RSS 1.0Theses and Dissertations School of Medicine
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Full text for dissertations and theses included in this collection dates back to 2011. For older dissertations, check the library’s catalog CatalogUSMAI or Dissertations and Theses database.
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Identifying transglutaminase 2 (TG2) downstream mediators that are required to maintain an aggressive epidermal squamous cell carcinoma cancer phenotypeEpidermal squamous cell carcinoma is among the most common cancers in humans. It is typically treated by surgical resection and chemotherapy. However, 5 – 10% of resected cases relapse as aggressive cancer. We attribute the reason as the existence of epidermal cancer stem-like cells (ECS cells). Transglutaminase 2 (TG2) is a key ECS cell survival protein. However, how TG2 maintains the aggressive ECS cell cancer phenotype is not well understood. Thus, our goal is to identify TG2-stimulated downstream mediators that are important to maintain the ECS cell cancer phenotype. In the present studies, we show that inhibition of TG2 reduces MET tyrosine kinase receptor expression and activity, and that this is associated with attenuated cancer phenotype. Inhibition of TG2 or HGF/MET function reduces downstream MEK1/2-ERK1/2 activity, and this is associated with reduced cancer cell spheroid formation, invasion, migration, and reduced EMT and stem cell marker expression. HGF partially restores the aggressive cancer phenotype, confirming that MET signaling is downstream of TG2. MET knockdown reduces ERK1/2 signaling, doubles the time to initial tumor appearance and reduces overall tumor growth. In addition, we show that TG2 knockdown or inactivation results in a reduction in mTOR level and activity in ECS cells which are associated with reduced spheroid formation, invasion, migration, and reduced stem cell and EMT marker expression. mTOR knockdown or treatment with the mTOR inhibitor rapamycin phenocopies the reductions in cancer phenotype and stem cell and EMT marker expression. Moreover, forced expression of constitutively active mTOR in TG2 knockdown cells partially restores the aggressive cancer phenotype and stem cell and EMT marker levels, suggesting that mTOR is a necessary mediator of TG2 action. Together, the present studies suggest that TG2 maintains HGF/MET/ERK1/2 signaling and mTOR signaling to maintain the aggressive ECS cell cancer phenotype and drive aggressive tumor formation, and that TG2-dependent MET or mTOR signaling may be useful anti-cancer targets.
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Relationship between kidney function and cognitive decline and moderating effects of race and SESThere is limited research as to whether pre-clinical chronic kidney disease (CKD) measured by a combination of kidney function markers such as the estimated glomerular filtration rate (eGFR), albuminuria, and Cystatin-C have an impact on cognition. Understanding kidney function decline as a potential early marker for cognitive decline has important public health implications given the high societal burden of clinically diagnosed kidney disease and cognitive disorders. The Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study wave 1 and 3 datasets were used for this analysis. HANDLS Wave 1 data were collected from year 2004 to 2009, and wave 3 from year 2009 to 2013. In Aim 1, mixed effects models with interaction terms were constructed to examine the effects of time-varying eGFR on several cognitive outcomes. A backward elimination method was used to trim the models of non-significant four-way interactions of eGFR*Age*Race*Poverty and all lower-order interactions. In Aim 2, mixed effects models with interaction terms similar to those used in Aim 1 were constructed to examine the effects of baseline eGFR, urine albumin and Cystatin-C on the same set of cognitive outcomes. All significant interactions were examined by interaction plots for racial and SES subgroups. The analytical cohorts included 1,206 participants for aim 1 and 638 participants for aim 2 at baseline. The main hypothesis that African Americans living below poverty would have the worst impact of lower kidney function on cognition was not supported. Over about 5 years, eGFR over time was associated with decline in verbal learning and memory among Whites below poverty. In addition, the associations between baseline kidney function markers and cognitive decline were highly equivocal. Higher baseline Cystatin-C were found to be associated with cognitive decline in attention, and this association was no longer significant after adjusting for cardiovascular factors and inflammation. It is possible that kidney function is broadly related to performance in the domains of attention, working memory and executive function. These findings suggest that functional changes in the kidney precedes cognitive decline among Whites, especially those living below poverty.
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Skeletal muscle properties, gross motor performance, and quality of life in survivors of childhood hematologic and oncologic health conditionsBackground: Gross motor skills such as running, hopping, and jumping are important for age-appropriate activities and sports throughout the lifespan. Difficulty with gross motor skills negatively affects one’s quality of life (QoL). Gross motor skills require the activation of the large lower-extremity skeletal muscles and the muscle properties of these muscles such as strength, size, and neuromuscular activation contribute to gross motor performance. Children with chronic hematologic and oncologic health conditions, such as sickle cell disease (SCD) and musculoskeletal sarcoma (MSS), are at risk for impairments in skeletal muscle properties, limitations in gross motor performance, and reduced QoL. However, there remains a lack of knowledge of skeletal muscle properties and their relationships to gross motor performance and QoL in children with chronic hematologic and oncologic health conditions. Methods: Quadriceps skeletal muscle properties (strength, size, and neuromuscular activation), gross motor performance, and quality of life were measured in children with SCD and adolescent, young adult MSS survivors of childhood cancer (CCS), and healthy controls. The effect of functional strengthening (PT-STRONG) was assessed in a sub-population of MSS CCS. Results: Children with SCD and adolescent and young adult MSS CCS presented with impairments in muscle properties including decreased knee extension strength and lower quadriceps rate of muscle activation (RoA), poorer gross motor performance, and reduced QoL compared to controls. MSS CCS demonstrated decreased surgical limb knee extension strength, and quadriceps muscle thickness and RoA compared to their non-surgical limb, and decreased bilateral knee extension strength, gross motor performance, and physical QoL compared to normative values. In children with SCD, positive relationships between RoA, strength, gross motor performance, and quality of life were identified. In MSS CCS, positive correlations between muscle thickness and strength, and between strength and gross motor performance were identified. In response to PT-STRONG, MSS CCS participants demonstrated individual improvements in neuromuscular activation, gross motor performance, and physical QoL. Conclusions: Children with SCD and adolescent and young adult MSS CCS demonstrate changes in muscle properties that are associated with limitations of gross motor performance and reduced quality of life.
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Care Interactions Between Staff and Nursing Home Residents with DementiaBackground: The quality of staff-resident care interactions is critical to residents living with dementia. Limited work has focused on understanding the quality of care interactions among nursing home (NH) residents and examining what factors are associated with the quality of staff-resident care interactions. Purpose: The purpose of this dissertation was to: (1) comprehensively describe staff-resident care interactions; (2) assess racial and gender differences in the quality of care interactions between staff and residents living with dementia; and (3) test the resident and community factors that are associated with the quality of care interactions between NH residents living with dementia and staff. Methods: Utilizing baseline data from a randomized pragmatic trial that included 553 residents from 55 NH facilities, Aim 1 used descriptive statistics to describe the characteristics of care interactions in NHs and a multiple linear regression to determine differences in the quality of care interactions between actively engaged and passively engaged residents, Aim 2 used analyses of covariance to examine racial and gender differences in the quality of care interactions, and Aim 3 used structural equation modeling to test the resident and community factors that were associated with quality of care interactions and test for invariance between model fit based on resident race and gender. Results: Although the majority of care interactions were positive, 21% of the interactions were negative and neutral. Active engagement was significantly associated with more positive care interactions than passive engagement. There was a racial difference in the quality of care interactions such that Black residents received significantly more positive care interactions than White residents. Increased pain and comorbidities were associated with more negative care interactions, while higher community star rating and for-profit communities were associated with more positive care interactions. Conclusions: Understanding the quality of staff-resident care interactions in NHs and the factors that are related to the quality of care is important to guide future interventions and training curricula for NH care staff. Using this information to improve care interactions is important so that all NH residents living with dementia experience positive interactions regardless of pain, comorbidities, race, or community characteristics.
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Telehealth Care: How reliable are our physical therapy outcome measures?Background: Changes in global health have prompted a transition from the in-person healthcare model towards the telehealth care model for patients with a variety of health conditions, including patients with cerebellar impairments and patients with Parkinson’s disease (PD). While it is known that patient satisfaction is high in the telehealth care setting, it is unknown whether the tools that clinicians utilize in the in-person setting are reliable and valid in the virtual setting. Thus, the goal of this project is to evaluate the psychometric properties of two physical therapy (PT) outcome measures, that have been previously established in the clinical in-person setting, in the telehealth setting. Methods: Nineteen individuals with cerebellar impairments and nineteen individuals with PD were recruited on a voluntary basis. The Scale for Assessment and Rating of Ataxia (SARA) was used to assess the degree of cerebellar damage and the Berg Balance Scale (BBS) was used to assess the impact of PD. Participants completed two testing sessions (one virtual and one in-person) during which the outcome measure of choice was performed. Outcome measure performance was video recorded in both environments. Videos were independently scored by four raters with varying levels of PT experience. Concurrent validity was assessed via Spearman’s Rank Order Correlation Coefficient, α<0.05, comparing the virtual SARA and BBS scores to their “gold standard” in-person scores. Inter-rater reliability for four raters was evaluated with an Intraclass Correlation Coefficient (ICC) (2,4), α<0.05. Results: The SARA and the BBS were found to have large concurrent validity with Spearman’s rho significant at the two tailed, alpha <0.01, 0.90, n=14; 0.87, n=18, respectively. Similarly, the SARA and the BBS had excellent inter-rater reliability in the clinic ICC (2,4) 0.97 and 0.90, n=19 for both measures. Virtually, the BSS had moderate inter-rater reliability (ICC (2,4) 0.72, n=18) and the SARA had excellent inter-rater reliability (ICC (2,4) 0.98, n=14). Conclusion: Our study shows that both the SARA and the BBS can be used in the virtual telehealth setting. Additionally, clinicians with varying years of PT experience can accurately score the SARA and the BBS for patients with cerebellar impairments and PD, respectively.
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Healthcare Epidemiology of Emerging Infectious DiseasesBackground: Readmission within 30-day in coronavirus disease 2019 (COVID-19) is an important indicator of patient safety and quality of care. Conflicting evidence exists regarding in-hospital mortality in solid organ transplant (SOT) recipients with COVID-19. Furthermore, during the pandemic, an increase in hospital-acquired infections (HAI) potentially partially due to environmental contamination was observed. Objective: This thesis evaluated i) the impact of comorbidities on the odds of 30-day readmission following COVID-19 hospitalization, ii) the risk of in-hospital mortality among SOT recipients with COVID-19, and iii) the efficacy of a novel continuously active disinfectant (CAD) to reduce bioburden and infectious pathogens on environmental surfaces in a critical-care setting. Methods: A retrospective cohort study was conducted using the Premier database of COVID-19 in-patients in the United States. The association between comorbidities and readmission, and the relationship between SOT and in-hospital mortality were examined using logistic regression and log-binomial models respectively. General linear models and logistic regression were used to evaluate the efficacy of the CAD in a randomized controlled trial. Results: Among 331,136 COVID-19 patients, 36,827 (11.1%) were readmitted within 30 days. Each additional comorbidity category was associated with increased odds of all-cause readmission (adjusted odds ratio [aOR], 1.18; 95% confidence interval (CI):1.17–1.19) and readmission for COVID-19 (aOR, 1.10; 95% CI:1.09–1.11). Among 378,111 COVID-19 patients, 3,527 (0.9%) SOT recipients had an increased risk of in-hospital mortality (adjusted relative risk, 1.26; 95% CI, 1.17–1.35). The mean difference in environmental bioburden between the new CAD and the standard disinfectant was -0.59 (95% CI: -1.45 , 0.27) and the odds of detection of epidemiologically important pathogens was 14% lower in rooms cleaned with the CAD compared to the standard disinfectant (OR 0.86, 95% CI: 0.31 -2.32). Conclusions: In COVID-19 patients, readmission was common within 30-days. SOT recipients with COVID-19 are at an increased risk of mortality. Larger trials may be warranted to further evaluate CAD to limit the transmission of infectious pathogens. A better understanding of these risk factors will facilitate hospital epidemiologists to better manage emerging infectious diseases by improving bed-flow planning, resource allocation, and implementing appropriate measures to deliver high-quality patient care.
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Single-Nucleus Multi-Omic Characterization of Age-Specific Effects of Traumatic Brain Injury in the Mouse HippocampusTraumatic brain injury (TBI) has been characterized as a silent epidemic with growing incidence rates worldwide. Recently, single cell genomics has revealed multiple microglial subtypes activated during inflammation, and emerging evidence suggests that distinct subtypes contribute to age-related differences in neurodegeneration and neuroprotection. In this thesis, I test the hypothesis that differential microglial activation states contribute to the worsened outcomes to TBI associated with aging. Using single-nucleus RNA and ATAC sequencing, I describe cell type specific transcriptional and epigenomic changes in microglia in the context of TBI in young vs. aged mice. I found substantial transcriptional effects of TBI in microglia in both young and aged mice. Regulatory network models predict common repression of homeostatic genes but differential activation of regulatory activity with transcription factors like Nfe2l2 and Runx1 in rescuing and repressing a neuroprotective state of microglia.
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Investigating Fn14 as a cell surface portal for targeted nanotherapeutic delivery to triple-negative breast cancer (TNBC) cellsMetastatic triple-negative breast cancer (TNBC) is associated with a dismal prognosis due to a lack of known therapeutic targets and several difficulties in treating disseminated disease. As a result, patients with this diagnosis have an average median survival time of just 13 months. Treatment challenges are further exacerbated in the 30-55% of metastatic TNBC patients that develop brain metastases (BMs), where therapeutic agent efficacy is further limited by the blood-brain barrier. Nanotherapeutics may be especially poised for overcoming such treatment barriers, particularly if specifically targeted to TNBC cells within the brain lesions. The TWEAK receptor, Fn14, is minimally expressed by healthy breast and brain tissues but overexpressed in primary TNBC tumors and TNBC BMs. We previously demonstrated that Fn14-targeted, paclitaxel-loaded polymeric nanoparticles (NPs), termed ‘DARTs’, outperform Abraxane—an FDA-approved paclitaxel nanoformulation—following intravenous delivery in mouse models of primary TNBC and TNBC BM. In Part 1 of this work, we investigated the impact of using different Fn14 targeting moieties and moiety surface densities on NP binding affinity and uptake by Fn14-expressing tumor cells. Specifically, we found that DARTs functionalized with ~13 ITEM4 monoclonal antibodies, which are specific for Fn14, exhibit significantly greater uptake by TNBC cells compared to NPs with other ITEM4 surface densities or those with ITEM4 fragment antigen binding (Fab) regions for targeting. In addition, we determined that DARTs are predominantly internalized via clathrin-mediated endocytosis and traffic via the endolysosomal pathway. In Part 2 of this project, we further examined the utility of this formulation as a drug delivery platform in complementary xenograft and syngeneic models of TNBC BM. First, we determined that tumor cells were the predominant source of Fn14 expression in the TNBC brain tumor-immune microenvironment, with minimal Fn14 expression by microglia, infiltrating macrophages, monocytes, or lymphocytes. We then showed that although DARTs, non-targeted PLGA-PEG NPs and Abraxane exhibit similar accumulation in brains harboring TNBC BMs following systemic delivery, only DART NPs specifically associated with TNBC cells. Collectively, these findings deepen our understanding of the potential use of the DART NP formulation for drug delivery to TNBC patients with BMs.
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The Anti-tumor Action and Skeletal Toxicity of Palovarotene, Retinoid Nuclear Receptor Gamma Agonists in Multiple Osteochondroma Mouse ModelOsteochondromas are cartilage-capped tumors that arise near growing physis and are the most common benign bone tumor in children. They can lead to skeletal deformity, pain, loss of motion and neurovascular compression. Multiple osteochondromas (MO) can occur from a hereditary cancer syndrome in which EXT1 and EXT2 are the major causative genes. Currently, treatment is limited to surgical resection only. There are no available FDA-approved drug therapies for MO. Previous translational research suggested that retinoic acid nuclear receptor gamma agonist (RARγ) suppresses ectopic cartilage formation including osteochondromas in rodent models. A clinical trial of the systemic treatment of Palovarotene, a RARγ agonist for MO (NCT03442985) trial was terminated due to concerns of skeletal toxicity observed in pediatric patients in a different clinical trial. The purposes of this project are to determine whether refining systemic and local RARγ agonist treatment inhibits pre-existing osteochondroma growth, to minimize the adverse actions of the RARγ agonists on adjacent growth plate, and to elucidate the molecular action of RARγ agonists on osteochondromas. A mouse model involving the conditional deletion of EXT1 in cartilage was used as MO animal model. Palovarotene (1.76 mg/Kg, daily) treatment for 2 weeks, strongly suppressed osteochondroma development in the wrists and ribs. Osteochondromas became evident under the treatment with the same dose for an additional 2 weeks, however, was significantly reduced with the increasing the Palovarotene dose to 4.0 mg/Kg. However, this increase in the drug dose exhibited skeletal toxicity, including changes in trabecular bone, thinning of the cortical bone and articular cartilage deformity. Local delivery is an alternative theory to overcome systemic exposure concerns. Two-week local application of RARγ-loaded nanoparticles inhibited osteochondroma volume in the writs of our osteochondroma model without causing effects on limb lengths compared to vehicle control. Mechanistic studies demonstrated that RARγ agonist treatment of human osteochondroma explants inhibited matrix synthesis, stimulated matrix degradation and induced cell death while systemic treatment inhibited matrix synthesis and stimulated matrix degradation in our osteochondroma mouse model. These findings indicate that RARγ agonist exerts anti-tumor function, and that local drug therapy may be an alternative to avoid systemic toxicity.
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Characterization of a Lipid Receptor in Tick Innate ImmunityInnate immunity in metazoans relies on an arsenal of pattern recognition receptors. These molecules activate signaling pathways that direct antimicrobial defense. The arthropod immune system, which has been largely studied in the fruit fly Drosophila melanogaster, utilizes the immune deficiency (IMD) pathway to defend against Gram-negative bacteria. Recent studies using the blacklegged tick Ixodes scapularis determined that immune signaling in chelicerates is wired differently from dipteran insects. Notably, ticks and other chelicerates do not encode several components of the canonical IMD pathway, including classical receptors. Here, we report that the I. scapularis homolog of Croquemort (Crq), a CD36-like lipid scavenger receptor, initiates immune pathways against the Lyme disease spirochete Borrelia burgdorferi. Crq binds the lipid and IMD pathway agonist 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and exhibits plasma membrane localization. Nymphs silenced for crq display impaired fitness, an inability to reach full repletion, and delayed molting to adulthood due to deficient ecdysteroid synthesis. Importantly, Crq relays antimicrobial signals, regulates immune gene expression, and limits acquisition of B. burgdorferi through the IMD and jun N-terminal kinase (JNK) pathways. Collectively, our findings reveal an ancient antibacterial immune response in non-insect arthropods and establish a new scientific paradigm in tick-borne diseases.
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The Allostery and Specificity of EF Hand Calcium-Binding ProteinsEF-hand Ca2+-binding proteins (CBPs), such as calmodulin (CaM) or those belonging to the S100 protein family (S100s) undergo conformational changes upon increasing intracellular Ca2+, facilitating interactions with protein targets and inducing important biological responses. In the absence of target, the Ca2+-binding affinity of CaM and most S100 proteins is weak (CaKD >1 μM). However, when bound to effectors, allosteric mechanisms increase the Ca2+ affinity of these CBPs (CaKD <1 μM) to allow for proper Ca2+ homeostasis and maintenance of Ca2+-signaling. The Ca2+-tightening of these CBPs is described here by the “binding and functional folding” framework for detailing this physiologically relevant phenomenon. This research seeks to elucidate both the mechanisms of allostery and the basis of ligand- and target- specificity for S100 proteins and CaM. First, molecular fragments were used to differentiate between two highly similar S100 proteins, S100B and S100A1, with the goal of designing S100-specific inhibitors to block these elevated S100 proteins in various disease states. This provided insight into the specificity of S100A1 versus S100B for small molecules and will enable improved S100 protein-based drug design efforts. S100A1 and S100B binding to ions was also compared and differences between ion binding sites within the two highly similar proteins were determined. Another study revealed an allosteric mechanism in which a peptide termed BP2, derived from the STRA6 vitamin A transporter, increased the Ca2+-binding affinity of CaM upon binding. CaM-STRA6 complex formation was observed at physiologically relevant free Ca2+ concentrations (<1 μM), suggesting that retinol transport by full-length CaCaM-STRA6 may be regulated by Ca2+-signaling. The effect of CaM on multiple full-length target proteins was then discussed to further describe CaM allostery. Together, these studies lead to an improved understanding of Ca2+ signaling, CBP allostery and CBP-target- and ligand- interaction specificity.
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Diagnostic Stewardship to Improve the Diagnosis of Urinary Tract InfectionsUrinary tract infections (UTIs) are common infections, however diagnosis remains challenging. Asymptomatic bacteriuria (positive urine cultures in the absence of true infection) represent a major driver of inappropriate diagnosis and unnecessary antimicrobials. Curbing the impact of inappropriate diagnosis of UTIs often falls upon antimicrobial stewardship (AMS) programs, however AMS interventions occur after diagnosis. In contrast, diagnostic stewardship is an innovative approach that intervenes at across the diagnostic pathway (urine culture ordering, processing, and reporting), upstream of traditional AMS activities, and has the potential to significantly impact diagnosis and management decisions. Best practices and optimal implementation of diagnostic stewardship, however, have yet to be fully described. Specific Aim 1 evaluated the feasibility and impact of conditional urine testing. This study compared three Veterans Affairs (VA) hospitals that had implemented this diagnostic stewardship intervention compared to three control sites. Conditional urine testing, particularly with more restrictive requirements, was associated with significantly fewer urine cultures performed. Importantly, there were no serious unintended consequences, such as secondary bacteremia, related to missed diagnosis of UTI at the population level. Specific Aim 2 sought to comprehensively review available diagnostic stewardship interventions and create expert guidance for best practices to improve diagnosis of UTI. Traditionally, guidelines for diagnosis and management of infections do not address diagnostic stewardship interventions, particularly those at the institutional or health system level. This Aim distilled available literature into best practices for urine culture stewardship through expert guidance and will serve as a guide for clinicians in the future. Specific Aim 3 applied implementation science and user-centered design principles to develop urine culture diagnostic stewardship interventions for three geographically diverse VA medical centers. This included interventions focused on urine culture ordering, processing, and reporting. Prototype intervention tools were refined using qualitative methods to facilitate local implementation at each participating site and ensure long-term intervention success. Patient-centered diagnostic stewardship can lead to improved patient outcomes and less antimicrobial misuse. The results of these three aims will be used to demonstrate the impact of urine culture diagnostic stewardship best practices on these outcomes and ultimately lead to an implementation toolkit to be used nationwide.
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The Clinical Translation of Cardiac XenotransplantationPatients with end-stage heart failure requiring heart transplantation die simply because allografts are in short supply. Cardiac xenotransplantation from genetically-modified pig source animals has been proposed to bridge the gap between supply and demand. However, there are two predominant barriers to clinical translation and are poorly understood. The first, is a type of primary graft dysfunction, termed perioperative cardiac xenograft dysfunction (PCXD), which causes failure of the xenograft within 48 hours after transplantation. The second, post-transplantation xenograft growth, which causes a life-limiting diastolic heart failure within the first month after transplantation. We demonstrate that PCXD can be overcome with cardiac preservation techniques that minimize ischemia, either with blood-based cardioplegia induction or non-ischemic continuous preservation (NICP). In a heterotopic PCXD model, we further demonstrate that PCXD is likely a phenomenon rooted in xenograft dysfunction resulting from activation of innate immunity within the xenograft. While further studies need to be done, we demonstrate evidence that PCXD is augmented by the synergistic inflammatory effects of both cardiopulmonary bypass and cross-species transplantation. We also provide evidence that polymorphisms from TLR4 of S. scrofa (swine) compared to H. sapiens, may explain a possible target for the unique inflammatory signaling, that results in xenograft dysfunction after cardiac xenotransplantation. We also demonstrate that post-transplantation xenograft growth is multifactorial, but can be limited by growth hormone receptor knockout donors, along with genetic modifications that reduce immunogenicity of the xenograft. This growth, within 6 months after transplantation, is not a result of physiologic mismatch or cardiomyocyte hypertrophy. Therefore, recipients do not need to be treated for tachycardia and hypertension as previously thought. Lastly, we demonstrate the clinical translation of cardiac xenotransplantation by applying knowledge and expertise obtained from these studies. Expanded access (“compassionate use”) FDA authorization was based on demonstration of principle from our preclinical model. An ECMO-dependent patient without other therapeutic options was transplanted a genetically-modified cardiac xenograft, with 10-gene edits, combined with non-ischemic cardiac preservation and anti-CD40 monoclonal antibody-based immunosuppression. The patient was able to successfully wean from ECMO, participate in active rehabilitation and survived 60 days after transplantation without evidence of rejection.
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Investigating the Role of Microglia and Extracellular Vesicles in Spinal Cord Injury-Induced Brain DysfunctionSpinal cord injury (SCI) causes brain neurodegeneration leading to cognitive and affective changes, including memory loss and mood alterations. Using SCI models, we and others have demonstrated that progressive neurodegeneration is accompanied by neuroinflammation, including sustained microglial activation. The primary goal of this dissertation was to test the hypothesis that SCI triggers brain microglia-mediated neuroinflammation and secondary neurological dysfunction and to study the underpinning mechanisms including changes in systemic and central extracellular vesicles (EVs). First, we probed the mechanisms responsible for microglia activation and examined the effect of pharmacological depletion of microglia on posttraumatic neuropathology and cognitive/depressive-like behavior in a mouse SCI model. Microglial depletion significantly improved neuronal cell loss in key brain regions and associated cognitive/depressive-like behavioral outcomes after SCI. The transcriptomes of the spinal cord and brain were also substantially altered, supporting our hypothesis that microglia significantly contribute to changes related to inflammation, neurotransmission, and apoptosis after SCI. Second, we studied changes in circulating EVs after SCI. EVs are biological nanoparticles released from cells that contribute to intercellular communication and can become altered with disease. We found a significant increase in plasma tetraspanin CD81+ EVs after SCI at 1d post-injury. Surface CD81 was decreased on astrocytes at the injury site, suggesting that these cells may release CD81+ EVs into circulation. Total plasma EV microRNA content was also significantly modified, similar to the profile previously described in inflammatory astrocyte EVs. Notably, when injected into the cerebroventricular system, plasma EVs from SCI mice increased brain expression of several inflammatory genes, including markers of astrocyte reactivity. Finally, we examined the brain transcriptional profile and EV changes 19 months post-SCI in male and female mice. While we observed strong sex-dependent differences in the overall brain transcriptome after SCI, the homeostatic microglial phenotype was reduced in both sexes. Chronic SCI increased EV count in the brain and modified their microRNA content, which may explain the observed transcriptional changes. Plasma EV markers were also elevated late after injury, especially in males. Collectively, these experiments are the first to characterize EV dynamics after SCI and suggest that EVs may be involved in posttraumatic brain inflammation.
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Characterization of Temperature Dependent Activity in a Model Polyextremophilic Beta-galactosidase Enzyme Through Kinetic and Structural AnalysisThe Antarctic haloarchaeon, Halorubrum lacusprofundi, contains a polyextremophilic family 42 β-galactosidase, which we are using as a model for polyextremophilic enzymes. Divergent amino acid residues in this 78 kDa protein were identified through comparative genomics and hypothesized to be important for cold activity. Six amino acid residues were previously mutated and five were shown by steady-state kinetic analysis to have altered temperature-dependent catalytic activity profiles via effects on Km and/or kcat compared to the wild-type enzyme. Double-mutated enzymes were constructed and tested for temperature effects from 0-50 ºC, including two new tandem residue pairs, two potential loop insertions, and pairwise combination of the single residue mutations. The observed temperature and salinity dependent kinetic effects were compared to root-mean-square fluctuations to determine structural mechanisms of polyextremophilic activity. All the mutated enzymes were found to be more catalytically active at higher and/or less active at colder temperatures compared to the wild-type, with both Km and kcat effects observed for the two tandem mutations. For pairwise combinations, a Km effect was seen when the surface-exposed F387L mutation located in a domain A TIM barrel α helix 19 Å from the active site was combined with two internal residues, N251D or V482L. When another surface-exposed residue, I476V, was paired with N251D or V482L, primarily a kcat effect was observed. The temperature dependent kinetic effects were continued to 25-50 ºC with a dominant Km effect observed in all mutated enzymes. By deleting the identified insertions/loops, we are able to determine the kinetic effects of these insertions. The domain B deletion resulted in a less active enzyme overall converging with the wild-type at higher temperatures while the domain C deletion resulted in reduced cold-activity and improved activity at higher temperatures. Domain B may confer cold-activity without changing the thermal stability while domain C confers cold activity, at least in part, at the expense of activity at higher temperatures. With molecular dynamics simulations, increased flexibility was observed with higher temperature and salinity and in the mutations, indicating that the enzyme’s function is related to its flexibility and there is a balance required for optimal polyextremophilic activity.
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Visual Deficits in a Model of Gestational HypothyroidismHypothyroidism prevalence among pregnant women is between 0.5 to 4% (Carney et al., 2014). Most studies look at the prolonged or severe reduction in thyroid hormone (TH) levels. We looked at how the reduced levels of TH during the third trimester of human gestation and the first weeks after birth in rodents impact the visual system. Additionally, we try to answer whether PTU treatment would affect neuronal plasticity in the visual cortex. We used the Visual Evoked Potential recordings to assess contrast sensitivity, spatial frequency acuity, and ocular dominance plasticity. In addition, we look into the expression of the photoreceptors in the retina. PTU exposure impacts the contrast sensitivity but not the spatial frequency acuity or ocular dominance plasticity. The expression level of the photoreceptor Opsin-M was also impacted. The reduced levels of the thyroid hormones during this crucial time have long-lasting consequences for the proper visual system processing.
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The Role of Colonization Factors CFA/I and CS21 in Enterotoxigenic E.coli (ETEC) Pathogenesis in the Human Enteroid ModelEnterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and in young children in low-to-middle income countries (LMICs). ETEC adhere to intestinal epithelia via colonization factors (CFs) and secrete heat-stable toxin (ST) and/or heat-labile toxin (LT), causing dysregulated cellular ion transport and water secretion. ETEC isolates often harbor genes encoding more than one CF and are prime targets as vaccine antigens. Many clinical isolates express CFA/I and CS21; however, a role for CS21 alone or with CFA/I has not been defined. We hypothesize that expression of both CFs confers increased adherence and toxin delivery to the human enteroid. Clinical strains expressing CFA/I and/or CS21 were evaluated, and CF-deficient mutants were engineered. After confirming CF expression using western blot and electron microscopy, assays demonstrated CFA/I was important for CFA/I-CS21 ETEC adherence, as CFA/I-deficient mutants and strains pre-incubated with anti-CFA/I antibody had significantly reduced adherence to enteroid monolayers compared to wildtype. In contrast, CS21 was not required as CS21-deficient mutants and strains pre-incubated with anti-CS21 antibody adhered at similar levels as wildtype. These data demonstrate that targeting CFA/I in CFA/I-CS21 ETEC is sufficient for significant adherence reduction. Delivery of ST by CFA/I-CS21 ETEC was evaluated. Strain-specific levels of toxin delivery were detected but CF-dependent ST delivery was not observed, which may reflect the lack of flow and stretch in the current enteroid model. Upon investigation of host responses to ETEC, the enteroid monolayer integrity was not disrupted, as shown by the increase in transepithelial electrical resistance and the lack of inflammatory cytokines produced. Infection with ETEC strains resulted in decreased mucus (MUC2) production, but this was not CF-dependent. Further studies of strain-specific CFA/I expression revealed that it may be transcriptionally or post-transcriptionally regulated, following observation of nearly identical CFA/I operon sequences and many shared CF-specific regulators at the genomic level. Overall, these data support the role of CFA/I in CFA/I-CS21 ETEC adherence and reinforces CFA/I as a main target for vaccines. These data also highlight the human enteroid model to study ETEC pathogenesis and for evaluation of preclinical therapeutics.
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Novel Modules in the T Cell Signaling Circuit Which Enable Synergy Between Responses to Self and Foreign PeptidesT cell activation occurs when a T cell receptor (TCR) engages with cognate agonistic peptides in the context of Major Histocompatibility Complexes (pMHCs) on antigen presenting cells (APCs). This initiates a series of intracellular signaling events proximal to the TCR and associated CD3 complexes, mediated by unique kinases together with scaffolding and lattice molecules. The downstream cascades ultimately lead to transcriptional changes that promote the cellular program of conventional T cell activation—for example, cytoskeletal rearrangement, cytokine production, cellular proliferation, and differentiation. Understanding signaling mechanisms not only allows us to decipher the regulation of T cell activation but also to manipulate immune responses pharmacologically. TCR signaling by agonistic pMHCs is well studied, but much less is known about signaling by a parallel universe of self-peptides that also engage TCRs in vivo. The focus of this thesis is to understand how T cells perceive these signals without fully acquiring effector responses as a result. The central hypothesis of my thesis is that self-peptide ligands signal uniquely through the TCR to alter the fate and function of a T cell both with and without presence of their cognate agonist. We evaluated this hypothesis using approaches that globally increased self-peptide presentation in vivo (using FLT3L to generate more DCs), deprived T cells of self-peptide (by culturing away from APCs) or stimulated a TCR with a known self-peptide in the presence or absence of the strong agonist. The significant findings from these studies are that (i) boosting self-peptide presentation transiently increases a narrow T cell effector subset; (ii) depriving T cells of self-engagement lowers basal phosphorylation in the key signaling adapter LAT; (iii) self-peptides do not trigger cellular activation on their own, but synergize to enhance activation as measured by CD69, pERK, and several other parameters (iv) self-peptides initiate TCR signaling up to the level of pMEK and (v) self-peptides elicit a unique transcriptional profile. Together, these results not only define the unique contributions of self-peptides to T cell activation but also demonstrate a distinct wiring profile in the TCR-signaling network that limits self-peptide sensing at the ERK step.
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The Role of Granzyme B in Tumor Migration and MetastasisThe granule exocytosis pathway has classically been described as our most potent response to pathogens, cancers, as well as autoreactive disorders. This is executed by the action of perforin and a family of granule-associated serine proteases known as granzymes (Gzm). Many studies have shown that these proteases exert their effects by killing affected cells, creating an obvious correlation with cytotoxic T cells (CTLs) and natural killer cells (NK) greatly expressing GzmB. Studies have found that the tumor cells ability to create a suppressive environment is dependent on Gzm expression by regulatory T lymphocytes (Treg). Research has shown GzmB can target and cleave extracellular molecules such as fibronectin, vitronectin and laminin. Bird et al published data showing that GzmB aids in the transmigration of CTLs, however the ability of GzmB to aid in the migration of non-T cells has not been investigated. We hypothesize that GzmB secretion may cause extracellular matrix (ECM) remodeling in the tumor microenvironment, aiding in the outgrowth of the tumor cells via promoting higher rates of invasion and/or metastasis. First, we designed experiments to test in-vivo and in-vitro conditions that generate GzmB production in multiple cell types. We were able to induce GzmB production by Tregs both in-vitro and in-vivo. We also identified multiple mechanisms that allow for CD8 T cells to produce GzmB. We next investigated mechanisms that would allow for GzmB to be found in the extracellular environment. We found that CD8 T cells have the ability to secrete GzmB without the need of an immunological synapse. In fact, GzmB secretion is not dependent on exogenous IL-2. GzmB improves the migration and invasive potential of tumor cells. In Treg-specific GzmBko (Foxp3creGzmBfl/fl) mice, GzmB+ Tregs increase the rate of tumor metastasis in the lungs of the mice. In addition, GzmB+ CD8 T cells can potentiate tumor metastasis, in contrast to GzmB-deficient CD8 T cells. These findings shed light on the protumorigenic potential of secretory GzmB, which could serve as a unique biomarker for predicting metastasis.
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Using PacBio SMRT platform to assess the genetic variation of Plasmodium falciparum Circumsporozoite Protein (PfCSP) in Kalifabougou, MaliObjectives: In this study, we assessed the genetic variation in CSP sequences from Kalifabougou, Mali, by evaluating the diversity of haplotypes in the N- and C- Terminal region, the repeat regions, and the T- and B-cell epitopes. Methods: Sequences used for the study were generated from 59 sequences collected during a malaria transmission season conducted in Kalifabougou, Mali. Generated using the PacBio SMRT platform. Results: Results showed that the central repeat and C-terminal Th2R/Th3R epitope regions were highly polymorphic in this study, whereas the N-terminal non-repeat region was less variable. Furthermore findings highlighted CSP polymorphism and suggested that the repeat and B-cell epitope of our samples is similar to those of 7G8 PfCSP, whereas the C-terminal is similar to that of the 3D7 PfCSP strain. Finally, findings the CSP sequences from the Kalifabougou site are more similar to those from Navrongo than to those from Cape Coast.
