Acoustic Reporter Genes for Noninvasive Imaging of Cellular Function

The study of cellular function within the context of intact living organisms is a grand challenge in biological research. Addressing this challenge requires imaging tools that can visualize cells inside the body. If successful, this would greatly increase our ability to study a battery of processes from brain development to tumorigenesis, to monitoring cell-based therapeutics. To date, most common methods for imaging cellular processes such as gene expression have relied on optical reporters, such as fluorescent or luminescent proteins, which provide high molecular precision for studies in petri dishes and transparent organisms, but have limited performance in large animals due to the poor penetration of light in biological tissue. Conversely, magnetic resonance imaging (MRI) and ultrasound can image tissues at depth with high spatial and temporal resolution, but they lack molecular reporters analogous to the green fluorescent protein (GFP). As a result, they have made limited impact on biological research. To address this, we focus on developing biomolecular reporters for MRI and ultrasound — based on a unique class of air-filled protein nanostructures called gas vesicles — using them to image the location and function of cells deep inside the body. This thesis begins with a brief review of genetically encoded materials for noninvasive imaging, highlighting key advances over the past two decades and providing context for the work below. We discuss the development of increasingly sophisticated tools starting from early efforts to engineer single molecule reporters to recent work on multi-component genetic machinery (including gas vesicles) with multi-modality capabilities. In Chapter 2, we present a platform for engineering the surface of gas vesicles to modulate their acoustic, surface charge, and molecular- targeting properties as injectable acoustic biomolecules. In Chapter 3, we present the recombinant expression of gas vesicles as injectable contrast agents in common lab strain bacteria to facilitate the genetic engineering of the entire gas vesicle gene cluster and to assist this technology’s adoption by other (non-specialist) research groups. This work characterized the ultrasound and hyperpolarized 129Xenon-MRI contrast of gas vesicles as nanoscale contrast agents. In a parallel effort, we developed a hybrid gene cluster that when introduced to microbes enables the imaging of their gene expression using ultrasound. These bacterial acoustic reporter genes were used to image the location of probiotic cells inside the gastrointestinal tract of mice. However, the ability for these genes to be expressed in mammalian cells had not been demonstrated and presented a major challenge in synthetic biology. In Chapter 4, we addressed this by introducing the first mammalian acoustic reporter genes — a genetic program whose introduction to mammalian cells resulted in the expression of gas vesicles that can be visualized by ultrasound. These mammalian acoustic reporter genes will enable previously impossible approaches to monitoring the location, viability and function of mammalian cells in vivo. In Chapter 5, we explore a new paradigm in MRI by taking advantage of the acousto-magnetic property of gas vesicles. Here, we present background-free MRI to address a longstanding challenge in untangling the signal of exogenous contrast agents from the endogenous MRI contrast produced by biological tissues. Chapter 6 explores the optical properties of gas vesicles as genetically encodable phase contrast agents in digital holographic imaging. Chapter 7 is a brief discussion of the potential future directions for this work. The data presented in this thesis lays the ground for exciting new research on developing noninvasive biomolecular tools that will enable the discovery of novel biological processes.</p

Energy Optimization in Commercial FPGAs with Voltage, Frequency and Logic Scaling

This paper investigates the energy reductions possible in commercially available FPGAs configured to support voltage, frequency and logic scalability combined with power gating. Voltage and frequency scaling is based on in-situ detectors that allow the device to detect valid working voltage and frequency pairs at run-time while logic scalability is achieved with partial dynamic reconfiguration. The considered devices are FPGA-processor hybrids with independent power domains fabricated in 28 nm process nodes. The test case is based on a number of operational scenarios in which the FPGA side is loaded with a motion estimation core that can be configured with a variable number of execution units. The results demonstrate that voltage scalability reduces power by up to 60 percent compared with nominal voltage operation at the same frequency. The energy analysis show that the most energy efficiency core configuration depends on the performance requirements. A low performance scenario shows that serial computation is more energy efficient than the parallel configuration while the opposite is true when the performance requirements increase. An algorithm is proposed to combine effectively adaptive voltage/logic scaling and power gating in the proposed system and application

Comparative Outcomes of Pulpotomy in Mature Molars with Irreversible Pulpitis: A Non-Randomized Trial Evaluating Calcified and Non-Calcified Pulp Chambers

Introduction: This non-randomized clinical trial investigated the outcomes of full pulpotomy in adult molars with irreversible pulpitis, comparing those with calcified and non-calcified pulp chambers over 6 and 12 months. Materials and Methods: A total of 101 adult permanent molars with irreversible pulpitis, in individuals over 12 years old, were categorized based on pulp chamber calcification observed in radiographic images by two endodontists. Subsequently, full pulpotomy procedures were performed, achieving hemostasis, and applying a 2 mm layer of calcium-enriched mixture (CEM) cement as a pulp covering agent. After 48 hours, the setting of the CEM cement was verified, followed by the application of a layer of resin-modified glass-ionomer. The tooth was then restored using amalgam. Clinical and radiographic evaluations were conducted at 6-month and 1-year follow-ups by blinded endodontists. Success rates were compared using Fisher's exact test and logistic regression tests with a significance level of 0.05. Results: Among the 97 patients with 6-month and 1-year follow-ups, all achieved clinical success. Radiographic success rates were 99% at 6 months and 96.9% at 1 year, regardless of pulp calcification. In the 6-month follow-up, success rates were 98.07% for non-calcified pulp chambers and 100% for calcified pulp chambers. At the 1-year follow-up, success rates were 96.1% and 97.8%, respectively. Statistical analysis showed no significant difference in radiographic success rate between the two groups at both follow-ups (P&gt;0.05). Conclusions: Full pulpotomy using CEM cement is a successful treatment for adult permanent teeth with calcified and non-calcified pulp chambers presenting signs and symptoms of irreversible pulpitis up to a 1-year follow-up. This study provides compelling evidence that vital pulp therapy can be effectively employed in the pulpotomy of calcified teeth, at least in the short term

Ultrasound Imaging of Gene Expression in Mammalian Cells

The study of cellular processes occurring inside intact organisms requires methods to visualize cellular functions such as gene expression in deep tissues. Ultrasound is a widely used biomedical technology enabling noninvasive imaging with high spatial and temporal resolution. However, no genetically encoded molecular reporters are available to connect ultrasound contrast to gene expression in mammalian cells. To address this limitation, we introduce mammalian acoustic reporter genes. Starting with a gene cluster derived from bacteria, we engineered a eukaryotic genetic program whose introduction into mammalian cells results in the expression of intracellular air-filled protein nanostructures called gas vesicles, which produce ultrasound contrast. Mammalian acoustic reporter genes allow cells to be visualized at volumetric densities below 0.5% and permit high-resolution imaging of gene expression in living animals