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Aging is a degenerative process characterized by a decline in physiological functions and cellular activities. Environmental and pharmacological interventions affecting longevity pathways have been extensively studied in model organisms. This study investigated the effect of chronic mild intermittent hypoxia (4 mg O2/L) or mild mitochondrial uncoupling with three doses of 0 (control), 0.1, 1, and 5 μM of 2,4-Dinitrophenol (DNP), on life history and gene expression in four clones of Daphnia magna. Interestingly, clones from intermittent ponds displayed better tolerance to hypoxia and DNP. Although neither treatments extended longevity, hypoxia increased fecundity and body size, and decreased food consumption and respiration rate. We uncovered 12 candidate genes that were differentially expressed in hypoxia-tolerant and sensitive clones in response to hypoxia. Unexpectedly, DNP increased fecundity and mitochondrial membrane potential without affecting food intake. This work opens up an opportunity for genomic determination of the potentially important phenotypes in a model organism.

In this study we build solutions to three common challenges in the fields of bioinformatics through utilizing statistical methods and developing computational approaches. First, we address a common problem in genome wide association studies, which is linking genotype features within organisms of the same species to their phenotype characteristics. We specifically studied FHA domain genes in Arabidopsis thaliana distributed within Eurasian regions by clustering those plants that share similar genotype characteristics and comparing that to the regions from which they were taken. Second, we also developed a tool for calculating transposable element density within different regions of a genome. The tool is built to utilize the information provided by other transposable element annotation tools and to provide the user with a number of options for calculating the density for various genomic elements such as genes, piRNA and miRNA or for the whole genome. It also provides a detailed calculation of densities for each family and subamily of the transposable elements. Finally, we address the problem of mapping multi reads in the genome and their effects on gene expression. To accomplish this, we implemented methods to determine the statistical significance of expression values within the genes utilizing both a unique and multi-read weighting scheme. We believe this approach provides a much more accurate measure of gene expression than existing methods such as discarding multi reads completely or assigning them randomly to a set of best assignments, while also providing a better estimation of the proper mapping locations of ambiguous reads. Overall, the solutions we built in these studies provide researchers with tools and approaches that aid in solving some of the common challenges that arise in the analysis of high throughput sequence data.

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Polyclonal plantings of Populus deltoides are expected to display increased site resource use, productivity, and tolerance to stress through plasticity changes leading to niche differentiation (i.e changes to crown/canopy structures). In the present study, P. deltoides Clones S7C8, 110412, and polyclonal plots were tested for differentially expressed genes and enriched biological pathways between planting schemes. Transcriptomic analysis of leaves revealed upregulation of an active growth gene and gene family members that play important roles in plant stress and stress tolerance in polyclonal plantings. A gene associated with oxidative stress was upregulated in polyclonal plantings across all treatments. Secondary metabolic pathways including arginine and proline metabolism were upregulated in monoclonal plantings and downregulated in polyclonal plantings. Phenotypic results displayed greater aboveground biomass in polyclonal plantings. Results suggested a potential increased tolerance in polyclonal plantings to water and heat stress, including increased productivity and resource usage.

Indiana University-Purdue University Indianapolis (IUPUI) / Triple-negative breast cancers (TNBCs) are negative for the expression of estrogen (ER), progesterone (PR), and HER-2 receptors. TNBC accounts for 15% of all breast cancers and results in disproportionally higher mortality compared to ER & HER2-positive tumours. Moreover, there is a paucity of therapies for this subtype of breast cancer resulting primarily from an inadequate understanding of the transcriptional differences that differentiate TNBC from normal breast. To this end, we embarked on a comprehensive examination of the transcriptomes of TNBCs and normal breast tissues using next-generation whole transcriptome sequencing (RNA-Seq). By comparing RNA-seq data from these tissues, we report the presence of differentially expressed coding and non-coding genes, novel transcribed regions, and mutations not previously reported in breast cancer. From these data we have identified two major themes. First, BRCA1 mutations are well known to be associated with development of TNBC. From these data we have identified many genes that work in concert with BRCA1 that are dysregulated suggesting a role of BRCA1 associated genes with sporadic TNBC. In addition, we observe a mutational profile in genes also associated with BRCA1 and DNA repair that lend more evidence to its role. Second, we demonstrate that using microdissected normal epithelium maybe an optimal comparator when searching for novel therapeutic targets for TNBC. Previous studies have used other controls such as reduction mammoplasties, adjacent normal tissue, or other breast cancer subtypes, which may be sub-optimal and have lead to identifying ineffective therapeutic targets. Our data suggests that the comparison of microdissected ductal epithelium to TNBC can identify potential therapeutic targets that may lead to be better clinical efficacy. In summation, with these data, we provide a detailed transcriptional landscape of TNBC and normal breast that we believe will lead to a better understanding of this complex disease.

Les myopathies héréditaires représentent un large groupe de pathologies neuromusculaires progressives affectant l’intégrité générale, structurelle et fonctionnelle du muscle squelettique. Elles engendrent une myriade de symptômes, ternissant qualité de vie et autonomie, et pouvant même s’avérer mortelles. La pose d’un diagnostic juste peut être difficile, entravée notamment par une faible prévalence de certaines myopathies, l’importante hétérogénéité clinique existante, et le chevauchement symptomatique des diverses formes. Malgré les avancées récentes faites dans le domaine des techniques de séquençage qui contribuent grandement au dépistage, au moins 25% des individus atteints de myopathies demeurent sans diagnostic génétique. Suivant l’investigation clinique d’un patient (Z46) atteint d’une myopathie distale à apparition tardive, l’analyse par séquençage ARN (RNA-seq) a révélé un variant non-sens homozygote de signification inconnue (VUS) à la fin de l’exon 5 du gène MLIP. Les niveaux d’expression génique altérés de « Protéine musculaire interagissant avec LMNA » (MLIP) et son partenaire « Lamine de type A » (LMNA) ont poussé à approfondir l’investigation. Davantage d’altérations -omiques furent identifiées par les techniques de RT-PCR, qPCR et WB, renforçant l’effet pathogénique du variant. Consolidant tous les résultats, le séquençage de longues lectures (LRS) a révélé un mécanisme d’épissage alternatif compensatoire de MLIP, qui tend à contourner et minimiser la production de transcrits arborant l’exon 5 muté. La présente étude vise à : 1) apporter un diagnostic génétique définitif au patient Z46, posant le variant MLIP comme causatif de la myopathie distale; 2) démontrer le pouvoir diagnostique du RNA-seq dans la résolution de ce cas complexe par l’identification et l’élucidation du VUS; 3) témoigner de l’étendue de la caractérisation transcriptomique offerte par les longues lectures du LRS. Couplé à cela, la modélisation du variant pathogénique par CRISPR/Cas9 dans une lignée cellulaire de myoblastes humains immortalisés permettra l’évaluation des impacts morpho-fonctionnels; conférant un supplément d’informations relatives aux fonctions musculaires normales et pathologiques de MLIP, faiblement caractérisées jusqu’à présent. / Hereditary myopathies represent a large group of progressive neuromuscular disorders affecting the general, structural and functional integrity of skeletal muscle. They cause a myriad of symptoms, impairing quality of life and autonomy, and can even be fatal. Making an accurate diagnosis can be difficult, hampered in particular by the low prevalence of certain myopathies, the significant clinical heterogeneity that exists, and the symptomatic overlap of the various forms. Despite recent advances in sequencing techniques that greatly assist in screening, at least 25% of individuals with myopathies remain without a genetic diagnosis. Following the clinical investigation of a patient (Z46) with a late-onset distal myopathy, RNA-sequencing (RNA-seq) analysis revealed a homozygous nonsense variant of unknown significance (VUS) at the end of exon 5 of the MLIP gene. Altered gene expression levels of ‘’Muscular LMNA-Interacting Protein’’ (MLIP) and its partner ‘’A-type Lamin’’ (LMNA) prompted further investigation. More -omic alterations were identified by RT-PCR, qPCR and WB technics, reinforcing the pathogenic effect of the variant. Consolidating all results, Long-Read Sequencing (LRS) revealed a compensatory alternative splicing mechanism of MLIP, which tends to bypass and minimize the production of transcripts carrying the mutated exon 5. The present study aims to: 1) provide a formal genetic diagnosis for patient Z46, positing the MLIP variant as causative of the distal myopathy; 2) demonstrate the diagnostic power of RNA-seq in resolving this complex case through identification and elucidation of the VUS; 3) testify to the breadth of transcriptomic characterization afforded by the long reads of LRS. Coupled with this, CRISPR/Cas9 modeling of the pathogenic variant in an immortalized human myoblast cell line will allow assessment of morpho-functional impacts; conferring additional information related to the normal and pathological muscles functions of MLIP, poorly characterized thus far.

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