This study aimed at providing a thorough understanding of temporal and spatial variations of VOCs and underlying factors in different microenvironments in two different urban atmospheres, with different degrees of regulatory enforcement. The VOC data were collected in field campaigns conducted in Ankara, Turkey, and Ottawa, Canada over the years 2000-2004. Insight into the sources of VOCs in different urban atmospheres was sought by using three commonly used receptor models namely / Positive Matrix Factorization (PMF), Chemical Mass Balance (CMB) Model and Conventional Factor Analysis (CFA). Motor vehicle related source profiles were developed to use in receptor modeling. Motor vehicles are the most abundant VOC sources with about 60% and 95% contributions to ambient levels in Ankara and Ottawa, respectively. Residential heating (31%) during winter season, biogenic (9%) and architectural coating (12%) emissions during summer season and solvent use (about 12%) emissions are the next abundant VOC sources in Ankara. In addition, a new method to estimate the contribution of sources from wind sectors in urban atmosphere was developed and implemented in this study. The comparison of the results of these two cities demonstrated the influence of control measures on ambient levels and sources of VOCs observed in different urban atmospheres. VOC levels in Ankara exceed EU levels and they are about factor of two higher than that are measured in Ottawa owing to lack of implementation of emission control regulations for VOCs in Ankara compared to well adopted regulations in Ottawa.

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国立情報学研究所で電子化したコンテンツを使用している。

Antibiotics are defined as chemical substances that inhibit or limit the growth of microorganisms. Since the second world war, antibiotics have been widely used to reduce the morbidity and mortality associated with serious bacterial infections caused by organisms such as Staphylococcus aureus. However, it has become increasingly difficult to treat bacterial infections due to the emergence of antibiotic resistant strains. The first clinical case of drug resistant bacteria was observed in S. aureus in 1947, just four years after the mass production of penicillin. Since then, resistance has been reported to every antibiotic ever employed. According to the Centres for Disease Control and Prevention of the United States, more than 70% of hospital-acquired infections show resistance to at least one commonly used antibiotic. Coupled with the paucity of therapeutic agents in the pipeline, there is now an urgent demand for new antibiotics. One of the strategies employed to combat drug resistant bacteria requires new chemical entities that work through novel drug targets for which there is no pre-existing resistance. This thesis focuses on the essential metabolic enzyme biotin protein ligase (BPL) as one such new drug target. BPL is the enzyme responsible for covalently attaching the cofactor biotin prosthetic group onto the biotin-dependent enzymes such as the carboxylases, decarboxylases and transcarboxylases. Enzymatic biotinylation proceeds via a two-step reaction whereby biotinyl-5'-AMP is synthesized from biotin and ATP before the biotin moiety is transferred onto the side chain of one specific lysine present in the active site of the biotin-dependent enzyme. One example of an important biotin-dependent enzyme is acetyl CoA carboxylase (ACC). ACC catalyzes the first committed step in fatty acid biosynthesis. Through genetic studies, it has been demonstrated that BPL activity is essential for bacterial survival. The aim for this project was to develop a convenient, high-throughput assay to measure BPL activity. This assay would permit 1) quantitative kinetic analysis of ligands and inhibitors and 2) screening of compound libraries for new BPL inhibitors. We propose that BPL inhibitors can be developed into new antibiotic agents. The novel BPL assay was developed employing fluorescence polarization (FP). FP is a light based technique which uses plane polarized light for the detection of tumbling motion of fluorescent molecules in solution. As polarization of the emitted light is relative to the apparent molecular mass of the fluorophore, this technique can be use for quantitation of changes in molecular mass of target molecules. This enabled 1) rapid kinetic analysis, 2) a minimal number of handling steps, 3) no washing steps and 4) automation by robotics. A first generation assay was developed for Escherichia coli BPL using peptide 85-11 that has been shown to be a convenient substrate. Following the BPL reaction, biotinylated peptides will form large molecular mass complexes with avidin. The amount of product could then be quantitated using FP. Here, kinetic analysis of MgATP (Km 0.25 ± 0.01 mM) and biotin (Km 1.45 ± 0.15 μM) binding produced results consistent with published data. We validated this assay with inhibition studies with end products of the BPL reaction, AMP and pyrophosphate, and a compound, biotinol-5'-AMP. Statistical analysis, performed upon both intraassay and interassay results (n = 30), showed the coefficient of variance to be <10% across all data sets. Furthermore, the Z' factors between 0.5 and 0.8 demonstrated the utility of this technology in high-throughput applications. However, the use of peptide 85-11, a substrate specific to E. coli BPL, does limit the application of this methodology to E. coli. In the second generation FP assay, I adapted this technology for S. aureus BPL by employing the biotin domain of S. aureus pyruvate carboxylase. Insertion of a fluorescein label was achieved by first engineering a cysteine residue into the domain by site directed mutagenesis then incubation with fluorescein-5'-maleimide. A series of mutants was created to investigate optimal positioning of the label into the substrate. Furthermore, the minimal size of the functional domain was determined. Our data showed that the placement of the fluorescein label is an important aspect of this project. Using this approach, I identified that a 90 amino acid domain with the label at position 1134 was optimal. Kinetic analysis of ligand binding showed SaBPL had a Km for biotin at 3.29 ± 0.37 μM and Km for MgATP at 66 ± 16.08 μM. This was in good agreement with data obtained from our previous assay measuring ³H-biotin incorporation. Inhibitor studies with pyrophosphate and analogues of biotin and biotinyl-5'-AMP further validated the assay. Various studies have shown cross-species biotinylation activities by a diverse range of BPLs. Therefore, using this methodology with a biotin domain as the substrate potentially provides a convenient assay for all BPLs. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374330 / Thesis (M.Sc.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Antibiotics are defined as chemical substances that inhibit or limit the growth of microorganisms. Since the second world war, antibiotics have been widely used to reduce the morbidity and mortality associated with serious bacterial infections caused by organisms such as Staphylococcus aureus. However, it has become increasingly difficult to treat bacterial infections due to the emergence of antibiotic resistant strains. The first clinical case of drug resistant bacteria was observed in S. aureus in 1947, just four years after the mass production of penicillin. Since then, resistance has been reported to every antibiotic ever employed. According to the Centres for Disease Control and Prevention of the United States, more than 70% of hospital-acquired infections show resistance to at least one commonly used antibiotic. Coupled with the paucity of therapeutic agents in the pipeline, there is now an urgent demand for new antibiotics. One of the strategies employed to combat drug resistant bacteria requires new chemical entities that work through novel drug targets for which there is no pre-existing resistance. This thesis focuses on the essential metabolic enzyme biotin protein ligase (BPL) as one such new drug target. BPL is the enzyme responsible for covalently attaching the cofactor biotin prosthetic group onto the biotin-dependent enzymes such as the carboxylases, decarboxylases and transcarboxylases. Enzymatic biotinylation proceeds via a two-step reaction whereby biotinyl-5'-AMP is synthesized from biotin and ATP before the biotin moiety is transferred onto the side chain of one specific lysine present in the active site of the biotin-dependent enzyme. One example of an important biotin-dependent enzyme is acetyl CoA carboxylase (ACC). ACC catalyzes the first committed step in fatty acid biosynthesis. Through genetic studies, it has been demonstrated that BPL activity is essential for bacterial survival. The aim for this project was to develop a convenient, high-throughput assay to measure BPL activity. This assay would permit 1) quantitative kinetic analysis of ligands and inhibitors and 2) screening of compound libraries for new BPL inhibitors. We propose that BPL inhibitors can be developed into new antibiotic agents. The novel BPL assay was developed employing fluorescence polarization (FP). FP is a light based technique which uses plane polarized light for the detection of tumbling motion of fluorescent molecules in solution. As polarization of the emitted light is relative to the apparent molecular mass of the fluorophore, this technique can be use for quantitation of changes in molecular mass of target molecules. This enabled 1) rapid kinetic analysis, 2) a minimal number of handling steps, 3) no washing steps and 4) automation by robotics. A first generation assay was developed for Escherichia coli BPL using peptide 85-11 that has been shown to be a convenient substrate. Following the BPL reaction, biotinylated peptides will form large molecular mass complexes with avidin. The amount of product could then be quantitated using FP. Here, kinetic analysis of MgATP (Km 0.25 ± 0.01 mM) and biotin (Km 1.45 ± 0.15 μM) binding produced results consistent with published data. We validated this assay with inhibition studies with end products of the BPL reaction, AMP and pyrophosphate, and a compound, biotinol-5'-AMP. Statistical analysis, performed upon both intraassay and interassay results (n = 30), showed the coefficient of variance to be <10% across all data sets. Furthermore, the Z' factors between 0.5 and 0.8 demonstrated the utility of this technology in high-throughput applications. However, the use of peptide 85-11, a substrate specific to E. coli BPL, does limit the application of this methodology to E. coli. In the second generation FP assay, I adapted this technology for S. aureus BPL by employing the biotin domain of S. aureus pyruvate carboxylase. Insertion of a fluorescein label was achieved by first engineering a cysteine residue into the domain by site directed mutagenesis then incubation with fluorescein-5'-maleimide. A series of mutants was created to investigate optimal positioning of the label into the substrate. Furthermore, the minimal size of the functional domain was determined. Our data showed that the placement of the fluorescein label is an important aspect of this project. Using this approach, I identified that a 90 amino acid domain with the label at position 1134 was optimal. Kinetic analysis of ligand binding showed SaBPL had a Km for biotin at 3.29 ± 0.37 μM and Km for MgATP at 66 ± 16.08 μM. This was in good agreement with data obtained from our previous assay measuring ³H-biotin incorporation. Inhibitor studies with pyrophosphate and analogues of biotin and biotinyl-5'-AMP further validated the assay. Various studies have shown cross-species biotinylation activities by a diverse range of BPLs. Therefore, using this methodology with a biotin domain as the substrate potentially provides a convenient assay for all BPLs. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374330 / Thesis (M.Sc.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Antibiotics are defined as chemical substances that inhibit or limit the growth of microorganisms. Since the second world war, antibiotics have been widely used to reduce the morbidity and mortality associated with serious bacterial infections caused by organisms such as Staphylococcus aureus. However, it has become increasingly difficult to treat bacterial infections due to the emergence of antibiotic resistant strains. The first clinical case of drug resistant bacteria was observed in S. aureus in 1947, just four years after the mass production of penicillin. Since then, resistance has been reported to every antibiotic ever employed. According to the Centres for Disease Control and Prevention of the United States, more than 70% of hospital-acquired infections show resistance to at least one commonly used antibiotic. Coupled with the paucity of therapeutic agents in the pipeline, there is now an urgent demand for new antibiotics. One of the strategies employed to combat drug resistant bacteria requires new chemical entities that work through novel drug targets for which there is no pre-existing resistance. This thesis focuses on the essential metabolic enzyme biotin protein ligase (BPL) as one such new drug target. BPL is the enzyme responsible for covalently attaching the cofactor biotin prosthetic group onto the biotin-dependent enzymes such as the carboxylases, decarboxylases and transcarboxylases. Enzymatic biotinylation proceeds via a two-step reaction whereby biotinyl-5'-AMP is synthesized from biotin and ATP before the biotin moiety is transferred onto the side chain of one specific lysine present in the active site of the biotin-dependent enzyme. One example of an important biotin-dependent enzyme is acetyl CoA carboxylase (ACC). ACC catalyzes the first committed step in fatty acid biosynthesis. Through genetic studies, it has been demonstrated that BPL activity is essential for bacterial survival. The aim for this project was to develop a convenient, high-throughput assay to measure BPL activity. This assay would permit 1) quantitative kinetic analysis of ligands and inhibitors and 2) screening of compound libraries for new BPL inhibitors. We propose that BPL inhibitors can be developed into new antibiotic agents. The novel BPL assay was developed employing fluorescence polarization (FP). FP is a light based technique which uses plane polarized light for the detection of tumbling motion of fluorescent molecules in solution. As polarization of the emitted light is relative to the apparent molecular mass of the fluorophore, this technique can be use for quantitation of changes in molecular mass of target molecules. This enabled 1) rapid kinetic analysis, 2) a minimal number of handling steps, 3) no washing steps and 4) automation by robotics. A first generation assay was developed for Escherichia coli BPL using peptide 85-11 that has been shown to be a convenient substrate. Following the BPL reaction, biotinylated peptides will form large molecular mass complexes with avidin. The amount of product could then be quantitated using FP. Here, kinetic analysis of MgATP (Km 0.25 ± 0.01 mM) and biotin (Km 1.45 ± 0.15 μM) binding produced results consistent with published data. We validated this assay with inhibition studies with end products of the BPL reaction, AMP and pyrophosphate, and a compound, biotinol-5'-AMP. Statistical analysis, performed upon both intraassay and interassay results (n = 30), showed the coefficient of variance to be <10% across all data sets. Furthermore, the Z' factors between 0.5 and 0.8 demonstrated the utility of this technology in high-throughput applications. However, the use of peptide 85-11, a substrate specific to E. coli BPL, does limit the application of this methodology to E. coli. In the second generation FP assay, I adapted this technology for S. aureus BPL by employing the biotin domain of S. aureus pyruvate carboxylase. Insertion of a fluorescein label was achieved by first engineering a cysteine residue into the domain by site directed mutagenesis then incubation with fluorescein-5'-maleimide. A series of mutants was created to investigate optimal positioning of the label into the substrate. Furthermore, the minimal size of the functional domain was determined. Our data showed that the placement of the fluorescein label is an important aspect of this project. Using this approach, I identified that a 90 amino acid domain with the label at position 1134 was optimal. Kinetic analysis of ligand binding showed SaBPL had a Km for biotin at 3.29 ± 0.37 μM and Km for MgATP at 66 ± 16.08 μM. This was in good agreement with data obtained from our previous assay measuring ³H-biotin incorporation. Inhibitor studies with pyrophosphate and analogues of biotin and biotinyl-5'-AMP further validated the assay. Various studies have shown cross-species biotinylation activities by a diverse range of BPLs. Therefore, using this methodology with a biotin domain as the substrate potentially provides a convenient assay for all BPLs. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374330 / Thesis (M.Sc.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Thesis (Ph.D.) -- McMaster University, 1998. / Includes bibliographical references (leaves 185-191). Also available via World Wide Web.

More than a decade after the completion of the human genome project, the function of a large number of genes remains to be elucidated. Forward and reverse genetic approaches have proven to be powerful tools to study gene function and have provided insights into fundamental biological processes. Furthermore, functional genetic screening can lead to a better understanding of the action of endogenous and exogenous stimuli such as hormones or drugs on biological systems. Thus far, systematic and unbiased studies have largely been limited to model organisms. However, complex disease-relevant genotypes and phenotypes cannot be studied in entirety in lower organisms creating a need for systematic approaches in human cells. This thesis describes a series of studies using forward and reverse genetic approaches combined with state-of-the-art technology in haploid human cells. The first chapter describes the development of a quantitative phenotypic read-out using a novel application of RNA-sequencing that allows the functional annotation of genes in signalling pathways. The presented data demonstrate that the employed shallow RNA-sequencing method is scalable and suitable as a read-out for reverse genetic screening. The second chapter focuses on the implementation of this method in a large reverse genetic study in human cells to functionally annotate tyrosine kinases in signalling pathways upon stimulation with a set of ten polypeptides and small molecules. The screens revealed known and unexpected interactions between different signalling molecules and pathways, validating the technical approach in a biological context. The third chapter presents a pilot study describing the set-up of a forward genetic technique for compound mode-of-action screening using a pooled human mutant cell line collection. The chemical genetic approach displayed sufficient sensitivity and allowed to monitor thousands of gene-drug interactions simultaneously. Together, this thesis combines elements to advance technological and biological aspects of functional genomics and chemical genetics.

Text in Afrikaans / An account of certain functional aspects of Afrikaans exocentric compounds is presented in this dissertation. This study builds on the preliminary survey presented in the dissertation 'n Korpusanalise van Afrikaanse eksosentriese komposita (Van Niekerk, 2001). Exemplary material is obtained from an extensive corpus, consisting of lexicographical and academic matter, as well as colloquial spoken language. Language is man's primary means of communication, used to convey knowledge and information. Lexical items are used to name and refer to all kinds of concepts, aspects, objects, persons and other references. Of particular importance to this study, however, is the expressive functionality of language, whereby it is used as an instrument to voice affect, judgement, opinion, perception and other emotional aspects. Exocentric compounds are singled out as lexemes of particular importance, utilized by Afrikaans speakers/writers to express themselves referentially and emotionally. In this study the researcher has endeavored to describe and explain certain aspects of exocentric compounds in terms of the cognitive process of conceptual blending, as explained in The way we think by Fauconnier and Turner (2003). Exocentric compounding is highly functional with regard to etnobiological naming of botanic and zoological references, especially as bahuvrihi compounds. More prominent, however, is the use of compounds to voice a wide variety of expressive values and connotations, both positive and negative. Humor is constantly referred to as probably the most important function of exocentric compounds. Other expressive functional aspects of exocentric compounds are discussed, such as insult, scorn and ridicule in nicknames and slurs, the softening effect of euphemism in contrast to the intensifying effect of dysphemism, idiomaticity, irony, et cetera, some of these aspects overlapping significantly. Exocentric compounds are creatively used as highly descriptive expressions in the informal register of colloquial Afrikaans, as well as in different dialects and sociolinguistic varieties. Based on observations in connection with the diverse use and optimal functionality of exocentric compounds in domains of every possible kind, the conclusion is reached that exocentric compounds is an essential part of the Afrikaans lexicon. / Afrikaans and Theory of Literature / D.Litt. et Phil.(Afrikaans)

Ce travail porte sur la mise en forme par compression des Sheet Moulding Compounds (SMC), matériaux composites à matrice polymère renforcés par des fibres courtes. Il s’inscrit dans un plus vaste projet visant à la création d'un outil de simulation du procédé de compression des SMC. Il se focalise sur le développement d’outils et de méthodes de caractérisation de la rhéologie et des microstructures dédiés à aux formulations de SMC allégées ou à fort taux de fibres, dans le but de construire un modèle rhéologique prenant en compte la forte compressibilité de ces matériaux. Dans un premier temps, des essais de compression sur site industriel sont réalisés en utilisant des flans de différentes couleurs. Couplés à des images 3D obtenues par microtomographie à rayons X, ils permettent de mettre en évidence la complexité des mécanismes de déformation vus par les SMC au cours de leur mise en forme par compression. En parallèle, des essais de compression sur rhéomètre transparent mettent en évidence une forte compressibilité des SMC qui se traduit par un écoulement en deux phases : une cinématique de consolidation suivie d’un écoulement isovolume. La consolidation et ses liens avec la porosité initiale des SMC est ensuite étudiée grâce à des essais de compression avec observations 3D in situ et en temps réel réalisées dans un microtomographe à rayons X synchrotron. De là, différents dispositifs expérimentaux permettant de caractériser les SMC dans des sollicitations représentatives du procédé ont été développés. Ainsi, un dispositif de traction, couplé à des mesures de champs cinématiques par corrélation d’images ainsi qu’à des observations des évolutions microstructurelles par microtomographie à rayons X a été mis au point, il permet de mieux comprendre le comportement des SMC au cours de la phase d’estampage. Un dispositif de compression œdométrique permettant une mesure des états de contrainte au cours de la compression ainsi qu’un dispositif de cisaillement simple permettant d’appliquer une contrainte normale variable ont également été développés afin d’étudier les liens entre la consolidation des SMC et leur cisaillement. Les différentes campagnes expérimentales réalisées au cours de ces travaux mettent d’une part en évidence des différences importantes au niveau des microstructures des formulations étudiées et de leurs évolutions en écoulement. D’autre part, les résultats expérimentaux mettent également en évidence un comportement mécanique viscoélastoplastique non linéaire, anisotrope et compressible, fortement dépendant de la microstructure initiale des SMC. Sur la base de tous ces essais un modèle rhéologique visqueux, compressible, isotrope transverse est proposé et ses paramètres constitutifs sont identifiés. / This study focuses on the compression moulding Sheet Moulding Compounds (SMCs), i.e., short fibre-reinforced thermoset polymer composites. It is part of a wider project aiming at the development of software to simulate the SMC compression moulding. It focuses on the development of tools and methods to characterise the rheology and the microstructures high fibre content and ultralight SMCs in order to build a rheological model that takes into account the noticeable compressibility of these materials. For that purpose, industrial compression using colored sheets were carried out and coupled with 3D images obtained from X-ray microtomography. They highlighted the complexity of flow-induced mechanisms during compression moulding. Compression experiments were also performed on a transparent rheometer to highlight the high compressibility of SMCs which resulted in a two steps flow: a compaction followed by an isovolume flow. The consolidation stage and its links with the initial porosity is then studied through 3D in situ and real time observations of compression tests carried out inside an X-ray synchrotron microtomograph. In addition, various experimental an dedicated rheometers were developed following flow modes close to those encountered during the compression moulding process. Hence, a tensile device was developed and coupled with (i) local kinematic strain field measurements using digital image correlation as well as (ii) microstructural observations obtained from X-ray microtomography. It allowed a better understanding of the stamping phase during the early stage of the SMC compression moulding process. Furthermore, an oedometric compression device equipped with sensors to measure the stress state during compression, together with a simple shear device allowing normal stress to be tuned, were developed in order to study the link between SMC consolidation and their shear behaviour. First of all, experimental results showed strong differences in the microstructures of the studied formulations and in their evolutions during the SMC flow. They also highlighted an anisotropic, compressible, nonlinear viscoelastoplastic mechanical behavior that strongly depended on the initial SMC microstructure. On the basis of these experimental results, a viscous, compressible and transversely isotropic rheological model was proposed and its consitutitve parameters were identified.