Membrane proteins are essential for life, and roughly one-quarter of all open reading frames in sequenced genomes code for membrane proteins. Unfortunately, our understanding of membrane proteins lags behind that of soluble proteins, and is best reflected by the fact that only 0.5% of the structures deposited in the protein data-bank (PDB) are of membrane proteins. This discrepancy has arisen because their hydrophobicity - which enables them to exist in a lipid environment - has made them resistant to most traditional approaches used for procuring knowledge from their soluble counter-parts. As such, novel methods are required to facilitate our knowledge acquisition of membrane proteins. In this thesis a generic approach for rapidly obtaining information on membrane proteins from the classic bacterial encyclopedia Escherichia coli is described. We have developed a Green Fluorescent Protein C-terminal tagging approach, with which we can acquire information as to the topology and ‘expressibility’ of membrane proteins in a high-throughput manner. This technology has been applied to the whole E. coli inner membrane proteome, and stands as an important advance for further membrane protein research.

Cancer is one of the major causes of death in the western world. Resistance to anti-cancer drugs and diagnostic difficulties are major obstacles to successful treatment. This thesis describes studies based on microarray expression analysis and high-throughput compound screening for identification of resistance mechanisms, drug targets and diagnostic markers. In paper I-IV, we applied global expression analysis and measurements of drug response in a human tumor cell line panel to identify drug targets and resistance mechanisms. In paper I, we identified gene transcript levels that correlate with drug resistance and sensitivity. Both well known and new potential markers and mechanisms were identified. In paper II, we showed that STAT1 activity is associated with cross-resistance to both doxorubicin and radiation in vitro and that fludarabine can counteract STAT1 activity and reduce resistance. In Paper III-IV, cell lines were exposed to a compound library consisting of more than thousand different substances in a high-throughput screening effort. These studies revealed that cell line models of squamous cell carcinoma (Paper III) and drug resistant myeloma (Paper IV) are sensitive to phosphodiesterase inhibitors and glucocorticoids respectively. The target molecules for these drugs were over-expressed at the mRNA level and constitute likely explanations for the observed drug potency. In paper V, we identified mRNA markers for the distinction between two types of thyroid tumors, thyroid follicular adenomas and thyroid follicular carcinomas, by means of microarray expression analysis. Our results indicated that distinction between the two tumor types is possible with a small number of markers.

Until recently, multiple SPME fibres could not be automatically evaluated in a single sequence without manual intervention. This drawback had been a critical issue until recently, particularly during the analysis of numerous on-site samples. Recently, GERSTEL® has developed and commercialized a Multi-Fibre Exchanger (MFX) system designed to overcome this drawback. In this research, a critical evaluation of the MFX performance in terms of storage stability and long term operation is presented. It was established in the course of our research that the MFX can operate continuously and precisely for over 200 extraction/injection cycles. However, when the effect of residence time of commercial fibres on the MFX tray was evaluated, the results have shown that amongst the evaluated fibre coatings, carboxen/polydimethylsiloxane (CAR/PDMS) was the only coating capable of efficient storage on the MFX tray for up to 24 hours after field sampling without suffering significant loss of analytes. Additionally, the MFX system capability for high-throughput analysis was demonstrated by the unattended desorption of multiple fibres after on-site sampling of two different systems, indoor air and biogenic emissions. Subsequently, a protocol based on a new, fast, reproducible, reusable and completely automated method that enables quick assessment of SPME coatings was developed. The protocol consists of an innovative in-vial standard generator containing vacuum pump oil doped with McReynolds probes and subsequently mixed with a polystyrene-divinylbenzene resin. According to our results, the protocol has proven to be a useful tool for the quick assessment of inter-fibre reproducibility prior to their application in on-site analysis. The implications of such protocols include, but are not limited to: time-saving, assurance of reliable and reproducible data, and a dependable guide for novice users of the technique. Finally, an innovative, reusable and readily deployable pen-like diffusive sampler for needle traps (PDS-NT) is proposed. Results have shown that the new PDS-NT is effective for air analysis of benzene, toluene, and o-xylene (BTX). In addition, no statistically significant effects of pen geometry on the uptake of analytes were found.

Fatty acid profiling provides information on dietary intakes and an understanding of lipid metabolism. High throughput techniques such as fingertip prick (FTP) sampling has gained popularity in recent years as a simplified method for basic research, and could be further used to assess disease risk in the population, and other similar high-throughput techniques have the potential to assist in the monitoring and labeling of fatty acids in the food supply. With the advancement of high-throughput sample analysis techniques, a more complete understanding of storage stability is required as a larger volume of samples are produced with equal amounts of time to analyze them. Energy-assisted analysis techniques have the potential to help ameliorate some of these issues. Presently, FTP blood, whole blood and salmon storage stability is assessed under various storage conditions, and both microwave-assisted direct transesterification and indirect ultrasound-assisted extraction techniques are assessed. It is determined that storage of FTP blood and whole blood samples at -20°C results in significant and nearly complete highly unsaturated fatty acid (HUFA) degradation compared to all other temperatures examined. This degradation is determined to be the result of hemolysis and subsequent iron release from erythrocytes initiating fatty acid peroxidation reactions. Direct transesterification of FTP blood is reduced from as long as three hours to one minute with microwave-assisted energy and fatty acid extraction from ground flaxseed is reduced to 40 minutes from as long as 24 hours without compromising fatty acid profiles. Results of the current study provides insight into the storage stability of food sample and blood samples collected via high-throughput techniques, and provides support for the utilization of further high-throughput energy-assisted analytical methods that can help to minimize the potentially detrimental effects that long-term storage can have on fatty acid profiles.

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Recombinant antibodies and antibody fragments have become powerful tools for therapy, in vivo and in vitro diagnostics, and laboratory research. However, the production of antibody fragments in high yield for preclinical and clinical trials can be a serious bottleneck in drug discovery. This dissertation describes the development of novel screening systems for isolating antibody fragments and alternatively, E. coli genes, that facilitate expression in E. coli. In the first part of this work, we have developed a screening system for isolating Fab mutants exhibiting 4~5 fold higher expression level at 37oC compared to the parental Fab, by utilizing the APEx 2-hybrid system and multi-color FACS as a screening tool. In the APEx 2-hybrid system, the bacterial periplasm constitutes the milieu for the association of membrane-anchored bait protein and solubly expressed, epitope-tagged prey protein. Upon disruption of the outer membrane, only prey proteins that bind to the bait remain cell-associated and are detected by flow cytometry using fluorescently labeled anti-epitope antibodies. In the second part of this work we developed a new strategy to engineer scFv that can be expressed in soluble and active form in the absence of disulfide bonds. This was achieved using a strain incapable of forming disulfide bonds in proteins expressed in its periplasm in combination with the APEx 2-hybrid system. The selected clones exhibited higher solubility, activity, and stability than that of the wild type scFv in the reducing condition of the cytoplasm. Finally, we sought to isolate E. coli gene fragments that can enhance IgG production in the periplasm of E. coli by a newly developed screening system based on soluble expression of IgG and E. coli genomic fragments. The isolated gene fragments, which are located between moeA and iaaA in the E. coli chromosome, improved the total expression of polypeptides of IgG and also assembly of IgG. / text

Dimethylarginine dimethylaminohydrolase (DDAH) indirectly regulates nitric oxide production by hydrolyzing methylated arginines, which are endogenous nitric oxide synthase inhibitors. This enzyme is a member of the mechanistically diverse pentein superfamily, which contains hydrolase, dihydrolase, and amidinotransferase enzymes. These enzymes are proposed to use the same first catalytic step, followed by partitioning into their respective activities. Here, variants of DDAH that can catalyze the dihydrolase and amidinotransfer reactions are presented, as well as a variant of succinylarginine dihydrolase which catalyzes a single hydrolysis reaction. The results experimentally demonstrate that the proposed common catalytic intermediate can be used for several different reactions. The results suggest that enzymes in the pentein superfamily may have evolved divergently from a catalytically promiscuous ancestor. The control DDAH asserts over nitric oxide production makes it an attractive drug target for disease states marked by pathological overproduction of nitric oxide. Only a limited number of inhibitors different from substrate are reported, due in part to lack of robust assays for high-throughput screening of compound libraries. Therefore, high-throughput assays were developed, optimized, and validated to screen for inhibitors of Pseudomonas aeruginosa DDAH and human DDAH-1. These assays were used to screen three commercial libraries totaling 6,466 compounds. One drug in phase III clinical trials, ebselen, was identified and characterized as a bioavailable, rapid covalent inactivator of DDAH both in vitro and in cultured cells. Four "fragment-sized" inhibitors were also identified and characterized in the screening, including 4-halopyridines and benzimidazole-like compounds. The 4-halopyridines, not previously known to modify proteins, act as quiescent affinity labels to selectively inactivate DDAH, and the benzimidazole-like compounds are competitive, rapidly reversible inhibitors of DDAH. These diverse molecules serve as starting points for the development of molecular probes and therapeutic drugs to reduce pathological overproduction of nitric oxide. / text

Engineering protease substrate specificity and selectivity has the potential to yield entirely new possibilities in the analytical, biotechnological, and therapeutic domains. For example, therapeutic applications can be envisioned in which engineered proteases could replace antibodies by irreversibly inactivating a large excess of disease-associated target proteins in a catalytic fashion. Technological advances in molecular biology have made laboratory-based evolution techniques for protein engineering readily accessible. However, the ability to interrogate the activities and substrate preference of large numbers of protease variants is predicated on the availability of quantitative high-throughput assays that maintain the essential link between genotype and phenotype. In this work we have investigated a variety of novel single cell fluorescence assays and selections for engineering protease substrate specificity and selectivity, and demonstrated the utility of some of these systems for the engineering of novel enzymes. The second chapter of this dissertation reports the isolation of a highly active ([chemical formula]) variant of the Escherichia coli endopeptidase OmpT that selectively hydrolyzes peptides after 3-nitrotyrosine while effectively discriminating against similar peptides containing unmodified tyrosine, sulfotyrosine, phosphotyrosine and phosphoserine. The isolation of protease variants that can discriminate between substrates based on the posttranslational modification of Tyr was made possible by implementing a multi-color flow cytometric assay using multiple simultaneous counter-selection substrates for the screening of large mutant libraries. While primary sequence recognition may suffice for proteomic applications, many therapeutic applications of engineered proteases will require the cleavage of folded protein targets. Unfortunately, we have found that engineered proteases that can cleave peptides very efficiently are often unable to digest the same sequences inserted into the loop regions of a folded protein. The logical conclusion, then, is that an entire target protein or at least a protein domain, rather than peptide segments, must be incorporated into protease engineering screening assays. As a critical first step toward the development of next generation, single cell screening systems for therapeutic protease engineering we have developed novel assays that exploit cell surface capture of exogenous protein substrates. One assay (Chapter 3) relies on an autoinhibited protein fusion that capitalizes on the p53 antagonist MDM2 as a detector of protease activity in addition to its utility as a counter-selection substrate. Using this system we successfully isolated OmpT variants that selectively cleave a designed site within our autoinhibited substrate. A second high-throughput screen (Chapter 4) monitors native protein cleavage. Target proteins are captured at the cell surface using a polycationic tail, incorporating counter-selection, and the proteolytic state of the substrate can be monitored using epitope tags fused to the N-and C-termini and fluorescently labeled anti-epitope tag antibodies. / text

Genome-wide association studies (GWAS) of common diseases have identified hundreds of genomic regions harboring disease-associated variants. Translating these findings into an improved understanding of human disease requires identifying the causal variants(s) and gene(s) in the implicated regions which, to date, has only been accomplished for a small number of associations. Several factors complicate the identification of mutations playing a causal role in disease. First, GWAS arrays survey only a subset of known variation. The true causal mutation may not have been directly assayed in the GWAS and may be an unknown, novel variant. Moreover, the regions identified by GWAS may contain several genes and many tightly linked variants with equivalent association signals, making it difficult to decipher causal variants from association data alone. Finally, in many cases the variants with strongest association signals map to non-coding regions that we do not yet know how to interpret and where it remains challenging to predict a variants likely phenotypic impact. Here, we present a framework for the genetic and functional study of intergenic regions identified through GWAS and describe application of this framework to chromosome 9p21: a non-coding region with associations to type 2 diabetes (T2D), myocardial infarction (MI), aneurysm, glaucoma, and multiple cancers. First, we compare methods for genetic fine-mapping of GWAS associations, including methods for creating a more comprehensive catalog of variants in implicated regions and methods for capturing these variants in case- control cohorts. Next, we describe an approach for using massively parallel reporter assays (MPRA) to systematically identify regulatory elements and variants across disease-associated regions. On chromosome 9p21, we fine-map the T2D and MI associations and identify, for each disease, a collection of common variants with equivalent association signals. Using MPRA, we identify hundreds of regulatory elements on chromosome 9p21 and multiple variants (including MI- and T2D-associated variants) with evidence for allelic effects on regulatory activity that can serve as a foundation for further study. More generally, the methods presented here have broad potential application to the many intergenic regions identified through GWAS and can help to uncover the mechanisms by which variants in these regions influence human disease.

Enzyme engineering relies on changes in the amino acid sequence of an enzyme to give rise to improvements in catalytic activity, substrate specificity, thermostability, and enantioselectivity. However, beneficial amino acid substitutions in proteins are difficult to rationally predict. Large numbers of enzyme variants containing random amino acid substitutions are screened in a high throughput manner to isolate improved enzymes. Identifying improved enzymes from the resulting library of randomized variants is a current challenge in protein engineering. This work focuses on the development of high-throughput screens for a class of enzymes called hydrolases, and in particular, proteases and esterases. In the first part of this work, we have developed an assay for detecting protease activity in the cytoplasm of Escherichia coli by exploiting the SsrA protein degradation pathway and flow cytometry. In this method, a protease-cleavable linker is inserted between a fusion protein consisting of GFP and the SsrA degradation tag. The SsrA-tagged fusion protein is degraded in the cell unless a co-expressed protease cleaves the linker conferring higher cellular fluorescence. The assay can detect specific cleavage of substrates by TEV protease and human caspase-8. To apply the screen for protease engineering, we sought to evolve a TEV protease variant that has altered P1 specificity. However, in screening enzyme libraries, the clones we recovered were found to be false positives in that they did not express protease variants with the requisite specificities. These experiments provided valuable information on physiological and chemical parameters that can be employed to optimize the screen for directed evolution of novel protease activities. In the second part of this work, single bacterial cells, expressing an esterase in the periplasm, were compartmentalized in aqueous droplets of a water-in-oil emulsion also containing a fluorogenic ester substrate. The primary water-in-oil emulsion was then re-emulsified to form a water-in-oil-in-water double emulsion which was capable of being analyzed and sorted by flow cytometry. This method was used to enrich cells expressing an esterase with activity towards fluorescein dibutyrate from an excess of cells expressing an esterase with no activity. A 50-fold enrichment was achieved in one round of sorting, demonstrating the potential of this method for use as a high-throughput screen for esterase activity. This method is suitable for engineering esterases with novel catalytic specificities or higher stabilit / text