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EV Fingerprinting: Resolving Extracellular Vesicle Heterogeneity using Multiparametric Flow Cytometry
No full textCancer metastasis remains a major unsolved clinical challenge as over 90% or cancer-related deaths are attributed to metastasis. Dynamic regulation of cell adhesion is crucial for successful metastatic dissemination. Cell adhesion can be fine-tuned through direct modulation of cell adhesion molecules or the functional contributions of extracellular vesicle (EVs). We have previously identified Activated Leukocyte Cell Adhesion Molecule (ALCAM) as a dynamic regulator of cell adhesion by expression of alternatively spliced isoform with differential proteolytic susceptibility that impacts metastasis. EVs are lipid bilayer enclosed particles secreted by cells that mediate of cell-cell communication in normal physiology as well as disease pathology. In cancer, EVs transfer bioactive cargoes (proteins, lipids, and nucleic acids) to facilitate directional migration, cell motility, and speed. Characterization of cargoes and biogenesis is therefore needed to identify populations of EVs with a distinctive function. ALCAM-mediated cell adhesion and EV biogenesis are linked through a common molecular factor, Syntenin-1. ALCAM is connected to the actin cytoskeleton through Syntenin-1 to form stable adhesion complexes. Syntenin-1 also participates in a distinctive class of EV biogenesis where it supports intraluminal vesicle budding through associations with Syndecan and ALIX on maturing endosomal compartments. This overlap in molecular machinery suggests that Syntenin-1 availability through ALCAM sequestration could affect EV biogenesis. Using a novel single-EV flow cytometry method called “EV Fingerprinting”, we showed that ALCAM shedding alters EV biogenesis by enhancing production of larger EVs. Additionally, cargo loading into EVs was affected by ACLAM expression. Examination of EV Fingerprints from bladder cancer cell lines cultured in a 3D organotypic model also showed similar profiles according to ALCAM shedding. In order to better understand the results of EV Fingerprinting, further validation of the method was required. Technical validation of EV Fingerprinting demonstrated that the lipophilic dye, di-8-ANEPPS, enables characterization of EVs based on relative fluorescence through size and liquid order by generating multiparametric data. Dimensional reduction and clustering of data revealed resolved populations of EVs. We used EV Fingerprinting to identify population-specific changes upon molecular perturbation of EV secretion through Rab27a knockdown and CD63 overexpression according to liquid order. Additionally, we determined that cargo partitioning is a non-random process by using multiplex analysis of CD63 and CD81 EV cargoes. In summary, this dissertation identifies a relationship between cell adhesion and EV biogenesis as well as establishes a tool to resolve heterogeneous EV populations
Strongly Coupled Light-Matter Phenomena Investigated with Algorithimic and Formalistic Density Functional Coupling Schemes
No full textNanostructures and quantum emitters in optical cavities are examined using novel light-matter coupling approaches. Treating light and matter on equal first-principles footing has not be necessary to advance the fields of optical and condensed matter physics; however, advancements in nanoplasmonics, quantum chemistry, and quantum optics present opportunities for more complex light-matter schemes, presenting questions not approachable using classical light propagation methods or many-body theories. Among the investigative opportunities are charge transfer observations in hybrid nanoparticles, ground state calculations for cavity-coupled atoms and molecules, and the time-dependent behavior of quantum emitters in optical cavities. In the "quantum-classical" regime, the current and density transfer dynamics of jellium and atomically-detailed nanoparticles are examined using coupled Maxwell and orbital-free density functional equations, in which all effective energy functionals are density-dependent, the Maxwell equations are propagated using the time evolution operator, and the back-reaction of the induced current is detectable in the classical net field. In the "quantum-quantum" framework, many-electron systems, atoms, and molecules, in and out of ground state equilibrium while coupled to optical cavities, are examined using density functional schemes constructed from the Pauli-Fierz Hamiltonian of non-relativistic quantum electrodynamics
"A Connected Idea of the Lesson": Teaching and Learning in African American Literature
No full text“A Connected Idea of the Lesson”: Teaching and Learning in African American Literature, examines depictions of the classroom in African American literature in order to understand how well-known writers and activists from Ida B. Wells and Kiese Laymon also offer important pedagogical lessons that foreshadow contemporary theories of culturally relevant, responsive, and sustaining pedagogies. I argue that Black teachers have long used literacy to develop students’ sense of agency while simultaneously helping students to recognize their obligation to one another as part of a mutually constitutive learning community. This research intervenes in the fields of African American literature and pedagogical praxis, as I demonstrate that the teachers I study are the forerunners of contemporary theorists of culturally relevant, responsive, and sustaining pedagogy, and that their writings represent a critical trove of knowledge for preservice and inservice educators today.
My dissertation moves roughly chronologically through the twentieth century, with each chapter centering a particular text or author central to the history of critical pedagogy in the United States. In my first chapter, I argue for the importance of connection and care to the work of nineteenth century teacher-authors Fanny Jackson Coppin and Frances E.W. Harper, both of whom depict the revolutionary potential of the classroom for helping students to find their individual voices in order to speak back to power. Also in that chapter, I discuss why their contemporary Ida B. Wells came to such a different assessment of the political potential of classroom teaching and what her more pessimistic perspective might teach teachers and policy makers today. In chapter two of my project, I argue that Septima Poinsette Clark and her contemporary, Mamie Garvin Fields, drew upon Black musical traditions to bring improvisation into the classroom in furtherance of their students’ projects of emancipation. Chapter three analyzes student-produced poetry and song from the Mississippi Freedom Schools. In these lyrical texts, I argue that students navigate their complex and often contradictory understandings of the pedagogies they gleaned from teachers such as Charlie Cobb and Stokely Carmichael. In chapter four, I delve into the relationship between masculinity and pedagogy in Ernest Gaines’ A Lesson Before Dying (1993) and J. Saunders Redding’s Stranger and Alone (1950), arguing that these novels show the importance of unlearning a heteropatriarchal conception of masculinity in order to teach for freedom. Finally, chapter five examines Kiese Laymon’s recent memoir Heavy (2018) in order to explicate how Black students of the late twentieth century revised deficit discourses in order to affirm each other’s sense of self. Far from a linear progress narrative, these chapters depict the extraordinary and sometimes futile efforts of Black students and teachers to succeed at effecting politically consequential acts of self- and collective transformation in a context of extreme violence and constraint.
This project examines representations of teaching and learning in African American literature, arguing for the existence of a Black radical pedagogical tradition which foreshadows contemporary theories of culturally relevant, responsive, and sustaining pedagogies (CRP/CSP). The educators whom I discuss help students to actualize their transformative potential, heightening students’ sense of individual agency while simultaneously helping students to recognize their obligation to one another as part of a mutually constitutive community.
In my first chapter, I discuss the depiction of connection and care in the works of teacher-authors Fanny Jackson Coppin and Frances E.W. Harper. I also discuss why their contemporary Ida B. Wells came to a different assessment of the political potential of classroom teaching and what her more pessimistic perspective might teach teachers and policy makers today. In Chapter Two, I argue that Septima Poinsette Clark and her contemporary, Mamie Garvin Fields, drew upon Black musical traditions to bring improvisation into the classroom in furtherance of their students’ projects of emancipation. Chapter Three analyzes student-produced poetry and song from the Mississippi Freedom Schools and the Children’s Development Group of Mississippi in which students navigate their complex and often contradictory understandings of the freedom, subjection, and subjectivity. In Chapter Four, I delve into the relationship between masculinity and pedagogy in Ernest Gaines’s A Lesson Before Dying (1993) and J. Saunders Redding’s Stranger and Alone (1950). Finally, Chapter Five examines Kiese Laymon’s recent memoir Heavy (2018) in order to explicate how Black students of the late-twentieth century revised deficit discourses in order to affirm each other’s sense of self. My project concludes with the results of my research study, in which I worked with an interdisciplinary team of MAT candidates at Vanderbilt’s Peabody College to qualitatively measure changes in their pedagogy after reading works by Clark, Gaines, and Laymon
Investigating the Requirement for the Gene TSC2 in Early Human Neurodevelopment
No full textTuberous sclerosis complex (TSC) is a multi-organ genetic disorder caused by mutations in either TSC1 or TSC2 which encode hamartin and tuberin, respectively. Hamartin and tuberin form a protein complex that indirectly, negatively regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1), a signaling hub that can regulate several cellular processes. When mTORC1 is hyperactive during development like in TSC, there can be severe consequences for patients such as benign brain tumors and debilitating neurological symptoms like epilepsy. It is thought that the epilepsy can be due to the presence of tubers located in the cerebral cortex which arise during development. Tubers are characterized by cortical mislamination, dysmorphic neurons, and giant cells that can co-express multiple, fate exclusive identity protein markers. These tuber characteristics indicate aberrant development of the cerebral cortex with changes in cell fate decisions that potentially are linked to abnormal epigenetic regulation. While TSC neurological phenotypes are well-documented, it is not yet known how early in neural development TSC1/2-mutant cells diverge from the typical developmental trajectory, whether such phenotypes are seen in the heterozygous-mutant populations comprising the majority of cells in patients, and if changes in neurodevelopment are accompanied by aberrant epigenetic regulation.
To examine early neurodevelopmental phenotypes, TSC patient-derived induced pluripotent stem cells (iPSCs) harboring a disease-causing heterozygous microdeletion mutation in TSC2 were utilized. The following dissertation uses these TSC iPSC models derived into neural progenitor cells (NPC) to explore atypical neurodevelopment seen in TSC patients. Protein expression, RNA transcripts, and DNA methylation were studied in TSC2 mutant NPCs. Further, non-canonical roles for tuberin in mitotic division in iPSCs were explored
Planning for Organizational Influence on Epilepsy Stigma
No full textLeadership and Learning in Organizations capstone projectROW Foundation is a funding focused 501(c)(3) private foundation that relies on successful domestic and international partnerships to reach people with epilepsy in LMICs. As those partners have identified stigma as a barrier to their projects, ROW recognizes an opportunity to help equip their partners to address stigma or to better identify potential partners with existing stigma strategies. This project explored three sources of data (document analysis, survey data, and semi-structured interviews) collected from ROW Foundation, ROW's current partners, and experts in the field to answer the project questions. Overall, three major themes emerged from the inductive and deductive coding process that may help ROW improve their ability to influence interventions aimed at stigma mitigation: 1) listening to partners and influencing interventions, 2) collecting and organizing data, 3) leveraging reputation among funders. Recommendations were developed and implementation strategies were suggested based off the findings
Beer Country: Anatomy of a Cultural Commodity in Postwar Central Europe
No full textThis dissertation explores the many aspects of what it meant for beer to be a cultural commodity in German and Czech societies of postwar Central Europe. By demonstrating how leaders and ordinary individuals treated this beverage in their words and actions, I argue that beer and its constitutive natural ingredients held tremendous power in postwar West Germany, East Germany, and Czechoslovakia owing to their hybrid cultural and economic importance. The “hard” business aspects of the beer market drew great attention on matters such as employment, tax revenue, and export value not only for beer itself, but for the industry’s second and third order impacts on other sectors. That, combined with the “soft” power of beer culture, especially its social role in reinforcing popular notions of gender or national identity, gave German and Czech beer an unusual degree of gravity in politics and everyday life. The influence of beer on such disparate and varied historical developments as the process of postwar justice and retribution, the emergence and struggles of welfare states, and the political and economic integration of Europe into two competing supranational blocs both demonstrates the validity of the argument that I put forward in this study and reflects the remarkable continuity of beer’s cultural and economic importance across the second half of the twentieth century. Only an investigation of the extent and scope that I present here, covering more than four decades of history in three countries on both sides of an ideological Cold War with scales of analysis from the micro-local to the multinational, can fully display the underrecognized importance of a product such as beer by showing the diffuse but steady pressure that it exerted on events across such a wide breadth of circumstances. Even as political and social life in Central Europe experienced extreme ruptures, to put it mildly, from 1945 to 2000, beer’s role as a cultural commodity and its ability to warp political power to its benefit shows remarkable continuity
Data Quality-Aware Graph Machine Learning
No full textGraph-structured data is ubiquitous in real-world applications (e.g., social networks, infrastructure, biomedical) and Graph Machine Learning (GML) has become a prominent method for handling such data. However, despite GML’s remarkable achievements, its reliance on node features makes it susceptible to conventional data quality issues, and the additional consideration of graph topology adds novel complexity. Inspired by these challenges, my research in this dissertation focuses on data-quality-aware graph machine learning, systematically examining issues related to topology, imbalance, bias, and limited data issues in graph data. This includes proposing data/model-centric solutions to handle them and applying my proposed data issue solutions for real-world applications, such as de-noising unrelated interactions in personalization learning for recommender systems, overcoming the inherent class-imbalance issue in computer-aided drug discovery, mitigating hallucination bias in multi-document question answering, and boosting limited training data for graph generations. Lastly, to further advance the field of data quality-aware GML, I have outlined multiple future directions to make data quality-aware GML more inclusive, intelligent, and trustworthy
The role of neuronal ensembles in the nucleus accumbens in cocaine use disorder
No full textWhile significant effort has been made to understand the neural basis of addiction, it remains unclear how drugs of abuse alter the neural systems involved in motivated behavior to control drug taking and seeking. The goal of this dissertation was to understand how cocaine and associated stimuli are encoded in the brain and act to drive drug seeking. In a given brain region, only a small percentage of cells are activated to any stimulus - termed an “ensemble”. Here we aimed to determine if ensembles activated by cocaine in the nucleus accumbens (NAc) – a brain region central to reward encoding - could drive motivated behaviors. Using transgenic animals that allowed for the temporally specific tagging of ensembles in a transcriptional activity-dependent manner, we were able to record from, identify, and manipulate the neural ensembles that are selectively activated by cocaine experience. Repeated cocaine experience inhibited NAc neuronal activity, causing decreased ensemble size; simultaneously, this smaller population of neurons were more excitable and had fundamentally different physiological properties. Mice were motivated to optically reactivate all ensembles, including saline, acute cocaine, and repeated cocaine. However, lesioning of repeated cocaine ensemble attenuated cocaine-associated behaviors, such as locomotor sensitization and cocaine self-administration. Further, we show that there is a critical effect of sex on cue learning, that may be critical in governing how animals respond to drugs of abuse. Overall, we hypothesize that a history of cocaine alters the physiological properties of the ensemble neurons, refining them and causing them to play a key role in cocaine-related behaviors and reinforcement learning
3D-2D Representation Learning for Longitudinal Analysis Using Single-Slice CT Scans
No full textAbdominal computed tomography (CT) offers high-resolution tissue maps that facilitate the quantification of body composition. The existing longitudinal CT dataset from the Baltimore Longitudinal Study of Aging (BLSA) enables us to explore the relationship between body composition and cognitive decline. To minimize radiation exposure, 2D axial scans are used instead of 3D volumetric scans, presenting several challenges. Notably, 2D scans lack the contextual information inherent in 3D data, complicating body composition segmentation. Furthermore, difficulties in consistently positioning cross-sectional scans mean that 2D slices may not always be taken at the same vertebral level, resulting in variability in the abdominal slices obtained from patients over different years. In this dissertation, we utilize existing 3D volumetric data for representation learning to mitigate the anatomical limitations present in single-slice data. We apply advanced deep learning techniques for segmentation and generation to address these challenges. Ultimately, we identify a potential underlying phenotype associated with cognitive decline using the derived body composition metrics, providing new insights into the connection between the body and the brain
