Retinal neuron degenerations of an eye can result in permanent photoreceptor or retinal ganglion cell loss which leads to permanent blindness. One potential approach to restore vision loss is through transplanting stem cell or stem cell-derived retinal neurons into the sub-retinal space of the injured retina. However, these transplanted cells often fail to integrate or form a functional connections with the neighboring retinal neurons. Another alternative approach is referred to as the endogenous regeneration whereby new retinal neurons are generated from Müller glia to replace those lost photoreceptors and retinal ganglion cells. Müller glia is the only cell in the retina that shares a common origin with retinal neurons. Thus it has the potential to convert into retinal neurons. Studies have shown that Müller cells in fish, amphibians, and chickens in early developmental stage respond to injury, triggering in vivo regeneration of retinal neurons. In mammals this form of regeneration is less common but does appear to occur as well, however, it remains to be determined whether human Müller glia can dedifferentiate into retinal progenitor and regenerate retinal neurons. Therefore, my project aims to establish Müller cell specific fluorescent reporter systems to aid in future Müller Glia endogenous studies. One approach is to build a dual reporter system that tags both retinal ganglion cells and Müller Glia, tracking the exact time point at which Müller cells are developed from iPSCs. The other approach is to use a lineage tracing system and permanently tag Müller cells once they are born. The advantage of the lineage tracer system is that even if Müller cells convert into other cell types, the fluorescent markers will continue to be expressed, therefore indicating their Müller Glia origin.

The loss of hair cells (HCs) and spiral ganglion neurons (SGNs) in the inner ear can lead to irreversible hearing loss in mammals. Because these cells are few in number and difficult to access, studies on their development, degeneration, and reprogramming capabilities in response to injury have been limited. Previous studies have analyzed the gene expression profiles of HCs and SGNs in mice through bulk and single-cell RNA sequencing, with some identifying transcription factor (TF) genes, such as Atoh1, Pou4f3, Gfi1, Neurog1, and Neurod1 as significant for HC and SGN development and maturation. However, it is not known whether these findings apply to the human system. Thus, a reliable and accurate in vitro model of human HCs and SGNs has yet to be generated to study their development and injury responses. In this study, we overexpressed two unique sets of TF gene cassettes in IMR90.4 cells with an endogenous POU4F3-p2A-eGFP reporter: (1) TetO-ATOH1-p2A-POU4F3-p2A-GFI1 (APG1) and (2) TetO-NEUROG1-p2A-ATOH1-p2A-NEUROD1 (NAN1) for generation of HC- and SGN-like cells, respectively. These modified hiPSCs were differentiated and collected for bulk RNA-sequencing after 6 and 14 days. Sequencing analysis revealed that after six days, APG1 induced cell gene expression was enriched for auditory processes, but this enrichment was less significant by day 14. Though day 6 NAN1 induced cells appear more neuronal than day 6 APG1 cells, possibly leading to an SGN-like cell fate, they do not take on a definitive cell type identity, instead expressing some HC- and neuronal genes at days 6 and 14.

We have determined the mineral composition of statoliths in 169 species or subspecies (256 populations) of the family Mysidae on a worldwide scale. Including previously published data, the crystallographic characteristics are now known for 296 extant species or subspecies: fluorite (CaF₂) in 79%, vaterite (a metastable form of crystalline CaCO₃) in 16%, and non-crystalline (organic) components in 5%, the latter exclusively and throughout in the subfamilies Boreomysinae and Rhopalophthalminae. Within the subfamily Mysinae vaterite or fluorite were found in three tribes, whereas other three tribes have fluorite only. The exclusive presence of fluorite was confirmed for the remaining seven subfamilies. Hotspots of vaterite were found in Amazonia and the Pontocaspis, in each case with reduced frequencies in main and tributary basins of the Atlantic and N-Indian Ocean. Vaterite is completely absent in the remaining aquatic regions of the world. In accordance with previous findings, fluorite occurred mainly in seawater, vaterite mostly in brackish to freshwater. Only vaterite was found in electrolyte-poor Black Water of Amazonia, which clearly cannot support the high fluorine demand for renewal of otherwise large fluorite statoliths upon each moult. Vaterite prevails in Diamysini, distributed over most of the area once occupied by the Tethyan Sea. It also prevails in Paramysini with main occurrence in the Pontocaspis, where fossil calcareous statoliths in the stable form of calcite are known from Miocene sediments of the brackish Paratethys. Four Recent genera from three tribes are heterogeneous in that they comprise both vaterite- and fluorite-precipitating species. Previous hypotheses are expanded to cover greater geographic and time scales, proposing that fluorite-bearing marine ancestors penetrated freshwaters in Tethyan and Paratethyan basins, where they developed precipitation of vaterite. This gave their successors predispositions for shifting into separate evolutionary lines from fluorite to vaterite precipitation and vice versa.

Growing concerns over the scalability of air traffic operations, air transportation fuel emissions and prices, as well as the advent of communication and sensing technologies motivate improvements to the air traffic management system. To address such improvements, in this thesis a hybrid dynamical model as an abstraction of the air traffic system is considered. Wind and hazardous weather impacts are included using a stochastic model. This thesis focuses on the design of algorithms for verification and control of hybrid and stochastic dynamical systems and the application of these algorithms to air traffic management problems.

In the deterministic setting, a numerically efficient algorithm for optimal control of hybrid systems is proposed based on extensions of classical optimal control techniques. This algorithm is applied to optimize the trajectory of an Airbus 320 aircraft in the presence of wind and storms. In the stochastic setting, the verification problem of reaching a target set while avoiding obstacles (reach-avoid) is formulated as a two-player game to account for external agents' influence on system dynamics. The solution approach is applied to air traffic conflict prediction in the presence of stochastic wind. Due to the uncertainty in forecasts of the hazardous weather, and hence the unsafe regions of airspace for aircraft flight, the reach-avoid framework is extended to account for stochastic target and safe sets. This methodology is used to maximize the probability of the safety of aircraft paths through hazardous weather.

Finally, the problem of modeling and optimization of arrival air traffic and runway configuration in dense airspace subject to stochastic weather data is addressed. This problem is formulated as a hybrid optimal control problem and is solved with a hierarchical approach that decouples safety and performance. As illustrated with this problem, the large scale of air traffic operations motivates future work on the efficient implementation of the proposed algorithms.

The loss of retinal ganglion cells (RGCs) causes irreversible vision loss in glaucoma and optic neuropathies. Some vertebrate species, such as zebrafish, possess a robust ability to regenerate parts of their body, including the retina through a process of endogenous regeneration. However, this ability is limited in mammals including humans. To understand endogenous regeneration and work towards restoring RGCs, we need to gain a better understanding of the developmental mechanisms leading to RGC formation. Our approach relies on the generation of RGC reporters that express fluorescent proteins coupled to the endogenous BRN3A and BRN3B genes. By ectopically overexpressing four transcription factors (Neurogenin-2, Atonal homolog 7, Islet-1, and Brn3b) involved in RGC specification and differentiation, we seek to directly convert the human pluripotent stem cell reporters into RGCs. If the differentiated neurons become bonafide BRN3A/BRN3B expressing RGCs, this would represent an important first step in developing and improving novel therapeutic strategies for retinal diseases.

While some vertebrate species such as zebrafish have demonstrated a robust repair and regeneration ability, the extent of this regenerative capacity in mammals is limited. In humans, it is unknown whether this process even occurs. Vision loss resulting from retinal ganglion cell (RGC) death is a leading cause of irreversible blindness. There are several areas of active research aimed at restoring retinal ganglion cells. One of them is endogenous repair. However, for this approach to work, we need to gain a better control of the developmental mechanisms leading to RGC formation. In this study, we use ectopic overexpression of four specific transcription factors involved in ganglion cell specification and differentiation to bring about the direct conversion of ganglion cells from human pluripotent stem cells. If these newly differentiated neurons turn out to be truly bonafide ganglion cells, then the idea of generating new ganglion cells to improve the prognosis of retinal diseases will be better understood.

Retinogenesis, the development of the retina, is regulated by dynamic changes in transcriptional expression and the chromatin landscape. While model organisms have contributed significantly to understanding retinal development, they do not allow studies of human-specific conditions. We utilized a SIX6-eGFP/VSX2-tdTomato dual fluorescent pluripotent stem-cell (PSC) line and grew retinal organoids (ROs) through 45 days. RNA- and ATAC-seq analyses through 45 days revealed transcriptomic and chromatin-landscape regulatory changes that drive retinal development and the activation of retinal specific pathways. To study an early modifier of retinal development we also investigated the Sonic Hedgehog (Shh) signaling pathway by modulating the timing and concentration of a smoothened agonist (SAG) in SIX6-eGFP/POU4F2-tdTomato dual reporter ROs. Early SAG treatment led to the development of non-retinal tissue while later treatment favored retinal development, indicated by robust POU4F2-tdTomato expression. To investigate factors governing the formation of retinal ganglion cells (RGCs; the first cell born in the eye), we developed an inducible transgene cassette expressing the transcription factors (TFs) NEUROG2-ATOH7-ISL1-POU4F2 which, in combination with Bone Morphogenic Protein (BMP) inhibition, led to robust formation of RGC-like neurons in as little as one week. An RGC-like identity was validated via fluorescent reporter expression, transcriptomic analysis, and electrophysiology. Our findings provide a better understanding of the complex interplay of transcriptional regulation, chromatin dynamics, and microenvironmental cues in RO development and advancing our understanding of retinal biology. Generating RGC-iNs illuminates factors governing RGC formation and provides insight into the early formation of the eye.

Retinal degeneration (RD) is a leading cause of vision impairment and blindness worldwide. Diseases which cause RD, including glaucoma, retinitis pigmentosa (RP) and age-related macular degeneration (AMD) are characterized by dysfunction and/or loss of key retinal neurons. Studying these human diseases in model organisms is useful but often approaches a limit at which the model organism does not sufficiently capture the human context. Many groups are utilizing in vitro methods to study the human retina, the most prominent being human induced-pluripotent stem cell (iPSC) derived neurons and iPSC-derived 3D retinal organoids (rORGs). A key first step to studying diseases like AMD and RP is establishing accurate in vitro models that can recapitulate the complexity of the retina and its many cell types. To address this need, I explored transcriptomic patterns in rORGs focusing on photoreceptors (PRs). I found that the rORG-derived PRs (O-PRs) exhibited a PR-like identity with both rod and cone markers detected. Importantly, I also compared these in vitro O-PRs with in vivo datasets, and found that they share similarities in their transcriptomic profiles. This work is intended to serve as a resource for scientists seeking to perform similar analyses on retinal datasets as well as cell types beyond the nervous system. Toward this goal, I have documented all the code used to generate results in a publicly available repository with guides on how to perform the analysis regardless of prior bioinformatics experience.