Browsing by Subject "Transcriptome"
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Item Characterizing the Cervical Response to Inflammation During Infection-Mediated Preterm Birth(2017-11-08) Willcockson, Alexandra Rae; Kraus, W. Lee; Patel, Vishal; Mendelson, Carole R.; Mahendroo, MalaPreterm birth, a delivery that occurs prior to 37 weeks of a 40 week gestation in women, is a leading cause of infant morbidity and mortality. Additionally, children born preterm who make it through their first year of life are at increased risk of medical complications throughout their lives. Costs associated with prematurity exceed $26 million each year in the United States alone, where nearly 1 in 10 babies is born preterm. The fact that preterm birth rates have only slight declined overall in the past 20 years can be attributed to the multiple, and most yet-to-be-identified, etiologies of the syndrome. More than 65% of all preterm births have no clinically identifiable cause. Of the premature deliveries with an identifiable cause, infection contributes to 40%. Regardless of the cause or timing of delivery, changes in the cervix precede the onset of labor. A better understanding of the pathological processes involved in premature cervical remodeling will allow for development of detection technologies and therapeutic approaches to preventing preterm birth. The goal of this study was to identify cervical pathways, distinctly regulated in response to inflammation, that lead to premature changes in extracellular matrix components, decreasing tissue biomechanical integrity and leading to preterm birth. Cervical elastic fiber ultrastructure becomes acutely disrupted in response to inflammation but not at term. RNA-seq studies identified enrichment of inflammasome activation and protease pathways in the cervix, both also exclusive to inflammation. Inflammasome-induced protease upregulation and subsequently increased activity targeting elastic fibers are potential mechanisms of premature cervical remodeling in response to inflammation, leading to preterm birth. These findings add to the understanding of how the tissue responds to inflammation and how this response can induce extracellular matrix changes that impact the biomechanical integrity of the cervix. Future investigations will focus on potential therapeutic approaches that target mechanisms upstream of protease activation to prevent disrupted extracellular matrix architecture. The effects of these studies have the potential to extend beyond first pregnancies impacted by infection; risk of preterm birth in subsequent pregnancies, which increases exponentially as the number of preterm deliveries a woman experiences increases, may also be lessened.Item Gene Regulatory Networks in Striated Muscle Pathologies(2023-05-01T05:00:00.000Z) Shah, Akansha Mahavir; Munshi, Nikhil; Olson, Eric N.; Mendell, Joshua T.; Kliewer, Steven A.The striated musculature, comprised of skeletal muscle and cardiac muscle tissues, is essential for vertebrate life. Skeletal muscles are composed of bundles of long and parallel multinucleated myofibers that constitute approximately 40% of the human body mass. The cardiac muscle is much smaller and consists of a branched network of short, mononucleated or binucleated cardiomyocytes that are connected by intercalated discs. Both these tissues partake in force generation through contractile units called sarcomeres to support movement, respiration (skeletal muscle), and to pump oxygenated blood throughout the body (cardiac muscle). Gene regulatory networks, the interactions between lineage or stage-determining transcription factors and the mRNAs they govern, tightly control cell function and striated tissue development and homeostasis. A disruption or change in these complex networks underlies most skeletal and cardiac muscle-related diseases. In this dissertation, we used transcriptomic and epigenomic approaches at single cell or bulk tissue resolution to provide a molecular framework for the function of TWIST2 in rhabdomyosarcoma pathogenesis as well as the mechanisms of heart remodeling and repair following myocardial infarction. In this process, we elucidated critical nodes in complex pathways that can be manipulated to derail the pathological process of FN-RMS or stimulate regeneration and repair in postnatal hearts.Item Hippocampal Subfield Transcriptome Analysis in Schizophrenia Psychosis(2018-11-29) Perez, Jessica Marie; Eisch, Amelia J.; Tamminga, Carol; Kim, Tae-Kyung; Zinn, Andrew R.Schizophrenia is one of the thirty most incapacitating conditions in the world and affects tens of millions of people worldwide. Devastatingly, suicide occurs in 10% of those diagnosed with schizophrenia. Symptoms are persistent and often severe and available treatments are not curative. In fact, 20-33% of people with schizophrenia are entirely resistant to treatment. The complex symptom manifestations of schizophrenia lack a molecular pathology. Consequently, advances in novel treatment directions are limited. Schizophrenia is recognized as a polygenic disorder influenced by environmental factors. This dissertation aims to examine this polygenic nature of this disorder. Genome wide association studies have identified hundreds of common genetic variants, which individually confer a small risk for schizophrenia. However, all identified genetic variants combined only account for a modest amount of the total heritability of schizophrenia. In this dissertation, I capitalize on the unique ability of next-generation sequencing to identify in a global and unbiased manner molecular changes, which have not been previously hypothesized, but may contribute to the origin of the missing heritability of schizophrenia and play a role in schizophrenia symptomatology. The Tamminga lab has particular interest in schizophrenia psychosis, conceptualizing it as a disorder of learning and memory, critically involving dentate gyrus (DG), CA3, and CA1 of the hippocampus. Therefore, this doctoral dissertation examines the transcriptome of all three subfields, DG, CA3, and CA1 in human postmortem tissue of controls and individuals diagnosed with schizophrenia, using RNA-seq to identify additional psychosis-mediating molecular candidates and produce plausible targets for therapeutic treatment. After Chapters 1, 2, and 3 introduce the significance and contribution of this dissertation to the field of neuroscience in psychiatry, I show (Chapter 4) that each hippocampal subfield in schizophrenia has a unique molecular identity based on its transcriptome profile. As well, I show only slight effects of antipsychotic medication on schizophrenia-dependent gene changes in DG, CA3, and CA1. Taken together, my data identify molecular candidates and specific cell populations that we previously did not hypothesize as potential contributors to schizophrenia pathology. Finally, in Chapter 5, I outline future directions based on the contributions of my doctoral dissertation to the field.Item Molecular Dissection of Hand2 During the Formation of Pacemaker-Like Myocytes During Direct Reprogramming(2019-03-06) Fernandez-Perez, Antonio; Zhang, Chun-Li; Cleaver, Ondine; Olson, Eric N.; Munshi, NikhilDirect reprogramming of one cell type into another has great promise for regenerative medicine, disease modeling, and lineage specification. Currently, the conversion of fibroblasts into induced cardiomyocytes (iCM) by Gata4, Mef2c, and Tbx5 (GMT) represents an important avenue for generating de novo cardiac myocytes. Recent evidence has shown that iCM formation and diversity can be enhanced by the addition of Hand2 to GMT (GHMT). These four transcription factors give rise to a heterogenous CM population, consisting of atrial (iAM), ventricular (iVM), and pacemaker myocytes (iPM). However, the molecular mechanisms that drive this plastic fate conversion remain poorly understood. Although chromatin and single-cell studies in GMT-iCM have shown the existence of a set of temporal steps that orchestrate iCM formation, little is known about how Hand2 enhances this process. In the present study, we seek to characterize these Hand2-dependent mechanisms. We hypothesize that Hand2 regulates a discrete pacemaker regulatory network that becomes active during GHMT-iCM reprogramming. To test this, we compared the transcriptional and genomic profiles of fibroblasts, GMT, GHMT, and endogenous mouse Pacemaker cells. We observe similar chromatin landscape and gene expression profiles between Hand2-iPM and endogenous sinoatrial node (SAN), however several known key PM pathways are not active. Activation of these networks further enhances iCM-iPM fo Moreover, we show that Hand2 enhances chromatin accessibility in regions related to sarcomere function and electrical coupling, as well as promoting the closing of regions related to alternative fates. Utilizing integrative genomics between ATAC-seq and RNA-seq datasets, we identify the desmosome machinery as an important feature of iPM formation. In parallel, we define a novel Hand2 domain region that regulates cardiac subtype diversity. Taken together, our results showcase Hand2-dependent mechanisms for iPM formation and gives insight into the improvement of future iPM engineering.Item Molecular Underpinnings of Human Brain Evolution and Cognition at Cellular Resolution(December 2023) Caglayan, Emre; Chahrour, Maria; Hon, Gary C.; Madabhushi, Ram; Sun, Lu O.; Konopka, GenevieveMolecular and functional characterization of the human brain is challenging due to its experimental inaccessibility. Most of our understanding about human brain function relies on the assumption that biological processes uncovered in model organisms are conserved in humans. Comparisons of the humanii brain with non-human primate brains offer to both uncover the novelties in human brain evolution and better evaluate the insights obtained from model organisms about human brain function. To achieve this, highthroughput sequencing methods on post-mortem brain tissues provide a rewarding readout to understand human brain evolution at the molecular level. In addition to their use in comparative studies, these technologies were also utilized with a hope to understand molecular underpinnings of measurable human brain activity metrics. During my dissertation, I read relevant literature extensively (Chapter 1) and sought to understand human-specific epigenomic and transcriptomic changes at cellular resolution in the cortical brain (Chapter 2). Additionally, after in-depth analysis of many human brain single-nuclei RNA-seq datasets, I found a pervasive ambient RNA contamination problem, and devised in silico solutions to tackle this problem. My efforts improved the analytical approach in the field as well as in my research (Chapter 3). I have also been involved in efforts to identify transcriptomic correlates of brain activity in human subjects (Chapters 4-5). After detailing these efforts, I discuss the implications of these findings, weigh their impact on our understanding of human brain function and offer ideas for further research (Chapter 6).Item Novel Roles for BET Bromodomain Protein 4 (BRD4) in Cardiac Physiology and Disease(2020-08-01T05:00:00.000Z) Kim, Soo Young; Munshi, Nikhil; Chiang, Cheng-Ming; Rothermel, Beverly A.; Gupta, Rana K.; Hill, Joseph A.; Gillette, Thomas G.Bromodomain (BRD) protein of the BET (Bromodomain and Extra-Terminal) family are epigenetic reader proteins that have emerged as novel therapeutic targets in cardiovascular disease as well as in a variety of cancers. Small molecule BET inhibitors, such as JQ1, have demonstrated efficacy in reversing cardiac hypertrophy and heart failure in preclinical models. Yet, genetic studies elucidating the biology of BRD proteins in the heart have not been conducted to validate pharmacological findings and unveil potential side effects. Focusing on BRD4, we tested the hypothesis that cardiomyocyte BRD4 drives pathological cardiac remodeling in the setting of disease-related stress. To facilitate these studies, we engineered a cardiomyocyte-specific BRD4 knockout mouse. Using this model, we investigated the role of BRD4 in cardiac physiology and disease. To our surprise, loss of BRD4 protein triggered a spontaneous and progressive decline in myocardial contractile performance, culminating in dilated cardiomyopathy. Transcriptome analysis of BRD4 knockout mouse hearts showed early and specific disruption of genes essential to mitochondrial energy production and homeostasis. Functional analysis of isolated mitochondria confirmed that BRD4 ablation results in specific changes in protein levels and activity of the mitochondrial electron transport chain. Comparative analysis of the JQ1-altered transcriptome suggests that a BRD4-dependent effect of BET inhibition includes changes in transcription of nucleus-encoded mitochondrial genes, raising concerns for cardiotoxicity with potent pharmacological BET inhibition. Furthermore, we tested the roles of BRD4 isoforms, BRD4-L and BRD4-S(a), in cardiomyocyte biology. Isoform-specific knockdown of BRD4 using siRNAs in primary cardiomyocyte culture demonstrated that BRD4-S(a) is required for cardiomyocyte hypertrophy. BRD4-S(a) expression was low in naïve hearts, but it increased significantly in remodeling and failing hearts. Moreover, transgenic over-expression revealed that the BRD4-S(a) isoform is sufficient to induce hypertrophic remodeling and heart failure. In contrast, restoring BRD4-L expression partially rescued systolic dysfunction in animals with a cardiomyocyte specific deletion of BRD4. In conclusion, present study provides evidence that BRD4 regulates cardiomyocyte mitochondrial homeostasis, and that it is required for maintaining normal cardiac function in rodents. Moreover, we demonstrated that the BRD4 short isoform is a driver of pathological cardiac hypertrophy, whereas the long isoform may have a homeostatic role. In aggregate, we have identified novel roles of BRD4 in cardiomyocyte biology, unveiling critical insights - as well as caveats - regarding the therapeutic targeting of BRD proteins in heart failure.