The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1.
I hypothesize that duplication events grant miRNA families with enhanced regulatory capabilities, specifically through distinct targeting preferences by family members. This has relevance for our understanding of vertebrate evolution, as well disease detection and personalized medicine. To test this hypothesis, I apply a conjunction of bioinformatic and experimental approaches, and design a novel high-throughput screening platform to identify human miRNA targets. Combined with conventional approaches, this tool allows systematic testing for functional targets of human miRNAs, and the identification of novel target genes on an unprecedented scale.
In this dissertation, I explore evolutionary signatures of 62 deeply conserved metazoan miRNA families, as well as the targeting preferences for several human miRNAs. I find that constraints on miRNA processing impact sequence evolution, creating evolutionary hotspots within families that guide distinct target preferences. I apply our novel screening platform to two cancer-relevant miRNAs, and identify hundreds of previously undescribed targets. I also analyze critical features of functional miRNA target sites, finding that each miRNA recognizes surprisingly distinct features of targets. To further explore the functional distinction between family members, I analyze miRNA expression patterns in multiple contexts, including mouse embryogenesis, RNA-seq data from human tissues, and cancer cell lines. Together, my results inform a model that describes the evolution of metazoan miRNAs, and suggests that highly similar miRNA family members possess distinct functions. These findings broaden our understanding of miRNA function in vertebrate evolution and development, and how their misexpression contributes to human disease.
The conservation of the dystrophin gene across metazoans suggests that both vertebrate and invertebrate model systems can provide valuable contributions to the understanding of DMD initiation and progression. Specifically, the invertebrate C. elegans possesses a dystrophin protein ortholog, dys-1, and a mild inflammatory response that is inactive in the muscle, allowing for the characterization of transcriptome rearrangements affecting disease progression independently of inflammation. Furthermore, C. elegans do not possess a satellite cell equivalent, meaning muscle regeneration does not occur. This makes C. elegans unique in that they allow for the study of dystrophin deficiencies without muscle regeneration that may obscure detection of subtle but consequential changes in gene expression.
I hypothesize that gaining a comprehensive definition of both the structural and signaling roles of dystrophin in C. elegans will improve the community’s understanding of the progression of DMD as a whole. To address this hypothesis, I have performed a phylogenetic analysis on the conservation of each member of the dystrophin associated protein complex (DAPC) across 10 species, established an in vivo system to identify muscle-specific changes in gene expression in the dystrophin-deficient C. elegans, and performed a functional analysis to test the biological significance of changes in gene expression identified in my sequencing results. The results from this study indicate that in C. elegans, dystrophin may have a signaling role early in development, and its absence may activate compensatory mechanisms that counteract disease progression. Furthermore, these findings allow for the identification of transcriptome changes that potentially serve as both independent drivers of disease and potential therapeutic targets for the treatment of DMD.
Domestic dogs have assisted humans for millennia. However, the extent to which these helpful behaviors are prosocially motivated remains unclear. To assess the propensity of pet dogs to spontaneously and actively rescue distressed humans, this study tested whether sixty pet dogs would release their seemingly trapped owners from a large box. To examine the causal mechanisms that shaped this behavior, the readiness of each dog to open the box was tested in three conditions: 1) the owner sat in the box and called for help (“Distress” test), 2) an experimenter placed high-value food rewards in the box (“Food” test), and 3) the owner sat in the box and calmly read aloud (“Reading” test).
Dogs were as likely to release their distressed owner as to retrieve treats from inside the box, indicating that rescuing an owner may be a highly rewarding action for dogs. After accounting for ability, dogs released the owner more often when the owner called for help than when the owner read aloud calmly. In addition, opening latencies decreased with test number in the Distress test but not the Reading test. Thus, rescuing the owner could not be attributed solely to social facilitation, stimulus enhancement, or social contact-seeking behavior.
Dogs displayed more stress behaviors in the Distress test than in the Reading test, and stress scores decreased with test number in the Reading test but not in the Distress test. This evidence of emotional contagion supports the hypothesis that rescuing the distressed owner was an empathetically-motivated prosocial behavior. Success in the Food task and previous (in-home) experience opening objects were both strong predictors of releasing the owner. Thus, prosocial behavior tests for dogs should control for physical ability and previous experience.
We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.
MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene output at the post-transcriptional level by targeting degenerate elements primarily in 3′untranslated regions (3′UTRs) of mRNAs. Individual miRNAs can regulate networks of hundreds of genes, yet for the majority of miRNAs few, if any, targets are known. Misexpression of miRNAs is also a major contributor to cancer progression, thus there is a critical need to validate miRNA targets in high-throughput to understand miRNAs' contribution to tumorigenesis. Here we introduce a novel high-throughput assay to detect miRNA targets in 3′UTRs, called Luminescent Identification of Functional Elements in 3′UTRs (3′LIFE). We demonstrate the feasibility of 3′LIFE using a data set of 275 human 3′UTRs and two cancer-relevant miRNAs, let-7c and miR-10b, and compare our results to alternative methods to detect miRNA targets throughout the genome. We identify a large number of novel gene targets for these miRNAs, with only 32% of hits being bioinformatically predicted and 27% directed by non-canonical interactions. Functional analysis of target genes reveals consistent roles for each miRNA as either a tumor suppressor (let-7c) or oncogenic miRNA (miR-10b), and preferentially target multiple genes within regulatory networks, suggesting 3′LIFE is a rapid and sensitive method to detect miRNA targets in high-throughput.