Clinically tractable diagnostics must be low-cost, rapid, sensitive, easy to use, and adaptable to new targets. With its rational design, synthetic biology holds promise for developing diagnostic technologies that can address these needs. In particular, progress in synthetic biology has led to improved circuit-building abilities and a large collection of biomolecular sensors. However, these technologies fundamentally require transcription and translation, limiting their applicability to cellular contexts In vitro cell-free expression systems that contain transcription and translation machinery provide the environment necessary for biologically-based technologies to function independently of living cells. Our lab recently developed a paper-based system for cell-free gene expression, which utilizes cell-free extracts that are freeze-dried on to paper and other porous substrates to allow for long-term preservation of synthetic circuits at room temperature. Our platform represents a scalable, cost-effective technology that is easy to use and is compatible with synthetic biology tools. In this dissertation, I present several advancements to this diagnostic platform that are geared towards improving the system’s clinical tractability. In the context of developing a diagnostic for Zika virus that could be deployed in low-resource settings, I demonstrate improvements to diagnostic sensitivity and rapid sample processing that allow for detection of low femtomolar quantities of active virus directly from blood plasma samples. I also describe preliminary results towards a streamlined one-pot amplification-sensing reaction, and propose the development of a paper-based diagnostic for antibiotic susceptibility testing.

Modern cancer immunotherapy involves the conditioning of endogenous T cells to fight cancerous bodies that have managed to resist or avoid detection. Recently approved antibody drugs target the immune checkpoint pathway in T cells to prevent their tolerance to cancer antigens. There exists a compelling need, especially in the drug discovery world, to develop better assays for screening and to study the underlying mechanisms of these new antibody drugs. The core motivation of my work is to develop a primary cell assay for the immune checkpoint pathway using 1-D imaging cytometry. The assay is focused on high throughput and high content screening. It takes advantage of our novel 1-D imaging cytometer platform. The assay is designed to artificially induce anergy in primary human T cells and systemically study their drug response. An automated statistical method quantifies the functional phenotypes of both healthy and anergic T cells into a single descriptive readout. Reducing localization of biomarkers into a single ‘activity score’ readout has many advantages for drug screening and characterization. Additional assays were developed to study T cell activation dynamics and other signaling events during the immune checkpoint pathway. Our 1-D instrument leverages both the high throughput aspects of traditional flow cytometry and the high spatial content of 2-D imaging cytometers. The PMC data analysis emphasizes an unbiased approach to analyze flow cytometry data, which eliminates the subjective manual gating of current cytometric methods. This is crucial to developing more accurate and reliable assays with minimal supervision and need for expert operators. The high-throughput and high-content capabilities presented enable new types of assays previously not possible with human primary T cells. Adoption of physiological relevant primary cell assays has potential to revolutionize large-scale drug screening and future applications in personalized medicine.

Changes in birefringence (or dynamic birefringence) provide an arguably cleaner method of measuring IOS as compared to scattering methods. Other imaging methods have substantial limitations. Nerves inherently exhibit a static (rest condition) birefringence that is associated with the structural anisotropies of axonal protein filaments, membrane phospholipids and proteins, as well as surrounding tissues, which include Schwann cells and axon sheaths. The dynamic birefringence, or “crossed-polarized signal” (XPS), in neurons arises from activity in axons and occurs with a rapid momentary change, typically a decrease, in the birefringence when action potentials (APs) propagate along them. We improved the signal-to-noise to make detecting this signal an easier task, and present the XPS as a viable candidate for detecting AP activity in anisotropic nervous tissue. Our data collectively serves as a strong indication that there is a capacitive-charging-like effect directly inducing a gradual recovery (long tail) of the XPS to baseline, and also causing a smoothing of the XPS trace. A setup was constructed that successfully demonstrated the feasibility of tracking propagating compound APs in a peripheral nerve using the XPS. We made significant progress in the attempt to investigate birefringence of myelination. For the first time, the XPS in a myelinated tissue was detected, and it appears to be bipolar in nature. Further work in investigating the nature of this signal is needed, and is currently underway. Since changes in birefringence in neurons are associated instantaneously with electrophysiological phenomena, they are well-suited for fast imaging of propagating action potentials in neuronal tissue. In summary, imaging based on polarization sensing of changes in birefringence offers promise for an improved noninvasive method of detecting and tracking AP activity in myelinated and unmyelinated nerves and could be designed for pre-clinical and surgical applications.

Treatments using a patient’s own T cells to target cancer have applied advances in genetic engineering and cancer immunotherapy as the basis for a powerful, targeted cell-based therapy. Tumor-infiltrating lymphocytes and T cells that have been genetically modified to express cancer antigen-specific receptors—such as T cell receptors (TCRs) and chimeric antigen receptors (CARs)—leverage the innate targeted cytotoxicity of T cells against cancer. Despite promising results in clinical trials, these therapies have elicited serious and sometimes fatal responses. These toxicities include killing of healthy tissue expressing lower levels of antigen, as well as an overstimulation of the immune response called cytokine release syndrome. These outcomes reflect the double-edged nature of T cell-based therapies: the same powerful capability that makes them strong therapeutic candidates can become fatal if modified cells are left to their own devices. T cell therapies would greatly benefit from the development of tools that enable doctors to have bedside control over a cell’s behavior and truly respond to each patient’s needs. The work of this thesis aims to develop genetic circuits that control T cell activity. This platform has been adapted to control when CARs are expressed and at what level. In contrast to the current approach where patients are treated to one therapeutic “state,” these genetic circuits will allow doctors to decide between multiple states defined by CAR expression through the addition of a drug. These circuits contain memory such that long-term administration of the drug is not required to maintain a change. I have designed an ON switch and an OFF switch to control when a CAR is expressed, and an EXPRESSION switch to increase CAR expression. I characterized the performance of these circuits to demonstrate their dynamics over time, as well as their ability to control T cell behavior. I also demonstrate that these circuits contain memory, and that they are tunable. / 2020-07-02T00:00:00Z

Protein interactions regulate gene expression, cell signaling, catalysis, and many other functions across all of molecular biology. We must understand them quantitatively, and experimental methods have provided the data that form the basis of our current understanding. They remain our most accurate tools. However, their low efficiency and high cost leave room for predictive, computational approaches that can provide faster and more detailed answers to biological problems. A rigid-body simulation can quickly and effectively calculate the predicted interaction energy between two molecular structures in proximity. The fast Fourier-transform-based mapping algorithm FTMap predicts small molecule binding 'hot spots' on a protein's surface and can provide likely orientations of specific ligands of interest that may occupy those hot spots. This process now allows unique ligands to be used by this algorithm while permitting additional small molecular cofactors to remain in their bound conformation. By keeping the cofactors bound, FTMap can reduce false positives where the algorithm identifies a true, but incorrect, ligand pocket where the known cofactor already binds. A related algorithm, ClusPro, can evaluate interaction energies for billions of docked conformations of macromolecular structures. The work reported in this thesis can predict protein-polysaccharide interactions and the software now contains a publicly available feature for predicting protein-heparin interactions. In addition, a new approach for determining regions of predicted activity on a protein's surface allows prediction of a protein-protein interface. This new tool can also identify the interface in encounter complexes formed by the process of protein association—more closely resembling the biological nature of the interaction than the former, calculated, binary, bound and unbound states.

An abnormal multicellular architecture and a stiffened extracellular matrix (ECM) are defining characteristics of breast cancer, and yet, most in vitro tumor models fail to recapitulate the aberrant tumor microenvironment or accurately predict in vivo cellular responses to therapeutics. This dissertation aims to fill this gap in knowledge by developing and applying a suite of novel in vitro tools to investigate how the physical properties of the tumor microenvironment drive cancer progression. Our approach to develop and apply in vitro tools rests on three independent, but synergistic pillars. First, we established a 3D in vitro tumor model that mimics critical cell-cell and cell-ECM interactions by embedding multicellular spheroids within 3D collagen matrices. We assessed the in vivo relevance of our 3D collagen embedded spheroid model by quantifying the presence of highly malignant cancer stem cells (CSCs) before and after chemotherapeutic treatment with either paclitaxel or cisplatin. By characterizing the CSC response within two other commonly used in vitro models—a 2D monolayer and a 3D collagen model in which single cells are diffusely embedded—we found the CSC response to be model-dependent. Our results therefore highlight the need to screen potential CSC-specific chemotherapy drugs within in vitro models that recapitulate the in vivo 3D multicellular tumor architecture. Second, through integrating computational and experimental approaches, we developed a mathematical model of the transcriptional regulators—Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ)—which are often overactive in late-stage cancers despite mutations within their upstream signaling pathway being rare. Here, dysregulated cytoskeletal tension and disrupted apical-basal polarity, two defining characteristics of breast cancer, have been suggested to promote overactive YAP/TAZ signaling. Therefore, we developed a computational model to study how overactive cytoskeletal tension, due to increased ECM stiffness, leads to aberrant YAP/TAZ signaling in cancer. The model revealed that simultaneous alterations in cell mechanics and cell-cell adhesion signaling synergistically converge on YAP/TAZ activity and lead to its overactivation, a process poorly understood in cancer progression. Finally, in an effort to decouple the effects of collagen fiber density and network mechanics on cancer cell behavior, we developed a highly tunable in vitro 3D interpenetrating network (IPN) platform consisting of a primary collagen network reinforced by a secondary visible-light-mediated thiol-ene PEG network. The IPN platform is cytocompatible, inherently bioactive, and mechanically tunable, which makes it a useful tool for studying mechanotransductive signaling pathways. Moreover, while this thesis work focused on in vitro applications, our approach raises the interesting possibility of altering the physical properties of the tumor microenvironment as a potential therapeutic. In summary, this work addresses the question of how the physical properties of the tumor microenvironment affect cancer progression by deploying three distinct, but complementary approaches, and suggests that addressing the physical aspects of cancer progression may improve clinical outcomes. / 2020-02-20T00:00:00Z

<p> The overall goal of this thesis work is to study the effects of biomodulations on Alzheimer's disease (AD) related cellular pathways, using biophysical and photobiological methods, including secretory phospholipase A2, various fatty acids treatments and low energy light irradiation. By increasing membrane fluidity in neuronal cells, secretory phospholipase A2 and unsaturated fatty acids with 4 or more double bonds are able to increase the secretion of neuroprotective and neurotrophic &alpha;-secretase-cleaved soluble APP (sAPP_&alpha;). Low energy laser at 632.8 nm is able to suppress amyloid-&beta; peptide (A&beta;)-induced oxidative and inflammatory responses in primary astrocytes, suggesting it has neuroprotective effects against oxidative stress and inflammation in AD. This thesis work provides insights into potential therapeutic treatments and prevention of AD.</p><p>

Cervical cancer is a major problem in the developing world and low- resource settings where standard screening techniques are not accessible. Cervical cancer is one of the few cancers that can be successfully treated when detected early. Therefore, there exists a high clinical need to screen for cervical cancer early. The etiological agent of cervical cancer is the human papillomavirus (HPV), with 70% of cases related to HPV genotypes 16 and 18. I sought to increase access to screening by developing a fully integrated and multiplexed molecular diagnostic assay to extract, amplify, detect, and distinguish HPV16 and HPV18 DNA on a low-cost paperfluidic platform for point- of-care (POC) applications. Isothermal (one temperature) loop-mediated amplification (LAMP) was used to amplify HPV DNA instead of the traditional polymerase chain reaction (PCR) that requires multiple temperatures. The amplified HPV16 and HPV18 DNA were differentially detected on a simple lateral flow strip – similar to that used in a pregnancy test – generating a visible colorimetric readout for each specific genotype. LAMP amplification is difficult to characterize and current methods were insufficient in providing specificity at the level needed for a multiplexed assay. Therefore, a novel characterization strategy was developed based on fluorescence to distinguish positive LAMP amplification products. This workflow used differential fluorescent tags to identify whether HPV16 DNA or HPV18 DNA was present and simplified complex nonspecific LAMP smears to a specific band pattern. After singleplex HPV16 and HPV18 LAMP assays were optimized with the new workflow, the two singleplex assays were successfully combined into one multiplex reaction with 12 primers, a nontrivial feat. Each assay step – DNA extraction, amplification, and detection – was optimized and integrated into a single chip that can control the timing of each step. Several chip configurations were tested to determine the optimal chip form factor, and a small subset of clinical samples were tested to demonstrate feasibility in low-resource settings. With this diagnostic platform, asymptomatic patients positive for HPV16 DNA and HPV18 DNA can be screened more closely, allocating precious resources to those most at risk, a beneficial use in both low-resource settings and the USA.

Cardiovascular diseases remain a leading cause of morbidity and mortality in the world, despite advances in drug and therapeutic developments. The complex nature of cardiac diseases such as myocardial infarction and heart failure require innovative approaches to elucidate disease mechanisms, identify molecular targets and develop novel therapies. The advent of human pluripotent stem cell (hPSC) technologies allowed for robust and reliable generations of contracting human cardiomyocytes (CMs) in vitro. hPSC-CMs hold great promise for a broad range of research and clinical applications including studying myocardial physiology, modeling cardiac diseases, and transplanting healthy cells to repair the damaged heart. However, one major limitation of hPSC-CMs differentiated in vitro is that they are relatively immature and resemble embryonic CMs. These cells lack well defined cellular edges and mature sarcomeres, which makes it difficult to quantitatively assess contractile functions using traditional edge detection technologies. In addition, hPSC-CMs cultured in traditional glucose rich media lack metabolic and functional maturity, utilizing mainly glycolysis for energy production, similar to the embryonic heart. To address these limitations, we first devised a novel technology to simultaneously quantify hPSC-CMs’ contractile kinetics, force generation and electrical activities at the single cell resolution. This methodology allowed us to examine the impact of energy substrates and metabolic pathway utilization on CM physiology and function. We identified that Hypoxia Inducible Factor 1 alpha (HIF1α) and its transcriptional target Lactate Dehydrogenase A (LDHA) are aberrantly upregulated in hPSC-CMs cultured in traditional glucose rich media. By using small molecules and siRNA, we demonstrated that inhibition of HIF1α/LDHA shifts hPSC-CMs’ metabolism from glycolysis to oxidative phosphorylation, which resulted in improved CM structural and functional maturation. Furthermore, we investigated the energy substrate dependency of hPSC-CMs in response to in vitro hypoxic and ischemia-reperfusion injuries. We observed that hPSC-CMs cultured in glucose rich media lack physiological responses to hypoxic insults. On the other hand, in vitro coverslip ischemia-reperfusion resulted in CM death and apoptosis, independent of glucose cultures. These findings highlighted the importance of bioenergetics in modeling cardiac diseases in vitro and provided us with the basis for a potential drug screening platform using hPSC-CMs. / 2020-07-02T00:00:00Z

The completion of the Human Genome Project has increased the need for investigation of genetic sequences and their biological functions, which will significantly contribute to the advances in biomedical sciences, human genetics and personalized medicine. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers an attractive option for DNA analysis due to its high accuracy, sensitivity and speed. In the first part of the thesis, we report the design, synthesis and evaluation of a novel set of mass tagged, cleavable biotinylated dideoxynucleotides (ddNTP-N3-biotins) for DNA polymerase extension reaction and its application in DNA sequencing and single nucleotide polymorphism (SNP) genotyping by mass spectrometry. These nucleotide analogs have a biotin moiety attached to the 5 position of the pyrimidines (C and U) or the 7 position of the purines (A and G) via a chemically cleavable azido-based linker, with different length linker arms serving as mass tags that contribute large mass differences among the nucleotides to increase resolution in MS analysis. It has been demonstrated that these modified nucleotides can be efficiently incorporated by DNA polymerase, and the DNA strand bearing biotinylated nucleotides can be captured by streptavidin coated beads and efficiently released using tris(2-carboxyethyl) phosphine in aqueous solution which is compatible with DNA and downstream procedures. Reversible solid phase capture (SPC) mass spectrometry sequencing using ddNTP-N3-biotins was performed, and various DNA templates, including biological samples, were accurately sequenced achieving a read-length of 37 bases. In mass spectrometric SNP genotyping, we have successfully exploited our reversible solid phase capture (SPC)-single base extension (SBE) assay and been able to detect as low as 2.5% heteroplasmy in mitochondrial DNA samples, with interrogation of human mitochondrial genome position 8344 which is associated with an important mitochondrial disease (myoclonic epilepsy with ragged red fibers, MERRF); we have also quantified the heteroplasmy level of a real MERRF patient and determined several mitochondrial MERRF mutations in a multiplex approach. These results demonstrated that our improved mass spectrometry genotyping technologies have great potential in DNA analysis, with particular applications in sequencing short-length targets or detecting SNPs with high accuracy and sensitivity requirements, such as DNA fragments with small indels, or SNPs in pooled samples. To truly implement this mass spectrometry-based genotyping method, we further explored the use of a lab-on-a-chip microfluidic device with the potential for high throughput, miniaturization, and automation. The microdevice primarily consists of a micro-reaction chamber for single base extension and cleavage reactions with an integrated micro heater and temperature sensor for on-chip temperature control, a microchannel loaded with streptavidin magnetic beads for solid phase capture, and a microchannel packed with C18-modified reversed-phase silica particles as a stationary phase for desalting before MALDI-TOF analysis. By performing each functional step, we have demonstrated 100% on-chip single base incorporation, sufficient capture and release of the biotin-ddNTP terminated single base extension products, and high sample recovery from the C18 reverse-phase microchannel with as little as 0.5 pmol DNA molecules. The feasibility of the microdevice has shown its promise to improve mass spectrometric DNA sequencing and SNP genotyping to a new paradigm. DNA sequencing by synthesis (SBS) appears to be a very promising molecular tool for genome analysis with the potential to achieve the $1000 Genome goal. However, the current short read-length is still a challenge. Therefore, the second part of the thesis focuses on strategies to overcome the short read-length of SBS. We developed a novel primer walking strategy to increase the read-length of SBS with cleavable fluorescent nucleotide reversible terminators (CF-NRTs) and nucleotide reversible terminators (NRTs) or hybrid-SBS with cleavable fluorescent nucleotide permanent terminators and NRTs. The idea of the walking strategy is to recover the initial template after one round of sequencing and re-initiate a second round of sequencing at a downstream base to cover more bases overall. The combination of three natural nucleotides and one NRT effectively regulated the primer walking: the primer extension temporarily paused when the NRT was incorporated, and resumed after removing the 3' capping group to restore the 3'-OH group. We have successfully demonstrated the integration of this primer walking strategy into the sequencing by synthesis approach, and were able to obtain a total read-length of 53 bases, nearly doubling the read-length of the previous sequencing. On the other hand, we explored the sequencing bead-on-chip approach to increase the throughput of SBS and hence the total genome coverage per run. The various prerequisite conditions have been optimized, allowing the accurate sequencing of several bases on the bead surface, which demonstrated the feasibility of this approach. Both of these approaches could be integrated into current SBS platforms, allowing increased overall coverage and lowering overall costs. As a step beyond genotyping, the in vivo visualization of biomolecules, like DNA and its encoded RNA and proteins, provides further information about their biological functions and mechanisms. The third part of the thesis focuses on the development of a novel quantum dot (QD)-based binary molecular probe, which takes advantage of fluorescent resonance energy transfer (FRET), for detection of nucleic acids, aiming at their eventual use for detection of mRNAs involved in long term memory studies in the model organism Aplysia californica. We reported the design, synthesis, and characterization of a binary probe (BP) that consists of carboxylic quantum dot (CdSe/ZnS core shell)-DNA (QD-DNA) conjugated donor and a cyanine-5 (Cy5)-DNA acceptor for the detection of a sensorin mRNA-based synthetic DNA molecule. We have demonstrated that in the absence of target DNA, the QD fluorescence is the main signal observed (605 nm); in the presence of the complementary target DNA sequence, a decrease of QD emission and an increase of Cy5 emission at 667 nm was observed. We have demonstrated the distance dependence of FRET, with the finding that the target with 16 base separation between the QD and Cy5 after probe hybridization gave the most efficient FRET. Further studies are in progress to evaluate the effectiveness of this QD-based probe inside a cell extract and in living cells.