Epithelial Reparative Capacity Regulates Extracellular Matrix Dynamics and Innate Immunity

The mammalian lung supports the transport and diffusion of inspired and expired gasses that are critical for aerobic life. With every inspiration the lung is exposed to environmental agents including microbes, virus, and environmental pollutants. In the event that injury occurs the epithelium is repaired by an abundant facultative progenitor pool and a sequestered population of adult tissue stem cells. Chronic lung diseases, such as asthma, chronic obstructive pulmonary disease, and bronchopulmonary dysplasia, are characterized by extensive epithelial remodeling resulting in a reduction to the number of non-ciliated bronchiolar Clara cells. Given the established role for Clara cells as abundant facultative progenitors, these data suggest that epithelial repair has been compromised. In addition to affects on the epithelium, these diseases are also accompanied by extensive subepithelial fibroproliferation, mesenchymal remodeling, and elevated extracellular matrix deposition as well as a profound increase to lung inflammation. It has been postulated, but never tested in vivo that mesenchymal remodeling and uncontrolled deposition of extracellular matrix may be a result of impaired airway epithelial reparative capacity. Moreover, the finding that airway epithelial cells are essential for modulation of innate immunity suggests that the enhanced inflammatory response described in chronic lung disease may be a result of attenuated airway epithelial cell function. Therefore, this dissertation tests the hypothesis that airway epithelial reparative capacity moderates extracellular matrix deposition and innate immunity. Through the use of in vivo models of injury, inflammation, and attenuated Clara cell function, this dissertation research work identifies a previously uncharacterized process in which extracellular matrix is dynamically and reversibly regulated during productive epithelial repair and severely disrupted by blocking stem cell mediated repair. In addition, the use of mouse models of decreased Clara cell abundance and secretion demonstrate airway epithelium modulates pulmonary innate immunity through regulation of macrophage behavior and inhibition of pulmonary inflammation. This work defines two phenotypes that are the result of attenuated epithelial repair and supports the paradigm that epithelial reparative capacity may be a principal determinant of lung disease

The effects of hemodynamic lag on functional connectivity and behavior after stroke

Stroke disrupts the brain's vascular supply, not only within but also outside areas of infarction. We investigated temporal delays (lag) in resting state functional magnetic resonance imaging signals in 130 stroke patients scanned two weeks, three months and 12 months post stroke onset. Thirty controls were scanned twice at an interval of three months. Hemodynamic lag was determined using cross-correlation with the global gray matter signal. Behavioral performance in multiple domains was assessed in all patients. Regional cerebral blood flow and carotid patency were assessed in subsets of the cohort using arterial spin labeling and carotid Doppler ultrasonography. Significant hemodynamic lag was observed in 30% of stroke patients sub-acutely. Approximately 10% of patients showed lag at one-year post-stroke. Hemodynamic lag corresponded to gross aberrancy in functional connectivity measures, performance deficits in multiple domains and local and global perfusion deficits. Correcting for lag partially normalized abnormalities in measured functional connectivity. Yet post-stroke FC-behavior relationships in the motor and attention systems persisted even after hemodynamic delays were corrected. Resting state fMRI can reliably identify areas of hemodynamic delay following stroke. Our data reveal that hemodynamic delay is common sub-acutely, alters functional connectivity, and may be of clinical importance

Structural Effects on Nonadiabatic Photocyclization in ortho-Arenes

Nonadiabatic photocyclization is the chemical dynamic relevant to the function of many photoswitchable materials as well as photochemical synthesis of polyaromatic hydrocarbons by cyclodehydrogenation. ortho-arenes are an under-studied class of molecular photoswitch owing to their low cyclized product stabilities that otherwise provide a unique opportunity for the experimentalist to study photocyclization mechanisms in detail. Through the use of time-resolved absorption spectroscopy on femtosecond to microsecond timescales the entire photocyclization process can be monitored from “birth” to “death,” i.e. ring-fusion to ring-fission. Following ultraviolet photoexcitation OTP undergoes cyclization to form DHT. Although global spectral analysis with simple kinetic models adequately fits spectral dynamics, signatures of DHT formation are obscured by spectral overlap with the excited-state that has a lifetime of ~3 picoseconds. Thermal ring-reopening of DHT to regenerate OTP occurs with a 38 nanosecond lifetime and an activation energy of 0.27 eV. Following the study of OTP a variety of other ortho-arenes were examined by systematic substitution, including phenyl substituted analogs, 1,2,3-triphenylbenzene, ortho-quaterphenyl and hexaphenylbenzene, as well as boron-nitrogen substituted analogs, including hexaphenylborazine and 1,2:3,4:5,6 tris(o,o’-biphenylylene) borazine. Generally these substitutions increased the excited-state lifetime relative to OTP due to an increase in either electronic delocalization or structural hindrance within excited-state geometries while the stability of the corresponding photoproducts decreased relative to DHT due to entropic effects. A notable exception is hexaphenylbenzene, which exhibits a 2 microsecond lifetime for ring-reopening of the photoproduct tetraphenyl-DHT that is a consequence of entropic stabilization due to increased phenyl-phenyl steric interactions that constrain thermally activated relaxation to the transition state. Furthermore, excited hexaphenylborazine decays within 3 picoseconds due to the localized electronic character of the borazine ring. No direct spectroscopic observation of cyclization was observed for any boron-nitrogen substituted system due to the increase in charge localization that reduces the stability of the conjugated DHT photoproduct. More recent experiments utilizing pump-repump-probe spectroscopy have determined that the observed excited-state decay is kinetically decoupled from photocyclization, which occurs in less than 200 femtoseconds. The results presented in this thesis provide insight for the improvement of photoswitch and photosynthetic efficiency through the generalization of these structure-dynamics relationships

Integrating Clinical Trial Imaging Data Resources Using Service-Oriented Architecture and Grid Computing

Clinical trials which use imaging typically require data management and workflow integration across several parties. We identify opportunities for all parties involved to realize benefits with a modular interoperability model based on service-oriented architecture and grid computing principles. We discuss middleware products for implementation of this model, and propose caGrid as an ideal candidate due to its healthcare focus; free, open source license; and mature developer tools and support

A comparison of resting state functional magnetic resonance imaging to invasive electrocortical stimulation for sensorimotor mapping in pediatric patients

Localizing neurologic function within the brain remains a significant challenge in clinical neurosurgery. Invasive mapping with direct electrocortical stimulation currently is the clinical gold standard but is impractical in young or cognitively delayed patients who are unable to reliably perform tasks. Resting state functional magnetic resonance imaging non-invasively identifies resting state networks without the need for task performance, hence, is well suited to pediatric patients. We compared sensorimotor network localization by resting state fMRI to cortical stimulation sensory and motor mapping in 16 pediatric patients aged 3.1 to 18.6 years. All had medically refractory epilepsy that required invasive electrographic monitoring and stimulation mapping. The resting state fMRI data were analyzed using a previously trained machine learning classifier that has previously been evaluated in adults. We report comparable functional localization by resting state fMRI compared to stimulation mapping. These results provide strong evidence for the utility of resting state functional imaging in the localization of sensorimotor cortex across a wide range of pediatric patients

On the role of the corpus callosum in interhemispheric functional connectivity in humans

Resting state functional connectivity is defined in terms of temporal correlations between physiologic signals, most commonly studied using functional magnetic resonance imaging. Major features of functional connectivity correspond to structural (axonal) connectivity. However, this relation is not one-to-one. Interhemispheric functional connectivity in relation to the corpus callosum presents a case in point. Specifically, several reports have documented nearly intact interhemispheric functional connectivity in individuals in whom the corpus callosum (the major commissure between the hemispheres) never develops. To investigate this question, we assessed functional connectivity before and after surgical section of the corpus callosum in 22 patients with medically refractory epilepsy. Section of the corpus callosum markedly reduced interhemispheric functional connectivity. This effect was more profound in multimodal associative areas in the frontal and parietal lobe than primary regions of sensorimotor and visual function. Moreover, no evidence of recovery was observed in a limited sample in which multiyear, longitudinal follow-up was obtained. Comparison of partial vs. complete callosotomy revealed several effects implying the existence of polysynaptic functional connectivity between remote brain regions. Thus, our results demonstrate that callosal as well as extracallosal anatomical connections play a role in the maintenance of interhemispheric functional connectivity

Disruption of mesoderm formation during cardiac differentiation due to developmental exposure to 13-cis-retinoic acid.

13-cis-retinoic acid (isotretinoin, INN) is an oral pharmaceutical drug used for the treatment of skin acne, and is also a known teratogen. In this study, the molecular mechanisms underlying INN-induced developmental toxicity during early cardiac differentiation were investigated using both human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). Pre-exposure of hiPSCs and hESCs to a sublethal concentration of INN did not influence cell proliferation and pluripotency. However, mesodermal differentiation was disrupted when INN was included in the medium during differentiation. Transcriptomic profiling by RNA-seq revealed that INN exposure leads to aberrant expression of genes involved in several signaling pathways that control early mesoderm differentiation, such as TGF-beta signaling. In addition, genome-wide chromatin accessibility profiling by ATAC-seq suggested that INN-exposure leads to enhanced DNA-binding of specific transcription factors (TFs), including HNF1B, SOX10 and NFIC, often in close spatial proximity to genes that are dysregulated in response to INN treatment. Altogether, these results identify potential molecular mechanisms underlying INN-induced perturbation during mesodermal differentiation in the context of cardiac development. This study further highlights the utility of human stem cells as an alternative system for investigating congenital diseases of newborns that arise as a result of maternal drug exposure during pregnancy