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Background: Avocado (Persea americana) is an economically important tropical fruit considered to be a good source of fatty acids. Despite its importance, the molecular and cellular characterization of biochemical and developmental processes in avocado is limited due to the lack of transcriptome and genomic information. Results: The transcriptomes of seeds, roots, stems, leaves, aerial buds and flowers were determined using different sequencing platforms. Additionally, the transcriptomes of three different stages of fruit ripening (pre-climacteric, climacteric and post-climacteric) were also analyzed. The analysis of the RNAseqatlas presented here reveals strong differences in gene expression patterns between different organs, especially between root and flower, but also reveals similarities among the gene expression patterns in other organs, such as stem, leaves and aerial buds (vegetative organs) or seed and fruit (storage organs). Important regulators, functional categories, and differentially expressed genes involved in avocado fruit ripening were identified. Additionally, to demonstrate the utility of the avocado gene expression atlas, we investigated the expression patterns of genes implicated in fatty acid metabolism and fruit ripening. Conclusions: A description of transcriptomic changes occurring during fruit ripening was obtained in Mexican avocado, contributing to a dynamic view of the expression patterns of genes involved in fatty acid biosynthesis and the fruit ripening process.

Maternal immune activation (MIA) disrupts the central innate immune system during a critical neurodevelopmental period. Microglia are the primary innate immune cells in the brain and can mediate neurodevelopment, but the direct influence of microglia on the MIA phenotype remains largely unknown. Here, we show that MIA can lead to long-lasting effects on microglial phenotype, neuronal circuitry, and behaviors. Transcriptomic analysis revealed aberrant expression of neurogenic genes in MIA microglia. We found that microglia repopulation by colony-stimulating factor receptor 1 (CSF1R) inhibition reversed MIA-induced social deficits and corrected expression of the newly identified MIA-associated neuritogenic molecules in microglia. In vitro whole-cell patch-clamp recording and immunohistochemistry revealed that microglia repopulation restored MIA-induced changes in intrinsic excitability, dendritic spine density, and microglia-neuron interactions of layer V intrinsically bursting pyramidal neurons in the prefrontal cortex. Maternal inflammation therefore alters microglial phenotypes and changes neuronal functions by mediating microglia-neuron interactions. We found that Wingless-related MMTV integration site 5a (WNT5a) is a critical regulator of this microglia-neuron communication. Studies have shown that the neurotrophic factor WNT5a plays a critical role in neurodevelopment, and here we demonstrate that WNT5a is one of the neuritogenic genes significantly upregulated in embryonic MIA microglia. We showed using microarray analysis that the microglial secretome can promote neural stem cell differentiation through various pathways, including Wnt pathways. Live imaging of neuron-microglia co-culture demonstrated that microglia enhanced neurite development and dendritic spine density and that this was diminished by microglial Wnt5a silencing using siRNA transfection. Multi-electrode array recordings revealed that microglia co-culture increased spontaneous neuronal firing rate. Thus, microglia can secrete WNT5a and regulate dendritic spine development, maintenance, and neural circuitry. These results indicate that altered expression of microglial WNT5a due to pathogenic states such as inflammation can lead to abnormal neuronal activity. To further elucidate microglia biology, we developed an inducible immortalized murine microglial cell line using a tetracycline expression system. The addition of doxycycline can induce rapid cell proliferation for the expansion of cell colonies. Upon withdrawal of doxycycline, this monoclonal microglial cell line can differentiate and resemble in vivo microglia physiology as assessed by expression of microglial genes, innate immune response, chemotaxis, and phagocytic capabilities. This cell line becomes a convenient and useful method to study microglia in vitro. / 2024-02-03T00:00:00Z

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Silencing phytochrome A1 gene (PHYA1) by RNA interference in upland cotton (Gossypium hirsutum L. cv. Coker 312) had generated PHYA1 RNAi lines with increased fiber length, strength and low micronair (finer fiber). In order to identify and characterize mRNAs and miRNAs that are differentially expressed in the RNAi plants, transcriptome and miRNAome analyses via high-throughput RNA sequencing were performed. Total RNA isolated from 10-DPA (days post anthesis) fibers and small RNAs isolated from 5-, 10-, and 15-DPA fibers of RNAi and Coker 312 lines were used to construct 6 RNA libraries and 18 small RNA libraries, respectively, which were sequenced using the Illumina HiSeq system. A total of 142 differentially expressed genes (DEGs) were identified in PHYA1 RNAi compared to Coker 312. GO analysis showed that these DEGs were mainly involved in metabolic pathways, binding and regulating enzymes (hydrolase, transferase, and oxidoreductase activities), and cell structures which were reported to play important roles in fiber development. Twenty-eight KEGG pathways were mapped for 142 DEGs, and the pathways related to glycolysis/gluconeogenesis and pyruvate metabolism were the most abundant, followed by cytochrome P450-involved pathways. Sixty-one conserved miRNA families and thirtyive novel miRNAs were identified in upland cotton. The targets of 6 conserved miRNAs, which expressed differentially in the RNAi line, were reported to participate in primary cell wall synthesis and phytohormone signaling pathways. The 35 novel miRNAs were identified in cotton for the first time, and their target genes were predicted. Nine novel miRNAs were identified to target cytochrome P450 TBP. Together, the results imply that miRNAs involved in fine-tune gene regulation might confer to the phenotype of the RNAi line with improved fiber quality. Besides characterizing mRNAs and miRNAs, the CpG site methylation status within coding regions of phytochrome genes in RNAi line in leaves and 10-DPA fibers was determined using bisulfite genomic sequencing. The PHYA1, PHYC and PHYE in RNAi line had higher methylation levels in leaves than those in Coker 312, but PHYB had lower methylation levels. In fibers, the methylation levels of PHYB also decreased in RNAi plants. However, the methylation of other phytochrome genes showed no significant changes.

The ability of populations to continually respond to directional selection even after many generations instead of reaching response plateaus suggests the presence of mechanisms for rapidly generating novel adaptive variation within organismal genomes. The contributions of cis regulation are now being widely studied. This study details the contributions of one such mechanism capable of generating adaptive genetic variation through transcribed microsatellite mutation. Microsatellites are abundant in eukaryotic genomes, exhibit one of the highest known mutation rates; and mutations involve indels that are reversible. These features make them excellent candidates for generating variation in populations. This study explores the functional roles of transcribed microsatellites in Helianthus annuus (common sunflower). More specifically, I explored the role of microsatellites as agents of rapid change that act as “tuning knobs” of phenotypic variation by influencing gene expression in a stepwise manner by expansions and contractions of the microsatellite tract. A bioinformatic study suggests that selection has favored expansion and maintenance of transcriptomic microsatellites. This inference is based on the non-random distribution of microsatellites, prevalence of motifs associated with gene regulation in untranslated regions, and the enrichment of microsatellites in Gene Ontologies representing plant response to stress and stimulus. A population genetics study provides support for selection on these transcribed microsatellites when compared to anonymous microsatellites that were assumed to evolve neutrally. The natural populations utilized in this study show greater similarity in allele frequencies, mean length, and variance in lengths at the transcribed microsatellites relative to that observed at anonymous microsatellite loci. This finding is indicative of balancing selection, and provides evidence that allele lengths are under selection. This finding provides support for the tuning knob hypothesis. The findings of a functional genomic study with regard to the tuning knob hypothesis are ambiguous. No correlation between allele lengths and gene expression was detected at any of three loci investigated. However, the loci utilized exhibited narrow ranges in length. The tuning knob hypothesis implies that similar allele lengths are likely to exhibit similar gene expression levels. Hence, variation in the populations studied may be tracking the optimal gene expression levels.

Viral infection severity often varies with host factors such as age and sex. The pathogenesis of infections caused by a broad range of viruses, from neurotropic viruses like Rabies and Zika to respiratory viruses such as Influenza and SARS-CoV-2, differ between the sexes and across the lifespan. Typically, older males are more susceptible to severe acute outcomes, while females are more vulnerable to the post-acute sequelae of infections. All of these complications can include neuroinflammation, stroke, cognitive dysfunction, and delirium. While these symptoms can be secondary to infection, recent studies suggest that even peripheral infections can lead to neuropathological changes in the brain. However, few studies have characterized the expression of viral receptors in the human brain or examined age- or sex-related differences in such expression. In this study, we used a publicly accessible transcriptomic database to assess the impact of age and sex on the expression of 67 viral host factor genes, associated with ten virus families. Analyzing data from 15 brain areas (n=33, F=14, M=19, age:4 mo-80 yrs), we determined the lifespan trajectory for each gene in each area via LOESS regressions. We used unsupervised hierarchical clustering to determine if a brain-wide pattern or virus family pattern can be detected. Using Dense-tSNE, a dimension-reduction and visualization technique, we discovered four distinct developmental trajectories, clustering the areas into two mixed-sex subcortical clusters and one each of male and female cortical clusters. Applying Differential Expression Sliding Window Analysis (DeSWAN), we identified the genes driving these age- and sex-related differences. Many sex differences were noted in childhood, potentially impacting the brain's susceptibility to viral infections and underscoring a broader dimorphic organization of male and female brains. These insights contribute to our understanding of sex-specific responses to viral infections, offering the potential for more personalized treatment strategies. / Thesis / Master of Science (MSc) / Viral infections, like Influenza and SARS-CoV-2, vary in severity based on a person's age and sex. Generally, older men suffer severe immediate symptoms such as stroke and seizures, while women endure long-term effects, including brain inflammation and cognitive issues. Recent research suggests even non-brain-related infections can lead to changes within the brain. Yet, our understanding of how our brains' viral receptors - key to infection - change with age and between sexes is limited. We used a public database to explore these changes, studying receptor-related genes in different brain areas across various ages and sexes. Our analysis revealed unique patterns of gene expression, grouping the brain into different regions based on development and sex. We noticed many differences between men and women during childhood, potentially influencing how their brains respond to viruses. This research aids our understanding of why viral infections impact individuals differently based on age and sex, offering insights that could help develop personalized treatments.

Phylogenetic research on non-model organisms has been hindered by limited marker availability. Next generation sequencing techniques are eliminating that barrier. Using Illumina sequencing technology, Trinity assembly software, custom Perl reciprocal BLAST scripts, and Primer3 primer prediction software, we produced and analyzed 7 Plecopteran transcriptomes, representing 7 of the 16 total families, in an attempt to identify and develop conserved orthologous genetic markers. The transcriptomes were used to reconstruct a gene content phylogeny using a simple distance matrix generated from reciprocal blastn data. By producing and filtering a reciprocal blast network we identified and aligned over 450 putative orthologs. Out of these, 25 primer pairs were selected that showed 100% conserved primer sites across all the transcripts from which they were created. Of those 25, 3 loci (PlecSK1, Perl534, and PvC2190) show very positive phylogenetic potential. These 3 markers may also be suitable and even highly useful in population genetic studies in which the populations have had sufficient time to develop significant genetic separation. The rapid and affordable nature of this study demonstrates the ease by which non-model organism phylogenetics can be expanded and made more robust.

Chronic lung diseases including Chronic Obstructive Pulmonary Disease (COPD), Idiopathic Pulmonary Fibrosis (IPF) and lung cancer are major causes of morbidity and mortality in the United States due to high incidence and limited therapeutic options. In order to address this critical issue, I have leveraged RNA sequencing and integrative genomics to define disease-associated transcriptomic changes which could be potentially targeted to lead to new therapeutics. We sequenced the lung transcriptome of subjects with IPF (n=19), emphysema (n=19, a subtype of COPD), or neither (n=20). The expression levels of 1770 genes differed between IPF and control lung, and 220 genes differed between emphysema and control lung (p<0.001). Upregulated genes in both emphysema and IPF were enriched for the p53/hypoxia pathway. These results were validated by immunohistochemistry of select p53/hypoxia proteins and by GSEA analysis of independent expression microarray experiments. To identify regulatory events, I constructed an integrative miRNA target prediction and anticorrelation miRNA-mRNA network, which highlighted several miRNA whose expression levels were the opposite of genes differentially expressed in both IPF and emphysema. MiR-96 was a highly connected hub in this network and was subsequently overexpressed in cell lines to validate several potential regulatory connections. Building upon these successful experiments, I next sought to define gene expression changes and the miRNA-mRNA regulatory network in never smoker lung cancer. Large and small RNA was sequenced from matched lung adenocarcinoma tumor and adjacent normal lung tissue obtained from 22 subjects (8 never, 14 current and former smokers). I identified 120 genes whose expression was modified uniquely in never smoker lung tumors. Using a repository of gene-expression profiles associated with small bioactive molecules, several compounds which counter the never smoker tumor signature were identified in silico. Leveraging differential expression information, I again constructed an mRNA-miRNA regulatory network, and subsequently identified a potential never smoker oncomir has-mir-424 and its transcription factor target FOXP2. In this thesis, I have identified genes, pathways and the miRNA-mRNA regulatory network that is altered in COPD, IPF, and lung adenocarcinoma among never smokers. My findings may ultimately lead to improved treatment options by identifying targetable pathways, regulators, and therapeutic drug candidates. / 2017-02-01T00:00:00Z

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide and is a risk factor for lung cancer development. COPD encompasses both emphysema and chronic bronchitis, the pathogenesis of which are unclear. In this dissertation, I leveraged genome-wide gene-expression studies of emphysema and lung cancer to investigate pathogenesis and carcinogenesis in COPD. Tobacco smoke is the primary cause of emphysema. The most severe form is also associated with alpha1-antitrypsin deficiency (AATD) resulting from a mutation. In this study, I leveraged multiple lung samples from patients with emphysema, with or without AATD. While genes involved in tissue repair decreased with emphysema severity, the unfolded protein response (UPR) was uniquely changed in AATD lungs. AATD may play multiple roles in emphysema and UPR activation suggests AAT replacement therapy may be insufficient to treat this form of emphysema. Emphysema is a progressive disease, and the mean linear intercept (Lm) can serve as a surrogate of progression. I evaluated whether Lm increases in non-diseased lungs may represent similar processes to those occurring in emphysema, and could offer insight into early stages of disease or homeostasis. Genes involved in tissue repair increased with Lm in controls but decreased in disease. Tissue repair processes may be active in even the non-insulted lung, suggesting their activity is necessary for lung homeostasis and their deficiency may drive emphysema progression. Finally, COPD patients are at increased lung cancer risk, and transcriptomic changes common to both diseases could explain this risk. In both COPD and lung cancer, I discovered that H3K27Me3 regulated genes are repressed, and that the methyltransferase responsible for H3K27me3, EZH2, is induced. H3K27Me3, an oncogenic histone modification, may drive carcinogenesis and pathogenesis in COPD. Though usual and AATD emphysema share transcriptomic signatures associated with tissue repair, which may be active in the normal homeostatic lung, the UPR changes in AATD emphysema only; successful therapeutic strategies in emphysema will need to account for this difference. In COPD, H3K27Me3 may play a role in both pathogenesis and carcinogenesis, making it an attractive target for therapeutic interventions, but one that would need further augmentation in AATD. / 2019-11-01T00:00:00Z