Serotonin modulates a wide array of neurobehavioral and physiologic processes in mammals, and it has been implicated in the pathophysiology of clinical disorders ranging from primary pulmonary hypertension to irritable bowel syndrome. Yet, the specific cellular and molecular mechanisms underlying the modulatory role of serotonin is still largely obscure, in part due to the multiple receptors and release sites for serotonin, and in part due to the lack of highly specific pharmacologic tools for these receptors. Indeed, serotonin signals thru some 15 receptors, 14 of which are G-Protein Coupled Receptors (GPCRs).

Here, I have used mouse genetic techniques to examine how 5-HT receptors and other similar GPCRs regulate two diverse biological processes: pancreatic islet development and novelty-related behavior. My results suggest that serotonin and Gi-coupled receptor signaling play a critical role in the former by regulating cell division during pancreatic development, and that serotonin and the 5-HT2C receptor play a critical role in the latter by regulating dopamine release in the dorsal striatum. Taken together, these results highlight the wide array of biological processes that are orchestrated by serotonin and its receptors, and they shed light on how evolution has co-opted an ancient neurotransmitter into regulating some of the most complex mammalian physiological and behavioral processes.

The ancient philosophers emphasized the virtues of balance and moderation, which resonate across cultures. This principle extends to scientific realms, notably evident in oxygen homeostasis. While insufficient or excessive oxygen levels can be detrimental, the optimal threshold remains elusive. Throughout Earth's history, oxygen levels have fluctuated significantly, prompting the questions how organisms respond to varying oxygen tensions. This thesis examines the impacts of oxygen in diverse disease settings and investigates cellular and organismal responses to aberrant oxygen levels.

First, we delve into the “optimal” oxygen level for mitochondrial disorders. Mitochondria diseases are devastating conditions without effective therapies, affecting thousands of children globally. Hypoxia emerges as a potential therapeutic avenue, previously explored in Leigh Syndrome, showcasing extended survival and mitigation of associated deficits. This study investigates hypoxia's generalizability across mitochondrial disorders, focusing on thiamine and pyruvate dehydrogenase deficiencies, highlighting its therapeutic potential beyond Leigh Syndrome. This study also reveals that for certain disease setting, the ambient oxygen level (21% oxygen) can cause toxicity and exacerbate disease, prompting a thorough investigation into the potential toxic effects of oxygen.

While excess oxygen is toxic to all known organisms, the underlying molecular mechanisms remain largely unexplored. This work delves into the effects of excess molecular oxygen, uncovering its effect on destabilizing a subset of iron-sulfur cluster (ISC)-containing proteins. This disruption hampers crucial processes such as diphthamide synthesis, purine metabolism, nucleotide excision repair, and electron transport chain (ETC) function. Our findings extend to a mouse model and primary human lung cells. In particular, we reveal that the ETC is the “Achilles' heel” in hyperoxia, leading to decreased mitochondrial oxygen consumption and subsequent tissue hyperoxia. This cyclic damage affects additional ISC-containing pathways. Notably, primary ETC dysfunction exacerbates lung tissue hyperoxia.

After observing the destabilizing effects of hyperoxia on ISC proteins, our subsequent step involves a systematic exploration of the influence of diverse oxygen tensions on protein turnover. We demonstrate that the lung proteome shows the most significant changes. Remarkably, extracellular matrix (ECM) proteins are stabilized in the lung under both hypoxia and hyperoxia, implying post-translational regulation in ECM remodeling. Furthermore, we identify MYBBP1A as a potential hyperoxia transcriptional regulator, specifically involved in rRNA homeostasis. Overall, our study underscores the intricate interplay between oxygen tensions and protein turnover, highlighting key mediators of oxygen-dependent signaling pathways. Our work has broad implications for disease including hyperoxia-related pathologies such as bronchopulmonary dysplasia, ischemia-reperfusion injury, aging, and mitochondrial disorders.

Lastly, we investigate the nexus between impaired proteostasis, varying oxygen tensions, and cardiac diseases. Leveraging human iPSC-derived cardiomyocytes, this research reveals a significant reduction in global protein turnover rates under hypoxia, particularly affecting biosynthetic and catabolic pathways. Notably, the study identifies potential targets for prolyl hydroxylases, suggesting their role in regulating responses to chronic hypoxia. This comprehensive exploration illuminates the nuanced effects of oxygen levels on proteostasis in cardiac pathology, paving the way for deeper insights into therapeutic interventions for cardiovascular diseases.

Collectively, these comprehensive investigations offer insights into the intricate interplay between oxygen tensions, cellular responses, and the pathophysiology of disease. They pave the way for interventions across diverse disease states impacted by oxygen homeostasis.

Background: G-protein signaling pathways regulate many aspects of cardiovascular development and disease. A-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate these signaling events into specific physiological processes. One family member, AKAP13, integrates Gs, Gq/11, and G12/13 signals through binding of protein kinase A, C (PKC), and D (PKD) and encoding an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. AKAP13 is required for mouse development as null embryos die by E10.5 and exhibit cardiovascular defects. Additionally, the Rho-GEF and PKC-PKD binding domains mediate cardiomyocyte hypertrophy in isolated cardiomyocytes. However, the specific developmental processes regulated by AKAP13 and the requirements for the Rho-GEF and PKC-PKD binding domains during development and cardiac hypertrophy are unknown.

Methodology/Principal Findings: We used an RNAi, loss-of-function approach, to determine if AKAP13 mediates differentiation of mouse embryonic stem (ES) cells or angiogenic processes in human umbilical vein endothelial cells (HUVECs). We found that AKAP13 knockdown did not decrease ES cell differentiation into Nkx2.5-GFP-positive cardiac mesodermal cells or functionally beating cardiomyocytes. Similarly, the expression of endodermal, endothelial, or cardiac structural marker genes was not affected. We also found that AKAP13-deficient HUVECs had normal tube formation, VEGF-induced wound healing, and VEGF-induced PKD phosphorylation. We then generated AKAP13-truncation mutant mice to determine if the Rho-GEF and PKC-PKD binding domains are required for development. Surprisingly, homozygous mutant mice were born at normal Mendelian ratios and had normal survival, fertility, and cardiac structure. Finally, to determine if these domains mediate β-adrenergic-induced cardiac hypertrophy, we stressed the mice with isoproterenol. We found that heart size increased normally in mice lacking the Rho-GEF and PKC-PKD binding domains. However, these hearts failed to increase cardiac ejection fraction or fractional shortening.

Conclusions: These results indicate that AKAP13 is not required for differentiation into cardiovascular tissue, the formation of functional cardiomyocytes, or angiogenic processes. They also indicate that the AKAP13 Rho-GEF and PKC-PKD binding domains are not required for mouse development or the cardiac structural response to β-adrenergic-induced hypertrophy. However, these domains are required for the proper cardiac functional response to β-adrenergic stimulation.

The development of an organism from conception to adulthood requires the specification of cell types to distinct fates. In an adult organism, tissues can similarly undergo dramatic transformation, altering their structure, physiology, and overall biochemical properties. In both of these paradigms, the study of gene transcription and its contribution to protein content in the cells has been the primary focus. While clearly important, more recently, alternative splicing and microRNA regulation have been recognized as significant processes that can have crucial regulatory influences on the diversity of mRNAs produced and their over-all expression in the cell.

In this dissertation, I have set out to characterize the molecular changes that occur in two distinct cellular paradigms, muscle remodeling in the uterus throughout pregnancy and the differentiation of embryonic stem cells to distinct fates using DNA microarray technology. To achieve this goal, I developed several new software applications, designed specifically to assess the relevance of coordinated gene regulatory events along biological pathways (GenMAPP and GO-Elite) and characterize sequence level functional attributes of proteins and mRNAs regulated by alternative splicing (AltAnalyze).

Studies of the mouse uterus during gestation reveal novel coordinated transcriptional networks regulating quiescence, contraction, and involution when multiple time-points are considered. Analysis of alternative splicing in mouse and human embryonic stem cell differentiation uncovered novel mechanisms for the regulation of protein domain and microRNA binding site inclusion and at least for one gene, Tcf3, the requirement of splicing to properly promote the early steps of embryonic lineage commitment down multiple paths. In summary, I have used novel computational methods and genomic resources to uncover new regulatory networks and potential biological mechanisms from both differentiating and transitioning cells, that involve regulation at the level of transcription, alternative splicing, and translational regulation by micoRNAs.

Genetic association studies have yielded a wealth of information on specific variants that lead to the development of disease, but our mechanistic understanding for most of them is lacking. Unbiased, comparative study of protein-protein interactions (PPI) for protein coding variants is a very promising approach to fill this gap. To yield data that can inform us on tissue-specific pathologies (e.g. cardiovascular, neurological disease) we need to study PPIs in the cell types affected by the disease. In a similar fashion, studying proteins expressed endogenously would allow us to overcome issues related to gene expression or dose-dependent protein function, which is a feature of many of these pathologies.

Here I present my work trying to unravel the mechanism mutations in the BAG3 chaperone gene lead to disease. To address this, I used a combination of induced pluripotent stem (iPS) cell technology (to generate cardiac myocytes), genome engineering (to induce specific disease-modifying mutations and protein fusions) and affinity purification coupled to mass spectrometry (to compare PPIs between variants). The results show that disease-related variants of BAG3 display a different profile of protein partners, some of them cardiac-specific, pointing towards interactions that potentially underlie the disease mechanism and tissue specificity. We also observed a cell culture phenotype for BAG3 deficiency and for some of the protein-coding variants, which sets the ground for the exploration of the role of BAG3 interactions in disease.

Overall, this piece of work proves the value of studying cardiac disease-related genetic variability using a disease-relevant iPS-derived model and genome engineering to explore multiple variants. In particular, we provide new clues on the role of BAG3 in the heart, and how that role is compromised by disease-related mutations. We hope that the knowledge gained on BAG3 genetics and mechanism will be useful for the development of therapeutic strategies in the future.

G-protein-coupled receptors (GPCRs) signal through a limited number of G-protein pathways and are crucial in many biological processes. The molecular and functional diversity of GPCRs and the lack of ligands with specific signaling effects have complicated studies of their in vivo functions. To better compare the effects of activating different G-protein-signaling pathways through agonist-mediated or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) activating through different G-protein-signaling pathways. These RASSLs are based on the human 5-HT4 receptor, a GPCR with a high level of constitutive Gs signaling and strong agonist-mediated G-protein activation of Gs and Gs/q pathways. The first receptor in this series, 5-HT4-D100A or Rs1 (RASSL serotonin 1), is not activated by its endogenous agonist, serotonin, but is selectively activated by the small synthetic molecules GR113808, GR125487, and RO110-0235 (antagonists and inverse agonists for the 5-HT4 receptor). All agonists potently induced Gs signaling, but only a few (e.g., Zacopride) also induced signaling via the Gq pathway. Zacopride-induced Gq signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT2C receptor. Additional point mutations (D66A and D66N) blocked constitutive Gs signaling and lowered agonist-mediated Gq signaling. Finally, replacing the third intracellular loop of Rs1 with that of human 5-HT1A conferred ligand-mediated Gi signaling. This Gi-coupled RASSL, Rs1.3, exhibited no measurable signaling to the Gs or Gq pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection. Here, we describe a new series of RASSLs developed to modify the agonist-mediated and constitutive signaling of the human 5-HT4 receptor. Since none of the inverse agonists for the 5-HT4 receptor works in the presence of the D100A mutation, I also engineered a series of synthetic internal ribosomal entry sites and a single plasmid tetracycline-inducible (tet) system to better control GPCR expression. Combined with a tissue-specific promoter, these RASSLs can be expressed in a tissue-specific manner and at specific levels. With these additional tools, RASSLs will help us better study the effect of constitutive Gs signaling and agonist-mediated Gs, Gs/Gq, and Gi signaling in vivo.

A variety of new procedures have been devised to handle the two sample comparison (e.g., tumor versus normal tissue) of gene expression values as measured with microarrays. Such new methods are required in part because of some defining characteristics of microarray-based studies: (i) the very large number of genes contributing expression measures which far exceeds the number of samples (observations) available, and (ii) the fact that by virtue of pathway/network relationships, the gene expression measures tend to be highly correlated. These concerns are exacerbated in the regression setting, where the objective is to relate gene expression, simultaneously for multiple genes, to some external outcome or phenotype. Correspondingly, several methods have been recently proposed for addressing these issues. We briefly critique some of these methods prior to a detailed evaluation of gene harvesting. This reveals that gene harvesting, without additional constraints, can yield artifactual solutions. Results obtained employing such constraints motivate the use of regularized regression procedures such as the lasso, least angle regression, and support vector machines. Model selection and solution multiplicity issues are also discussed. The methods are evaluated using a microarraybased study of cardiomyopathy in transgenic mice.