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Die Intensivierung des Rapsanbaus in den letzten Jahren hat zu einem verstärkten Aufkommen des bodenbürtigen Gefäßpathogens V. longisporum geführt. Die für den Pilz charakteristischen Mikrosklerotien können langjährig im Boden überdauern, akkumulieren und somit zur fortdauernden Bodenkontamination führen. Eine Infektion mit V. longisporum kann bereits im Herbst erfolgen, wenn durch Wurzelexsudate stimulierte Mikrosklerotien auskeimen und direkt die Wurzelepidermis der Rapspflanze penetrieren. Einer sowohl intra-als auch interzellulär gerichteten Ausbreitung bis zu den Gefäßelementen schließt sich eine langanhaltende Phase des Pilzes im Gefäßsystem an. In dieser latenten Phase zeigen sich keine auffälligen Symptome an der Pflanze, erst zum Ende der Pflanzenentwicklung zeigt sich halbseitige Stängelverbräunung und vorzeitige Abreife kann zu Ertragseinbußen führen. Der Pilz bleibt so lange auf die Gefäße beschränkt bis die Pflanze in die Seneszenzphase eintritt. Dann erfolgt eine Besiedelung der angrenzenden parenchymatischen Zellen und die Bildung von Mikrosklerotien. Mit Pflanzenresten können diese wieder in den Boden gelangen. Da derzeit keine adequaten Pflanzenschutzmittel zur Verfügung stehen, ist der Anbau resistenter Sorten eine wirkungsvolle Maßnahme die Verbreitung des Pilzes einzudämmen und der Anreicherung von Mikrosklerotien im Boden entgegenzuwirken. Im Rahmen dieser Arbeit wurde ein entscheidender Beitrag zur Züchtung neuer resistenter Genotypen geleistet. Phänotypisierungen zur Identifizierung resistenter B. napus-Linien (darunter auch DH-Linien) erfolgten unter kontrollierten Bedingungen im Gewächshaus in Göttingen. Darüber hinaus wurde die Resistenz ausgewählter B. napus-Linien in zwei aufeinander folgenden Jahren anhand von Feldversuchen in Göttingen, an verschiedenen Standorten in Norddeutschland und an einem Standort in Südschweden evaluiert. Eine Untersuchung der von 2004 bis 2009 im Gewächshaus getesteten B. napus Akzessionen wurde hinsichtlich der Häufigkeitsverteilungen der berechneten normierten AUDPC-Werte betrachtet. So konnte deutlich gezeigt werden, dass sich das Resistenzlevel in den aktuellsten Screenings deutlich verbessert hat im Vergleich zum Beginn der Screenings. Die Reproduzierbarkeit der Screenings wurde deutlich durch die Betrachtung der normierten AUDPC-Werte der Referenzsorten ‘Falcon’ und ‘Express’. So waren die normierten AUDPC-Werte der mittelgradig resistenten Referenzsorte ‘Express’ durchgängig niedriger im Vergleich zu der anfälligen Sorte ‘Falcon’, was für die Robustheit der Methodik spricht. Der Vergleich zwischen Gewächshaus- und Feldversuchen zeigte, dass eine geringe Korrelation zwischen den im Feld und Gewächshaus getesteten Akzessionen besteht und macht die Komplexität der Untersuchungen deutlich. Ein Screening von Genotypen kann jedoch nur schnell und in großem Umfang unter Gewächshaus-Bedingungen erfolgen. Die erweiterte Testung im Feld ist dann jedoch nötig, um die Resistenz unter zusätzlichem abiotischem Stress zu evaluieren. Neben der Bewertung des Befallsgrades (Befallshäufigkeit, Befallsstärke) mittels Stoppelbonitur wurde eine alternative Bewertungsmethode zur Evaluierung der Resistenz im Feld kultivierter Rapspflanzen gegenüber V. longisporum entwickelt. Die Entwicklung einer sensitiven real-time PCR (qPCR)-Methode zur Detektion von V. longisporum in Rapsstängeln beinhaltete die Bewertung zweier unterschiedlicher Primer, abzielend auf die internal transcribed spacer (ITS) Region bzw. auf die β-Tubulin-Region, die hinsichtlich ihrer Sensitivität und Spezifität analysiert wurden. Die hier getesteten ITS-Primer wiesen eine hohe Sensitivität gegenüber genomischer Pilz-DNA auf, jedoch wurde keine Spezifität gegenüber V. longisporum Isolaten festgestellt; vielmehr wurden V. dahliae Isolate und zwei weitere Verticillium Arten mit ITS-Primern detektiert. Das zweite getestete Primerpaar zeigte hingegen eine hohe Spezifität gegenüber V. longsiporum Isolaten, lediglich 3 von 15 getesteten V. longisporum Isolaten wurden nicht erfasst. Die Sensitivität dieser Primer war jedoch im Vergleich zu den ITS-Primern stark verringert. Die ITS-basierte qPCR Analyse führte zur Detektion des Pathogens noch vor der Symptomausbildung im Feld. So konnte in der Saison 2008/09 am Standort Göttingen gezeigt werden, dass frühe Infektionen bereits zu BBCH 65 auftraten und innerhalb weniger Wochen eine massive Besiedelung anfälliger Sorten erfolgte. Zudem konnte die pilzliche DNA-Konzentration in infizierten Rapsstängeln verschieden anfälliger Sorten quantifiziert und eine Korrelation zwischen der herkömmlichen Stoppelbonitur und dem Screening im Gewächshaus hinsichtlich der Einordnung der Resistenzniveaus hergestellt werden. Dies unterstützt die Verwendung der molekularen Methode als Alternative zur Stoppelbonitur. Neben der Verbesserung der Detektion von V. longisporum im Feld wurde die Pathogen-Wirt-Interaktion hinsichtlich der Ausbildung von Resistenzmechanismen charakterisiert. Dazu wurden zwei verschieden anfällige B. napus-Linien nach Inokulation mit V. longisporum sowohl auf histologischer als auch auf molekularbiologischer Ebene im Hypokotylbereich untersucht. Dieser Abschnitt, der den Bereich vom Wurzelhals bis zum Keimblattansatz markiert, konnte in vorangegangenen Untersuchungen als Schlüsselgewebe für die Ausbildung von Resistenzstrukturen identifiziert werden (Eynck et al., 2009). Anknüpfend an diese Untersuchungen wurden mittels Transmissionselektronenmikroskopie (TEM) genotypabhängige Resistenzstrukturen wie Gefäßverschlüsse und morphologische Veränderungen des Gefäßbereiches untersucht und begleitende qPCR-Messungen dokumentierten die Pathogenausbreitung. Diese ließen erkennen, dass der anfällige Genotyp im Vergleich zum resistenten Genotyp schneller besiedelt wird. Jedoch zeigten beide mit V. longisporum inokulierten Genotypen ähnliche ultrastrukturelle Veränderungen im vaskulären Bereich. So konnten Veränderungen an vaskulären Zellwänden wie elektronendichte Ablagerungen und Degradation primärer Zellwände im Bereich der Tüpfel beobachtet werden. Zudem konnte das Verschließen von Gefäßelementen mittels gelartiger Strukturen nachgewiesen werden. Unsere Untersuchungen lassen vermuten, dass der resistente Genotyp fähig ist Infektionen schneller zu erkennen und Resistenzmechanismen zielgerichteter und intensiver zu aktivieren. Da eine V. longisporum-Infektion in dem untersuchten resistenten Genotyp SEM 05-500256 u. a. zu einer verstärkten Bildung von Gefäßbarrieren im Hypokotylbereich führt (Eynck et al., 2009), wurde eine Beeinträchtigung des pflanzlichen Wassertransportes vermutet. Zur Klärung dieser Frage wurde der resistente Genotyp zusätzlich zu einer Infektion mit V. longisporum Trockenstressbedingungen (30% Feldkapazität) ausgesetzt und physiologische Parameter (Gaswechselmessungen), Befallswerte (AUDPC, Stauchung) und agronomische Parameter (Phänologisches Entwicklungsstadium, Anzahl Seitentriebe, Ertragsparameter) erfasst und im Vergleich zu der anfälligen Sorte ‘Falcon’ evaluiert. Weder die Befallsparameter noch die agronomischen Parameter zeigten eine Beeinträchtigung der Resistenz von SEM bei V. longisporum-Infektion in Kombination mit Trockenstress an.

Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-27Bitstream added on 2014-06-13T20:35:48Z : No. of bitstreams: 1 moreira_wmq_me_jabo.pdf: 763719 bytes, checksum: 21d0bf3b9a629afa4e42b015a9f29722 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O Brasil é o segundo maior produtor de soja no mundo, cuja cultura requer o elemento nitrogênio em quantidades elevadas para manutenção do alto teor protéico dos grãos. A entrada de nitrogênio nos sistemas agrícolas pode ocorrer pela adição de fertilizantes nitrogenados ou por processos naturais como a Fixação Biológica do Nitrogênio, que se constitui como supridor de nitrogênio mais viável para a cultura da soja, tanto economicamente como ecologicamente. Este processo ocorre graças à simbiose que ocorre entre esta leguminosa e as bactérias do gênero Bradyrhizobium, resultando na formação de nódulos radiculares onde se dá a obtenção de todo o nitrogênio que a cultura necessita para alta produtividade. A introdução destas bactérias no solo se dá através da utilização de inoculantes comerciais, que incluem as bactérias Bradyrhizobium elkanii e Bradyrhizobium japonicum em sua composição. Aspectos relacionados à formulação e fabricação dos inoculantes comerciais reúnem os fatores mais importantes para a obtenção de um produto de qualidade, obedecendo à legislação vigente. Tendo em vista a análise de qualidade de inoculantes comerciais para soja em diferentes períodos de armazenamento, um estudo do metabolismo respiratório de bradirrizóbios foi realizado in vitro e em simbiose. Inoculantes comerciais com 12, 27 e 48 meses de idade foram analisados quanto suas características bioquímicas e fisiológicas, assim como testados em casa de vegetação. Adicionalmente, os mesmos testes foram realizados com Bradyrhizobium elkanii sob diferentes condições de oxigênio. Análise da expressão gênica indicou que um processo de expressão de genes relacionados à fixação biológica de nitrogênio (genes sensores a baixas tensões de oxigênio) foram expressos, entretanto não ocorrendo expressão do gene nifH, este só expresso em condições... / Brazil is the second major soybean producer in the world, whose culture requires the element nitrogen in large amounts for the maintenance of the high protein content in grains. The input of nitrogen in agricultural systems can occur by adding nitrogen fertilizer or by natural processes such as biological nitrogen fixation (BNF). BNF is the most feasible way to delivery nitrogen for the soybean crop, both economically and ecologically. This process occurs through the symbiosis between the legume and the bacteria of the genus Bradyrhizobium, resulting in the formation of root nodules where all nitrogen that the crop needs for high productivity is acquired. The introduction of these bacteria in soil is given by use of commercial inoculants, which include the bacteria Bradyrhizobium japonicum and Bradyrhizobium elkanii in its composition. Aspects related to formulation and manufacture of inoculants include the most important factors affecting the product quality, according to law. In order to analysis of the quality of commercial inoculants for soybean in different storage periods, a study of respiratory metabolism of bradyrhizobia was performed in vitro and in symbiosis. Inoculants with 12, 27 and 48 months old were analyzed accessing their biochemical and physiological characteristics, and tested at greenhouse. In addition, the same tests were conducted on cultures of Bradyrhizobium elkanii under different oxygen conditions. Analysis of gene expression have indicated that genes related to biological nitrogen fixation (genes sensors at low oxygen tension) were intensively expressed, but not occurring nifH gene expression which is only expressed under symbiosis. Similar data were found for the expression of these genes when B. elkanii grown at different oxygen tensions. These data indicate that as the oxygen level is reduced some genes related to nitrogen fixation are expressed... (Complete abstract click electronic access below)

This work is aimed at the study of the effect of 17α-ethinylestradiol (EE2) on the production and characteristics of ligninolytic enzymes (laccase, Mn-dependent peroxidase and lignin peroxidase) in I. lacteus, T. versicolor, P. chrysosporium and P. ostreatus cultures grown on two types of liquid media. Enzyme activity production in fungal cultures was affected by the composition of culture medium. In the case of P. chrysosporium, the addition of EE2 to the complex- medium cultures led to a MnP activity stimulation and simultaneously LiP production was partially repressed in these cultures. In the mineral MM medium, no effect of EE2 on enzyme production by P. chrysosporium was observed. In EE2 treated MM cultures of P. ostreatus lower MnP activities were found when compared to biotic controls. In the case of T. versicolor cultures, the addition of EE2 to the complex medium caused laccase and LiP stimulation in the cultures. In the MM medium, however, only laccase production was affected by EE2. I. lacteus MnP production was partially repressed by EE2 in MM medium. In contrast to that, significantly higher MnP activities were detected in complex- medium I. lacteus cultures after the treatment with EE2. Further EE2 degradation by the fungal cultures was studied. The highest degradation effeciency was...

MicroRNAs (miRNAs) are small noncoding RNAs of length 18 to 25 nucleotides that regulate gene expression posttranscriptionally. Expression of some miRNAs is tissue specific. I assumed that pregnancy induced complications associated with placental insufficiency could be characterized by a unique profil of placental-specific miRNAs in maternal circulation. I measured concentration and gene expression of selected miRNAs (miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) in the plasma of patients with preeclampsia (PE), fetal growth restriction (FGR) and gestational hypertension (GH). The control group consisted of patients with a normal course of pregnancy (FG). I processed 168 plasma samples, the representation of individual diagnosis were as follows: PE 63, FGR 27, GH 23, FG 55. Detection and quantification was carried out by quantitative real-time PCR. I identified three miRNAs with elevated levels in a group of preeclamptic patients: miR-517*, miR-520a* and miR-525. The severity of the PE, which was characterized by a form of the disease (mild or severe PE) and term (before or after the 34th week of pregnancy), did not have a statistically significant effect on the levels of miRNAs. More than a quarter of patients had PE superimposed on previous hypertension. Previous history of...

Teak tree (Tectona grandis L.f.) has a high value in the timber trade for fabrication of woody products due to its extraordinary qualities of color, density and durability. Despite the importance of this species, genetic and molecular studies available are limited. Also, the lack of molecular information about secondary xylem and tree maturation has hindered genetic exploration of teak. Therefore, gene expression studies and transcriptomic profiling are essential to explore wood formation and lignin biosynthesis through the development and aging of vascular plants. Aiming the gene expression studies, it was essential to identify and clone reference genes for teak. Eight genes were tested, commonly used in qRT-PCR, including TgRP60S, TgCAC, TgACT, TgHIS3, TgSAND, TgTUB, TgUBQ and TgEF1a. Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem). Stability validation by NormFinder, BestKeeper, geNorm and Delta Ct programs showed that TgUBQ and TgEF1a are the most stable genes to use as qRT-PCR reference genes in teak in the conditions tested. Due to the availability of 12- and 60-year-old teak trees, RNA-seq was performed in diferent organs (seedlings, leaves, flowers, root, stem and branch secondary xylem). A total of 462,260 transcripts were obtained by assembling with \"Trinity\" software. Also, 1,502 and 931 genes differentially expressed were identified for stem and branch secondary xylem, respectively, using DESeq program, and MYB transcription factors, which were characterized. TgMYB1 amino acid sequence displayed a predicted coiled-coil (CC) motif while TgMYB2, TgMYB3 and TgMYB4 showed R2R3-MYB domain. All of them were phylogenetically grouped with several gymnosperms and flowering plants. High expression of TgMYB1 and TgMYB4 in lignified tissues of 60-year-old trees was observed. In this work, the Cinnamyl Alcohol Dehydrogenase (CAD) gene family was also studied. One complete (TgCAD1) and three partial (TgCAD2 to TgCAD4) members were characterized. The four enzymes presented residues for catalytic and structural zinc action, NADPH binding and substrate specificity, consistent with the mechanism of alcohol dehydrogenases. TgCAD3 and TgCAD4 were highly expressed in young and mature sapwood and seem to be duplicated and highly related with lignin biosynthesis. Tree genetic improvement, marker-assisted selection and plant transformation seem to be promising lines of research for the data obtained from this research. This is the first study addressing gene characterization and expression, phylogeny and transcriptomic profiling in teak. / A árvore de teca (Tectona grandis L.f.) tem alto valor no comércio de madeira para a fabricação de produtos lenhosos, devido às suas qualidades extraordinárias de cor, densidade e durabilidade. Apesar da importância desta espécie, são poucos os estudos genéticos e moleculares disponíveis. Também, a falta de informação molecular sobre xilema secundário e maturação da árvore tem dificultado a exploração genética de teca. Assim, estudos de expressão gênica e perfis transcricionais são relevantes para explorar a formação da madeira e a biossíntese de lignina durante o desenvolvimento e envelhecimento das plantas vasculares. Visando os estudos de expressão gênica, foi essencial identificar e clonar genes de referencia para a teca. Foram testados oito genes comumente usados em qRT-PCR, TgRP60S, TgCAC, TgACT, TgHIS3, TgSAND, TgTUB, TgUBQ e TgEF1a. Perfis de expressão destes genes foram avaliados por qRT-PCR em seis amostras de tecidos e órgãos (folhas, flores, plântulas, raiz, xilema secundário de caule e ramo). A validação da estabilidade pelos programas NormFinder, BestKeeper, geNorm e Delta CT mostrou que TgUBQ e TgEF1a são os genes mais estáveis para usar como genes de referência em teca nas condições testadas. Em virtude da disponibilidade de árvores de teca de diferentes idades, entre 12 e 60 anos, foi realizado o RNAseq de diferentes órgãos (plântulas, folhas, flores, raiz, ramos e caules de árvores de 12 e 60 anos). Obteve-se um total de 462.260 transcritos pela montagem com o software \"Trinity\". Foram identificados 1.502 e 931 genes diferencialmente expressos para xilema secundário de caule e ramo, respectivamente, utilizando o programa DESeq e fatores de transcrição MYB, que foram posteriormente caracterizados. A sequência de aminoácidos do TgMYB1 exibiu um motivo \"coiled-coil\" (CC), enquanto TgMYB2, TgMYB3 e TgMYB4 mostraram domínio R2R3-MYB. Todos eles foram filogeneticamente agrupados com várias gimnospermas e angiospermas. Observou-se alta expressão do TgMYB1 e TgMYB4 em tecidos lignificados de árvores de 60 anos de idade. Neste trabalho também foi estudada a família gênica Cinamil álcool desidrogenase (CAD). Foi caracterizado um membro completo (TgCAD1) e três parciais (TgCAD2 a TgCAD4). As quatro enzimas apresentaram resíduos de ação catalítica e estrutural de zinco, de ligação ao NADPH e de especificidade de substrato, em conformidade com o mecanismo conservado de álcool desidrogenases. TgCAD3 e TgCAD4 foram altamente expressos no alburno jovem e maduro e parecem estar duplicados e relacionados com a biossíntese de lignina. O melhoramento genético de árvores, a seleção assistida utilizando marcadores moleculares e a transformação de plantas parecem ser linhas promissoras de pesquisa, a partir dos dados obtidos nesta pesquisa. Este é o primeiro estudo sobre caracterização e expressão gênica, filogenia e perfis transcricionais em teca.

Les épisodes de mortalité massive de l’huître creuse Crassostreae gigas observés sur les côtes françaises depuis 2008 ont été associés à certaines espèces appartenant au genre bactérien Vibrio. Ces mortalités, particulièrement intenses et rapides au cœur des lagunes méditerranéennes, atteignent 80 à 100% de la production ostréicole remettant ainsi en cause la pérennité de cette activité. Une surveillance environnementale de ces bactéries apparait donc essentielle et nécessite la mise au point de méthodes d’analyse innovantes, alternatives aux techniques couramment employées, afin de permettre un suivi rapide et en temps réel des Vibrio dans les milieux aquatiques côtiers.Dans ce contexte, l’objectif de cette thèse a été de concevoir des outils analytiques de type génocapteurs pour la détection et la quantification des Vibrio dans les écosystèmes aquatiques. Dans un premier temps, un système basé sur un format d’hybridation « sandwich » reposant sur l’intercalation des acides nucléiques cibles entre une sonde capture immobilisée et une sonde signal marquée, couplé à une détection optique, a été élaboré. Après optimisation des conditions expérimentales, le test développé s’est avéré très sensible avec une limite de détection de 5 ng.µL-1 d’acides nucléiques, ainsi qu’hautement spécifique du genre Vibrio. La méthode a ensuite été appliquée avec succès à la détection des Vibrio dans des échantillons environnementaux, collectés dans la lagune de Salses-Leucate. Un second format d’hybridation, basé sur la compétition entre les acides nucléiques cibles et la sonde capture pour la sonde signal, a ensuite été envisagé en utilisant aussi bien une transduction optique qu’électrochimique. En parallèle, des méthodes de PCR quantitative en temps réel ont été mises au point afin de servir de références pour la validation des génocapteurs. / Mass mortality events affecting the Pacific oyster Crassostreae gigas on French coasts since 2008 have been associated to some Vibrio species. These mortalities, particularly severe and sudden in the mediterranean lagoons, can reach 80 to 100% of the oyster production threatening the sustainability of this activity. An environmental monitoring of these bacteria appears essential and, for this purpose, innovative analytical methods have to be developed as alternative to classical techniques, in order to allow the rapid and in real time monitoring of Vibrio in the coastal aquatic environments. In this context, the objective of the thesis was to design genosensors as analytical tools for Vibrio detection and quantification in aquatic ecosystems. In a first step, a system based on a « sandwich » hybridization format, in which nucleic acid targets were bound between an immobilized capture probe and a labeled signal probe, coupled with an optical detection method, was developed. After experimental condition optimization, the test showed high sensitivity with a limit of detection of 5 ng.µL-1 of nucleic acids and was highly specific to Vibrio spp. The method was then successfully applied to Vibrio detection in environmental samples collected in Salses-Leucate lagoon. A second hybridization format, based on a competition between the targeted nucleic acids and the capture probe for the signal probe has been considered using both optical and electrochemical transductions. Concurrently with the development of genosensors, quantitative real-time PCR have been designed as reference methods.

Helicoverpa armigera is an insect, causes important economical losses in crops. To reduce this loss, chemical insecticides such as pyrethroids have been commonly used against H. armigera in farming areas all over the world. However, excess and continuous usages of them cause resistance development in H. armigera. Insects develop resistance against applied insecticides by following three main mechanisms / by reducing the amount of insecticide entering into the insect body, developing insensitivity of the insecticide effective site and increasing detoxification metabolism of insecticides such as increased metabolism of them in midgut tissue of H. armigera. Therefore, changes in differentially expressed midgut proteins were analysed at protein level with two-dimensional gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) together with examine biochemical activity changes of certain detoxification enzymes such as esterases (EST) and glutathione S-transferases (GST). Moreover, transcriptional level analysis of certain genes from EST and GST systems together with cytochrome P450 monooxygenases (CYP450) system were done with quantitative real-time PCR method, too. According to the comparative proteome analysis, it was found that H. armigera field samples overcome pyrethroid stress mainly by increasing energy metabolism related proteins expressions such as ATP synthase, Vacuolar ATPase A and B and arginine kinase proteins. Furthermore, certain detoxification enzymes such as thioredoxin peroxidase and NADPH cytochrome P450 reductase were up-regulated in Mardin population, suggesting that they were actively participating in response to pyrethroid stress. NADPH cytochrome P450 reductase could play a role in detoxification of toxic pyrethroid metabolites such as 3-phenoxybenzaldehyde. However, while glutathione S-transferases (GSTs) were not found up-regulated in the comparative proteome analysis, biochemical assays (GST-CDNB, GST-DCNB and GST-PNBC) showed significant increases in enzyme activities in the Adana and in the Mardin field population, as compared to the susceptible strain. Furthermore, GST-DCNB and GST-PNBC activities showed significant increase in &Ccedil / anakkale population. As overcoming energy crisis may lead to an increase in oxidative stress, detoxification enzymes (GSTs and thioredoxin peroxidase) might be involved in pathways for eliminating toxic reactive oxygen species such as H2O2. Similarly, although esterases (EST) were not found as differentially expressed, biochemical assays for ESTs showed significant increases in enzymatic activities in the Adana and the Mardin field populations. Thus, ESTs are also proposed to be involved in developing resistance as an initiator of pyrethroid metabolism in H. armigera from Turkey. Quantitative real-time PCR results showed that while CYP9A14 gene expression was up-regulated in all analyzed field populations, CYP9A12 gene expression was up-regulated in both &Ccedil / anakkale and Mardin populations. CYP4S1 gene expression was also up-regulated only in Mardin field population. However, while CYP6B7 gene expression together with CYP9A12 and CYP4S1 genes expressions were down-regulated in Adana population, CYP6B7 gene expression was not significantly changed in both &Ccedil / anakkale and Mardin populations. In addition, GST, GSTX01 and ESTX018 gene expressions were not significantly changed in all field populations in comparison to susceptible population. Therefore, CYP9A14, CYP9A12 and CYP4S1 genes proposed to be involved in detoxification of toxic pyrethroid metabolites possibly through regulation of NADPH cytochrome P450 reductase. In conclusion, it is suggested that one of the main mechanisms of resistance development is increased energy metabolism in the midgut tissue of H. armigera which may be a general prerequisite for compensating the costs of energy-consuming detoxification processes.

The factors influencing senescence in white clover (Trifolium repens L.) are of considerable importance to the pastoral sector of New Zealand’s economy. The plant hormones, ethylene and the cytokinins, have been implicated as having opposing influences on senescence. This project focused on the cytokinins. The rate limiting step in cytokinin biosynthesis is catalysed by isopentenyl transferase (IPT) and the primary enzyme in the degradation of cytokinins is cytokinin oxidase/dehydrogenase (CKX). Both IPT and CKX genes are present as multi-gene families. A reduction in the level of active cytokinins either via a decrease in IPT expression, or an increase in CKX expression, or both, would implicate the cytokinins in developmental leaf senescence in white clover. White clover grows in a sequential pattern with leaves at all stages of development making it a good model for studying leaf development and senescence. A decrease in leaf chlorophyll is used as a marker for the onset of senescence. A micro-scale chlorophyll analysis was developed using the NanoDrop™ thus allowing tissue from the same leaflet to be used for gene expression and chlorophyll measurements. The pattern of chlorophyll changes was similar to that shown by Hunter et al.(1999) and Yoo et al.(2003) in white clover stolons used for ethylene research. Reverse transcriptase PCR (RT-PCR) and BLAST analysis was used to identify five putative IPT genes and seven putative CKX genes from white clover. RT-PCR demonstrated the expression of seven of these genes (TrIPT1. TrIPT13, TrIPT15 TrCKX1, TrCKX2, TrCKX6). Analysis with quantitative real-time PCR showed expression of TrCKX2 increased markedly during leaf expansion and was consistently high during senescence, suggesting a potential role for CKX in facilitating the progression of senescence.

La néphropathie à BK virus (BKV) est l'une des complications les plus fréquentes de la transplantation rénale. La prise en charge consiste en la réduction préemptive de l'immunosuppression basée sur le suivi de la charge virale, mais cette stratégie n’est pas complètement efficace et augmente le risque de rejet. Dans un premier volet, nous avons évalué la mesure de la charge virale par PCR quantitative en temps réel, permettant de mettre en évidence des facteurs de variabilité comme le polymorphisme du BKV et de valider la technique utilisée pour le suivi de notre cohorte. L’intérêt des anticorps neutralisants (AcNs) anti-BKV en tant que marqueur prédictif de la réplication BKV a ensuite été évalué dans une cohorte de 168 transplantés rénaux. Nous avons montré i) que le virus responsable de l’infection provenait du donneur ; ii) que les AcNs jouent un rôle dans la prévention de la réactivation et le contrôle de la réplication virale et iii) qu’un seuil d’AcNs de 4 log10 permettait de stratifier le risque de réplication BKV. Ce travail ouvre la voie à un suivi personnalisé en fonction du risque de réplication BKV et à de nouvelles approches immunothérapeutiques. / BK virus (BKV)-associated nephropathy is one of the major causes of graft dysfunction and loss in kidney transplant recipients. Since no BKV-specific antiviral therapies are available, management relies on preemptive immunosuppression reduction based on viral load monitoring. However, this strategy does not fully eliminate the risk of nephropathy and can increase the risk of graft rejection. In this work, we evaluated viral load measurement by quantitative real-time PCR in an interlaboratory comparison. Variability factors such as BKV polymorphism or pre-PCR steps have been highlighted and the method used for monitoring our cohort has been validated. The role of anti-BKV neutralizing antibodies (NAbs) as a predictive marker of BKV replication has been investigated in a cohort of 168 kidney transplant recipients. We showed that i) viral infection is caused by the donor strain; ii) NAbs play an essential role in viral replication prevention and control and iii) a NAbs cutoff of 4 log10 allows to stratify BKV replication risk. This work paves the way for personalized monitoring according to BKV replication risk and for new preventive or therapeutic strategies.