48670 research outputs found
Sort by
The Relational Role Of Place In The Production Of Racial Stratification
Get PDFIn this dissertation, I examine how we quantify the dynamic, cumulative effects of relational social exposures with longitudinal survey data. In Chapter 1, I demonstrate a new mediation framework for describing what are often conceptualized problematically as “neighborhood effects.” Findings from this study clarify the reciprocal, life-course process through which neighborhood is implicated in the early production of social inequality. In Chapter 2, I extend this mediation framework to respond to theoretical critiques of how variables for race are used in common regression frameworks in attempts to study structural racism. I demonstrate an alternative counterfactual approach to explain how multiple racialized systems dynamically shape health over time, examining racial inequities in cardio-metabolic risk. I decompose the observed disparity into three types of effects: a controlled direct effect (“unobserved racism”), proportions attributable to interaction (“racial discrimination”), and pure indirect effects (“emergent discrimination”). I discuss the limitations of counterfactual approaches while highlighting how they can be combined with critical theories to quantify how interlocking systems produce racial health inequities. In Chapter 3, I use this framework to examine the Black-white wealth gap in the United States. Descriptive and qualitative analyses have identified many mechanisms underlying wealth correlations across successive generations, but few studies have quantified the relative contributions of these interconnected and racialized systems of reproduction to the total gap we observe today. I define a wealth gap in 2015-17 between the grandchildren of those racialized as Black and the grandchildren of those racialized as white in 1968-70. I use a fully interacted counterfactual mediation framework to decompose this disparity into the historical, racialized contributions of 1) effects of home values in 1968-70 on home values in successive generations and 2) effects via educational attainment in successive generations. Findings from this study contribute to our understanding of the dynamic, racialized process of multigenerational place-based wealth accumulation and support the importance of historically contingent social policy centered on reparative justice
Profiling Of Protein Post-Translational Modifications Through Proteomic And Genomic Approaches Reveals Critical Biological Regulation
No full textPost-translational modifications (PTMs) regulate biological outcomes by influencing protein structure, localization, function, and interactions. While numerous PTMs have been detected to date, often the dynamics and role of these PTMs in specific biological contexts remains unclear. This dissertation highlights my work interrogating the dynamics and role of two PTMs: acetylation on a proteome-wide scale, and methylation on lysine 27 of the histone variant H3.3 in the context of stem cell differentiation.
We performed a comprehensive characterization of protein acetylation dynamics using mass spectrometry based proteomics through utilization of 13C-glucose or D3-acetate, which are metabolized into acetyl-CoA, labeling acetyl groups through subsequent incorporation into proteins. We characterized around 1,000 sites with significantly increasing acetylation trends. Faster rates were enriched on proteins associated with chromatin and RNA metabolism, while slower rates were more typical on proteins involved with lipid metabolism. We identified sites catalyzed at faster rates with potential critical roles in protein activation, including the histone acetyltransferase p300 acetylated in its activation loop. This study highlights the dynamic nature of protein acetylation, and how metabolism plays a central role in this regulation.
Histone variants, such as histone H3.3, replace canonical histones within the nucleosome to alter chromatin accessibility and gene expression. We demonstrate through methylation dynamics studies that methylation on H3.3K27 is maintained more than on canonical H3K27 over stem cell differentiation. Using a custom-made antibody, we identify a distinct enrichment of H3.3K27me3 at lineage-specific genes, such as olfactory receptor genes, and at binding motifs for the transcription factors FOXJ2/3. REST, a predicted FOXJ2/3 target that acts as a transcriptional repressor of terminal neuronal genes, was identified with H3.3K27me3 at its promoter region. H3.3K27A mutant cells confirmed an upregulation of FOXJ2/3 targets upon the loss of methylation at H3.3K27. Thus, while canonical H3K27me3 has been characterized to regulate the expression of transcription factors that play a general role in differentiation, our work suggests H3.3K27me3 is essential for regulating distinct terminal differentiation genes. This work highlights the importance of understanding the effects of PTMs not only on canonical histones but also on specific histone variants, as they may exhibit distinct roles
Discovery And Application Of Cytosine Carboxymethylation
No full textEvolution molds biological function. DNA, the scaffold upon which natural evolution progresses, is composed of four nucleobases: adenine, cytosine, guanine, and thymine. As the genome propagates across generations of replication, these nucleobases must find a way to balance their primary function of maintaining trans-generational fidelity while still leaving room for purposeful mutation and adaptation. Within generations, additional opportunities to encode new functions exist through the intentional chemical modification of DNA. In particular, DNA methylation, found within Cytosine-Guanine (CpG) dinucleotides, represents a canonical “epigenetic” mark, whereby covalent modification to cytosine perturbs gene expression. In our attempt to understand the catalytic determinants of 5-methylcytosine (5mC) generation by DNA methyltransferase (MTase) enzymes, we created an unexpected and previously unknown DNA modification, 5-carboxymethylcytosine (5cxmC). We show that only a single point mutation in the active site is responsible for conferring neomorphic activity on this enzyme family, effectively turning DNA MTases into DNA carboxymethyltransferases (CxMTases). In E. coli, these CxMTases can shunt the sparse but natural metabolite carboxy-S-adenosyl-L-Methionine (CxSAM) to be directly used as a substate to make 5cxmC in genomic DNA. Our discovery of this new CxMTase/CxSAM, enzyme/substrate pair further enabled the development a new biotechnology, termed Direct-Methylation Sequencing (DM-Seq), which can directly localize 5mCpGs at single base resolution using limited DNA input. Our findings advance both synthetic biology and biotechnology, whereby our laboratory-based evolution characterizes the biochemical circumstances limiting the natural evolution of DNA modifications and simultaneously reveal the power of structure-guided protein engineering in unlocking a transformative epigenetic sequencing technology
In The Time Of Disaster: Representations Of Hurricane Katrina In African American Literature And Culture
No full textIn the Time of Disaster: Representations of Hurricane Katrina in African American Literature and Culture examines Black literary and cultural works that employ Hurricane Katrina as a poetic persona, narrative subject, geographical index, and temporal signal. In the months that followed its landfall, “Hurricane Katrina” functioned discursively as a loaded metonym for interrelated crises including the levee breaches, the failure of the state to adequately prepare for and respond to the needs of citizens, the deluge of media images centered on Black suffering, and the incompetence of the Federal Emergency Management Agency. Media efforts to rationalize the government’s culpability inspired an abundance of scholarship in the humanities and social sciences about Hurricane Katrina’s economic, social, and political implications. In the Time of Disaster turns to literature and art to consider Hurricane Katrina’s significations through the works of Spike Lee, Terence Blanchard, Mat Johnson, Natasha Trethewey, Nikky Finney, Kiese Laymon, and Jesmyn Ward. This project examines how Black writers and artists disrupt and redraw the temporal and spatial boundaries of disaster to revise our collective cultural memories of the storm and suggest new ways of reading and writing our relationship to the environment in the wake of catastrophic loss
Privacy-Preserving Distributed Regression Algorithms For Analysis Of Multi-Site Real-World Data
No full textReal-world data, including electronic health records and administrative claims data, are widelyused in modern healthcare research to generate real-world evidence for improving patient care. The widespread availability of observational data from a variety of institutions has prompted many large-scale, multi-site studies in recent years. Studies incorporating data from multiple institutions often attain results more generalizable than those from single-site studies and offer improved power for studying rare outcomes or exposures. Various challenges concerning patient-level data sharing, primarily those related to data privacy, have made distributed data analysis a practical alternative to analyzing centralized data in multi-site studies. Under a distributed data analysis framework, patient-level data are not shared across institutions. Instead, aggregated data are shared and communicated to a coordinating site to obtain analysis results. While methods for performing distributed analyses are increasingly available, analytical methods for analyzing binary and count outcomes are limited. In this work, we propose two distributed regression algorithms for modeling count outcomes in multi-site studies. The first algorithm uses distributed quasi-Poisson regression to model counts while accounting for institution-specific heterogeneity in the outcome. The second uses distributed hurdle regression to model counts subject to zero-inflation. Both algorithms are communication efficient and highly accurate, requiring at most two or three rounds of communication among participating institutions and achieving results close to those obtained using pooled regression of all patient-level data, a method usable only if data are centralized. We evaluate the performance of each method through simulations and applications to real-world clinical research networks. Finally, we illustrate a novel application of a distributed generalized linear mixed modeling algorithm with binary outcomes to study the effect of admitting hospital on racial disparities in mortality for patients hospitalized with COVID-19 via counterfactual modeling
Glial Contributions To Traumatic Brain Injury
Get PDFWhy the brain continues to undergo functional and histopathological decline years and decades after a traumatic injury is a central question in the field of traumatic brain injury (TBI) research. Despite the heterogeneity of TBIs, sustained and aberrant activation of glia is found in nearly all injuries and likely contributes to long-term pathology. This notion is supported by emergent evidence in classical degenerative diseases, such as Alzheimer’s and ALS. In early TBI, glia are thought to have a supportive role in recovery but over time, their continued activation may foment inflammation and decline, though mechanistic understanding of this process is limited. To define disease mechanisms, we performed an unbiased time course RNA-sequencing experiment in a Drosophila model of head injury (dTBI). We identified a rapid and sustained transcriptional response mediated by the conserved complex, AP1. We determined that AP1 activates in glia, in an injury-dose dependent manner. Our functional studies determined that AP1 drives distinct glia behavior in early and late injury. Acutely, glial AP1 is essential for injury recovery; in its absence, a survivable TBI becomes lethal. However, continued activation of AP1 promotes human tau pathology, degeneration, and death. We extend our work in the fly to humans and uncover evidence of chronic AP1 activity in survivors of moderate TBI. In these subjects, AP1 activity positively correlates with microglial activation and tau pathology. Curiously, in flies, we find that glial AP1 also activates during healthy brain aging, suggesting that TBI may accelerate age-onset processes in glia. In summary, transcriptional insights gained from a Drosophila model of TBI uncovered a potentially conserved mechanism by which glia initially protect but in time, promote disease. Our work has broad implications for injury, aging, and disease
Structral And Biochemical Insights Into The Transition From Transcription Initiation To Elongation
Get PDFTranscription by RNA polymerase II (Pol II) is a complex process that requires timely and coordinated regulation at multiple steps for proper gene expression. Initiation is the first step in transcription and decades of biochemical and genome-wide studies have identified proteins involved in the process and revealed their functions. Additionally, technological advancements in cryo-EM enabled researchers to visualize initiation complexes and provide mechanistic insights into initiation processes in the last several years. However, the mechanistic understanding of the transition from transcription initiation to elongation has been limited in part due to the lack of an efficient transcription initiation system in vitro. We purified yeast general transcription factors (GTFs: TFIIA, TFIIB, TBP(a component of TFIID), TFIIE, TFIIF, and TFIIH) and Pol II, all of which are necessary and sufficient for basal transcription initiation, and optimized the initiation system. Using this system, we biochemically re-examined effects of two elongation factors (Cet1-Ceg1 and Spt4/5) on promoter escape, a process in which Pol II dissociates from GTFs except TFIIF for elongation. We find that inclusion of these elongation factors has positive effects on promoter escape. Furthermore, we took advantage of our efficient system, and generated and isolated post-initiation complexes in vitro for structural characterization by cryo-EM. Our structure of the initially-transcribing complex (ITC) stalled +26 shows a large conformational change of TFIIH in the way that it is much closer to TFIIE than in the pre-initiation complex (PIC) and it loses contacts with Pol II. These changes most likely prime for Pol II to escape the promoter. In addition, the structural studies of post-initiation complex stalled +49 reveal two elongation complexes (ECs) colliding to each other as well as show the presence of EC+ITC. In the structure the colliding ECs, the trailing EC contained RNA of ~25 nt in length but has backtracked by ~10 nt upon colliding. These studies together provide a model of the process of promoter escape, where TFIIH can get kicked out by the preceding promoter-proximal EC
Analysis Of Matched Primary And Recurrent Brca1/2 Mutation-Associated Tumors Identifies Recurrence-Specific Drivers
Get PDFPatients with inherited germline mutations in BRCA1/2 carry a drastically increased risk of early-onset breast and ovarian cancers. BRCA1/2 mutation-associated tumors respond to therapies that exploit their inherent homologous recombination deficiency, including platinum-based chemotherapy and poly(ADP-ribose) polymerase inhibitors (PARPi). However, these tumors frequently return as lethal, therapy-resistant recurrences. Outside of somatic BRCA1/2 reversions, the mechanisms underlying acquired therapeutic resistance and recurrence remain unknown. To address this gap in knowledge, we performed whole exome, targeted, and RNA sequencing on paired primary and recurrent breast and ovarian tumors from 27 BRCA1/2 mutation carriers. The main outcomes of the study were somatic variants, copy number variation, BRCA1/2 loss of heterozygosity, differential gene expression, and differential transcript usage. One key finding was a high prevalence of copy number gains and amplifications in PARP1. We detected PARP1 gains across primary and recurrent BRCA1/2 mutation-associated breast and ovarian tumors, with increased expression observed at the mRNA and protein levels. Our results suggest that PARP1 gains could be an under-appreciated mechanism of endogenous PARPi resistance in BRCA1/2 mutation-associated and sporadic breast and ovarian tumors. We also assessed allele-specific BRCA1/2 loss of heterozygosity (LOH) across the cohort. In general, LOH status was concordant between paired primary and recurrent tumors. However, seven tumors underwent LOH transitions over the course of recurrence, suggesting that selective pressure drove most cancers towards biallelic BRCA1/2 loss but some towards BRCA1/2 proficiency. Lastly, we found that recurrent tumors express a shorter BRCA2 transcript. This non-canonical isoform is protein-coding and differs only in the 3’ UTR. Expression of the alternative BRCA2 transcript was significantly associated with reduced overall survival in BRCA1/2 mutation carriers with breast cancer (median 87 vs. 121 months). Our results indicate that the shorter BRCA2 isoform may represent a novel driver of recurrence in BRCA1/2 mutation-associated breast tumors. Ultimately, these findings improve our understanding of tumor evolution in BRCA1/2 mutation-associated cancers, including conserved drivers and other features that may contribute to therapeutic resistance. Ultimately, this work will improve our understanding of late-stage disease in BRCA1/2 mutation carriers, as well as inform new treatment options for these patients
Cultivating Communities On The Eastern Frontier: Agrarian Landscapes And Life In Downeast Maine (1760–1860)
Get PDFThis dissertation explores how people transform “new” and unfamiliar environments through colonization. While adaptationist perspectives typically stress how the environment shapes human behavior and communities, I integrate historical ecology and settlement ecology to examine how people mold, maintain, and manage landscapes. Rather than passive backdrops, landscapes are dynamic, produced as humans actively modify the environment. Boundaries, whether stable or fluid, divide and structure landscapes. As a type of boundary, frontiers can be understood as centers of social interaction and exchange, but more often are viewed as peripheries, remote but ripe for settlement, or wild zones where pioneers struggled to survive. Given factors like severe winters, poor soils, and warfare, the latter portrayal dominates narratives of America’s Eastern frontier during the 18th and 19th centuries. To interrogate notions of a largely static, intractable frontier environment, I assess how Euroamericans transformed the Downeast Maine region through settlement and enclosure. To determine how they colonized, cleared, bounded, and cultivated the landscape, I analyze archival, archaeological, and geospatial data from nine towns. First, I trace changes in the landscape and agricultural production between 1792 and 1811 using historical tax valuations. Statistical and geospatial analyses of this data suggest some town landscapes were more thoroughly improved and refined through agriculture than others. Initial parallels between frontier agricultural production and that of southern New England challenge notions of the intractable frontier environment. Second, I juxtapose 18th- and 19th-century maps with Google Earth and Light Detection and Ranging imagery to explore how the frontier landscape was settled, divided, and enclosed. By identifying historical landscape features that endure in the modern landscape, I chart continuity and change in the structure of these towns through the present. Finally, I examine the Foster Farmstead in Deer Isle, Maine as a case study to investigate how settlement and agrarian activities became physically embedded in the landscape at a small scale. My archaeological survey and excavation reveal settlement and landscape features like foundations, stone walls, and stone piles, which attest to how descendants of the Fosters continued to inhabit, transform, and enclose the land through time
Brd4 As A Regulator Of Tissue-Specific Gene Program And As An Orchestrator Of Genome Folding
No full textCell fate decisions and the maintenance of cellular identity relies on cell-specific coordination of gene regulatory networks. Besides the known role of transcriptional regulators in this process, the spatial organization of chromatin has emerged as additional layer of transcriptional control, and mutations in proteins mediating genome folding have been associated with developmental disorders known as cohesinopathies. However, how ubiquitously expressed transcription regulators achieve cell-specificity, and the complex relationship between higher-order structure and physiologic development remains unclear. Here, we define two uncharacterized roles for Bromodomain-containing protein 4 (BRD4) in the regulation of a cardiomyocyte-specific gene program and in the maintenance of genome folding. To understand the cell-specific role of BRD4, we used conditional mouse genetics to show that cardiomyocyte-specific deletion of Brd4 in adult mice leads to impaired cardiac contractility accompanied by decreased expression of genes critical for mitochondrial bioenergetics. Genome-wide occupancy data show that BRD4 enriches at downregulated genes and preferentially co-localizes with GATA4, a lineage-determining cardiac transcription factor not previously implicated in regulation of cardiac metabolism. BRD4 and GATA4 form an endogenous complex in cardiomyocytes, revealing a new interaction partner for BRD4 that directs its locus and tissue specificity to regulate a gene program governing bioenergetic homeostasis in the adult heart. In an interrelated work, we also show that genetic deletion of the BRD4 in murine neural crest cells recapitulates key features observed in cohesinopathies. We leverage on this finding and demonstrate that BRD4 interacts with NIPBL, a positive cohesin regulator and commonly mutated in patients with cohesinopathies. Acute depletion of BRD4 or loss of the BRD4-NIPBL interaction reduces NIPBL-occupancy, elucidating the importance of BRD4 in stabilizing NIPBL on chromatin. Genome-wide chromatin interaction mapping and quantitative imaging studies demonstrate that BRD4-depletion results in aberrant genome folding, specifically loss of a subset of chromatin loops, weakening of TADs, and compromised loop extrusion. Finally, loss of BRD4 or the interaction with NIPBL attenuates neural crest differentiation. Remarkably, differentiation defects resulting from BRD4 depletion can be rescued by concomitant loss of WAPL, a negative cohesin regulator. Collectively, our data elucidate mechanisms of tissue-specific gene regulation and the physiological relevance of genome organization on differentiation