PhD, University of Cincinnati, 2022, Medicine: Molecular and Developmental Biology

Soluble N-ethylmaleimide-sensitive factor protein receptors (SNAREs) comprise a universally conserved complex of proteins that are key components of the cellular machinery required for intracellular membrane trafficking. Although the mechanism by which SNAREs mediate neurotransmitter release at the synaptic membrane is well-studied, their requirements during normal vertebrate development have only recently become appreciated. Indeed, mutations in SNAREs are now known to result in developmental syndromes, primarily synaptopathies—these disorders have been collectively termed “SNAREopathies”. However, SNAREs ubiquitously regulate vesicle fusion in virtually all cell lineages; therefore, extra-neuronal disease pathologies may also plausibly manifest as “SNAREopathies”. Cardiomyocytes are among the most specialized cell types, owing to their substantial organization and the dynamic requirements for their diverse function. While it is known that cardiomyocytes heavily rely on intracellular membrane trafficking, to date remarkably few bona fide trafficking proteins have been identified as having a specific function in cardiac tissues. SNAREs are a promising candidate for elucidating how cardiac intracellular trafficking is regulated. Consequently, herein, we endeavored to understand the in vivo requirement for Syntaxin 4 (STX4), a target-SNARE, during normal vertebrate development, cardiac conduction, and cardiomyocyte vesicular transport. This work was initiated upon the identification of two patients with damaging variants in the STX4 locus: One patient, homozygous for a R240W missense variant, presented with sensorineural hearing loss, global developmental delay, hypotonia, and biventricular dilated cardiomyopathy; ectopy; and runs of non-sustained ventricular tachycardia, requiring an orthotopic heart transplant, while a second patient with compound heterozygous truncating alleles presented with severe pleiotropic abnormalities that resulted in perinatal letha (open full item for complete abstract)

Committee: Joshua Waxman Ph.D. (Committee Member); Steve Danzer Ph.D. (Committee Member); Juan Sanchez-Gurmaches Ph.D. (Committee Member); Douglas Millay Ph.D. (Committee Member); Carlos Prada M.D. (Committee Member); Raphael Kopan Ph.D. (Committee Member) Subjects: Developmental Biology

Doctor of Philosophy, Case Western Reserve University, 2016, Biomedical Engineering

Coordinated cardiac conduction plays an important role in cardiogenesis, not only for initiating rhythmic contractions of cardiac myocytes for efficient blood pumping, but also for maintaining normal cardiac development. Optical mapping (OM), which uses fluorescent voltage-sensitive dyes to measure membrane potential is currently the most effective method for electrophysiology studies in early embryonic hearts due to its noninvasiveness and large field-of-view. OM has two major limitations: 1) it projects signals from part of a 3D sample to a 2D map therefore the electrophysiological information is incomplete, orientation-dependent and ignorant of 3D topology of the sample; 2) it requires excitation-contraction (EC) uncoupling drugs to stop the contraction of the heart, yet EC-uncouplers may affect calcium handling, ion channel kinetics and action potential characteristics. This dissertation focuses on overcoming the limitations of OM and improving cardiac conduction imaging in embryonic hearts. First, OM was integrated with optical coherence tomography (OCT), which is capable of capturing the 3D topology of the looped embryonic heart. A 3D conduction velocity correction algorithm was developed. This eliminated underestimation bias in 2D conduction velocity calculation and provided more accurate 3D corrected conduction velocity measurements. Second, 4D OM in the embryonic heart was achieved with light-sheet fluorescence microscopy. We built a fast light-sheet system that illuminated the sample with a sheet of light generated by a cylindrical lens and collected OM signals from an orthogonal direction. OM data from multiple slices throughout the looping stage quail embryonic hearts were acquired. With this imaging system, complete, orientation independent, four-dimensional transmembrane potentials were demonstrated. Next, correction of motion artifacts in freely beating embryonic hearts was demonstrated using a B-spline nonrigid registration algorithm. Activation maps (open full item for complete abstract)

Committee: Andrew Rollins (Advisor); Xin Yu (Committee Chair); Michiko Watanabe (Committee Member); Kenneth Singer (Committee Member); Michael Jenkins (Committee Member) Subjects: Biomedical Engineering