Bioaugmentation with Dehalococcoides-containing cultures is a successful technology for the remediation of chlorinated ethene-contaminated roundwater. The overall goal of this research was to identify and characterize genes that are used by a Dehalococcoides-containing culture, KB-1, during degradation of Trichloroethene (TCE) to ethene, via cis-Dichloroethene (cDCE) and vinyl chloride. Firstly, the diversity and dynamics of the microbial populations within KB1 was assessed using 16S rRNA clone libraries and quantitative PCR analyses. Secondly, reductive-dehalogenase-homologous- (RDH) genes in KB1 were identified, sequenced and their transcription compared through RNA-generated RDH cDNA clone libraries. Finally, to elucidate functionally important genes within the community, Shotgun metagenome microarrays were constructed and used to investigate transcription during dechlorination. Results of the phylogenetic analyses indicated that KB1 is a diverse community of microorganisms whose stability is enhanced by functional redundancy within the culture. To fully understand this diverse community of uncultivated microorganisms a metatranscriptome approach was used. Experiments with shotgun metagenome microarrays identified spots which were statistically significantly differentially expressed during dechlorination. These spots were then sequenced, revealing Dehalococcoides and non-Dehalococcoides-genes which are important during dechlorination. These results demonstrated that shotgun microarrays can be constructed without prior sequence knowledge and used to effectively examine differential transcription within an uncultivated community. Subsequently, all of the spots of the array were sequenced, and additional array experiments were conducted. Sequencing identified 24 reductive dehalogenase genes in the culture, and analysis of the microarray results indicated that many of these RDH genes were differentially expressed in response to certain chlorinated compounds. Interspecies interactions were also highlighted as results suggested that non- Dehalococcoides microorganisms provide partial corrinoids which Dehalococcoides salvages to synthesize cobalamin which is essential for reductive dehalogenation. Transcription of CRISPR-associated genes also indicated interaction between phage and other microorganism in the KB1 community. Overall these results provided sequence and transcription information about possible biomarkers for reductive dechlorination by KB1 and can be used for more effective design and monitoring of bioremediation technologies.

Differential scanning calorimetry (DSC) melting analysis was performed on 27 short double stranded DNA duplexes containing 15 to 25 base pairs and short single stranded overhangs from one to 10 bases, on both ends. Molecules have two 5' dangling ends or one 5' and one 3' dangling end. For these molecules the duplex region was incrementally reduced from 25 to 15 base pairs with increased length of the dangling ends from one to 10 bases. A third set of molecules contained 21 base pair duplexes with a four base dangling end on either the 5' or 3' end. Blunt ended duplexes from 15 to 25 base pairs were also examined and served as control duplexes. DSC melting curves were measured in solution containing 85 mM, 300 mM or 1.0 M Na+. From these measurements, thermodynamic parameters for 5' and 3' dangling ends as a function of end length were evaluated. Results showed the 5' ends were slightly stabilizing, and this stability was essentially constant with end length, while the 3' ends were generally destabilizing with increasing length of the end. This finding of lower stability for the 3' ends is consistent with results of published studies that have found 5' dangling ends to be more than or equally as stabilizing as 3' dangling ends. Our finding that 3' dangling ends are actually destabilizing for duplex DNA contrasts with published results. The 3' ends also display a stronger dependence on the [Na+]. In the lower Na+ environment the 3' ends are more destabilizing than at the higher salt environments. Analysis of the thermodynamic parameters of the dangling-ended duplexes as a function [Na+] indicated the 3' dangling end molecules behave differently compared to 5' dangling ended and blunt ended duplexes. The net counterion release per phosphate upon melting the molecules having one 5' and one 3' end was approximately 15% smaller as a function of end length compared to the duplex having two 5' ends. Further analysis of the DSC evaluated thermodynamic transition parameter, ΔHcal, and its relationship to the measured transition temperatures of the DNA molecules, provided an estimate on the excess heat capacity differences, ΔCp, between duplex and melted single strands for the dangling-ended molecules. The analysis revealed the molecules with one 5' and one 3' dangling end had very different ΔCp values compared to the blunt-ended molecule; while the molecules with two 5' ends have ΔCp that are essentially the same as the blunt-ended duplex. These observations are interpreted as differences in the interactions with Na+, solvent and the terminal base pairs of the duplex for the 5' versus 3' dangling ends.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisors: Babatunde Ogunnaike and Jeremy S. Edwards, Dept. of Chemical Engineering. Includes bibliographical references.

This dissertation explores the genetic basis of gene expression in Fundulus heteroclitus by focusing on the role of the environment and its effects on gene expression and by making direct estimates of heritability using cDNA microarrays. The second chapter describes the utility of F. heteroclitus cDNA microarrays for studies of F. heteroclitus which seek to understand the genetic variation in gene expression. Measurements of mRNA fluorescence and concentration as well as differences in sample preparation and sampling of blood from a single individual over time demonstrate that F. heteroclitus cDNA microarrays are quantitative, reproducible and consistent. The third chapter examines the effect of the environment and genetic factors on the variation of gene expression. F. heteroclitus cDNA microarrays are used to determine whether a genetic component of gene expression can describe the variation in gene expression between inbred and outbred individuals from the same population. The results show that variation in mRNA expression is related to the genetic variation among individuals within a group. While chapter three reveals that there is a genetic component of variation in gene expression, the percentage of genes that are significantly heritable was not known. In the fourth chapter, the heritability of the variation in gene expression is estimated to determine the genetic basis of gene expression in F1 individuals from natural, outbred populations of F. heteroclitus. The data presented in chapter 4 are the first to formally estimate the genetic component of gene expression in F. heteroclitus. The estimates of heritability range from 0.25 to 0.86 depending on the estimation method with approximately 6.5% of genes having significant heritability. The results presented in this dissertation support the concept that genetic variation affects variation in mRNA expression among natural populations of F. heteroclitus. Natural, heritable variation in gene expression is important for understanding evolutionary adaptation and the role of natural selection in evolutionary processes.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

La thèse s’inscrit dans un projet de recherche global visant à caractériser les tumeurs thyroïdiennes sur le plan moléculaire, afin de mieux comprendre leur physiopathologie et afin d’identifier des biomarqueurs (signatures moléculaires) qui pourront être utilisés pour le diagnostic, le pronostic et leur traitement. Parmi celles-ci, nous distinguons les adénomes autonomes (AA) et folliculaires (FTA) tumeurs bénignes encapsulées, et les carcinomes, tumeurs malignes. Ceux-ci sont eux-mêmes subdivisés en carcinomes différenciés, folliculaires (FTC) ou papillaires (PTC), et peuvent évoluer en carcinomes anaplasiques (ATC), totalement dédifférenciés. Un autre type de tumeurs bénignes différenciées, très rares, existe: l’hyperthyroïdie non auto-immune familiale (FNAH). Ces tumeurs sont causées pour la plupart par des mutations qui activent de manière constitutive des cascades de signalisation, essentiellement la cascade de l’AMPc et la cascade des MAPK. Le but de notre thèse était de valider un système expérimental in vitro des PTC, d’étudier les profils d’expression génique des FNAH et de les comparer avec ceux des AA, et de définir les profils ARNm et miRNA des ATC pour les comparer à ceux des PTC pour identifier de nouvelles cibles thérapeutiques potentielles. Pour réaliser ces objectifs, nous avons utilisé la technologie des microarrays qui permet d’analyser simultanément l’expression de milliers de gènes dans différentes conditions. Nous avons donc utilisé une approche multidisciplinaire alliant une partie expérimentale et une partie bioinformatique. La première partie du travail a consisté à réaliser un modèle d’étude in vitro pour caractériser les PTC au niveau moléculaire. A cet effet, des cultures primaires de thyrocytes ont été traitées avec de l’EGF et du sérum pendant différents temps (1,5h, 3h, 16h, 24h et 48h) ce qui stimule la cascade des MAPK, activée constitutivement dans les PTC. Nous avons hybridé sur des lames microarrays maison les différents échantillons et nous avons montré que les cultures primaires stimulées pendant des temps longs (24h et 48h) ont des profils d’expression génique qui ressemblent à ceux des PTC et constituent donc un bon modèle d’étude de cette tumeur. La seconde partie a pour objectif de définir les phénotypes moléculaires et fonctionnels des FNAH et de les comparer aux AA. Ces deux pathologies résultent d’une mutation dans le récepteur de la TSH (TSHR) activant de manière constitutive la cascade de l’AMPc. Dans le cas des FNAH, la mutation est héréditaire et toute la glande est affectée contrairement aux AA où la mutation survient plus tard, généralement à l’âge adulte, et où seule une partie de la glande est affectée. Nous avons comparé le profil d’expression génique des FNAH avec celui des AA, par hybridation sur des lames microarrays HEEBO. L’intégration de ces différentes données montre que les AA et les FNAH sont deux sous-types différents de la même maladie: l’hyperthyroïdie génétique. Les caractéristiques de chacun de ces sous-types dépendent de l’intensité de la mutation, du nombre de cellules initialement affectées et du stade de développement au moment duquel la mutation survient. Dans la dernière partie de ce travail, nous avons caractérisé les ATC au niveau du profil d’expression des ARNm et des miRNA par hybridation respectivement sur lames Affymetrix ou sur lames miRNA maison et au niveau de leur état mutationnel du gène p53. Le profil d’expression génique des ATC a été comparé avec celui des PTC afin de mettre en évidence des gènes différentiellement exprimés entre les 2 types de cancers, que nous avons ensuite tenté d’invalider par siRNA, dans un modèle in vitro de lignée cellulaire thyroïdienne dérivée d’un ATC (8505C). Les résultats obtenus jusqu’ici ne sont malheureusement pas prometteurs. Le profil d’expression des miRNA nous a permis d’identifier une signature de 34 miRNA caractéristique des ATC.

Thesis Erasmus University Rotterdam. / ook verschenen in gedrukte versie. With bibliogr., with a summary in Dutch.

Thesis (M.S. in App. Bio.)--School of Biology, Georgia Institute of Technology, 2004. Directed by Roger M. Wartell. / Includes bibliographical references (leaves 34-37).

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