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EXPLORING P2X7 RECEPTOR ANTAGONISM AS A POTENTIAL THERAPEUTIC INTERVENTION IN ALS USING A HUMAN INDUCED PLURIPOTENT STEM CELL MODEL
Get PDFExtracellular adenosine triphosphate (ATP) binds to metabotropic and ionotropic purine receptors (P2) on the surface of cells, triggering a variety of intracellular signaling cascades involved in both physiological and pathophysiological processes. Included in the family of ionotropic P2 receptors is P2X receptor 7 (P2X7R), a non-specific cation channel with unique functional and structural properties that suggest it has distinct roles in pathological conditions marked by increased extracellular ATP. Previous work has demonstrated that human iPSC-derived astrocytes (hiPSC-A) generated from patients with amyotrophic lateral sclerosis (ALS) release more ATP into the extracellular milieu through connexin 43 (Cx43) hemichannels compared to healthy controls, suggesting that ALS astrocytes may contribute to increased extracellular ATP through pathological changes in Cx43. This work hypothesizes that increased extracellular ATP may be neurotoxic to motor neurons through the activation of P2X7R on their surface, positioning extracellular ATP as a potential toxic factor in astrocyte-mediated motor neuron death in ALS.
The role of P2X7R has previously been explored in microglia and astrocytes within the context of neuroinflammation, however the presence of P2X7R on human motor neurons has not been the focus of the current literature. This work leverages the use of human iPSC-derived spinal motor neurons (hiPSC-MN) as well as human and rodent tissue to demonstrate the expression of P2X7R on motor neurons. Further extending this observation, this work demonstrates that these receptors are functionally active on hiPSC-MN and that ATP can directly induce motor neuron death as a result of P2X7R activation, in a dose-dependent manner. Finally, using a highly specific P2X7R blocker, this work demonstrates how modulation of P2X7R activation on motor neurons is neuroprotective and could provide a unique pharmacologic target for ATP-induced motor neuron death
DEVELOPMENT OF A BIOSTIMULATORY NANOFIBER-HYDROGEL COMPOSITE FOR STEM CELL DELIVERY TOWARD SOFT TISSUE REMODELING
No full textSoft tissues, including muscle, fat, blood vessels, nerves, and tendons, play crucial roles in supporting and protecting various body organs and tissues. The significant loss of soft tissue due to factors such as tumor resection, trauma, aging, congenital malformations, and chronic diseases like diabetes and inflammatory bowel disease, poses substantial clinical challenges. Current soft tissue restoration techniques, both autologous and prosthetic-based, are hindered by issues including donor site defects, unpredictable tissue survival, unsatisfactory restoration duration and volume, and complications related to prosthetic use. This underscores the urgent need for an off-the-shelf solution that not only immediately restores lost soft tissue volume but also promotes natural tissue remodeling over time, thereby easing the challenges faced in soft tissue transplantation and chronic wound closure.
In this thesis, a novel nanofiber-hydrogel composite (NHC) is developed, combining polyester nanofibers (poly (ε-caprolactone), PCL) and hyaluronic acid (HA) hydrogel components. This chemically defined, biostimulatory NHC is designed to facilitate host cell infiltration, create a pro-regenerative microenvironment, and progressively remodel the implanted construct into vascularized soft tissue. Overarching objective of this work is to optimize biostimulatory activity of the NHC co-delivering with allogeneic adipose-derived stem cells, delineating the material properties and cellular responses essential for effective soft tissue restoration in established translational models.
This thesis is structured as follows:
Chapter One introduces the major aims of this thesis and offers background information on utilizing hydrogels for soft tissue reconstruction.
Chapter Two details the synthesis of a biodegradable NHC, employing glycidyl acrylate modified hyaluronic acid hydrogel and maleimide functionalized PCL nanofiber, ensuring tunable degradation and tailored in-vivo response.
Chapter Three presents the conversion of NHC bulk gel into microparticles for enhanced stem cell (adipose-derived stem cells, ADSC) delivery and soft tissue remodeling.
Chapter Four discusses the development of microRNA-laden lipid nanoparticles within the NHC microgel for augmented soft tissue remodeling.
Chapter Five applies the stem cell co-delivered NHC microgel in a chronic inflammatory wound repair model in a rat Crohn’s disease model.
Chapter Six outlines the isolation of exosomes from ADSC and their integration with NHC microgel, demonstrating an improved healing ratio in treating Crohn’s disease perianal fistula model in rats.
Chapter Seven proposes future development, focusing on selection and optimization of stem cell type and advancements in NHC composition for enhanced soft tissue engineering, and a scaffold design incorporating stem cell-derived exosomes in fistula model repair with nanofiber and hydrogel.
This comprehensive study provides a robust foundation for the advancement of soft tissue restoration, offering innovative insights and methodologies for enhancing soft tissue regeneration and remodeling
CANCER ASSOCIATED FIBROBLASTS REGULATE EPITHELIAL AND T CELL STATES IN PANCREATIC DUCTAL ADENOCARCINOMA
No full textPancreatic ductal adenocarcinoma (PDAC) presents a formidable challenge with grim prognosis and limited treatment options, worsened by late-stage diagnoses. The tumor microenvironment (TME) is central to PDAC progression and therapy resistance, with cancer-associated fibroblasts (CAFs) crucial in shaping this landscape. This thesis explores CAF-tumor interactions in PDAC, revealing CAF impact on tumor and immune cells. Dynamic CAF-driven signaling pathways create an immunosuppressive milieu, emphasizing the need for innovative therapies.
Utilizing a convergence approach, we created a single cell ATLAS resulting in discovery and validation of novel CAF-tumor interactions. We identified VEGFa-NRP1 axis that mediates CAF-epithelial cell crosstalk and is associated with inflammation and epithelial-mesenchymal transition (EMT). This work expands our understanding of CAF-driven EMT and inflammation regulation and introduces transfer learning for single-cell data analysis from patient tissues and patient derived organoid cocultures.
We built on this discovery by employing a CAF targeted CAR-T cell approach, targeting CAFs and tumor cells simultaneously. We investigated a novel mesothelin CAR-T that, when bound, secretes a bicistronic T cell engager molecule targeting FAP and CD3, directing the CAR-T to targeted and specific killing of FAP+ CAFs and mesothelin+ tumor cells. mesoFAP CAR-TEAM cells demonstrate superior efficacy in modifying stroma, sustaining CAR-T cell activation and function, and eliminating both the primary tumor and metastases.
We enhance this further by investigating how CAFs shape the TME through crosstalk. We leveraged high dimensional approaches to discover distinct basal and classical tumor cell neighborhoods and show associations with specific CAF populations by Imaging Mass Cytometry. We expand on this finding by creating a novel in vitro platform to explore complex interactions between these cells by coculture and RNA sequencing. We show CAFs drive a classical-to-basal gene transition in tumor cells through mechanisms of both cell-cell contact and secreted factors.
Collectively, our studies highlight CAFs' impact on the TME and tumor cell function, the role of spatial distribution and cellular crosstalk in driving tumor cell phenotype. Specifically, our results suggest CAFs drive a worse tumor type and critically regulate the TME. This work provides justification for further defining and identifying approaches to reprogram the CAF compartment
MICROMECHANICAL MODELING OF DEFORMATION AND FRACTURE IN POLYCRYSTALLINE MICROSTRUCTURES
No full textThe mechanical behavior of metallic alloys at structural scales is significantly influenced by their underlying microstructures, characterized by grains with varying crystallographic orientations and sizes. This work employs a rate- and size-dependent crystal plasticity finite element (CPFE) model as the foundational micromechanical tool for studying the deformation behavior of Titanium alloys. The crystal plasticity parameters for Ti-7Al are calibrated and validated against experimental data from cruciform dwell tests, incorporating Digital Image Correlation (DIC) surface strain measurements and in-situ measurements of individual grain-averaged lattice strains using far-field high-energy diffraction microscopy (ff-HEDM). To accurately predict microstructural lattice strain evolution, this thesis introduces a novel method for initializing the CPFE model with a distribution of equilibrated residual stresses in the initial microstructure.
Addressing the concerns of fatigue failure in the widely used Ti-6Al-4V alloy within the aerospace industry, this research endeavors to predict microstructurally short crack growth. For accurate prediction of microstructrally short crack growth in metallic microstructures, this thesis develops a thermodynamically consistent coupled crystal plasticity phase-field model enhanced with adaptive wavelet-based hierarchical finite element framework. This framework efficiently simulates crack propagation in polycrystalline microstructures, providing accurate representations of fracture processes at the micro-scale. To prevent contributions of compressive stresses in crack growth under cyclic loading conditions, a spectral decomposition of elastic stored energy is developed, applicable to anisotropic materials undergoing finite deformation. Microstructural sensitivity of short crack growth is studied using the model to develop an understanding of how crystallographic orientation of crystals affect crack growth rate and paths.
Expanding the application of the CPFE-PF framework, this study extends its scope to simulate deformation and crack growth in Nitinol (NiTi) polycrystalline microstructures. An important characteristic response of Nitinol is superelasticity a.k.a psuedoelasticity. The superelastic behavior of NiTi is attributed to a reversible solid-solid, diffusionless phase transformation under mechanical loading. A rate dependent crystal plasticity based constitutive model is implemented in this work for modeling stress induced phase transformation in NiTi. The CPFE-PF framework is deployed to study the crack growth rate in NiTi microstructure-based statistically equivalent representative volume element(M-SERVE) subjected to cyclic loading
Truth in the District: A Collection of Novel Chapters
Get PDFTruth in the District is a satirical novel, represented here in three selected chapters and an introduction, that explores Washington through the eyes of an outsider: a gay freshman representative who is loathed for his habit of telling the truth. Alexander Fox needs to learn what his politicians privately believe to understand why gun legislation is impossible. After witnessing an unarmed young Black man murdered by police, he runs for Congress and is now faced with the puzzle of discovering the truth behind the public personas of the TV talking heads. The novel follows Alexander as he unearths Capitol Hill’s skeletons to discover a bit of truth in the District of Columbia. As a representative, Alexander discovers more about his own life than he does about Congress, while questioning how Capitol Hill has devolved into an inept body barely capable of tying its own shoes
MULTIETHNIC PERSPECTIVES OF SHARED DECISION MAKING IN HYPERTENSION
Get PDFBackground: There is underrepresentation of racially and ethnically minoritized populations and limited evidence regarding the influence of patient race and ethnicity on shared decision making (SDM). SDM has been associated with improved patient outcomes and could improve cardiovascular care quality and reduce disparities. The aim of this study is to describe SDM in the hypertension management context in relation to blood pressure (BP) levels.
Methods and results: An explanatory sequential mixed methods design (quantitative followed by qualitative) was used. Quantitative data was sourced from participants (n=1212) in the RICH LIFE Project, a cluster randomized pragmatic trial comparing the effectiveness of health system only vs. health system plus a collaborative/stepped care intervention to reduce hypertension disparities; qualitative data was collected from semi-structured individual interviews with RICH LIFE participants (n=36) selected based on their SDM scores and systolic BP level at 12 months follow up. Multinomial logistic regression analysis showed that predictors of SDM scores and BP outcome were race and ethnicity (p=0.028), age (p=0.003), educational level (p=0.014), patient activation (p=0.043), and hypertension knowledge (p=0.049). Qualitative and mixed methods findings highlight facilitators, barriers and racial and ethnic factors related to SDM varied by participants’ SDM scores and BP outcomes. Communication was the factor most emphasized by participants as both a facilitator and a barrier to SDM. Other facilitators were patient’s understanding of hypertension; clinician’s interest in the patient and clinician’s
personality and attitudes; and barriers included perceived lack of compassion, relationship hierarchy, and time constraints.
Conclusions: Our study found that participants with different SDM scores and BP outcomes varied in determinants of SDM and BP outcome, and their description of contextual factors influencing SDM. Results are novel and expand evidence in the field through a comprehensive understanding of the issues related to SDM in hypertension from the perspective of participants from diverse backgrounds. Results provide actionable information that can be used to enhance policy development and clinical practice, establish effective clinician and patient training, and create decision aids
Toward targeting DXP synthase function in metabolic adaptation
Get PDFThe rising rate of antimicrobial resistance continues to threaten global public health. Further hastening antimicrobial resistance is the lack of new antibiotics against new targets. The bacterial enzyme, 1-deoxy-D-xylulose 5-phosphate synthase (DXPS), represents a new target for antibiotic development. DXPS functions at a critical metabolic branchpoint to produce the metabolite DXP, a precursor to pyridoxal-5-phosphate (PLP), thiamin diphosphate (ThDP) and isoprenoids presumed essential for metabolic adaptation in nutrient-limited host environments.
Chapter 2 investigates DXPS’s role in bacterial adaptations relevant to urinary tract infections (UTIs). Uropathogenic E. coli growth is inhibited when a DXPS inhibitor is combined with D-serine (D-Ser), a bacteriostatic host metabolite that is present at high concentrations in the urinary tract. UPEC adapt to D-Ser by producing a PLP-dependent deaminase, DsdA, that converts D-Ser to pyruvate, pointing to a role for DXPS-dependent PLP synthesis in this adaptation. Using a DXPS-selective probe, butyl acetylphosphonate (BAP), and leveraging the toxic effects of D-Ser, we reveal a link between DXPS activity and D-Ser catabolism. We find that UPEC are sensitized to D-Ser and produce sustained higher levels of DsdA to catabolize D-Ser in the presence of BAP. This BAP-dependent sensitivity to D-Ser marks a metabolic vulnerability that can be exploited to design combination therapies. We show that combining inhibitors of DXPS and CoA biosynthesis displays synergy against UPEC grown in urine.
In Chapter 3, the efficacy of alkylAPs (alkyl acetylphosphonates) in an in vivo model of UTI is investigated. AlkylAPs exhibit an excellent safety and pharmacokinetic (PK) profile in mice for UTI with no acute toxicities observed and significant accumulation found in the urine. The alkylAPs also demonstrate a modest protective effect in lowering UTI bacterial burden in mice, with a prodrug of homopropargyl acetylphosphonate (hpAP), a structural homolog of BAP, able to protect mice at a dose 10-times lower than BAP.
Lastly, Chapter 4 investigates the broader impacts of DXPS inhibition in UPEC through metabolomic studies. The metabolomic results are consistent with our hypothesis that DXPS plays a role in bacterial adaptation to UTI as several metabolites known to be critical for urinary tract colonization are at lower concentrations upon BAP-treatment. Cellular levels of TCA cycle intermediates, nucleotide precursors, and polyamines were all found to be significantly impacted by BAP treatment. These results also reveal intriguing avenues to investigate for future studies.
Overall, this work demonstrates that DXPS plays a role in bacterial adaptation in the urinary tract and is a promising target for antibiotic development
BUILDING SUSTAINABLE AND RESILIENT FOOD SYSTEMS: CHALLENGES AND OPPORTUNITIES FOR LOCAL GOVERNMENTS
No full textBackground: Food systems are a major contributor to anthropogenic climate change and are significantly affected by climate-related disruptions. Recognizing this, some local governments in the United States (US) have taken actions to reduce food system greenhouse gas (GHG) emissions or begun preparing for future disruptions. The outcomes of these initiatives are not well understood. This dissertation works with six local governments in the US to evaluate the effectiveness of local government strategies aimed at building healthy, sustainable, and resilient food systems.
Methods: Chapter one provides a rationale for this dissertation and an overview of the local government food system sustainability and resilience literature. Chapter two presents a policy evaluation of the nutrition and climate change co-benefits and tradeoffs of the New York City (NYC) Food Standards for Meals and Snacks Purchased and Served (Food Standards). Chapter three explores the sources and opportunities for GHG emissions mitigation for the largest food-serving agency in NYC, the Department of Education, through a lifecycle assessment of high school entrées. Chapter four explores resilience. Using survey and interview data, this chapter investigates how key attributes were linked to local governments' COVID-19 food responses and ways to strengthen these for future disruptions.
Results: Our evaluation of the NYC Food Standards showed a significant reduction in GHG emissions and stable or slightly improving nutrition indicators (e.g., increased fiber), except for a shift towards more ultra-processed foods. Our lifecycle assessment indicated that post-farmgate GHG emissions are important to consider when serving vegetarian food items and that some but not all post-farmgate policy interventions will reduce emissions. Outside NYC, we found that preparedness, diversity, redundancy, flexibility, capital reserves, and equity were crucial factors impacting cities’ COVID-19 food responses, and specific actions could be taken to foster these attributes.
Conclusions: The findings underscore the vital role of local governments in mitigating food system GHG emissions and enhancing resilience. They emphasize the need for careful evaluation to ensure that policy actions promote co-benefits for human health and equity while mitigating unintended consequences. This research contributes valuable insights to guide future efforts in building sustainable and resilient food systems
THE IMPACT OF 3D EXTRACELLULAR MATRIX VISCOELASTICITY ON NEURAL PROGENITOR STEM CELL FATE, REACTIVE ASTROCYTE RESPONSE, AND MICROGLIA ACTIVATION
No full textBrain tissue is soft and has fast stress-relaxing viscoelastic properties. Development of mechanical characterization techniques in the field further points towards the idea that brain viscoelasticity is a dynamic property that changes in the context of pathology and aging. However, we do not fully understand the effect of brain viscoelasticity in the cellular and biomolecular levels. Brain is constituted of multiple types of cells like neurons, astrocytes, microglia, oligodendrocyte, neural progenitor cells, and etc., and they are all known to have different functionalities in the brain tissue. To start understanding the effects of brain viscoelasticity with such complex cellular heterogeneity, this dissertation focused on studying three different types of neural cells; neural progenitor/stem cell, astrocyte, and microglia. Tunable viscoelastic hydrogel system is used to study these neural cells on the effects of 3D viscoelastic extracellular environment. In brief this dissertation will show; 1) matrix stress-relaxation regulates neural progenitor-stem cell stemness and differentiation, 2) extracellular matrix stress-relaxation modulates reactive astrocyte phenotypes, 3) brain tissue stiffness increased with Alzheimer disease model and 3D matrix stiffness alters microglia activation