The active human retrotransposon L1 is the most prevalent human retroelement, constituting 17% of the mass of the human genome and contributing significantly to mutagenesis. L1 mutagenizes human genomes in a number of ways including insertional mutagenesis of itself and other retrotransposons, creating of DNA double strand breaks, and induction of non-allelic homologous recombination. Through these processes, the activity of L1 is responsible for approximately 0.5% of all new genetic diseases. All L1-derived mutagenesis stems from the activity of a small number of intact full-length L1 loci that remain capable of mobilization. A smaller subset of these active L1s are called hot L1s and are responsible for the vast majority of all L1 activity. Hot L1s are polymorphic in the population and represent evolutionarily recent L1 insertion events. Here, we show that potentially active full length L1 elements are more prevalent in individual genomes than previously believed. We find that the typical individual likely harbors approximately 60 active and 50 hot L1s. However, we also find that there is significant variation between individuals in numbers of potentially active L1s. As a result, the mutagenic burden associated with L1 likely varies between individuals. / acase@tulane.edu

G–Protein Coupled Receptors are one of the most important targets in drug development, making up over 60% of drug targets. Recent studies have implicated a role of Regulator of G–Protein Signaling (RGS) proteins in the development and progression of pathologies, including some cancers. RGS17, the most–recently identified family member of the RZ family of RGS proteins, has been implicated in the growth, proliferation, metastasis and migration of prostate tumors as well as small–cell and non–small cell lung cancers. In neoplastic tumor tissues RGS17 is up–regulated 13 fold over patient–matched normal tissues in prostate cancer. Studies have shown that RGS17 RNAi knockdown inhibits colony formation and decreases tumorigenesis in nude mice. Based on these findings, this thesis explores the research undertaken to develop small molecule inhibitors of the RGS17: Gαo protein: protein interaction. In this thesis, we implemented AlphaScreen® technology to develop a high–throughput screening method for interrogating small molecule libraries for inhibitors of RGS17. Chapter 3 focuses on the initial results of the AlphaScreen® in 384–well format. The screen utilizes a measurement of the Gα: RGS17 protein: protein interaction (PPI) and with an excellent Z–score exceeding 0.73, a signal to noise ratio >70 and a screening time of 1,100 compounds per hour. Chapter 3 presents the development, validation and initial high–throughput screening for inhibitors of Gα: RGS17 interaction as well as preliminary characterization of the RL series of hits. In this pilot screen the NCI Diversity Set II was interrogated, yielding 35 initial hits of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 ΜM in dose–response experiments for inhibiting the Gα: RGS17 interaction. Four exhibited IC50 values <6 ΜM while inhibiting the Gα: RGS17 interaction >50% when compared to a biotinylated GST control (TrueHits). Compounds RL–1 and RL–2 were confirmed by flow cytometry protein interaction assay (FCPIA) while RL–3 and RL–4 were unable to disrupt this PPI in FCPIA. All four compounds were tested using the differential scanning fluorimetry (DSF) method, which is based on energetic coupling between ligand binding and protein unfolding and found compounds RL–1 to RL–4 all slightly increased protein stability upon ligand binding. Chapter 4 focuses on the miniaturization and optimization of AlphaScreen® to a 1536–well format and screening of the MicroSource SPECTRUM and NDL3000 small molecule libraries. This increased throughput 11–fold and decreased our working volumes from 45 ΜL to 10 ΜL, which reduced reagent cost. After optimization, we retained in an excellent Z–factor ≥0.70 with S/N>5.77 and increased the screening rate to more than 12,000 compounds per hour. In this format, the initial screening of the SPECTRUM and NDL3000 libraries was completed and filtered the initial hits by counter screening and PAINs filtering as well as developing four powerful orthogonal assays for the characterization of potential lead molecules. Chapter 6 focuses on the future directions, which include the screening the in–house 50,000 compound library in the University of Iowa HTS Core facility as well as the development of cell based assays to determine the activity of these leads in the cellular milieu. These screens are the first step to developing novel pharmacophores for further optimization of structure with the focus on RGS17 activity in enzymatic, whole cell, xenograft and whole animal models as well as providing new avenues for the development of anticancer therapies.

La microbiologie est la science qui s'attache à l'étude des microorganismes et de leurs propriétés. Depuis ses débuts au XV II siècle, les méthodes développées par les microbiologistes ont permis de révéler un énorme potentiel de connaissances et d'applications. Dans les dernières décennies, les industriels ont vu un intérêt tout particulier dans ce domaine d'étude. Le besoin de prévenir les contaminations en inhibant le développement microbien, ou au contraire la volonté de l'optimiser pour profiter des capacités de transformations chimiques des microorganismes a fait naître une demande croissante de tests microbiologiques. Le faible rendement des méthodes traditionnelles ne permettant pas de satisfaire à cette demande, la recherche de nouvelles méthodes de test focalise les intérêts. Les outils fluidiques miniaturisés ont d'ores et déjà fait preuve de leur potentiel pour ce type d'application, bien que leur validation vis-à-vis des méthodes classiques manque souvent.Dans ce travail de thèse, nous avons développé des incubateurs miniaturisés et des méthodes de suivi de populations de microorganismes optimisés. L'objectif est d'aborder l'impact de la réduction d'échelle d'incubation sur la croissance par rapport aux dispositifs de culture traditionnel, pour ensuite aboutir à un outil haut-débit pour la caractérisation de biocides. / Microbiology is the part of science linked with the study of microorganisms and their properties. Since its beginnings in the XV IIth century, the methods developped by the microbiologists revealed a huge potential of knowledge and applications.In the last decades, industrials realized the interests of this study areaThe need to prevent contaminations by inhibiting microbial development, or on the contrary the will to improve it to enjoy the chemical transformations capacities of the microorganisms gave birth to an increasing demand for microbiological tests. The poor yield of traditionnal methods does not allow to satisfy this need, and the search for new test methods is thus focalizing interests. Miniaturized fluidic tools have already proven their potential for this kind of applications, and yet, their validation towards traditionnal methods often lacks.In this work, we aim at developping miniaturized cultivation techniques and optimized growth analysis methods, to study the scale reduction impact of incubator's size on growth, in order to end up with a high-throughput tool for biocide caracterization.

Die Deregulation des Transkriptionsfaktors Myc ist ein charakteristisches Merkmal für eine Vielzahl von humanen Tumoren. Durch die transkriptionelle Aktivierung von Genen, die im Zusammenhang mit Metabolismus, Translation und Proliferation stehen, wird dadurch das Tumorwachstum begünstigt. Myc bildet zudem mit dem Zinkfinger-Protein Miz1 einen Komplex, der hemmend auf die Transkription von Zielgenen wirkt. Bisher sind nur wenige Myc/Miz1-reprimierte Zielgene bekannt. In der vorliegenden Arbeit konnten genomweit die DNA-Bindestellen von Myc und Miz1 durch Chromatin-Immunpräzipitationen gefolgt von Hochdurchsatzsequenzierung in einer Zervixkarzinomzelllinie bestimmt werden. Es konnte gezeigt werden, dass Myc an Promotoren aller drei RNA-Polymerasen sowie in enhancer-Regionen bindet, während Miz1 Kernpromotoren von RNA-Polymerase II- und III-transkribierten Genen besetzt. reChIP-Experimente zeigten, dass Myc und Miz1 als Komplex an Promotoren von Zielgenen binden. Zudem wurde ein Miz1-DNA-Bindemotiv identifiziert und der transaktivierende Einfluss von Miz1 auf Gene mit diesem Motiv nachgewiesen. Das überwiegende Vorhandensein von Myc/Max-Komplexen führt zu einer Transaktivierung von E-Box-haltigen Promotoren. Andererseits erfolgt die transkriptionelle Repression von Myc/Miz1-Zielgenen an Promotoren, an denen der Myc/Miz1-Komplex vorherrscht. In aktuellen Publikationen konnte gezeigt werden, dass nach mitogener Stimulation von Lymphozyten es zu einer Erhöhung der Myc-Expression kommt, wodurch Myc als ein genereller Transkriptionsaktivator fungiert, der alle Gene gleichermaßen induziert. Trotz hoher Myc-Mengen in Tumorzellen konnte die generelle Myc-vermittelte Transaktivierung nicht nachgewiesen werden. Zusätzlich zur Myc-abhängigen Transaktivierung von E-Box-haltigen Genen, z. B. beteiligt an Translation und RNA-Prozessierung, und der Miz1-vermittelten transkriptionellen Aktivierung von Genen mit Miz1-Motiv (z. B. involviert in Autophagie), konnte entgegen dem Modell der generellen Genamplifikation durch Myc eine Myc/Miz1-abhängige Repression von Zielgenen belegt werden. Die neu gewonnenen Erkenntnisse des Bindeverhaltens des Myc/Miz1-Komplexes und der daraus resultierenden transkriptionellen Regulation von Myc/Miz1-Zielgenen ermöglichen ein besseres Verständnis der Myc-Funktion in Tumorzellen und könnte zur Verbesserung von Tumortherapien führen. / Deregulation of the transcription factor Myc is a characteristic feature of a variety of human tumors. The Myc-dependent transcriptional activation of genes involved in metabolism, translation and proliferation therefor promotes tumor growth. Additionally, Myc forms a complex with the zinc finger protein Miz1, which represses transcription of target genes. So far, only a limited number of Myc/Miz1-repressed genes is known. Within the present thesis DNA binding sites of Myc and Miz1 were mapped genome-wide using chromatin immunoprecipitations followed by high-throughput sequencing in a cervical cancer cell line. It could be shown that Myc binds to promoters of all three RNA polymerases as well as to enhancer regions, whereas Miz1 binding sites could be found only in core promoters of RNA polymerase II and III transcribed genes. reChIP experiments illustrated binding of Myc and Miz1 as a complex on DNA. Additionally, a DNA binding motif of Miz1 was identified and furthermore it was possible to verify the transctivating influence of Miz1 on genes carrying that motif in the promoter. On E-box containing promoters the predominantly existence of Myc/Max complexes resulted in transactivation of the respective genes. Otherwise, transcriptional repression of Myc/Miz1 target genes occured at promoters where the Myc/Miz1 complex dominates. Recent publications have illustrated that after mitogenic stimulation of primary lymphocytes, Myc expression is enhanced, whereby Myc serves as a general transcriptional activator that induces the expression of virtually all genes. Although Myc levels are high in tumor cells that general mechanism of Myc-mediated transactivation could not be verified. Additionally to the Myc-dependent transactivation of E-box-containing genes, e. g. involved in translation and RNA processing, and Miz1-mediated transcriptional activation of genes containing a Miz1 binding motif (e. g. autophagy-related genes), and in opposition to the general amplifier model a Myc/Miz1-dependent repression of target genes could be proven. The obtained evidences concerning DNA binding properties of the Myc/Miz1 complex as well as the resulting transcriptional regulation of Myc/Miz1 target genes facilitates a better understanding of Myc function in tumor cells and could leed to better anti-tumor therapies.

Cette thèse a pour objectif de développer une méthode électrochimique permettant de cribler à haut débit des inhibiteurs d'enzymes. Pour cela, une enzyme cible a été sélectionnée pour son intérêt thérapeutique, la transcétolase (TK) : elle est impliquée dans de nombreuses pathologies (cancer, maladies neurodégénératives, diabète…) et dans la survie de certains parasites pathogènes. Afin de mesurer l'activité de la TK, deux systèmes rapporteurs ont été développés, à l'aide de plaques de criblage électrochimique (PCE) constituées de 96 électrodes indépendantes. Le premier système rapporteur repose sur un système bienzymatique nécessitant l'intervention d'une enzyme auxiliaire, la galactose oxydase (GAOx) sous forme libre ou immobilisée dans la laponite. Cette enzyme est capable d'oxyder les produits de réaction de la TK et de produire du peroxyde d'hydrogène. Le format 96 des PCE a permis d'optimiser rapidement la détection électrochimique par ampérométrie pulsée par intermittence (IPA) d'activité oxydase et seulement 10 minutes sont nécessaires pour réaliser 96 mesures simultanées. Parallèlement, afin d'exploiter au maximum le système à 96 électrodes, cette détection d'activité oxydase a également été réalisée, en 10 minutes par électrochimiluminescence. Cette méthode offre l'avantage d'être plus sensible et moins variable que par IPA, mais limite l'utilisation de matrice d'immobilisation d'enzymes. La GAOx a ensuite été utilisée pour mesurer l'activité TK, cependant, les conditions réactionnelles n'étant pas optimales en présence du système bienzymatique (TK-GAOx), des temps d'incubation longs sont nécessaires et sont peu adaptées au criblage d'inhibiteurs de la TK. Un second système rapporteur ne nécessitant plus d'enzyme auxiliaire et faisant intervenir uniquement un seul substrat de la TK a été optimisé. Ce système repose sur l'oxydation de l'intermédiaire réactionnel formé par la fixation du cofacteur (la thiamine pyrophosphate) et du substrat sur l'enzyme par le ferricyanure. Cette méthode permet de mesurer 96 activités TK en seulement 7 minutes et a aisément été utilisée pour cribler une bibliothèque de molécules. Le criblage de 1360 molécules a permis d'identifier un nouvel inhibiteur de cet enzyme, présentant une CI50 de 63 μM. Le système électrochimique a également été utilisé pour déterminer le mécanisme d'inhibition (non compétitive pure partielle) et la constante d'inhibition associée (3,4 μM). Ces résultats sont inédits dans le domaine de l'électrochimie et offrent un large éventail d'applications que ce soit pour le criblage d'activité enzymatiques ou d'inhibiteurs d'enzymes / This thesis focuses on the development of a new electrochemical method allowing the high throughputscreening of enzyme inhibitors. For this purpose, a target enzyme has been selected for its therapeutic interest,transketolase (TK): this enzyme is involved in many diseases (cancer, neurodegenerative diseases, diabetes ...) and inthe survival of pathogenic parasites. To measure TK activity, two reporter systems have been developed by usingelectrochemical plates, composed of 96 independent electrodes.The first one is based on a bienzymatic system, requiring the use of an auxiliary enzyme, galactose oxidase(GAOx), in its soluble form or immobilized in laponite. This enzyme is able to oxidize TK products and producehydrogen peroxide. The 96-well format allowed to quickly optimize the electrochemical detection of oxidase activityby intermittent pulse amperometry (IPA) of oxidase activity and only 10 minutes are required to perform 96simultaneous measurements. In parallel, in order to harness the 96-well electrochemical system, this detection ofoxidase activity was also carried out in 10 minutes using electrochemiluminescence. This method is more sensitiveand less variable than IPA but is limited to the use of soluble enzymes. However, the reaction conditions are notoptimal for the bienzymatic system (TK-GAOx): long incubation times are required and are poorly adapted for thescreening of TK inhibitors.A second reporter system no longer requiring an auxiliary enzyme and involving only one TK substrate hasbeen optimized. This system relies on the oxidation by ferricyanide of the reactional intermediate resulted of thebinding of the cofactor (thiamine pyrophosphate) and the substrate. This method allows to measure 96 TK activitiesin only 7 minutes and was easily used to screen an in-house chemical library. The screening of 1360 molecules leadto the identification of a new TK inhibitor with an IC50 of 63 μM. This electrochemical system was also used todetermine the mechanism of inhibition (partial non-competitive mechanism) and the associated inhibition constant(3.4 μM). These results are innovative in the field of electrochemistry and offer a wide range of applications forenzymatic activity screening or the screening of enzymes inhibitors

<p>The main objective of the reported study was to investigate how biomedical ontologies, logically structured representations of various aspects of the biomedical reality, can help researchers in analyzing experimental data. The dissertation reports two attempts to construct tools for the analysis of high-throughput experimental results using explicit domain knowledge representations. Furthermore, integrative efforts made by the community of Open Biomedical Ontologies (OBO), in which the author has participated, are reported, and a framework for consistently connecting the Gene Ontology (GO) with the Taxonomy of Species is proposed and discussed.</p>

Biological data derived from high-throughput microarrays can be transformed into finite, simple, undirected graphs and analyzed using tools first introduced by the Langston Lab at the University of Tennessee. Transforming raw data can be broken down into three main tasks: data normalization, generation of similarity metrics, and threshold selection. The choice of methods used in each of these steps effect the final outcome of the graph, with respect to size, density, and structure. A number of different algorithms are examined and analyzed to illustrate the magnitude of the effects. Graph-based tools are then used to extract putative gene networks. These tools are loosely based on the concept of clique, which generates clusters optimized for density. Innovative additions to the paraclique algorithm, developed at the Langston Lab, are introduced to generate results that have highest average correlation or highest density. A new suite of algorithms is then presented that exploits the use of a priori gene interactions. Aptly named the anchored analysis toolkit, these algorithms use known interactions as anchor points for generating subgraphs, which are then analyzed for their graph structure. This results in clusters that might have otherwise been lost in noise. A main product of this thesis is a novel collection of algorithms to generate exact solutions to the maximum clique problem for graphs that are too large to fit within core memory. No other algorithms are currently known that produce exact solutions to this problem for extremely large graphs. A combination of in-core and out-of-core techniques is used in conjunction with a distributed-memory programming model. These algorithms take into consideration such pitfalls as external disk I/O and hardware failure and recovery. Finally, a web-based tool is described that provides researchers access the aforementioned algorithms. The Graph Algorithms Pipeline for Pathway Analysis tool, GrAPPA, was previously developed by the Langston Lab and provides the software needed to take raw microarray data as input and preprocess, analyze, and post-process it in a single package. GrAPPA also provides access to high-performance computing resources, via the TeraGrid.

The concept of the gene has been constantly challenged by new discoveries in the life sciences. Recent challenging observations include the high frequency of alternative splicing events and the common transcription of non-protein-coding-RNAs (ncRNAs) from the genome. The latter has long been considered noise in biological systems. Multiple lines of evidence from genomic studies indicate that alternative splicing and ncRNA play important roles in expanding proteome diversity in eukaryotes. Here, the aim is to find the link between alternative splicing and ncRNAs by studying the expression profile of the spliceosomal snRNAs (U snRNA). Spliceosomal snRNAs are essential for pre-mRNA splicing in eukaryotes. They participate in splice site selection, recruitment of protein factors and catalyzing the splicing reaction. Because of this, both the abundance and diversity of U snRNAs were expected to be large. In our study we deeply analyzed the U snRNA population in primates using a combination of bioinformatical, biochemical and high throughput sequencing approaches. This transcriptome profiling has revealed that human, chimpanzee and rhesus have similar U snRNA populations, i.e. the vast majority of U snRNAs originate from few well-defined gene loci and the heterogeneity observed in U snRNA populations was largely due to the presence of SNPs at these loci. It seems that the gene loci that could potentially encode a significantly heterogeneous population of U snRNAs are mostly silent. Only few minority transcripts were detected in our study, and among them three U1-like snRNAs might play a role in the regulation of alternative splicing by recognizing non-canonical splicing sites. Mutations of U snRNA have been shown to impact the splicing process. Therefore, our study provides a reference to study the biological significance of SNPs in U snRNA genes and their association with diseases.

The main objective of the reported study was to investigate how biomedical ontologies, logically structured representations of various aspects of the biomedical reality, can help researchers in analyzing experimental data. The dissertation reports two attempts to construct tools for the analysis of high-throughput experimental results using explicit domain knowledge representations. Furthermore, integrative efforts made by the community of Open Biomedical Ontologies (OBO), in which the author has participated, are reported, and a framework for consistently connecting the Gene Ontology (GO) with the Taxonomy of Species is proposed and discussed.

Throughout this thesis work, various catalysts were investigated with combinational approach to develop highly active and selective novel catalysts for direct epoxidation of propylene to PO using molecular oxygen. The promoted and un-promoted silver (Ag), copper (Cu), ruthenium (Ru), manganese (Mn) mono and multimetallic catalytic systems over different silica supports were prepared via sol-gel method and incipient wetness method. In addition to support effect, the effects of different promoters on the catalytic performances of these catalyst candidates were investigated. The study showed that commercial silica (c-SiO2) is the most effective support when compared to silica (SiO2) and silica synthesized with templete (t-SiO2). Among bimetallic catalytic systems containing Ag, Ru, Mn and Cu metals, c-SiO2 supported Cu-Ru catalyst was determined as the most active catalytic system. In addition, the most effective v catalyst and promoter in the epoxidation reaction was determined as NaCI promoted Cu-Ru catalyst supported over c-SiO2 with 35.98% selectivity&amp / 9.55% conversion (3.44% yield) at 3000C and 0.5 feed gas ratio (C3H6/O2).. In the study, the selected catalysts showed low and high PO productivity were also investigated by characterization techniques such as XRD, XPS, BET and FTIR.It was inferred from characterization tests that bimetallic systems reveal a synergistic behavior by exposing more active sites on the silica support material with respect to their monometallic counterparts. Besides, NaCl catalytic promoter has a strong interaction particularly with the Cu sites on the Cu/Ru/SiO2 catalyst surface, altering the electronic structure of Cu sites that favors to PO production.