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Immune Modulation At The Maternal-Fetal Interface Regulates Perinatal Outcomes
Get PDFThe decidua and placenta are immune organs, as most of their functions are mediated by immune cells: implantation, vascular remodeling, and maternal-fetal tolerance. However, how their resident immune cells accomplish each element and promote the growth of a semi-allogeneic fetus is unknown. Research in reproductive immunology has largely focused on the role of systemic maternal T cells in maintaining maternal-fetal tolerance or descriptive studies of the maternal-fetal interface at a single time point in gestation. Furthermore, how these immune cells may contribute to diseases of pregnancy is less understood. The goal of this study is to understand the changes that occur over gestation in the immune populations of the decidua and placenta and how they affect pregnancy outcomes. In humans, these organs are only available for research at two time points: early in gestation after a spontaneous or elective abortion, or postpartum after the massive physiologic change of parturition. As such, we utilize three mouse models of common pregnancy outcomes: (1) parturition, (2) intrauterine fetal demise (IUFD), and (3) fetal neuronal injury. In each model, extensive flow cytometry was completed to characterize alterations in immune composition and function both systemically and at the maternal-fetal interface, in addition to other experimental tools. We find that term parturition involves a loss of immune regulation, and conversely that preterm parturition recruits inflammatory populations. In a model of IUFD, we utilize known information about maternal systemic regulatory T cells (Tregs) to rescue this adverse outcome and then investigate how systemic changes impact the local immune composition using single-cell RNA-sequencing. Finally, using a model of fetal brain injury, we identify a plausible mechanism by which inflammation progresses through tissue compartments at the maternal-fetal interface, ultimately causing pathologic IFN production in the fetal brain. Together these findings demonstrate the diverse roles of the immune populations in the decidua and placenta, and their impact on perinatal outcomes
Development Of Nanoparticle-Based Contrast Agents For Applications With Conventional And Photon-Counting Ct Imaging
Get PDFX-ray computed tomography (CT) is one of the most widely used clinical imaging modalities, and recent developments in CT detectors and reconstruction methods are fueling its rapid innovation and expansion of its diagnostic values. However, despite the advancement in nanotechnology and nanoparticle-based contrast agents, small molecule-based iodinated contrast agents with several drawbacks, such as short blood half-lives, low CT contrast generation at high tube potential, and potential adverse effects, remain to be the only FDA approved CT contrast agents for intravascular administration. Development of novel nanoparticle-based CT contrast agents will not only resolve these drawbacks, but also facilitate the emergence of new CT technologies, such as photon-counting CT (SPCCT), and novel CT imaging applications. In this thesis, we present the development of nanoparticles that are specifically designed for numerous CT and SPCCT imaging applications. Sub-5 nm tantalum oxide nanoparticles (TaONP) were developed after investigating CT contrast generation properties and material differentiation of several candidate elements for SPCCT-specific contrast agent. To prolong blood circulation time and improve CT contrast production, TaONP were encapsulated in polymeric nanoparticles along with other sub-5 nm nanoparticles made of cerium (CeONP) and gold (AuNP). These polymeric nanoparticles produced consistently high CT attenuation across multiple clinical settings and were efficiently degraded into small nanoparticles within 7 days in biological fluids. Our CT contrast generation results also revealed that CeONP were able to produce higher CT contrast when compared to other experimental contrast agents especially at low tube potentials. Understanding that CeONP can also have immunomodulatory properties, we further investigated them as both CT contrast agents and therapeutic agents for targeted imaging and treatment of inflammatory diseases, expanding the potential CT imaging applications. Moreover, 85 % of the injected dose were excreted within 24 hours of intravenous injection, indicating CeONP’s feasibility of clinical translation. To further expand the CT imaging applications to cell tracking, sub-5 nm AuNP were encompassed in lipid-based transfection reagents to label chimeric antigen receptor (CAR)-T cells to monitor their behavior in cancer immunotherapy against solid tumors. This work highlights key considerations in the development of CT and SPCCT-specific contrast agents and the potential use of nanoparticle-based contrast agents for broadening CT and SPCCT imaging applications
Flying Modular Robots: From Self-Assembling Structures In Midair To Embedding Grasping Capabilities
Get PDFFlying modular robots offer a suitable autonomous platform for multiple applications such as: search and rescue, cargo lifting, and object transportation. In addition, modular robots in a swarm can use their own bodies as building units to assemble large structures. This thesis introduces ModQuad, the self-assembly structure that can cooperatively fly based on autonomous modules. With these modules it is possible to assemble structures with rigid connections in multiple configurations. In contrast to related work, instead of assembling on the ground or on water, a midair approach to assemble structures is proposed. Docking modules in midair offer relevant advantages by the cost of the complexity to adapt the conglomerate controller at each docking step. Assembling structures in midair usually requires a relative localization system in between modules. A vision-based self-assembly method is proposed with structures composed of two modules. Scaling these flying modular robotic structures is a challenging problem usually limiting the benefits of modularity. A novel yaw actuation for quadrotor-based modules using individuals rolling angles by a one degree of freedom cage design is proposed for a more effective controllability around the structure z-axis. The resulting implementation enlarges the configuration space in a line configuration. Expanding the one degree of freedom cage design to arbitrary configurations allowed the development of algorithms capable of generating optimal and near-optimal configurations through a computational efficient search that first classifies and groups modules within the structure. Lastly, structures composed of non-rigid connections are explored leading the whole conglomerate to change its shape dynamically in-flight. Instead of adding extra components and mechanisms, versatility of modularity is utilized to embed grasping capabilities through a shape change addressed to a four-bar linkage configuration
Elucidating The Pathological Mechanism Of Histone H3.3 Mutations In Neurodevelopment
Get PDFIn eukaryotic cells, octamers of histone proteins intricately organize DNA, forming a macromolecule known as nucleosomes. A nucleosome is comprised of two copies of histone H2A, H2B, H4, and H3. Histone H3.3 (H3.3), a histone variant, is often found at actively transcribed loci. H3.3 plays a role in cellular inheritance as ablation of H3.3 expression leads to loss of active gene states and dysfunction of heterochromatin telomeric structures. H3F3A and H3F3B, the two genes are known to encode H3.3, are expressed in all human cells with higher expression in the gonads and brain. A recent publication detailed H3.3 as the causative gene in a neurodevelopmental disorder with craniofacial abnormalities. The underlying cause of the disease is unknown; however, H3.3 is involved in a wide range of central nervous system functions. This dissertation highlights work that aimed to profile the effects of these germline mutations. We performed a comprehensive screen on pathogenic H3.3 variants utilizing a combination of techniques and methodology, including tissue culture, bottom-up proteomics, and neurodevelopmental assays in Xenopus laevis. We found global alterations to histone post-translational modifications with significant alterations to histone acetylation on histone H2a, H3, and H4 peptides. In addition, performing quantitative proteomics in the 293T cells led us to determine that these mutations affect several cellular processes, such as RNA splicing, cell motility, neurofilament maintenance, folic acid metabolism, and post-synaptic density, all processes known to be dysregulated in other neurological syndromes. Utilizing the model organism Xenopus, we introduced two of the pathogenic variants into embryos. We observed reduced craniofacial cartilage, abnormal head shape, and impaired motility due to kinked tails in the mutant tadpoles. We performed quantitative proteomics on these tadpoles and found pathways related to carbon metabolism and amino acid degradation upregulated. Lastly, our transcriptomic analyses corroborated some of these finds and an upregulation of the TGF-beta signaling pathway in one of the mutants. This work provides the first mechanistic study of these germline H3.3 mutations, introduces pathways dysregulated that can be further studied. Understanding the basic biology of these mutations will shed light on the molecular mechanisms of H3.3 in neurodevelopment
The Role Of Interleukin-4 Receptor Signaling In Neonatal Adipocyte Precursors
Get PDFAdipose tissue has multiple functions including defense against the cold through thermogenesis of brown and beige adipocytes. The type 2 immune cytokine Interleukin-4 (IL-4) can increase beige adipogenesis in mature rodents. However, developing animals use a distinct adipocyte precursor compartment for adipogenesis compared to adults. In this body of work, we analyze the effects of IL-4 on the adipose tissue of developing rodents. In rats, we found that exogenous IL-4 induced an acute increase in beige adipogenesis and a persistent decrease in overall adipogenesis in neonatal inguinal white adipose tissue (iWAT). We determined that DPP4+ progenitors and ICAM1+ preadipocytes were the populations expressing IL-4 receptor in neonatal mice. Isolating neonatal ICAM1+ preadipocytes for RNA-seq, we discovered no significant differentially regulated genes in IL-4RKO compared to WT. After adipogenic differentiation of IL-4RKO and WT ICAM1+ preadipocytes, we also found no changes in adipocyte gene expression. However, using neonatal WT ICAM1+ preadipocytes, we show that IL-4 pretreatment increases Ucp1 expression. We also identified that IL-4 directly interferes with the process of adipogenesis, decreasing adipocyte maturation. Overall, these findings describe the role of IL-4 in developing iWAT, identify the precursor population altered by IL-4 receptor signaling and characterize long term effects of IL-4 that may influence the later onset of obesity
Disentangling T Cell Heterogeneity In Human Ovarian Cancer: A Focus On Tumor Specific And Early Memory T Cells.
No full textDespite first-line therapy, ovarian cancer (OC) is the 5th leading cause of cancer death in women and is the most lethal gynecological cancer, underscoring the need for improved forms of therapy. Immunotherapies exhibit profound improvements for some cancers; however, efficacy is unimpressive for OC. Both tumor-specific and memory T cells are crucial for efficacious immunotherapies and interrogating their biology in human OC may improve immunotherapeutic strategies for ovarian cancer.Identifying tumor-specific T cells in ovarian tumors is challenging and it is unclear which biomarker (CD137, CD39, CD103, and PD-1) is the most selective in identifying tumor-specific TILs. Mass cytometry analysis found that CD137+ TILs expressed the greatest levels of effector molecules, compared to PD-1+, CD103+ or CD39+ TILs. Removal of CD137+ TILs from PD-1+, CD103+, or CD39+ TILs results in lower secretion of IFNg in response to autologous tumor stimulation, while CD137+ TILs highly secrete IFNg in an HLA-dependent manner. CD137+ TILs exhibited an exhausted phenotype and co-expressed CD28, indicating receptiveness to reinvigoration via immune checkpoint blockade. Overall, our results show that the antitumor abilities of PD-1+, CD103+, and CD39+ TILs are mainly derived from CD137 expressing TILs, supporting that CD137 is a more selective biomarker for tumor-specific TILs.
Furthermore, despite evidence underscoring the importance of stem-cell memory (Tscm) T cells, it is uncertain whether canonical stem cell memory T cells exist in human OC and whether they exhibit antitumor features. Our findings demonstrated that human OC harbors stem cell memory (Tscm) tumor-infiltrating lymphocytes (TILs) and a novel CD45RO expressing Tscm TIL subset. The CD45RO+ Tcsm T cell subset is hierarchically positioned in between CD45RO- (canonical) Tscm T cells and central memory T cells. Notably, the CD45RO+ Tscm T cell subset exhibits a profile indicative of tumor-specificity and effector capabilities, and shares similarities in gene expression and phenotype to T cell subsets in the literature that mediate successful immunotherapy treatment. Taken together, these findings lay the foundation to improve immunotherapy treatments for ovarian cancer patients
Structural Biology Of Oligomeric Assembly-Line Terpene Synthases
No full textTerpene cyclase enzymes catalyze the most complex chemical reactions found in nature, forming products used as fragrances, biofuels and drugs. Most famously, taxadiene synthase and amorphadiene synthase catalyze principal chemical reactions in the biosynthesis of anti-cancer drug Taxol and anti-malarial artemisinin, respectively. Additionally: Fusicoccin A is a 5-8-5 tricyclic phytotoxin with anti-metastatic and axon regenerative properties; mangicdiene is a 5-5-6-5 tetracycle with antibiotic properties; ophiobolin F is a 25-carbon 5-8-5 tricycle with potential anti-tumor activities. The first committed steps of biosynthesis of these molecules are catalyzed by fusicoccadiene synthase (PaFS), mangicdiene synthase (FgMS), and ophiobolin F synthase (AcOS).Terpene cyclization is a major branch point in terpenoid biosynthetic pathway, following the condensation of 5-carbon substrates dimethylallyl diphosphate and isopentenyl diphosphate to form linear isoprenoid substrates. A small handful of fungal terpene synthase enzymes remarkably catalyze both prenyltransferase and cyclization reactions in independent catalytic domains of a single polypeptide chain. The elusive structural nature of these so-called bifunctional assembly-line terpene biosynthesis enzymes is described here. PaFS is the first assembly-line terpene synthase to be discovered; PaFS forms high-order oligomers and exhibits substrate channeling, in which the isoprenoid intermediate is transported to the cyclization domain without diffusion into bulk solution. While interactions between cyclase and prenyltransferase domains are transient, I use cryo-electron microscopy (cryo-EM) aided by combinatorial crosslinking studies to provide snapshots of condensed positions that occur at low abundance. I argue that these interactions, while wobbly, may be consequential to substrate channeling. I also consider orthologs of PaFS, including FgMS, and use cryo-EM and x-ray crystallography to study the extent to which properties of PaFS are conserved in assembly-line biosynthesis. I discuss ‘dynamic cluster channeling’ as a modified version of cluster channeling operative here, in which combined agglomeration of active sites and covalent tethering of consecutive domains enhances product flux at this metabolic branch point. These findings will inform the synthetic biological production of terpenoid-based drugs
Embedded Energy Landscapes in Soft Matter for Micro-Robotics and Reconfigurable Structures
No full textThe ability to manipulate microscale objects with precision to form complex structures is central to the field of micro-robotics and to the realization of reconfigurable systems. Understanding and exploiting the forces that dominate at the microscale in complex environments pose major challenges and open untapped opportunities. This is particularly the case for micro-particles in soft milieu like fluid interfaces or nematic liquid crystalline fluids, which deform or reorganize around dispersed colloids or near bounding surfaces. These energetically costly deformations can be designed as embedded energy landscapes, a form of physical intelligence, to dictate emergent colloidal interactions. The fluid nature of these soft milieu allows colloids to move to minimize the free energy and externally forced robotic structures to re-write the embedded energy landscapes in the domain. Such physically intelligent systems are of great interest at the intersection of materials science and micro-robotics. Micro-particles on fluid interfaces deform the interface shape, migrate, and assemble to minimize the capillary energy. In the first part of my thesis, I design and fabricate a magnetic micro-robot as a mobile curvature source to interact with passive colloids on the water/oil interface. An analytical expression that includes both capillary and hydrodynamic interactions is derived and captures the main feature of experimental observations. I further demonstrate multiple micro-robotic tasks including directed assembly, cargo carrying, desired release and cargo delivery on the interface. Micro-particles in confined nematic liquid crystals (NLCs) distort the nematic director field, generating interactions. These interactions depend strongly on the colloids shape and surface chemistry, geometric frustration of director field and behavior of dynamic topological defects. To probe far-from-equilibrium dynamics, I fabricate a magnetic disk with hybrid anchoring. Upon controlled rotation, the disk’s companion defect undergoes periodic rearrangement, executing a complex swim stroke that propels disk translation. I study this new swimming modality in both high and low Ericksen number regimes. At high rotation rates, the defect elongates significantly adjacent to the disk, generating broken symmetries that allow steering of the disk. This ability is exploited in path planning. Thereafter, I design a four-armed micro-robot as a mobile distortion source to promote passive colloids assembly at particular sites via emergent interactions in NLCs whose strengths are characterized and found to be several orders of magnitude larger than thermal energies. While the strength of theses interactions allows colloidal cargo to be carried with the micro-robot during translation, it poses challenges for cargo release. We find that rotation of this micro-robot generates a complex dynamic defect-sharing event with colloidal cargo that spurs cargo release. Thereafter, I demonstrate the ability to exploit NLC elastodynamics to construct reconfigurable colloidal structures in a micro-robotics platform. At the colloidal scale, rotation dynamics are easier to generate, and this motivated me to exploit the topological swimming modality of the micro-robot. Using programmable rotating fields to direct the micro-robot’s motion, I achieve fully autonomous cargo manipulations including approach, assembly, transport and release. The ability to dynamically manipulate micro-particles and their structures in soft matter systems with embedded energy landscapes, as demonstrated in this thesis, creates new possibilities for micro-robotics and reconfigurable systems
Imagining Un Futuro Digno: Indigenous Youth Striving For Non-Migration In Guatemala
No full textABSTRACTIMAGINING UN FUTURO DIGNO: INDIGENOUS YOUTH STRIVING FOR NON-MIGRATION IN GUATEMALA Briana Nichols Deborah Thomas Kathleen Hall Imagining un Futuro Digno: Indigenous youth striving for non-migration in Guatemala analyses youth aspirations for non-migratory futures in contexts of extensive migration. This dissertation asks: how does immobility becomes desired and sought after by young people in social-geographic contexts of extensive mobility? What does it mean to live and make futures in these contexts? What do practices of immobility look like and how do they relate to the larger social and material fields in which youth are embedded? Integrating seventeen months of multi-sited ethnographic participant observation with archival research and media analysis, this project examines how Indigenous Guatemalan youth reckon with, and leverage, the changing material and social realities that migration produces. Bringing into conversation anthropologies of migration and education, critical diaspora studies, and the work of critical indigenous and feminist scholars, I demonstrate how aspirations of non-migration come into being, the ways in which youth work towards their actualization, and the achievability of these aspirations. I argue for the productive reframing of migration as a problem-space through which people evaluate migration as a future-making technique and I demonstrate how (im)mobility emerges as a desired modality. Through this work I show how young people come to focus on the lack of dignity afforded their migrating loved ones in the United States and use this diasporic knowledge to contest taken-for-granted spatiotemporalities of future making within their communities. I conclude that these practices are a form of pragmatic futurity through which Indigenous youth navigate the problem space of migration with the goal of animating the repressed potentials of their communities. Through their diasporic knowledge production and pragmatic futurity youth reframe their communities as spaces in which a future is not only possible, but desirable, and themselves as the people who can and will build this future, contesting the pathways of opportunity represented both by transnational migration and regional development interventions. The findings of this study inform future research on the sociocultural contexts of extreme migration and migration prevention in global settings
From \u27-Omics\u27 To Biomarkers And Mechanisms In Parkinson\u27s Disease: Growth Hormone Receptor And Gpnmb
No full textParkinson’s disease (PD) is a devastating neurodegenerative disorder that affects an estimated ~5 million people worldwide and is associated with intracellular α-synuclein protein inclusions and progressive neuronal cell loss. There are currently no disease-modifying therapies for PD due in part to lack of clinically useful biomarkers and clearly intervenable therapeutic targets. Despite recent advances made with genome-wide association studies (GWAS), there is an unmet need in the PD field for (1) generation of diagnostic and prognostic biomarkers that can distinguish PD from non-PD and parse considerable clinical heterogeneity, and (2) mechanistic follow-up of GWAS-identified leads to elucidate their role in neurodegeneration. This dissertation addresses these two gaps in therapeutic development in PD. First, we perform an unbiased, ~1000-protein screen in plasma samples from over 500 PD patients and neurologically normal controls across three different clinical sites, identifying novel proteins associated with PD. We show that our top PD-associated biomarkers – particularly, plasma levels of growth hormone receptor (GHR) – replicate their associations with disease status across multiple cohorts, are robust to common sources of variability in a clinical setting (specifically, sample handling and dopaminergic medication), and predict future cognitive decline across cohorts, cognitive measures, and statistical test used. Second, to “widen the net” for potential pathophysiological mechanisms and therapeutic targets in PD, we dissect the mechanism behind the non-coding chromosome 7 locus first associated with PD by GWAS. We identify the glycoprotein non-metastatic melanoma protein B gene (GPNMB) as the target of this locus by mining existing “-omic” datasets and analyzing the probability of a shared causal variant behind both the association to PD risk and expression levels of potential target genes. We then confirm these in silico analyses with allele-specific expression experiments using patient-derived brain samples. We explore interactions between GPNMB and α-synuclein by immunofluorescence, co-immunoprecipitation, immunoblotting, and RNA-seq-based transcriptomic profiling in immortalized cell lines and iPSC-derived neurons with normal levels, partial loss, or full loss of GPNMB. We find that GPNMB interacts with α-synuclein and that loss of GPNMB leads to decreased synaptic α-synuclein and broad transcriptomic changes among synaptic genes. Lastly, we test the clinical utility of GPNMB as a biomarker in plasma and CSF samples from \u3e800 PD patients and controls, finding that plasma GPNMB is elevated in PD and higher levels are found in PD patients with more severe disease. These results suggest a model whereby the rs199347 risk allele results in increased GPNMB expression which, through interactions with α-synuclein, leads to synaptic changes, thereby impacting risk for neurodegeneration. More broadly, this dissertation exemplifies an investigative approach combining computational analyses of large datasets with targeted bench-based experiments. Through this work, we nominate GHR as a promising PD biomarker and GPNMB as both a biomarker and potential therapeutic target in PD