This dissertation introduces novel machine learning methods based on parallelized cellomics to analyze interactions between cells, bacteria, and chemical compounds while reducing the use of fluorescent reagents. Machine learning analysis using image-based high-content screening (HCS) data is compartmentalized into three primary components: (1) \textit{Image Analytics}, (2) \textit{Phenotypic Analytics}, and (3) \textit{Compound Analytics}. A novel software analytics tool called the Insights project is also introduced. The Insights project fully incorporates distributed processing, high performance computing, and database management that can rapidly and effectively utilize and store massive amounts of data generated using HCS biological assessments (bioassays). It is ideally suited for parallelized cellomics in high dimensional space.
Results demonstrate that a parallelized cellomics approach increases the quality of a bioassay while vastly decreasing the need for control data. The reduction in control data leads to less fluorescent reagent consumption. Furthermore, a novel proposed method that uses single-cell data points is proven to identify known active chemical compounds with a high degree of accuracy, despite traditional quality control measurements indicating the bioassay to be of poor quality. This, ultimately, decreases the time and resources needed in optimizing bioassays while still accurately identifying active compounds.
Background: Chemistry and particularly enzymology at surfaces is a topic of rapidly growing interest, both in terms of its role in biological systems and its application in biocatalysis. Existing protein immobilization approaches, including noncovalent or covalent attachments to solid supports, have difficulties in controlling protein orientation, reducing nonspecific absorption and preventing protein denaturation. New strategies for enzyme immobilization are needed that allow the precise control over orientation and position and thereby provide optimized activity.
Methodology/Principal Findings: A method is presented for utilizing peptide ligands to immobilize enzymes on surfaces with improved enzyme activity and stability. The appropriate peptide ligands have been rapidly selected from high-density arrays and when desirable, the peptide sequences were further optimized by single-point variant screening to enhance both the affinity and activity of the bound enzyme. For proof of concept, the peptides that bound to β-galactosidase and optimized its activity were covalently attached to surfaces for the purpose of capturing target enzymes. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activity and stability, as well as controlled protein orientation.
Conclusions/Significance: A simple method for immobilizing enzymes through specific interactions with peptides anchored on surfaces has been developed. This approach will be applicable to the immobilization of a wide variety of enzymes on surfaces with optimized orientation, location and performance, and provides a potential mechanism for the patterned self-assembly of multiple enzymes on surfaces.
