Ingestion of photoreceptor outer segments (POS/OS) by the retinal pigment epithelium (RPE) is a fundamentally important process for vision. The RPE form an actin based phagocytic cup that extends along the OS and engages in trogocytosis to break off a piece of the OS for subsequent ingestion. This process is highly dynamic, and so we have used live cell imaging with high temporal resolution to understand the kinetics of ingestion and the localization of different relevant proteins throughout the ingestion process. We have found that the phagocytic cup is initiated by mDia1 formin activity. The Arp2/3 complex creates branched actin around the rim of the cup and beneath phagosomes to hold them in their proper location for scission and ingestion. Once excised, Myosin 7a traffics the phagosome basally into the cell for subsequent degradation. When photoreceptor-RPE interaction stops, such as during a retinal detachment, there are consequences to the photoreceptor synapse. Utilizing the high resolution capabilities of electron microscope (EM) tomography, we then explored the ultrastructure of the rod photoreceptor synapse (or rod spherule, RS) after 7 days of retinal detachment. 7 days of retinal detachment results in significant degeneration to the synaptic ultrastructure at the outer plexiform layer, including a loss of rod bipolar dendrites, simplification of horizontal cell axon terminals, fragmentation of the synaptic ribbon, and a simplification of the rod spherule invagination membrane. Following up, we then explored the timescale of these degenerative changes, performing tomography on synapses in retinas detached for 30min., 1hr., 24hrs., 48hrs., and 72hrs., in order to understand how long of a detachment may be too late for full recovery. By 24hrs. of detachment, there is significant remodeling including elongated ribbons and simplified horizontal cell processes; at least one rod bipolar cell process per rod spherule retracts by 24hrs. of detachment in a majority of cases. 24hrs. is likely the latest time point for reattachment without incurring long term visual deficits due to synaptic degeneration. By better understanding the way that OS are phagocytosed, as well as the consequences to the rod synapse when they are not, we can learn significant additional information about the health and maintenance of the rod photoreceptor.

Very high energy (VHE; E>100 GeV) blazars are a type of active galaxy detected above 100 GeV with a jet pointed toward the observer. This work investigates VHE blazars through broadband observations, starting with a description of the VHE-discovery and time-independent modeling of the non-thermal emission from RX J0648.7+1516. Additionally, synchrotron self-Compton models are applied to six non-VHE blazars, with the VHE flux of each blazar being constrained by non-detection during observation by VERITAS. The general lack of physical measurements of model parameters is highlighted and a scheme of supplementary observations involving millimeter carbon monoxide (CO) luminosity and soft X-ray absorption measurements is explored for three VHE blazars. The limited sample supports a possible connection between the existence of CO in the vicinity of the blazar and additional soft X-ray absorption beyond what can be attributed to the Milky Way. RGB J0710+590 and W Comae both lack a significant level of CO and do not require additional absorption for the description of the soft X-ray emission as observed by Swift XRT. 1ES 1959+650, on the other hand, shows a significant level of CO in the vicinity of the blazar and requires additional absorption to describe the soft X-ray emission. The positive detection of CO in the vicinity of 1ES 1959+650 is used as motivation to apply a mirrored emission scenario to broadband variability data. Limits on the redshifts of the two VHE blazars 3C 66A and PKS 1424+240 are derived from HST/COS observations of intervening Lyman absorption. These observations show 3C 66A to reside at a redshift below the tentative z=0.444 at 99.9% confidence and reveal PKS 1424+240 to be the most distant VHE-detected blazar thus far. The redshift information is paired with VERITAS and Fermi Large Area Telescope gamma-ray observations to probe the density of the extragalactic background light and correct the observed gamma-ray spectra to the intrinsically emitted spectra, allowing insight into the acceleration mechanisms at work within the blazar jets and the propagation of gamma-ray photons through intergalactic space.

Purpose of review

Recent findings

Summary

The RPE is a polarized epithelium, whose apical surface faces the outer segments of photoreceptor cells in the neurosensory retina, and performs numerous functions that are essential for healthy vision. In this dissertation, I investigated aspects of RPE biology that are critical for cellular homeostasis and, when perturbed, may contribute to RPE dysfunction and retinal degeneration. In particular, I focused on the RPE’s phagocytic function in which the cells internalize shed disks of photoreceptor outer segments and subsequently degrade the resultant phagosomes, thereby maintaining photoreceptor health. Using in vitro models of RPE, phagocytosis assays were performed to test the kinetics of outer segment degradation using state-of-the-art live-cell imaging. The kinetics of this process were further tested in a mouse model of dominant Stargardt 3 macular degeneration, which showed that the digestion of mutant outer segments is delayed and thus contributes to photoreceptor degeneration. This data implicated the impaired motility of phagosomes in the RPE as a major contributor to their delayed degradation, thereby identifying a sensitive measurement of a parameter that is critical to overall RPE health. Given that the RPE is a primary site of insult in retinal degeneration, cell transplantation may be a viable treatment to replace damaged RPE cells in human patients. To that end, we generated human RPE cells from induced pluripotent stem cells and extensively characterized the critical aspects of their cell biology. Importantly, we demonstrated that when these cells manifest phagosome degradation kinetics, comparable to that in vivo, and, when transplanted into a mouse model of retinal degeneration, they are capable of integrating into the host monolayer and partially rescue the photoreceptor from degenerating. Lastly, we developed a rapid method to differentiate a human immortalized RPE cell line such that the cultures exhibit many of the characteristics of their in vivo counterparts, and we propose that when these cells are properly differentiated, they can be used to draw meaningful inferences about RPE physiology and pathology.

Blazars, active galactic nuclei with their jets aligned along our line of sight, emit radiation in all bands of the electromagnetic spectrum. Many questions remain about the emission mechanisms, and this dissertation investigates how thermal emission originating outside of the jet plays a role in producing the non-thermal radiation created in the jet. We present multi-wavelength observations of the very-high-energy emitting blazar 3C\,66A taken from 2007 to 2016 by VERITAS, the \textit{Fermi}-Large Area Telescope, the Neil Gehrels \textit{Swift} Observatory, and ground-based optical observatories including the Tuorla Observatory and Fred Lawrence Whipple Observatory. 3C\,66A is a TeV-emitting intermediate-synchrotron-peaked blazar, a relatively small class of TeV-detected AGN. We construct the multi-wavelength spectral energy distribution during multiple epochs of observation and present the first spectral energy distributions of 3C\,66A in quiescent flux states. To explain the emission, we develop and implement a Markov Chain Monte Carlo method to fit single-zone gamma-ray emission models. Additionally, we study the gamma-ray flux and optical spectral features of five TeV-detected blazars: Mrk 501, Mrk 421, 3C\,279, PKS\,1441+25 and PKS\,1222+216. Correlations between optical spectral features and gamma-ray flux can give insight to the interplay between the non-thermal and thermal emission in the blazar environment.