Advances in combinatorial chemistry have facilitated the production of large chemical libraries that can be used as tools to discover biological probes and therapeutics. High-throughput screening (HTS) strategies have emerged as the standard method to assess the biological activity of small molecules. These screens involve the individual analysis of each small molecule in multi-well plates, often requiring expensive automated methods and development of robust assays that may not translate to physiologically relevant contexts. This problem of evaluating large numbers of reagents in physiologically relevant cell and animal models has been addressed for genetic reagents such as RNAi, CRISPR, and cDNA by creating barcoded retroviral libraries that can be used to infect target cells in culture or in animal models. Using these tools, effective reagents can be selected and decoded using a rapid and inexpensive procedure compared to testing of individual reagents one at a time in an arrayed fashion. In order to more efficiently analyze small molecules, a pooled approach would similarly be useful. This dissertation describes the studies towards developing a pooled screening strategy for small molecules in cellular contexts. Through an initial screen, we set to phenotypically profile small molecule biological activity in a pooled fashion, while simultaneously gain insight about an individual, active molecule’s mechanism of action. I first describe the design of the pooled screen and define the goals necessary for successful application. Next, I outline the steps taken and challenges encountered during the invention of each component of the technology. Finally, I discuss a computational, target-based approach to design small molecules appropriate for future applications of the new screening technology.

Le travail présenté dans ce manuscrit porte sur la recherche de nouveaux membres de la famille des dioxygénases α-cétoglutarate et fer dépendantes (α-KAO) et leur application en synthèse organique. Dans un premier, ce travail a consisté à chercher de nouvelles enzymes selon une approche génomique basée sur l’homologie de séquence et le partage d’un motif InterPro. Deux criblages haut débit avec 79 et 127 enzymes candidates ont ensuite été effectués sur des panels constitués respectivement de 23 et 36 substrats, structurellement plus ou moins proches des substrats métaboliques. Huit nouvelles α-KAO ont ainsi pu être découvertes. Parmi ces huit nouvelles α-KAO, quatre ont été étudiées plus en détail. Après optimisation des conditions de réaction pour chaque enzyme, des montées en échelle ont été réalisées pour caractériser les produits formés. A partir de ces quatre enzymes, la (3S)-3-hydroxy-L-lysine, un dérivé cyclisé de la (4R)-4-hydroxy-L-lysine, (3S)-3-hydroxy-L-ornithine et un dérivé de la (3S)-3-hydroxy-L-arginine ont pu être produits. Nous avons proposé une synthèse biocatalytique de mono et dihydroxydiamines en couplant une ou deux α-KAO avec une décarboxylase. Les (2S)-1,5-diamino-2-pentanol, 1,5-diamino-3-pentanol, (2S)-1,4-diamino-2-butanol et (2S,3S)-1,5-diamino-2,3-pentanediol ont ainsi été obtenus avec de bonnes conversions. / The work described in this manuscript deals with the search of new members of the α-ketoglutarate and Iron-dependent dioxygenases family (α-KAO) and their applications in organic synthesis. The first part of this work presents the search of new enzymes through a genomic approach based on sequence homology and InterPro motif sharing. Two high-throughput screenings with 79 and 127 candidate enzymes have been performed on 23 and 36 substrates more or less structurally close to known metabolic substrates. 8 new α-KAOs have been discovered. Among these new enzymes, four were studied in more details. After optimization of the enzymatic reaction conditions for each enzyme, scale-up allowed to obtain compounds for isolation and characterization. With these four enzymes, (3S)-3-hydroxy-L-lysine, (4R)-4-hydroxy-L-lysine as its cyclic derivative, (3S)-3-hydroxy-L-ornithine and a derivative of (3S)-3-hydroxy-L-arginine were produced. Two of the new α-KAO were combined in a cascade process to afford the (3R,4R)-3,4-dihydroxy-L-lysine as its cyclic derivative. We proposed a biocatalytic synthesis of mono and hydroxydiamines by coupling one or two α-KAO with a decarboxylase enzyme. (2S)-1,5-diamino-2-pentanol, 1,5-diamino-3-pentanol, (2S)-1,4-diamino-2-butanol and (2S,3S)-1,5-diamino-2,3-pentanediol were obtained with good overall conversions.

Transkriptionsfaktoren (TF) sind wichtige Regulatoren der Genexpression. In Arabidopsis kodieren ca. 1500-2000 Gene für TF, von denen die Mehrheit bis heute nicht funktionell charakterisiert ist. Um die Aufklärung der TF-Funktionen weiter voranzutreiben, werden daher Analyse-Plattformen für Hochdurchsatzverfahren immer wichtiger. In den letzten Jahren sind umfangreiche Gateway® -kompatible ORF (open-reading-frame)-Kollektionen für Arabidopsis aufgebaut worden, die nun als nützliche Ressourcen für genetische Analysen zur Verfügung stehen. Auf Grundlage dieser Kollektionen wurde in dieser Arbeit eine neue Screening-Plattform etabliert, mit der trans-regulatorische Eigenschaften von TF in einem Hochdurchsatzverfahren untersucht werden können. Ein Mikrotiterplatten-System für Protoplastentransformationen erlaubt die transiente Koexpression von 96 verschiedenen TF-Expressionsvektoren mit einem Promotor:Luciferase-Reporter der Wahl. Das Transaktivierungspotential jedes einzelnen TF kann über die Luciferaseaktivität bestimmt werden, indem emittierte Lumineszenz in einem Luminometer detektiert wird. Die Funktionalität des PTA (Protoplast Trans Activation)-Systems wurde anhand einer Transaktivierungsstudie der bereits gut charakterisierten Promotoren von RD29A und PDF1.2 und der ERF (Ethylene Response Factor)-TF-Familie überprüft, wobei bekannte Bindungsspezifitäten der TF bestätigt werden konnten. Für das System wurde eine umfassende Arabidopsis TF-Kollektion aufgebaut. Ca. 950 verschiedene Gateway® -kompatible TF-Expressionsvektoren stehen für Screening-Ansätze zur Verfügung. Für das PTA-System wurden verschiedene Anwendungen etabliert. Neben transaktivierenden, konnten beispielsweise auch repressive Eigenschaften von TF bestimmt werden. Darüber hinaus wurde gezeigt, dass es möglich ist, (I) die Expression von Promotoren gezielt durch verschiedene Stimuli, wie Salz oder Pflanzenhormone zu modulieren, (II) Protein-Protein-Interaktionen zu bestimmen, sowie (III) den Einfluss von Signalmolekülen (wie z. B. Kinasen) auf ihre Aktivierungseigenschaften zu untersuchen. Das PTA-System wurde in verschiedenen Screening-Ansätzen zur Identifizierung transkriptioneller Regulatoren pflanzlicher Stressantworten eingesetzt. In einer Analyse des Auxin-induzierbaren GH3.3-Promotors wurde dabei gezeigt, dass weit mehr bZIP-TF Einfluss auf die Auxin-vermittelte GH3.3-Expression haben, als bisher angenommen. Beispielsweise zeigten bZIP16 und bZIP68 ein höheres Transaktivierungspotential, als die bisher beschriebenen bZIP-Regulatoren der GH3.3-Expression. In einem zweiten Ansatz wurde die koordinierte Regulation der Biosynthese von Tryptophan-abgeleiteten antimikrobiellen Sekundärmetaboliten (Indol-Glukosinolate, Camalexin) untersucht. Dabei konnten ERF-TF der phylogenetischen Gruppen VIII und IX als potentielle Regulatoren mehrerer wichtiger Gene der Biosynthesewege identifiziert werden. Mit einem zusätzlichen Screening-Ansatz der gesamten TF-Expressionsvektor-Kollektion und einem Markerpromotor des Camalexin-Biosynthesewegs wurden weitere potentielle Regulatoren identifiziert, von denen einige bereits in der Pathogenantwort beschrieben sind. In einem weiteren Schwerpunkt dieser Arbeit wurde die von Weiste et al. (2007) etablierte Arabidopsis thaliana TF-ORF-Überexpressions-Kollektion (AtTORF-EX) erweitert. Mit Hilfe des dafür entwickelten Hochdurchsatzverfahrens zur Generierung stabil transformierter Pflanzenlinien wurden neue Überexpressionssamen-Kollektionen hergestellt und anschließend in einem Screening-Ansatz auf erhöhte Toleranz gegenüber oxidativem Stress getestet, wobei die Chemikalie Paraquat als oxidativer Stress-Geber eingesetzt wurde. Die TF bZIP1 und OBP1 konnten dabei als Resistenz-vermittelnd identifiziert werden. Zusammenfassend wurden in dieser Arbeit mit Hilfe beider Systeme neue potentielle Regulatoren pflanzlicher Stressantworten identifiziert. / Transcription factors (TFs) are important cellular regulators of gene expression. In Arabidopsis approximately 1500-2000 genes encode for TFs. Until now, the majority of these genes has not been functionally characterized. To further promote the evaluation of TF function, high-throughput tools are required. In recent years, comprehensive Arabidopsis open reading frame (ORF) collections have been established, which are valuable resources for functional genomics. Based on these collections a high-throughput microtiter plate based Protoplast Trans Activation (PTA) system has been established to screen for TFs which regulate a given promoter:Luciferase construct in planta. 96 protoplast transfection experiments can be performed simultaneously in a standard microtiter plate. Transactivation of a promoter:Luciferase reporter is measured via luciferase imaging. A screening collection of roughly 950 TFs expression vectors has been assembled using Gateway® technology and can be tested in various screening approaches. In this respect, it is possible to analyze transactivating as well as repressive properties. Moreover (I) stimulus induced transcription, (II) studies of protein-protein interaction and (III) the impact of signaling molecules (e.g. kinases) on the promoters activation potential can be measured. To demonstrate the feasibility of the high-throughput system, the transactivating properties of the Ethylene Response Factor (ERF) TF family were studied in combination with the well-characterized RD29A and PDF1.2 promoters. By this means, known binding specificities of the TF family were confirmed. Furthermore, the PTA-System was applied to identify transcriptional regulators involved in plant stress responses. In one approach the influence of bZIP TFs on the auxin-inducibility of the GH3.3-promoter was studied. In particular, bZIP16 and bZIP68 showed a stronger transactivation potential than those bZIPs which were previously described to regulate this auxin-responsive promoter. In an independent approach the transcriptional regulation of tryptophan-derived antifungal compounds (indol-glycosinolates, camalexin) biosynthesis has been studied. ERF TFs of the groups VIII and IX were identified as potential regulators of several biosynthetic genes. A subsequent screening approach of a key promoter of the camalexin biosynthetic pathway disclosed further potential regulators. Among these TFs, many have been described previously in plant pathogen responses. As a second approach to examine TF function the Arabidopsis thaliana TF ORF-EXpression-library (AtTORF-EX) established by Weiste et al. (2007) was extended. The developed high-throughput transformation procedure was used to generate new TF overexpression seed collections. Afterwards the library was applied in a screening approach to identify regulators which mediate enhanced tolerance towards the oxidative stress inducing chemical Paraquat. Thus, the TFs bZIP1 and OBP1 were found to promote resistance against Paraquat when overexpressed in Arabidopsis. In summary, using both approaches novel putative regulators of plant stress response signaling were identfied.

Ionisierende Strahlung (IR) ist in der medizinischen Diagnostik und in der Tumortherapie von zentraler Bedeutung, kann aber Genominstabilität und Krebs auslösen. Strahleninduzierte Genominstabilität (RIGI) ist in den klonalen Nachkommen bestrahlter Zellen zu beobachten, die zugrundeliegenden Mechanismen sind jedoch noch unverstanden. Zur Erforschung von verzögerten Strahleneffekten wurden primäre embryonale Fibroblastenkulturen mit 2 Gray bestrahlt und für 20 Populationsverdopplungen klonal expandiert. Zellen, die keiner Strahlung ausgesetzt waren, dienten als Kontrolle für normale Alterungsprozesse. Die Klone wurden durch klassische Chromosomenbänderungstechniken analysiert und in Abhängigkeit der Stabilität ihres Genoms in Gruppen eingeteilt. Ein Klon wurde als stabil gewertet, wenn die analysierten Metaphasen keinerlei Auffälligkeiten zeigten, während instabile Klone ein Mosaik aus normalen und abnormalen Metaphasen waren. Die Zellen von zwei Spendern wurden untersucht, um interindividuelle Strahleneffekte zu beurteilen. Nach Bestrahlung hatten mehr als die Hälfte der Klone Metaphasen mit strukturellen Aberrationen und wurden dementsprechend als instabil eingestuft. Drei Klone zeigten zudem numerische Aberrationen, die ausschließlich das Y Chromosom betrafen. Fluoreszenz in situ Hybridisierungen verifizierten diese Beobachtung in weiteren Klonen und deuteten an, dass der Verlust des Y Chromosoms mit RIGI assoziiert ist. Molekulare Karyotypisierungen mit SNP Arrays ergaben, dass IR in den Klonen Veränderungen der Kopienzahl auslöst. Ein Unterschied zwischen chromosomal stabilen und instabilen Klonen konnte jedoch nicht detektiert werden. Chromosomale Regionen, in denen sich bekanntermaßen fragile Stellen befinden, zeigten eine Anhäufung von CNVs. Ein RIGI Effekt konnte für die fragile Stelle 3B, in der sich das Gen FHIT befindet, identifiziert werden. Exom Sequenzierungen von Klonen und der entsprechenden Massenkultur zeigten eine alterungsassoziierte Entstehung von Varianten. Der Effekt wurde durch die Einwirkung von Strahlung erhöht. Auf Ebene von einzelnen Nukleotiden konnten ebenfalls Anhäufungen von Schäden in bestimmten genomischen Bereichen detektiert werden, dieser Effekt ging ohne die typischen RIGI Endpunkte einher. Die Ergebnisse der vorliegenden Arbeit zeigen, dass strahlenbedingte Veränderungen auf verschiedenen Ebenen (Chromosomen, Genkopienzahl und einzelnen Nukleotiden) beobachtet werden können, welche, unabhängig von RIGI, die Tumorentstehung begünstigen. Speziell Veränderungen im FRA3B Lokus und der Verlust des Y Chromosoms scheinen jedoch über die Destabilisierung des Genoms zur Krebsentstehung beizutragen. / Ionizing radiation is important in medical diagnosis and cancer treatment but can lead to genomic instability and secondary malignancies as a late effect. Radiation induced genomic instability (RIGI) can be observed in the progeny of irradiated cells but the underlying cellular processes remain to be elucidated. To study delayed genetic radiation effects, single cell fibroblast clones from two different male donors were established after a single X ray dose of 2 Gray. Non irradiated cells were used as controls to account for aging related effects. Clones were characterized using chromosome banding analysis. Stable clones were endowed with no karyotype abnormalities in all analysed metaphases after 20 Population doublings, whereas unstable clones showed both normal and abnormal metaphases. Two fibroblast strains were used to detect differences in radiation sensitivity. More than half of the irradiated clones were chromosomally abnormal and thus classified as unstable. Both banding analysis and fluorescence in situ hybridization revealed an increased rate of Y chromosome loss in irradiated clones which might be associated with RIGI. Using SNP Arrays, an increased rate of de novo copy number variations (CNVs) was detected in the progeny of irradiated cells when compared to non irradiated controls. However, a significant difference between chromosomally stable and unstable clones was not found. CNVs clustered in chromosomal regions that are associated with known fragile sites. The fragile site 3B, which encompasses the gene FHIT, was found to be affected by CNVs in unstable clones suggesting a RIGI related effect. Exome sequencing of clones and the respective primary cultures revealed an increased rate of variants during in vitro aging. This effect was further increased by radiation exposure. Analysis of single nucleotides showed a RIGI independent accumulation of damage in specific regions. The results of the present study show that radiation induced changes can manifest as chromosomal abnormalities, copy number variations and DNA sequence mutations promoting tumorigenesis independent from RIGI. However, changes in FRA3B and loss of Y chromosome might trigger cancer through destabilizing the genome.

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.

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