Heat stress poses a serious threat to plant survival and productivity, and has a direct influence on crop yield stability. Plants response to high temperature is tightly controlled by complex genetic networks. Plants can be acclimated through gradual pre-exposure to increasing temperatures and that in turn causes higher survival in subsequent and otherwise lethal heat stress conditions. To investigate the physiological and molecular processes underlying heat acclimation and recovery, we examined changes in Arabidopsis thaliana transcriptome throughout the acclimation and the subsequent heat shock treatment. Groups of differentially expressed genes and enriched biological pathways that constitute the heat transcriptional memory were identified. The function of flavonoids in plant heat stress were further explored experimentally. In addition, we observed altered stomata density and aperture responses in heat acclimated plants, and this might be partially controlled by AGAMOUS-LIKE16 (AGL16) transcription factor and its negative regulator microRNA824 (miR824). By utilizing an automated non-invasive phenotyping facility, we have developed a protocol to record plant growth and photosynthetic performance after heat stress in wild type Arabidopsis thaliana and mutant lines at daily intervals. Through an imaging-based analysis of plants growth, we confirmed impaired thermotolerance of hsp101 compared to wild type plants by a time-series growth, morphology and chlorophyll responses. This offers a novel experimental setup for thermotolerance screenings in Arabidopsis, with defined digital markers linking the function of selected genes in heat stress responses to phenotypic traits.

The advancement of Nucleic acids (DNA and RNA) sequencing technology has enabled many projects targeted towards the identification of genome structure and transcriptome complexity of organisms. The first conclusions of the human and mouse projects have underscored two important, yet unexpected, findings. First, while almost the entire genome is transcribed, only 5% of it encodes for proteins. Thereby, most transcripts are noncoding RNA. This includes both short RNA (<200 nucleotides (nt)) comprising piRNAs; microRNAs (miRNAs); endogenous Short Interfering RNAs (siRNAs) among others, and includes lncRNA (>200nt). Second, a significant portion of the mammalian genome (45%) is composed of Repeat Elements (REs). RE are mostly relics of ancestral viruses that during evolution have invaded the host genome by producing thousands of copies. Their roles within their host genomes have yet to be fully explored considering that they sometimes produce lncRNA, and have been shown to influence expression at the transcriptional and post-transcriptional levels. Moreover, because some REs can still mobilize within host genomes, host genomes have evolved mechanisms, mainly epigenetic, to maintain REs under tight control. Recent reports indicate that REs activity is regulated in somatic cells, particularily in the brain, suggesting a physiological role of RE mobilization during normal development. In this thesis, I focus on the analysis of ncRNAs, specifically REs; piRNAs; lncRNAs in human and mouse post-mitotic somatic cells. The main aspects of this analysis are: Using sRNA-Seq, I show that piRNAs, a class of ncRNAs responsible for the silencing of Transposable elements (TEs) in testes, are present also in adult mouse brain. Furthermore, their regulation shows only a subset of testes piRNAs are expressed in the brain and may be controlled by known neurogenesis factors. To investigate the dynamics of the transcriptome during cellular differentiation, I examined deep RNA-Seq and Cap Analysis of Gene Expression (CAGE) data from time-course progression program of primary human skeletal muscle cell differentiation. I contrasted this program with Duchenne Muscular Dystrophy (DMD) donors. I identified novel candidates, protein-coding genes and lncRNAs, that may be involved in myogenesis and reaffirmed known myogenic players. Using RNA-Seq data, I designed a novel pipeline to identify possible de novo insertion sites during muscular differentiation, which I have also tested on embryonic mouse cerebral cortex.

Biofouling is a significant economic problem, particularly for marine and offshore oil industries. The acorn barnacle (Amphibalanus (Balanus) amphitrite) is the main biofouling organism in marine environments. Environmental conditions, the physiology of the biofouling organism, the surrounding microbial community, and the properties of the substratum can all influence the attachment of biofouling organisms to substrates. My dissertation investigated the biological processes involved in B. amphitrite development and attachment in the unique environment of the Red Sea, where the average water surface temperature is 34°C and the salinity reaches 41‰. I profiled the transcriptome and proteome of B. amphitrite at different life stages (nauplius II, nauplius VI, and cyprid) and identified 65,784 expressed contigs and 1387 expressed proteins by quantitative proteomics. During the planktonic stage, genes related to osmotic stress, salt stress, the hyperosmotic response, and the Wnt signaling pathway were strongly up-regulated, hereas genes related to the MAPK pathway, lipid metabolism, and cuticle development were down-regulated. In the transition from the nauplius VI to cyprid stages, there was up-regulation of genes involved in blood coagulation, cuticle development, and eggshell formation, and down-regulation of genes in the nitric oxide pathway, which stimulates the swimming and feeding responses of marine invertebrates. This system-wide integrated approach elucidated the development and attachment pathways important in B. amphitrite. Enzymes and metabolites in these pathways are potential molecular targets for the development of new antifouling compounds.

The mechanism by which plants synthesize and store high amounts of triacylglycerols (TAG) in tissues other than seeds is not well understood. The comprehension of controls for carbon partitioning and oil accumulation in nonseed tissues is essential to generate oil-rich biomass in perennial bioenergy crops. Persea americana (avocado), a basal angiosperm with unique features that are ancestral to most flowering plants, stores ~ 70 % TAG per dry weight in its mesocarp, a nonseed tissue. Transcriptome analyses of select pathways, from generation of pyruvate and leading up to TAG accumulation, in mesocarp tissues of avocado was conducted and compared with that of oil-rich monocot (oil palm) and dicot (rapeseed and castor) tissues to identify tissue- and species-specific regulation and biosynthesis of TAG in plants.

Neuroblastoma (NBL), the most common non-Central Nervous System (CNS) solid tumor of childhood, characteristically displays heterogeneous clinical presentation and biological behavior. Previous work has studied the genetic basis of the disease and revealed a low somatic mutation burden. In order to identify novel therapeutic targets and better understand the biology of high-risk NBLs, I investigated whole transcriptome profiles of two cohorts of metastatic NBLs using RNA sequencing. First, I studied changes in splicing pattern in a cohort of 29 patients. V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplified NBLs showed a distinct splicing pattern affecting multiple cancer hallmarks. Six splicing factors have altered expression patterns in MYCN-amplified tumors and cell lines, and binding motifs for these factors were significantly enriched in differentially-spliced genes. ChIP-seq analysis showed direct binding of MYCN to promoter regions of splicing factors PTBP1 and HNRNPA1, demonstrating that MYCN regulates splicing by directly regulating expression of key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage 4 NBL patients (p≤0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, a pro-tumor-growth isoform, resulted in repression of NBL cell growth. Second, I used whole transcriptome sequencing in a cohort of 150 patients to assess expressed mutations, fusion genes, and gene expression including long non-coding genes to provide clinically-relevant classification and to offer insights into NBL tumor biology. Twenty-four genes including ALK, ATRX and MYCN were recurrently mutated in NBL transcriptomes. In-frame FOXR1 fusions were detected in 4 samples, including 3 cases or 14% of stage 4S NBLs. Unsupervised gene expression analysis revealed four molecular subgroups. MYCN and tumor microenvironment were the primary discriminating signatures in these molecular subgroups. Fifty-eight percent of MYCN-not-amplified samples showed high MYCN signatures, which were potentially contributed by various genomic events such as MYCN activating mutations and FOXR1 fusions. High MYCN signature was significantly associated with poor overall survival in MYCN-not-amplified tumors (p=0.0017). In addition, the tumor microenvironment including stromal and immune cell infiltration significantly contributed to the NBL transcriptional landscape and tumor progression.

RAG1-/- mutant zebrafish lack T and B lymphocytes. However, when re-exposed to homologous bacteria, these fish mount a response that provides specific protection. To further define this response, we utilized microarray analyses to determine the mechanisms underlying innate immune system memory in zebrafish. We also analyzed interferon (IFN) gamma by qRT-PCR. It is produced by activated NK cells and could indicate if this cell mediates the protective response seen in lymphocyte deficient zebrafish. Pathological studies and in situ hybridizations were performed to observe tissue changes and location of the cells that produced IFN gamma. Following bacterial re-exposure, zebrafish transcripts in cell receptor activation, cell proliferation and cytotoxic function categories were differentially expressed. We found high expression of IFN gamma in the lymphocyte like cell population after bacterial exposure and this was induced to a higher level in fish that had been vaccinated. The phagocytic cell population showed no induction of INF gamma. Over-all, the pathological response was much less severe in the vaccinated (48 hps) fish. Our microarray and pathological findings indicate that the primary immune response of mutant zebrafish is not impaired, and they demonstrate an enhanced innate immune response following secondary bacteria exposure. Following homologous secondary exposure, mutant zebrafish have a cell population that is undergoing upregulated cell receptor activation, cell cytotoxic functions and cell proliferation. This cell population expresses INF gamma. Activated T cells, NK-T cells and NK cells express INF gamma. Since RAG1 deficient zebrafish do not have T or NK-T cells, this cell population is most likely NK cells.

Fireflies are some of the most captivating organisms on the planet. Because of this, they have a rich history of study, especially concerning their bioluminescent and visual behavior. Among insects, opsin copy number variation has been shown to be quite diverse. However, within the beetles, very little work on opsins has been conducted. Here we look at the visual system of fireflies (Coleoptera: Lampyridae), which offer an elegant system in which to study visual evolution as it relates to their behavior and broader ecology. They are the best-known case of a terrestrial organism that communicates through the use bioluminescence. The molecular basis for this communication is relatively simple: one gene-family (opsins) controls the detection of the signal, and one gene family (luciferase) controls the production of the signal. We use a transcriptomic approach to sample for and investigate opsin evolution in fireflies. We also present the first total evidence approach using both an extensive molecular matrix and a robust morphological matrix to reconstruct the lampyrid phylogeny. We then use this phylogeny to assess the hypothesis that adult use of bioluminescence occurred after the origin of Lampyridae. We find evidence for only two expressed opsin classes in each of the nine firefly species studied, one in the ultra-violet sensitive and one in the long-wavelength sensitive areas of the visible spectrum. Despite the need for most adult fireflies to respond to a clearly sexual and colorful visual signal (bioluminescence) to maximize fitness, their visual system is relatively simple, and does not match the trend for opsin duplication found in other insect groups. All subfamilies except for Lampyrinae are recovered as monophyletic; Pterotinae and Ototretinae are recovered within the Lampyridae. The ancestral state of adult bioluminescence is suggested to be non-bioluminescent, with at least three gains and at least three losses.

Channel catfish (Ictalurus punctatus) are raised for aquaculture in ponds characterized by dramatic swings in dissolved oxygen concentration. If morning dissolved oxygen concentration falls below approximately 3.0 mg/L catfish consume less feed, leading to a reduction in growth and production. Although the effects of low oxygen on channel catfish appetite have been described, the underlying mechanisms responsible for those effects are unknown. Increased production of the neuropeptides corticotropin-releasing factor (CRF) and urotensin I (UI) are implicated in other fishes as neuropeptides that reduce appetite once an environmental stressor is detected. This project characterizes the hematological responses and transcriptional response of the hypothalamus to acute, chronic, and cyclical (repeating periods of hypoxia and normoxia) hypoxia. During acute (12 hours) hypoxia, venous PO2 decreased within 6 hours, coupled with an increase in hematocrit and decreased blood osmolality. These changes reversed within 12 hours after returning to normoxia but were not coupled with a change in transcription of the genes for CRF and UI. If this pattern of hypoxia and normoxia is repeated cyclically for 5 days, the same physiological responses repeat continually. During chronic hypoxia up to 5 days in duration, channel catfish have a similar hematological response, but did not recover to normoxic control values over the duration of the challenge. Likewise, no significant change in gene expression of CRF or UI were detected during chronic hypoxia. The hypothalamic transcriptome was analyzed during a 12-hour exposure to hypoxia followed by a 12-hour normoxic recovery. Across all time points, 190 genes were differentially expressed, with the greatest numbers occurring during periods of hypoxia. Differentially expressed genes were grouped into Gene Ontology biological processes and were most overrepresented by the term “response to hypoxia,†which included genes involved with angiogenesis, red blood cell production, and negative feedback to hypoxia-inducible factor proteins. Although this study did not find a change in hypothalamic transcription of CRF and UI, it did identify multiple adaptive responses that work together to reduce the severity of hypoxia along with several gene candidates for future hypoxia studies.

Even under the best management strategies contemporary crops face yield losses from diverse threats such as, pathogens, pests, and environmental stress. Adding to this management challenge is that under current global climate projections these impacts are predicted to become even greater. Natural genetic variation, long used by traditional plant breeders, holds great promise for adapting high performing agronomic lines to these stressors. Yet, efforts to bolster crop plant resilience using wild relatives have been hindered by time consuming efforts to develop genomic tools and/or identify the genetic basis for agronomic traits. Thus, increasing crop plant resilience requires developing and deploying approaches that leverage current high-throughput sequencing technologies to more rapidly and robustly develop genomic tools in these systems. Here we report the integration of bioinformatic and statistical tools to leverage high-throughput sequencing to 1) develop a machine learning approach to determine factors impacting transcriptome assembly and quantitatively evaluate transcriptome completeness, 2) dissect complex physiological pathway interactions in Solanum pimpinellifolium under combined stresses—using comparative transcriptomics, and 3) develop a genome assembly pipeline that can be deployed to rapidly assemble a more contiguous genome, unraveling previously hidden complexity, using Phytopthora capsici as a model. As a result, we have generated strategic guidelines for transcriptome assembly and developed an orthologue and reference free, machine learning based tool "WWMT" to quantitatively score transcriptome completeness from short read data. Secondly, we identified "hub genes" and describe genes involved with "cross-talk" between drought and herbivore stress response pathways. Finally, we demonstrate a protocol for combining long-read sequencing from the Oxford Nanopore Technologies MinION, and short-read data, to rapidly assembly a cost-effective, contiguous and relatively complete genome. Here we uncovered hidden variation in a well-known plant pathogen finding that the genome was 92% bigger than previous estimates with more than 39% of duplicated regions, supporting a hypothesized recent whole genome duplication in this clade. This community resource will support new functional and evolutionary studies in this economically important pathogen. / Doctor of Philosophy / Meeting the food production demands of a burgeoning population in a changing environment, means adapting crop plants to become more resilient to environmental stress. One of the greatest barriers to understanding and predicting crop responses to future environmental change is our poor understanding of the functional and genomic basis of stress resistance traits for contemporary crops. This impediment presents a barrier for rapid crop improvement technologies, such as, gene editing or genomic selection, that is only partially overcome by generating large amounts of sequencing data. Here we need tools that allow us to process and evaluate huge amounts of data generated from next generation sequencing studies to help identify genomic regions associated with agronomic traits. We also need technical approaches that allow us to disentangle the complex genetic interactions that drive plant stress responses. Here we present work that used statistical analysis and recent advances of artificial intelligence to develop a bioinformatic approach to evaluate genomic sequencing data prior to downstream analyses. Secondly, we used a reductionist approach to filter thousands of genes to key genes associated with combined stress responses (herbivory and drought), in the most widely used vegetable in the world, tomato. Finally, we developed a method for generating whole genome sequences that is low-cost and time sensitive and tested it using a well-known plant pathogen genome, wherein we unraveled significant hidden complexity. Overall this work provides community-wide genomic tools and information to promote crop resilience.

Plants are sessile organisms adapted to cope with dynamic changes in their environment. Abiotic stresses, such as heat, drought, or nutrient deficiency must be overcome simultaneously with biotic threats such as pathogens and herbivores. Oomycete pathogens represent a significant threat to global food production and natural ecosystems. Novel modes of oomycete disease control could increase crop yield and reduce pesticide application. Overlaps between the plant response to iron deficiency and pathogens have been documented, but the impact of simultaneous imposition of both stresses on the plant have not been studied. Additionally, nothing is known about the impact of iron deficiency on oomycete infection, or mechanisms of oomycete iron uptake. We adapted a hydroponic system to simultaneously impose iron deficiency and monitor pathogen infection. The oomycete pathogens Hyaloperonospora arabidopsidis, and Phytophthora capsici grew less well on iron-deficient Arabidopsis thaliana, at least in part because of observed activation of immunity due to iron stress. We screened A. thaliana T-DNA insertion mutants defective in iron metabolism and transport and identified potential mechanisms of H. arabidopsidis iron acquisition. We conducted RNA sequencing to understand how A. thaliana responds to iron deficiency and root infection of P. capsici. 323 genes were differentially upregulated in iron-starved plants over three days, irrespective of pathogen infection, representing a core iron deficiency response. This group of core genes included the primary A. thaliana iron uptake pathway and genes for coumarin biosynthesis. Salicylic acid responsive genes were observed in both treatments consistent with this defense hormone's previously identified role in iron deficiency. Genes related to glucosinolate production – shown to be important in defense against P. capsici – were down regulated during infection, potentially due to the activity of virulence effectors. Our work demonstrates crosstalk between the iron deficiency response and plant immunity, and that iron acquisition remains important to the plant even after pathogen invasion. These new insights provide a first step in developing novel resistance strategies to control oomycetes in agronomically important crops. / Doctor of Philosophy / Oomycetes can cause diseases in plants resulting in loss of crops and requiring application of chemical pesticides. Better understanding of how oomycetes interact with plants will lead to new strategies to control them and more efficient agriculture. In this study, we investigated the role of iron in plant-oomycete interactions, to see what this important metal nutrient might be doing to help or hurt the plants response to infection. We developed a growth system to limit iron to the plant and simultaneously observe oomycete infection. We studied the leaf pathogen Hyaloperonospora arabidopsidis or downy mildew, and Phytophthora capsici, a root pathogen that infects many types of vegetable crops. In rice, iron restriction hurt the plant's ability to fight off disease, but we observed the opposite: iron limitation caused the plant to be more resistant to both oomycete pathogens. Microscopic observation revealed that the plants ability to fight off downy mildew was not compromised by iron deficiency. Our results suggest that iron limitation triggers an immune response in the plant, which limits pathogen growth. We performed RNA sequencing on iron-deficient roots also infected with the root pathogen. This allowed us to observe how the plant responded to both stresses. The plant balances the response to iron deficiency and infection. Again, we found that iron deficiency triggers immune activation, and observed that iron-deficient plants are more resistant to infection.