Interdisciplinary Design Studio: Programming Document Visioning for a Robotic Demonstration, Research, and Engagement Dairy

The 2022 COLLABORATE Design Studio brought together students from various disciplines to address a complex, real-world project which required collaborative input from different perspectives. The studio worked to advance the co-creation of knowledge between external stakeholders, students, and instructors. The course was co-taught by faculty from different disciplines, and areas of expertise. During the semester, Nate Bicak and Steven Hardy worked with students from Architecture and Interior Design in collaboration with students in Dr. Tami Brown-Brandl’s students in Biological Systems Engineering and Animal Science to explore the values, spatial qualities, and area requirements of a Robotic Demonstration, Research, and Engagement Dairy. Students organized a series of meetings and participatory activities to gather information from a range of project stakeholders including: Heather Akin (Agricultural Leadership, Education & Communication), Kris Bousquet (NE Dairy Association), Paul Kononoff (Animal Science), Eric Markvicka (Mechanical and Material Engineering), Julia McQuillan (Sociology), Santosh Pitla (BioSystems and Agricultural Engineering), Ling Ling Sun (NE Public Media), and Rosanna Villa Rojas (Food Science & Technology). The information gathered helped to frame the overall problem - both quantitative and qualitative - to be addressed during the design visioning stage (not included in this document). Student contributors included: Sarah Alduaylij, Noor Al-Maamari, Devyn Beekman, Kelsey Belgum, Lauren Chubb, Nicholas Forte, Mitchell Hill, Joshua Holstein, Dylan Lambe, Phuong Le, Mia LeRiger, Elizabeth Loftus, Josh Lorenzen , Megan Lovci, Alex Martino, Zade Miller, Hannah Morgan , Annabelle Nichols , Collin Shearman, Rebecca Sowl, Nalin Theplikhith, Angela Vu, Shaylee Wagner, Ethan Watermeier, Trever Zelenk

Lessons from non-canonical splicing

Recent improvements in experimental and computational techniques that are used to study the transcriptome have enabled an unprecedented view of RNA processing, revealing many previously unknown non-canonical splicing events. This includes cryptic events located far from the currently annotated exons and unconventional splicing mechanisms that have important roles in regulating gene expression. These non-canonical splicing events are a major source of newly emerging transcripts during evolution, especially when they involve sequences derived from transposable elements. They are therefore under precise regulation and quality control, which minimizes their potential to disrupt gene expression. We explain how non-canonical splicing can lead to aberrant transcripts that cause many diseases, and also how it can be exploited for new therapeutic strategies

High-throughput computation to uncover novel mechanisms of RNA regulation

My thesis work is aimed at integrating high-throughput biochemical readouts to understand the effect that particular RNA-binding proteins have on their targets' metabolism. I studied several RNA-binding proteins in diverse model organisms. Along the way I have gained insight into the characteristics of target sites for two families of proteins: Argonaute and RBFOX proteins and learned about novel mechanisms they use to control their targets' fate.In this dissertation, I will present three of my articles that summarize the methods and content of my work. In the first chapter I give an overview of methods I used to quantify the ideal depth of coverage for sequencing experiments in order to sufficiently capture a desired level of complexity. This included the development of novel computational approaches to quantify RNA abundance and splicing with sequencing tools. This was applied to a human model of prostate cancer, LNCaP cells upon stimulation with an androgen compound.To closely examine mechanisms of miRNA regulation, we generated genome wide maps of the Argonaute protein ALG-1's binding in C. elegans. We found that there exists a large potential for non-canonical associations of ALG-1/miRNA complexes with their targets. Among the surprises we encountered, ALG-1 binds in coding exons but does not seem to repress gene expression when bound there, and we also found an auto-regulatory repression by ALG-1 on miRNA pathway components. Finally, I turned my attention to the RBFOX family of proteins that is known for their role in RNA splicing regulation. In the work presented in chapter 4, we elucidated a new molecular mechanism whereby RBFOX proteins can regulate RNA splicing from very distant sites. These RNA-bridges, as we have called them because they link RNA regulators with regulated sites via RNA structures, appear to be a common feature of alternatively spliced exons and the regulation of RNA structures like this may be important for dictating splicing outcomes. The application of this knowledge that distant binding sites are functional and that they are mediated by RNA structures is immediately relevant to the design of novel therapeutics for diseases that arise from defects in RNA splicing. To put the goals of my thesis broadly, I approach two questions: what are the mechanisms of RNA-binding protein targeting, and what are the effects of RNA-binding proteins on their direct targets

Post-translational Modifications And Rna-binding Proteins

RNA-binding proteins affect cellular metabolic programs through development and in response to cellular stimuli. Though much work has been done to elucidate the roles of a handful of RNA-binding proteins and their effect on RNA metabolism, the progress of studies to understand the effects of post-translational modifications of this class of proteins is far from complete. This chapter summarizes the work that has been done to identify the consequence of post-translational modifications to some RNA-binding proteins. The effects of these modifications have been shown to increase the panoply of functions that a given RNA-binding protein can assume. We will survey the experimental methods that are used to identify the presence of several protein modifications and methods that attempt to discern the consequence of these modifications.90729731

Pairing beyond the Seed Supports MicroRNA Targeting Specificity

To identify endogenous miRNA-target sites, we isolated AGO-bound RNAs from Caenorhabditis elegans by individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP), which fortuitously also produced miRNA-target chimeric reads. Through the analysis of thousands of reproducible chimeras, pairing to the miRNA seed emerged as the predominant motif associated with functional interactions. Unexpectedly, we discovered that additional pairing to 3' sequences is prevalent in the majority of target sites and leads to specific targeting by members of miRNA families. By editing an endogenous target site, we demonstrate that 3' pairing determines targeting by specific miRNA family members and that seed pairing is not always sufficient for functional target interactions. Finally, we present a simplified method, chimera PCR (ChimP), for the detection of specific miRNA-target interactions. Overall, our analysis revealed that sequences in the 5' as well as the 3' regions of a miRNA provide the information necessary for stable and specific miRNA-target interactions in vivo

Deep sequencing identifies new and regulated microRNAs in Schmidtea mediterranea

MicroRNAs (miRNAs) play important roles in directing the differentiation of cells down a variety of cell lineage pathways. The planarian Schmidtea mediterranea can regenerate all lost body tissue after amputation due to a population of pluripotent somatic stem cells called neoblasts, and is therefore an excellent model organism to study the roles of miRNAs in stem cell function. Here, we use a combination of deep sequencing and bioinformatics to discover 66 new miRNAs in S. mediterranea. We also identify 21 miRNAs that are specifically expressed in either sexual or asexual animals. Finally, we identified five miRNAs whose expression is sensitive to γ-irradiation, suggesting they are expressed in neoblasts or early neoblast progeny. Together, these results increase the known repertoire of S. mediterranea miRNAs and identify numerous regulated miRNAs that may play important roles in regeneration, homeostasis, neoblast function, and reproduction

Functional genomic analysis of the let-7 regulatory network in Caenorhabditis elegans.

The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7-dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development

Hartmann_etal_MEC_Fig_1_data

The gene expression data obtained from Orbicella faveolata larvae and used to generate Figure 1. The data matrix was filtered to only the genes that successfully hybridized (i.e., produced data) in at least three replicates for each treatment. Missing values were imputed and all values were normalized to a mean of zero and the same unit variance in each gene. The 'Name' column provides the gene annotation, if there is one. The subsequent columns contain data for the 20 samples. The sample names refer treatment group, which includes light status (L = Light, D = Dark), symbiont status (S = Symb, NS = NoSymb), and replicate number (1-5)