identify the products, the technique of nuclear magnetic resonance espectroscopy, NMR H1. In accord to the study, the reduction of KC occur between the C-N bond on the imidazole ring. / No presente trabalho foi investigado o comportamento eletroquÃmico do antifÃngico Cetoconazol (CTZ) sobre o Eletrodo SÃlido de AmÃlgama de Prata (p-AgSAE). A otimizaÃÃo das condiÃÃes de reduÃÃo foi obtida por voltametria de onda quadrada (VOQ), onde foram analisadas as condiÃÃes mais adequadas para a realizaÃÃo dos experimentos. O meio de trabalho escolhido foi TampÃo BR 0,04 mol L-1. Foi avaliada a influÃncia do pH, onde o valor escolhido para estudar a reduÃÃo do analito foi 12. Os parÃmetros da VOQ otimizados foram frequÃncia (100 s-1), amplitude (25 mV) e incremento de potencial (2 mV). De acordo com os resultados, foi observado que o CTZ sofre um processo de reduÃÃo em -1,5 V vs. Ag/AgCl/Cl-. A concentraÃÃo de CTZ utilizada nos estudos de otimizaÃÃo foi de 1,0x10-5 mol L-1. ApÃs estabelecimento dos parÃmetros experimentais, foram estudadas as figuras de mÃrito especificadas pela ANVISA para validaÃÃo de mÃtodos analÃticos na anÃlise de fÃrmacos. Assim, foram avaliados regiÃo de linearidade (4,97x10-7 a 4,30x10-6 mol L-1 com R = 0,9993), limites de detecÃÃo(1,19x10-7 mol L-1 - 63,4 μg L-1) e de quantificaÃÃo (3,98x10-7 mol L-1 - 211,5 μg L-1), precisÃo e robustez. Os estudos de exatidÃo da metodologia foram feitos em diferentes produtos comerciais: comprimido, creme e xampu. Para fins de comparaÃÃo e avaliaÃÃo de possÃvel influÃncia do meio eletroquÃmico na resposta, foi feito o estudo de exatidÃo utilizando curvas de recuperaÃÃo para o eletrÃlito de suporte, onde o valor da taxa de exatidÃo foi de 100,1%. Assim, os valores das taxas de exatidÃo para as amostras foram de 91% para a amostra de comprimido, 104% para a amostra de creme e 103% para a amostra de xampu. Os valores de RSD para as amostras foram inferiores a 5%. CÃlculos quÃmico-quÃnticos tambÃm foram feitos com o objetivo de estudar as caracterÃsticas eletrÃnicas da molÃcula, e prever qual regiÃo tem maior probabilidade de sofrer a reduÃÃo. De fato, foram investigadas as distribuiÃÃes de carga na molÃcula, bem como os orbitais de fronteira (HOMO e LUMO), que sÃo possÃveis sÃtios de oxidaÃÃo e reduÃÃo, respectivamente. O LUMO foi localizado sobre o anel imidazÃlico. Na sequÃncia da elucidaÃÃo mecanÃstica, foi feita uma eletrÃlise exaustiva (E = -1,8V durante seis horas) com acompanhamento por espectroscopia UVVis, sendo utilizada nos estudos de identificaÃÃo do produto a tÃcnica de espectroscopia de ressonÃncia magnÃtica nuclear, RMN H1. Com base neste estudo, foi constatado que o CTZ sofreu uma reduÃÃo na dupla ligaÃÃo entre C-N do anel imidazÃlico da molÃcula de CTZ.

Public performance auditing is a type of auditing government departments’ use to review three specific aspects (efficiency, effectiveness and economy) of a sector, department or project. Upon completion of a public performance audit, the relevant supreme audit institution releases a performance audit report. The purpose of this study is to determine the level content compliance in these public performance audit reports with the international performance audit standards ISSAI 3000-3001. This study compares the level of content compliance in South Africa, a developing countries against Australia, a developed country. A type of text analysis was used to collate the levels of content compliance in a total of fifty reports, twenty-five reports published by the Auditor General of South Africa (AGSA) and twenty-five reports published by the Australian National Audit Office (ANAO), during the period 2006 – 2013. The finding revealed that public performance audit reports published in Australia have a greater level of content compliance than reports published in South Africa. The results of the report further detail the content shortcomings in the reports reviewed. This study will assist both the AGSA and ANAO in highlighting content concerns in these performance audit report, which is the first step towards improvement.

Small heat shock proteins (sHSPs) are a ubiquitous family of molecular chaperones that play a vital role in maintaining protein homeostasis in cells. They are the first line of defence against the detrimental effects of cellular stress conditions like fluctuations in temperature, pH, oxidative and osmotic potentials, heavy metal toxicity, drought and anoxia. Many sHSPs are also constitutively expressed during developmental stages of different plant tissues. Members of this family are ATP-independent chaperones, with monomeric masses varying from 12-40 kDa. A characteristic feature of sHSPs is their ability to assemble into large oligomers, ranging from dimers to 48-mers. Under stress conditions, these oligomers dissociate and/or undergo drastic conformational changes to facilitate their binding to misfolded substrate proteins in the cell. This interaction prevents the substrate from aggregating during stress. When physiological conditions are restored, the substrates are transferred to other ATP-dependent heat shock proteins for refolding. Thus sHSPs do not refold their substrates, but instead prevent them from aggregating and maintain them in a „folding-competent‟ state. The clientele of sHSPs includes proteins with a wide range of molecular masses, secondary structures and pIs. This promiscuity has led to sHSPs occupying key positions in the protein quality control network. As molecular chaperones that protect proteins, sHSPs prevent disease. Concomitantly, mutations in sHSPs have also been linked to various human diseases. Till date, high resolution crystal structures are available only for 3 sHSP oligomers. This insufficiency of structural information has hindered our understanding of the mechanism of chaperone function, the link between the oligomeric status and chaperone activity, identification of substrate binding sites and the role of the flexible terminal segments in mediating both the oligomerization and chaperone function. We undertook structural and functional characterization of plant and bacterial sHSPs in order to address some of these questions. Chapter 1 of this thesis gives an overview of the sHSP family, with special emphasis on the oligomeric assemblies of sHSPs of known structures. We highlight what we know about this family through mutational studies, what is as yet unknown, and why it is important to study this family. Chapter 2 describes our efforts at structural and functional characterization of 5 sHSPS in rice, each targeted to a different organelle. We probed the role played by the N-terminal region in mediating oligomer assembly and in the chaperone activity of the protein. Rice sHSPs displayed a wide range of hydrodynamic radii, from 4 nm to 14 nm, suggesting that their oligomeric assemblies are likely to be diverse. In chapter 3, we discuss our attempts at the structural characterization of a bacterial sHSP, Aggregation suppressing protein A, or AgsA from Salmonella enterica. We obtained a high resolution crystal structure of the dimer of the core sHSP domain. We compared this dimer with other known sHSP dimers, reported the deviations that we observed and analysed the structure to account for these differences. We used this dimer structure to successfully obtain solutions for low resolution X-ray diffraction data for oligomers of different truncated constructs of AgsA. We observed that a C-terminal truncated construct formed an octahedral 24¬mer (4.5 Å resolution), whereas a construct truncated at both termini formed a triangular bipyramidal 18-mer (7.7 Å resolution), an assembly hitherto unobserved for any sHSP. A similar 18-mer was obtained when the C-terminal truncated construct was incubated with a dipeptide prior to crystallisation (6.7 Å resolution). The cryo-EM map of the wild type protein (12 Å resolution) could be fitted with a different 18-mer. The low resolution of the data pre-empted an atomic-level description of the interfaces of the assemblies. However, our work highlights the structural plasticity of this protein and probes the sensitivity of the oligomeric assembly to minor differences in construct length.