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.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 118-128).

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-16T16:45:04Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_DanieleMorgensternAimi.pdf: 5748053 bytes, checksum: d57b44570d4e19a760d7a8379dcc9315 (MD5) / Made available in DSpace on 2018-08-16T16:45:04Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_DanieleMorgensternAimi.pdf: 5748053 bytes, checksum: d57b44570d4e19a760d7a8379dcc9315 (MD5) Previous issue date: 2012-01-09 / The structural and electronic properties of biotin and cysteine molecules interacting with pure, carboxylated and vacancy-type defects carbon nanotubes was evaluated using ab initio simulations. Carbon nanotubes (NTC) have been used for a variety of applications. Among these, we highlight the use of NTC as sensors for biological molecules. Thus, the objective of this work is to use NTC pure carboxylated and defective molecules interacting with biotin and cysteine in the development of nanosensors for these molecules. Our results showed that the interaction between these molecules and the NTC occurs via a physical adsorption with binding energies ranging from 0.22 to 1.91 eV [0.12 to 1.14 eV] for NTC interacting with biotin [cysteine]. For the carboxylated nanostructure higher binding energies are observed. In this way, carboxylated NTC aren’t interesting for interaction with the molecules, because the eventual removal of these NTC had a high energy cost. The physical adsorption between the NTC and the molecules of biotin and cysteine is of great interest for a possible experimental application of NTC as a biological sensor for these molecules. / As propriedades eletrônicas e estruturais das moléculas de biotina e cisteína interagindo com nanotubos de carbono puros, carboxilados e com defeito do tipo vacância foram estudadas fazendo-se uso de simulações computacionais baseadas na Teoria do Funcional da Densidade (DFT). Os nanotubos de carbono (NTC) vem sendo utilizados para uma série de aplicações. Dentre estas, podemos destacar sua utilização como sensores para moléculas biológicas. O objetivo deste trabalho foi utilizar NTC puros, carboxilados e com defeito interagindo com as moléculas de biotina e cisteína no desenvolvimento de nanosensores para estas moléculas. Os resultados mostraram que a interação entre essas moléculas e os NTC ocorre via uma adsorção física, pois não altera as propriedades dos nanotubos, tendo energias de ligação que variam de 0,22 a 1,91 eV para NTC interagindo com a biotina e 0,12 a 1,14 eV para NTC interagindo com a cisteína, sendo que as energias mais altas, são aquelas às quais as moléculas interagem com os NTC carboxilados. Logo, NTC carboxilados não são os mais indicados para a interação com as moléculas, pois a remoção posterior destas do NTC teria um alto custo energético. A adsorção física entre os NTC e as moléculas de biotina e cisteína é de grande interesse para uma possível aplicação experimental destes NTC como sensores biológicos para estas moléculas.

Cancer vaccines represent a promising treatment modality for a world-wide health problem. Whether as an adjuvant or as a stand-alone therapy, cancer vaccines represent a tumor-specific and systemic treatment potentially capable of eliminating metastatic lesions without the severe side-effects often associated with chemotherapy. Specifically, whole cell tumor vaccines have shown promise in preclinical and clinical settings and the studies presented here represent the beginnings of an approach to improve the antitumor potency of these vaccines. This project demonstrates as "proof of concept" the feasibility of manufacturing tumor cell-particle hybrids. The coupled use of these two components, whole tumor cells and cargo-carrying biodegradable particles, as one entity in a cancer vaccine system is a new line of research. Stable cell-particle hybrids were assembled using avidin-biotin chemistry where cargo-carrying PLGA particles (500 nm diameter) were coated with streptavidin and allowed to bind to tumor cells that had been indirectly labeled with biotin (using an integrin-specific biotinylated antibody). That successful cell-particle hybrids were assembled was determined by multiple means, including flow cytometry, laser scanning confocal microscopy and scanning electron microscopy. Two murine tumor cell lines (representing melanoma and prostate cancer) were investigated in this study and successfully demonstrated the general applicability of the assembly method. Particles appeared to be localized on the cell surface (rather than endocytosed) as determined by microscopic imaging. The cell-particle hybrid was shown to be stable to irradiation, an important consideration since whole tumor cells need to be treated with ionizing radiation prior to being used as vaccines in order to render them nonproliferative and immunogenic. We also characterized loading and release profiles of CpG, a prospective vaccine adjuvant, into PLGA particles. We conclude that we have developed a method for manufacturing cell-particle hybrids comprising PLGA nanoparticles and irradiated tumor cells. The next step would be to use CpG-loaded particles in the assembled hybrid and test the anti-tumor immune efficiency of this cancer vaccine formulation in either a melanoma or prostate cancer model.

Novel biocatalysts are highly demanded in the white biotechnology. Hence, the development of highly stable and enantioselective biocatalysts with novel functionalities is an ongoing research topic. Here, an osmium ligating single-site ArM was created based on the biotinstreptavidin technology for the dihydroxylation of olefins. For the creation of the artificial catalytic metal center in the streptavidin (SAV) cavity, efficient osmium tetroxide (OsO4) chelating biotin-ligands were created. The unspecific metal binding of the host scaffold was diminished through genetical and chemical modification of the host protein. The created single-site OsO4 chelating ArM was successfully applied in the asymmetric cyclopropanation, revealing a stable and tunable catalytic hybrid system for application. The structural analysis of protein-ligand complexes is essential for the advanced rational design and engineering of artificial metalloenzymes. In previous studies, a SAV-dirhodium ArM was created and successfully applied in the asymmetric cyclopropanation reaction. To improve the selectivity of the SAV-dirhodium complex, the structural location of the organometallic complex in the SAV cavity was targeted and small-angle x-ray scattering (SAXS) was used to obtain the structural information. The SAXS analysis revealed valuable information of the molecular state of the complexes; hence, the method proved to be useful for the structural analysis of protein-ligand interactions. The discovery of novel enzymes from nature is still the major source for improved biocatalysts. One of the most important enzymes used in the molecular biology are DNA polymerases in PCR reactions. The halothermophilic brine-pool 3 polymerase (BR3 Pol) from the Atlantis II Red Sea brine pool showed optimal activities at 55 °C and salt concentrations up to 0.5 M NaCl, and was stable at temperatures above 95 °C. The comparison with the hyperthermophilic KOD polymerase revealed the haloadaptation of BR3 Pol due to an increased negative electrostatic surface charge and an overall higher structural flexibility. Engineered chimeric KOD polymerases with swapped single BR3 Pol domains revealed increased salt tolerance in the PCR, showing increased structural flexibility and a local negative surface charge. The understanding of the BR3 Pol haloadaptation might enable the development of a DNA polymerase tailored for specific PCR reactions with increased salt concentrations.

The aim of the work was the selective isolation of bacterial DNA with help of magnetic carriers covered by streptavidine (PGMA-NH2-STV, MPG® Streptavidin). Conditions of functionalisation of carriers using two biotinylated probes were optimized: the amount of carrier, the amount of probe, binding of biotinyled probe to streptavidine. Purified DNA Lactobacillus was used for hybridization. DNA binding to the probe (DNA/DNA hybridization) and nospecific adsorption of DNA to the carrier were tested. Target DNA eluted from the carrier was identified using PCR with primers R16-1 and LbLMA1-rev and with primers P_eub and F_eub. The amount of probe bound to the carrier was estimated using UV spectrophotometry. It was estimated that biotinyled probe can be used for functionalisation in concentration 5 pmol/µl added to the carrier in the ration carrier : probe 1:1. It was shown that nonspecific DNA adsorption to the MPG® Streptavidin is significantly lower than to the carrier PGMA-NH2-STV.Using DNA/DNA hybridization and the MPG® Streptavidin, DNA from pure culture Lactobacillus was isolated. Procedure was applicated for DNA isolation from milk products.

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