Development of a Non-Human Primate Model for Staphylococcus aureus Nasal Carriage

Staphylococcus aureus nasal carriage (SANC) is largely asymptomatic, but presents a risk of autoinfection and dissemination to new immunocompromised hosts. SA disease states range from mild cutaneous infections to life-threatening bacteremia. Historically utilized rodent models do not naturally carry SA in the nose, are insufficient in longitudinal SANC experimentation, and lack immune factors that are vital in human clearance of SA. The nasal passages of non-human primates are similar anatomically and histologically, and reproductive mucosal studies have shown similar immune responses to pathogens and human-relevant microbial profiles. Seventeen captive pigtailed macaques (Macaca nemestrina) were found to naturally carry SA in the nose and pharynx, while topical mupirocin ointment effectively decolonized SA, similar to humans. Colonization was established with a human-relevant inoculum of 104 SA CFUs per nostril in four independent experiments, including with a human isolate (ST398). Autologous and non-autologous macaque strains were carried similarly in load and duration, each surviving over 40 days. Animals that cleared SA showed a rapid neutrophilic innate response, with up-regulation of IL-8, MCP-1, and IL-1β following inoculation, as observed in human hosts. Assessment of the nasal microbiome of pigtailed macaques and humans demonstrated similar relative abundance of the most prevalent genera: Staphylococcus, Corynebacterium, and Acinetobacter. Collectively, these multidimensional analyses provide evidence that the pigtailed macaque is a novel physiological model of human SANC that may be useful for testing novel SA decolonization strategies

Non-coding genome contributions to the development and evolution of mammalian organs

Protein-coding sequences only cover 1-2% of a typical mammalian genome. The remaining non-coding space hides thousands of genomic elements, some of which act via their DNA sequence while others are transcribed into non-coding RNAs. Many well-characterized non-coding elements are involved in the regulation of other genes, a process essential for the emergence of different cell types and organs during development. Changes in the expression of conserved genes during development are in turn thought to facilitate evolutionary innovation in form and function. Thus, non-coding genomic elements are hypothesized to play important roles in developmental and evolutionary processes. However, challenges related to the identification and characterization of these elements, in particular in non-model organisms, has limited the study of their overall contributions to mammalian organ development and evolution. During my dissertation work, I addressed this gap by studying two major classes of non-coding elements, long non-coding RNAs (lncRNAs) and cis-regulatory elements (CREs). In the first part of my thesis, I analyzed the expression profiles of lncRNAs during the development of seven major organs in six mammals and a bird. I showed that, unlike protein-coding genes, only a small fraction of lncRNAs is expressed in reproducibly dynamic patterns during organ development. These lncRNAs are enriched for a series of features associated with functional relevance, including increased evolutionary conservation and regulatory complexity, highlighting them as candidates for further molecular characterization. I then associated these lncRNAs with specific genes and functions based on their spatiotemporal expression profiles. My analyses also revealed differences in lncRNA contributions across organs and developmental stages, identifying a developmental transition from broadly expressed and conserved lncRNAs towards an increasing number of lineage- and organ-specific lncRNAs. Following up on these global analyses, I then focused on a newly-identified lncRNA in the marsupial opossum, Female Specific on chromosome X (FSX). The broad and likely autonomous female-specific expression of FSX suggests a role in marsupial X-chromosome inactivation (XCI). I showed that FSX shares many expression and sequence features with another lncRNA, RSX — a known regulator of XCI in marsupials. Comparisons to other marsupials revealed that both RSX and FSX emerged in the common marsupial ancestor and have since been preserved in marsupial genomes, while their broad and female-specific expression has been retained for at least 76 million years of evolution. Taken together, my analyses highlighted FSX as a novel candidate for regulating marsupial XCI. In the third part of this work, I shifted my focus to CREs and their cell type-specific activities in the developing mouse cerebellum. After annotating cerebellar cell types and states based on single-cell chromatin accessibility data, I identified putative CREs and characterized their spatiotemporal activity across cell types and developmental stages. Focusing on progenitor cells, I described temporal changes in CRE activity that are shared between early germinal zones, supporting a model of cell fate induction through common developmental cues. By examining chromatin accessibility dynamics during neuronal differentiation, I revealed a gradual divergence in the regulatory programs of major cerebellar neuron types. In the final part, I explored the evolutionary histories of CREs and their potential contributions to gene expression changes between species. By comparing mouse CREs to vertebrate genomes and chromatin accessibility profiles from the marsupial opossum, I identified a temporal decrease in CRE conservation, which is shared across cerebellar cell types. However, I also found differences in constraint between cell types, with microglia having the fastest evolving CREs in the mouse cerebellum. Finally, I used deep learning models to study the regulatory grammar of cerebellar cell types in human and mouse, showing that the sequence rules determining CRE activity are conserved across mammals. I then used these models to retrace the evolutionary changes leading to divergent CRE activity between species. Collectively, my PhD work provides insights into the evolutionary dynamics of non-coding genes and regulatory elements, the processes associated with their conservation, and their contributions to the development and evolution of mammalian cell types and organs

Aerospace Medicine and Biology: A continuing bibliography with indexes

This bibliography lists 169 reports, articles and other documents introduced into the NASA scientific and technical information system in August 1984

Imaging the subthalamic nucleus in Parkinson’s disease

This thesis is comprised of a set of work that aims to visualize and quantify the anatomy, structural variability, and connectivity of the subthalamic nucleus (STN) with optimized neuroimaging methods. The study populations include both healthy cohorts and individuals living with Parkinson's disease (PD). PD was chosen specifically due to the involvement of the STN in the pathophysiology of the disease. Optimized neuroimaging methods were primarily obtained using ultra-high field (UHF) magnetic resonance imaging (MRI). An additional component of this thesis was to determine to what extent UHF-MRI can be used in a clinical setting, specifically for pre-operative planning of deep brain stimulation (DBS) of the STN for patients with advanced PD. The thesis collectively demonstrates that i, MRI research, and clinical applications must account for the different anatomical and structural changes that occur in the STN with both age and PD. ii, Anatomical connections involved in preparatory motor control, response inhibition, and decision-making may be compromised in PD. iii. The accuracy of visualizing and quantifying the STN strongly depends on the type of MR contrast and voxel size. iv, MRI at a field strength of 3 Tesla (T) can under certain circumstances be optimized to produce results similar to that of 7 T at the expense of increased acquisition time

Brain connectivity studied by fMRI: homologous network organization in the rat, monkey, and human

The mammalian brain is composed of functional networks operating at different spatial and temporal scales — characterized by patterns of interconnections linking sensory, motor, and cognitive systems. Assessment of brain connectivity has revealed that the structure and dynamics of large-scale network organization are altered in multiple disease states suggesting their use as diagnostic or prognostic indicators. Further investigation into the underlying mechanisms, organization, and alteration of large-scale brain networks requires homologous animal models that would allow neurophysiological recordings and experimental manipulations. My current dissertation presents a comprehensive assessment and comparison of rat, macaque, and human brain networks based on evaluation of intrinsic low-frequency fluctuations of the blood oxygen-level-dependent (BOLD) fMRI signal. The signal fluctuations, recorded in the absence of any task paradigm, have been shown to reflect anatomical connectivity and are presumed to be a hemodynamic manifestation of slow fluctuations in neuronal activity. Importantly, the technique circumvents many practical limitations of other methodologies and can be compared directly between multiple species. Networks of all species were found underlying multiple levels of sensory, motor, and cognitive processing. Remarkable homologous functional connectivity was found across all species, however network complexity was dramatically increased in primate compared to rodent species. Spontaneous temporal dynamics of the resting-state networks were also preserved across species. The results demonstrate that rats and macaques share remarkable homologous network organization with humans, thereby providing strong support for their use as an animal model in the study of normal and abnormal brain connectivity as well as aiding the interpretation of electrophysiological recordings within the context of large-scale brain networks

The Strength of the Mind: Essays on Consciousness and Introspection

I defend the view that mental states have degrees of strength. Our pains are more or less intense, our mental imagery is more or less vivid, our visual perceptions are more or less striking, and our desires and thoughts are more or less gripping. Mental strength is a phenomenal magnitude shared by all conscious experiences that determines their degree of felt intensity. Mental strength, however, has been largely ignored over other aspects of mental states such as their representational contents, phenomenology, or type. Considering mental strength is crucial for illuminating philosophical discussions related to representationalism, the transparency of experiences, cognitive phenomenology, attention, and the structure and function of consciousness. I use mental strength to develop in detail a neuropsychologically plausible theory of introspection and its limits that is inspired by a signal detection theoretic model of perception. In the second half of the dissertation, I look into methodological issues concerning the neural correlates of consciousness such as controlling for performance capacity and stimulus strength, and what these methodological concerns reveal about our theories of consciousness and its function

Fluorescence microscopy image analysis of retinal neurons using deep learning

An essential goal of neuroscience is to understand the brain by simultaneously identifying, measuring, and analyzing activity from individual cells within a neural population in live brain tissue. Analyzing fluorescence microscopy (FM) images in real-time with computational algorithms is essential for achieving this goal. Deep learning techniques have shown promise in this area, but face domain-specific challenges due to limited training data, significant amounts of voxel noise in FM images, and thin structures present in large 3D images. In this thesis, I address these issues by introducing a novel deep learning pipeline to analyze static FM images of neurons with minimal data requirements and demonstrate the pipeline’s ability to segment neurons from low signal-to-noise ratio FM images with few training samples. The first step of this pipeline employs a Generative Adversarial Network (GAN) equipped to learn imaging properties from a small set of static FM images acquired for a given neuroscientific experiment. Operating like an actual microscope, our fully-trained GAN can then generate realistic static FM images from volumetric reconstructions of neurons with added control over the intensity and noise of the generated images. For the second step in our pipeline, a novel segmentation network is trained on GAN-generated images with reconstructed neurons serving as “gold standard” ground truths. While training on a large dataset of FM images is optimal, a 15\% improvement in neuron segmentation accuracy from noisy FM images is shown when architectures are fine-tuned only on a small subsample of real image data. To evaluate the overall feasibility of our pipeline and the utility of generated images, 2 novel synthetic and 3 newly acquired FM image datasets are introduced along with a new evaluation protocol for FM image ”realness” that incorporates content, texture, and expert opinion metrics. While this pipeline's primary application is to segment neurons from highly noisy FM images, its utility can be extended to automate other FM tasks such as synapse identification, neuron classification, or super-resolution