Im Rahmen der EU-Richtlinie REACH müssen industrierelevante Chemikalien bezüglich des Risikos für Mensch und Umwelt (neu) untersucht und bewertet werden, wobei die Anzahl an Tierversuchen zu minimieren ist und neue toxikologische Prüfmethoden, Bewertungsstrategien sowie alternative Testsysteme entwickelt und optimiert werden sollen. Ubiquitär vorkommende Testorganismen, welche ähnliche Eigenschaften und eine vergleichbare Sensitivität bezüglich äußerer Einflüsse wie komplexere Organismen (Fisch, Säugetier, Mensch) besitzen, sind hier besonders gefragt um die Belastung abzuschätzen und Trendaussagen über die Wirkung zu formulieren. Durch den Einsatz vieler dieser Testorganismen sowie durch die Ermittlung der chemischen Reaktivität und der Strukturmerkmale einer Verbindung kann ein globales Bild über die Wirkung erhalten werden. Ein Organismus, der diese Charakteristika aufweist, ist der eukaryotische Einzeller Tetrahymena pyriformis GL. In der vorliegenden Dissertation wird zum einen die jeweilige Toxizität elektrophiler Substanzen unterschiedlicher Stoffklassen und Reaktions-mechanismen (α,β-ungesättigte Carbonyl- und Carboxylverbindungen, heterozyklische Drei- und Vierringe, α-halogenierte Carbonyle, Harnstoffe und Thioharnstoffe, aromatische Disulfide sowie aliphatische und aromatische Nitroverbindungen) nach den Expositionszeiten 24 h, 48 h und 72 h mit Hilfe eines etablierten Wachstumshemmtests (Müller, 2001) bestimmt und Struktur-Toxizitäts-Beziehungen bzw. Strukturalarme abgeleitet. Neben der methodischen Optimierung werden die substanzspezifischen Eigenschaften, Flüchtigkeit und Sorptionsfähigkeit, quantitativ erfasst und durch neu aufgestellten Modellgleichungen für jede Substanz ermittelt. Mit der Bestimmung der Toxizitätserhöhung, Te, welche auch als ein Maß für die Reaktivität angesehen werden kann, werden Organismen-spezifische Narkose-Basis-Geraden generiert, exzesstoxische Verbindungen identifiziert und Strukturalarme für die jeweiligen Stoffklassen aufgestellt. Zum anderen werden binäre Mischungen untersucht, welche aus den Einzelstoffen bestehen, deren Toxizität im ersten Abschnitt der Arbeit ermittelt wurden und bei denen theoretische Annahmen über die Reaktionsmechanismen bestehen. Dieser Abschnitt identifiziert, ob gleiche bzw. unähnliche primäre Wechselwirkungen zweier Substanzen mit den biologischen Targets vorliegen und welcher Mechanismus, der reaktive Mechanismus exzesstoxischer Substanzen oder die Wechselwirkungen mit Membran-Bestandteilen, dominierend ist. Für die Auswertung der Mischungsergebnisse werden die etablierten biometrischen Modelle Konzentrations-Additivität und Unabhängigen Wirkung verwendet. Es wird dargestellt, dass die Mischungstoxizitäten der binären Mischungen ähnlich wirkender Substanzen zwar relativ genau mithilfe beider Modelle berechnet werden, jedoch keine eindeutige Charakterisierung der Mechanismen möglich ist. Die Ergebnisse der untersuchten binären Mischungen unterschiedlich wirkender Substanzen zeigen dagegen, dass die Mischungstoxizitäten unähnlicher Substanzen relativ genau mithilfe des Modells der Unabhängigen Wirkung berechnet werden und eine Charakterisierung der Mechanismen möglich ist.

Le cortex de la plupart des protistes ciliés contient un squelette membranaire dont le principal élément est l'épiplasme, une structure apposée à la membrane alvéolaire interne. Chez la paramécie, l'épiplasme se présente sous forme d'écailles indépendantes disposées autour de chaque appareil ciliaire ; il est composé d'une famille multigénique de protéines appelées épiplasmines. Nous avons réalisé une étude structurale de cette famille multigénique. L'analyse phylogénétique a permis de confirmer l'existence de 5 groupes d'épiplasmines divisé chacun en deux sous-groupes a et b. L'utilisation de la méthodologie HCA nous a permis de montrer que ces protéines sont modulaires et présentent un arrangement de leurs domaines structuraux. Elles peuvent ainsi être regroupées en trois classes symétriques, asymétriques et atypiques. L'analyse des régions 5'UTR des membres de cette famille multigénique montre la présence d'éléments putatifs de régulation d'expression. La comparaison des épiplasmines de la paramécie avec leurs orthologues de Tetrahymena montre une relation structurale entre les groupes 1,2,3 et 5 et les EpiT 1,2,3, et 5 respectivement suggérant l'existence d'un ancêtre commun pour l'épiplasme de Paramecium et Tetrahymena. Nous avons réalisé l'analyse fonctionnelle des épiplasmines à partir d'approche par ARN interférence et par localisation couplée à la GFP. La perturbation de l'expression des épiplasmines symétriques et asymétriques, aboutit à une réponse cellulaire commune qui se traduit par un changement de la forme cellulaire, un blocage de la cytocinèse et enfin l'apparition de formes plasmodiales. L'analyse du cortex par microscopie à fluorescence montre une altération des unités corticales qui est fonction des types structuraux des épiplasmines. Les épiplasmines se localisent de manière différentielle autour du corps basal définissant un territoire dont le modèle d'organisation est centrifuge. On définit alors des épiplasmines cinétomosales, péricinétomosales, core et enfin périphériques. Ce modèle est discuté en relation avec les phénotypes obtenus par l'analyse fonctionnelle. Il permet d'intégrer les différents niveaux de relation entre le corps basal, son territoire et l'ensemble de l'épiplasme.

No description available.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 90-99).

Many functional RNAs are required to fold into specific three-dimensional structures. A fundamental property of RNA is that its secondary structure and even some tertiary contacts are highly stable, which gives rise to independent modular RNA motifs and makes RNAs prone to adopting misfolded intermediates. Consequently, in addition to stabilizing the native structure relative to the unfolded species (defined here as stability), RNAs are faced with the challenge of stabilizing the native structure relative to alternative structures (defined as structural specificity). How RNAs have evolved to overcome these challenges is incompletely understood. Self-splicing group I introns have been used to study RNA structure and folding for decades. Among them, the Tetrahymena intron was the first discovered and has been studied extensively. In this work, we found that a version of the intron that was generated by in vitro selection for enhanced stability also displayed enhanced specificity against a stable misfolded structure that is globally similar to the native state, despite the absence of selective pressure to increase the energy gap between these structures. Further dissection suggests that the increased specificity against misfolding arises from two point mutations, which strengthen a local tertiary contact network that apparently cannot form in the misfolded conformation. Our results suggest that the structural rigidity and intricate networks of contacts inherent to structured RNAs can allow them to evolve exquisite structural specificity without explicit negative selection, even against closely-related alternative structures. To explore further how RNAs gain stability from intricate architectures, we examined a novel group I intron from red algae (Bangia). Biochemical methods and computational modeling suggest that this intron possesses general motifs of group IC1 introns but also forms an atypical tertiary contact, which has been reported previously in other subgroups and helps position the reactive helix at the active site. In the Bangia intron, the partners have been swapped relative to known group I RNAs that include this contact. This result underscores the modular nature of RNA motifs and provides insight into how structured RNAs can arrange helices and contacts in multiple ways to achieve and stabilize functional structures. / text

Group I introns are structured RNAs that have been used extensively as model systems for RNA folding because they are experimentally tractable, yet complex enough to have folding challenges associated with larger RNAs. The Tetrahymena group I intron consists of a set of conserved core helices and a set of peripheral elements. Peripheral elements surround the core helices and form long range tertiary contacts between each other and to the core. Interestingly, a long-lived misfolded state is populated that has the same long range tertiary contacts as the native state but differs locally within the core. Our lab showed that the intact periphery is necessary to specify the correct core structure, as mutating tertiary contacts or removing the P5abc peripheral element dramatically destabilized the native ribozyme relative to the misfolded form. However, we also showed that the thermodynamic benefit peripheral structure provided is accompanied by kinetic liability in folding, apparently because native tertiary contacts formed by peripheral elements around the misfolded core must come apart to allow refolding of the misfolded RNA to the native state. In addition to peripheral elements, proteins also play a role in stabilizing the native structures of many group I introns. The CYT-18 protein, which occupies the same binding site as P5abc, stabilizes the functional structures of certain group I introns by using a set of insertions that are absent in other related bacterial and mitochondrial aminoacyl tRNA synthetases. Using the P5abc deletion variant of the Tetrahymena ribozyme, I sought to further define CYT-18 roles in RNA folding by probing its thermodynamic and kinetic effects on the native state formation relative to the misfolded state. I demonstrated that CYT-18, like P5abc, provided thermodynamic stability to the native state. However, unlike P5abc, CYT-18 had no apparent effect on the refolding kinetics, suggesting that a protein co-factor can stabilize the functional structure without acquiring the associated costs in RNA folding kinetics. Furthermore, I found that the mechanism of CYT-18 action appears to be distinct from P5abc. Disruption of the long-range contact P14, which is formed between P5c and L2 and is part of the network of peripheral contacts, dramatically weakened P5abc binding to the native ribozyme core by ~10⁸ fold. Interestingly, CYT-18 maintained specific and tight binding to these mutants, which suggests that CYT-18 does not rely on a circular network of contacts to specifically stabilize the native state. Instead, the specificity may arise from a more direct and intimate contact of CYT-18 with the ribozyme core. This study gives insight into an evolutionary advantage of protein co-factors in RNA folding; proteins may offer thermodynamic assistance without inhibiting folding kinetics. / text

Currently, bacterial movement in karst aquifers is not well understood. Use of stable isotopes to label non-pathogenic Escherichia coli as a particulate groundwater tracer in karst systems has been examined in previous studies. Loss of the stable isotope signal is anticipated in traces greater than 500 m in length. Potential loss of 15N due to predation by protozoa was examined. Filter-sterilized water from Royal Spring in Georgetown, Kentucky, was inoculated with a mixture of either Tetrahymena pyriformis or Colpoda steinii and 15N-enriched E. coli and stored in the dark at 14°C. Samples were analyzed for their nitrogen isotope composition (as δ15N values), and for population counts of bacteria and protozoa in a time course experiment, on days zero and seven after inoculation. Protozoan populations increased in the presence of E. coli, while bacterial populations decreased. δ15N values increased in T. pyriformis fed enriched E. coli but did not show values as high as the bacteria themselves, indicating that attenuation via predation may be a concern in future groundwater traces.

Msh2 and Mlh1 proteins from Tetrahymena thermophla are homologues of MutS and MutL from Escherichia coli respectively. MutS and MutL are DNA mismatch repair proteins. In eukaryotes, MutS homologues recognize the replication errors and MutL homologues interact with MutS homologues and other proteins to make the repair occur. Biolistic transformation has been done to make the msh2 and mlh1 single knockouts in the macronuclei of different strains and the knockouts were verified complete. Two strains of WT crossing KO or KO crossing KO, with different mating types, were induced to conjugate. The processes were studied by microscopy using DAPI staining. For the msh2 knockouts, there were no crescent micronuclei formed throughout the conjugation of two knockout cells, and the pairing level was reduced severely. However, a knockout cell and a wild-type cell could conjugate normally at a high level pairing efficiency. Msh2 protein seems to be important to cell pairing and indispensible for the formation of the crescent micronuclei during cell conjugation. For the mlh1 knockouts, the pairing level of a knockout and a wild-type was reduced by half and the pairing level of two knockouts was reduced more than 80%; however, the paired cells in both could complete the conjugation with delay. Pms2 protein may have redundant roles in the MutL heterodimer (Mlh1-Pms2). In addition, chemical mutagens treated knockout was crossed with non-treated wild-type and the conjugation was compared with treated wild-types. Most of the treated knockout cells could not pair after starvation and mixing with non-treated wild-type cells, which means most of the cells could not enter meiotic phase. It is probable that G2/M checkpoint arrested the meiotic cell cycle and the intra-S phase was inactivated. Thus, Msh2 protein may have a role in the meiotic intra-S phase checkpoint system.

The DNA of all organisms is continuously exposed to exogenous and endogenous genotoxic agents. Fortunately, through the concerted actions of several DNA repair and mutation avoidance pathways, DNA damage can be removed and an organism’s genomic stability maintained. DNA base-base mismatches are generated as a result of the inherent replication errors made by the DNA replication machinery, as well as during the meiotic pairing of homologous but non-identical chromosomes. Through the coordinated actions of the highly conserved DNA mismatch repair (MMR) system, these errors are detected, removed and corrected, thus restoring the integrity of the DNA. In the absence of DNA MMR, genetic instability is unavoidable, resulting in the accumulation of mutations, and in mammals, a susceptibility to cancer. To better understand the roles of the MMR system in mutation avoidance during DNA replication, meiosis, and in nuclear apoptosis, we have utilized the nuclear dimorphic, ciliate protozoan Tetrahymena thermophila. We have identified seven putative MMR homologues; two are similar to eukaryotic MLH1 and PMS2, respectively, and five are similar to eukaryotic MutS homologues, one with eukaryotic MSH2 and four with MSH6. Our studies demonstrate that during conjugation, the relative transcript abundance of each MMR homologue is increased compared to vegetatively growing or nutritionally deprived (starved) cells. Also, the expression profile throughout conjugation is bimodal, corresponding to micronuclear (MIC) meiosis and macronuclear (MAC) anlagen development, both periods in which DNA replication occurs. Cells containing macronuclear knockouts of the PMS2, MSH2 and MSH6_1 genes were unable to successfully pair and complete conjugation, but were viable throughout vegetative growth. Cells in which the macronuclear MSH6_2 gene was knocked out had a phenotype that was similar to wild-type cells, during conjugation and vegetative growth. Interestingly, we observed that the MIC of cells containing MAC knockouts of the PMS2 and TML1 genes appear to have decreased copy number of specific “target sequences”, as determined by qPCR using the Random Mutation Capture (RMC) assay. This decrease reflects neither a loss of micronuclei nor a reduction in total micronuclear DNA content. These studies demonstrate that the PMS2, TML1, MSH2, and MSH6_1 homologues are necessary for the maintenance of micronuclear function and stability during conjugal development and vegetative growth, whereas the remaining MSH6 homologues have less pronounced roles in DNA repair and development. Additionally, macronuclear development in Tetrahymena appears less reliant on the DNA mismatch repair system and perhaps uses alternate surveillance mechanisms to maintain genomic stability during asexual and sexual development. / Graduate / 0306 / 0379 / 0307

In der vorliegenden Arbeit sollte die Eignung von T. pyriformis GL für den Nachweis von Botulinumneurotoxinen als biologische Alternative zum Maus-Bioassay untersucht werden. Dazu wurden funktionelle Tests für die Quantifizierung der mutmaßlich SNARE-abhängigen Prozesse Phagozytose und Exozytose entwickelt. Die Botulinumneurotoxine wurden durch Kultivierung der C. botulinum-Toxovare A bis G in einem Caseinpepton-Glukose-Hefeextrakt-Medium mit und ohne Zusatz von Trypsin herge-stellt. Die Neurotoxinkonzentrationen wurden mit Hilfe des Maus-Bioassays bestimmt. Für den Phagozytosetest wurde E. coli K12 als Beutekeim gewählt. Es konnte gezeigt werden, dass die KbE/ml von E. coli K12 allein durch die Phagozytoseaktivität von T. pyriformis reduziert wurde. Für den Exozytosetest wurde die saure Phosphatase als Leitenzym gewählt. Die neurotoxinhaltigen Kulturüberstände wurden mit Neurotoxinendkonzentrationen von 1,50E+02 MLD/ml (Maus letale Dosis/ml) bei den trypsinisierten und nicht trypsinisierten Ansätzen der Toxovare A bis D, F und G bzw. von 1,50E+00 MLD/ml bei dem trypsinisierten Ansatz des Toxovars E in den entwickelten Tests eingesetzt und auf ihren Einfluss untersucht. Die eingesetzten Neurotoxinkonzentrationen erwiesen sich als nicht ausreichend. Zudem wurde eine erhebliche Anfälligkeit der Phagozytose- und Exozytoseleistung von T. pyriformis gegen die Proteinkonzentrationen der eingesetzten Kulturüberstände nachgewiesen. Aufgrund dessen ist die Eignung von T. pyriformis im Rahmen der entwickelten Tests für den Nachweis von Botulinumneurotoxinen aus Proben wie biologischen Substraten oder mit ähnlichen, komplexen Matrizes nicht gegeben.