Translational Medicine Graduate Theses

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    Understanding the Neural Correlates of Recovery in a Non- Human Primate Model of Ischemic Stroke
    Nashed, Joseph Y.; Translational Medicine; Cook, Douglas J.
    Stroke is a leading cause of long-term disability worldwide, and understanding the neural substrates that underlie stroke recovery is of paramount importance for developing effective interventions. However, stroke recovery in humans exhibits substantial variability across individuals, posing challenges for treatment customization and prediction of outcomes. While small animal models, such as rodents, have enhanced our understanding of stroke pathophysiology and recovery, their translational relevance to humans remains limited due to anatomical, physiological, and behavioral disparities. Non-human primate (NHP) models of stroke represent a bridge this gap due to their immense similarities to humans. These NHP models allow for whole-brain imaging before and after precisely controlled stroke lesions, which is not possible in humans. Thus, the model could provide novel insights into neural substrates underlying recovery unseen in humans or rodents. The following chapters of this doctoral thesis aim to investigate the complex nature of stroke recovery, by utilizing a previously validated non- human primate (NHPs) stroke model and cutting edge structural and functional neuroimaging techniques. The first topic of this thesis examining the brain’s structural correlates of recovery in a middle cerebral artery occlusion model of non-human primates. We show that preservation of sensory and association cortices plays a key role in the recovery process of animals. The second topic of this thesis is concerned with elucidating the functional brain changes that occur pre- to post-stroke. We highlight that considerable functional changes occur throughout the brain, and that sensorimotor and cognitive networks may play a critical role in the degree of recovery that animals exhibit. Lastly, the final topic of this thesis is aimed at determining if pre-stroke brain variation can be used to predict outcomes following an ischemic lesion. We show that we can predict recovery with considerable accuracy (>70%) using only individual variability of the pre-stroke functional scans, suggesting that there are inherent connections throughout the brain that may be exploited during the recovery process following stroke.
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    Comparing Carotid and Coronary Plaque Composition Using Noninvasive Imaging Modalities for Cardiovascular Risk Stratification
    Kersche, Georgia; Translational Medicine; Johri, Amer
    Noninvasive imaging and early identification of atherosclerosis is one of the most important tools for the prevention of cardiovascular events. Coronary artery calcium (CAC) scoring and stress echocardiography are widely used for the assessment of coronary artery disease (CAD) and to stratify patients for their risk of experiencing adverse cardiac events. Patients that have a low or intermediate pretest probability of CAD are at the greatest risk of being misclassified and experiencing an unexpected event. We looked into whether carotid ultrasound shared associations with CAC scoring. In a sample of low-intermediate risk patients with no history of CAD we assessed relationships using Spearman correlation coefficients between carotid plaque composition and burden with their CAC scores. We found that plaque burden measures including total plaque area (TPA) and carotid intima media thickness (CIMT) share a moderate and significant association with CAC score. Carotid composition measures, including calcium and fibrous tissue content, did not align with CAC scores. This suggests that carotid ultrasound plaque assessment may be useful for predicting CAC scores and the presence of CAD, and that CAC scoring can underestimate the presence of soft plaques vulnerable to rupture. To further understand how carotid ultrasound changes physician assessments of risk and to identify barriers to implementing the tool for this population we surveyed staff cardiologists. We identified that the presence of plaque tends to increase their perceptions of patient risk compared to stress echo alone. The majority of respondents reported that carotid ultrasound would be feasible to add to all stress echoes and would be useful when considering the initiation of statin therapy. Our results suggest that the addition of carotid ultrasound to cardiovascular risk assessment algorithms does have potential to identify subclinical atherosclerosis and inform patient stratification. Using carotid plaque measures may improve sensitivity and reduce the occurrence of cardiovascular events in the low and intermediate risk population.
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    Evaluating the Role of the Ocular Surface Microbiome in Dry Eye Disease Mediated Neural Dysregulation
    Brand, Cassandra; Translational Medicine; Rullo, Jacob; Lomax, Alan
    Dry eye disease (DED) is a multifactorial ocular surface disorder arising from a loss of homeostasis at the corneal-tear interface. This leads to visual and neurosensory abnormalities which are regulated by the corneal-trigeminal ganglion pathway. An important contributor to ocular homeostasis is the ocular surface microbiome (OSM). In DED, the OSM has been postulated to be dysregulated, exhibiting a loss of diversity and increase in bacterial abundance. Along with evidence of roles for microbiota in regulating pain and neural activity, this has led to an emerging role for the OSM in the pathogenesis of DED. We sought to investigate how shifts in the OSM may be associated with neural dysregulation in DED. Using a newly described chronic mouse model of DED, we characterized the OSM using conjunctival swabs and culture-based techniques. Accompanying changes in neural activity were assessed using Ca2+ imaging on trigeminal ganglion (TG) neuron cultures derived from healthy and DED mice. Capsaicin, a transient receptor potential vanilloid 1 (TRPV1) agonist, was used to evaluate nociceptive changes in DED. Functional studies were complemented with morphological analyses of TRPV1 in the TG and cornea using qPCR and/or immunofluorescence. To assess the impact of microbes in DED mediated neural dysregulation, we manipulated the OSM in DED mice with topical administration of Gatifloxacin. Effects on the OSM and neural activity were monitored using conjunctival swabs and Ca2+ imaging, respectively. Our study showed that the OSM and neural activity were dysregulated in DED. In DED, we showed that the OSM was more abundant than controls and had a greater presence of potentially pathogenic strains of bacteria. Nociceptive responses in DED mice were diminished although no significant changes in the expression of TRPV1 were noted in the TG or cornea. This suggests that desensitization of TRPV1 may contribute to symptom presentation of DED. Although microbial changes support a role for the OSM in DED, a reduction in bacterial abundance was not shown to influence neural activity. Further manipulation studies are required to fully elucidate the role of the OSM in neural dysregulation with the hope of identifying the OSM as a novel therapeutic target.
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    The Roles of Midzone and Peripheral Mitochondrial Fission in Pulmonary Arterial Hypertension
    Colpman, Pierce; Translational Medicine; Archer, Stephen
    Mitochondrial fission is vital for maintaining mitochondrial health, regulating cellular processes like proliferation and apoptosis. Disrupted fission is associated with development of cardiovascular diseases, including pulmonary arterial hypertension (PAH), ischemia-reperfusion injury, cardiac hypertrophy, and heart failure. PAH is characterized by unrestricted proliferation and impaired apoptosis of pulmonary artery smooth muscle cells (PASMC), resulting in an obstructive pulmonary vasculopathy. Accelerated mitotic fission, the processes of mitochondrial fission coordinated with nuclear division, contributes to the pseudoneoplastic phenotype of PAH. Inhibiting mitochondrial fission causes cell cycle arrest halting proliferation of PASMC and triggering apoptosis. Thus, inhibiting pathologic fission is a potential therapeutic strategy for hyperproliferative diseases such as PAH. Mitochondrial fission is mediated by the large GTPase, dynamin-related protein 1 (Drp1), which upon posttranslational activation translocates near the mitochondrial outer membrane (OMM) where it binds partner proteins of the OMM (Mid49, MiD51, MFF and Fis1) creating the fission apparatus that divides the mitochondrion. Recent research by Kleele et al., identified two types of mitochondrial fission, peripheral fission, associated with mitophagy, and midzone fission, linked to mitochondrial biogenesis and cellular proliferation. Drp1 is required for both forms of fission, but these processes involve different binding partners: mitochondrial fission factor (MFF) for midzone fission and fission protein 1 (Fis1) for peripheral fission. In this study, we investigate the role of MFF and Fis1 in the pathogenesis of PAH. To do so we developed Fission GPS, based on a tool called MitoMeter by Austin Lefebvre et al. MitoMeter has the capability to quantify fission events, track, and segment mitochondria, but cannot report positional data on where these fission events take place. Fission GPS allows fast, unbiased, and automated localization of mitochondrial fission in live cells segmented by MitoMeter. We gained novel insights into the spatial regulation of mitochondrial fission under cellular stress and better defined the role of MFF and Fis1 in fission in human PAH and healthy cells. Understanding the role of MFF and Fis1 in PAH provides valuable insights into mitochondrial mechanisms contributing to cellular proliferation and apoptosis. This knowledge has implications for developing targeted therapeutic interventions aimed at modulating mitochondrial fission pathways.
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    Preclinical Investigation of Mitochondrial Fission Inhibitors in Pulmonary Arterial Hypertension and Non-Small Cell Lung Cancer
    Breault, Nolan; Translational Medicine; Archer, Stephen
    Background: Dynamin-related protein 1 (DRP1)-mediated mitochondrial fission is coordinated with nuclear division. This phenomenon, known as mitotic fission, is increased in hyperproliferative diseases, such as non-small cell lung cancer (NSCLC) and pulmonary arterial hypertension (PAH), due to increased DRP1 expression and activity. PAH is a progressive pulmonary vasculopathy that culminates in right ventricular failure (RVF). We identified two putative DRP1 inhibitors via in silico screening predicted to bind DRP1's guanosine triphosphatase (GTPase) domain. We hypothesize that inhibition of DRP1's GTPase activity by the novel DRP1 inhibitor, 2-(2-Phenyl-2-propanyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (Compound 6) can suppress NSCLC and PAH phenotypes in vitro, and Drpitor1a, previously shown to reduce NSCLC hyperproliferation in vitro and ex vivo, can prevent PAH progression in vivo. Methods & Results: Only Drpitor1a was found to inhibit DRP1's GTPase activity in inorganic phosphate assays. While Compound 6 did not inhibit GTPase activity under the conditions tested, 4-hour treatment of PAH pulmonary artery smooth muscle cells (PASMC) and A549 adenocarcinoma cells with either Compound 6 or Drpitor1a inhibited mitochondrial fission as determined using an imaging-based machine learning algorithm. xCELLigence and EdU assays showed inhibition of cell proliferation in both PAH PASMC and A549 by Drpitor1a, though Compound 6 only reduced proliferation in A549. Drpitor1a also induced apoptosis in A549 as observed via annexin V assays, though Compound 6 did not. The monocrotaline (MCT) model of PAH (MCT-PAH) was created in female Sprague-Dawley rats by a single subcutaneous injection of MCT (60 mg/kg). 1 mg/kg Drpitor1a treatment in MCT-PAH over 12 days regressed vascular remodeling, improved hemodynamics, and reduced RV hypertrophy as shown by quantitative staining of lung tissue and RVs, echocardiography, and right heart catheterization. Conclusion: Drpitor1a is an efficacious treatment for PAH in female rodents. Drpitor1a prevents disease progression at the level of vascular remodeling, providing hemodynamic relief that benefits the structural and functional characteristics of the RV. Compound 6, though with an undetermined mechanism of action, inhibits mitochondrial fission in both cancer and PAH and additionally prevents cell proliferation in A549. Drpitor1a has promise for human study, while Compound 6 may have merit in NSCLC animal studies.