Thesis (Ph.D.)--Aberdeen University, 2009. / Title from web page (viewed on July 8, 2009). Includes bibliographical references.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 113-121). Also available in electronic version.

Techn. Universiẗat, Diss., 2002--Freiberg (Sachsen).

Universiẗat, Diss., 2004--Stuttgart.

Techn. Hochsch., Diss., 2005--Aachen.

Mitochondria exist in a dynamic network whereby fusion and fission events are critical to the health of the mitochondria, the cell, and the organism. Dysfunctional mitochondrial dynamics underlie a plethora of diseases including various cancers, heart diseases, diabetes, neurodegenerative diseases, and a number of mitochondrial disorders. Despite a strong molecular knowledge of a handful of functional mediators of mitochondrial dynamics, much less is known about how this process is regulated at a cellular level, and what genes are involved in signaling pathways. A previously completed mitochondrial morphology genome screen was repeated with an automated confocal microscope resulting in the identification and validation of MTX2 as a novel regulator of mitochondrial dynamics. Functional characterization of the role of MTX2 in mitochondrial dynamics will further our understanding of mitochondrial biology, and has the future potential to inform therapies for some of the many diseases underscored by dysfunctional mitochondrial dynamics.

Concomitant with antibiotic use is the development of bacterial strains that are resistant to such compounds. Presently, the rate at which antibiotic-resistant pathogenic bacteria are emerging is outpacing our resupply of new antibacterials; therefore, renewed efforts to identify novel therapies are urgently needed. Transition metals are required by all life forms and, for bacteria, an adequate supply of nutrient metal is necessary to establish infection in a host. Indeed, as an antibacterial defense mechanism, eukaryotes have developed various means by which to restrict the availability of metal to the invading pathogen, thereby limiting its chances for successful colonization. As such, bacterial metal acquisition and homeostasis have been suggested as potential antibiotic targets to explore for the identification of new antibacterial small molecules. In this thesis I discuss my development of a high-throughput screening assay that specifically selects for compounds that perturb bacterial iron homeostasis. The results of this work led to the identification of a series of spiro-indoline-thiadiazole compounds that are toxic to bacteria via iron chelation. In addition to molecules that perturb the availability of bacterial intracellular iron, we present a series of spiro-indoline-thiadiazole analogs that inhibit bacterial growth by limiting zinc availability. Furthermore, we show that the respective zinc-perturbing analogs re-sensitize an otherwise drug-resistant strain of NDM-1-harbouring Klebsiella pneumoniae to carbapenem antibiotics. We discuss the potential for this class of compounds to serve as carbapenem adjuvants for treating infections caused by metallo-β-lactamase-containing pathogens. / Thesis / Doctor of Philosophy (PhD)

This dissertation describes the development of a system for the automated, high-throughput screening of organic reactions. This system utilizes a liquid handling robot for reaction mixture preparation combined with desorption electrospray ionization mass spectrometry (DESI-MS) for reaction mixture analysis. With an analysis speed of ~1 second per reaction mixture, this system is capable of screening thousands of reactions per hour. Reaction mixtures are prepared in 384-well microtiter plates using a liquid handling robot. A sample of each reaction mixture (50 nL) is then transferred to a PTFE coated, glass slide using a pin tool. By offsetting the placement of the pin tool during each transfer, up to 6,144 unique reaction mixtures can be placed on each slide. The slide is then transferred to the DESI stage by a robotic arm, and the DESI-MS analysis begins, taking as little as 7 minutes for 384 reaction mixtures. We utilize a scheduling software to control each component of the system, which automates the entire process from reaction mixture preparation to DESI-MS analysis. In order to efficiently analyze and visualize the extremely large data sets generated by the system, we developed a custom software suite to automatically process each data set. We have used this system to screen several classes of industrially relevant reactions including Suzuki coupling, nucleophilic aromatic substitution, reductive amination, and Sonogashira coupling. We have validated both positive and negative results from the system using flow chemistry, and we have observed excellent agreement between the two methodologies. By being capable of screening thousands of reactions per hour, requiring only microliter quantities of reaction mixtures, and consuming less than a milliliter of solvent during the DESI-MS analysis, this system significantly reduces the time and costs associated with organic reaction screening.

Master of Science / Department of Biochemistry and Molecular Biophysics / Phillip Klebba / Iron acquisition is a component of Gram-negative bacteria pathogenesis, therefore as a form of 'nutritional immunity' host organisms sequester iron. To obtain iron bacteria secrete siderophores that scavenge iron. The E. coli outer membrane protein FepA actively transports the siderophore ferric enterobactin into the periplasm. We observe this uptake reaction by fluorescently labeling FepA in live bacteria, monitoring quenching that occurs upon binding of FeEnt, and then fluorescence recovery during transport. Energy poisons azide, arsenate, and 2,4-dinitrophenol were evaluated to determine sensitivity to known transport inhibitors. We developed and optimized methods to screen for iron transport inhibitors using a cell-based high-throughput screening platform. These inhibitors may have broad spectrum bacteriostatic antibiotic properties.

The colony formation assay (CFA) is the gold standard for measuring cytotoxic effects on cells. To increase efficiency, the CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation (IR) on the FaDu and A549 cancer cell lines. Its ability to evaluate combination therapies was investigated using cisplatin and IR. The 96-well CFA was transferred to a robotic platform for evaluation as a high-throughput screen (HTS) readout for the discovery of novel anti-cancer compounds, and radiosensitizers. Screening yielded eight putative anti-cancer hits, and five putative radiosensitizing hits. Secondary screening confirmed 6/8 anti-cancer compounds, and 0/5 radiosensitizing compounds. Thus, the 96-well CFA can be adopted as an alternative assay to the 6-well CFA in the evaluation of cytotoxicity in vitro, providing a possible readout to be utilized in HTS for discovering anti-cancer compounds, but with limited applicability in discovering radiosensitizers.