Degree

Doctor of Philosophy (PhD)

Department

Chemical Engineering

Document Type

Dissertation

Abstract

Enzymes regulate nearly all cellular and biochemical processes governing cellular proliferation, metabolism, and migration. One example is the ubiquitin-proteasome system (UPS), a highly complex, tightly regulated pathway responsible for the recognition and degradation of misfolded or damaged proteins inside the cell. Protein degradation requires posttranslational addition of a polyubiquitin chain to target the protein to the proteasome for degradation which can be reversed by deubiquitinating enzymes (DUBs) which remove the chain. Dysregulation of the UPS is associated with many diseases including cancer which has resulted in the development of inhibitors, like Bortezomib and Carfilzomib, driving higher median patient survival rates in multiple myeloma (MM), a cancer of the blood cells. Unfortunately, due the heterogeneous nature of cancer, patients have been found to respond differently to proteasome-targeted therapeutics motivating the need for a personalized treatment approach. Unfortunately, there are limited options to measure proteasome activity with nearly all available methods being restricted to cell lysates. Bulk analysis of cell lysates fails to detect small populations of drug resistant cells which limits the ability to tailor a personalized treatment protocol. The result is a significant need for new bioanalytical techniques capable of quantifying enzyme activity in intact single cells in a high throughput manner. The goal for this thesis was to address this need through the development of several facets of a high-throughput platform including the development of peptide-based biosensors and microfluidic devices capable of isolating and studying intact single cells. Most of the thesis is focused on a new approach to quantifying enzymes associated with the UPS. In the first part, a thorough study was carried out investigating the effects of adding a cargo of different characteristics to a cell penetrating peptide (CPP) on its uptake efficiency. CPPs are an effective method to deliver peptide-based biosensors to intact cells and this study demonstrated that increasing net positive charge on the cargo can enhance the cellular internalization of the CPP/cargo complex. Leveraging these findings, the next component of the thesis described the synthesis and characterization of a proteasome biosensor consisting of four equally important parts: (i) a β-hairpin CPP, (ii) a short 4 amino acid proteasome recognition sequence, (iii) a fluorescent readout, and (iv) a short CPP enhancer. The proposed biosensor was shown to be selective towards the proteasome, and capable of quantifying its activity directly in intact cells with great efficiency and sensitivity. Next, a droplet microfluidic device incorporated with a previously developed live-cell compatible DUB biosensor was utilized to study single-cell DUB activity to identify subpopulations with enhanced or diminished DUB activity. This platform was able to index and dynamically analyze single-cell DUB activity. The final component of the thesis targeted a different type of enzyme called alkaline phosphatase (AP) which algae use to fix available phosphorous during growth. AP activity has been suggested as a better metric to characterize algal growth during a harmful algal bloom (HAB); however, there are no existing bioanalytical methods to perform single cell analysis. In this chapter a trap-based microfluidic device was used to study AP activity at the single cell-level, and provide insight on the advantages of single-cell over bulk analysis in identifying distinct cellular subpopulations and unmasking valuable data that would be otherwise overlooked. Together, this thesis offers an all-around versatile experimental and analytical platform for single-cell analysis of enzyme activity with significant potential for addressing healthcare and environmental challenges.

Date

11-15-2023

Committee Chair

Melvin, Adam T.

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