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Mechanical properties of rodent brains and neural cell response in brain aging and Alzheimer’s Disease pathogenesis
No full textMechanical properties of mammalian brains have been studied to understand brain aging and diagnose brain diseases, including Alzheimer's Disease. On cellular level, mechanical properties are involved in the mechanism of regulating cellular behaviors by cell-cell and cell-matrix interaction in neural cells. However, existing studies are limited by their focus solely on elasticity, the use of mechanical characterization techniques that are not accurate or suitable for viscoelastic living tissues, and a lack of comprehensive analysis of the relationship between mechanical properties and biological processes.
This dissertation examines the elasticity, viscoelasticity, and plasticity of rodent brains as well as neural cell responses in brain aging and the pathogenesis of Alzheimer’s Disease, using both bulk and localized mechanical tests. It also studies regulation of mechanical properties on neural cell functions related to aging and Alzheimer's Disease pathogenesis, including neural progenitor stem cell (NPSC) differentiation, and microglia and astrocyte recruitment.
After a brief introduction of the importance of mechanical properties of mammal brains on both tissue and cellular levels, and the limitations in the existing research, this dissertation discusses three main projects. Chapter 2 explores the impact of Alzheimer’s Disease on the elasticity of mouse brains. Stiffening of the entire brain, the isocortex region, and the CA1 region of the hippocampus was observed in Alzheimer's Disease mice at 6-month- and 12-month-old, but not at 1-month-old, which aligns with the progression of Alzheimer's Disease pathogenesis. This stiffening is associated with symptoms and pathological characteristics of Alzheimer's Disease, including decreased cognition, accumulation of amyloid β, and the recruitment of glial cells.
Chapter 3 documents change in brain plasticity and local plasticity in the hippocampus of aging mice, another critical mechanical property that has been scarcely studied before. Viscoelastic recovery and elastic extension were also studied.
Chapter 4 discusses how matrix viscoelasticity regulates the stemness maintenance and differentiation of neural progenitor stem cells, including differentiation into neurons, astrocytes, and oligodendrocytes. Mechanotransduction studies on integrin biding, actomyosin contractility, actin polymerization and Piezo1 ion channel were performed
Ribosomes as Macromolecular Crowding Sensors
No full textMacromolecular crowding is largely understudied, and yet, may play a key role in many biological processes. The intracellular environment is packed with macromolecules of many different shapes and sizes that function and interact to maintain homeostatic conditions despite the coordinated chaos. Extracellular stressors cause biophysical changes that trigger intracellular signaling events and thus change the environment within the cell. Different stressors stimulate unique MAP3Ks that promote combinatorial activation of stress-activated MAPK (SAPK) via the three-tier, phosphorylation activating cascade that ultimately leads to cell fate decisions such as apoptosis, senescence, or proliferation. Artificially inducing macromolecular crowding, by using hyperosmotic stress, leads to the activation of all three SAPKs – p38, JNK, and ERK5 – but the underlying molecular mechanisms and functional roles of this activation are still not well understood.
Several MAP3Ks are known to be involved in the mammalian osmotic stress response. ZAKα MAP3K, in particular, has previously been shown to scale the intensity of cell death by sensing translating ribosomal collisions. However, we show that translating ribosomal collisions may not be responsible for SAPK signaling during exposure to treatments that induce macromolecular crowding. We hypothesized that macromolecular crowding increases the local concentrations of non-translating ribosomes, stabilizing stable stress granule cores which leads to activation of ZAKα MAP3K. This generates an intrinsic negative feedback loop between mature biphasic stress granules to mitigate MAPK signaling. Using cutting-edge single cell and single molecule bioimaging technology, along with traditional biochemical techniques, we uncovered a novel mechanism for ZAKα MAP3K signaling, independently of translation, that promotes cell survival in breast epithelial cells
In 'X' they distrust: Exploring institutional distrust as a determinant of healthcare engagement among women who use drugs amid the COVID-19 pandemic
Get PDFIntroduction: Women who use drugs (WWUD) experience multiple barriers to engaging with health services, such as high levels of material insecurity, which were amplified by the COVID-19 pandemic. Additionally, past experiences of stigma and discrimination within service settings contribute to service avoidance among WWUD. While institutional distrust, including healthcare distrust and government distrust, are frequently cited barriers to service engagement for WWUD, this relationship has rarely been quantitatively assessed.
Methods: We recruited 16 WWUD in Baltimore, Maryland between April and June 2021 to participate in semi-structured interviews about their healthcare experiences. We additionally recruited 226 WWUD for a quantitative survey from August 2021 and December 2022. Factor analysis was used to identify distinct dimensions of healthcare distrust and government distrust among WWUD. Exploratory structural equation modeling was used to explore the relationship between institutional distrust and vaccine hesitancy. Poisson regression with robust variance was used to examine the effect of institutional distrust on uptake of the COVID-19 vaccine.
Results: Integration of quantitative and qualitative findings identified trust- and distrust-generating mechanisms of healthcare distrust along dimensions of competence, communication, predictability, fairness, and fidelity. Confirmatory factor analysis identified dissatisfaction and disaffection as distinct latent factors underlying government distrust among WWUD. Exploratory structural equation modelling revealed significant correlations between vaccine hesitancy, healthcare trust and distrust, and government dissatisfaction. However, government disaffection was only correlated with healthcare distrust. In adjusted Poisson regression, only government disaffection (aIRR: 0.87, p<0.05) and vaccine hesitancy (aIRR: 0.75, p<0.01) were significantly associated with uptake of the COVID-19 vaccine.
Conclusions: The COVID-19 pandemic provided a unique context in which to examine institutional distrust as a determinant of healthcare engagement among WWUD. Institutional distrust among WWUD was shown to be influenced through experiences of material insecurity, access barriers, and stigma. Our findings highlight the multidimensional nature of institutional distrust, revealing dissatisfaction and cynicism as distinct aspects of distrust contributing to service disengagement and avoidance. Healthcare institutions and government agencies seeking to build trust with WWUD should consider removing barriers to access, improving coordination of services, and eliminating policies and practices that exclude, degrade, and detain on the basis of drug use
Cellular, molecular, and physiological organization of the Drosophila foregut
No full textThe animal foregut is the first tissue to encounter ingested food, bacteria, and viruses. Efficiently digesting and absorbing nutrients from this intake, while retaining beneficial microorganisms and rejecting pathogenic ones represents a critical task for this gatekeeper tissue. To delineate these intricate physiological processes, the adult Drosophila foregut was characterized as a model for dissecting how it triages consumed items for digestion or immune response. Major foregut cell types were identified, validated, and examined using single-cell RNAseq and >150 in vivo gene reporter fly lines. Transcriptome analysis revealed potential mechanisms that the foregut-associated neuroendocrine cells, including those found within the corpus cardiacum and corpus allatum, use to coordinate gut activity with metabolism, the microbiome, and circadian cycles. Multiple intestinal cell types differentially express juvenile hormone binding proteins that likely allow them to respond to changes in juvenile hormone produced by the corpus allatum. In addition to juvenile hormone biosynthetic enzymes, the corpus allatum expresses circadian clock genes. Studying all the cell types of the proventriculus, the central foregut organ that secretes the peritrophic matrix lining the intestinal tract, provided a detailed molecular view of the layered structure and dynamic character of the peritrophic matrix. Analyzing cell types synthesizing individual peritrophic matrix layers revealed abundant mucin production close to enterocytes, the same location as in mammalian intestinal mucus. Two previously unrecognized cell groups in the posterior proventriculus were found to produce abundant digestive enzymes likely secreted into the lumen of the peritrophic matrix to join ingested food for digestion. A commensal niche exists within the adult esophageal tissue that selectively binds microbiome species including Lactiplantibacillus and Acetobacter strains with strain-level specificity, stabilizing their colonization. The esophagus and salivary glands express secreted proteins, some of which may line the esophageal surface for the foregut commensal niche. Overall, the present study strongly supports the foregut's role as a central coordinator of intestinal activities, immune defense, and microbiome interactions by virtue of its location near the brain, sensory organs, and esophagus. The results also suggest that the cells, genes, and functions of the foregut have been significantly conserved throughout animal evolution
Pathogenesis of Human iPSC-Derived Spinal Motor and Sensory Axons
No full textOf the many neurological disorders in which distal axon degeneration is an early and prominent pathological sign, we are particularly interested in amyotrophic lateral sclerosis (ALS) and diabetic peripheral neuropathy (DPN). Early in disease progression, the distal axon degenerates from its target tissue and exhibits dying back axonopathy. The loss of connection between the nervous system and peripheral organs leads to the downstream symptoms seen in both diseases. To study this axonal pathogenesis, we cultured human induced pluripotent stem cell (iPSC)-derived neurons in a spatially separated microfluidic system to isolate axons from the neuronal cell bodies.
However, given early disease pathology, axonal mRNA specifically may contribute to disease. Using our microfluidic system, we extracted pure, high quality axonal RNA along with the corresponding somal RNA from SOD1+/A4V and SOD1+/+ iPSC-derived spinal motor neurons. We found differential expression of thousands of genes in SOD1+/A4V axons compared to SOD1+/+, while only about 250 genes were found when comparing bulk cell body RNA in each line. Many of the differentially expressed genes in axons are involved in processes or pathways known to be disrupted in ALS, but they were not found when comparing total cell body RNA, highlighting the importance of focusing on axonal pathogenesis in this disease.
It is not yet well understood why, in addition to initial axon degeneration, DPN patients have a diminished capacity for axonal regeneration within skin compared to healthy controls. We have developed a human-derived co-culture system to elucidate the relationship between the diabetic skin environment and sensory axon degeneration in DPN. Human sensory neurons were differentiated using an optimized mRNA-based protocol, generating mature sensory neurons within two weeks. Skin tissue slices were taken from patient biopsies and added to the axonal device compartment where they survived structurally intact. Axons could be visualized reaching the dermal and epidermal layers, a quantifiable way to measure axonal growth in DPN vs control skin environments. Isolating the sensory axon-skin interaction and comparing DPN patient vs control cell lines and tissue will allow us to ascertain what factors lead to the degeneration and reduced plasticity in DPN patients
DEVELOPMENT OF BIOMATERIALS SYSTEMS TO MODULATE CRISPR-CAS9 GENE EDITING
No full textCRISPR-Cas9 gene editing has become one of the fastest growing fields in scientific research. Its introduction has renewed hope for numerous genetic conditions once thought to be incurable. With a rapid transition towards translational applications, it has become evident that efficient delivery of the Cas9 cargo and significant gene editing efficiencies are critical requirements for potential therapies. Currently, many studies focus on either modifying Cas9 or developing new delivery systems. This thesis aims to take a more holistic approach by establishing a relationship between mechanobiology and CRISPR-Cas9 gene editing. Specifically, substrate rigidity provided by hydrogels for 2D cell culture, and their effects on gene editing efficiency were investigated. This principle is explored across multiple hydrogel systems, transfection methods, forms of Cas9, target genes, and cell lines. Both small molecule inhibitor and siRNA studies are performed so that the key mediators of mechanotransduction pathways such as cytoskeletal and nucleoskeletal components can be evaluated to discover their involvement in this phenomenon. Mechanistic studies were performed to better understand how 2D hydrogel culture improves Cas9 editing relative to conventional TCPS. Finally, hydrogel-based gene editing practices were applied in a more translational setting with adoptive cell therapies. Overall, the results demonstrate the feasibility of mechanical cues such as substrate stiffness to supplement established methods for gene editing while also establishing the use of biophysical cues as a tool for enhancing gene and cell therapies
ELUCIDATING MECHANISMS OF NERVE INJURY-INDUCED NEUROPATHIC PAIN
No full textNeuropathic pain is pain stemming from a damaged or diseased somatosensory nervous system. Clinically, treatments for neuropathic pain are often ineffective and thus, it is important to develop a better understanding of the underlying mechanisms of neuropathic pain. In this dissertation, we tackled two aspects of nerve injury-induced neuropathic pain development. First, utilizing genetic Merkel cell knockout (KO) mice, we investigated the requirement of Merkel cells for the development of mechanical hypersensitivity in the spared nerve injury (SNI) model. We found that mechanical allodynia was attenuated in male, but not in female, mice lacking Merkel cells after SNI, suggesting that intact Merkel cell-Aβ afferent complexes are necessary for the full extent of nerve injury-induced mechanical hypersensitivity in a sex-dependent manner. Second, we examined the contribution of sterile alpha and TIR motif containing 1 (SARM1) to nerve injury-induced neuropathic pain in three different surgical models: chronic constriction injury (CCI), SNI, and spinal nerve ligation (SNL). Using a recently published CRISPR-generated Sarm1 KO mouse line, we found that SARM1 was required for heat hyperalgesia but not mechanical or cold allodynia after CCI. In contrast, Sarm1 KO mice exhibited no deficits in the development of mechanical or heat hypersensitivity after either SNI or SNL. In addition, mice lacking SARM1 displayed a reduced increase in spontaneous pain behaviors after SNI but not after CCI. Importantly, Sarm1 KO mice showed less heat-evoked neuronal activity after CCI in both the dorsal root ganglia and in the spinal cord dorsal horn compared to wildtype controls, suggesting a SARM1-dependent physiological mechanism arising from the periphery that may contribute to the pain phenotype after nerve injury. In summary, our results bring forth new insights on the underlying mechanisms of nerve injury-induced neuropathic pain and more broadly, advances our knowledge in the field of pain
Administrative Discretion and Civil Servants’ Obligations: Accounting for Ethical Considerations in Policymaking
No full textCivil servants are granted circumscribed authority to implement legislation and their decisions can have important and wide-ranging effects on society. This project examines administrative discretion, which is the exercise of decision-making authority by civil servants, in the context of policymaking with the goal of understanding whether there are non-legal standards for judging the use of administrative discretion in specific instances of policymaking. I propose that a high-quality policymaking process should be informed by a broad range of perspectives and, for policy issues with salient ethical features, include ethical expertise as a policy-relevant domain of knowledge. This standard is applied to a case study, comparing human subjects research policy and animal research policy, which are two policy areas with obvious ethical dimensions but for which ethical expertise has been utilized quite differently for policy development. Based on this analysis, I argue that animal research policymakers have performed poorly in this aspect of their role because they have failed to engage with ethicists and ethical scholarship during policy deliberations and therefore failed to sufficiently consider relevant ethical features of policy during the policymaking process
New Methods for Robust and Reliability-based Topology Optimization: Simulation and Surrogate Approaches
No full textTopology optimization (TO) is a systematic, computational design tool capable of obtaining optimized distributions of material within a design domain, often producing new solutions that outperform those found using traditional design methods. While advancements in TO have addressed a wide variety of applications, the majority of work performed has assumed a deterministic environment and real-world underlying uncertainties have been largely neglected. Structures obtained from these deterministic TO techniques exhibit non-redundant features and can be susceptible to variations present in material properties and uncertainties occurring in the manufacturing process or during end-use conditions. As TO methods have advanced and interest from industry grown, the need for reliable and robust designs has elicited the development of algorithms capable of efficiently incorporating complex uncertainties into our existing frameworks. Traditional Monte Carlo simulations (MCS) become prohibitively expensive when considering sophisticated computational models, large random dimensions, or highly complex uncertainty structures, with cost further exacerbated by the iterative nature of TO. Other works have introduced techniques that alleviate the computational burden of uncertainty aware design, such as perturbation methods, spectral approaches, and approximations of the reliability. While these methods can drastically reduce the cost of uncertainty propagation, the savings often come at the cost of complex, challenging sensitivity calculations, reducing computational savings or affecting the stability of the optimization problem. Two well researched, general approaches exist that incorporate uncertainty in the design optimization process. One method, robust design optimization (RDO), seeks to reduce the magnitude of variability present in the performance of optimized designs by including statistical quantities (mean, variance, etc) within the objective and constraint functions. Reliability-based design optimization (RBDO) is an alternative approach in which the probability of failure is considered, resulting in designs resistant to failure. Robust topology optimization (RTO) and reliability-based topology optimization (RBTO) incorporate uncertainties into the topology optimization framework, allowing for the underlying optimization algorithm to account for these variations. This dissertation seeks to present general, computationally efficient, and non intrusive methodologies that inform RTO and RBTO frameworks of existing uncertainties. The work is divided into two chapters; the first focuses on robust designs obtained through a stochastic reduced order model and the latter targets a Polynomial Chaos Expansion approach coupled with an improved reliability constraint. For both the RTO and RBTO frameworks, a simulation-based approach is adopted, utilizing non-intrusive surrogates that relate samples of random variable inputs to the Quantities of Interest (QoI) referenced within the optimization problem. We first introduce the stochastic reduced order model (SROM) as a surrogate for the RTO problem due to its accurate estimation of low-order moments. We then shift focus to the RBTO formulation and, having identified the disadvantages of recent smooth Heaviside reliability constraints, propose an alternative version of the constraint with substantial improvements in optimization stability. Finally, we examine a recent multi fidelity approach utilizing a greedy Kaczmarz algorithm for efficiently obtaining a Polynomial Chaos Expansion (PCE), that is then successfully deployed as a surrogate within the RBTO framework. Numerical examples are presented throughout, emphasizing the effectiveness of the proposed methodologies while validating the accuracy of the propagated uncertainties. Several aspects of the approaches presented are discussed, with potential improvements and future adaptations highlighted in detail
OPTIMIZATION-BASED POWER SYSTEM PLANNING FOR LOW-CARBON ENERGY SYSTEM TRANSITIONS: IMPACTS OF OPERATIONAL DETAILS ON RESOURCE ADEQUACY ASSESSMENTS; MULTILEVEL GRID PLANNING UNDER HETEROGENEOUS REGIONAL CARBON POLICIES; AND LAND USE TRADEOFFS OF GRID EXPANSIONS
Get PDFThe changing landscape of the electricity system calls for enhancements in power system planning models to address emerging challenges alongside the low-carbon energy system transition. This dissertation aims to address three research questions in power system planning to support a reliable, clean, sustainable, and affordable electricity system. Chapter 1 starts with an overview of power system planning and introduces the challenges associated with resource adequacy assessments, heterogeneous subregional carbon policies, and land use tradeoffs for grid planning.
Chapter 2 proposes a new resource adequacy modeling framework to address the research question on how incorporating different operational details and assumptions in resource adequacy modeling impact system reliability assessments. The analysis explores the impacts of considering detailed operational representation, short-term information flow and uncertainty, and hybrid resource configurations on resource adequacy outcomes to support long-term reliability assessments.
Chapter 3 discusses a new proactive transmission planning work to analyze the question on whether proactive grid planning can mitigate carbon emissions at reasonable costs, especially given the existence of heterogeneous subregional carbon policies. The results for a western North America case study highlight the value of proactive transmission planning in reducing carbon emissions with asymmetrical subregional carbon policies. The analysis also demonstrates how transmission planning can proactively support decarbonization to achieve a clean grid.
Chapter 4 examines the coupling between the electricity system and the physical environment and addresses the question on how different resource siting and transmission expansion availabilities impact land use related tradeoffs under different clean energy policies and load growth futures in western North America. The planning model endogenously considers resource siting availabilities and explores the tradeoffs among system costs, land use, and emissions under these different assumptions into a clean and sustainable future.
Chapter 2, 3, and 4 each proposes enhancements to power system planning related models and provides analysis to facilitate better understanding to achieve different planning goals during this low-carbon energy system transition. Finally, Chapter 5 concludes this dissertation by presenting the limitations and future research opportunities in these topic areas