Practical resistance of Erysiphe necator to quinone outside inhibitors (QoIs) is now widespread, and resistance to demethylation inhibitors (DMIs) has also developed.  The goal of this research was to characterize fungicide resistance by elucidating resistance mechanisms and determining its stability.  QoI resistance persisted for several years in a field population after QoI application ended.  Resistant isolates were highly competitive in mixed populations in competition assays under laboratory conditions, indicating a lack of fitness cost.  In one competition trial under field conditions, resistance frequency declined, possibly due to spore migration and influx of background inoculum, but in a second trial, it did not decline.  Double resistance to QoI and DMI was detected and DMI application may have been partially responsible for maintaining QoI resistance in the field.  One isolate with QoI resistance but an undetectable level of the major QoI mutation was shown to be heteroplasmic -- resistant strains could be selected from this isolate. DMI resistance mechanisms in E. necator included the Y136F mutation in CYP51 and cyp51 over-expression.  The first mechanism was present in almost all isolates with substantial levels of resistance, and cyp51 expression level was correlated with resistance level.  Three cyp51 genotypes were detected.  Wildtype isolates with the TAT genotype were sensitive to DMIs, while isolates with increased resistance had either a TTT or TWT genotype; TWT indicated the presence of both wildtype and mutant alleles.  Cyp51 was expressed 1.4 to 19 times more in mutants than in wildtype.  It is not known whether the significant differences in cyp51 expression level among isolates and among genotype groups are due to gene copy number variation.  DMI resistance was found to decline after years of subculturing, and the decline appeared to occur after a few culture transfers of field samples on fungicide-free host leaves.  The observed decline, together with the finding that isolates could be "trained" to increase resistance, and may be slightly induced in cyp51 expression when successively challenged to grow in increasing fungicide concentration, indicate instability of DMI resistance. / Ph. D.

Die Strahlentherapie ist neben der Chemotherapie und einer operativen Entfernung die stärkste Waffe für die Bekämpfung bösartiger Tumore in der Krebsmedizin. Nach Herz-Kreislauf-Erkrankungen ist Krebs die zweithäufigste Todesursache in der westlichen Welt, wobei Prostatakrebs heutzutage die häufigste, männliche Krebserkrankung darstellt. Trotz technologischer Fortschritte der radiologischen Verfahren kann es noch viele Jahre nach einer Radiotherapie zu einem Rezidiv kommen, was zum Teil auf die hohe Resistenzfähigkeit einzelner, entarteter Zellen des lokal vorkommenden Tumors zurückgeführt werden kann. Obwohl die moderne Strahlenbiologie viele Aspekte der Resistenzmechanismen näher beleuchtet hat, bleiben Fragestellungen, speziell über das zeitliche Ansprechen eines Tumors auf ionisierende Strahlung, größtenteils unbeantwortet, da systemweite Untersuchungen nur begrenzt vorliegen. Als Zellmodelle wurden vier Prostata-Krebszelllinien (PC3, DuCaP, DU-145, RWPE-1) mit unterschiedlichen Strahlungsempfindlichkeiten kultiviert und auf ihre Überlebensfähigkeit nach ionisierender Bestrahlung durch einen Trypanblau- und MTT-Vitalitätstest geprüft. Die proliferative Kapazität wurde mit einem Koloniebildungstest bestimmt. Die PC3 Zelllinie, als Strahlungsresistente, und die DuCaP Zelllinie, als Strahlungssensitive, zeigten dabei die größten Differenzen bezüglich der Strahlungsempfindlichkeit. Auf Grundlage dieser Ergebnisse wurden die beiden Zelllinien ausgewählt, um anhand ihrer transkriptomweiten Genexpressionen, eine Identifizierung potentieller Marker für die Prognose der Effizienz einer Strahlentherapie zu ermöglichen. Weiterhin wurde mit der PC3 Zelllinie ein Zeitreihenexperiment durchgeführt, wobei zu 8 verschiedenen Zeitpunkten nach Bestrahlung mit 1 Gy die mRNA mittels einer Hochdurchsatz-Sequenzierung quantifiziert wurde, um das dynamisch zeitversetzte Genexpressionsverhalten auf Resistenzmechanismen untersuchen zu können. Durch das Setzen eines Fold Change Grenzwertes in Verbindung mit einem P-Wert < 0,01 konnten aus 10.966 aktiven Genen 730 signifikant differentiell exprimierte Gene bestimmt werden, von denen 305 stärker in der PC3 und 425 stärker in der DuCaP Zelllinie exprimiert werden. Innerhalb dieser 730 Gene sind viele stressassoziierte Gene wiederzufinden, wie bspw. die beiden Transmembranproteingene CA9 und CA12. Durch Berechnung eines Netzwerk-Scores konnten aus den GO- und KEGG-Datenbanken interessante Kategorien und Netzwerke abgeleitet werden, wobei insbesondere die GO-Kategorien Aldehyd-Dehydrogenase [NAD(P)+] Aktivität (GO:0004030) und der KEGG-Stoffwechselweg der O-Glykan Biosynthese (hsa00512) als relevante Netzwerke auffällig wurden. Durch eine weitere Interaktionsanalyse konnten zwei vielversprechende Netzwerke mit den Transkriptionsfaktoren JUN und FOS als zentrale Elemente identifiziert werden. Zum besseren Verständnis des dynamisch zeitversetzten Ansprechens der strahlungsresistenten PC3 Zelllinie auf ionisierende Strahlung, konnten anhand der 10.840 exprimierten Gene und ihrer Expressionsprofile über 8 Zeitpunkte interessante Einblicke erzielt werden. Während es innerhalb von 30 min (00:00 - 00:30) nach Bestrahlung zu einer schnellen Runterregulierung der globalen Genexpression kommt, folgen in den drei darauffolgenden Zeitabschnitten (00:30 - 01:03; 01:03 - 02:12; 02:12 - 04:38) spezifische Expressionserhöhungen, die eine Aktivierung schützender Netzwerke, wie die Hochregulierung der DNA-Reparatursysteme oder die Arretierung des Zellzyklus, auslösen. In den abschließenden drei Zeitbereichen (04:38 - 09:43; 09:43 - 20:25; 20:25 - 42:35) liegt wiederum eine Ausgewogenheit zwischen Induzierung und Supprimierung vor, wobei die absoluten Genexpressionsveränderungen ansteigen. Beim Vergleich der Genexpressionen kurz vor der Bestrahlung mit dem letzten Zeitpunkt (00:00 - 42:53) liegen mit 2.670 die meisten verändert exprimierten Gene vor, was einer massiven, systemweiten Genexpressionsänderung entspricht. Signalwege wie die ATM-Regulierung des Zellzyklus und der Apoptose, des NRF2-Signalwegs nach oxidativer Stresseinwirkung und die DNA-Reparaturmechanismen der homologen Rekombination, des nicht-homologen End Joinings, der MisMatch-, der Basen-Exzision- und der Strang-Exzision-Reparatur spielen bei der zellulären Antwort eine tragende Rolle. Äußerst interessant sind weiterhin die hohen Aktivitäten RNA-gesteuerter Ereignisse, insbesondere von small nucleolar RNAs und Pseudouridin-Prozessen. Demnach scheinen diese RNA-modifizierenden Netzwerke einen bisher unbekannten funktionalen und schützenden Einfluss auf das Zellüberleben nach ionisierender Bestrahlung zu haben. All diese schützenden Netzwerke mit ihren zeitspezifischen Interaktionen sind essentiell für das Zellüberleben nach Einwirkung von oxidativem Stress und zeigen ein komplexes aber im Einklang befindliches Zusammenspiel vieler Einzelkomponenten zu einem systemweit ablaufenden Programm. / The use of radiotherapy in addition to chemotherapy and surgical removal is the most powerful instrument in the fight against malignant tumors in cancer medicine. After cardiovascular diseases, cancer is the second leading cause of death in the western world, in which prostate cancer is the most frequent male cancer. Despite continuous technological improvements in radiological instruments and prognosis, it may occur a recurrence up to many years after radiotherapy due to a high resistance capability of individual malignant cells of the locally occurring tumor. Although modern radiation biology has studied many aspects of the resistance mechanisms, questions are largely unanswered especially in regards to prognostic terms and time response of tumor cells to ionizing radiation. As cellular models four prostate cancer cell lines with different radiation sensitivities (PC3, DuCaP, DU-145, RWPE-1) were cultured and tested for their ability to survive after exposure to ionizing radiation by a trypane blue and MTT viability assay. The proliferative capacity of the four cell lines was determined using a colony formation assay. The PC3 cell line (radiation-resistant) and the DuCaP cell line (radiation-sensitive) showed the maximal differences in terms of radiation sensitivity. Based on these results the two cell lines were selected to allow identification of potential prognostic marker for predicting the effectiveness of radiation therapy via their transcriptome-wide gene expression. Furthermore, a time series experiment with the radiation-resistant PC3 cell line was performed. At 8 different time points, during the period from 00:00 - 42:53 (hh:mm) after exposure with 1 Gy, the mRNA was quantified by next generation sequencing to investigate the dynamic behavior of time-delayed gene expression and to discover resistance mechanisms. Of 10,966 expressed genes 730 were significant differentially expressed, determined by setting a fold change threshold in conjunction with a P-value < 0.01. Of those 305 were more strongly expressed in PC3 cell line and 425 were more strongly expressed in the DuCaP cell line. Within these 730 genes many known stress-associated genes could be found, such as the two trans-membrane protein genes CA9 and CA12, which are associated with increased radiation resistance. By calculating a network score interesting networks were derived by the GO and KEGG databases. In particular the GO categories aldehyde dehydrogenase [NAD(P)+] activity (GO:0004030) as well as the KEGG pathway of O-glycan biosynthesis (hsa00512) seems to be remarkably relevant. An interaction analysis revealed two promising networks with the transcription factors JUN and FOS as central elements. High expression of the JUN network would be stand as indicator for radiation resistance whereas a high expression of the FOS network is equated with radiation sensitivity. Interesting insights could be achieved by analyzing the 10,840 expressed genes of the PC3 cell line and its expression profile over the 8 time points. Shortly after irradiation (00:00 - 00:30) a transcriptome-wide down-regulation occurred, within the next three, short time periods (00:30 - 01:03; 01:03 - 02:12; 02:12 - 04:38) a predominant increase of gene expression and the activation of protective networks followed, such as the up-regulation of DNA repair systems or the arresting of cell cycle. In the ensuing three time periods (4:38 - 09:43; 09:43 - 20:25; 20:25 - 42:35) a balance between gene induction and suppression was present and the absolute gene expression change was increased. When comparing the gene expression prior to irradiation with the last time point (00:00 - 42:53) 2,670 genes were differentially expressed, suggesting a massive and system-wide change of gene expression. Signaling pathways such as the ATM-regulated cell cycle and apoptosis, the Nrf2 pathway after oxidative stress exposure, the DNA repair mechanisms of homologous recombination, the non-homologous end joining, the mismatch repair, base-excision repair and strand-excision repair play a major role. Very interesting are the high activity of RNA-driven events, especially activities of small nucleolar RNAs and pseudouridine processes. This suggests that these RNA-modifying networks could have a hitherto unknown functional and protective effect on cell survival after exposure to ionizing radiation. All these protective networks and their time-specific interactions are essential for the survival of cells after exposure to oxidative stress and show a complex but consistent interaction of many individual components to a system-wide running program.

Francisella tularensis est une bactérie à Gram négatif intracellulaire facultative, agent causal de la tularémie. Cette zoonose est induite principalement par deux sous espèces : F. tularensis subsp. tularensis (type A) et F. tularensis subsp. holarctica (type B) retrouvées respectivement en Amérique du Nord et dans tout l’hémisphère Nord. Cette seconde sous espèce, moins virulente que la première induit majoritairement des formes cliniques de sévérité moyenne à modérée dites ganglionnaires. Leur traitement est basé sur l’utilisation des antibiotiques de la classe des fluoroquinolones ou des tetracyclines, l’utilisation des aminosides étant réservée aux formes graves. Les adénopathies évoluent cependant souvent vers la suppuration et la chronicité (20 à 40% des cas), malgré l’administration d’un traitement antibiotique adapté.Les travaux réalisés visent à étudier l’hypothèse de l’émergence de la résistance bactérienne chez Francisella, expliquant ces échecs thérapeutiques. Ils sont basés sur le développement et l’étude d’un modèle d’évolution in vitro de la bactérie en présence de ciprofloxacine, une fluoroquinolone. Nos travaux ont confirmé la capacité de la bactérie à évoluer vers un haut niveau de résistance à ces antibiotiques, corrélée à l’accumulation de mutations dans les gènes codant pour les topoïsomérases de type II. De plus, nous avons observé la présence sur l’ensemble des souches de F. tularensis subsp. holarctica d’un niveau de résistance cliniquement significatif induit par des mutations modifiant la sous-unité GyrA de l’ADN gyrase sur les acides aminés en position 83 et 87. La recherche de ce marqueur dans des prélèvements de patient en échec thérapeutique suite à divers traitements antibiotiques s’est avérée infructueuse.Après avoir vérifié l’action de l’antibiotique sur les bactéries dans le compartiment intracellulaire (fibroblates), nous avons recherché les autres mutations induites lors de l’évolution de Francisella en présence de fluoroquinolones. Cette étude a permis l’implication de plusieurs systèmes de transports transmembranaires dans la résistance antibiotique. Nous avons également révélé l’existence d’une seconde cible majeure impliquée dans le métabolisme du fer de la bactérie. L’altération de cette cible (FupA/B) en plus d’être associée à une augmentation de la résistance aux fluoroquinolones est corrélée à une forte diminution de la capacité de la bactérie à se multiplier dans les cellules phagocytaires. / Francisella tularensis is a gram-negative facultative intracellular bacterium, causing tularemia. This zoonosis is mainly related to two subspecies: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) in North America and throughout the Northern Hemisphere, respectively. Infections with this second subspecies, less virulent than the first one, predominantly induce glandular clinical forms of mild to moderate severity. Their treatment is based on antibiotherapy using a fluoroquinolone or a tetracycline. The use of aminoglycosides is reserved for severe clinical forms. The lymph nodes infection, however, often become chronic (20 to 40% of cases), despite administration of an appropriate antibiotic treatment.The aim of this study was to verify the hypothesis of the emergence of bacterial resistance in Francisella, which could explain treatment failures. It is based on the development and study of an in vitro evolutionary experiment of the bacterium in the presence of ciprofloxacin, a fluoroquinolone. Our work confirmed the bacterium's ability to evolve towards a high-level of resistance to fluoroquinolones, this evolution being correlated with the accumulation of mutations in the genes encoding for type II topoisomerases. In addition, we observed in all strains of F. tularensis subsp. holarctica resistant to fluoroquinolones at a clinically significant level, the presence of mutations altering the GyrA subunit of DNA gyrase at amino acids positions 83 and 87. The research of this marker in clinical samples from patients with treatment failure following appropriate antibiotic treatment was however unsuccessful.After checking the action of antibiotics on bacteria internalized in the intracellular compartment in fibroblast cells, we looked for other mutations induced during the evolution of Francisella to resistance to fluoroquinolones. This study unveiled the involvement of several transmembrane transport systems in antibiotic resistance. We also revealed the existence of a second major target involved in Francisella iron metabolism. The alteration of this target (FupA/B), in addition to being associated with an increase in fluoroquinolone resistance, is correlated with a sharp decrease in the ability of the bacteria to multiply in phagocytic cells.

Although A. fumigatus strains are generally susceptible to azoles, recently, acquired resistance to a number of antifungal compounds has been reported, especially to triazoles possibly due to widespread clinical use of triazoles or through exposure to azole fungicides in the environment. The significant clinical problem of azole resistance has led to study the antifungal resistance mechanisms for developing effective therapeutic strategies. Of 230 clinical A. fumigatus isolates submitted during 2008 and 2009 to the Mycology Reference Centre Manchester, UK (MRCM), 64 (28%) were azole resistant and 14% and 20% of patients had resistant isolates, respectively. Among the resistant isolates, 62 of 64 (97%) were itraconazole resistant, 2 of 64 (3%) were only voriconazole resistant and 78% were multi-azole resistant. The gene encoding 14-α sterol demethylase (cyp51A) was analyzed in 63 itraconazole resistant (ITR-R) and 16 ITR-susceptible clinical and environmental isolates of A. fumigatus respectively. Amino acid substitutions in the cyp51A, the commonest known mechanism of azole resistance in A. fumigatus, were found in some ITR-R isolates. Fifteen different amino acid substitutions were found in the cyp51A three of which, A284T, M220R and M220W, have not been previously reported. In addition, several mutations were found in the cyp51A gene in one of the A. fumigatus environmental isolates. Importantly, a remarkably increased frequency of azole-resistant isolates without cyp51A mutations was observed in 43% of isolates and 54% of patients. Other mechanisms of resistance must be responsible for resistance. In order to assess the contribution of transporters and other genes to resistance, particular resistant isolates that did not carry a cyp51A mutation were studied. The relative expression of three novel transporter genes; ABC11, MFS56 and M85 as well as cyp51A, cyp51B, AfuMDR1, AfuMDR2 AfuMDR3, AfuMDR4 and atrF were assessed using real-time RT-PCR in both azole susceptible and resistant isolates, without cyp51A mutations. Interestingly, deletion of ABC11, MFS56 and M85 from a wild-type strain increased A. fumigatus susceptibility to azoles and these genes showed changes in expression levels in many ITR-R isolates. Most ITR-R isolates without cyp51A mutations showed either constitutive high-level expression of the three novel genes or induction of expression upon exposure to itraconazole. One isolate highly over-expressed cyp51B, a novel finding. Our results are most consistent with over-expression of one or more of these genes in ITR-R A. fumigatus without cyp51A mutations being at least partially responsible for ITR resistance. Multiple concurrent possible resistance mechanisms were found in some isolates. My work probably explains the mechanism(s) of resistance in A. fumigatus isolates with cyp51A mutations. Other ITR resistance mechanisms are also possible. To determine taxonomic relationships among A. fumigatus clinical and environmental isolates, the sequences of the ITS, β-tubulin, actin and calmodulin gene of 23 clinical and 16 environmental isolates were analyzed phylogenetically. Actin and calmodulin sequences proved to be good for species differentiation of A. fumigatus while both ITS, β-tubulin regions did not, in this dataset. Many cryptic species of A. fumigates (complex) were found. All environmental A. fumigates complex isolates were ITR susceptible and no cross resistance was found.

Yes / One of the main obstacles to therapeutic success in colorectal cancer (CRC) is the development of acquired resistance to treatment with drugs such as 5-fluorouracil (5-FU). Whilst some resistance mechanisms are well known, it is clear from the stasis in therapy success rate that much is still unknown. Here, a proteomics approach is taken towards identification of candidate proteins using 5-FU-resistant sublines of human CRC cell lines generated in house. Using a multiplexed stable isotope labelling with amino acids in cell culture (SILAC) strategy, 5-FU-resistant and equivalently passaged sensitive cell lines were compared to parent cell lines by growing in Heavy medium with 2D liquid chromatography and Orbitrap Fusion™ Tribrid™ Mass Spectrometry analysis. Among 3003 commonly quantified proteins, six (CD44, APP, NAGLU, CORO7, AGR2, PLSCR1) were found up-regulated, and six (VPS45, RBMS2, RIOK1, RAP1GDS1, POLR3D, CD55) down-regulated. A total of 11 of the 12 proteins have a known association with drug resistance mechanisms or role in CRC oncogenesis. Validation through immunodetection techniques confirmed high expression of CD44 and CD63, two known drug resistance mediators with elevated proteomics expression results. The information revealed by the sensitivity of this method warrants it as an important tool for elaborating the complexity of acquired drug resistance in CRC. / Sadr ul-Shaheed and the University of Bradford Proteomics Facility were supported by Yorkshire Cancer Research, UK (Cancer Medicine Discovery II, grant B381PA).

Vibrio cholerae, l’agent étiologique du choléra, est une bactérie adaptée à l’environnement aquatique et, dans le cadre infectieux, à la colonisation du petit intestin humain. L’environnement intestinal est un milieu a priori hostile aux bactéries externes, à cause de son environnement anaérobie et de la présence des effecteurs du système immunitaire. Capable de produire un biofilm dans ces deux niches écologiques, V. cholerae est capable de persister dans des conditions défavorables et de résister aux molécules antimicrobiennes, y compris les peptides antimicrobiens (PAM). J’ai d’abord étudié la résistance aux antimicrobiens de V. cholerae dans des conditions expérimentales représentatives de l’intestin. Ensuite, j’ai caractérisé, quantitativement et qualitativement, la résistance à la polymyxine B (PmB) des biofilms de V. cholerae, exposés à des concentrations sous-inhibitrices ou létales de ce PAM. Nos résultats suggèrent que la résistance aux PAM de V. cholerae est influencée par la disponibilité de l’oxygène dans le milieu. Je propose également que des concentrations létales de PmB peuvent stimuler un mécanisme de résistance exclusif aux biofilms matures. Les différences soulevées par nos investigations mettent en perspective l’importance d’adapter les conditions expérimentales aux caractéristiques réelles de l’environnement infectieux lors des études de résistances aux antimicrobiens. / Vibrio cholerae, the etiological agent of cholera, is a bacterium that is adapted to the aquatic environment and, in the infectious setting, to the colonization of the small intestine. The intestinal environment is at first glance hostile to external bacteria, due to its anaerobic conditions and the presence of the effectors of the immune system. Capable of producing a biofilm in its two ecological niches, V. cholerae is quite capable of persisting in unfavorable conditions and of resisting antimicrobial molecules, including antimicrobial peptides (AMPs). I first investigated the antimicrobial resistance of V. cholerae under experimental conditions representative of the intestine. Then, I characterized, quantitatively and qualitatively, the antimicrobial resistance of V. cholerae biofilms to Polymyxin B (PmB), at sub-inhibitory or lethal concentrations of this AMP. Our results suggest that resistance to PmB in V. cholerae is influenced by oxygen availability in the medium. I also propose that lethal concentrations of PmB may promote a resistance mechanism exclusive to mature biofilms. The differences raised by our investigations put into perspective the importance of adapting laboratory conditions to the actual features of the infectious environment when investigating antimicrobial resistance.

No description available.