The placenta remains poorly studied despite being implicated in many pregnancy and chronic disorders. Trophoblast invasion and nutrient transport are critical placental processes where defects can lead to preeclampsia and other diseases. We set out to characterize protein pathways underlying those processes, using mouse as a model. We profiled the placenta proteome and phosphoproteome at embryonic day (e)7.5, when trophoblast invasion peaks, and at e9.5, when nutrient transport is occurring, and integrated this data with RNA-seq.

Comparing the unmodified proteome and the transcriptome revealed that most upregulated proteins are not the result of transcript changes. However, genes upregulated at both levels reflected expected functions, such as enrichment of migration processes at e7.5 and of metabolic processes at e9.5. Proteins that were only upregulated at the protein level contained potentially novel genes involved in migration and patterning, and indicated that the placenta at e9.5 is under stress.

The phosphoproteome revealed novel phosphosites on placental transcription factors (TFs) that were conserved in human and differentially phosphorylated in our dataset, indicating an important role for the sites in modulating TF function. When we combined the phosphoproteome with the other datasets, we found further clues that e9.5 placenta is a stressful environment, and we identified a posttranscriptionally and posttranslationally regulated network at this timepoint.

This analysis provides a systems-level view of gene expression patterns at two critical timepoints of placental development, and opens the door for experimental validation of potentially novel proteins, phosphosites, and pathways that may be critical for normal placental function.

Plant epidermal cells express unique molecular machinery that juxtapose the assembly of intracellular lipid components and the unique extracellular cuticular lipids that are unidirectionally secreted to plant surfaces. In maize (Zea mays), mutations at the glossy2 (gl2) locus affect the deposition of extracellular cuticular lipids. Sequence-based genome scanning identified a new Gl2 homolog in the maize genome, namely Gl2-like. Both the Gl2-like and Gl2 genes are members of the BAHD superfamily of acyltransferases, with close sequence similarity to the Arabidopsis (Arabidopsis thaliana) CER2 gene. Transgenic experiments demonstrated that Gl2-like and Gl2 functionally complement the Arabidopsis cer2 mutation, with differential influences on the cuticular lipids and the lipidome of the plant, particularly affecting the longer alkyl chain acyl lipids, especially at the 32-carbon chain length. Site-directed mutagenesis of the putative BAHD catalytic HXXXDX-motif indicated that Gl2-like requires this catalytic capability to fully complement the cer2 function, but Gl2 can accomplish complementation without the need for this catalytic motif. These findings demonstrate that Gl2 and Gl2-like overlap in their cuticular lipid function, but have evolutionarily diverged to acquire nonoverlapping functions.

With every newly sequenced species we discover hundreds of novel protein coding genes. Many of these "orphan" genes have been experimentally proven to have dramatic functions in development, sexual dimorphism, pathogen resistance, and social traits like symbiosis. Whereas in the past, researchers viewed genes as the product of continuous variation acting on ancient material, we now know that novel genes may arise de novo from non-genic sequence. Thus evolutionary experimentation is not limited to tweaking existing genes or their regulatory patterns. Any orphan genes that arose in the distant past, should appear today as lineage-specific genes (or gene families). The search for genes by their relative time of origin is called "phylostratigraphy". However, phylostratigraphy has proven to be a challenging task with different methodologies often yielding contradictory conclusions. Standard phylostratigraphy infers the age of a gene by finding the most distant species that has an inferred homolog. However, this approach is highly sensitive to annotation quality and cannot easily distinguish between rapidly evolving genes and genes of de novo origin.

This dissertation contributes a suite of tools for more accurately determining the phylostratigraphic age of genes and the level of support for the classification. First, we developed phylostratr to automate standard phylostratigraphy. Second, we developed a program, synder, to infer syntenic-homologs of query features using a synteny map. Third, we developed fagin, a package that builds on synder to search query genes against related species for traces of genic or non-genic orthology. The pipeline can distinguish orphans with high-confidence data support from orphans identified due to bad assembly or missing data. We traced many orphans to their non-genic cousins, identifying the non-genic footprint from which they arose. We linked others to putative genes in related species from which they diverged beyond recognition. Knowing the approximate location of each gene across species and the amount of data support provides a launching point for future orphan studies.

The unfolded protein response (UPR) is activated in plants in response to endoplasmic reticulum stress and plays an important role in mitigating stress damage. Multiple factors act in the UPR, including the membrane-associated transcription factor, BASIC LEUCINE ZIPPER 17 (bZIP17), and the membrane-associated RNA splicing factor, INOSITOL REQUIRING ENZYME1 (IRE1). We have analyzed an Arabidopsis (Arabidopsis thaliana) ire1a ire1b bzip17 triple mutant, with defects in stress signaling, and found that the mutant is also impaired in vegetative plant growth under conditions without externally applied stress. This raised the possibility that the UPR functions in plant development in the same manner as it does in responding to stress. bZIP17 is mobilized to the nucleus in response to stress, and through the analysis of a mobilization-defective bZIP17 mutant, we found that to support normal plant development bZIP17 must be capable of mobilization. Likewise, through the analysis of ire1 mutants defective in either protein kinase or RNase activities, we found that both must be operative to promote normal development. These findings demonstrate that the UPR, which is associated with stress responses in plants, also functions under unstressed conditions to support normal development.

The Association of American Colleges and Universities ranks multiple process of science (POS) skills among the top-10 skills employers seek in college graduates. As part of an effort to explore and align the emphasis on POS skills in our science departments, we sought three things: (a) to determine if faculty and students felt enough time was devoted to POS skills, (b) to identify the skills that science students and faculty felt were important to acquire from an undergraduate education, and (c) to evaluate whether there were differences in these views among disciplines. We found that faculty and students agreed on the amount of time spent in class on POS skills, but students thought that amount of time was sufficient, whereas faculty did not. Further, students and faculty placed a high importance on the POS skills of problem solving/critical thinking, communicating results, and interpreting data. We did not find differences among faculty of different science disciplines on the most highly ranked POS skills, but we did in students. The findings of this study have informed curricular discussions and decisions.

Background: The impact of extreme changes in weather patterns in the economy and humanity welfare are some of the biggest challenges that our civilization is facing. From the anthropogenic activities that contribute to climate change, reducing the impact of farming activities is a priority, since its responsible for up to 18% of greenhouse gases linked to such activities. To this end, we tested if the ruminal and fecal microbiomes components of 52 Brazilian Nelore bulls, belonging to two experimental groups based on the feed intervention, conventional (A) and byproducts based diet (B), could be used as biomarkers for methane (CH₄) emission.

Results: We identified a total of 5,693 Amplicon Sequence Variants (ASVs) in the Nelore bulls microbiomes from the experimental group B. Statistical analysis showed that the microbiome populations were significantly different among treatment groups. Differential abundance (DA) analysis with the ANCOM approach identified 30 bacterial and 15 archaea ASVs as DA among treatment groups. Random forest models, using either bacteria or archaea ASVs as predictors, were able to predict the treatment group with high accuracy (r2>0.85). Association analysis using Mixed Linear Models indicate that bacterial and archaea ASVs are linked to the CH₄ emission phenotype, of which the most prominent were the ruminal ASV 40 and fecal ASV 35. These ASVs contributed to a 9.7% increase and 7.3% decrease of the variation in CH₄ emission, respectively, which indicated their potential as targets for feed interventions and/or biomarkers.

Conclusion: The feed composition induced significant differences in abundance and richness of ruminal and fecal microbial populations. The dietary treatment based on industrial byproducts applied had an impact on the microbiome diversity of bacteria and archaea, but not on protozoa. Microbiome components (ASVs) of bacteria and archaea can be successfully used to predict the treatment group, thus giving support to the hypothesis that the feed intervention modulate microbiome abundance and diversity. Microbiome components were associated with CH₄ emission in both microbiomes. Therefore, both ruminal and fecal ASVs can be used as biomarkers for methane production and emission.

Inflorescence architecture in Zea mays is affected by a large collection of interrelated genes. The ramosa genes are some of the most prominent and well-studied of these genes due to their overtly branched ear and tassel mutant phenotype. ramosa1 confers determinate, short-branch identity on branch meristems during their initiation. Several new genes are proposed to work directly with ramosa1. ail6 was identified from a Quantitative Trait Locus study to identify genes which enhance or suppress the ramosa1 branching phenotype. A Yeast-2-Hybrid study identified several genes as potential interactors with ramosa1. A CRISPR/Cas9 knockout study was performed to produce novel mutants to analyze this pathway. Two unique CRISPR expression arrays were utilized targeting 12 sites in five genes. Agrobacterium tumefaciens mediated transformation was used for transfection of Hi-II immature embryos. Both arrays functioned, and mutations were acquired in all five genes. Specifically, thirteen unique mutations have been detected in the ramosa1 gene, and in the other four genes we detected eight, 16, four and one mutation. A total of 112 plants were recovered and crossed into a B104, B73 or B73 ra1-63 background.

The eukaryotic alga, Chlamydomonas reinhardtii, acclimates to limiting CO2 conditions by the induction of the CO2-concentrating mechanism (CCM) – a complex system of changes in its metabolism, gene expression patterns, and physiology – to compensate for the reduction in the amount of available CO2 and counter the hindrance to its ability to photosynthesize and grow. LCIB, a gene upregulated in such conditions, encodes a protein potentially involved in uptake of CO2 into the cell and in preventing the leakage of CO2 out of the cell. This protein is indispensable for growth in air-level CO2 (~350-400 ppm), since mutants in this gene are unable to grow (hence they are called air dier mutants). Several mutants that have second-site alterations that restore growth in air-level CO2 (i.e., suppress the air dier phenotype) have been isolated. Identifying the genes that are mutated in these suppressors and the functions of the encoded proteins will help us better discern the role of LCIB and comprehend the workings of the entire mechanism.

To identify the locus of the mutated gene in one suppressor mutant, crosses were performed between the mutant strain and a non-mutant, polymorphic wild-type strain, and a large population of recombinant progeny that segregated against the mutation of interest was amassed. Using a strain that has unique single nucleotide polymorphisms (SNPs) as the non-mutant parent allowed us to seek a particular characteristic (the polymorphisms) in the region of interest in the genome of the progeny. With a sequenced genome, a library of SNPs in the polymorphic strain, and a pool of the genomic DNA from the entire population, we mapped the mutation to a specific region of the genome and narrowed potential candidates down to a small number of genes. By cosegregation analysis, we were able to confirm one of the candidates, LCI15, as the implicated gene. Preliminary functional analyses with semi-quantitative RT-PCR and Western immunoblots reveal the LCI15 protein as possibly playing an overarching role in regulation in the CCM and offer terms for discussing potential methods by which the lack of LCI15 might potentially mask the deleterious effects of the absence of LCIB.

Ortholog inference is a key step in understanding the evolution and function of a gene or other genomic feature. Yet often no similar sequence can be identified, or the true ortholog is hidden among false positives. A solution is to consider the sequence's genomic context. We present the generic program, synder, for tracing features of interest between genomes based on a synteny map. This approach narrows genomic search-space independently of the sequence of the feature of interest. We illustrate the utility of synder by finding orthologs for the Arabidopsis thaliana 13-member gene family of Nuclear Factor YC transcription factor across the Brassicaceae clade.

Macroautophagy/autophagy is a conserved process in eukaryotes that contributes to cell survival in response to stress. Previously, we found that endoplasmic reticulum (ER) stress induces autophagy in plants via a pathway dependent upon AT5G24360/IRE1B (INOSITOL REQUIRING 1–1), an ER membrane-anchored factor involved in the splicing of AT1G42990/BZIP60 (basic leucine zipper protein 60) mRNA. IRE1B is a dual protein kinase and ribonuclease, and here we determined the involvement of the protein kinase catalytic domain, nucleotide binding and RNase domains of IRE1B in activating autophagy. We found that the nucleotide binding and RNase activity of IRE1B, but not its protein kinase activity or splicing target BZIP60, are required for ER stress-mediated autophagy. Upon ER stress, the RNase activity of IRE1B engages in regulated IRE1-dependent decay of messenger RNA (RIDD), in which mRNAs of secreted proteins are degraded by IRE1 upon ER stress. Twelve genes most highly targeted by RIDD were tested for their role in inhibiting ER stress-induced autophagy, and 3 of their encoded proteins, AT1G66270/BGLU21 (β-glucosidase 21), AT2G16005/ROSY1/ML (MD2-related lipid recognition protein) and AT5G01870/PR-14 (pathogenesis-related protein 14), were found to inhibit autophagy upon overexpression. From these findings, IRE1B is posited to be a ‘licensing factor’ linking ER stress to autophagy by degrading the RNA transcripts of factors that interfere with the induction of autophagy.