Biotin is an essential vitamin in plants and mammals functioning as the carbon dioxide carrier within central lipid metabolism. Biotin is composed of a fused bicylic ring system and a five carbon, carboxylic acid chain. Biotin biosynthesis in bacteria is catalysed by a series of enzymes that use fatty acid, amino acid and sulfur-containing substrates. In Bacillus subtilis, pimeloyl-CoA synthetase (PCAS, EC 6.2.1.14, UNIPROT code: P53559, 29.6 kDa) is the first enzyme in the biotin biosynthetic pathway and acts as a highly specific substrate selection gate ensuring the integrity of the carbon chain in biotin synthesis. PCAS catalyses the synthesis of the key acyl-thioester, pimeloyl-CoA in two steps; the first involves activation of pimelic acid (C7 dicarboxylic acid) using ATP to give an acyl-adenylate, enzyme-bound intermediate and pyrophosphate (PPi), and in the second step, this pimeloyl-adenylate reacts with coenzyme A (CoASH) to form the pimeloyl-CoA thioester. This thesis describes the results of biochemical, structural and mechanistic studies of B. subtilis PCAS. Recombinant PCAS was prepared by expressing the B. subtilis BioW gene in E. coli in various hexa-histidine affinity-tagged forms and the enzyme purified in high purity and yield. Enzyme activity and kinetic constants were measured using reverse-phase HPLC and enzyme coupled spectroscopic assays. These revealed the enzyme to have a strict carboxylic acid specificity. In collaboration with colleagues at the University of St. Andrews various commercial and in-house screens were used to obtain diffraction-quality crystals suitable for X-ray crystallography. This also included the generation of seleno-methionine (SeMet) labelled PCAS, as well as heavy-metal derivatives. Structures of B. subtilis PCAS in complex with the substrate pimelic acid and the pimeloyl-adenylate intermediate and product PPi were determined at 2.04 Å and 2.34 Å resolution respectively. The B. subtilis PCAS displays a novel 3D fold and defines a new class (Class IV) in the ANL superfamily of adenylate forming enzymes. The enzyme is a homodimer composed of two domains, a short N-terminus and a large C-terminal domain and the ligand-bound structures revealed the residues potentially involved in substrate specificity and enzyme catalysis. The enzyme uses an internal ruler composed of a number of conserved arginine residues (Arg213, Arg227 and Arg170) to select the correct dicarboxylic acid substrate. The X-ray structures guided the production of a number of site directed mutants to identify residues involved in the catalytic mechanism and stabilising the acyl-adenylate intermediate. This also allowed rational engineering of the PCAS active site to generate mutants with altered substrate specificity. Mutant PCAS Y211F was shown to synthesise both heptanoyl (C7) and octanoyl (C8) mono carboxylic acid-CoA and C8 dicarboxylic-CoA thioester products, highlighting the synthetic potential of PCAS. The PCAS pimeloyl-CoA product is the substrate for the next enzyme in the biotin pathway, a pyridoxal 5'phosphate (PLP)-dependent 8-amino 7-oxononanoate synthase (AONS). AONS catalyses the condensation of pimeloyl-CoA with L-alanine to give AON which is converted to biotin by the action of three other enzymes. We used genome mining to identify a putative ~66 kDa, bi-functional PCAS/AONS enzyme with an N-terminal PCAS domain fused to C-terminal AONS domain in the organism Corynebacterium amycolatum. A recombinant C. amycolatum PCAS/AONS fusion protein was expressed and purified from E. coli and initial studies suggest that it forms a functional, fused, dimeric enzyme.

Chlorinated paraffins (CPs) are ubiquitous environmental contaminants. They fulfill all of the criteria (persistent, toxic, and subject to long-range transport) for persistent organic pollutants (POPs) according to the United Nations Economic Commission for Europe (UNECE). CPs are also under consideration for inclusion in the Stockholm Convention on POPs. Their presence has been shown in various environmental matrices in the industrialized parts of the world, as well as in remote regions such as the Arctic. The aim of this thesis was to increase the limited knowledge of the presence of CPs in the environment, their sources to the environment, and the resulting human exposure. An analytical procedure for the determination of CPs in environmental samples based on gas chromatography coupled to electron capture detection (GC-ECD) has been developed. GC-ECD is a relatively inexpensive instrument that is fast and easy to operate. These advantages open up the possibility for a comprehensive screening of the occurrence of CPs in the environment, including developing countries. Furthermore, the occurrence of CPs in ambient air and in indoor air and dust was studied. Elevated CP concentrations in indoor air (<5-210 ng/m3) were observed compared to ambient air (0.7-33 ng/m3), which is indicative of the presence of indoor emission sources. Indoor air and dust concentrations were used to estimate the human exposure to CPs via the indoor environment. Comparison of the estimates to available dietary intake estimates indicated that the indoor exposure pathways are not negligible. CP concentrations in ambient air from urban Stockholm were higher than in rural Aspvreten, Sweden. This indicates the presence of additional (emission) sources in urban areas compared to rural sites. Additionally, a seasonal variation of air concentrations was observed at both locations, suggesting temperature dependent emission sources for CPs. These observations were supported by a substance flow analysis of CPs performed for Stockholm. This study estimated the major emission sources of CPs to the Stockholm environment to be emissions from painted surfaces and in-place sealants. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Manuscript.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-15T12:20:57Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese SILVA AMRB.pdf: 10197611 bytes, checksum: dfa95dac75e87b0ffef8a344cb8d9996 (MD5) / Made available in DSpace on 2016-07-15T12:20:57Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese SILVA AMRB.pdf: 10197611 bytes, checksum: dfa95dac75e87b0ffef8a344cb8d9996 (MD5) Previous issue date: 2015-01-30 / CNPq / Petrobrás / A aplicação de técnicas de Análises de Componentes Principais (ACP), Redes Neurais Artificiais (RNA), Lógica Fuzzy e Sistema Neurofuzzy para investigar as alterações da característica da água das barragens de Utinga e do Bita que abastecem de água bruta a ETA Suape é de fundamental importância em função do grande número de variáveis utilizadas para definir a qualidade. Neste trabalho, foram realizadas 10 coletas de água em cada área, no período de novembro de 2007 a agosto de 2012, totalizando 120 amostras. Ainda que o conjunto de dados experimentais obtidos seja reduzido, houve múltiplos esforços em demanda da aquisição de informações da qualidade da água junto aos órgãos oficiais de monitoramento ambiental. Os resultados mostraram uma tendência à degradação da propriedade da água das barragens em decorrência da presença de microrganismos, sais e nutrientes, responsáveis pelo processo de eutrofização, o que se configurou pela maior concentração de fósforo total, Coliformes termotolerantes, e diminuição de pH e OD, provavelmente devido à ocorrência de descarte de efluentes da agroindústria canavieira, industrial e doméstico. A ACP caracterizou mais 76% das amostras permitindo visualizar a existência de mudanças sazonais e uma pequena variação espacial d`água nas barragens. A condição da água das duas barragens foi modelada satisfatoriamente, razoável precisão e confiabilidade com os modelos estatístico e computacionais, para uma quantidade de parâmetros e dados ambientais, que embora limitados foram suficientes para realização deste trabalho. Ainda assim, fica evidente a eficiência e sucesso da utilização do Sistema Neurofuzzy (coeficiente de regressão de 0,608 a 0,925) que combina as vantagens das Redes Neurais e da Lógica Fuzzy em modelar o conjunto de dados da qualidade da água das barragens de Utinga e Bita. / The application of techniques such as the Principal Components Analysis (PCAs), Artificial Neural Networks (ANNs), Fuzzy Logic and Neuro-fuzzy Systems for investigating the changes in the water quality characteristics in the Utinga and Bita dams, which supplies raw water to the Suape Wastewater Treatment Plant (WWP), is of great importance due to the high number of variables used to define water quality. In this work were collected 10 water samples used to define water quality, in a period ranging from November 2007 to August 2012, with a total of 120 samples. Although the experimental dataset was limited, there were multiple efforts in gathering information from the Environmental Control Agencies. The results showed a tendency of degradation of the water properties in the dams studied due to the presence of microorganisms, salts and nutrients, responsible for the eutrophication process; result of the higher concentration of total phosphorus, Thermotolerant Coliforms and decrease in pH and DO, probably from the discharge of the sugarcane agroindustry and domestic waste. The PCAs characterised more than 76% of the samples collected, and consequently observing the existence of seasonal changes and small spatial variation of water levels in the dams. The water quality conditions in both dams were satisfactorily modelled, obtaining a reasonable precision and statistical and computational reliability for a certain amount of parameters and environmental data that, even though considered limited, were enough to run this trial. Nonetheless, it becomes evident the efficiency and success in using the Neuro- Fuzzy System (regression coefficient of 0.608 to 0.925), which combines the advantages of both the Neural Networks and Fuzzy Logic in modelling the water quality dataset in the Utinga and Bita dams.

Emissions from airport operations are of significant concern because of their potential impact on local air quality and human health. The currently limited scientific knowledge of aircraft emissions is an important issue worldwide, when considering air pollution associated with airport operation, and this is especially so for ultrafine particles. This limited knowledge is due to scientific complexities associated with measuring aircraft emissions during normal operations on the ground. In particular this type of research has required the development of novel sampling techniques which must take into account aircraft plume dispersion and dilution as well as the various particle dynamics that can affect the measurements of the aircraft engine plume from an operational aircraft. In order to address this scientific problem, a novel mobile emission measurement method called the Plume Capture and Analysis System (PCAS), was developed and tested. The PCAS permits the capture and analysis of aircraft exhaust during ground level operations including landing, taxiing, takeoff and idle. The PCAS uses a sampling bag to temporarily store a sample, providing sufficient time to utilize sensitive but slow instrumental techniques to be employed to measure gas and particle emissions simultaneously and to record detailed particle size distributions. The challenges in relation to the development of the technique include complexities associated with the assessment of the various particle loss and deposition mechanisms which are active during storage in the PCAS. Laboratory based assessment of the method showed that the bag sampling technique can be used to accurately measure particle emissions (e.g. particle number, mass and size distribution) from a moving aircraft or vehicle. Further assessment of the sensitivity of PCAS results to distance from the source and plume concentration was conducted in the airfield with taxiing aircraft. The results showed that the PCAS is a robust method capable of capturing the plume in only 10 seconds. The PCAS is able to account for aircraft plume dispersion and dilution at distances of 60 to 180 meters downwind of moving a aircraft along with particle deposition loss mechanisms during the measurements. Characterization of the plume in terms of particle number, mass (PM2.5), gaseous emissions and particle size distribution takes only 5 minutes allowing large numbers of tests to be completed in a short time. The results were broadly consistent and compared well with the available data. Comprehensive measurements and analyses of the aircraft plumes during various modes of the landing and takeoff (LTO) cycle (e.g. idle, taxi, landing and takeoff) were conducted at Brisbane Airport (BNE). Gaseous (NOx, CO2) emission factors, particle number and mass (PM2.5) emission factors and size distributions were determined for a range of Boeing and Airbus aircraft, as a function of aircraft type and engine thrust level. The scientific complexities including the analysis of the often multimodal particle size distributions to describe the contributions of different particle source processes during the various stages of aircraft operation were addressed through comprehensive data analysis and interpretation. The measurement results were used to develop an inventory of aircraft emissions at BNE, including all modes of the aircraft LTO cycle and ground running procedures (GRP). Measurements of the actual duration of aircraft activity in each mode of operation (time-in-mode) and compiling a comprehensive matrix of gas and particle emission rates as a function of aircraft type and engine thrust level for real world situations was crucial for developing the inventory. The significance of the resulting matrix of emission rates in this study lies in the estimate it provides of the annual particle emissions due to aircraft operations, especially in terms of particle number. In summary, this PhD thesis presents for the first time a comprehensive study of the particle and NOx emission factors and rates along with the particle size distributions from aircraft operations and provides a basis for estimating such emissions at other airports. This is a significant addition to the scientific knowledge in terms of particle emissions from aircraft operations, since the standard particle number emissions rates are not currently available for aircraft activities.