The recently-emerged field of chemical genomics is centered on the use of small molecules to perturb biological systems as a means of investigating their function. In order to employ this approach for the study of plant-pathogen interactions, I established an assay in which Arabidopsis thaliana seedlings are grown in liquid media in 96-well plates. Inoculation of these seedlings with a virulent strain of the bacterial phytopathogen Pseudomonas syringae resulted in macroscopic bleaching of the cotyledons of these seedlings. This symptom was used as the basis for high-throughput chemical genomic screens aimed at identifying small molecules that protect Arabidopsis seedlings from infection. One of the first chemicals identified through this screen was the sulfanilamide compound sulfamethoxazole (Smex). This compound was later shown to also reduce the susceptibility of both Arabidopsis and wheat to infection by the fungal pathogen Fusarium graminearum, suggesting a broad spectrum of activity. More detailed investigations of Smex indicated that the protective activity of this compound did not derive from antimicrobial effects, and that this activity was not executed through common defence-related signalling pathways. The folate biosynthetic pathway enzyme dihydropteroate synthase is a known target of sulfanilamides, and it does appear to contribute to Smex-induced disease resistance, albeit in a folate-independent manner. In order to identify downstream mediators of Smex activity, I initiated two forward genetic screens intended to recover mutants with altered sensitivity to Smex in a seedling growth assay. Interestingly, while these screens yielded mutants with striking Smex sensitivity phenotypes, disease resistance phenotypes were not altered. Gene expression profiling of Arabidopsis tissues treated with Smex prior to bacterial inoculation suggested that this compound generally affects lipid signalling. Altogether, it is evident that Smex elicits a complex set of responses in Arabidopsis with apparently non-overlapping phenotypic outputs.

The recently-emerged field of chemical genomics is centered on the use of small molecules to perturb biological systems as a means of investigating their function. In order to employ this approach for the study of plant-pathogen interactions, I established an assay in which Arabidopsis thaliana seedlings are grown in liquid media in 96-well plates. Inoculation of these seedlings with a virulent strain of the bacterial phytopathogen Pseudomonas syringae resulted in macroscopic bleaching of the cotyledons of these seedlings. This symptom was used as the basis for high-throughput chemical genomic screens aimed at identifying small molecules that protect Arabidopsis seedlings from infection. One of the first chemicals identified through this screen was the sulfanilamide compound sulfamethoxazole (Smex). This compound was later shown to also reduce the susceptibility of both Arabidopsis and wheat to infection by the fungal pathogen Fusarium graminearum, suggesting a broad spectrum of activity. More detailed investigations of Smex indicated that the protective activity of this compound did not derive from antimicrobial effects, and that this activity was not executed through common defence-related signalling pathways. The folate biosynthetic pathway enzyme dihydropteroate synthase is a known target of sulfanilamides, and it does appear to contribute to Smex-induced disease resistance, albeit in a folate-independent manner. In order to identify downstream mediators of Smex activity, I initiated two forward genetic screens intended to recover mutants with altered sensitivity to Smex in a seedling growth assay. Interestingly, while these screens yielded mutants with striking Smex sensitivity phenotypes, disease resistance phenotypes were not altered. Gene expression profiling of Arabidopsis tissues treated with Smex prior to bacterial inoculation suggested that this compound generally affects lipid signalling. Altogether, it is evident that Smex elicits a complex set of responses in Arabidopsis with apparently non-overlapping phenotypic outputs.

Biocatalysts offer advantages over their chemical counterparts in terms of their high enantioselectivity and the opportunity to develop more environmentally friendly processes. However, the widespread adoption of biocatalytic processes is hampered by the long development times for enzymes with novel and sufficient activity and adequate stability under operating conditions. Protein engineering, while extremely useful for modifying the properties of protein catalysts in select cases, still cannot be performed rapidly enough for many applications. In order for biocatalysts to become a competitive alternative to chemical catalysts, new tools to make the tailoring of biocatalysts by protein engineering methods speedier and more efficient are necessary. The aim of this work was to develop methods to aid in the faster production of novel biocatalysts. Protein engineering involves two steps: the generation of diversity and the screening or selection of variants with the desired properties. Both of these must be targeted to create a faster protein engineering process. In the case of the former, this work sought to clone and overexpress some template enzymes which would create smaller, more manageable libraries of mutants with a higher likelihood of function by the manipulation of a few focused amino acid residues. For the latter, this work developed and validated a Monte-Carlo simulation model of pooling to increase screening throughput and created a set of vectors to aid in high-throughput screening by eliminating unwanted mutants from the assay procedure entirely.

Thesis (M.S.)--Ball State University, 2009. / Title from PDF t.p. (viewed on Nov. 30, 2009). Includes bibliographical references (p. 74-80).

Ricin and Shiga toxin type 1 are potent cytotoxins known as ribosome inhibition proteins, abbreviated RIPs. Proteins of this family shut down protein synthesis by removing a critical adenine in the conserved stem-loop structure of 28S rRNA. Due to its exquisite cytotoxicity, the plant toxin ricin has been used as a biological warfare agent. Although great achievement has been made on ricin research, including catalytic mechanism and structure analysis, there is still no specific treatment available for ricin exposure. In addition, ricin A chain inhibitors may also be useful against the homologous bacterial proteins shiga toxins, which are responsible for dysentery, and diseases related to food poisoning, including hemolytic uremic syndrome. Previous study on RTA inhibitor search has provided a number of substrate analog inhibitors, all of which, however, are weaker inhibitors. Therefore, the goal of this work is to improve the binding affinity of known inhibitors and to discovery new scaffolds for inhibitor discovery and development. In this work, multiple approaches were employed for this purpose, including optimizing known inhibitors and searching new inhibitors by Virtual Drug Screening (VDS) and High Throughput Screening (HTS). A number of new RTA inhibitors were discovered by these strategies, which provide a variety of pharmacophores for RTA inhibitor design, and also added a new line of evidence for VDS as an advanced technology for drug discovery and development. / text

Asymmetric synthesis has become an important tool to prepare enantiomerically pure compounds because it avoids the wasteful discarding of the undesired enantiomer. Combinatorial libraries allow for much faster screening for new and better asymmetric catalysts/auxiliaries, but they generate a large number of samples whose enantiomeric excess (ee) cannot be determined rapidly. This bottleneck currently limits the applicability of such approaches. We propose here the use of faster optical techniques for the determination of ee using common instrumentation, such as UV-vis spectrophotometers, and circular dichroism (CD) spectrophotometers. Our methods are easily transitioned to the microwell format commonly used in parallel/combinatorial chemistry endeavors, just by using common microplate readers: this allows for an even more rapid analysis of samples and a seamless integration in a high-throughput workflow. We have shown that enantioselective indicator displacement assays can be developed to determine ee in a high-throughput fashion utilizing either a UV-vis spectrophotometer or a 96-well plate reader. Two chiral receptors and a commercial pH indicator were used to enantioselectively discriminate α-amino acids by monitoring the degree of indicator displacement. The two receptors were able to enantioselectively discriminate 13 of the 17 analyzed α-amino acids and accurately determine ee values of independent test samples with the use of ee calibration curves. Moreover, a sample of valine was synthesized through an asymmetric reaction, whose ee was then determined with our assay and compared to chiral HPLC and 1H NMR chiral shift reagent analysis, with excellent correlation. An artificial neural network was also successfully employed in the analyses, as an alternative to ee calibration curves. Both techniques consistently produced results accurate enough for preliminary determination of ee in a rapid manner, allowing for high throughput screening (HTS) of asymmetric reactions. The use of circular dichroism spectroscopy with chiral BINAP was also explored to enantioselectively discriminate α-chiral ketones. The ketones were derivatized with pyridyl hydrazines to produce hydrazones, which were then bound to enantiomerically pure [Cu(I)(BINAP)]+, forming diastereomeric complexes with differential steric interactions leading to different degrees of twist in the BINAP moiety and characteristic signatures in the CD spectrum, as a function of sample ee. / text

EZH2 is a SET domain-containing methyltransferase and the catalytic component of the multimeric Polycomb- group (PcG) protein complex, PRC2. When in complex with other PRC2 members (EED, SUZ12, AEBP2, and RBBP4), EZH2 catalyzes methylation of H3K27, a histone modification associated with transcriptional repression and developmental regulation. As several PRC2 components are upregulated or mutated in a variety of human cancers, efforts to discover small-molecule modulators of PRC2 and understand its regulation may yield therapeutic insights. Identification of small-molecule probes with distinct chemotypes, MOAs, and selectivity profiles are not only of great value, but necessary in establishing comprehensive probe sets capable of illuminating the various roles of EZH2 in oncogenesis. Here we describe efforts to identify and characterize small-molecule modulators of PRC2 and further understand its regulation. Chapter II outlines the expression and purification of 5-component PRC2 (EZH2-EED-SUZ12-AEBP2-RBBP4) and the establishment of biochemical and cellular HTS assays. These assays were used to screen a diverse set of small molecules (>120,000), identifying biochemical PRC2 inhibitors and activators (described in Chapter III). One biochemical PRC2 inhibitor, BRD1835, appeared to inhibit PRC2 activity through a novel artifactual mechanism involving interaction with peptide substrate, leading to apparent peptide-competitive behavior and putative cellular activity (described in Chapter IV). The characterization of novel biochemical PRC2 activators, BRD3934 and BRD8284, is discussed in Chapter V. Chapter VI describes the use of an HCS assay to identify known bioactive compounds that alter intracellular levels of H3K27me3 through modulating H3K27me3-connected regulatory nodes or by targeting PRC2 directly. These efforts led to the discovery that an antifungal agent, miconazole, is capable of activating PRC2 activity in vitro, while a mucolytic agent, bromhexine, selectively ablates cellular H3K27me3 levels through targeting an activity distinct from PRC2. Finally, Chapter VII discusses novel PRC2-connected crosstalk mechanisms identified through screening libraries of uniquely modified histone peptides for their ability to bind or support methylation by PRC2. These studies enhance our understanding of PRC2 regulation by revealing the effects of H3R26 and H3K23me1 modifications on enzymatic activity, implicating their respective methyltransferases in PRC2 regulation.

Plant hormones act in a complex network where their pathways regulate and interact to control different mechanisms, such as development and stress responses. This crosstalk between hormones can be exploited by pathogens to suppress plant defense responses and thereby increase pathogen growth. Pseudomonas syringae pathogenicity is reliant on a Type III secretion system (TTSS) that acts as a specialized injection apparatus to deliver virulence proteins, known as type III effectors (TTEs), into the plant cell cytosol. In my work, I have screened hormone inducible promoter::GUS transgenic Arabidopsis thaliana lines against a P. syringae TTE library in order to identify TTEs involved in the perturbation of hormone signaling in planta. Through this screen I identified two P. syringae TTEs, HopAK1 and HopAL1, both belonging to the same bacterial strain P. syringae pv. maculicola ES4326. I found that HopAK1 can sensitize A. thaliana plants to auxin. On the other hand, HopAL1 activates auxin signaling. Monitoring of auxin signaling was done using transgenic DR5::GUS plants. Both TTEs render the plant susceptible to bacterial infection, highlighting a potential relationship between increased auxin signaling and virulence.

Determining how biological cells respond to external factors in the environment can aid in understanding disease progression, lead to rational design strategies for tissue engineering, and contribute to understanding fundamental mechanisms of cellular function. Dynamic mechanical forces exist in vivo and are known to alter cellular response to other stimuli. However, identifying the roles multiple external factors play in regulating cell fate and function is currently impractical, as experimental techniques to mechanically stimulate cells in culture are severely limited in throughput. Hence, determining cell response to combinations of mechanical and biological factors is technically limited. In this thesis, microfabricated systems were designed, implemented and characterized to screen for the effects of mechanical stimulation in a high-throughput manner. Realizing these systems required the development of a fabrication process for precisely-aligned multilayer microstructures, and the development of a method to integrate non-traditional and clinically-relevant biomaterials into the microfabrication process. Three microfabricated platforms were developed for this application. First, an array was designed for experiments with high mechanical throughput, in which cells cultured on a surface experience a range of cyclic, uniform, equibiaxial strains. Using this array, a novel time- and strain-dependent mechanism regulating nuclear β-catenin accumulation in valve interstitial cells was identified. Second, a simpler system was designed to screen for the effects of combinatorially manipulated mechanobiological parameters on the pathological differentiation of valve interstitial cells. The results demonstrate functional heterogeneity between cells isolated from different regions of the heart valve leaflet. Last, a microfabricated platform was developed for high-throughput mechanical stimulation of cells encapsulated in a three-dimensional biomaterial, enabling the study of mechanical forces on cells in a more physiologically relevant microenvironment. Overall, these studies identified novel biological phenomena as a result of designing higher-throughput systems for the mechanical stimulation of cells.

Determining how biological cells respond to external factors in the environment can aid in understanding disease progression, lead to rational design strategies for tissue engineering, and contribute to understanding fundamental mechanisms of cellular function. Dynamic mechanical forces exist in vivo and are known to alter cellular response to other stimuli. However, identifying the roles multiple external factors play in regulating cell fate and function is currently impractical, as experimental techniques to mechanically stimulate cells in culture are severely limited in throughput. Hence, determining cell response to combinations of mechanical and biological factors is technically limited. In this thesis, microfabricated systems were designed, implemented and characterized to screen for the effects of mechanical stimulation in a high-throughput manner. Realizing these systems required the development of a fabrication process for precisely-aligned multilayer microstructures, and the development of a method to integrate non-traditional and clinically-relevant biomaterials into the microfabrication process. Three microfabricated platforms were developed for this application. First, an array was designed for experiments with high mechanical throughput, in which cells cultured on a surface experience a range of cyclic, uniform, equibiaxial strains. Using this array, a novel time- and strain-dependent mechanism regulating nuclear β-catenin accumulation in valve interstitial cells was identified. Second, a simpler system was designed to screen for the effects of combinatorially manipulated mechanobiological parameters on the pathological differentiation of valve interstitial cells. The results demonstrate functional heterogeneity between cells isolated from different regions of the heart valve leaflet. Last, a microfabricated platform was developed for high-throughput mechanical stimulation of cells encapsulated in a three-dimensional biomaterial, enabling the study of mechanical forces on cells in a more physiologically relevant microenvironment. Overall, these studies identified novel biological phenomena as a result of designing higher-throughput systems for the mechanical stimulation of cells.