Chicken Caecal Microbiome Modifications Induced by Campylobacter jejuni Colonization and by a Non-Antibiotic Feed Additive

Campylobacter jejuni is an important zoonotic foodborne pathogen causing acute gastroenteritis in humans. Chickens are often colonized at very high numbers by C. jejuni, up to 109 CFU per gram of caecal content, with no detrimental effects on their health. Farm control strategies are being developed to lower the C. jejuni contamination of chicken food products in an effort to reduce human campylobacteriosis incidence. It is believed that intestinal microbiome composition may affect gut colonization by such undesirable bacteria but, although the chicken microbiome is being increasingly characterized, information is lacking on the factors affecting its modulation, especially by foodborne pathogens. This study monitored the effects of C. jejuni chicken caecal colonization on the chicken microbiome in healthy chickens. It also evaluated the capacity of a feed additive to affect caecal bacterial populations and to lower C. jejuni colonization. From day-0, chickens received or not a microencapsulated feed additive and were inoculated or not with C. jejuni at 14 days of age. Fresh caecal content was harvested at 35 days of age. The caecal microbiome was characterized by real time quantitative PCR and Ion Torrent sequencing. We observed that the feed additive lowered C. jejuni caecal count by 0.7 log (p<0.05). Alpha-diversity of the caecal microbiome was not affected by C. jejuni colonization or by the feed additive. C. jejuni colonization modified the caecal beta-diversity while the feed additive did not. We observed that C. jejuni colonization was associated with an increase of Bifidobacterium and affected Clostridia and Mollicutes relative abundances. The feed additive was associated with a lower Streptococcus relative abundance. The caecal microbiome remained relatively unchanged despite high C. jejuni colonization. The feed additive was efficient in lowering C. jejuni colonization while not disturbing the caecal microbiome.

La génétique inverse dans l'étude des réovirus: Progrès, obstacles et développements futurs

En génétique dite « classique », l’examen d’un phénotype conduit à l’étude des gènes impliqués dans son obtention. La génétique inverse est une méthode expérimentale très puissante dans laquelle, au contraire, le matériel génétique est modifié et utilisé pour reconstruire un organisme complet, afin de déterminer le résultat de ces modifications. Cette approche est spécialement bien adaptée à l'étude des virus, compte tenu de la relative simplicité et de la petite taille de leurs génomes; l’obstacle principal demeure de récupérer des virus infectieux à partir de génomes viraux clonés. Au cours des années, cet exploit a été accompli pour des représentants de presque toutes les familles de virus de mammifères. Jusqu’à récemment, les Reoviridae, virus à génome d'ARN bicaténaire segmenté, faisaient toutefois exception. Dans cette revue, les progrès réalisés vers la mise au point de la génétique inverse pour l'étude du réovirus seront discutés. La génétique inverse pourrait avoir un impact majeur dans l'optimisation de nouvelles souches de réovirus pour leur utilisation en thérapie comme agents oncolytiques et pour le développement de vaccins dans le cas des rotavirus et des orbivirus. Les travaux actuels font toutefois ressortir les limites de l'approche, la nécessité d’une analyse prudente des résultats obtenus, ainsi que le besoin de développer des systèmes plus efficaces et polyvalents.

Quantitative proteomics methods for the analysis of histone post-translational modifications

Les histones sont des protéines nucléaires hautement conservées chez les cellules des eucaryotes. Elles permettent d’organiser et de compacter l’ADN sous la forme de nucléosomes, ceux-ci representant les sous unités de base de la chromatine. Les histones peuvent être modifiées par de nombreuses modifications post-traductionnelles (PTMs) telles que l’acétylation, la méthylation et la phosphorylation. Ces modifications jouent un rôle essentiel dans la réplication de l’ADN, la transcription et l’assemblage de la chromatine. L’abondance de ces modifications peut varier de facon significative lors du developpement des maladies incluant plusieurs types de cancer. Par exemple, la perte totale de la triméthylation sur H4K20 ainsi que l’acétylation sur H4K16 sont des marqueurs tumoraux spécifiques a certains types de cancer chez l’humain. Par conséquent, l’étude de ces modifications et des événements determinant la dynamique des leurs changements d’abondance sont des atouts importants pour mieux comprendre les fonctions cellulaires et moléculaires lors du développement de la maladie. De manière générale, les modifications des histones sont étudiées par des approches biochimiques telles que les immuno-buvardage de type Western ou les méthodes d’immunoprécipitation de la chromatine (ChIP). Cependant, ces approches présentent plusieurs inconvénients telles que le manque de spécificité ou la disponibilité des anticorps, leur coût ou encore la difficulté de les produire et de les valider. Au cours des dernières décennies, la spectrométrie de masse (MS) s’est avérée être une méthode performante pour la caractérisation et la quantification des modifications d’histones. La MS offre de nombreux avantages par rapport aux techniques traditionnelles. Entre autre, elle permet d’effectuer des analyses reproductibles, spécifiques et facilite l’etude d’un large spectre de PTMs en une seule analyse. Dans cette thèse, nous présenterons le développement et l’application de nouveaux outils analytiques pour l’identification et à la quantification des PTMs modifiant les histones. Dans un premier temps, une méthode a été développée pour mesurer les changements d’acétylation spécifiques à certains sites des histones. Cette méthode combine l’analyse des histones intactes et les méthodes de séquençage peptidique afin de déterminer les changements d’acétylation suite à la réaction in vitro par l’histone acétyltransférase (HAT) de levure Rtt109 en présence de ses chaperonnes (Asf1 ou Vps75). Dans un second temps, nous avons développé une méthode d’analyse des peptides isomériques des histones. Cette méthode combine la LC-MS/MS à haute résolution et un nouvel outil informatique appelé Iso-PeptidAce qui permet de déconvoluer les spectres mixtes de peptides isomériques. Nous avons évalué Iso-PeptidAce avec un mélange de peptides synthétiques isomériques. Nous avons également validé les performances de cette approche avec des histones isolées de cellules humaines érythroleucémiques (K562) traitées avec des inhibiteurs d’histones désacétylases (HDACi) utilisés en clinique, et des histones de Saccharomyces cerevisiae liées au facteur d’assemblage de la chromatine (CAF-1) purifiées par chromatographie d’affinité. Enfin, en utilisant la méthode présentée précédemment, nous avons fait une analyse approfondie de la spécificité de plusieurs HATs et HDACs chez Schizosaccharomyces pombe. Nous avons donc déterminé les niveaux d’acétylation d’histones purifiées à partir de cellules contrôles ou de souches mutantes auxquelles il manque une HAT ou HDAC. Notre analyse nous a permis de valider plusieurs cibles connues des HATs et HDACs et d’en identifier de nouvelles. Nos données ont également permis de définir le rôle des différentes HATs et HDACs dans le maintien de l’équilibre d’acétylation des histones. Dans l’ensemble, nous anticipons que les méthodes décrites dans cette thèse permettront de résoudre certains défis rencontrés dans l’étude de la chromatine. De plus, ces données apportent de nouvelles connaissances pour l’élaboration d’études génétiques et biochimiques utilisant S. pombe.

Modifications post-traductionnelles des canaux calciques cardiaques de type L : identification des résidus asparagine qui participent à la glycosylation de la sous-unité auxiliaire CaVα2δ1

Les canaux calciques de type L CaV1.2 sont principalement responsables de l’entrée des ions calcium pendant la phase plateau du potentiel d’action des cardiomyocytes ventriculaires. Cet influx calcique est requis pour initier la contraction du muscle cardiaque. Le canal CaV1.2 est un complexe oligomérique qui est composé de la sous-unité principale CaVα1 et des sous-unités auxiliaires CaVβ et CaVα2δ1. CaVβ joue un rôle déterminant dans l’adressage membranaire de la sous-unité CaVα1. CaVα2δ1 stabilise l’état ouvert du canal mais le mécanisme moléculaire responsable de cette modulation n’a pas été encore identifié. Nous avons récemment montré que cette modulation requiert une expression membranaire significative de CaVα2δ1 (Bourdin et al. 2015). CaVα2δ1 est une glycoprotéine qui possède 16 sites potentiels de glycosylation de type N. Nous avons donc évalué le rôle de la glycosylation de type-N dans l’adressage membranaire et la stabilité de CaVα2δ1. Nous avons d’abord confirmé que la protéine CaVα2δ1 recombinante, telle la protéine endogène, est significativement glycosylée puisque le traitement à la PNGase F se traduit par une diminution de 50 kDa de sa masse moléculaire, ce qui est compatible avec la présence de 16 sites Asn. Il s’est avéré par ailleurs que la mutation simultanée de 6/16 sites (6xNQ) est suffisante pour 1) réduire significativement la densité de surface de! CaVα2δ1 telle que mesurée par cytométrie en flux et par imagerie confocale 2) accélérer les cinétiques de dégradation telle qu’estimée après arrêt de la synthèse protéique et 3) diminuer la modulation fonctionnelle des courants générés par CaV1.2 telle qu’évaluée par la méthode du « patch-clamp ». Les effets les plus importants ont toutefois été obtenus avec les mutants N663Q, et les doubles mutants N348Q/N468Q, N348Q/N812Q, N468Q/N812Q. Ensemble, ces résultats montrent que Asn663 et à un moindre degré Asn348, Asn468 et Asn812 contribuent à la biogenèse et la stabilité de CaVα2δ1 et confirment que la glycosylation de type N de CaVα2δ1 est nécessaire à la fonction du canal calcique cardiaque de type L.

Effects and Modifications in In/Se and In/Sb Systems by Swift Heavy Ion Irradiation

Department of Physics, Cochin University of Science and Technology

Effects of Thermal Modifications on the Rheological Characteristics and Protein Quality of Three Species of Fish of Varying Collagen Content

School of Industrial Fisheries, Cochin University of Science and Technology

Modifications of secondary reflector antenna patterns due to shaped beams from flanged primary horn feeds

With the advent of satellite communication and radio astronomy, the need for large and efficient reflector antennas had triggered a widespread investigation in reflector feed design techniques. Major improvements sought are reduction in spill-over, cross polarization losses and the enhancement of aperture efficiency. The search for such a feed culminated in the corrugated horn. The main idea behind the present work is to use the H-plane sectoral horns fitted with,corrugated flanges as feeds of a paraboloid and see how the secondary pattern of the reflector antenna varies with different parameters of the feed. An offset paraboloid is used as the secondary reflector in order to avoid the adverse effect of aperture ‘blocking by the feed horn structure on the secondary radiation pattern. The measurements were repeated for three different H-plane sectoral horns with the same set of corrugated flanges at various X-band frequencies. The following parameters of the whole system are studied: (a) Beam shaping. (b) Gain. (c) Variation of VSWR and (d) Cross polarization

“Reconstruction of Gene Regulatory Network from Expression Profile of Plasma RNA Data of Colorectal Cancer Patients using Soft Computing Techniques”

Microarray data analysis is one of data mining tool which is used to extract meaningful information hidden in biological data. One of the major focuses on microarray data analysis is the reconstruction of gene regulatory network that may be used to provide a broader understanding on the functioning of complex cellular systems. Since cancer is a genetic disease arising from the abnormal gene function, the identification of cancerous genes and the regulatory pathways they control will provide a better platform for understanding the tumor formation and development. The major focus of this thesis is to understand the regulation of genes responsible for the development of cancer, particularly colorectal cancer by analyzing the microarray expression data. In this thesis, four computational algorithms namely fuzzy logic algorithm, modified genetic algorithm, dynamic neural fuzzy network and Takagi Sugeno Kang-type recurrent neural fuzzy network are used to extract cancer specific gene regulatory network from plasma RNA dataset of colorectal cancer patients. Plasma RNA is highly attractive for cancer analysis since it requires a collection of small amount of blood and it can be obtained at any time in repetitive fashion allowing the analysis of disease progression and treatment response.

Magnetic and topographical modifications of amorphous Co–Fe thin films induced by high energy Ag7+ ion irradiation

We have investigated the effects of swift heavy ion irradiation on thermally evaporated 44 nm thick, amorphous Co77Fe23 thin films on silicon substrates using 100 MeV Ag7+ ions fluences of 1 1011 ions/ cm2, 1 1012 ions/cm2, 1 1013 ions/cm2, and 3 1013 ions/cm2. The structural modifications upon swift heavy irradiation were investigated using glancing angle X-ray diffraction. The surface morphological evolution of thin film with irradiation was studied using Atomic Force Microscopy. Power spectral density analysis was used to correlate the roughness variation with structural modifications investigated using X-ray diffraction. Magnetic measurements were carried out using vibrating sample magnetometry and the observed variation in coercivity of the irradiated films is explained on the basis of stress relaxation. Magnetic force microscopy images are subjected to analysis using the scanning probe image processor software. These results are in agreement with the results obtained using vibrating sample magnetometry. The magnetic and structural properties are correlated

Tailoring Magnetic Properties of Co-Fe Thin Films by Topographical Modifications Using Thermal Annealing and Swift Heavy Ion Irradiation and Fabrication of Magnetoelectric Multilayers for Device Applications

Magnetism and magnetic materials have been playing a lead role in improving the quality of life. They are increasingly being used in a wide variety of applications ranging from compasses to modern technological devices. Metallic glasses occupy an important position among magnetic materials. They assume importance both from a scientific and an application point of view since they represent an amorphous form of condensed matter with significant deviation from thermodynamic equilibrium. Metallic glasses having good soft magnetic properties are widely used in tape recorder heads, cores of high-power transformers and metallic shields. Superconducting metallic glasses are being used to produce high magnetic fields and magnetic levitation effect. Upon heat treatment, they undergo structural relaxation leading to subtle rearrangements of constituent atoms. This leads to densification of amorphous phase and subsequent nanocrystallisation. The short-range structural relaxation phenomenon gives rise to significant variations in physical, mechanical and magnetic properties. Magnetic amorphous alloys of Co-Fe exhibit excellent soft magnetic properties which make them promising candidates for applications as transformer cores, sensors, and actuators. With the advent of microminiaturization and nanotechnology, thin film forms of these alloys are sought after for soft under layers for perpendicular recording media. The thin film forms of these alloys can also be used for fabrication of magnetic micro electro mechanical systems (magnetic MEMS). In bulk, they are drawn in the form of ribbons, often by melt spinning. The main constituents of these alloys are Co, Fe, Ni, Si, Mo and B. Mo acts as the grain growth inhibitor and Si and B facilitate the amorphous nature in the alloy structure. The ferromagnetic phases such as Co-Fe and Fe-Ni in the alloy composition determine the soft magnetic properties. The grain correlation length, a measure of the grain size, often determines the soft magnetic properties of these alloys. Amorphous alloys could be restructured in to their nanocrystalline counterparts by different techniques. The structure of nanocrystalline material consists of nanosized ferromagnetic crystallites embedded in an amorphous matrix. When the amorphous phase is ferromagnetic, they facilitate exchange coupling between nanocrystallites. This exchange coupling results in the vanishing of magnetocrystalline anisotropy which improves the soft magnetic properties. From a fundamental perspective, exchange correlation length and grain size are the deciding factors that determine the magnetic properties of these nanocrystalline materials. In thin films, surfaces and interfaces predominantly decides the bulk property and hence tailoring the surface roughness and morphology of the film could result in modified magnetic properties. Surface modifications can be achieved by thermal annealing at various temperatures. Ion irradiation is an alternative tool to modify the surface/structural properties. The surface evolution of a thin film under swift heavy ion (SHI) irradiation is an outcome of different competing mechanism. It could be sputtering induced by SHI followed by surface roughening process and the material transport induced smoothening process. The impingement of ions with different fluence on the alloy is bound to produce systematic microstructural changes and this could effectively be used for tailoring magnetic parameters namely coercivity, saturation magnetization, magnetic permeability and remanence of these materials. Swift heavy ion irradiation is a novel and an ingenious tool for surface modification which eventually will lead to changes in the bulk as well as surface magnetic property. SHI has been widely used as a method for the creation of latent tracks in thin films. The bombardment of SHI modifies the surfaces or interfaces or creates defects, which induces strain in the film. These changes will have profound influence on the magnetic anisotropy and the magnetisation of the specimen. Thus inducing structural and morphological changes by thermal annealing and swift heavy ion irradiation, which in turn induce changes in the magnetic properties of these alloys, is one of the motivation of this study. Multiferroic and magneto-electrics is a class of functional materials with wide application potential and are of great interest to material scientists and engineers. Magnetoelectric materials combine both magnetic as well as ferroelectric properties in a single specimen. The dielectric properties of such materials can be controlled by the application of an external magnetic field and the magnetic properties by an electric field. Composites with magnetic and piezo/ferroelectric individual phases are found to have strong magnetoelectric (ME) response at room temperature and hence are preferred to single phasic multiferroic materials. Currently research in this class of materials is towards optimization of the ME coupling by tailoring the piezoelectric and magnetostrictive properties of the two individual components of ME composites. The magnetoelectric coupling constant (MECC) (_ ME) is the parameter that decides the extent of interdependence of magnetic and electric response of the composite structure. Extensive investigates have been carried out in bulk composites possessing on giant ME coupling. These materials are fabricated by either gluing the individual components to each other or mixing the magnetic material to a piezoelectric matrix. The most extensively investigated material combinations are Lead Zirconate Titanate (PZT) or Lead Magnesium Niobate-Lead Titanate (PMNPT) as the piezoelectric, and Terfenol-D as the magnetostrictive phase and the coupling is measured in different configurations like transverse, longitudinal and inplane longitudinal. Fabrication of a lead free multiferroic composite with a strong ME response is the need of the hour from a device application point of view. The multilayer structure is expected to be far superior to bulk composites in terms of ME coupling since the piezoelectric (PE) layer can easily be poled electrically to enhance the piezoelectricity and hence the ME effect. The giant magnetostriction reported in the Co-Fe thin films makes it an ideal candidate for the ferromagnetic component and BaTiO3 which is a well known ferroelectric material with improved piezoelectric properties as the ferroelectric component. The multilayer structure of BaTiO3- CoFe- BaTiO3 is an ideal system to understand the underlying fundamental physics behind the ME coupling mechanism. Giant magnetoelectric coupling coefficient is anticipated for these multilayer structures of BaTiO3-CoFe-BaTiO3. This makes it an ideal candidate for cantilever applications in magnetic MEMS/NEMS devices. SrTiO3 is an incipient ferroelectric material which is paraelectric up to 0K in its pure unstressed form. Recently few studies showed that ferroelectricity can be induced by application of stress or by chemical / isotopic substitution. The search for room temperature magnetoelectric coupling in SrTiO3-CoFe-SrTiO3 multilayer structures is of fundamental interest. Yet another motivation of the present work is to fabricate multilayer structures consisting of CoFe/ BaTiO3 and CoFe/ SrTiO3 for possible giant ME coupling coefficient (MECC) values. These are lead free and hence promising candidates for MEMS applications. The elucidation of mechanism for the giant MECC also will be the part of the objective of this investigation.

Modifications of DGEBA Using Epoxidised Resins

Modifications of DGEBA Using Epoxidised Resins

Das RNA-bindende Protein Orb2 und seine regulatorische Funktion in der Spermatogenese von Drosophila melanogaster

Das Protein Orb2, welches zum Xenopus CPEB homolog ist, erfüllt während der Spermatogenese von Drosophila melanogaster eine wesentliche Funktion. Das teilweise Ausschalten von orb2 führt zu Störungen in der Individualisierung der Spermatiden, Veränderung in der Morphologie und Lokalisation der Spermatidenkerne und damit verbunden zu männlicher Sterilität. Der weit gestreute Phänotyp spricht für eine regulatorische Funktion des Proteins, wie es aufgrund der Homologie zu CPEB zu erwarten ist. Orb2 mutante Weibchen zeigen dagegen keinen Phänotyp. Die Sterilität konnte mit spezifischen Rettungskonstrukten rückgängig gemacht werden, wobei die beiden Proteinformen in ihrer Funktion höchstwahrscheinlich äquivalent sind, da eine größere Menge an kleinem Protein das Fehlen des größeren ausgleichen kann. Beide Proteinformen lokalisieren in fast alle Stadien der Spermatogenese, wobei nur das kleinere auch in reifen Spermien persistiert. Zur Untersuchung der regulatorischen Funktion des Proteins Orb2 wurden zunächst drei mögliche Protein-Interaktionskandidaten analysiert. Obwohl ähnliche mutante Phänotypen in Gap und Cup ausgelöst wurden, lässt sich eine Interaktion bis jetzt mit diesen Kandidaten weder ausschließen noch bestätigen. Daneben zeigte das Protein Tob eine ähnliche Lokalisierung und einen deutlich ähnlicheren mutanten Phänotyp, wie er für Orb2 beschrieben wurde. Besonders auffällig ist die Lokalisation der Tob mRNA an die Spermatidenenden und die Verringerung der Transkriptmenge in der orb2-Mutante. Ob dieser Phänotyp durch den Verlust der regulatorischen Funktion von Orb2 hervorgerufen wird oder durch den späten Zeitpunkt der Transkription bedingt ist, muß in späteren Experimenten geklärt werden. Mit Hilfe eines Co-Immunpräzipitations-Experimentes wurde nach weiteren Proteininteraktionspartnern sowie nach Ziel-mRNAs gesucht, die durch Orb2 reguliert werden könnten. Dabei ergaben die massenspektrometrischen Analysen zwar Proteine, die mit der Translation selbst in Zusammenhang stehen, sowie einige regulatorische RNA-bindende Proteine, wiesen aber auch in Gestalt eines häufig nachgewiesenen Anhangsdrüsenproteins auf deutliche systematische Probleme hin. Auf genetischem Wege war bereits der Nachweis gelungen, dass die Protamine und mst77F, die strukturelle Komponenten der kompaktierten Kern-DNA sind, durch Orb2 in ihrer Translation reprimiert werden. Dieses Ergebnis wurde zum Teil bestätigt durch den Nachweis der Protamin mRNAs in den Eluaten aus dem Co-Immunpräzipitationsexperiment. Damit konnte zum ersten Mal in der Drosophila Spermatogenese das regulatorische Protein zu einer translationskontrollierten mRNA identifiziert werden.

Involvements of the Plant 3'-5' Exonuclease ERL1 in Chloroplast Ribosomal RNA Biogenesis and RNA Silencing Pathways

ERI-1 und ihm homologe Proteine sind 3‘-5‘ Exoribonukleasen mit konservierten Funktionen in der Regulation von RNA Silencing sowie der Prozessierung ribosomaler RNA. Caenorhabditis elegans ERI-1 (Enhanced RNAi 1) enthält eine konservierte ERI-1_3’hExo_like EXOIII-Domäne, die siRNAs in vitro bindet und degradiert, und deren Inaktivierung eine RNAi-Hypersensitivität zur Folge hat. ERI-1 ist phylogenetisch konserviert, und homologe Proteine wurden Reiche-übergreifend in einer Vielzahl von Modellorganismen identifiziert. RNA-Silencing-reprimierende Eigenschaften dieser Proteine wurden in einigen Fällen charakterisiert. Zusätzlich wurde für eine Untergruppe ERI-1-homologer Proteine eine Funktion in der Biogenese der 5.8S ribosomalen RNA aufgezeigt: Katalyse des letzten Prozessierungsschritts während der Reifung des 5.8S rRNA 3‘-Endes. Diese Doppelfunktion ERI-1-homologer Proteine schlägt eine interessante Brücke zwischen evolutionär weit entfernten auf nicht-codierender RNA basierenden Mechanismen. In dieser Arbeit werden Ergebnisse präsentiert, die Charakteristika des pflanzlichen ERI-1-Homologs ERL1 in verschiedenen regulatorischen Zusammenhängen zum Gegenstand haben. ERL1 lokalisiert in Chloroplasten und zeigt keinerlei messbare Aktivität in Bezug auf die Regulierung von RNA Silencing. Im Gegensatz dazu konnte gezeigt werden, dass ERL1 eine wichtige Rolle während der Reifung der chloroplastischen 5S rRNA spielt. ERL1-supprimierende bzw. -überexprimierende transgene Pflanzen, zeigen unterschiedliche phänotypische Aberrationen. Diese beinhalten vielfarbige Blätter, reduziertes Wachstum und Fruchtbarkeit, sowie den Verlust Photosynthese-kompetenter Chloroplasten in gebleichten Sektoren. Diese Defekte werden dadurch verursacht, dass die Plastid-Entwicklung in einem frühen Stadium blockiert wird. Dies führt zu defekten Plastiden, die keine kanonischen internen Strukturen, einschließlich Grana, bilden können. Die gestörte Plastid-Entwicklung ist ein Resultat fehlerhafter Prozessierung ribosomaler RNAs und dem daraus folgenden Verlust plastidärer Transkription und Translation. Wenn ERL1 runterreguliert oder überexprimiert ist, akkumulieren 3‘-elongierte 5S rRNA-Moleküle, was Störungen in der Produktion der Ribosomen hervorruft. Die Reifung der 5S rRNA ist leit langem als Prozess bekannt, der viele aufeinander folgende endonukleolytische Spaltungen sowie exonukleolytische Rezessionen beinhaltet. Bis dato war die Gesamtheit der Exonukleasen während dieser Reifung jedoch nur lückenhaft bekannt. Die Ergebnisse dieser Arbeit zeigen, dass ERL1 eine wichtige Rolle in der Plastid-Entwicklung spielt, indem ERL1 den finalen Reifungsschritt des 5S rRNA 3‘-Endes katalysiert.

Characterization of DnmA, the Dnmt2 homolog in Dictyostelium discoideum

Eukaryotic DNA m5C methyltransferases (MTases) play a major role in many epigenetic regulatory processes like genomic imprinting, X-chromosome inactivation, silencing of transposons and gene expression. Members of the two DNA m5C MTase families, Dnmt1 and Dnmt3, are relatively well studied and many details of their biological functions, biochemical properties as well as interaction partners are known. In contrast, the biological functions of the highly conserved Dnmt2 family, which appear to have non-canonical dual substrate specificity, remain enigmatic despite the efforts of many researchers. The genome of the social amoeba Dictyostelium encodes Dnmt2-homolog, the DnmA, as the only DNA m5C MTase which allowed us to study Dnmt2 function in this organism without interference by the other enzymes. The dnmA gene can be easily disrupted but the knock-out clones did not show obvious phenotypes under normal lab conditions, suggesting that the function of DnmA is not vital for the organism. It appears that the dnmA gene has a low expression profile during vegetative growth and is only 5-fold upregulated during development. Fluorescence microscopy indicated that DnmA-GFP fusions were distributed between both the nucleus and cytoplasm with some enrichment in nuclei. Interestingly, the experiments showed specific dynamics of DnmA-GFP distribution during the cell cycle. The proteins colocalized with DNA in the interphase and were mainly removed from nuclei during mitosis. DnmA functions as an active DNA m5C MTase in vivo and is responsible for weak but detectable DNA methylation of several regions in the Dictyostelium genome. Nevertheless, gel retardation assays showed only slightly higher affinity of the enzyme to dsDNA compared to ssDNA and no specificity towards various sequence contexts, although weak but detectable specificity towards AT-rich sequences was observed. This could be due to intrinsic curvature of such sequences. Furthermore, DnmA did not show denaturant-resistant covalent complexes with dsDNA in vitro, although it could form covalent adducts with ssDNA. Low binding and methyltransfer activity in vitro suggest the necessity of additional factor in DnmA function. Nevertheless, no candidates could be identified in affinity purification experiments with different tagged DnmA fusions. In this respect, it should be noted that tagged DnmA fusion preparations from Dictyostelium showed somewhat higher activity in both covalent adduct formation and methylation assays than DnmA expressed in E.coli. Thus, the presence of co-purified factors cannot be excluded. The low efficiency of complex formation by the recombinant enzyme and the failure to define interacting proteins that could be required for DNA methylation in vivo, brought up the assumption that post-translational modifications could influence target recognition and enzymatic activity. Indeed, sites of phosphorylation, methylation and acetylation were identified within the target recognition domain (TRD) of DnmA by mass spectrometry. For phosphorylation, the combination of MS data and bioinformatic analysis revealed that some of the sites could well be targets for specific kinases in vivo. Preliminary 3D modeling of DnmA protein based on homology with hDNMT2 allowed us to show that several identified phosphorylation sites located on the surface of the molecule, where they would be available for kinases. The presence of modifications almost solely within the TRD domain of DnmA could potentially modulate the mode of its interaction with the target nucleic acids. DnmA was able to form denaturant-resistant covalent intermediates with several Dictyostelium tRNAs, using as a target C38 in the anticodon loop. The formation of complexes not always correlated with the data from methylation assays, and seemed to be dependent on both sequence and structure of the tRNA substrate. The pattern, previously suggested by the Helm group for optimal methyltransferase activity of hDNMT2, appeared to contribute significantly in the formation of covalent adducts but was not the only feature of the substrate required for DnmA and hDNMT2 functions. Both enzymes required Mg2+ to form covalent complexes, which indicated that the specific structure of the target tRNA was indispensable. The dynamics of covalent adduct accumulation was different for DnmA and different tRNAs. Interestingly, the profiles of covalent adduct accumulation for different tRNAs were somewhat similar for DnmA and hDNMT2 enzymes. According to the proposed catalytic mechanism for DNA m5C MTases, the observed denaturant-resistant complexes corresponded to covalent enamine intermediates. The apparent discrepancies in the data from covalent complex formation and methylation assays may be interpreted by the possibility of alternative pathways of the catalytic mechanism, leading not to methylation but to exchange or demethylation reactions. The reversibility of enamine intermediate formation should also be considered. Curiously, native gel retardation assays showed no or little difference in binding affinities of DnmA to different RNA substrates and thus the absence of specificity in the initial enzyme binding. The meaning of the tRNA methylation as well as identification of novel RNA substrates in vivo should be the aim of further experiments.

Artificial micro RNA system in Dictyostelium discoideum

Dictyostelium discoideum is a social amoeba that serves as a model system for RNA interference and related mechanisms. Its position between plants and animals enables evolutionary snapshot of mechanisms and protein machinery involved in investigated subjects. MiRNAs are small regulatory RNAs that are evolutionary conserved and present in animals, plants, viruses and some prokaryotes. They have roles in development, cell growth and differentiation, apoptosis and their miss-regulation is associated with many diseases such as cancer, neurodegenerative disorders and diabetes. Recently, through sequencing of DNA libraries miRNAs have been discovered in D. discoideum. In this work, it has been shown that heterologues miRNA let-7 can be expressed and processed in D. discoideum. Expression of let-7 miRNA in social amoeba resulted in a strong developmental phenotype suggesting an overload of the processing/silencing system or/and endogenous targets. The various effects on prel-7 strain have been observed and characterized, serving as a background for postulation of miRNA roles. An artificial miRNA system has been established and imposed to D. discoideum, showing that miRNAs in Dictyostelium could mediate gene expression on the level of mRNA stability and on the posttranscriptional level. Furthermore, presence of translational inhibition as a type of gene control was shown for the first time in this organism. Due to it new structures representing co-localities of miRNA and target mRNA have been detected. Taken together, this work shows functional artificial miRNA system and postulates roles of endogenous small RNA in social amoeba.