The global re-emergence of TB and other mycobacterial infections have underscored the need for a thorough investigation of the biology of the causative agent, Mycobacterium tuberculosis, at the molecular level. The peculiar features of the bacterium such as slow growth rate, dormancy, unique cell wall composition and resistance towards phagocytosis by macrophages demands a detailed understanding of different essential molecular processes including transcription in this genus. Sequencing of several mycobacterial genomes provided an impetus for understanding the gene function and regulation of this formidable pathogen. Transcriptional regulation is one of the major mechanisms controlling gene expression. While a number of transcription units, promoters, sigma factors, and gene functions were identified and characterized, key features of transcription process are yet to be understood. The current study aims to understand some of the facets of transcription initiation and elongation in mycobacteria. The thesis is divided into five chapters. Chapter 1 introduces the bacterial transcription process. It starts with the description of the central molecule in transcription -the RNA polymerase (RNAP) and its catalytic mechanism. In the next section, each step of the transcription initiation, elongation and termination has been discussed. The mechanistic details as well as the different cellular factors involved in the regulation of the transcription have been discussed. The final part gives an overview of the transcription machinery of the mycobacteria, describing the promoter specificity and regulation of different sigma factors and other transcription factors known till date in mycobacteria. The scope and the objectives of the thesis are presented at the end of this chapter. In Chapter 2, a method of purification of RNAP from mycobacteria for optimized promoter -polymerase interactions is described. In vitro transcription analysis is important to understand the mechanism of transcription. Various assays for the analysis of initiation, elongation and termination form the basis for better understanding of the process. Purified RNAP with high specific activity is necessary to carry out a variety of these specific reactions. The RNAP purified from Mycobacterium smegmatis from exponential phase showed low σA-promoter specificity in promoter -polymerase interaction studies. This is due to the presence of a large number of sigma factors during exponential phase and under-representation of σA required for house - keeping transcription. In vivo reconstitution of RNAP holoenzyme with σA and its purification procedure which resulted in a holoenzyme with stoichiometric σA content is described in this chapter. The reconstituted holoenzyme showed enhanced promoter -specific binding and transcription activity compared to the enzyme isolated using standard procedure. Chapter 3 is aimed at the comparison of promoter - specific events during transcription initiation in mycobacteria. DNA -protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNAP binds to promoters in a sequence -specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. The first detailed kinetic analysis of promoter - RNAP interactions during transcription initiation in the σA-dependent promoters PrrnAPCL1, PrrnB and Pgyr of M. smegmatis are presented in this chapter. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to the distinct kinetic patterns, each one of the house -keeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation. Moving the focus of the thesis from transcription initiation to elongation, a transcript cleavage factor of M. tuberculosis has been characterized in Chapter 4. After initiation of transcription, a number of proteins participate during elongation and termination by modifying the properties of the RNAP. Gre proteins are one such class of transcription elongation factors which are conserved across bacteria. They regulate transcription by binding near the secondary channel of RNAP, projecting their N-terminal coiled-coil domain into the active center and stimulating hydrolysis of the newly synthesized RNA by RNAP in the backtracked elongation complexes. Rv1080c is a putative gre factor homolog (MtbGre) present in M. tuberculosis.The protein enhanced the efficiency of promoter clearance by lowering the abortive transcription and also rescued the arrested and paused elongation complexes efficiently in the GC rich mycobacterial template. The Gre factor of M. smegmatis encoded by the gene MSMEG_5263 also showed biochemical properties similar to the M. tuberculosis protein. Although the mycobacterial Gre is similar in domain organization and shared the key residues for catalysis and RNAP interaction with Escherichia coli Gre proteins, it could not complement the E. coli strain deficient in Gre factors. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein - protein interactions for transcript cleavage. Decrease in the level of MtbGre also reduced the bacterial survival by several fold indicating its essential role in mycobacteria and suggesting that a single Gre copes up with the burden of transcription fidelity of the genome. Chapter 5 describes the studies carried out to identify Gre factor homologs in mycobacteria and deciphering their function during transcription. Gre factors are members of a growing family of proteins which regulate RNAP through secondary channel. Apart from the Gre factor, putative members of this class of proteins are identified in both M. smegmatis and M. tuberculosis.The closest homologue of the canonical Gre factor of M. tuberculosis in its genome is Rv3788. The protein has Gre factor like domain organization and possess the key acidic residues required for transcript cleavage activity and the putative hydrophobic RNAP interacting residues in the C-terminus similar to MtbGre. Despite having these common features, Rv3788 did not stimulate transcript cleavage. In contrast, it turns out to be a transcription inhibitor by preventing the binding of NTPs to the enzyme. The transcription inhibition is not promoter specific, and is mediated by its binding to RNAP through the secondary channel with its N-terminus coiled coil domain. Like M. tuberculosis, the fast growing non-pathogenic mycobacteria M. smegmatis also has an ORF (MSMEG_6292) which is homologous to its canonical Gre factor and it interacts with RNAP in a similar manner. However, this protein did not exert any transcript cleavage or inhibitory activities but could compete with the Gre factor for binding to RNAP. The Gre factor homologs in mycobacteria may be involved in regulation by inhibiting transcription or by blocking the RNAP secondary channel from other RNAP active site modulators.

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est un virus à simple brin positif qui appartient au genre Lentivirus dans la famille retroviridae et qui constitue l’agent étiologique du SIDA pandémique.Pendant le cycle réplicatif du VIH-1, la traduction de protéines virales dépend exclusivement de la machinerie traductionnelle cellulaire. Pour cette raison, nous avons cherché à comprendre le rôle de quelques facteurs cellulaires qui pourraient contrôler la traduction du VIH-1 à différents nivaux. Nous avons centré nos recherches sur la traduction de l’ARN génomique (ARNg) du virus qui sert en même temps de génome pour être encapsidé et comme ARN messager pour la traduction des protéines virales Gag et Gag-Pol. 1) Le rôle de l’hélicase d’ARN DDX3 dans la traduction du VIH-1. L’ARNg du VIH-1 possède une région 5’ non traduite très structurée, raison pour laquelle nous avons spéculé sur un possible rôle de DDX3 dans la traduction du VIH-1. Nous avons utilisé une combinaison de techniques in vitro et ex vivo afin de pouvoir démontrer que DDX3 était capable de lier et faire des complexes avec l’ARN de la région 5’ non traduite pour promouvoir l’initiation de la traduction. Nous avons aussi pu démontrer que DDX3 formait des complexes avec les facteurs d’initiation de la traduction PABP, eIF4G et eIF4E. 2) Le changement programmé du cadre de lecture (PRF) dans l’ARN génomique du VIH-1. La traduction de la polyprotéine Gag-Pol du VIH-1 nécessite un décalage de phase de 1 nucléotide en arrière. Ce mécanisme permet la synthèse des protéines Gag et Gag-Pol avec des ratios de 95 et 5% respectivement à partir du même ARN. Cette proportion doit être conservée pour assurer la réplication du virus. Nous avons utilisé un système de double gène rapporteurs et un système de réplication complète du provirus pour montrer que la protéine associé aux granules de stress TIAR pouvait contrôler la réplication viral en régulant la proportion de ribosome qui assurent / Human Immunodeficiency virus type 1 (HIV-1) is a positive strand RNA virus belonging to the lentivirus genus of the retroviridae family and it is the etiological agent of the pandemic AIDS, which is a major health concern worldwide. Throughout HIV-1 replication cycle, the production of viral proteins depends exclusively on the cellular translational machinery. This is the reason why we have explored the role of some cellular factors that could control HIV-1 translation at different stages. We have focused our studies on the translation of the full length genomic RNA (gRNA), which serves both as genome for viral encapsidation and as a messenger for translation of Gag and Gag-Pol viral polyproteins.1) The role of the RNA helicase DDX3 in HIV-1 translation Initiation The fact that HIV-1 possesses a highly structured 5’ untranslated region (5’UTR) prompted us to speculate that DDX3 may be involved in HIV-1 translation. We used a combination of in vitro and ex-vivo approaches to show that DDX3 was able to bind and form complexes with the 5’-UTR of HIV-1 to assist translation initiation. We also demonstrated that DDX3 can form a complex with initiation factors such as PABP, eIF4G and eIF4E. 2) Programmed Ribosomal Frameshift (PRF) in the genomic RNA of HIV-1Translation of HIV-1 Gag-Pol polyprotein requires a -1 PRF. This mechanism allows the synthesis of Gag and Gag-Pol polyproteins, using the same mRNA template, at ratios of 95 and 5% respectively. Keeping the -1PRF ratio is important as any change leads to reduction in virus infectivity.By means of a dual reporter construct and full provirus replication system we were able to demonstrate that the stress granules associated protein TIAR, controls HIV-1 infectious progeny by regulating the ratio of the HIV-1 PRF.

Both the topics of prey-catching mechanism and visual perception are closely connected, affecting each other in many complex situations. The main subjects of this thesis were amphibians and reptiles and the many effects that impact their hunting abilities. We studied their hunting patterns in a few species of frogs and the leopard gecko (Eublepharis macularius) in an arena. We did not find any universal hunting pattern for neither of those groups, moreover even the quantity of individual sequences differed among them. However, hunting in nature is often engaged in habitats that are far from the flat calm arena. Often the animals have to adapt to different conditions, for example an unsteady surface underneath them. In such conditions they need to compensate for the passive movement with their heads and eyes to stabilize the image on their retina. The ability to compensate while hunting in frogs is affecting their behaviour and the success rate of their prey-catching. Another aspect that is influencing frog's hunting efficiency are their protrusible tongues that are commonly divided into three categories: mechanical, inertial and hydrostatic. The last goal of this thesis was looking for the evolution of this trait in frog's phylogenesis and their different effects on hunting movements.

La construction du cerveau et de ses connexions pendant le développement reste une question ouverte dans la communauté scientifique. Des efforts fructueux ont été faits pour élucider les mécanismes de la croissance axonale, tels que la guidance axonale et les molécules de guidage. Cependant, des preuves récentes suggèrent que d'autres acteurs seraient impliqués dans la croissance des neurones in vivo. Notamment, les axones se développent dans des environnements mécaniquement contraints. Ainsi, pour bien comprendre ce processus dynamique, il faut prendre en compte les mécanismes collectifs et les interactions mécaniques au sein des populations axonales. Néanmoins, les techniques pour mesurer directement cela à partir de cerveaux vivants sont aujourd'hui insuffisantes ou lourdes à mettre en œuvre. Cette thèse résulte d'une collaboration multidisciplinaire, pour faire la lumière sur le développement axonal in vivo et les morphologies complexes des axones adultes. Notre travail a été inspiré et validé à partir d'images d'axones y individuels chez la drosophile, de type sauvage et modifiés génétiquement, que nous avons segmentés et normalisés. Nous avons d'abord proposé un cadre mathématique pour l'étude morphologique et la classification des groupes axonaux. A partir de cette analyse, nous avons émis l'hypothèse que la croissance axonale dérive d'un processus stochastique et que la variabilité et la complexité des arbres axonaux résultent de sa nature intrinsèque, ainsi que des stratégies d'élongation développées pour surmonter les contraintes mécaniques du cerveau en développement. Nous avons conçu un modèle mathématique de la croissance d'un axone isolé fondé sur des chaînes de Markov gaussiennes avec deux paramètres, représentant la rigidité axonale et l'attraction du champ cible. Nous avons estimé les paramètres de ce modèle à partir de données réelles et simulé la croissance des axones à l'échelle de populations et avec des contraintes spatiales pour tester notre hypothèse. Nous avons abordé des thèmes de mathématiques appliquées ainsi que de la biologie, et dévoilé des effets inexplorés de la croissance collective sur le développement axonal in vivo. / How the brain wires up during development remains an open question in the scientific community across disciplines. Fruitful efforts have been made to elucidate the mechanisms of axonal growth, such as pathfinding and guiding molecules. However, recent evidence suggests other actors to be involved in neuron growth in vivo. Notably, axons develop in populations and embedded in mechanically constrained environments. Thus, to fully understand this dynamic process, one must take into account collective mechanisms and mechanical interactions within the axonal populations. However, techniques to directly measure this from living brains are today lacking or heavy to implement. This thesis emerges from a multidisciplinary collaboration, to shed light on axonal development in vivo and how adult complex axonal morphologies are attained. Our work is inspired and validated from images of single wild type and mutated Drosophila y axons, which we have segmented and normalized. We first proposed a mathematical framework for the morphological study and classification of axonal groups. From this analysis we hypothesized that axon growth derives from a stochastic process, and that the variability and complexity of axonal trees result from its intrinsic nature, as well as from elongation strategies developed to overcome the mechanical constraints of the developing brain. We designed a mathematical model of single axon growth based on Gaussian Markov Chains with two parameters, accounting for axon rigidity and attraction to the target field. We estimated the model parameters from data, and simulated the growing axons embedded in spatially constraint populations to test our hypothesis. We dealt with themes from applied mathematics as well as from biology, and unveiled unexplored effects of collective growth on axonal development in vivo.

Der Einsatz von Fasergefügen und insbesondere von Papier geht heute über dessen ursprüngliches Anwendungsgebiet als Informationsträger weit hinaus. Mit alternativen und neuen Aufgabenfeldern des Papiers kommen auch weitere, qualitative Anforderungen hinzu, welche es während der Herstellung, Weiterverarbeitung und Nutzung erfüllen muss. In der Vergangenheit stand verstärkt z.B. die Verbesserung der statischen und dynamischen Festigkeitseigenschaften im Vordergrund. Für viele Anwendungsfälle spielt jedoch auch die Dehnung eine entscheidende Rolle. Beispiele sind Sackpapier oder Elektroisolationspapier. Darum verfolgt diese Arbeit das Ziel, systematisch und anhand eines neuen Dehnungsmodells, qualitativ und quantitativ die Einflüsse der Faser- und Gefügeeigenschaften anhand ausgewählter Prozessbedingungen auf das Kraft-Dehnungsverhalten, aber insbesondere auf dessen Dehnung zu untersuchen. Des Weiteren wurde eine Methode entwickelt, mit der es unter Nutzung eines Röntgen-Computertomographen möglich ist, weitere Gefügeparameter, auch während einer semi-dynamischen Zugprüfung in-situ zu ermitteln. Für die Bewertung der Fasereigenschaften wurden vier Faserstoffe ausgewählt. Zum Einsatz kam ein ungebleichter Nadelholzsulfatzellstoff (UKP), ein gebleichter Eukalyptuszellstoff, Baumwolllinters und Tencel, eine synthetische Cellulosefaser. Diese Faserstoffe sind chemisch und morphologisch analysiert worden, bevor sie sowohl überwiegend fibrillierend als auch überwiegend kürzend in einem Refiner gemahlen wurden. Nach unterschiedlich hohem Eintrag an massenspezifischer Mahlarbeit in den Faserstoff wurden aus der Suspension Papiermuster gebildet, schrumpfungsbehindert getrocknet und charakterisiert. Durch die Mahlung der Faserstoffe erfolgte eine Reduktion deren mittlerer längengewichteter Faserkonturlänge, der Feinstoffanteil konnte gesteigert werden und das Wasserrückhaltevermögen nahm zu. Es konnte ein unterschiedliches Verhalten der Entwicklung des Wasserrückhaltevermögens zwischen dem ungebleichten Nadelholzsulfatzellstoff und dem gebleichten Eukalyptus gegenüber den Baumwolllinters und den Tencelfasern gefunden werden. Das Wasserrückhaltevermögen von Baumwolllinters und den Tencelfasern blieb, unabhängig von der Mahlstrategie, fast bis zum maximalen Eintrag an massenspezifischer Mahlarbeit von ca. 770 kWh/t unbeeinflusst. Mit den erhaltenen Kraft-Dehnungsdiagrammen der Papiermuster, welche durch eine uniaxiale Zugprüfung mit konstanter Dehnungsgeschwindigkeit messtechnisch erfasst wurden, konnte durch eine Kurveneinpassung mit dem entwickelten Dehnungsmodell das jeweilige Kraft-Dehnungsverhalten mathematisch nachgebildet werden. Dieser neue Modellansatz wurde gewählt, nachdem die Auswertung des Ansatzes von Paetow [77;78;79;90] zu große Abweichungen bei bestimmten Papiermustern aufzeigte. Dies ermöglichte die quantitative Auswertung relevanter Parameter der Kraft-Dehnungskurven. Dabei wurden der Elastizitätsmodul, die Reißlänge und die Dehnung bei maximaler Reißlänge bewertet. Eine sehr hohe Reißlänge konnte mit einem fibrillierend gemahlenem, ungebleichtem Nadelholzsulfatzellstoff und eine hohe Dehnung mit einem, ebenfalls fibrillierend gemahlenem Eukalyptuszellstoff erreicht werden. Des Weiteren sind die Reißlänge am Übergang von einem initial linearen in den nicht-linearen Kurventeil, ein Abknickfaktor sowie der weitere Kurvenverlauf nach dem nicht-linearen Bereich, bis zur maximalen Reißlänge des Gefüges bewertet worden. Der letzte Kurvenbereich wurde entweder durch den weiteren, nicht-linearen Verlauf oder durch einen sekundären Linearbereich charakterisiert. Der von Seth und Page [22] dargestellte Einfluss der Faserstoffmahlung auf den Verlauf der Kraft-Dehnungskurve von Papier konnte nicht nachgebildet werden. Dies zeigte auch, dass die in dieser Arbeit gewonnenen Erkenntnisse durch eine Korrektur des Elastizitätsmoduls mit den Kraft-Dehnungskurven nicht mit den Ergebnissen aus [22] übereinstimmen. Die Faserstoffmahlung hat demnach nicht nur einen Einfluss auf die maximal erreichbare Reißlänge und Dehnung, sondern beeinflusst auch den qualitativen Verlauf der Kraft-Dehnungskurve von Papier. Es konnten keine individuellen Einflussgröße der Fasermorphologie und der Prozessparameter auf die Dehnung oder das Kraft-Dehnungsverhalten festgestellt werden, da sich die meisten dieser Eigenschaften direkt mit der eingebrachten, massenspezifischen Mahlarbeit verändern, die Papierdehnung jedoch schon nach Erreichen einer moderaten massenspezifischen Mahlarbeit von ca. 100 – 200 kWh/t nicht weiter steigern ließ. Für eine weitere Bewertung der Einflüsse auf das Kraft-Dehnungsverhalten von Papier wurden Messwerte aus [143] analysiert. Dabei zeigte sich, dass ein Anstieg an Fasern mit einer hohen Faserkräuselung die Reißlänge des Papiers sowie den Elastizitätsmodul signifikant reduziert. Die Reißlänge am Kurvenübergang vom initial linearen in den nicht-linearen Teil bleibt dabei jedoch konstant. Ein anderes Verhalten, welches mit den Ergebnissen von Seth und Page [22] sowie Lowe [12] übereinstimmt, ist die Auswirkung eines kationischen Additivs wie z.B. Stärke auf die Entwicklung des Kraft-Dehnungsverhaltens. Es konnte nachgewiesen werden, dass das Additiv keinerlei Einfluss auf den initial linearen sowie den nicht-linearen Teil der Kraft-Dehnungskurve hat, sondern nur den sekundären, linearen Kurvenbereich in Abhängigkeit der Dosiermenge beeinflusst. Dabei wurde die Steigung im sekundären Linearbereich bestimmt. Dieses Verhalten führte zu einer Erweiterung der Theorie von Kallmes [82], welcher nach einem Anstieg der Festigkeit und der Dehnung, ab einer kritischen, relativen Bindungsfläche nur noch einen Anstieg der Festigkeit vorhersagte, jedoch nicht mehr der Dehnung. Auf Grund der in dieser Arbeit gewonnenen Erkenntnisse müssen drei Fälle der Entwicklung des Kraft-Dehnungsverhaltens von Fasergefügen unterschieden werden, welche primär von der Homogenität der Spannungsverteilung im Fasergefüge abhängig sind und z.B. durch die Faserkräuselung oder Blattformation beeinflusst werden kann. Diese neue Ansicht basiert auf dem Verhältnis zwischen der Bindungsenergie der Faser-Faserbindung und dem formbasierten sowie dem längenbasierten Arbeitsaufnahmevermögen der Fasern. Der erste Fall der Gefügedehnung beschreibt das Verhalten, wenn die Bindungsenergie geringer ist als das formbasierte Arbeitsaufnahmevermögen. Dies führt zu einem Auseinandergleiten des Fasergefüges. Dieses Verhalten konnte mit der Analyse von Papierproben aus Linters im Röntgen-Computertomograph qualitativ nachgewiesen werden. Steigt die Bindungsenergie an, wie es der zweite Fall voraussetzt, kann das formbasierte Arbeitsvermögen der Fasern überwunden werden und steht als Dehnvermögen zur Verfügung. Um auch, wie es der dritte Fall beschreibt, das längenbasierte Dehnvermögen der Fasern nutzen zu können, muss die Bindungsenergie zusätzlich durch z.B. ein Additiv oder hohe Drücke in einer Nasspresse weiter steigen. Die erweiterte Theorie bildet nun das gesamte Kraft-Dehnungsverhalten von Fasergefügen ab und muss in weiteren Arbeiten zur Papieranalyse verifiziert werden. Eine wertvolle Ergänzung der zu ermittelnden Gefügeparameter kann durch die entwickelte Methode mit der Röntgen-Computertomographie geleistet werden.

RNA polymerase II-mediated transcription of HIV-1 genes depends on positive transcription elongation factor b (P-TEFb), the complex of cyclin T1 and CDK9. Recent evidence suggests that regulation of transcription by P-TEFb involves chromatin binding and modifying factors. To determine how P-TEFb may connect chromatin remodeling to transcription, we investigated the relationship between P-TEFb and histone H1. We show that P-TEFb interacts with H1 and that H1 phosphorylation in cell culture correlates with P-TEFb activity. Importantly, P-TEFb also directs H1 phosphorylation during Tat transactivation and wild type HIV-1 infection. Our results also show that P-TEFb phosphorylates histone H1.1 at a specific C-terminal site. Expression of a mutant H1.1 that cannot be phosphorylated by P-TEFb disrupts Tat transactivation as well as transcription of the c-fos and hsp70 genes in HeLa cells. P-TEFb phosphorylation of H1 also plays a role in the expression of muscle differentiation marker genes in the skeletal myoblast cell line C2C12. Additionally, ChIP experiments demonstrate that H1 dissociates from the HIV-1 LTR in MAGI cells, stress-activated genes in HeLa cells, and muscle differentiation marker genes in C2C12 cells under active P-TEFb conditions. Our results overall suggest a new role for P-TEFb in both cellular and HIV-1 transcription through chromatin.

Early planting is one technique to avoid or reduce heat and drought problems that negatively affecting grain crop production. If producers adopt early planting, cold temperatures may negatively affect corn yield. It is important to select hybrids that are suited for planting earlier in the southern United States. Experiments were conducted by imposing low temperatures during seed germination. Twenty commercially available corn hybrids were evaluated for seed germination and root elongation. The first objective was: 1) To determine if some hybrids germinate better at cooler temperatures than others; and 2) Determine variation in root elongation at cold temperatures among commercially available hybrids. Corn hybrids varied significantly for seed germination and root traits under cold temperatures. Some hybrids have significantly surpassed others in seed germination traits, and they germinated earlier as well having longer radicle length. Also, there were significant differences across temperatures for all traits measured. A second objective was: 1) To quantify the effects of cold temperature on seed germination rate; 2) To evaluate the effects of different cold temperatures on seed germination behavior of corn hybrids under laboratory conditions to determine how fast they germinated; and 3) To classify hybrids for response to cold temperature using cumulative seed germination. The results showed that standard germination performance occurred at 10ºC for all hybrids, but these hybrids performed well under other cold treatments (7.2°C and 8.6° C). There were no big differences between early hybrids 93 to 105 RM (Relative Maturity) and full season 115 to 120 RM in germination % and rate in both experiments, so that means that it pretty much depends on the hybrid. Therefore, the temperature had a major influence on seed germination parameters. These findings are useful for hybrid selection with respect to cool soil temperature conditions during early planting.

No description available.

The objective of this research was to investigate the occurrence of mycoflora in rice plants and rice seeds in South Africa and their negative impact. A total of six species of Fusarium were isolated from diseased rice plants and rice seeds and identified as F. anthophilum, F. chlamydosporum, F. compactum, F. equiseti, F. fujikuroi and F. semitectum. In the translation elongation factor data set, Fusarium equiseti isolates grouped together within the F. incarnatum - equiseti Species Complex (FIESC). The isolates from rice clustered together in a single clade with the F. equiseti and F. incarnatum isolates forming two separate sub-clades.The isolates of F. equiseti present a new phylogenetically distinct species in FIESC. In the pathogenicity tests, isolates of both F. anthophilum and F. fujikuroi caused bakanae disease to rice plants. Fifty four rice cultivars and lines were tested by the standardized test tube inoculation method for resistance and susceptibility against bakanae isolate of F. anthophilum and the bakanae isolate of F. fujikuroi. None of the rice cultivars and lines was found to be resistant to bakanae isolates of Fusarium spp. The fungicide, benomyl was found to be most effective as a seed treatment for controlling bakanae disease of rice due to isolates of both F. anthophilum and F. fujikuroi. Thiram was found to be the least effective fungicide for controlling bakanae disease of rice caused by isolates of both the Fusarium spp. Apart from Fusarium species, other fungi that were also isolated from diseased rice plants and rice seeds were identified as Alternaria alternata, Alternaria longipes, Cochliobolus miyabeanus, Nigrospora sphaerica, Phoma eupyrena, Phoma jolyana, Phoma sorghina and Pithomyces sp. In mycotoxin tests, the isolates of both F. anthophilum and F. fujikuroi produced moniliformin. None of the isolates of F. anthophilum and F. fujikuroi produced fumonisins. This research is important as it identifies many fungal species in rice plants and seeds in South Africa for the first time. Currently, there is very little literature that makes reference to such findings under South African conditions. In addition, this investigation unravels previously unknown information on the resistance of rice to bakanese disease. Finally, information is provided on the effectiveness of commonly used fungicides (benomyl and thiram) to control rice diseases. This knowledge is crucial information that is useful to plant pathologists, the farming community and the scientists that are involved in strategies of fighting or reducing rice diseases so as to help contribute to food security. / Environmental Sciences / D. Phil. (Environmental Science)

La régulation de la transcription est un processus complexe qui a évolué pendant des millions d’années permettant ainsi aux cellules de s’adapter aux changements environnementaux. Notre laboratoire étudie le rôle de la rapamycine, un agent immunosuppresseur et anticancéreux, qui mime la carence nutritionelle. Afin de comprendre les mécanismes impliqués dans la réponse a la rapamycine, nous recherchons des mutants de la levure Saccaromyces cerevisiae qui ont un phenotype altérée envers cette drogue. Nous avons identifié le gène RRD1, qui encode une peptidyl prolyl isomérase et dont la mutation rend les levures très résistantes à la rapamycine et il semble que se soit associé à une réponse transcriptionelle alterée. Mon projet de recherche de doctorat est d’identifier le rôle de Rrd1 dans la réponse à la rapamycine. Tout d’abord nous avons trouvé que Rrd1 interagit avec l’ARN polymérase II (RNAPII), plus spécifiquement avec son domaine C-terminal. En réponse à la rapamycine, Rrd1 induit un changement dans la conformation du domaine C-terminal in vivo permettant la régulation de l’association de RNAPII avec certains gènes. Des analyses in vitro ont également montré que cette action est directe et probablement liée à l’activité isomérase de Rrd1 suggérant un rôle pour Rrd1 dans la régulation de la transcription. Nous avons utilisé la technologie de ChIP sur micropuce pour localiser Rrd1 sur la majorité des gènes transcrits par RNAPII et montre que Rrd1 agit en tant que facteur d’élongation de RNAPII. Pour finir, des résultats suggèrent que Rrd1 n’est pas seulement impliqué dans la réponse à la rapamycine mais aussi à differents stress environnementaux, nous permettant ainsi d’établir que Rrd1 est un facteur d’élongation de la transcription requis pour la régulation de la transcription via RNAPII en réponse au stress. / Transcriptional regulation is a complex process that has evolved over millions of years of evolution. Cells have to sense environmental conditions and adapt to them by altering their transcription. Herein, we study the role of rapamycin, an immunosuppressant and anticancer molecule that mimics cellular starvation. To understand how the action of rapamycin is mediated, we analyzed gene deletion mutants in the yeast Saccharomyces cerevisiae that have an altered response to this drug. Deletion of RRD1, a gene encoding a peptidyl prolyl isomerase, causes strong resistance to rapamycin and this was associated with a role of Rrd1 in the transcriptional response towards rapamycin. The main focus of my PhD was therefore to unravel the role of Rrd1 in response to rapamycin. First, we discovered that Rrd1 interacts with RNA polymerase II (RNAPII), more specifically with its C-terminal domain and we showed that in response to rapamycin, Rrd1 alters the structure of this C-terminal domain. This phenomenon was confirmed to be directly mediated by Rrd1 in vitro, presumably through its peptidyl prolyl isomerase activity. Further, we demonstrated that Rrd1 is capable of altering the occupancy of RNAPII on genes in vivo and in vitro. With the use of ChIP on chip technology, we show that Rrd1 is actually a transcription elongation factor that is associated with RNAPII on actively transcribed genes. In addition, we demonstrate that Rrd1 is indeed required to regulate the expression of a large subset of genes in response to rapamycin. This data let us propose a novel mechanism by which Rrd1 regulates RNAPII during transcription elongation. Finally, we provide evidence that Rrd1 is not only required for an efficient response towards rapamycin but to a larger variety of environmental stress conditions, thus establishing Rrd1 as a transcriptional elongation factor required to fine tune the transcriptional stress response of RNAPII.