Lysosome Turnover in the Retinal Pigment Epithelium in Health, Ageing and Age- related Macular Degeneration

The Retinal Pigment Epithelium (RPE) phagocytoses and degrades spent Photoreceptor Outer Segments (POS) every day, placing an unparalleled burden on lysosomes in RPE cells, which require well-regulated turnover to maintain degradative capacity. With age and in Age-related Macular Degeneration (AMD), debris accumulates inside and outside RPE cells leading to the hypothesis that lysosome dysfunction could contribute to AMD pathology. This project aimed to identify the functional relevance of different subpopulations of lysosome within the heterogenous lysosomal compartment of RPE cells. Models of lysosome dysfunction were then generated in order to determine whether they recapitulate hallmarks of aging and AMD. Electron microscopy (EM) and electron tomography of primary porcine RPE (pRPE) revealed morphologically distinct subpopulations of lysosomes that form a complex network. Pulse-chase experiments showed that multilamellar Cathepsin D-rich lysosomes transition to electron dense lysosomes that stained poorly for Cathepsin D, suggesting that these subpopulations represent different stages of the lysosome cycle. Tracking of phagocytosed POS suggests that POS degradation occurs by both full fusion and kiss-and-run interactions between phagosomes and lysosomes. Correlative light and electron microscopy, and high content imaging, demonstrated that POS loading induces the gradual formation of lipofuscin-like autofluorescent granules. Loading cells with degradation- resistant UV-irradiated POS induced the formation of more and larger autofluorescent granules, thus recapitulating one of the hallmarks of aging and AMD. Treatment with the acidotropic compound chloroquine caused accumulation of undigested phagocytic, endocytic and autophagic cargo, consistent with lysosome dysfunction. This was accompanied by accumulation of lamellar lysosomes containing unprocessed lysosomal Cathepsin D, a feature 4 recapitulated by direct protease inhibition and prevented by inhibiting protein synthesis. Together, the findings in this PhD project shed new light on the identity, function and fate of lysosomes in the RPE and suggest that lysosome dysfunction could contribute to the pathology of AMD, as well as to chloroquine-induced retinopathy

Utilizing Magnetic Tunnel Junction Devices in Digital Systems

The research described in this dissertation is motivated by the desire to effectively utilize magnetic tunnel junctions (MTJs) in digital systems. We explore two aspects of this: (1) a read circuit useful for global clocking and magnetologic, and (2) hardware virtualization that utilizes the deeply-pipelined nature of magnetologic. In the first aspect, a read circuit is used to sense the state of an MTJ (low or high resistance) and produce a logic output that represents this state. With global clocking, an external magnetic field combined with on-chip MTJs is used as an alternative mechanism for distributing the clock signal across the chip. With magnetologic, logic is evaluated with MTJs that must be sensed by a read circuit and used to drive downstream logic. For these two uses, we develop a resistance-to-voltage (R2V) read circuit to sense MTJ resistance and produce a logic voltage output. We design and fabricate a prototype test chip in the 3 metal 2 poly 0.5 um process for testing the R2V read circuit and experimentally validating its correctness. Using a clocked low/high resistor pair, we show that the read circuit can correctly detect the input resistance and produce the desired square wave output. The read circuit speed is measured to operate correctly up to 48 MHz. The input node is relatively insensitive to node capacitance and can handle up to 10s of pF of capacitance without changing the bandwidth of the circuit. In the second aspect, hardware virtualization is a technique by which deeply-pipelined circuits that have feedback can be utilized. MTJs have the potential to act as state in a magnetologic circuit which may result in a deep pipeline. Streams of computation are then context switched into the hardware logic, allowing them to share hardware resources and more fully utilize the pipeline stages of the logic. While applicable to magnetologic using MTJs, virtualization is also applicable to traditional logic technologies like CMOS. Our investigation targets MTJs, FPGAs, and ASICs. We develop M/D/1 and M/G/1 queueing models of the performance of virtualized hardware with secondary memory using a fixed, hierarchical, round-robin schedule that predict average throughput, latency, and queue occupancy in the system. We develop three C-slow applications and calibrate them to a clock and resource model for FPGA and ASIC technologies. Last, using the M/G/1 model, we predict throughput, latency, and resource usage for MTJ, FPGA, and ASIC technologies. We show three design scenarios illustrating ways in which to use the model

The proficiency of the original host species determines community-level plasmid dynamics

Plasmids are common in natural bacterial communities, facilitating bacterial evolution via horizontal gene transfer. Bacterial species vary in their proficiency to host plasmids: Whereas plasmids are stably maintained in some species regardless of selection for plasmid-encoded genes, in other species, even beneficial plasmids are rapidly lost. It is, however, unclear how this variation in host proficiency affects plasmid persistence in communities. Here, we test this using multispecies bacterial soil communities comprising species varying in their proficiency to host a large conjugative mercury resistance plasmid, pQBR103. The plasmid reached higher community-level abundance where beneficial and when introduced to the community in a more proficient host species. Proficient plasmid host species were also better able to disseminate the plasmid to a wider diversity of host species. These findings suggest that the dynamics of plasmids in natural bacterial communities depend not only upon the plasmid's attributes and the selective environment, but also upon the proficiency of their host species

Source-sink plasmid transfer dynamics maintain gene mobility in soil bacterial communities

Horizontal gene transfer is a fundamental process in bacterial evolution that can accelerate adaptation via the sharing of genes between lineages. Conjugative plasmids are the principal genetic elements mediating the horizontal transfer of genes, both within and between bacterial species. In some species, plasmids are unstable and likely to be lost through purifying selection, but when alternative hosts are available, interspecific plasmid transfer could counteract this and maintain access to plasmid-borne genes. To investigate the evolutionary importance of alternative hosts to plasmid population dynamics in an ecologically relevant environment, we established simple soil microcosm communities comprising two species of common soil bacteria, Pseudomonas fluorescens and Pseudomonas putida, and a mercury resistance (Hg R) plasmid, pQBR57, both with and without positive selection [i.e., addition of Hg(II)]. In single-species populations, plasmid stability varied between species: although pQBR57 survived both with and without positive selection in P. fluorescens, it was lost or replaced by nontransferable Hg R captured to the chromosome in P. putida. A simple mathematical model suggests these differences were likely due to pQBR57's lower intraspecific conjugation rate in P. putida. By contrast, in two-species communities, both models and experiments show that interspecific conjugation from P. fluorescens allowed pQBR57 to persist in P. putida via source-sink transfer dynamics. Moreover, the replacement of pQBR57 by nontransferable chromosomal Hg R in P. putida was slowed in coculture. Interspecific transfer allows plasmid survival in host species unable to sustain the plasmid in monoculture, promoting community-wide access to the plasmid-borne accessory gene pool and thus potentiating future evolvability

Rapid compensatory evolution promotes the survival of conjugative plasmids

Conjugative plasmids play a vital role in bacterial adaptation through horizontal gene transfer. Explaining how plasmids persist in host populations however is difficult, given the high costs often associated with plasmid carriage. Compensatory evolution to ameliorate this cost can rescue plasmids from extinction. In a recently published study we showed that compensatory evolution repeatedly targeted the same bacterial regulatory system, GacA/GacS, in populations of plasmid-carrying bacteria evolving across a range of selective environments. Mutations in these genes arose rapidly and completely eliminated the cost of plasmid carriage. Here we extend our analysis using an individual based model to explore the dynamics of compensatory evolution in this system. We show that mutations which ameliorate the cost of plasmid carriage can prevent both the loss of plasmids from the population and the fixation of accessory traits on the bacterial chromosome. We discuss how dependent the outcome of compensatory evolution is on the strength and availability of such mutations and the rate at which beneficial accessory traits integrate on the host chromosome

Monitoring Aircraft Cabin Particulate Matter Using a Wireless Sensor Network

The semi-enclosed and pressurized nature of the aircraft cabin results in a highly dynamic environment. The dynamic conditions establish spatiotemporal dependent environmental characteristics. Characterization of aircraft cabin environmental and bleed-air conditions have traditionally been done with stand-alone measurement systems which, by their very nature, cannot provide the necessary sensor coverage in such an environment. To this purpose, a prototype wireless sensor network system has been developed that can be deployed in the aircraft cabin environment. Each sensor node in the system incorporates the ability to measure common aircraft contaminants such as particulate matter and carbon dioxide, along with other key environmental factors such as temperature, air pressure, humidity, and sound pressure level. The wireless sensor network enables the collection of time-correlated results from the aircraft cabin, passing sensor data to a central collection point for storage or real-time monitoring. This paper discusses the results of testing this sensor system in a mockup of the Boeing 767 aircraft cabin environment. In this series of tests, both particulate matter and carbon dioxide were introduced into the simulated aircraft environment and measured using an array of 16 wirelessly connected sensor nodes. Two different arrangements of sensor nodes targeted both a two-dimensional plane across the aircraft cabin space and a localized three-dimensional space centered on two rows of the cabin. The test results show successful simultaneous tracking of the particulate matter and carbon dioxide concentrations as they disperse over time

A Low-Cost Wireless Portable Particulate Matter Monitoring System

This paper describes a portable sensor system design for environmental research. The designed system has been deployed in many environments and has been EMI/EMC approved for operation in commercial aircraft cabins. The sensor suite includes particulate matter, CO, CO2, temperature, humidity, pressure, light intensity, and sound sensors. Multiple sensor systems can be deployed at once to form a wireless sensor network. Each sensor node is powered either with six AA batteries or via a transformer and standard outlet. The data collected by a node can be stored locally on the device and/or sent through the integrated ZigBee mesh network to a central coordinator node. As of this writing, single nodes have been carried by passengers to monitor the normal airliner cabin flight conditions, and networks have been used to monitor indoor and outdoor air quality conditions

The Impact of Mercury Selection and Conjugative Genetic Elements on Community Structure and Resistance Gene Transfer

Carriage of resistance genes can underpin bacterial survival, and by spreading these genes between species, mobile genetic elements (MGEs) can potentially protect diversity within microbial communities. The spread of MGEs could be affected by environmental factors such as selection for resistance, and biological factors such as plasmid host range, with consequences for individual species and for community structure. Here we cultured a focal bacterial strain,Pseudomonas fluorescensSBW25, embedded within a soil microbial community, with and without mercury selection, and with and without mercury resistance plasmids (pQBR57 or pQBR103), to investigate the effects of selection and resistance gene introduction on (1) the focal species; (2) the community as a whole; (3) the spread of the introducedmerresistance operon. We found thatP. fluorescensSBW25 only escaped competitive exclusion by other members of community under mercury selection, even when it did not begin with a mercury resistance plasmid, due to its propensity to acquire resistance from the community by horizontal gene transfer. Mercury pollution had a significant effect on community structure, decreasing alpha diversity within communities while increasing beta diversity between communities, a pattern that was not affected by the introduction of mercury resistance plasmids byP. fluorescensSBW25. Nevertheless, the introducedmerAgene spread to a phylogenetically diverse set of recipients over the 5 weeks of the experiment, as assessed by epicPCR. Our data demonstrates how the effects of MGEs can be experimentally assessed for individual lineages, the wider community, and for the spread of adaptive traits.Peer reviewe