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Synthesis of Sulfonyl Fluorides and the Total Synthesis of the Rhodocorane Natural Products
No full textThe selective reactivity of different functional groups is at the forefront of modern synthetic chemistry. As predictable and controllable reactivity are the key to the synthesis of many bioactive and structurally challenging molecules, synthetic methodologies to install and understand these reactive moieties is always of interest to organic chemists. However, there is a fine line between too reactive and not reactive enough. Our studies on the development of new catalytic methods to synthesize sulfonyl fluorides via two separate photocatalytic methods and the synthesis of the rhodocorane family of bioactive natural products are described.
In the first chapter, an overview of the development and the understanding of the reactivity of sulfonyl fluorides as a second generation "click" reaction is discussed. Various studies from the literature show that these moieties can undergo a selective activation and become reactive in both chemical and biological instances. The potential applications of these interesting functionalities are limitless, with the only hindrance being the lack of easily accessible syntheses. The understanding of that activation is discussed along with a variety of ways to synthesize both aliphatic and aryl sulfonyl fluorides.
The second chapter describes our studies in the development of a photocatalytic method to convert anilines into sulfonyl fluorides through an activated and isolated substrate class of diazonium salts. Our approach allows for a mild and selective method to synthesize aryl sulfonyl fluorides from a widely available functional group while being tolerant of a range of other functionalities. We also present various experimental and theoretical studies to analyze the mechanistic pathways involved in the methodology.
In the third chapter, we describe our effort to develop a separate organophotocatalytic methodology to synthesize aliphatic sulfonyl fluorides from potassium trifluoroborate salts. Our synthetic strategy leverages the activated boronate species to generate aliphatic radicals with the highly oxidizing organophotocatalyst, which undergoes a three-component coupling with a sulfur dioxide source and a fluorine source. The challenges associated with this methodology along with photophysical measurements and evidence for the mechanism are discussed.
In the fourth chapter, we discuss our approach to the asymmetric synthesis of the rhodocorane family of bioactive natural products. These natural products possess a range of spirocyclic, fused rings, and highly oxygenated systems that allow for structurally diverse and complex molecules. We propose a common synthetic intermediate that can access a majority of this family through the addition of other commercially available molecules. The challenges and optimization of this synthesis and our approaches to solve these problems are discussed
Explaining Racial Discrepancies in Verbal Memory Assessment with Joint Estimation of Life Course Social Inequalities and Measurement Bias
No full textDecades of research in cognitive test performance have demonstrated that racial group differences appear on a wide variety of tests and samples. The development of new tests, statistical adjustments, and incorporation of additional sociocultural information have contributed to varying degrees of success in the resolution of these differences. At the same time, racially minoritized communities are affected by higher rates of dementia, and yet require, on average, greater levels of severity in cognitive impairment to be diagnosed. To improve the detection of dementia at earlier stages where interventions are more likely to be useful and to facilitate better research into treatments where cognitive test performance is the standard outcome of interest, solutions to measurement differences between racial groups need to be identified. This study proposes a novel theoretically informed psychometric approach to separate cognitive test performance into factors related to life course disparities and those related to measurement bias. Utilizing the Consortium to Establish a Registry for Alzheimer's Disease list learning test administered as part of the national Harmonized Cognitive Assessment Protocol (HCAP) study conducted in partnership with the Health and Retirement Study, the pervasive bias model is used to identify sources of measurement differences, quantify the remaining impact of social inequities on performance differences, and evaluate the potential clinical applications of the approach. Utilizing a sample of 2074 cognitively normal adults over the age of 65 who identified as either racially White or Black, the pervasive bias model revealed that race had minimal direct contributions to measurement bias. In contrast, educational attainment was found to cause measurement bias on the list learning test, and since there were significant differences between the racial groups' years of education, this effect explained apparent racial group differences. Importantly, accounting for education's effects on the measurement of memory not only removed evidence for racial group differences but provided positive evidence overall that the two racial groups had equivalent overall memory abilities. While promising as a new approach to understanding test data, additional evaluation, particularly with clinical samples, is needed before the potential of this novel approach is truly understood
Dynamic Shuttling of AGO2 to the Nucleus Relieves MicroRNA Repression
No full textThe Argonaute (AGO) and the Trinucleotide Repeat Containing 6 (TNRC6) family proteins are the core components of the mammalian microRNA-induced silencing complex (miRISC). This machinery mediates post-transcriptional silencing of mRNA targets by sequence-specific hybridization with microRNAs (miRNAs) in the cytoplasm. Localization to subcellular compartments is critical to understanding miRNA action, and our lab and others have shown that miRISC complexes are also present and active in mammalian nuclei. miRISC has several proposed nuclear functions, yet the biological significance and regulatory mechanisms of action of nuclear miRISC in mammalian cancer cells remain poorly understood. We observed that three different model systems that mimic colon tumor microenvironments: (1) 2D colorectal cancer cell, HCT116, cultures grown beyond a monolayer to high cell density, (2) 3D HCT116 tumor spheroid cultures grown in collagen matrix, and (3) primary tissue samples from malignant colon tumor and normal adjacent colon tissue, show significant nuclear enrichment of core RISC effector protein, AGO2.
We tested the consequences of nuclear AGO2 localization on global microRNA regulation using a multipronged transcriptomics approach. We combined AGO2-eCLIP-sequencing, small-RNA-sequencing, and whole transcriptome sequencing to identify candidate gene targets bound by AGO2 that may be regulated by the most abundant microRNA families. We observed that the majority of AGO2-bound cytoplasmic 3'UTR miRISC targets were significantly upregulated, potentially de-repressed, when AGO2 is nuclear-enriched. Using an AGO2 construct with a nuclear localization sequence (NLS-AGO2-GFP), we were able to phenocopy the de-repression of cytoplasmic candidate genes and observe a correlation with a higher rate of migration. Our findings reveal dynamic relocalization of AGO2 to the nucleus in colon cancer culture systems and primary colon tumor tissue, which may impact our understanding of spatial regulation of microRNA action and reveal therapeutic targets that contribute to colon tumor progression.
Another study from my thesis research provides a systemic evaluation of the effects of depleting microRNA biogenesis enzyme, DROSHA, and microRNA effector proteins, AGO1-4 and TNRC6A-C, on microRNA expression. Our findings help to define the boundaries of RNAi in a widely used cell line, HCT116, by refining a subset of abundant microRNAs that are DROSHA-dependent, associated with Argonaute proteins, and most likely to carry out robust gene regulation
The Role of Metabolic Inflexibility in Heart Failure
No full textHeart failure (HF) is a syndrome defined by the inability of the heart to supply adequate nutrients and oxygen to peripheral tissues. One of the most common causes of HF is chronic pressure overload due to hypertension, which leads to hypertrophic remodeling, accompanied by metabolic alterations. These changes are initially compensatory but lead to worse outcomes in the long term, making them targets for therapeutic intervention. When hypertension and diabetes are present simultaneously, a type of HF with preserved ejection fraction (HFpEF) ensues. HFpEF is distinct from HF with reduced ejection fraction (HFrEF) and diabetes in clinical presentation and physiology, necessitating alternative treatments. Understanding metabolic alterations which contribute to the development and progression of HFpEF can aid us greatly in creating these treatments.
This study aimed to replicate the cellular metabolic profile in diabetic heart to 1) determine if energy deficiency plays a causative role in HFpEF progression, and 2) test the necessity of glucose oxidation for left ventricular hypertrophy development and progression to HF. Since pyruvate dehydrogenase (PDH) kinase 4 (PDK4) is the predominant PDK found in the heart and PDK4 expression in the heart increases with high circulating fat levels, I employed a genetic approach to modulate its expression in cardiomyocytes specifically. This animal model allows us to control the activity level of PDH by its phosphorylation as seen in diabetic patients while bypassing complications of altering metabolism systemically.
Deletion of PDK4 in cardiomyocytes of adult mice prior to a high-fat diet prevented PDH inactivation in response to increased free fatty acids, allowing continued use of glucose for energy production by the TCA cycle. The impact on HFpEF development was unclear as the control group failed to fully develop the phenotype. In a separate experiment, constitutive overexpression of PDK4 gene was used to persistently inhibit PDH before surveying cellular metabolism and its effect on cardiac function at baseline as well as in response to increased cardiac work due to pressure afterload. This revealed that cardiac energetic deficiency did not require hypertrophic growth, and hypertrophic growth was not blunted by lack of glucose oxidation. Taken together, these findings suggest that metabolic inflexibility in cardiomyocytes plays a role in HFrEF development, independently of LVH. Furthermore, the metabolic profile in our animal model resembles what little is known of in HFpEF patients more closely than in diabetic cardiomyopathy, paving the way for future studies
Systemic Impact of Insulin-Sensitized Adipocytes and Their Precursors
No full textMaintenance of healthy adipose, especially when challenged with a prolonged unhealthy diet, is crucial for the prevention of diabetes and metabolic disease. Previous studies have shown that healthy expansion of adipose tissue occurs via hyperplasia, rather than hypertrophy. Therefore, a better understanding of the adipocyte precursor can potentially lead to interventions that favor new adipocyte formation over unhealthy, hypoxic adipocyte enlargement. Insulin signaling is central to the development of type 2 diabetes mellitus; resistance within this pathway leads to global metabolic dysfunction and cardiometabolic disease. Downregulation of adipocyte PTEN, a downstream inhibitor of the insulin signaling pathway, can lead to insulin sensitization of the adipose tissue and, as a result, in whole body insulin sensitivity. Previous work from our lab has shown that insulin sensitization of mature adipocytes, or of only thermogenic adipocytes, is sufficient to improve global metabolic health. We now report that by sensitizing murine adipocyte precursor cells to insulin action, we can induce durable improvements in glucose tolerance, lower circulating insulin levels, and eliminate steatohepatitis, without a change in overall body weight, even in high fat diet-fed mice. Remarkably, transplantation of small amounts of murine gonadal adipose tissue, containing insulin-sensitized precursors, is sufficient to induce improvements of global glucose handling and insulin signaling. The transplantation of such an insulin-sensitized fat pad can induce a durable reduction in circulating insulin even in high fat diet-fed mice. These studies demonstrate that targeting the relatively small cell population of adipocyte precursors is sufficient to induce widespread improvements in metabolic health, and that these cells may hold the key to future therapies for diabetes and metabolic disease
Delineating Mechanisms of Signal-Induced RNA Polymerase II-Transcription
No full textSignal-induced transcriptional programs regulate critical biological processes through the precise spatio-temporal activation of inducible gene programs. Understanding the dynamics by which RNA Polymerase II precisely induces transcription to activate these programs is important for dissecting the basis for their role in cell fate responses and disease progression. Here, we utilize high-resolution genomic approaches coupled with temporal signal induction to characterize how individual transcription steps contribute to the gene expression cycle in signal-induced transcription activation. Our first story (chapter 2) utilizes acute depletion approaches to reveal that the KAP1 protein is a positive regulator of transcription of immediate early genes, a class of signal-induced genes that regulate diverse biological processes including cancer and development. Mechanistically, KAP1 negatively regulates elongation rate at the early stages of transcription, which allows for proper kinetic progression through the transcription cycle by bolstering new initiation and full activation of gene expression. Overall, this study is the first report to link KAP1 "repressive" control of transcription to a positive role in gene activation and has implications for transcription-induced cell fate responses. Our second story (chapter 3) centers on ligand-induced transcription activation of the HIV-1 provirus. Decades of research has shown that HIV-1 transcription is primarily activated through pause release and transcription elongation by the HIV-1 transactivator Tat. Here, we use a novel Tat depletion approach to show that Tat function in pause release is the catalyst that promotes sustained RNA Polymerase II recruitment to the HIV-1 promoter. This recruitment of RNA Polymerase II is the mechanism that robustly induces "logarithmic" HIV-1 expression and viral replication. These data reveals a new significance for Tat function in transcription initiation and explains how Tat can sustain extended levels of HIV-1 transcription for proviral fate. Overall, the two studies have provided important mechanistic insights for understanding transcription dynamics in gene expression programs that have implications for diverse biological phenomena and pathogenesis
From Genetics to Neurodevelopment: Identifying the Role of the Chromatin Regulator KDM5A in Autism Spectrum Disorder
No full textAutism spectrum disorder (ASD) is a constellation of neurodevelopmental disorders with high phenotypic and genetic heterogeneity, complicating the discovery of causative genes. Through a forward genetics approach, we identified KDM5A as a candidate ASD gene, regulating vocalization and nest building in mice. We subsequently analyzed whole exome sequencing data from a clinical cohort and identified pathogenic KDM5A variants in patients with ASD. KDM5A encodes a chromatin regulator that belongs to the KDM5 family of lysine-specific histone H3 demethylases. Epigenetic chromatin regulation is essential for establishing and maintaining cellular identity and differentiation. It is required for normal brain development and proper gene expression as well as wiring of neuronal circuits. Disruptions in chromatin regulators lead to several diseases, including ASD. In fact, it is one of the top pathways disrupted in ASD (e.g., ARID1B, CHD8, KMT5B). To characterize the in vivo function of KDM5A, we developed a Kdm5a knockout mouse model (Kdm5a-/-) and showed that loss of KDM5A leads to severe social communication and interaction deficits, repetitive behaviors, and learning and memory deficits. Kdm5a-/- also showed an abnormal neuronal phenotype in the cortex and hippocampus, as well as disruption of transcriptional networks essential for normal brain functions. Patients with ASD in many cases present with cognitive deficits, which are often mediated by the cortex and the hippocampus. These two brain regions are composed of a variety of different cell types, each unique in its functions and transcriptome. However, the specific cell types that are affected in ASD as well as the cell-type specific transcriptional programs that are disrupted in this disease are unknown. To investigate this, we performed single-nuclei RNA sequencing from wildtype (WT) and Kdm5a-/- hippocampal tissue, and single-nuclei RNA and ATAC multiome sequencing from the cortex. We found that KDM5A is essential in establishing hippocampal and cortical cell identity, where specific subtypes of excitatory, inhibitory, and glial cells are disrupted. Our findings advance our knowledge of the role of epigenetic chromatin regulation in dictating cellular identities in the brain and help inform future efforts to develop therapeutic strategies in this genetic subtype of ASD
Pre- and Postsynaptic MEF2C Promote Experience-Dependent, Input-Specific Development of Local Cortical Excitatory Synapses
No full textComplex and specific neocortical circuits mature postnatally through a combination of genetic factors and sensory experience-driven neural activity. Experience- and activity-dependent transcriptional factor activation is a candidate mechanism for the development and refinement of these circuits. Synapses form the basis of neurons. Robust synapse proliferation during development is closely followed by experience-dependent pruning and modification to preserve and strengthen circuits. Neurodevelopmental disorders, including Autism Spectrum Disorder, are characterized by synaptic and circuit properties that give rise to behavioral and cognitive deficits and symptoms. Transcription factor Myocyte Enhancer Factor 2 C (MEF2C) is highly expressed in the cortex during development and into adulthood and has been identified as a regulator of synaptic strength and transmission in a sensory experience-dependent manner. MEF2C has been shown to function in both repressive and activator roles in postsynaptic compartments; however, little is known about presynaptic regulation by MEF2C. Additionally, the mechanisms by which MEF2C regulates synapses in an activity- and input-specific manner are still largely unknown.
My work provides evidence that the activity-dependent transcription factor MEF2C is required for experience-dependent development of inputs from Layer (L) 4 to L2/3 neurons in the mouse primary somatosensory barrel cortex (S1). Importantly, MEF2C is required in both presynaptic L4 and postsynaptic L2/3 neurons during the first two postnatal weeks for L4–L2/3 synapse development. MEF2C plays a local L4 input-specific role in postsynaptic L2/3 cells through the mechanism of reduced probability of presynaptic neurotransmitter release for L4 presynaptic MEF2C. Constitutively active MEF2C-VP16 can rescue the lack of whisker sensory input but does not rescue the loss of MEF2C in presynaptic neurons. Together, these results suggest that the activity-dependent transcriptional activation of MEF2C promotes the development of L4–L2/3 synapses. MEF2C is necessary for the activity-dependent expression of genes encoding pre-, post-, and transsynaptic proteins in cortical neurons. I examined the protein tyrosine kinase PYK2, which was found to be elevated in the cortex of MEF2C KO mice but appeared to be insufficient in affecting or regulating synaptic strength, like MEF2C. Altogether, this work provides insights into the mechanisms of MEF2C-mediatied, experience-dependent development of specific cortical circuits
Enhancing Nanoparticle Drug Delivery to Brain Tumors with Focused Ultrasound
No full textGlioblastoma mulitforme (GBM) is regarded as an incurable and universally fatal disease, characterized by its physical inaccessibility to most therapies and ultimately serve as a source for tumor recurrence which leads to the patient's demise. Furthermore, the prognosis for GBM has not improved significantly over the last 20 years. To achieve meaningful advances in the treatment of GBM, novel strategies must be devised to effectively treat these invasive glioma cells, therein halting the progression of the disease. Our laboratory's research centers on a nanoparticle biologic with the ability to selectively kill cancer cells despite uptake into normal healthy cells. This novel biologic therapy is based on plasma‐derived low‐density lipoprotein (LDL) reconstituted with the natural omega‐3 fatty acid, docosahexaenoic acid (DHA) (herein referred to as LDL‐DHA). However, therapeutic delivery to the brain is infamous due to the BBB that regulates the homeostasis of the brain by cell tight junctions with limited passage to certain molecules and nutrients. To bypass the BBB, Focused Ultrasound (FUS) combined with microbubbles are used to mechanically open the BBB to allow transient and targeted delivery of the brain. FUS in combination with LDL-DHA for delivery in normal rat brains showed safety of FUS and no evidence of cytotoxicity, respectively. However, delivery of the LDL particle by FUS to the brain has not been done in a tumor bearing animal model as well as the evaluation of LDL-DHA toxicity, uptake, and mechanisms in normal and malignant brain cell lines. In this dissertation, we focused on the development of a GBM tumor-bearing mouse model, delivery of LDL particles using FUS to the tumor, and assessment of particle cellular uptake and cytotoxicity by LDL-DHA in normal and malignant murine brain cells lines. In the following chapters we discuss accumulation of LDL particles in the surrounding mouse tumor by FUS, assessed mechanisms of particle uptake in normal and malignant brain cells, and showed the selective toxicity of LDL-DHA to GBM cell lines
The Role of Target-Directed MicroRNA Degradation in Mammalian Development
No full textPages vi-xvi are misnumbered as pages vii-xvii.MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. In animals, miRNAs canonically bind to partially complementary sites in messenger RNA 3′ untranslated regions, resulting in target repression. However, specialized targets, typically exhibiting extensive complementarity to the miRNA, can invert the regulatory logic and trigger degradation of the miRNA. Although this pathway, known as target-directed miRNA degradation (TDMD), has emerged as a potent mechanism of controlling miRNA levels, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8, which encodes an essential TDMD factor. Loss of ZSWIM8 resulted in developmental defects in heart and lung, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in co-transcribed clusters and examples in which TDMD underlies 'arm switching', a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two TDMD-regulated miRNAs, miR-322 and miR-503, rescued growth of Zswim8 null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. Together, these data reveal the broad landscape of TDMD in mammals and demonstrate that regulation of miRNA abundance by this pathway is essential for normal mammalian development