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Patterned neuronal activity driven by the sensory environment plays a key role in the development of precise synaptic connectivity in the brain. It is well established that the action potentials (‘spikes’) generated by individual neurons are crucial to this developmental process. A neuron’s spiking activity is jointly determined by its synaptic inputs and its intrinsic excitability. It is therefore important to ask how a neuron develops these attributes, and whether the emergence of spiking might itself be governed by activity-dependent processes. In this thesis, I address these questions in the retinotectal system of Xenopus laevis. First, I investigate the extent to which visuospatial information is available to the developing retinotectal system. I show that the eyes of developing Xenopus larvae are hyperopic at the onset of vision, but rapidly grow towards correct vision. Despite its imperfect optics, the Xenopus eye is able to generate spatially restricted activity on the retina, which is evident in the spatial structure of the receptive fields (RFs) of tectal neurons. Using a novel method to map the visually driven spiking output and synaptic inputs of the same tectal neuron in vivo, I show that neuronal spiking activity closely follows the spatiotemporal profile of glutamatergic inputs. Next, I characterise a population of neurons in the developing optic tectum that does not fire action potentials, despite receiving visually evoked glutamatergic and γ-aminobutyric acid (GABA)ergic synaptic inputs. A comparison of visually spiking and visually non-spiking neurons reveals that the principal reason these neurons are ‘silent’ is that they lack sufficient glutamatergic synaptic excitation. In the final section of the thesis, I investigate whether visually driven activity can play a role in the ‘unsilencing’ of these silent neurons. I show that non-spiking tectal neurons can be rapidly converted into spiking neurons through a visual conditioning protocol. This conversion is associated with a selective increase in glutamatergic input and implicates a novel, spike-independent form of synaptic potentiation. I provide evidence that this novel plasticity process is mediated by GABAergic inputs that are depolarising during early development, and can act in synergy with N-methyl-D-aspartate receptors (NMDARs) to strengthen immature glutamatergic synapses. Consistent with this, preventing the depolarising effects of GABA or blocking NMDARs abolished the activity-dependent unsilencing of tectal neurons. These results therefore support a model in which GABAergic and glutamatergic transmitter systems function synergistically to enable a neuron to recruit the synaptic excitation it needs to develop sensory-driven spiking activity. This represents a transition with important consequences for both the functional output and the activity-dependent development of a neuron.

Cells have evolved proteins that detect foreign DNA, RNA, or proteins which then activate cellular pathways to combat bacterial or viral infection.1–4 Apolipoprotein B editing complex 3 (APOBEC3 or A3) is a host cytidine deaminase that deaminates cytidine to uridine in viral DNA in the cytoplasm causing hyper G→A mutation leading to destabilization and degradation of the viral genome.5–8 HIV viral infectivity factor (vif) has evolved to regulate A3 degradation of viral DNA.9 Vif hijacks host ubiquitination machinery to degrade A3s and prevent packaging of A3 into the viral particle.10,11 Vif hijacks the cotranscription factor core factor binding unit beta (CBF-beta), Elongin B (ELOB) and Elongin C (ELOC) to form the VCBC complex.12–15 VCBC binds A3s and Cullin 5 (Cul5) for ubiquitination and degradation of A3 thereby preventing packaging.12,16 Antigen binding fragments (Fabs) were generated against VCBC using a naïve B-cell Fab phage display library to isolate biological tools that are specific for the host-virus interaction.17 Two high affinity Fabs were found to bind at distinct epitopes on VCBC and produced distinct phenotypes when expressed in cells as single chain variable fragments (scFvs).17 The Fab 3C9 shields A3F from ubiquitination and restores packaging of A3F into the viral particle. The Fab 1D1 blocks binding of Cul5 and ubiquitination in vitro. The scFv 1D1 prevented ubiquitination of A3F but did not restore packaging. Affinity purification mass spectroscopy (AP-MS) in HEK293Ts with 3C9 scFv and 1D1 scFv showed different interactomes in the presence of vif. AP-MS with 1D1 scFv did not interact with CBF-beta, an important component of the VCBC complex. Spatial and temporal elucidation of proteins interacting with the complex will further determine events effecting viral infectivity.

Serological tests are conducted to assess humoral response against viral protein antigens, to assess viral exposure and protection from pathogens. The rapid development and modularity of serological assays have proven critical to managing the current SARS-CoV-2 pandemic. The receptor binding domain of SARS-CoV-2 is within the trimeric Spike protein and serves as a highly immunogenic target for potentially neutralizing antibodies. Current receptor binding domain serological assays use recombinant monomers or dimers of the receptor binding domain. The receptor binding domain is presented to the immune system natively in the context of the Spike protein trimer. Therefore, a recombinant trimeric receptor binding domain may be predictive of protection and improve antibody binding. For this thesis, using the trimerization domain from Bacteriophage T4 Fibritin, called Foldon, fused to the receptor binding domain, a novel antigen was produced. The antigen was expressed in and extracted from Nicotiana benthamiana plants, purified through immobilized metal affinity FPLC, and used to develop a serological ELISA. The antigen was tested with hospitalized (n=46), non-hospitalized (n=36), and negative (n=46) patient sera sample lots and batch reproducibility was examined. From these studies, it was concluded that this trimeric antigen can be consistently expressed, extracted, and purified and can be used to reliably detect responses to SARS-CoV-2 in sera. Additionally, fusion of the Foldon trimerization domain to trimeric viral proteins serves as a platform for the development of plant produced viral antigens to better detect host response.

Extracellular vesicles (EVs) are lipid-bilayer enclosed, cell-derived nanoparticles released constitutively by most cells. EVs carry proteins and nucleic acids from their cells of origin, facilitating intercellular communication through a variety of different pathways. These include signaling through cellular receptors, triggering immune responses, and delivering bioactive cargos after EV internalization. Viruses share many characteristics with EVs, including size, structure, and biogenesis pathways. Virally infected cells continue to release EVs, resulting in the production of virus-induced EVs that carry viral elements like proteins or RNAs. Viral fusion proteins – viral envelope glycoproteins that mediate virus/cell membrane fusion – are often incorporated by virus-induced EVs. In this work, we investigated the effect that viral fusion protein incorporation has on EV communication and functionality. First, we investigated the spike protein of the current pandemic virus SARS-CoV-2. Interestingly, we found that EVs released from spike expressing cells did incorporate this type I viral fusion protein. These spike(+) EVs displayed the spike protein in a conformation available for antibody binding, allowing the EVs to serve as decoy targets for neutralizing antibodies. This held true with both convalescent patient serum and a commercial neutralizing antibody, with the effect of reducing virus neutralization and increasing infection. The second component of this work concerned the development of EV/cell membrane fusion assays to monitor EV cargo delivery. This EV communication pathway is particularly important in disease, where EVs are thought to be capable of influencing cells through membrane fusion and delivery of bioactive cargos to cell cytosol. We successfully developed two EV fusion assays, based on delivery of the proteins beta-lactamase and Cre-recombinase. In the third and final component of this work, we utilized these fusion assays to investigate the incidence of EV/cell membrane fusion and cargo delivery. Surprisingly, we found that most EVs were incapable of fusion. Supplementation of EVs with viral fusion proteins, however, could confer fusogenicity to EVs. These viral fusion proteins could originate from circulating viruses, or endogenous retroviral elements encoded in the human genome. These findings alter the current understanding of EV-mediated intercellular communication, demonstrating that captured viral proteins play a significant functional role.

The basic functions of living things are predominantly carried out by uniquely adapted protein nanomachines. Molecular phenomenology is an approach to biology driven by the most basic questions about these species: what are their atomic structures, and how do they move? The past two centuries have seen great strides in biology, yet we are only beginning to understand its fundamental units of function. In the past decade, revolutionary advances in electron microscopy have enabled direct imaging of biological molecules in solution, opening what may be the final frontier of molecular phenomenology. My doctoral work has focused especially on applying single-particle electron microscopy to the dynamics of those protein machines that lie at the plasma membrane that divides cells from their environment.In the first chapter of this dissertation I introduce a previously unpublished method for modeling protein motions through multi-body refinement and principal component analysis with geodesic kernels. While multi-body analysis is not new, other methods have not considered the intrinsic nature of rigid-body motions as a non-linear manifold. As such, they have been forced to rely on ad-hoc parameter scaling, and are valid only over very small motions where manifold curvature can be neglected. The kernel approach, in contrast, eliminates the parameter scaling problem and is applicable to arbitrary rigid-body motions. The basic method may be of use outside cryo-EM of biological molecules, for example in 3D animation of articulated figures or controlling swarms of drones or satellites. In the second chapter, I and others apply molecular phenomenology to uncover the mechanisms of action underlying antibody modulation of the fusogenic activity of the SARS-CoV-2 Spike protein. This work was the first study demonstrating that Spike-reactive antibodies can either inhibit or enhance Spike-mediated membrane fusion leading to viral entry or pathological cell-cell fusion and formation of syncytia (giant, multinucleated cells). The results epitomize the deep connection between basic biophysical understanding and biological function, by tying molecular structure and conformation to the virulence of an emergent pathogen responsible for millions of deaths and hundreds of millions of injuries. In the third chapter, I and others report high resolution structures of the murine Dispatched and its complex with Sonic hedgehog. I then use 3D variability analysis, a state-of-the-art phenomenological method, to uncover coupling between transmembrane ion flux and release of Sonic hedgehog from organizer cells during animal development. While multi-body refinement and 3D variability analysis both visualize interdomain motions, the latter also captures intradomain conformational changes by direct 3D reconstruction of orthogonal axes of particle covariance. This approach enables the production of true molecular movies that go beyond animations based on multi-body refinement. Dispatched bioactivity is characterized by subtle conformational rearrangements in its transmembrane domain that direct the energy of the plasma membrane Na+ gradient towards extraction of lipid-modified Sonic hedgehog from the plasma membrane and activation of Sonic hedgehog for efficient recruitment of its critical adapter protein SCUBE2. High-resolution molecular movies of active Dispatched, validation through traditional 3D classification, and detailed mutational analysis using a direct read-out of protein function yield one of the most complete studies in molecular phenomenology to-date, foreshadowing a new era of biophysics in which modeling dynamic structural ensembles may become de rigueur.

The coronavirus spike protein is a critical determinant of cell tropism and pathogenicity. It is expressed on the viral surface and is responsible for binding the host cell receptor and initiating fusion of the viral and host cell membranes. The work presented herein is an investigation into the genetic and biochemical properties of spike that influence the in vivo pathogenesis of feline and canine coronaviruses. A targeted sequencing approach was undertaken to identify mutations in a key viral activation site that correlate with the development of systemic disease after feline coronavirus infection. A similar strategy was used to characterize the spike proteins of canine coronaviruses associated with fatal enteritis in dogs and a thorough histopathologic characterization was performed in an attempt to independently confirm reports pantropic canine coronavirus variants.

The following thesis presents an experimental study observing and measuring the change in two of the key features of the Richtmyer-Meshkov (RM) instability in a shock-accelerated, initially cylindrical gas column; that is, the counter-rotating vortex pair and the central spike; and the observation of secondary instabilities within the primary instability. The formation of the instabilities is the result of a standing normal shock wave of air interacting with a cylindrical column of sulfur hexafluoride saturated with acetone. The experimental study is performed at two Mach numbers, 1.7 and 2.1, both with a maximum variation of 10% of either Mach number. The measurements of the size of these features of interest were compared to two external characteristics of the experiment: the actual timing after shock when the instability was measured and the distance downstream of the initial position of the cylindrical gas column. The development of the instability is tracked from the moment of shock impact until transition to turbulence when the flow becomes well-mixed. It was observed that the development of the instabilities with respect to the downstream distance point of observation was weakly correlated to the experimental Mach number. In contrast, a stronger correlation between the downstream distance and the feature size was evident. This behavior was previously observed for the counter-rotating vortex pairs, but it is a new observation for the growth of the spike that forms due to shock focusing.

This thesis is concerned with the development of techniques for analyzing the sequences of stereotypical electrical impulses within neurons known as spikes. Sequences of spikes, also called spike trains, transmit neural information; decoding them often provides details about the physiological processes generating the neural activity. Here, the statistical theory of event arrivals, called point processes, is applied to human muscle sympathetic spike trains, a peripheral nerve signal responsible for cardiovascular regulation. A novel technique that uses observed spike trains to dynamically derive information about the physiological processes generating them is also introduced. Despite the emerging usage of individual spikes in the analysis of human muscle sympathetic nerve activity, the majority of studies in this field remain focused on bursts of activity at or below cardiac rhythm frequencies. Point process theory applied to multi-neuron spike trains captured both fast and slow spiking rhythms. First, analysis of high-frequency spiking patterns within cardiac cycles was performed and, surprisingly, revealed fibers with no cardiac rhythmicity. Modeling spikes as a function of average firing rates showed that individual nerves contribute substantially to the differences in the sympathetic stressor response across experimental conditions. Subsequent investigation of low-frequency spiking identified two physiologically relevant frequency bands, and modeling spike trains as a function of hemodynamic variables uncovered complex associations between spiking activity and biophysical covariates at these two frequencies. For example, exercise-induced neural activation enhances the relationship of spikes to respiration but does not affect the extremely precise alignment of spikes to diastolic blood pressure. Additionally, a novel method of utilizing point process observations to estimate an internal state process with partially linear dynamics was introduced. Separation of the linear components of the process model and reduction of the sampled space dimensionality improved the computational efficiency of the estimator. The method was tested on an established biophysical model by concurrently computing the dynamic electrical currents of a simulated neuron and estimating its conductance properties. Computational load reduction, improved accuracy, and applicability outside neuroscience establish the new technique as a valuable tool for decoding large dynamical systems with linear substructure and point process observations.

Fabrication and operation of carbon nanotube (CNT) based electronic devices called "synapstors," with the goal of emulating the functions of biological synapses, are reported. These synapstors have a structure akin to field-effect transistors, utilizing a random network of single-wall semiconducting CNTs as its conducting channel. Analog spike signal processing with low power consumption was demonstrated. These synaptic devices are capable of carrying out logic, learning, and memory functions, all in a single element. Analog spike neuromorphic circuits, composed of CNT synapstors and integrate-and-fire (I&F) circuits, are also reported. A positive voltage spike, applied on the gate electrode of a synapstor, could generate a postsynaptic current (PSC) via the CNT channel of the device. Multiple input spikes could be applied on individual CNT synapstors, which could be either excitatory or inhibitory and be connected in a neuromorphic circuit, inducing excitatory or inhibitory PSCs, respectively. These PSCs flow collectively to an I&F circuit, triggering output spikes. Such neuromorphic circuits are capable of emulating biological neural networks, enabling parallel signal processing, dynamic learning, and memory functions with low power consumption.

This research focuses on empirical observations of horizontal series arc faults. These faults differ from ground faults, for series faults encompass the electromagnetic transient effects of arc formations in series with sustained current flow when there is a break in the circuit. This may happen intentionally (as in a breaker) or unintentionally (as in a loose, damaged, or severed cable). This paper studies some of those transient effects during arc ignition, propagation, and cessation. Emphasis is on dc systems, for series faults present some of the more challenging safety concerns relating to widespread dc micro-grid acceptance and proliferation. However, arc behavior is also compared to that of ac systems under “quasi-equivalent”, passive circuit parameters. Variables of study primarily include arc voltage, current, and their relationship to electrode spacing under dynamic conditions. Results indicate that interruptions in dc current, while appearing more chaotic from a localized standpoint, do not produce the fast-acting transients associated with ac disturbances. Additionally, if dc arcs propagate over a slowly increasing distance of separation, they can be modeled as quasi-static in nature. An equation model is developed and curve-fitting parameters match well with historically tabulated constants.

This project has developed an activity-dependent intracortical microstimulation (ICMS) system-on-chip (SoC) fabricated in a 0.35-µm two-poly four-metal CMOS process that converts extracellular neural spikes recorded from one brain region to electrical stimuli delivered to another brain region in real time in vivo. The 10.9-mm2 SoC incorporates two identical 4-channel modules, each comprising an analog recording front-end with total input noise voltage of 3.12 µVrms and noise efficiency factor (NEF) of 2.68, 5.9-µW 10b successive approximation register analog-to-digital converters (SAR ADCs), 12.4-µW digital processor for spike discrimination based on threshold crossing and two user-adjustable time-amplitude windows, and a programmable constant-current microstimulating back-end that delivers up to 94.5 µA with 6b resolution to stimulate the cortical tissue when triggered by neural activity. For autonomous operation, the SoC also integrates biasing and clock generation circuitry, frequency-shift-keyed (FSK) transmitter at 433 MHz, and dc-dc converter that generates a power supply of 5.05 V for the microstimulating back-end from 1.5 V.The fabricated SoC has been assembled and packaged on a miniature rigid-flex substrate together with a few external components for programming, supply regulation, and wireless operation. The resulting microdevice operates autonomously from a single 1.55-V battery, measures 3.6 cm x 1.3 cm x 0.6 cm, weighs 1.7 g (including the battery), and is capable of stimulating as well as recording the neural response to ICMS in biological experiments with anesthetized laboratory rats. Moreover, it has been interfaced with silicon microelectrodes chronically implanted in the cerebral cortex of an ambulatory rat and successfully delivers electrical stimuli to one cortical region when triggered by neural activity recorded from another distant cortical region with a user-adjustable spike-stimulus time delay. The spike-triggered ICMS is further shown to modulate the neuronal firing rate, indicating that it is physiologically effective.

Neurons receive large amount of synaptic inputs in vivo, which may impact the coupling between EPSPs and spikes. We mimicked the in vivo synaptic activity of the cell with the dynamic clamp system. We recorded from pyramidal cells in neocortical slices in vitro to investigate how timing and probability of spike generation in response to an EPSP is affected by background synaptic conductance under these conditions. We found that near threshold, background synaptic conductance improved the precision of spike timing by reducing the depolarization-related prolongation of the EPSP. In cells with ongoing spike activity and background synaptic conductances, an EPSP rapidly increased the probability of firing. The time window of the spike probability increase was comparable to the EPSP rise time and was followed by a long period of reduced firing. We found that the net synaptic gain was determined not only by the amplitude of the EPSP, but also by the firing frequency of the cell. In addition, a background fluctuating conductance reduced the time window of perturbation of spike patterns generated by EPSP related spikes. Taken together, these results indicate that in vivo, the level of the background synaptic activity may regulate spike-timing precision and affect synaptic gain.