Freshwater sculpins (Cottus) are common throughout temperate regions of the Northern Hemisphere. Their broad distribution in the Western North America makes them a good model for understanding phylogeographic relationships among western fishes. Within much of the interior west three lineages, C. bairdii, C. confusus, and the C. beldingii complex, are most prevalent. The distribution of these three overlap in the Snake River Basin. All occur below Shoshone Falls on the Snake River. However, only two currently reside in the Upper Snake River above the falls. An exception are the Lost River streams of central Idaho. While these streams are technically part of the Upper Snake River Basin, they do not directly connect with the Snake River. Preliminary studies with a single mitochondrial DNA (mtDNA) gene suggested multiple pathways for Cottus introduction into the Lost River stream complex. Here, three mitochondrial and five nuclear genes were examined to investigate the phylogenetic relationships of these three lineages. Sequences were obtained from 71 different populations in the Lost River streams and surrounding basins. Maximum Likelihood (ML) phylogenies were constructed using these data. Our data indicate that relationships among populations within these species are complex and that no single invasion into the Lost River streams and surrounding regions can account for the phylogenetic signals detected. Instead, it appears that multiple invasions in an evolving landscape played a significant role in the modern distribution of species in this region.

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Salvia hispanica L. (commonly known as chia) is gaining popularity worldwide and specially in US as a healthy oil and food supplement for human and animal consumption due to its favorable oil composition, and high protein, fiber, and antioxidant contents. Despite these benefits and its growing public demand, very limited gene sequence information is currently available in public databases. In this project, we generated 90 million high quality 150 bp paired-end sequences from the chia leaf and root tissues. The sequences were de novo assembled into 103,367 contigs with average length of 1,445 bp. The resulted assembly represented 92.2% transcriptome completeness. Around 69% of the assembled contigs were annotated against the uniprot database and represented a diverse array of functional and biological categories. A total of 14,267 contigs showed significant expression difference between the leaf and root tissues, with 6,151 and 8,116 contigs upregulated in the leaf and root, respectively. The sequence data generated in this project will provide valuable resources for future functional genomic research in chia. With the availability of transcriptome sequences, it would be possible to identify genes involved in the important metabolic pathways that give chia its unique nutritional and medicinal properties. Finally, the generated data will contribute to the genetic improvement efforts of chia to better serve the public demand.

Mycobacterium tuberculosis (Mtb) remains a pathogen of significant importance with respect to global health. Although approximately one third of the world is infected with TB, only 5-10% develop clinical manifestations of active TB within 2 years post exposure. Infection with Mtb can cause active tuberculosis (ATB), inactive latent infection (LTBI) and be reactivated. The immune response is contained within the formation of a collection of immune cells, a granuloma, in the infected individual’s lungs, which is seen in all disease states. The granuloma functions both as an immune response to contain the bacterium from surrounding lung parenchyma as well as a site for the bacterium to remain in the individual; therefore, providing an environment in which the bacterium maintains the ability to reactivate. We currently lack a complete understanding of the physiology and the metabolic state of Mtb in this granulomatous environment during different states of infection. Leveraging a novel technique known as mesodisection, we microdissect TB granulomas from various infective stages as well as from different sections of the granuloma from non-human primate (NHP) derived formalin fixed paraffin embedded (FFPE) lung tissue. From these extracted tissue sections, RNA is extracted, amplified and subsequent microarray and nCounter analysis is performed; consequently, allowing us to uncover the Mtb specific transcriptomic profiles. First, our findings reveal statistically significant Mtb genes induced in ATB and LTBI in various granuloma types. Of the genes induced, many belong to the following categories: PE/ PPE, sigma factors, toxin-antitoxin complexes and the DosR regulon. In addition, our findings reveal a core group of genes commonly identified in both ATB and LTBI induced in ATB and LTBI in various granuloma types. Of the genes induced, many belong to the following categories: PE/ PPE, sigma factors, toxin-antitoxin complexes and the DosR regulon. In addition, our findings reveal a core group of genes commonly identified in both ATB and LTBI in all lesion types. Further, we propose that these findings improve our understanding of the physiology of the pathogen as well as its virulence which in turn can be used for the development of improved therapeutics, diagnostics and vaccines. / acase@tulane.edu

Chronic consumption of, and acute intoxication from, alcohol can have profound effects on the functional integrity of the central nervous system (CNS). The resident immunomodulatory cells of the CNS, microglia, provide signaling factors with both pro- and anti-inflammatory effects for protection. Microglial activation ranges through a multiplex of phases, of which have yet to be defined when induced by exposure to alcohol, and how the activation impacts surrounding cells. Exposure of alcohol has been revealed to induce an immune response in microglia, which can exhibit characteristics unique to a pro-inflammatory response depending on dose and time of alcohol exposure. To define the activation state produced by microglia in response to alcohol, ethanol-induced microglial protein and microRNA (miRNA) global profile expression changes were obtained in vitro, using the BV2 murine microglial cells, using mass spectrometry (MS)-based proteomics and microarray-based transcriptomic approaches, respectively, revealing potential regulatory miRNAs for inflammation mediation. The 2,277 protein groups identified through mass spectrometry and 3,195 miRNA genes identified using microarray analysis provided a strong foundation to determine miRNA-mRNA regulators and the pathways in which they are involved, that potentially play a role in microglial activation. The comparison of the miRNA expressed in microglia after lipopolysaccharide (LPS) and ethanol (EtOH) exposure, indicate that EtOH influenced miRNA does not signify having a pro-inflammatory activation phenotype, but the miRNA expressed under the influence of LPS does support this phenotype. The global pathway regulation evidence and defined proteins and miRNA-mRNA interactions upon microglial activation have the possibility to unite the pathways described in previous studies and further our understanding of EtOH-induced microglial activation, and their role in neuroinflammation and neurodegeneration. Further research to determine and validate the extent of gene regulation by miRNAs and subsequent impact on specific protein levels should be employed to define the miRNA transcriptome influence on pathways relevant to microglial function.

<p>This review article presents the splicing process during messenger RNA maturation and how it is regulated by different <em>Cis</em>-regulatory RNA-sequence elements and splicing factors. A more detailed description of the process alternative splicing and its importance to the function of genes from the model organism <em>Arabidopsis thaliana</em> is also given. A single eukaryotic gene can by the process alternative splicing (AS) give rise to a number of functionally mature mRNA-molecules, which in turn encodes for structurally and/or functionally different proteins. During the course of evolution, the process alternative splicing has thus shown to be effective in increasing transcriptome and proteome diversity of most eukaryotic organisms. This suggests therefore that the dominant theory in molecular biology, a gene encodes for a protein, needs to be corrected. A future challenge is to determine the function of the proteins obtained from a given gene by alternative splicing.</p>

L'enjeu de l'étude du transcriptome est de proposer de nouveaux biomarqueurs pronostiques. Cette<br />étude soulève cependant de nombreuses questions statistiques dues à l'analyse simultanée de l'expression<br />de milliers de gènes pour un nombre restreint de patients. Nous nous sommes intéressés<br />à deux aspects de l'évaluation des modèles pronostiques issus de l'analyse du transcriptome. Dans<br />un premier temps, l'utilisation complémentaire de jeux de données simulés et publics nous a permis<br />de montrer que le choix de la méthode d'analyse la plus adaptée repose sur la manière dont ses<br />propriétés théoriques s'adaptent à la structure des données. Cette réflexion a été appliquée aux<br />analyses discriminante et inter-groupes. Dans un second temps, des simulations nous ont permis<br />d'estimer les contributions respectives des variables clinico-biologiques classiques et transcriptomiques<br />dans des modèles de survie. Les paramètres associés à la surestimation de la contribution<br />des biopuces ont été identifiés.

Novel technologies for genome analysis have provided almost unlimited opportunities to uncover structural gene variants behind human disorders. Whole exome sequencing (WES) is especially useful for understanding rare Mendelian conditions, because it reduces the requirements for a priori clinical data, and can be applied on a small number of patients. However, supporting functional data on the effect of specific gene variants are often required to power these findings. A variety of methods and biological model systems exists for this purpose. Among those, induced pluripotent stem cells (iPSCs), which are capable of self-renewal and differentiation, stand out as an alternative to animal models. In papers I and II we took advantage of WES to identify gene variants underlying autosomal recessive pure hair and nail ectodermal dysplasia (AR PHNED) as well as autosomal dominant familial visceral myopathy (FVM). We identified a homozygous variant c.821T&gt;C (p.Phe274Ser) in the KRT74 gene as the causative mutation in AR PHNED, supported by the fact that Keratin-74 was undetectable in hair follicles of an affected family member. In a family segregating FVM we found a heterozygous tandem base substitution c.806_807delinsAA (p.(Gly269Glu)) in the ACTG2 gene in the affected members. This novel variant is associated with a broad range of visceral symptoms and a variable age of onset. In Paper III we explored the similarity between clonally derived iPSC lines originating from a single parental fibroblast line and we highlighted the necessity to use lines originating from various donors in disease modeling because of biological variation. Paper IV focused on how the genomic integrity of iPSCs is affected by the choice of reprogramming methods. We described several novel cytogenetic rearrangements in iPSCs and we identified a chromosome 5q duplication as a candidate aberration for growth advantage. In summary, this doctoral thesis brings novel findings on unreported disease-causing variants, as supported by extensive genetic analysis and functional data. A novel molecular mechanism behind AR PHNED is presented and the phenotypic spectrum associated with FVM is expanded. In addition, the thesis brings novel understanding of benefits and limitations of the iPSC technology to be considered for disease modeling.